Objective: Plant extracts are widely used, especially in food supplements and cosmetics. Their complexity and, for cosmetics, that animal tests are no longer possible, warrants an innovative approach based on natural molecules to allow for their risk assessment. This study outlines new methodologies developed in the frame of the NCSTOX project to undertake safety assessments of alkaloids based on in silico tools and text mining.

Methods: Instead of assessing each extract, information was gathered on all plant constituents.Focusing on alkaloids, which are present in numerous plants and known to exhibit safety concerns, we validated the methodology.

An innovative multi-step text mining approach, using the integrative ARGO workbench, was combined with the use of various in silico models, including those of VEGA platform, to predict safe levels of use based on 3 key endpoints: genotoxicity, systemic toxicity and sensitization. Critical compounds were identified as those associated with genotoxicity or classified as regulated allergens. Other molecules were classified according to a Threshold of Toxicological Concern (TTC) approach.

Results: Information on 3500 alkaloids was compiled. Out of them, 300 underwent a full safety assessment and the others were screened in silico. Read across was further performed among the 24 different molecular groups.

Around 30% are classified as genotoxic, specially acridones. Non-genotoxic molecules were almost exclusively belonging to Cramer class III.

On a third endpoint: sensitization, half of the molecules are classified as skin sensitizers.

Conclusions: The safety assessment of selected alkaloids validated a new methodology to establish the toxicological profile of plant constituents. Using text mining methods, we curated a novel database to provide the scientific community with animal-free, safe levels of use for plant constituents.

As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Obtaining data for developmental and reproductive toxicity can be particularly difficult, and so a weight of evidence strategy for these endpoints will be illustrated which utilizes new in vitro approaches. Both receptor binding, enzyme activity assays and gene expression studies have been explored as tools to inform on modes of action. Several extracts of both botanicals suspected to have reproductive effects and herbs with a significant history of use were tested against a suite of receptors and enzyme activity assays at biologically relevant doses to probe developmental and reproductive activity at a molecular level. Additionally, gene expression changes in a number of different cell types were analysed using the connectivity mapping approach to identify major modes of action through a functional read across approach. Together, these two data streams have been shown to increase confidence in predictions of botanical biological mode of action, and allow for assessment of relative potency in the decision making process.

Polychlorinated biphenyls (PCBs) are widely spread in the environment, and commonly found in human blood. PCBs have been suggested to be linked to Parkinson’s disease (PD), and non-coplanar PCBs 138, 153, and 180 were shown to have accumulated to a higher level in the brains of female PD patients [1]. To elucidate the association between PCBs and PD risk we applied whole genome gene expression analysis in blood cells from PCB-exposed subjects.

The relationship between gene expression in blood cells and exposure levels of PCBs 118, 138, 153, 170, and 180, was studied in healthy individuals from two European cohorts (369 females, 280 males) using linear mixed model analysis. Significantly expressed gender-specific genes were selected for the individual PCBs, and compared to gene expression data available from the SN of deceased PD patients, to determine similarities.

In particular among females, blood levels of non-coplanar PCBs 153, 170 and 180 were associated with genes specifically related to PD, while none were detected among males. Among these females, 39 genes were discovered which were previously shown to display similar changes in expression levels in the SN of deceased PD patients. Especially among the down-regulated genes, transcripts of genes involved in neurotransmitter vesicle-related functions were predominant.

These investigations support the potential of PCB exposure as a PD-risk factor, and suggest an interesting overlap between the response of brain and blood cells.

[1] JM. Hatcher-Martin, M. Gearing, K. Steenland, AI. Levey, GW. Miller, KD. Pennell, NeuroToxicology. 33, (2012)

Toxicological evaluation of chemicals has been in place for many decades. A number of health based exposure limits (HBELs) such as Occupational Exposure Limits (OELs) for workers or Permitted Daily Exposure (PDE) limits for patients have been used to safely manage worker, patient, and general population health hazards. Conversely, ecotoxicological evaluation of chemicals is currently gaining momentum, especially when it comes to pharmaceuticals in the environment, for which, in the context of Marketing Authorization Applications (MAA), Environmental Risk Assessments (ERA) have to be performed. In contrast to the limits calculated for humans, the outcome of the ERA is a risk calculation based on a Predicted No Effect Concentration (PNEC) for aquatic species from three trophic levels but not considering the potential use of impacted water for drinking water production. There is a large data gap with respect to PNECs for the non-pharmaceuticals (e.g. chemicals), while OELs and even PDEs might be available.

We have evaluated data gaps for ecotoxicological and toxicological hazards of chemicals produced and used at a multipurpose manufacturing site in Switzerland. We are proposing a decision tree to identify priority substances based on existing toxicological and ecotoxicological data with the aim of reducing emissions and filling knowledge gaps for possible technical measures to reduce their emissions in wastewater in line with Swiss regulatory requirements. Additionally, we are presenting a case study for one of the large volume chemicals.

Objective Diphenylcyanarsine(DC) is a irritating agent. Subchronic inhaling toxicity study of DC in SD rats had been done and provided the basis for establishing the environmental protection standard. Methods The groups of 20 SD rats of each sex were inhaled exposure with 0, 0.00036, 0.0011 and 0.0031μg/L of DC for 6h/d, 5d/w for 13 weeks by dynamic inhaling exposure device. The general physical conditions of rats had been observed everyday. The body weight of rats were measured and the mean food intakes per 100g BW were calculated every week. The eyes and the main breath indexes of rats had been examinated in the end of exposure. Hematological and blood biochemical indexes were analyzed and histopathology observation were evaluated in one quarter of rats every month. Results The results indicated that a few rats emerged irritating effects, such as scratching mouth, washing face, shedding tears and blood trace in eyelids side after 15-40 days of exposure in 0.0031μg/L group. The individual animals emerged blood trace in eyelids side after 60 days of exposure in 0.0011μg/L group. The results of the eyes check indicated that 12/19 rats emerged median conjunctival congestion in 0.0031μg/L group and 5/20 rats emerged slight conjunctival congestion in 0.0011μg/L group. The tidal volume of rats were significantly declined in 0.0031μg/L group compared with that of the control group. The average red blood cell volume, hemoglobin content and the thrombocytocrit were significantly increased in 0.0011 and 0.0031μg/L group. The concentrations of K, TP, ALP and the activity of LDH of serum were increased in high and median dose group, the concentration of glucose was significantly declined and the concentration of BUN was significantly increased in high dose group after 90 days of exposure. The female showed profound toxic effects compared with the male. The damages of throat, windpipe, lung, liver, kidney were more obvious in the rats exposured to high dose DC for more than 30d, while the damages of these organs were obvious in the rats exposured to the median dose of DC for 90d. These damages of organs were reversible. Conclusions The main target organs of toxic effects of DC on the rats (ih) were throat, windpipe, lung, liver, kidney. The estimated dose of NOAEL for DC was 0.00036μg/L and LOAEL was 0.0011μg/L in SD rats inhaled exposure for 13 weeks.

An integrated approach to the understanding of effects, including the significance of metabolic processes, is highly desirable in a regulatory context such as REACH, for reasons explained in this poster. The integration of health and environmental regulatory endpoints can help to maximise the scientific credibility of chemical safety assessment. It is a common problem that data used for regulatory purposes have not been integrated across the endpoints.

This poster shows how there can be substantial overlap in the scientific issues affecting human and environmental toxicology; at an overview level, the example of alkanes and aliphatic alcohols is presented, alongside wider discussion. They are considered together due to the common metabolic issues: alkanes and alcohols metabolise via aliphatic acids. The metabolites are of significance biosynthetically.

The pathways themselves are shown, taken from standard literature sources. These pathways are highly conserved in mammals and micro-organisms alike. Bacterial degradation is critical for the understanding of fate of the substances in waste water treatment, surface water and soil.

Where hazard exists, the REACH Regulation calls for the setting of a derived no-effect level (DNEL) for comparison with human exposure levels. A predicted no-effect concentration (PNEC) to protect ecosystems is derived from available laboratory studies. The toxic effects of alkanes and alcohols in mammals are (with specific exceptional compounds) limited to local effects and are reviewed briefly. For aquatic organisms, hazards are found in laboratory studies. The significance of these observations, given the essentiality of the substances, is reviewed.

Given the essentiality of alcohols and acids it is highly questionable as to whether the standard ‘assessment factor’ approach is relevant to substances such as alkanes or alcohols. An alternative approach applicable to all aspects of (eco)toxicology, is suggested. In summary, this requires the assessor to consider the significance of the hazard and to limit how low a DNEL or PNEC can be, based on the natural exposure concentrations in the bodies of organisms.

Rapid agricultural, medical and industrial development is occurring on a global scale and bringing with it emerging environmental threats for humans. Contamination by rare earth elements (REE) has emerged as a public health concern due to their numerous applications in the modern industry. However, little is known about their toxicological effects despite their accumulation in different organs including brain and bone. To determine exposure to these contaminants in a young Spanish population, scalp hair samples were collected in 37 young adults (20 to 24 years-old; 28 female and 9 male) from different towns in the Community of Madrid (Spain). Despite being controversial, human hair could be an appropriate tool to determine environmental exposure to inorganic metal contaminants and to estimate the chemical burden in the individual. Lanthanum (La), cerium (Ce), praseodymium (Pr), erbium (Er) and gadolinium (Gd) were analysed in these samples by ICP-MS following appropriate methodologies. The limits of detection were (in ng/g): La (1.87), Ce (4.29), Pr (0.47), Er (0.06) and Gd (0.24 ng/g). Gd was detected in only one of the monitored samples (2.66 ng/g). The concentrations were as follows (median and interquartile range are provided in ng/g): La 5.30 (4.22, 7.13), Ce 11.18 (8.97, 15.45), Pr 1.28 (1.04, 1.72) and Er 0.19 (0.14, 0.28). In general, the presence of these metals in the Spanish group’s hair samples were lower than those reported in environmentally exposed groups, which may indicate that the studied group would have a low exposure to REE. None of these elements showed influence due to sex, although slightly higher levels were observed for La (5.57 vs. 5.17 ng/g), Pr (1.40 vs. 1.27 ng/g), Nd (2.48 vs. 2.29 ng/g) and Er (0.21 vs. 0.19 ng/g) in men’s hair and in women’s hair for Ce (11.58 vs 10.30 ng/g). Our results would be in agreement with those studies that have suggested that men may be more sensitive to REE than women.

Acknowledgement: This work was supported by Project FP-16-RESIDUA (IMIDRA, Comunidad de Madrid).

Fine particulate matter (PM_2.5) has been paid much attention in recent years. This study aims to analyze the scientific output and investigate the trend of PM_2.5 research. PM_2.5 related publications from 2007 to 2016 were retrieved from the Web of Science Core Collection database. Microsoft Excel 2016 and CiteSpace V software analyzed publication trend, countries/territories, journals, institutions, research areas, authors, citation counts and research frontiers. As a result, a total of 13681 papers on PM_2.5 research were published by with a time limit of December 1, 2017, and the quantity of publications on PM_2.5 continued to grow. The USA had the most publication (5941), citation (217,009) and H-index (172), followed by China, considered as the leading countries in this field. The journal Atmospheric Environment had the highest number of publications. Querol X has published the most papers (187), and Pope CA had the highest co-citation counts (5019) in this field. Furthermore, “climate-change,” “cardiovascular mortality,” and “long-term exposure” are becoming popular and should be closely observed in this field. In conclusion, PM_2.5 is closely related to people's long-term health and we should understand it better, study it deeper, and try our best to reduce it.

A key event of the adverse outcome pathway (AOP) for skin sensitization is the activation of dendritic cells. In an attempt to integrate the impact of the skin environment created by stratified keratinocyte layers on the activation of dendritic cells, we investigated whether available 3D reconstructed human epidermis (RHE) models can be cocultured together with THP-1 cells, as surrogate dendritic cells, to assess chemical-induced dendritic cell activation in vitro. For this purpose, THP-1 cells were placed underneath the RHE, which was topically exposed to increasing concentrations of chemicals with variable physicochemical properties and with different reaction mechanisms and sensitizing potential (eugenol, coumarin, ascaridole, cumine hydroperoxide and dibutylanaline). Cell surface expression of the costimulatory molecule CD86 and the adhesion molecule CD54 on THP-1 cells were analyzed by flow cytometry. Preliminary results are promising and we conclude that the coculture THP-1/RHE may allow the investigation of compounds with varying physicochemical properties and exposure at the air liquid interface.

In silico approaches and read-across are recognized as preliminary concept to perform a safety assessment (SA) without animal testing. To facilitate read-across, various in silico tools were developed in recent years, providing the basis for a next generation SA strategy. In addition, several authorities (ECHA, OECD and ECETOC) published related guidance documents. However, there is limited consensus in how to select and use each tool. Taking the first step towards clarification of this issue, several case studies were performed using raw materials such as hydrogenated polyisobutene and 2-ethylhexyl 12-hydroxystearate as well as a complex mixture of fatty acids. The aim was to establish a feasible SA strategy based on a combined use of available in silico tools. The general toxicity of compounds was assessed by means of the three tools AMBIT, COSMOS and ToxRead in order to select similar chemicals based on their chemical structure. The tools use different chemical descriptors and algorithms to calculate the pairwise similarity of compounds. Physico-chemical properties were evaluated by EPI-SUITE. The sensitization potential was predicted via TIMES-SS, followed by a quantitative risk assessment (QRA) using literature-based EC3 values. Although there is no clear definition for the threshold used to define an acceptable pairwise similarity of chemicals, retrospective analysis of published read-across case studies revealed that a similarity of 70% seems to be acceptable. AMBIT, ToxRead and COSMOS proposed differential but also overlapping analogues for the different compounds. The most conservative toxicological profile (e.g. lowest NOAEL) was selected among the identified analogues to perform the SA. TIMES-SS evaluation of the complex mixture identified nine of twelve fatty acids as non-sensitizer, while the remaining three fatty acids were predicted to have a weak to strong sensitizing potential. QRA based on the EC3 values of these three fatty acids indicated that no sensitization risk must be considered under the expected consumer exposure level. In summary, our case studies showed that a combined use of different in silico tools can result in a sufficient conservative SA. Moreover, several known chemical spaces can be covered complementarily by a combination of tools.

Consumer exposure to cosmetic (personal care) products is mostly by dermal contact. However, additional considerations with regards to potential inhalation exposure from some cosmetics, such as sprays and powders, may be needed for a robust and reliable safety assessment.

To get a deeper understanding of the exposure to airborne particles from loose or pressed powders during product application, sufficient data on their diameter and typical doses are key. Our work describes the principles for inhalation exposure to such solids, and the derivation of safe exposure levels for cosmetic powder products. The general principles described herein are the same as for spray products as published earlier (Steiling et al. 2014). However, in contrast to liquids, where droplet maturation is important when the product is sprayed, airborne powders do not typically exhibit maturation, although aggregation and/or agglomeration of particles may occur. Moreover, certain characteristics of the powder like its dustiness also need to be considered. We have developed a decision tree with the basic elements of a tiered inhalation safety assessment approach for the different kinds of powder formulations. Furthermore, an overview of existing available inhalation in silico prediction models and real-time measurement approaches is given, as well as typical habits and practices data which are needed for the risk assessment of powder formulations following the tiered approach.

The research project aims at the development of an animal-free testing strategy for the prediction of respiratory toxicity in vitro (funded by German Federal Ministry of Education and Research; Acronym: AeroSafe, 031L0128A-D, 2017-2020). Overall goal is to reduce the number of animal experiments needed for safety assessment by establishing a standardized in chemico/in vitro test strategy. Based on available in vivo data of the test materials and mechanistic knowledge, AeroSafe focuses on inflammatory effects, modulation of epithelial barrier function and activation of the innate immune system. AeroSafe pursues to establish the simplest and most convenient but appropriate test strategy. We include approaches for analyzing protein reactivity and effects on target cells, imitating the complex in vivo situation. Starting from the first line of defense in the alveolar region, AeroSafe is focusing on macrophages followed by coculture of macrophages with alveolar epithelial cells, and consequently further target cells, both using cell lines and primary cells. Add on, delivery of test materials via exposure chambers is included. The Research strategy is clearly structured in a bottom-up approach – from in chemico and/or single cell cultures to complex cocultures. In doing so, we will select the number of models required to assess respiratory toxicity.

In sum, AeroSafe includes testing of representative structures of three different classes with in total 30 different materials consisting of nanomaterials, pharmaceuticals and low molecular weight respiratory allergens. Obtained in chemico/in vitro data will be used for comparison with available in vivo findings. Consequently, single or multiple test strategies will be deduced. The outcome of AeroSafe is expected to considerably contribute to significantly reduce the currently high number of so far indispensable animal experiments.

Aflatoxins (AF) are toxic, immunosuppressive and carcinogenic compound; AF often contaminate agricultural commodities intended for human and animal consumption. AF are produced as secondary metabolites of Aspergillus spp., which can be influenced by water-temperature stress. The objective of study was to evaluate the association between climatic factors and the occurrence of AF contamination in the dairy feed and their residues in milk. Three dairy farms (DF) located in Aguascalientes, Mexico (21°48’N, 102°03’ W; 2000 mamsl; semi-temperate climate) were selected by non-probabilistic convenience method. DF were monitored monthly for two years (2015-2016). AF were tested by HPLC and ELISA in 266 samples of total mixed feed and 156 samples of raw milk; samples were obtained directly from each feeder and from the milk collection tank for each batch of dairy cows. The climate information of the near (<15 km) Huizache Weather Station (Num. 48291) of the National Network of Stations of the INIFAP was accessed; data were analyzed by one-way ANOVA, logistic regression and correlation using statistical software. AF contamination were detected in 99% of feed samples and 27% of milk samples; one sixth of them (17.3 and 16.7%) with AF concentrations above the maximum permissible limit (MPL, 20 µg/kg and 50 ng/kg). The AF concentration in the cows diet were positively associated (P<0.05) with the daily average temperature (DAT) and precipitation (DAP; P<0.01, R-square 37.6%); DAT and DAP, in turn, were associated with relative humidity and potential evapotranspiration. Other participating factors (cold hours, radiation, wind, and annual or DF-associated effects) did not show statistical significance. Compared to autumn and winter, the summer season recorded a higher DAT (18.2-18.7^a, 15.4-15.9^b and 13.9-14.5^c °C, IC 95%, Tukey´s HSD test); but due to lower rainfall, spring had the highest DAT (19.6-20.2°C, P<0.05). Consequently, the incidence of milk and feed samples with AF above MPL were higher in the summer than in autumn and winter (Relative Risk: 1.4, 12.9 and 1.6, 5.4, respectively), but not that spring (0.9 and 1.0). These results suggest that the AF occurrence in the dairy food chain is related to DAT-DAP combination, so it is expected that the risk of exposure and damage may increase in predictable global warming scenarios.

The TTC is a pragmatic risk assessment screening tool to establish a safe exposure level for substances without an adequate toxicological data set. This approach has found acceptance among regulators globally. However, since the Munro database for non-cancer endpoints, on which the TTCs for non-genotoxic compounds were based, did not contain any organosilicon compounds, some regulators exclude this substance class from the TTC approach.

We evaluated all public available robust study summaries of oral repeated dose and reproductive/developmental toxicity studies of organosilicon compounds to confirm whether the TTC approach can also be applied to this chemistry. Only studies that were found to be adequate for the use as stand-alone studies for human health risk assessment were taken into consideration. If more than one study was available per substance, the one providing the most human relevant point of departure for risk assessment was chosen as the key study for this project.

Reliable and relevant high-quality key studies for 46 different organosilicon compounds could be identified, representing the chemical space of low molecular weight siloxanes, silanes and silanols. For repeat dose studies uncertainty factors were used to extrapolate to chronic exposure.

All identified NOAELs, with appropriate uncertainty factors, were compared to the Munro Cramer Class III TTC threshold, the default substance class for silicon-containing substances. Based on this comparison, in all cases, the derived human risk values for silicon-containing substances were greater than the Cramer Class III threshold.

The health-based values for lifetime exposure of contaminants in drinking water have been established as “Drinking Water Quality Standards (DWQS)” and notified as target values for “Complimentary Items” of the DWQS under the Japanese Water Supply Act. In addition to these two criteria lists, chemicals that can be detected in natural water sources but not yet managed are listed as “Items for Further Study” because of provisional risk assessment. We previously derived Subacute Reference Dose (saRfD) for 35 chemicals from the above three criteria lists to propose the subacute guidance values. We present the latest updates for 8 chemicals listed as “Items for Further Study” and an overview of our work. To derive the saRfD, the NOAEL or BMDL₁₀ of a one to three-month study was basically used, and a total uncertainty factors of 100 was generally applied. Three out of 8 chemicals were genotoxic carcinogens. Therefore, 10 times of the Virtual Safe Dose at 10^-5 risk was defined as the saRfD for these chemicals. The saRfD for perchloric acid was evaluated as same as a TDI, because the TDI was based on the human short-term effect. By using the saRfDs, subacute guidance values were culculated. The most of subacute guidance values for 43 chemicals became three to several dozen times as high as the corresponding health-based values for lifetime exposure. Especially, as for genotoxic chemicals or reproductive toxic chemicals, the subacute guidance values are less than ten times. Sources of water supply can be transiently influenced by local weather or chemical accidents. In such cases, the subacute reference values we proposed are considered to be useful for risk management. ACKNOWLEGMENT: This study was supported by a Health and Labour Sciences Research Grant (H28-Kenki-Ippan-005) from the Ministry of Health, Labour and Welfare, Japan.

Due to the rapid development of the aviation industry and high demand for air transportation, the concomitant airport pollution has attracted increasing attention in recent years. Airport particulate matter (PM) emissions are the known source of air pollution in the proximity of an airport. Often large airports are located near metropolises, and airport emissions may have a potentially considerable impact on public health in the surrounding urban areas. However, little is known about the sources that are relevant to air quality and health in the vicinity of airports. Therefore, the effect of the chemical composition of airport-related particulate matter (PM) on adverse health risks was investigated in comparison to urban traffic emissions.

PM_0.25were collected at the Los Angeles International Airport (LAX) and at a central Los Angeles site (University of Southern California, USC campus), along with PM_2.5collected directly from turbine and diesel engines. The chemical composition, oxidative potential (OP) (measured by means of ascorbic acid (AA), and electron spin resonance (ESR)) of particles as well as the reactive oxygen species (ROS) activity, inflammatory potential (interleukin (IL) 6 and 8 and tumor necrosis factor (TNF) –α release) and cytotoxicity on human bronchial epithelial (16HBE) cells were assessed. From the results we obtained, chemical composition measurements confirmed that aircraft emissions were the major source to LAX PM_0.25, while the sources of USC samples were more complex, including traffic emissions, suspended road and soil dust, and secondary sulfate. The traffic-related transition metals (Fe and Cu) in LAX and USC samples mainly affected OP values of particles, while multiple factors such as compositions, size distribution and internalized amount of particles contributed to the promotion of ROS generation in 16HBE cells during 4 h exposure to 10 μg/mL particles. Internalized particles in cells might also play an important role in activating inflammatory responses during cell recovery period, with LAX particles being more potent.

Our results demonstrate considerable toxicity of airport-related particles, even at low exposure concentrations, which suggests that airport emission as source of PM_0.25 may also contribute to the adverse effects on public health attributable to PM. The potency of such particles is in the same range as those collected at a site in urban area with dense traffic.

As a group of emerging unregulated disinfection by-products (DBPs) in drinking water, N-nitrosamines are perceived to pose a greater health risk due to their strong carcinogenicity and mutagenicity. In this study, the presence of nine nitrosamines in source water and finished water from eleven drinking water treatment plants in Nanjing with a low incidence and Huaian with a high incidence of esophageal cancer was evaluated by a solid phase extraction-gas chromatograph-mass spectrometry (SPE-GC-MS) method in March, October 2017 and January 2018. Overall, N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosopyrrolidine (NPyr) topped the first three commonly detected nitrosamines list and the detection rate was 71.0, 48.4 and 37.1%. The highest concentrations of them in source water were up to 119.8, 57.3, 22.9 ng/L, and in finished water were 58.3, 51.0 and 21.0 ng/L, respectively. The occurrence of N-nitrosodimethylamine (NDMA) was in 19.3% of water samples and the average and maximum value were 2.2 and 49.4 ng/L. Meanwhile, the average exposure level of total nitrosamines in source water was 2.9 times as high as that in finished water. Seasonal distribution of total nitrosamines showed that samples in October had the highest level, followed by January and March. It was obviously high to compare detection rate and concentrations of NDPA, NDBA, N-nitrosodiethylamine (NDEA) and total nitrosamines in water samples from Huaian with these of water samples from Nanjing. The risk level indicated that the average carcinogenic risk of total nitrosamines in finished water from Huaian district was 4.8 times that of Nanjing. Among them, NDPA, NDBA and NDEA were the main carcinogenic risk contributor for waters from Huaian, and NDMA was the major risk contributor for waters from Nanjing. These findings showed that N-nitrosamines were popular in drinking water and may be a potential health risk factor for population and the local water environment. It is very important for regulators to reinforce evaluation of the health risk with long-term exposure to N-nitrosamines in drinking water. (Supported by National Natural Science Foundation of China, 81573191).

Lithium-ion battery storage is becoming more frequent as the use of this technology increases due to their high energy and power densities. Exceeding the thermal stability limits of a lithium-ion cell can result in thermal runaway and such failures can generate intense heat, smoke and toxic gases. A human health and toxicological risk assessment of hydrogen fluoride (HF) was undertaken for a lithium-ion battery testing facility to assess potential HF exposures to staff if thermal runaway of lithium-ion battery occurred during testing. While instances of thermal runaway occurring are rare, assessment of risk to worker health was still recommended. The project required consideration of acute exposure duration (seconds) under a defined workplace scenario. The key exposure pathway assessed was inhalation as HF is readily absorbed in the upper respiratory tract which presents the most sensitive target of acute toxicity.

The assessment required estimation of emission rates for HF and subsequent conversion to HF indoor air concentrations. Emission rates of HF depended on the state of charge of the batteries and this was also incorporated in modelled scenarios adding to the complexity and range of results. Due to the acute exposure durations assessed (5 to 15 seconds) obtaining appropriate short-term acute exposure standards or toxicological guidelines was a challenge. Occupational exposure limits and emergency guidelines were considered and Haber’s Law was used for time-scale downward extrapolations of the selected acute guidelines.

The results showed that the modelled indoor air concentrations exceeded the selected guideline and the potential health consequences required further analysis. A toxicological assessment of the concentration-time response was undertaken to characterise potential risks to workers. Combining risk assessment principles with toxicological understating of HF behaviour allowed a more detailed assessment of the actual (likely) risks. Consideration of mitigation measures was then undertaken to provide an aid in reducing the risks inferred by the study. The project illustrated the challenges and complexities in assessing rare emergency exposure scenarios that may not fall within the routine guidance and methodologies employed in environmental contaminants risk assessment.

The concern about container-content interactions has really increased this last decade, especially with the growing preoccupation about endocrine disruptors. In fact, lots of plasticizers are endocrine disruptors and are able to migrate from packagings into products. In cosmetic industry, these studies are complicated since no protocol exists in actual regulations. Moreover, there are no acceptance thresholds available for potential contaminants. In this context, a strategy of risk evaluation were developed, combining migration tests and safety evaluation for container-content interactions. Due to the complexity of cosmetic matrices, the study were made on simulants, as for studies in food industry. In a previous work, aqueous simulants such as water or ethanol were used to develop the strategy. Positive results led to extend this work to oily simulants that represent an important part of beauty products. Unlike aqueous simulants, oily simulants cannot be directly injected into the analytical system. An extraction step is necessary. In order to obtain the best results possible, two extraction methods were compared: liquid-liquid extraction and solid phase micro-extraction (SPME). Liquid paraffin and glycerin were chosen as oily simulants, and put in contact with eleven selected plastic packagings. As for aqueous simulants, 12 potential contaminants were screened: 10 phthalates, bisphenol A and distearyl thiodipropionate (European Pharmacopoeia plastic additive n°17). After one month at 50°C and extraction steps, the oily simulants were analyzed by GC-MS and obtained results were evaluated using a safety evaluation developed specifically for this study. This work complete the container-content interactions evaluation realized on cosmetic simulants and corroborate the strategy developed.

Among metallic trace elements that can be present in food, some are known as toxic and must be monitored. Regarding food safety, it is relevant to study the case of the pita since it represents a large part of the daily diet of the Lebanese population. The objective of this study was primarily to quantify As, Cd, Co, Cr, Hg, Ni and Pb in Lebanese pita. Then, to evaluate the consumer exposure to metal contaminants via white pita consumption, a survey was carried out in supermarkets of 5 different Lebanese regions. Thousand individuals (half men, half women) were grouped as follows: 800 adults (15 years and above) and 200 children divided in two categories (6-9 and 10-14 years). The exclusion criteria were chronic or severe pathology patients and adults who had stayed in Lebanon for less than 15 years. Different bread brands, sizes and numbers of pita consumed per day were taken into consideration.

Pitas were sampled according to EU regulation No 836/2011. Three bags of bread were collected from 3 bakeries (B1, B2 and B3), and 3 pitas out of 8 were taken from each bag. The mineralization of samples was carried out in nitric acid and hydrogen peroxide using microwave digestion. As, Cd, Co, Cr, Ni and Pb were analyzed by atomic absorption spectrometry and Hg was analyzed by a direct mercury detector. To ensure the methods reliability, a certified reference material (BCR-191) was tested at the same time as samples.

The metal contents were different depending on the selected bakeries. The highest quantities of Ni and Co were found in bread of B1, As, Pb and Cd in bread of B2, and Cr and Hg in bread of B3. Maximum limits in Lebanese bread are only defined for Pb and Cd. The content of these elements in breads respect the Lebanese standard.

The human dietary exposures of the population categories were compared to toxicological reference values, when available, to characterize the risk for the Lebanese consumer. The population between 6 and 9-years-old was the most exposed to As, Pb and Co. Concerning these elements, the highest exposures (95^th percentile) were above toxicological reference values or with a margin of exposure too low.

Many molecules are found under the generic name of silicones. Applications are numerous and silicones are found in industrial, pharmaceutical, medical device and personal care products.

Revisiting the concept of biodegradation and degradation in such a complex family is challenging. For the purpose of this study fluid silicones with various viscosity levels were used, such as those found in the cosmetic industry.

Biodegradability was investigated using in silico tools (Epiwin and QSAR toolbox) together with in vitro OECD approaches.

In parallel, a non-conventional approach was used with the aim of studying potential relevant effects such as a solar simulator to study UV effects combined with abiotic degradation in wet and dry soils.

Combining the results of each approach lead to the following observations:

A low level of polymerisation leads to easily hydrolysed silicones by enzymatic and non-enzymatic reactions of the grafted carbon chain. A higher level of polymerisation provides greater protection against degradation.

The ultimate stage, represented by the siloxane backbone degradation, may be obtained by the combined effect of bacteria and UV light leading to formation of silicates.

Physico-chemical properties, in part driven by the polymerisation level, play a crucial role as they influence the physical state: solid, liquid, volatile, gel. Functionalisation of alkyl dimethicone compounds is also a key parameter for biodegradability.

Consequently (bio)degradability should be approached on a case-by-case basis.

Even if (bio)degradation, with respect to the OECD definition for very high molecular weight silicones, is not totally achieved, a degradation of lower molecular weight silicones, as used in the personal care industry, is achieved by other means such as UV and abiotic transformation.

The harmful effects of indoor and outdoor air pollution on health are of great concern in several regions of the world. Several epidemiological studies have established a correlation between exposure to atmospheric pollutants and the onset of respiratory pathologies. Dakar, the capital and biggest city of Senegal, is characterized by a vehicle fleet characterized by poorly efficient combustion technologies that largely contributes to the deterioration of air quality. The aim of this study was to assess individual exposure level to air pollution and its potential health impact on professionals (bus drivers and traders) and housewives in Dakar city center, where road traffic is particularly dense and in a rural area with minor traffic related emissions.

BTEX analysis revealed that toluene was the volatile pollutant with higher concentrations, ranging from 800 to 1950 µg/m³. Benzene and xylenes concentrations were from 150 to 650 µg/m³ while ethylbenzene was always around about 50 µg/m³. Benzene concentration was significantly higher for the housewives group (649 and 568 µg/m³ respectively for urban and rural sites) than for professionals (192 and 425 μg/m³ respectively for bus drivers and traders). The high concentrations found for housewives is probably due to the cooking habits (coal used in kitchens as combustible), local practices (important incense burning) and use of cleaning products that are important emitters of volatile organic compounds. Different urinary metabolites were selected to assess human exposure. t-t-MA was not detected (below the detection limit) in urine samples, while S-PMA values were higher for housewives (1.3 and 1.2 µg/g creatinine for urban and rural sites respectively) when compared to professionals (1.1 and 0.8 µg/g creatinine for traders and drivers respectively). The level of 1-HOP, used to assess a pyrene exposure, was slightly higher in drivers (0.4 µg/g creatinine) compared to others groups (0.2, 0.3 and 0.2 µg/g creatinine respectively for traders, city housewives and district semirural housewives). Statistical analysis revealed that urinary 1-HOP levels were significantly higher for urban site housewives compared to those at the semirural district. These results showed that indoor air pollution seems to be greater than outdoor air pollution, confirming previous observations on the BTEX exposure level and therefore indicating a non-negligible risk also for indoor exposure to benzene.

Classical analytical methods to monitor water quality have their limitations: i) not all chemicals of emerging concern can be measured sensitively enough in water, ii) they do not assess the presence and levels of all unknown chemicals (e.g. transformation products), and iii) they do not assess chemical mixture effects. For these reasons, the use of bioassays to assess the health risks associated with chemical contaminants in water has been promoted these past few years. We propose a battery of sensitive, 3R-based and high-throughput bioassays, covering ecologically- and human-relevant toxicity pathways. These effect-directed monitoring (EDM) tools will assess the ecological and human health risks of chemicals in different types of water bodies taking into account the whole complex mixture of compounds (known and unknown). To do so, we linked the protection goals for humans and ecosystems described in (supra)national guidelines and a list of priority chemicals with related adverse outcome pathway (AOP). The most critical and sensitive adverse outcomes (AO) were selected, from which we compiled a set of key events (KE) to test in 3R bioassays. We chose the most suitable bioassays to measure these KEs, predicting critical ecological or human endpoints. This way, a toxicological “fingerprint” of a water sample can be obtained by testing the sample in the proposed 3R test battery. Thanks to the integrative information provided by these EDM tools, the risks assessment of water bodies should be more relevant for water quality monitoring.

In 2014, a guideline of the EMA was published introducing the concept of Permitted Daily Exposure (PDE) values. The approach for setting PDE limits is the same outlined in ICH Q3C consensus guideline on residual solvents and in ICH Q3D consensus guideline on elemental impurities. The background for that was the request of the pharmaceutical industry to be able to decide itself on a risk-based approach whether a product can be produced in a multipurpose facility, or not.

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Medicinal plants are an integral part of traditional medicine since ancient era. Homeopathy is a natural form of medicine used by over 200 million people worldwide to treat both acute and chronic conditions. It is based on the principle of ‘like cures like’. In other words, a substance taken in small amounts will cure the same symptoms it causes if taken in large amounts.

The homeopathic ophthalmic ointment containing Echinacea purpurea (3%), Euphrasia officinalis (5%) and Calendula officinalis (5%) extracts is used for treatment of conjunctivitis and other inflammatory disorders of the eye and its annexes. A thin line of the ointment (corresponding to around 18 mg) has to be placed along the inside of the lower eyelid.

A common approach for the selection of the Point of Departure (POD) and Adjustment Factors (AFs) can be applied to the three extracts. The three extracts’ toxicological and pharmacological profile is poorly characterized and there are few useful quali-quantitative data to be used to allow a NOAEL or LOAEL determination, so the PDE for the three extracts is based on the lowest ocular recommended dose of 1.62 mg/day (Echinacea) or 2.7 mg/day (Euphrasia and Calendula) in adults.

Regarding selection of the AFs have to be considered: intra-species and inter-species variability, duration of therapy, adverse effects, therapeutic dose and bioavailability adjustment.

The resulting calculated ocular PDE values for each extract are the following: 162 µg/day for Echinacea purpurea extract; 270 µg/day for Euphrasia officinalis and Calendula officinalis extracts.

The goal of this research is to apply a category-based read-across approach toward identifying a point of departure (POD) for the alkyl-substituted urea compounds (ASUCs) with no relevant toxicological data. The ASUCs are commonly used as reaction intermediates in the production of both polymers and pesticide products, and nearly 20 ASUCs were identified without adequate data to derive a chemical-specific POD.

Boundaries for the chemical category were established based on the presence or absence of specific structural attributes, and in order to identify ASUCs with relevant toxicological data, 13 ASUCs previously detected as drinking water contaminants by NSF International were profiled in the OECD QSAR Toolbox software (version 4.1). In this manner, 15 additional ASUCs with relevant data from OECD guideline-compliant studies were identified, which were analyzed to determine systemic and reproductive/developmental NOAELs. Additionally, this software tool was utilized to address mutagenicity and clastogenicity data gaps for ASUCs with no relevant data for these endpoints.

The available OECD guideline-compliant mutagenicity and clastogenicity data on the evaluated ASUCs, combined with data gap filling using read-across, suggests low genotoxic concern. The most conservative point of departure (POD) identified was 30 mg/kg-day, a NOAEL derived from a developmental toxicity study on 1,3-dimethylurea, which is supported by 50 mg/kg-day NOAELs from 28-day and 90-day repeated dose studies on 1,3-dimethylurea and diethyldiphenylurea, respectively. The critical effects associated with this POD included reduced maternal food intake (21%), reduced maternal body weight gain (66%), and increased incidence of fetuses with hydroureter. As the POD derived from 1,3-dimethylurea is based most sensitive adverse effect for the entire category, it is therefore adequately health-protective for read-across to other ASUCs for which no chemical-specific POD can be derived.

The heavy metals coming from the effluents of industrial facilities, predominant of which are leather tanning and jewelry factories, render the Meycauayan River in Bulacan, Philippines polluted. Through bioaccumulation, the heavy metals in water are eventually retained in the tissues of fishes cultured in fishponds downstream of the river such as Nile tilapia (Oreochromis niloticus L.).

This study assessed the potential risks to human health that may result from consuming Nile tilapia exposed to heavy metal contaminated waters from four (4) fishpond sites located downstream of the Meycauayan River. Tilapia samples were collected in March 2017 and were analyzed for the presence of cadmium (Cd), lead (Pb), nickel (Ni), chromium (Cr) and manganese (Mn) using atomic absorption spectrometry; and for mercury (Hg) using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Target Hazard Quotient (THQ) was used to estimate the associated health risks on long-term consumption. Biomarkers were also measured to evaluate the level of oxidative stress affecting the Nile tilapia samples.

The heavy metals chromium, nickel, lead, and manganese were found to be bioaccumulated in the edible tissues of Nile tilapia samples wherein nickel and lead concentrations exceeded international quality standards for fish and fish products. Calculated THQs show that lead (Pb) contributes the greatest risk to human health from all the pollutants that were analyzed in the fish tissues. The activity of enzyme biomarkers, catalase and superoxide dismutase and the lipid peroxidation level in tilapia were also found to be significantly elevated.

From a public health perspective, long-term consumption of tilapia obtained from Meycauayan River is not recommended due to adverse effects which may be contributed by the highly elevated level of lead.

The default of dermal absorption is fixed as 10% in Korean guidance unlikely to EFSA guidance (2017) for operator risk assessment of plant protection products. In case of application of EFSA guidance, the risk was considered to increase because 10% dermal absorption is minimum default. Default of dermal absorption was classified for 169 products according to EFSA guidance. 24 products were classified as 25% dermal absorption in mixing and 75% in application, 107 products as 10% in mixing and 50% in application. It was investigated to change the operator risk through assessment by different default of dermal absorption with Ko-POEM in 223 cases of 169 products by crops. 33% of cases without PPE and 19% of cases with PPE were indicated the risk was high in Korean guidance. 61% of cases without PPE and 52% of cases with PPE were indicated the risk was high in EFSA guidance. The cases of using oral absorption were 40 and 5 cases among these were indicated that the risk was low. As a result, the high risk assessed by the different default of dermal absorption was increased 2.6 times compared with only 10% default

The world’s population is ageing rapidly. Elderly are a well-recognised susceptible population due to the decline of immune defences and the burden of multiple chronic diseases. As a susceptible population, the burden of environmentally induced disease and lifestyle factors in the elderly is an increasing concern. The major public health goal for older adults is to prevent disability and to enhance active life expectancy. Several studies established a link between genomic instability and age and age-related disorders. Physical activity, on the other hand, has been demonstrated to be effective in the prevention and treatment of different chronic conditions, including age-related disorders. Thus, it is hypothesised that physical exercise programs should impact some biological endpoints. The present study sought to investigate the impact of regular exercise in the evaluation of genomic biomarkers in the elderly. Lifestyle factors were also addressed to account for its possible influence in the results obtained. A group of around 70 older adults (≥65 years old) was engaged in this longitudinal study. A physical exercise programme of 9 months was ministered to the participants. Blood samples were collected before and after the physical exercise programme for genomic biomarkers evaluation. Primary DNA damage and oxidative DNA damage were measured through comet assay and H2AX phosphorylation was measured through gH2AX assay. A life-course exposure questionnaire was also included to characterise and identify lifestyle factors and key exposures. Data here obtained addresses the differences between the levels of primary DNA damage, oxidative DNA damage and H2AX phosphorylation before and after the physical exercise programme. The influence of key exposures and life-style factors, such as the consume of home-produced vegetables, living near traffic zones, smoking habits, etc, was also observed. The ageing phenomenon offers great challenges for all societies, being important to ensure that the increase in longevity is accompanied by the preservation of health and good quality of life. Obtaining other conclusions on the biological effect of regular exercise may help to clarify its role in prevention of age-related diseases. Acknowledgments:Armanda Gomes and Solange Costa are supported by FCT under the grants SFRH/BD/121802/2016 and SFRH/BPD/100948/2014, respectively. Vanessa Valdiglesias is supported by Xunta de Galicia postdoctoral fellowship (ref. ED481B 2016/190-0).

Purpose: Among anticancer agents, cytotoxic drugs display nonspecific activity towards both tumor and the normal cells which is responsible for their numerous and severe side effects. The toxicity of these agents depends on several parameters like their intrinsic carcinogenic character, concentration in tissues and their length of contact with the same tissues. Then, in case of fortuitous contamination of healthy people, cytotoxic agents can cause acute and/or delayed toxicity. Consequently, preparation of cytotoxic drugs by healthcare workers exposes them to a professional contamination risk. The aim of the study is reducing the healthcare workers duration of exposure to the cytotoxic agents without increasing the unused dose wasting during preparation by avoiding the use of small size vials of five different drugs that frequently used in oncology centers.

Methods: All antineoplastic agents are prepared in negative pressurized clean rooms with using closed system transfer devices throughout the study. Doxorubicin (10 and 50 mg), epirubicin (10 and 50 mg), cisplatin (10, 50 and 100 mg), oxaliplatin (50, 100 and 200 mg) and carboplatin (50, 150 and 450 mg) wasted doses and preparation time (potential exposure time) were measured for six months. The usage of small dose vials which causes prolongation in preparation time has been avoided and larger dosage forms were preferred as much as possible in last trimester. Wasted drug dose amounts and preparation periods were recorded. Subsequently, the data compared between the first and the last trimesters.

Results: The total preparation period of the selected antineoplastic agents with different size vials was 1407 minutes. In the second trimester, which avoided the small size vial usage as much as possible it is also prepared in 1058 minutes. There was no significant changes between the first and the last trimester of the study in terms of patients numbers that receiving chemotherapy ( 504 and 505 patients respectively) and the rate of unused drug doses extermination (totally 840 mg and 782 mg respectively) (p>0.05). On the other hand, drug preparation time, in other words the health employee's exposure period to cytotoxic drugs was reduced 24.81% (349 minutes).

In the study, it has been shown that by making rational choices about drug usage can reduce the healthcare workers duration of exposure to the cytotoxic agents without increasing the unused dose wasting during preparation.

The U.S. Forest Service Risk Assessment Worksheet Tool (FSWT) is a highly sophisticated Microsoft Suite based application developed to perform risk evaluations based on toxicity values, chemical properties, water and off-site soil contamination rates, and built-in exposure assumptions. The FSWT is a set of interlinked word documents, excel spreadsheets, and database that prompt and guide a user through the risk assessment process. Risk assessors are presented with the challenge of evaluating risk in a variety of situations. Computational tools such as the FSWT can expedite the risk evaluation process and allow comparisons to be made under multiple scenarios.

This talk compares the FSWT to other current risk assessment methodologies. The FSWT has unique features that provide both advantages and limitations. These advantages and limitations will be presented along with proposed enhancements to improve the FSWT.

Background: With the increase of unwanted pollutants in the environment, this study focuses on the abatement and release of pharmaceuticals in the wastewater stream by using the new plasma-activated-water (PAW) technology. PAW is a promising technique for the pre-treatment of contaminated waters, Electric gas discharges in air over water produces reactive hydroxyl and nitrogen species with oxidative degradation capacities.

Purpose: To evaluate the treatment effect of PAW and UV-C light combined with hydrogen peroxide (UV/H₂O₂) for the oxidative degradation of cyclophosphamide (CP).

Methods: Different CP concentrations were spiked in tap water and subsequently treated with the advanced oxidation processes (AOPs) PAW and UV/H₂O₂. Plasma activation of aqueous CP solution was applied at 50, 100 and 150 W electric power input. The UV/H₂O₂ oxidation process was carried out by adding approximately 10 mg/L 30% (v/v) H₂O₂ to the samples and by placing an UV-C lamp 20 cm above the solution for immediate radiation. Both AOPs were tested for 120 min. and analysed by LC-MS/MS to determine the removal rate and possible formation of degradation products.

Results and discussion: PAW removes CP at a rate of 94.6 % at 100 W and 100 % at 150 W within 120 minutes. With UV/H₂O₂ treatment, within 60 min. CP was almost completely degraded (> 0%). Although this rapid degradation of CP indicates that a photochemistry process is more efficient, it is demonstrated that multiple oxidative reaction by-products are formed compared to PAW treatment. UV/H₂O₂treatment produce compounds such as 4-keto-CP, 4-hydroxyperoxy-CP and carboxyphosphamide, whereas only 4-keto-CP was seen with PAW oxidation. Identification of by-products and the reaction kinetics indicate that two studied approaches results in distinct patterns of oxidative degradation. UV/H₂O₂ oxidation can be explained as a pseudo-first order reaction where PAW oxidation shows a bi-model degradation of the pseudo-first order reaction.

Conclusion: The oxidative degradation of CP in spiked tap water shows efficient removal rates indicating that PAW is a promising alternative methodology for the elimination of CP from wastewater. Further research should reveal whether PAW may be suitable for the removal of other persistent pharmaceuticals from wastewater.

For improving hazard and risk assessment processes, a paradigm switch in toxicity assessment from apical endpoint evaluation in rodents towards a mode-of-action (MoA)-based testing in human-relevant in vitro methods was proposed. This switch was inspired by the high attrition rates in new drug development when moving from the preclinical phase of animal experimentation into clinical trials with humans. Therefore, it is thought that in vitro methods based on human cells might increase predictivity of compound testing for humans due to the elimination of species differences. One field with amongst the highest attrition rates concerns CNS diseases. Thus, such human-relevant alternative methods are needed that have the ability to predict adverse effects on the nervous system. We are using human induced pluripotent stem cells (hiPSC) for neural induction into 3D neurospheres, which consist of neural progenitor cells (hiNPC). Upon plating on laminin-coated single- or multi-well microelectrode arrays (MEAs), hiNPC differentiate into neurons and astrocytes and thereby form electrically active neuronal networks. In contrast to neuronal networks derived from rat NPC, hiNPC-derived networks exhibit less electrical activity (spikes and bursts) and synchronicity over differentiation time. To improve hiNPC network activity and maturation we established a differentiation medium with maturation-supporting supplements (CINDA medium). HiNPC differentiated with CINDA medium need shorter times for first network activities, display higher spike as well as burst frequencies and show network synchronicity. Challenging of networks with pharmacological compounds indicates presence and functionality of synapses and inhibitory and excitatory receptors. On the molecular level, CINDA medium induces gene expression of NSE2, GFAP, as well as synapse-, receptor- and neuronal subtype-specific markers compared to medium without CINDA supplements. These data suggest that hiNPC-derived neuronal networks formed in presence of CINDA medium are a promising system for setting up test methods for neurotoxicity evaluation.

Purpose: Nowadays, personal care products are widely consumed by all categories of persons. The European Regulation (EC) N°1223/2009 concerning cosmetic products defines pregnant women as a vulnerable population. Thus, a specific risk assessment with accurate exposure data is required. However, exposure values from SCCS guidelines do not consider pregnant women and little information is available in the literature. The aim of this study was to obtain frequencies of use and to identify potential disparities in cosmetic habits during and outside pregnancy period.

Methods: 111 adult pregnant women (mean age = 32 y/o) from 7 different regions of France answered a web-based or paper form survey distributed by healthcare professionals (gynecologists, midwives, pharmacists and physiotherapists). The questionnaire was in-house developed, including items about socio-demographic and pregnancy characteristics and focusing on frequencies of use of 37 cosmetic products, selection criteria, purchasing places and safety perception of personal care products during and outside the pregnancy.

Results: The percentage of women expecting a baby who modified their frequency of use was obtain for each product. Some products were less frequently used during the pregnancy: nail polish remover (46%), nail polish (44%), perfume (32%), foundation (27%), face mask (26%), lipstick (25%), mascara (23%), hair dye product (23%) and eye pencil (22%). On the contrary, pregnant women increased their frequency of use of stretch-marks care (68%), hydrating body cream (40%), bust care (26%) and massage oil (23%) indicating these products are commonly used during this specific period. In addition, pregnancy appears to have an impact on selection criteria of cosmetic products. The most reported criterion of choice outside pregnancy is the price while during pregnancy it is the presence of natural ingredients. This study provides frequencies of use of cosmetic products and highlights the influence of pregnancy on usage patterns which could be useful for the risk assessment of products dedicated to pregnant women.

Flame retardants (FRs) inhibit or delay the spread of fire by suppressing the chemical reactions in the flame or by forming a protective layer on the surface of a material. In the past decades, FRs like brominated diphenyl ether-99 (BDE-99) have been identified as threats to human health, especially to the developing brain. Less persistent alternative flame retardants (aFRs) have been released onto the market, however, toxicities of these compounds have not been investigated yet. Therefore, it is essential to assess the developmental neurotoxicity (DNT) potential of these aFRs.

We have developed two in vitro 3D neurospheremodels, i.e. human second trimester neural stem/progenitor cells (NPCs) and human induced pluripotent stem cell (hiPSC)-derived NPCs. Both represent distinct processes of brain development mimicking specific neurodevelopmental key events (KE), e.g. proliferation, migration, differentiation into neural effector cells (astrocytes, neurons and oligodendrocytes), interference with thyroid hormone-dependent oligodendrocyte maturation and the formation of functional neuronal networks. The two FR Tetrabrombisphenyl A (TBBPA) and BDE-99 interfere with some of these neurodevelopmental KE within the testing battery. Currently, the adverse effects of selected aFRs (Tris(chloroethyl)phosphate (TCEP), Triphenylphosphate (TPHP), Tris(2-butoxyethyl)phosphate (TBOEP) and Bis-(2-butoxyethyl)phosphate (BBOEP)) are studied.

Hence, our assays represent an array of neurodevelopmental KE that can be used for the DNT assessment of aFRs. In addition, they are under discussion to be a part of an in vitro DNT testing battery for compound screening at the OECD level.

Cosmetics Europe’s Long Range Science Strategy (LRSS) focusses on developing approaches allowing non-animal safety assessments for cosmetic ingredients. Case study safety assessments are conducted to evaluate and gain experience of using new approaches when making safety decisions. We present an example of an exposure-based ab initio case study for a cosmetic ingredient associated with significant systemic exposure, with the purpose of replacing repeat dose toxicity animal testing.

The Scientific Committee on Consumer Safety (SCCS) assessed phenoxyethanol as safe in cosmetics at its current use level (up to 1% in rinse-off and leave-on cosmetics). This case study aims to deem if this conclusion can be drawn using only data from non-animal approaches.

We used the tiered SEURAT-1 ab initio workflow, and each tier can allow safety decisions. Information used includes traditional safety assessment approaches such as toxicological threshold of concern (TTC) and read across, and then moves into in silico predictions (Tier 0), internal TTC based on bioavailability and formation of mode of action hypotheses using available mechanistic data such as ToxCast and transcriptomics (Tier 1). Tier 2 focusses on targeted non-animal testing, biokinetic refinement and calculating points of departure.

In Tier 0 we considered the exposure scenario and available non-animal data. We determined that the level of exposure could not be supported using TTC but were able to perform a read across risk assessment for some health effects e.g. skin sensitisation, but not for systemic effects due to lack of data on analogues.

In Tier 1 we used an aggregate exposure input and predictions from physiologically-based biokinetic modelling to make predictions of systemic exposure. We considered which modes of action may be relevant to the case study ingredient by performing in silico predictions and using or generating high throughput & high content information.

Tier 2 involves assessing if modes of action are likely to be relevant at the systemic exposures experienced following the use of cosmetic products and determining whether further testing is needed to reach a final conclusion on the safety assessment.

We believe that this ab initio workflow is useful in guiding non-animal mechanistic and exposure-based safety assessment. Moreover, transparently documenting the logic of the risk assessment can improve confidence in decision making.

Disinfection of pool water is an extremely important process that ensures the safety of its use avoiding water-borne infections caused by microbial pathogens. However, the chlorine used as disinfectant, in the presence of organic matter, may generate disinfection by-products (DBPs), including trihalomethanes (THMs), a class that includes genotoxic compounds, which are inhaled and absorbed by the skin. Previous studies have shown that exposure to these DBPs are associated with adverse health outcomes.

The aim of this study was to assess DNA damage in swimming pool users exposed to THMs. To evaluate DNA damage, blood samples were collected from a group of approximately 150 swimmers in different indoor chlorinated pools located in north of Portugal. Levels of DNA strand breaks and oxidized purines (formamidopyrimidine DNA glycolase) sites were determined in lymphocytes using the comet assay. Individual exposure to THMs compounds was analysed using a predictive model; analysis included not only total THMs but also chloroform, dibromochloromethane, bromochloromethane and bromoform separately.

Data obtained will increase knowledge on the potential genotoxic risks associated to THMs exposure in indoor swimming pools. Furthermore, results may be useful to perform a human health risk assessment model essential for development of risk management strategies.

This work was supported by Project NORTE-01-0145-FEDER-000010 - Health, Comfort and Energy in the Built Environment (HEBE), cofinanced by Programa Operacional Regional do Norte (NORTE2020), through Fundo Europeu de Desenvolvimento Regional (FEDER)

Swimming is seen as an activity with innumerous wellbeing and health benefits. For the maintenance of the quality of swimming pools’ water, and consequently for the safety of its users, there is a need to add disinfection products. The interaction between these products and organic matter, naturally introduced in the water by users, leads to the formation of disinfection by-products (DBPs), many of them with mutagenic and carcinogenic characteristics. Despite the relevance of this issue in public health, there is limited research on the subject.

The aim of this study was to evaluate genetic damage levels in young federated swimmers exposed to pool DBPs. A sample of urine, buccal mucosa and blood was collected from 12 young federated swimmers at the end of season competition period. Samples of a control group of 12 healthy individuals, matched for age and gender were also collected. Genetic damage was analyzed using CBMN assay in peripheral blood lymphocytes and micronucleus assay in buccal and urothelial cells. Alkaline and enzyme (FPG) versions of comet assay were also performed in order to measure the primary and oxidative DNA damage in lymphocytes.

Younger individuals constitute a susceptible group and their exposure to genotoxic compounds may lead to health problems later in life. Despite some limitations, results here obtained contribute to the understanding of the possible genotoxic effects of DBPs in children providing support for possible implementation of coherent measures for disease prevention in early stages of life.

This work was supported by Project NORTE-01-0145-FEDER-000010 - Health, Comfort and Energy in the Built Environment (HEBE), cofinanced by Programa Operacional Regional do Norte (NORTE2020), through Fundo Europeu de Desenvolvimento Regional (FEDER).

The American Cleaning Institute’s (ACI) “Guidance for the Risk Assessment of Enzyme-Containing Consumer Products” provides valuable information on the potential health hazards of enzymes used in consumer products and provides a framework for manufacturers of these products to conduct risk assessments to help ensure the safety of new products containing enzymes. Enzymes like many other proteins can act as allergens and induce the production of enzyme-specific IgE antibody upon repeated inhalation or exposure to mucous membranes that may lead to allergy symptoms, including asthma. The primary challenge associated with enzyme use is preventing the generation of enzyme-specific antibody and the development of symptoms of respiratory Type 1 hypersensitivity. This hazard is the primary focus for risk assessment of enzymes, and it must be managed carefully. Another hazard that should be addressed is primary irritation of the eye and skin which can be caused by enzymes belonging to the class of proteases. Only enzymes of the class of protease will have this irritating effect.

It is recommended that companies using enzymes in consumer products responsibly consider how they are managing enzyme safety, including the conduct of appropriate risk assessments and risk management programs. Such programs will include measures to manage exposures to enzymes. Program designs should be developed on a case-by-case basis to address parameters specific to the type of product and its applications. Experience in the cleaning products industry demonstrates that the potential risk of adverse effects can be successfully managed by identifying the hazards, carefully assessing exposure, characterizing the risk and then applying appropriate risk management. An updated ACI guidance document provides risk assessment approaches and risk management strategies that have been used successfully by the cleaning products industry and other industries.

In case of urgent food and consumer product safety questions, it is important that the relevant authorities and ministries receive an independent and unambiguous assessment of the possible health risks within a short time frame. On the basis of these urgent risk assessments, authorities can take preventive and protective risk management measures.

In the Netherlands, the Front Office Food and Product Safety is operated by the National Institute of Public Health and the Environment (RIVM) and RIKILT – Wageningen University and performs ad hoc (rapid) risk assessments commissioned by the Office for Risk Assessment and Research (BuRO) of the Netherlands Food and Consumer Product Safety Authority (NVWA). These assessments are the basis of the advices of BuRO for the ministries or the Inspector General of the NVWA.

The Front Office has been established to answer ad hoc questions on the chemical and microbiological safety of food and feed, and on genetically modified organisms. In addition, questions regarding chemical substances in consumer products such as toys and cosmetics are answered. The Front Office also conducts chemical analysis and risk assessment of food supplements.

Approximately 30 ad hoc risk assessments are commissioned each year. In general, these requests result from specific cases or occasions. Examples are results of analyses by NVWA and/or RIKILT, questions from the ministries, information from industry and topics in the media.

The risk assessments usually need to be performed within a short time frame and are based on available analytical results, data on maximum (use) levels in food, feed and consumer products, legal criteria, (existing or newly derived) health-based guidance values and information on exposure. In some cases, a risk assessment has to be based on very limited information. Where applicable, limitations of the assessment and their impact on the outcome are specified.

Recent topics for ad hoc risk assessments include fipronil in eggs, mineral oils in cheese biscuits, recreational use of nitrous oxide (laughing gas) and the possible extension of shelf life for perishable products (eggs). More information on previous assessments can be found at rivm.nl.

Pesticides in Morocco are widely used in agriculture. Their usage may pose risk to human health and environment. The aim of this study was to monitor pesticides levels in groundwater from Sais plain in Morocco during summer 2017 and winter 2018. For this purpose, passive samplers were deployed between 14 to 20 days in 21 traditional wells from the study area. Twelve pesticide compounds were analyzed including fungicides, insecticides, herbicides and their metabolites. Moreover, based on the pesticide monitoring results we performed a human health risk assessment for both adults and children using the model described by Hu et al. (2011), as well as an ecotoxicological risk assessment using Risk Quotient (RQ) method (Vryzas et al., 2009). RQ was predicted based on the ratio between the measured environmental concentration and the predicted no effect concentration. In the study area, seasonal variations of pesticides concentrations were observed in all wells. The highest concentrations were recorded during summer campaign, right after pesticide application, especially chlorpyrifos methyl (0.1 μg/L) and chlorpyrifos ethyl (0.015 μg/L). Based on the human health risk assessment, pesticides in groundwater of Sais plain posed low potential non carcinogenic risk on human through drinking water as exposure route, except for chlorpyrifos methyl in the case of children. In this case, the ratio of the potential exposure dose to the chloprpyrifos methyl and the level at which no adverse effect are expected was greater than one (1.6), due to this compound adverse health effect were possible. In addition, simazine showed unacceptable carcinogenic risk estimated 4.51 x 10^-6. High ecotoxicological risk for two pesticides was observed according to the RQ approach, namely chlorpyrifos methyl and chlorpyrifos ethyl (i.e. 462.6 and 1.12 respectively). The coupling of environmental monitoring data to probabilistic human and ecotoxicological risk estimates showed that cancer risk is unlikely due to drinking water but there is a high ecotoxicological risk.

References:

Hu, Y., Qi, S., Zhang, J., Tan,L., Zhang, J., Wang, Y., Yuan, D., 2011. Assessment of organochlorine pesticides contamination in underground rivers in Chongqing, Southwest China. J. Geochem. 111, 47–55.

Vryzas, Z., Vassiliou, G., Alexoudis, C., Papadopoulou-Mourkidou, E., 2009. Spatial and temporal distribution of pesticide residues in surface waters in north- eastern Greece. Water Res. 43 (1), 1–10.

Recent evidence has shown an increasing presence and distribution of extended-spectrum beta-lactamases (ESBLs) in the environment, which could represent a threat to public health. However, their presence in open water systems in England remains unknown despite the significant role of water in disseminating contamination and as a reservoir for biological hazards. Therefore, three sets of 30 water samples each were collected from different open water environments in Leicestershire (UK) including Leicester city in three periods of time (summer, autumn and winter) in 2017. Water samples were collected in the same locations each season using a portable water pump connected to a foam filter module according to the 1623 method developed by the US Environmental Protection Agency. The environments monitored were: the course of the River Soar and the Grand Union Canal (a canalised section of the River Soar) throughout the city including the River Biam; lakes highly frequented for fishing or leisure (e.g. John Merricks' Lake, Kings Lear’s Lake; Bennion Pools Fishing Lake); and a marina. The River Soar is rich in wildlife and attracts large numbers of users. Water samples were concentrated using the IDEXX® Filta Max system according to manufacturer's instructions and 1623 method. The DNA was extracted from each concentrated water sample with a Fast DNA® Kit. Genotypic prevalence of ESBL-producing bacteria was investigated by means of multiplex PCR. The assay was performed to detect the ESBLs: bla_TEM, bla_SHV, bla_OXA and bla_CTX-M using a CTX-M-15 producing strain of Escherichia coli as a positive control. All 90 samples assessed for ESBLs were negative. However these results should be considered inconclusive, as different factors such as dilution in the surface water could have affected them, as we have previously detected the presence of ESBLs (specifically CTX-M-15) in animal faecal samples (6 waterfowls, 3 dogs and 2 foxes) collected in different parks in Leicester city highly frequented by the public in 2016. Further studies will be needed to determine the presence and distribution of ESBLs in the open water systems in England as the World Health Organisation has highlighted ESBLs as a priority to be controlled to response to the bacterial resistance phenomenon. Information about the presence and distribution of human-pathogenic bacteria harbouring ESBL enzymes is needed to develop national intervention strategies to protect the public.

Derek Nexus is an in silico, expert rule-based system capable of predicting for multiple toxicity endpoints. Accompanying the predictions are informative descriptions highlighting known toxicants and a plausible biological mechanism of action. Structural alerts are currently organised and presented to the user according to a hierarchy of toxicity endpoints, an approach which has proved useful for numerous use cases.

An alternative approach to organising this knowledge is the use of adverse outcome pathways (AOPs). An AOP is a mechanistic representation linking a molecular initiating event (MIE) to an adverse outcome (AO) through sequential key events (KEs). KEs are described at a defined level of biological organisation and linked together according to known biological mechanisms which are supported by empirical evidence. Similar knowledge is contained in Derek Nexus, having been collated by the alert writer.

In this work we sought to rearrange the knowledge already contained in Derek Nexus relating to the AO of carcinogenicity, into a format more compatible with an AOP framework. Accompanying information relating to the mechanism of action for structural alerts from endpoints related to carcinogenicity (carcinogenicity, chromosome damage, mutagenicity) was analysed and MIEs were assigned to the alerts where possible. MIEs linking a compound class to carcinogenicity were assigned to 268 alerts in this way. The alerts and their relationship with MIEs were then used to profile a carcinogenicity data set to establish which MIEs are most prevalently associated with the data and which may be the most predictive of the AO.

Organising knowledge in this way means that it can be reused and combined with other information sources such as experimental assay data and pharmacokinetic studies more effectively. The grouping of hazards according to MIE may also be more appropriate in current regulatory use cases. In the future we expect the reformatted alerts will ultimately support development of a network of MIEs and KEs that also relate to other toxicological endpoints and increase the application of Derek as a tool for risk assessment across multiple endpoints of concern.

This study was designed to provide an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake dust (from brakes manufactured with chrysotile) in a 28-day repeated multi-dose inhalation toxicology study (6 h/d, 5 d/wk. 4 wk) followed by 28-days of exposure free recovery and served as a range finding study for a subsequent ongoing 90-day repeated dose inhalation toxicology study with lifetime recovery. Comparative fiber control groups were included of a similar grade commercial chrysotile as used in the brakes and a commercial crocidolite asbestos sample. The aerosol fiber distribution of the chrysotile (chry) and crocidolite (croc) asbestos were similar (Fibers L>20 μm/cm³: Chry-LD 42, Chry-HD 62; Croc-LD 36, Croc-HD 55; WHO fibers/cm³ Chry-LD 192, Chry-HD 219; Croc-LD 211, Croc-HD 255). The total number of particles in the aerosol of the brake dust groups was similar to that in the chrysotile groups (Part/cm³: B-D 710 - 1065; Chry 532 - 1442). A macrophage dose-response to the brake dust groups was observed with no fiber related effects. In the fiber control groups, the study differentiated between similar exposures to chrysotile and crocidolite asbestos. The chrysotile exposure resulted in a dose-dependent particle laden macrophage response characterised as Wagner Grade 1 to 3. In contrast, following crocidolite exposure there was a dose-response accumulation of fiber laden macrophages and interstitial fibrosis during exposure (Wagner score Grades 3 to 4) which increased in incidence following exposure (Wagner score Grade 4). Confocal microscopy images (determined following deep freezing of the chest wall at sacrifice) revealed no difference between the air control, brake dust and chrysotile high-dose exposure groups and no difference in the visceral or parietal pleura thickness at 28 or 56 days. The crocidolite exposure resulted in more than double the thickness of the visceral pleura and parietal pleura, with associated extensive inflammatory response and collagen development observed including adhesions between the visceral and parietal surfaces. These results provided a basis for the design of the 90 day study. (Study funded by Honeywell International Inc).

Paraquat is a non-selective herbicide commonly used in the world to eliminate weeds in agricultural fields. Its use has benefits, however, it has been described that exposure to paraquat generates adverse consequences depending on the concentration and time of exposure. Acute exposure has reportedly caused the death of workers and the subacute exposure causes damage to the respiratory and central nervous system in humans and animals. It has not been fully described what the mechanisms of toxicity of paraquat are, but it has been shown that the herbicide can increase the production of ion superoxide (O2.-), which has resulted in an increase in the generation of reactive oxygen species capable of oxidizing molecules necessary (NADPH and reduced glutathione) for proper cell functioning. In addition, it has been described that paraquat is capable of generating nitric oxide (NO) that can interact with the superoxide ion, generating peroxynitrite (ONOO-) causing cellular damage. The application of herbicides contributes to an increase of productivity in the agricultural fields, however, the inappropriate use has negative consequences in cultivation sites and nearby bodies of water where many species live. The American Bullfrog (Lithobates catesbeianus) is an amphibian that develops both in aquatic and terrestrial environments so it can be affected by herbicides. The acute toxicity of paraquat was determined at 48 hours of exposure, as well as the antioxidant response that includes the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and Lipoperoxidation (TBARS) in a concentration non-lethal of 1.13 mg / L for 6, 12, 24 and 48 hours of exposure in bullfrog tadpoles. The median lethal concentration of paraquat during 48 hours of exposure was 11. 30 mg/L. To assess enzymatic activity it was observed that SOD activity increased after 12 hours and remains so until the end of the time of exposure; CAT activity increased only at 6 hours, GPx showed no changes; regarding the TBARS, an increase was observed from 6 hours until the end of the exposure time. It can be concluded that at the sublethal concentration of 1.13 mg / L of paraquat, oxidative stress is generated in bullfrog tadpoles.

The Local Lymph Node Assay (LLNA): BrdU-ELISA utilises 5-bromo-2-deoxyuridine (BrdU) is an ELISA-based (Enzyme-Linked Immunosorbent Assay) test system to measure lymphocyte proliferation. The LLNA: BrdU-ELISA has been validated and is recommended as useful method for identifying skin sensitizing and non-sensitizing test substances (at the last point of the AOP strategy). The current OECD Test Guideline no. 442B (2010) for the conduct of the LLNA recommends the use of only CBA/J strain mice for the assessment of skin sensitization potential for chemicals. However, other strains can also be used when sufficient data are generated to demonstrate that significant strain-specific differences response do not exist. Thus, this study was conducted to determine whether BALB/c strain mice can also be used in the LLNA:BrdU-ELISA.

In this study, two chemicals with known sensitizing properties (hexyl cinnamic aldehyde and eugenol; both in acetone: olive oil (4:1, v/v)) were examined at two concentrations (25 % and 10 %). Female CBA/J (Jackson Laboratory) and BALB/c (Medical University of Białystok) mice were randomly allocated to groups (four mice per group). Mice were topically treated with chemicals or vehicle on both ears for 3 days. Following intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone ELISA (Cell Proliferation ELISA, BrdU kit; Roche) to determine the level of BrdU incorporated to lymph node cells. The Stimulation Index (SI) was calculated and statistical analysis was performed.

The results revealed no significant difference between CBA/J and BALB/c at all concentrations. The average SI values were similar for both stains for hexyl cinnamic aldehyde (2.3, 1.7 for CBA/J mice and 2.8, 1.7 for BALB/c mice at the following concentrations: 25 %, 10 %, respectively) and for eugenol (2.3, 1.5 for CBA/J mice and 2.7, 1.6 for BALB/c mice at the concentrations 25 % and 10 %, respectively). The differences were not statistically significant for each concentration (p>0.05).

In conclusion, this study indicated that female BALB/c strain mice can be used as an alternative to CBA/J strain mice in the LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.

Epidemiological evidence links nitrogen dioxide and volatile organic compounds (VOC) trends to a wide range of health effects. In this context, a key issue is the ability of the monitoring to serve as an indicator of personal exposure to these atmospheric pollutants. In Morocco, only few regions have air quality monitoring stations. In Meknes where no air quality monitoring station exists, the use of diffusive sampling tubes for monitoring and mapping of air quality is relevant.

The purpose of this study was to evaluate the air quality in Meknes by measuring levels of VOC and NO₂ in relation with road traffic during summer 2014 and winter 2015. Thus, passive diffusion tubes were deployed in 16 near-road and residential sites for 14 to 30 days to measure BTX and NO₂. In parallel to the winter campaign, road traffic counting sessions were conducted at the main roads of the city. Collected samples were analyzed and pollutants concentrations were determined using spectrophotometry and gas chromatography techniques. Our study results show that the average concentrations of BTX and NO₂ are highest in the city center, from where we noticed a decreasing gradient of pollutant levels. No season impact was noticed. The average concentration of benzene in Meknes during the two measurement campaigns is equal to 2.065 ug / m³ which exceeds the quality objective set by the European Union (2 ug/m³) but still lower than the annual limit value set in Morocco (10 ug / m³). Concerning the average of NO₂ concentration in all sites and measurement campaigns, it is approximately 30.652 ug /m³, which lowers than the admissible limit value set by the European Union (40 ug/m³) . Sites with high levels of benzene have also shown high levels of nitrogen dioxide (R²= 0.8714). VOC and NO₂ present in the atmosphere of our study area are mainly generated by road traffic. This study shows that population is exposed to atmospheric pollutants which may lead to emerging health effects. Our results also invite to examine the various lines of setted limit values in different countries and to assess the collective evidence.

Under Biocidal Products Regulation (BPR) 528/2012, read-across is a possible adaptation to the standard testing and is listed in Annex IV of BPR. Read-across entails the use of results from tests on other (analogue) substances to predict properties for the active substance under evaluation. The use of read-across can mean that applicants do not need to carry out one-by-one active substance testing of each toxicological property to fulfil their data requirements and is therefore a way to avoid unnecessary animal testing. However, it also has to be ensured that such prediction of a property based on read-across is reliable, can be used for risk assessment and classification and labelling, and complies in general with the provisions in BPR for the source study proposed.

The current poster presents the use of read-across in the assessment of biocidal active substances approved under BPR and Biocidal Products Directive (BPD) 1998/8/EC. Specific characteristics of the read-across are presented such as the basis of read-across justification / hypothesis and the toxicological endpoint(s) for which read-across is applied. The analysis of data shows that read-across was used in one third of the assessments of the approved active substances, which makes read-across an extensively used adaptation to data requirements of BPR and BPD. Further guidance on the use and applicability of read-across in biocidal active substance approval is needed.

Formaldehyde occurs naturally in the environment and is ubiquitous in the environment. Formaldehyde occurs in very low levels in water as it is rapidly hydrated and therefore, predominantly is found as methylene glycol. Formaldehyde occurs naturally in food. Reported background levels vary considerable. Formaldehyde can also occur in food if released from melamine resin food contact materials. Being very reactive, formaldehyde is essentially present in food bound reversibly and irreversibly to different constituents. In humans, as in other animals, formaldehyde is an essential metabolic intermediate in the physiological one-carbon pool (central to many biological processes).

The general population is exposed to formaldehyde from many sources. The European Food Safety Authority has estimated that the contribution of formaldehyde from food does not exceed 100 mg/person/day. Drinking water is only a minor source of exposure.

The critical effects of formaldehyde following repeated oral exposure are considered to be the non-neoplastic histopathological changes observed in the forestomach and stomach (erosion, ulceration, inflammation and hyperplasia, most likely due to the irritative potential of formaldehyde) in experimental animals (long term studies drinking water). A NOAEL of 260 mg/l is considered.

Formaldehyde is genotoxic, with effects observed in vivo in cells from first site of contact tissues (i.e. nasal tissue); however, there is no evidence of genotoxicity locally in the gastro-intestinal tract. The weight of evidence indicates that formaldehyde is not carcinogenic by the oral route.

A tolerable concentration of formaldehyde in drinking water is estimated to 30 mg/l (rounded value) based on the NOAEL of 260 mg/l and assessment factors of 2.5 and 3.2 for interspecies and inter-individual variability, respectively, in toxicodynamics. Based on this tolerable concentration and an estimated (worst-case) concentration of formaldehyde in drinking water of 30 µg/l, no risk for adverse effects from intake of formaldehyde in drinking water is identified.

Based on the available data, a tolerable concentration of formaldehyde in food cannot be estimated. Therefore, the risk for adverse effects from intake of formaldehyde in beverages and foods could not be evaluated.

Consumers in real life are not exposed to single chemicals but to real-life chemical mixture at doses around toxicological reference values. The aim of this study was to assess the chronic toxicity of a real life mixture of pesticides with food additives and common consumer products chemicals using doses that simulate the real life exposure scenario. Four groups of 10 Sprague Dawley rats (5 males and 5 females) were treated with a mixture of dimethoate, glyphosate, triadimefon, methyl parathion, carbaryl, aspartame, benzoic acid, calcium disodium ethylene diamine tetra-acetate (EDTA), ethylparaben, butylparaben, bisphenol and acacia gum in doses of 0, 0.25 x acceptable daily intake (ADI), ADI and 5xADI doses for 12 months. The behavioural changes were assessed every 3 months using open field exploratory test and elevated plus-maze test. After 12 months of exposure there was an increase in locomotor activity, quantified as number of crossing over external squares in low dose group (46.8 ±11.57) compared with control (13.70±6.15) (p=0.038). The spatial orientation activity, quantified as number of rearings, was significantly increased in the low dose exposed rats (22.20 ±4.77) compared with the control rats (6.90 ±1.61) (p=0.015). In medium and high dose exposed rats the locomotor activity and special orientation activity decreased compared with the low dose group almost till the control levels in high dose exposed group. Mixture exposure determine a two-phase dose-dependent effect, the chronic use of a small dose has a stimulating effect on the organism while the higher doses have some additional negative effects that eliminate/mask the stimulating effect of the low dose.

Introduction: The rapid industrial growth coupled with the development of modern technologies has resulted in the introduction of diverse toxic substances, including toxic metals into the environment. Toxic metal ions are imperishable and have lethal effects on living beings when their quantity surpasses a certain threshold level. The presence of the metals like Cr, Cd, Pb, Mn and Ni above the threshold limits exhibit carcinogenic and genotoxic effects on humans. The industrial or harbor zone is typically more populated which is responsible for creating anthropogenic pressure on the coastal environment.

Material and method:

The toxic metal content of marine water and sediment and fishes from the five selected sites of the east coast of Bay of Bengal have been collected on a seasonal basis during Pre-monsoon, Monsoon and Post-monsoon during (2014-2016).

The enrichment level of metals in the different trophic level of marine ecosystem (Water/sediment → Phytoplankton→ zooplankton → benthic organisms and fishes) of Bay of Bengal has been quantified. Prediction of environmental risks, as well as risks associated with humans using different pollution indices from the available sources for toxicity information, has been calculated.

Results:

The outcome of this study clearly indicates that the bioaccumulation was lowest in phytoplankton compared to organisms of higher trophic levels. There is a clear evidence of the anthropogenic origin of all toxic metals. It was found that the selected study sites of the coastal Bay of Bengal are mostly polluted by Chromium followed by Arsenic and lead. Human health risk assessment indicated consumption of seafood could increase cancer risks. Among all the heavy metals analyzed, Cr is the most critical and possess highest carcinogenic and non-carcinogenic health threat for seafood consumers. The risk assessment of coastal sediments indicated, there are probabilities of developing cancer and other health-related issues in the coastal population after long-term exposure. The biomagnifications were observed only for Cr, Ni, and Pb in tissues and not in As, Hg, and Cd. THQ_Cr possesses highest non-carcinogenic risks, which contributes about 50% to HI. Likewise, the contribution of carcinogenic risk (CR _Cr) to TCR is about 60%. The non-carcinogenic risk was found to be beyond the safety limit in children, below 15 years, than adults in the study site (HI child >1). The sediment was profoundly polluted with Arsenic.

Pesticides used in agriculture have led to widespread occurrence in the aquatic environment that causes detrimental effects on the aquatic organisms even at very low concentrations. In this context, the present study was aimed to examine the impact of lower levels of isoprothiolane (2.7 and 27 µg/L; reported concentration in Yung San River, Korea) on the common carp Cyprinus carpio under 96 h exposure using various biomarkers (hematological, ionoregulatory, biochemical, and enzymological parameters). The concentrations used in this study significantly (p<0.05) decreased the hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC), serum glucose, cholesterol, triglycerides, aspartate aminotransferase, and gill Na⁺/K⁺-ATPase levels in fish exposed to isoprothiolane. In contrast, the white blood cell (WBC), serum sodium (Na⁺), chloride (Cl^-), and albumin levels were increased (p<0.05) when compared to that of their control groups. Further, there were slight changes in mean cellular volume (MCV), mean cellular hemoglobin (MCH), mean cellular hemoglobin concentration (MCHC), serum potassium (K⁺), protein, globulin, and alkaline phosphatase levels. Taken together, the present study suggests that alterations of hematological, ionoregulatory, biochemical, and enzymological parameters can be used as efficient biomarkers in monitoring the toxicity of pesticides in aquatic organisms. Further, the long-term effects of isoprothiolane need to be investigated in our future studies with more endpoints.

In Belgium the assessment of risk to public health in the framework of the International Health Regulations is coordinated by Sciensano (former Scientific Institute for Public Health). In July 2017, the Federal Agency for the Safety of the Food Chain communicated the finding of fipronil in eggs through a notification via the EU Rapid Alert System for Food and Feed (RASFF). Because of the high attention in the media and divergent interpretations on the risk for public health, the Belgian Risk Assessment Group (RAG) was requested by the Federal Public Service Health, Food chain Safety and Environment, to perform a risk assessment.
Fipronil is a broad spectrum insecticide/acaricide authorized for use in pets for the control of fleas, lice, ticks, cockroaches and mites, and, in the past, for use as a plant protection product (seed treatment). Fipronil is not authorized for use in food production animals. In the case of the 2017 crisis, fipronil entered the food chain via use of an illegal product containing fipronil (Dega-16) to decontaminate chicken farms from red lice, a frequently occurring and difficult to treat ectoparasite of laying hens. The product was sold to farms in several European countries. In Belgium, fipronil was detected in eggs from 21 companies, in concentrations ranging from “not detected” to 0.92 mg/kg egg. Except for one sample, the concentrations of fipronil measured in eggs were below the European threshold of health risk for the consumer of 0.72 mg/kg. This threshold is based on the acute reference dose (ARfD) and the P 97,5 % consumption of eggs by children (8.7 kg) in Europe (EFSA PRIMo2 model).
The health risk was primarily assessed for the highest reported level (0.92 mg/kg). For the acute exposure scenario, data on consumption of raw and cooked eggs in Belgium were used (Food consumption poll 2014-2015). For the repeated exposure scenario, the total daily consumption of eggs (raw, cooked and secundary products) was taken into account. According to WHO/EFSA, the estimate of acceptable daily intake (ADI) of fipronil in humans is 0.0002 mg/kg bw/day. The Acute Reference Dose (ARfD) for Fipronil is 0.009 mg/kg bw.
At a concentration of 0.92 mg/kg egg, all the calculated estimates were below the ARfD for all age groups. Taking into account a usual daily intake of eggs (and secondary products) that would all contain this high concentration of fipronil, the ADI would be exceeded for almost the entire population. However, this is a worst case scenario, very unlikely to have occurred.
We concluded that the fipronil eggs contamination incident was unlikely to have acutely affected public health. A risk to human health on a long-term cannot be formally excluded but is very unlikely.

Dendritic cells (DC) play a major role in the regulation of immune responses to a variety of antigens. Indeed, metallic haptens such as nickel and cobalt are able to induce DC maturation throughTLR4 activation leading to a T cell-mediated inflammatory skin disease called allergic contact dermatitis. We recently showed that nickel but not cobalt induced the production of IL-12p40 and IL-23 in human monocyte-derived dendritic cells (MoDCs) leading to Th17 cell polarization. Although both metals are considered to be TLR4 agonists, we wanted to better understand the particular mechanism behind metals-induced IL-12p40 production and we were interested in the NFIL-3 transcription factor, a recently described regulator of IL-12p40. Using an in vitro model, we showed that both nickel and cobalt were able to induce NFIL-3 expression, in human MoDCs. Pretreatment of MoDC with a TLR4 blocking antibody followed by metals stimulation did not modify NFIL-3 expression suggesting that NFIL-3 induction by nickel and cobalt was independent of the TLR4 pathway. However, this expression of NFIL-3 was dependent on the Jak-STAT pathway and oxidative stress since the pretreatment of MoDC with Jak Inhibitor I or N-acetylcysteine followed by metals stimulation induced a decrease in NFIL-3 expression. Moreover, we showed that cobalt is a strong inducer of reactive species, measured using dichlorofluorescin by flow cytometry, which may explain why cobalt is a strong inducer of the NFIL-3 pathway, compared to nickel, and thus its inability to induce IL-12p40 production. In summary, our data suggested that the NFIL-3 signaling pathway plays an important role in regulating the expression of IL-12 cytokine family in metals-exposed DC that may be implicated in ACD.

The integrity and function of the mucosal epithelial barrier plays an important role in maintaining the gut homeostasis. Research indicate a link between altered barrier function and the development of food allergy. Barrier disruptors, like cholera toxin (CT), house dust mite (HDM), and the mycotoxin deoxynivalenol (DON) have been shown to facilitate allergy development. We hypothesized that this is due to 1) damage to intestinal epithelial cells, leading to impaired barrier integrity and increased uptake of allergens, and/or 2) the production of cytokine ‘alarmins’ by intestinal epithelial cells. The aims of this project were a) to detect early markers of a disrupted gut barrier, and b) to investigate if the tested barrier disruptors could promote development of food allergy.

This study was based on a food allergy model in mice, being composed of two short-term experiments for gut barrier assessment and one allergy model experiment. In the short-term experiments, C3H/HeOuJ mice were exposed intragastrically to one of the compounds DON, HDM or CT, and the food allergen peanut (PE). In the second experiment, we excluded PE, and included the pesticide glyphosate. 4, 24 and 48 hours after exposure, intestine and draining lymph nodes and/or Peyer’s patches were excised, and serum was collected. The intestines were homogenized and cells from lymphatic tissue were stimulated ex vivo for determination of cytokine secretion. ELISA and flow cytometry were used to detect markers of disrupted barrier and allergy.

Preliminary results indicate that the barrier disruptors elicit early effects. In DON-treated groups, the levels of the pro-inflammatory cytokines TNF-α and IL-6 in the draining lymph nodes were higher than in the other groups at 4 hours in both experiments. The levels of IL-33 in ileum and TSLP in duodenum were higher at 4 hours in the first experiment, when PE was given together with DON. This trend was not detected in the second experiment when PE was excluded, suggesting a role of PE.
Preliminary analyses suggest that different barrier disruptors affect different early markers of barrier function as well as immune activation. Thus, no single marker but rather a set of markers, are needed to identify barrier disruptors with allergy-promoting capacity. Our results also suggest that co-exposure with PE had an effect on the local response in the intestine. Investigation of the allergy promoting capacity of the compounds is ongoing in the food allergy model.

Tattoo allergies are severe side effects related to the permanent deposition of pigments in the dermal layer of the skin. Even though most type IV delayed hypersensitivity reactions are observed in the red color spectrum, the true allergen is rarely identified. So far, just a few metallic allergens and one organic pigment were reliably linked to allergic tattoo reactions. Organic pigments are only thought to act as pro- or pre-haptens since they lack water solubility and therefore hardly interact with biomolecules in physiological media. Based on this, degradation products became the main suspects in focus.

Based on the analysis of more than 100 skin samples of patients with red tattoo allergies, we identified the most common organic tattoo pigments correlating with these reactions. Possible degradation products of these pigments were deduced from substances found after laser irradiation, sunlight exposure and pyrolysis. In addition, we identified UV degradation products using untargeted quadrupole time-of-flight tandem mass spectrometry (qTOF-MS/MS). We selected known sensitizers, irritants, carcinogens and other suspected substances for further testing. The Direct Peptide Binding Assay (DPRA) was applied to identify substances that are able to bind to peptides, which is a key event in sensitization. Additionally, the activation of immune cells was analyzed using the Human Cell Line Activation Test (hClat).

We found that the majority of organic pigments in the tattoo allergy skin samples belong to the azo pigment class. These pigments are known to be easily degraded upon sun light or laser irradiation. Also, cleavage of the azo bond by reductases in vivo may occur. Most common degradation products are primary aromatic amines cleaved from either azo or amide bonds present in the molecule. Among other substances, 2-aminophenol showed positive test results in the DPRA. This substance is as yet only classified as “suspected of causing genetic defects” by the harmonized classification of the European Chemicals Agency (ECHA). However, ECHA refers to data from manufacturers supporting that 2-aminophenol may cause skin sensitization.

The results derived from this study should lead to the development of a skin patch test series for identification of sensitizing substances in tattoo allergies. In the long run, tattoo pigments likely to release these substances should be banned for use in tattoo inks.

The ISO norm 10993 part 10 describes the procedure for the assessment of medical devices and their constituent materials with regard to their potential to produce irritation and skin sensitization. The success of the recent Round Robin Study to assess in vitro skin irritation of medical devices extracts, with Reconstructed Human Epidermis (RhE) models, opened the way to in vitro approaches for this endpoint. For skin sensitization of medical devices, animal methods are still requested. The goal of this study was to evaluate the capacity of the SENS-IS assay to predict skin sensitization of medical devices extracts.

Based on a comprehensive analysis of skin sensitization, the SENS-IS assay uses the quantitative analysis of multiple specific biomarkers, expressed in EPISKIN model, a 3D reconstructed human epidermis. ImmunoSearch, developed this assay after analyzing genes modulated during the sensitization process either on mice (LLNA) or human (blisters). The panel of sensitization biomarkers defined includes genes already identified as markers, such as the ARE family, and others not yet associated with the sensitization process (called SENS-IS gene subset).

Solvents used for extraction of medical devices, polar (physiological saline) and nonpolar

(sesame oil), were spiked with different concentrations of sensitizer and successfully detected with SENS-IS assay in the Episkin model and the SkinEthic RHE model. Then, samples of polymers used in medical device industry, MED-2000 silicone, were loaded with 6 known sensitizers (10% final concentration): 1-phenyl-1,2 propanedione, 1-Chroro-2,4-dinitrobenzene, Diethyl maleate, p-Benzoquinone, Propyl gallate and Phenyl Benzoate and extracted in polar and nonpolar solvents in accordance with ISO 10993-12:2012.

After optimization of the original protocol used for neat chemicals, the SENS-IS method was able to correctly classify the 6 polymers.

These preliminary results show that the EpiSkin model and the SkinEthic^TM-RHE model could be used in the SENS-IS assay to assess the sensitizing potential of medical devices extracts. Further studies are engaged with a more comprehensive set of molecules. If confirmed, these results would allow toxicologists, familiar with either the EpiSkin or the SkinEthic-RHE model to assess skin sensitization with the SENS-IS assay.

Our aim is to apply mechanistic and clinical understanding to develop a risk assessment approach for skin allergy that does not require new animal test data and addresses novel exposure scenarios, while better characterising uncertainty.

Our Integrated Strategy for Skin Allergy Risk Assessment (SARA) is a tiered model-based approach that predicts the probability of human skin sensitisation to an ingredient occurring following a given product exposure, with explicit uncertainty. The first tier is a probabilistic, weight of evidence (WoE) human potency model designed to use historical in vivo (HRIPT), in vitro (DPRA, Keratinosens, hCLAT, SENS-IS and U-SENS) and in silico (DEREK-NEXUS) hazard information to inform an initial prediction of human skin allergy risk which can be combined with an applied dose model. A skin toxicokinetic (TK) model may also be utilised as an additional tier that can further reduce uncertainty in the risk prediction from the clinical potency model by extrapolating from the HRIPT exposure to a market scenario.The Integrated Strategy for SARA has been evaluated using six case study ingredients included at 0.2% in two products types, face cream and shampoo. The results from the first-tier analysis are given as the probability of human skin sensitisation occurring following a given product exposure. The output from the Integrated Strategy for SARA allows a decision to be made using probability of skin sensitisation induction as a risk metric without the need for new animal data.

Introduction: Malignant Mesothelioma (MM) is a cancer of peritoneal and pleural serous membranes induced by asbestos and carbon nanotubes (CNT). MM remains a contemporary health problem, the WorldHealth Organisation (WHO) is expecting a peak of incidence in 2020. We suggested that carcinogenic fibers possess the intrinsic capacity to induce a preferential, rapid and sustained accumulation of macrophages that promote early immunosuppressive microenvironment, tumor cell evasion and mesothelioma development.

Aims: The goal of the present study is to determine by which mechanisms mesotheliomagenic macrophages are immunosuppressive and specifically suppress T lymphocyte functions after carcinogenic fiber exposure.

Methods and results: We analyzed the entire transcriptome profile (Illumina array) of peritoneal macrophages collected from Wistar rats treated with mesotheliomagenic (asbestos or CNT-7, i.p. injection) and non-mesotheliomagenic fibers (tangled carbon nanotubes or CNT-T) and identified specific sets of genes which may explain the immunosuppressive properties of mesotheliomagenic macrophages. We found that pathways related to the TGF-β superfamily could be used for distinguishing macrophages from peritoneal mesothelioma. In particular, Syndecan-1, a transmembrane heparan sulfate proteoglycan implicated in TGF-β signaling, is specifically expressed by peritoneal macrophages from CNT-7-treated rats. Mesotheliomagenic macrophages also express integrin β3 recently implicated in the activation of TGF-β. Transglutaminase-2, an extracellular matrix enzyme that plays a role in the interaction between latent TGF-β and extracellular matrix, represents an additional TGF-β activation signature. Moreover, we also noticed an increase of TGF-β levels in peritoneal fluid from rats exposed to CNT-7 comparatively to CNT-T.

Conclusions: Our data support an important role of TGF-β activation in immunosuppressive functions of macrophages during the development of experimental mesothelioma.

Patients with respiratory diseases in rural areas have been reported to have enhanced responsiveness to ambient particulate matter (PM) (Brunekreef et al 2002, Pope et al 2009). In addition to the physical and chemical components, ambient PM can contain microorganisms or parts thereof which is referred here as BioPM. The aim of this study is to characterize the microbial composition of BioPM originating from livestock, and to investigate whether these BioPM can trigger the activation of innate receptors and cells.

Coarse (2.5-10 μm) and fine (< 2.5 μm) indoor BioPM were collected from various animal farms using the versatile aerosol concentration enrichment system (VACES). The fungal and bacterial community was assessed with an amplicon based approach using Next Generation Sequencing (NGS). In parallel, HEK-Blue cells expressing different pattern recognition receptors (Toll like receptors (TLR) 2,3,4,5,7,8,9 and NOD 1,2) and a human monocytic cell line (MM6) were exposed to increasing concentrations of BioPM from these sites. The levels of secreted embryonic alkaline phosphatase (SEAP) and cytokine (Interleukin-6, IL-6) were measured indicating TLR and MM6 activation respectively. TLR antagonists or blocking antibodies were used to examine the role of specific TLR on MM6 activation.

Results indicate distinct airborne microbiota profiles associated with the corresponding animal farm. Moreover, the various BioPM contained mainly ligands for TLR2 and TLR4 resulting in a dose-dependent production of IL-6 by MM6 cells. In addition to TLR2 and TLR4 only the pig-derived BioPM induced TLR5 activation. Blocking experiments indicate that only blocking TLR4 interfered with the IL-6 production by the MM6 cells upon stimulation with these BioPM.

These findings indicate that BioPM from livestock can activate innate cells and therefore, trigger inflammatory responses. Knowledge on the microbial composition of BioPM derived from different farms and understanding what type of inflammatory responses they induce is crucial for future studies on the effect of that exposure to BioPM may have on respiratory symptoms. Such as asthma, which is expected to increase in the next two decennia, resulting in expenditures up to 900 million euros per year in the Netherlands.

This work was supported by the Dutch Ministry for Public Health and the Environment under grant S/121012/01.

Air pollution is known to be associated with increase morbidity and mortality. Atmospheric fine particulate matter (FP) is able to enter the lungs where some compounds can reach the bloodstream and enter in contact with immune cells. Ageing is accompanied by many changes in the body: immunosenescence involves changes in the organization and the functions of the immune system making elderly more vulnerable. The project aims to identify the effects of FP on human T lymphocytes while attempting to firstly determine biomarkers related to exposure and secondly to evaluate the variation of the cellular response as a function of age. T cells were isolated from blood samples of 90 healthy volunteers belonging to three age groups (20-30, 45-55, 70-85 years) then exposed ex vivo for 72h to 45μg/μL of FP collected in Dunkirk. Analyses of the exposed T cells were then performed using transcriptomics, metabolomics, DNA adductomics and cytokines quantification. We have demonstrated that T cells exposure to FP caused an induction of the genes CYP1A1, CYP2S1 and NQO1 coding for the enzymes involved in the metabolic activation of organic compounds such as PAHs identified in the PF sample. We could then detect DNA adducts of organic metabolites, BaP Diol Epoxyde (BPDE) and formaldehyde, and of oxidative stress markers, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde. The implication of oxidative stress in this toxicity was confirmed by the detection of a decrease of cystein level during the metabolomics analysis performed on T cells supernatants. Characterization of the T cells profile made it possible to propose a mixed Th1/Th2 profile caused by the exposure. The transcriptomic study, using Affymetrix array then confirmed by qPCR, finally revealed an overexpression of several miRNAs such as miR-124-3p involved in the regulation of several functions in the immune system and miR-1290 involved in several types of cancer. Concerning the influence of age, overexpression of the genes coding for the antioxidant enzymes (NQO1 and HMOX1), an increase in the concentration of cytokines (IL-4 and IL-13) as well as a modification of the expression profile of some miRNAs were noted in the elderly. These results showed that exposure of T cells to FP causes effects at the transcriptomics, metabolomics and DNA adductomics levels.

Environmental mercury exposure is an inevitable threat to human health. It has been proposed that the reduction in adenosine monophosphate activated protein kinase (AMPK), a regulator of cellular energy homeostasis, makes cells highly susceptible to methyl mercury toxicity. Glutamate activates AMPK through N-methyl-D-aspartate (NMDA) receptor, while metformin promotes phosphorylation of AMPK. Methyl mercury easily crosses the blood‑brain barrier, however it is not known whether the NMDA receptor inhibition by kynurenic acid (KynA) or stimulation of AMPK by metformin might reduce the mercury-induced damage in dopaminergic neurons. This study evaluated the effects of KynA and metformin on mercury-induced toxicity in SH-SY5Y cells. SH-SY5Y neuroblastoma cells were exposed to various concentrations of methyl mercury at different time periods. Mercury exposed cells were incubated with KynA or metformin. Total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined, and nitric oxide levels were measured spectrophotometrically. Mercury compound caused a remarkable increase in the mitochondrial oxidative stress of SH-SY5Y cells in glutamine-containing medium. By increasing dose and duration of mercury exposure, metformin exacerbated the neuronal oxidative stress and decreased mitochondrial metabolic activity. KynA supplementation presented a significant protective effect against methyl mercury toxicity with glutamate transmission. Surprisingly, despite being an AMPK activator, metformin strongly enhanced the toxic effect of 5µM methyl mercury on SH-SY5Y cells. Further studies should be made considering the association between AMPK and methyl mercury toxicity.

This study was partially supported by The Scientific and Technological Research Council of Turkey, 214S112.

Scorpion venom compounds are able to induce alteration and dysfunction of the host organs such as liver and kidneys. These effects can be attributed to the activation of an inflammatory response. Trace elements such as selenium are known to exert multiple beneficial effects including anti-inflammatory and antioxidant action related to various diseases (inflammatory bowel diseases, arthritis…). However, studies related to the protective effects of trace elements against the induced inflammatory response by scorpion venom are scarce. The aim of this study is to investigate the potential immunoprotective effects of selenium in the induced hepatorenal toxicity by Androctonus australis hector venom.

Selenium was therefore, administered to mice orally during 2 weeks prior to the injection of a sublethal dose of scorpion venom. The hepatorenal inflammation response was assessed twenty four hours after the envenomation of mice by the measurement of vascular permeability changes, edema formation, oxidative/nitrosative stress markers levels and also by histological examination.

The results showed that the venom induced inflammatory disorders characterized by an increase of levels of reactive oxygen/nitrogen species, lipid peroxidation, and a decreased antioxidant defense. Moreover, significant alterations of the hepatic tissue such as hemorrhages and cell degeneration were also observed. The administration of selenium prevented the induced hepatorenal toxicity by the venom, as evidenced by a decreased edema formation, leukocyte infiltration, nitric oxide and thiobarbituric acid-reactive substances levels in the liver and the kidneys, as well as a reduced incidence of hepatorenal tissue alteration.

These results indicate that the selenium is able to exhibit potent protective effects against the induced inflammation response and oxidative/nitrosative stress by the venom, in hepatic and renal tissues, probably by inhibiting oxidative damage and increasing antioxidant defense. However, more researches are needed to better understand the exact mechanism by which selenium prevents the induced hepatorenal toxicity by scorpion venom.

Rheumatoid arthritis (RA) is a long-term autoimmune disorder that is pathologically occurred by immune responses of many cell types, including T cells, B cells, and macrophages. B-cell activating factor (BAFF) plays a role in the maturation and maintenance of B cells, which is associated with RA. Fibroblast-like synoviocytes (FLS) also play a key role in producing inflammatory cytokines that contribute to cartilage destruction. Rheumatoid FLS develop a unique aggressive phenotype that increases invasiveness into the extracellular matrix and further exacerbates joint damage. We previously reported that BAFF expression was increased in MH7A synovial cells transfected with the SV40 T antigen. In addition, FLS could activate macrophages to increase BAFF expression. Bisphenol A (BPA), an organic synthetic compound, is widely used to make certain plastics such as water bottles, sports equipment, DVDs and CDs. Epoxy resins containing BPA are also used to line water pipes, as coatings on the inside of many food and beverage cans and in making thermal paper such as that used in sales receipts. BPA is a xenoestrogen, exhibiting estrogen mimicking, hormone-like properties. Here, we investigated whether BPA affect BAFF expression in macrophages to influence the damage of synovial tissues. We are also studying the polarization into M1- or M2-type macrophages by BPA. Taken together, these data demonstrate that macrophages were activated by the incubation with BPA, which up-regulated BAFF expression. This suggests that individuals at high risk for autoimmune disease should avoid long-term exposure to BPA and its analogues.

Introduction and purpose: rCollinein-1 is a recombinant snake venom serine protease (SVSP) derived from Crotalus durissus collilineatus venom. This SVSP has a thrombin-like effect and interferes in blood coagulation, being considered a promising molecule targeting the development of novel biopharmaceuticals to treat hemostatic disorders. However, heterologous proteins may be immunogenic. In this regard, PEGylating proteins emerges as a strategy targeting the suppression of the immune system response. Thus, this work proposed the comparison of the cytotoxicity in peripheral blood mononuclear cells (PBMC) of rCollinein-1 and its PEGylated form and the in silico epitope prediction within the structure of rCollinein-1. Methods: rCollinein-1 was expressed in Pichia pastoris system and purified employing immobilized metal affinity (IMAC) and ion exchange chromatographies. The protein was PEGylated using a 5 kDa PEG-derived polymer. Protein structure was evaluated using circular dichroism (CD). The degradation of a chromogenic substrate was employed to evaluate their enzymatic parameters. Cytotoxicity was investigated in human PBMC in five concentrations. In silico epitope prediction was performed using ABCpred server (imtech.res.in/raghava/abcpred/). SWISS-MODEL (swissmodel.expasy.org) was employed to build the molecular model. Results: PEGylated rCollinein-1 maintained its enzymatic properties and CD revealed the secondary structure contents were similar between rCollinein-1 and its PEGylated form. These results indicate the preservation of protein conformation after PEGylation. Both enzymes showed to be non-toxic to PBMC in all tested concentrations, with cell viability higher than 85%. Regarding epitopes prediction, four peptides with 16 amino acid residues presented a high score to be immunogenic (>0.85). They are located in the surface of the protein structure, near to possible sites of PEGylation. Conclusions: PEGylating rCollinein-1 did not change its structure and has no influence on its toxicity. The epitopes with highest score to be immunogenic lie close to sites of PEGylation. Consequently, PEGylating rCollinein-1 may be an interesting modification targeting the reduction of immunogenicity, allowing its therapeutic applications.

Inhalation of commonly present irritants, such as chlorine and chlorine derivatives, can cause adverse respiratory effects, including irritant-induced asthma (IIA). We hypothesize that due to airway barrier impairment, exposure to hypochlorite (ClO^-) can result in airway hypersensitivity.

C57Bl/6 mice received an intra-peritoneal (i.p.) injection of the airway damaging agent naphthalene (NA, 200 mg/kg body weight) or vehicle (mineral oil, MO). In vivo micro-computed tomography (CT) images of the lungs were acquired before and at regular time points after the i.p. treatment. After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO^-) or vehicle (distilled water, H₂O) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. Dendritic cells (DC) and innate lymphoid cells (ILC) of the lungs are measured using FACS.

In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO^-, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO^- mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO^- induced airway barrier impairment, as shown by increased broncho-alveolar lavage (BAL) protein and plasma surfactant protein (SP)-D concentrations; histology revealed epithelial denudation.

These data proves that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO^-, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.

The Russian system of genetically modified organisms (GMO) safety assessment within the framework of their state registration includes the study of toxic and allergenic properties of the new product in in vivo experiments. The study of immunotoxic and sensitizing features is carried out in experiments on mice of CВА and C57B1/6 lines in four tests: (1) the effect on the humoral immunity is evaluated with hemagglutination test; (2) the effect on the cellular immunity is evaluated with delayed-type hypersensitivity reaction; (3) the sensitizing effect is evaluated with histamine sensitivity test; (4) the resistance is evaluated on a model of Salmonella typhimurium (strain 415) intraperitoneal infection. The study of GMO allergenic potential is studied on a model of rats’ systemic anaphylaxis, induced by ovalbumin. The method includes the assessment (1) of systemic anaphylaxis severity and (2) of circulating antibodies (subclasses IgG1 + IgG4) level.

The advent of new genetic engineering technologies and new GM plants requires the existing safety assessment system improvement, not excepting the methodology of the immunotoxicity and allergenicity research.

As the most promising parameters that can describe the immune status, are being considered the main cytokines: pro - and anti-inflammatory (IL-1,IL-6, IL-8,TNF-α,IFN-γ;IL-4,IL-10,IL-13), cell regulators (IL-1, IL-2, IL-10,IL-12,IFN-γ) and humoral immune response regulators (IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ), the basic immunoregulatory chemokines, the adhesion molecules, and immunoglobulins (IgG, IgE) in serum, and the cell population structure (CD) markers on the cell surface.

Taking into account the wide range of indicators that can be determined with modern research methods, one of the main tasks of GMO safety assessment system improving is the formation of an optimal list of parameters that can be used in immunotoxicological studies.

This work was carried out within the state assignment of FASO Russia (theme No. 0529-2014-0047).

The older people are more susceptible by environmental factors. Organic solvents are used widely in industry and in everyday life; they are found in paints, cosmetics, drugs, and gasoline. We’ve been reported environmental neurotic metabolite, 1,2-diacetylbenzene (DAB), DAB causes central and peripheral neuropathies that lead to motor neuronal deficits. DAB increases oxidative stress and protein adducts formation along with impaired hippocampal neurogenesis in mice.

To explore the link between neurodegenerative disease and exposure to DAB, we treated the DAB (3.0 mg/kg) in young (6 months) and old (21 months) Sprague Dawley rats for 1 week. RNA was isolated and applied RNAseq technique. The transcriptome analysis of mRNA reveals that DAB has the different effects on young and old rats. Young rats showed increased the genes which are related to metabolism and excretion. However, old rats showed increased response in inflammatory and immune responses. The inflammatory responses in old rats by DAB was very similar with gene expression pattern which is regulated by the bacterial endotoxin lipopolysaccharide (LPS). Especially in old rat group showed increased prolactin level. Prolactin plays an essential role in metabolism, regulation of immune response. We also confirmed up-regulated genes that are related to inflammation-related pathway by real time PCR. These results might be helpful to elucidate the underlying mechanism of neurotoxicity by DAB especially in older people.

Novel mouse skin sensitization model with ovalbumin (OVA) as antigen has been developed which is sufficient to detect key immune pathway necessary for immediate hypersensitivity reaction. But more alternative and widely exposed antigen than OVA is needed to consider in this model since it is rear to be transcutaneously sensitized with OVA. We investigated immunological and histopathological changes of mice transcutaneously sensitized with acid-hydrolyzed wheat protein (HWP), which was reported to induce immediate hypersensitivity to human.

HWP at a dose of 0.22, 1.1, 5.4, 27 and 135 µg was applied in skin patches to the left flanks of 8-week old BALB/c female mice 3 days a week for 4 weeks, and immune responses were evoked with 270 µg of HWP given via intraperitoneal route. Rectal temperatures, scores on anaphylactic responses, plasma histamine levels and histopathological changes were evaluated. In over 5.4 µg HWP groups evocated via intraperitoneal injection, increased IgG1 and IgE levels after sensitization and with augmented anaphylaxis scores after evocation were noted as compared with vehicle control. In over 27 µg HWP groups and 135 µg HWP group, decreased rectal temperature and increased plasma histamine levels were observed as compared with vehicle control, respectively. Ki67-positive germinal center in the regional axillary lymph node was observed more frequently in over 5.4 µg HWP groups.

Thus when mice were sensitized transcutaneously to HWP, sufficient sensitization potential in skin sensitization model was showed at low dose. These results suggest that HWP is promising effective antigen at comparable low dose to OVA (2 µg).

We raise the important issue of understanding drug immunogenicity. There is much ignorance on the term and the current perception of the ‘immunogenicity’ of a drug is rather misguided and limited to its ‘antigenicity’ property only, hence the scope of understanding and testing the actual immunogenicity of a drug remains very much ignored and misunderstood. The immunology of a drug and its immunologic safety are critical matters in the development of a drug. One third of all drugs in development fail prior to reaching the market due to drug-induced organ toxicity of which immunotoxicity or immunogenicity could be a contributing factor. This poses a significant financial burden to drug-developing companies. One major shortcoming in the preclinical study is that the testing of ADAs is a measure of the antigenicity of a drug only rather than the full scale immunogenicity. The immune response against drug-induced damaged tissue is ignored and drugs with the greater risk of causing secondary pathology in vital organs such as lungs or nervous system pass onto clinical phase and even the market. Drugs can also injure non-target tissue generating serious pathology, such as lung fibrosis by amiodarone. Another shortcoming is the viability of preclinical studies due to the dependence on animal models with translation from animals to human being a serious risk. This leaves the possibility of injury or fatality in clinical trials. The FDA and EMA require better understanding of drug immuno-toxicology that can be achieved through integrated assays that analyse novel markers of damage and immune response. We are running a series of in vitro human cell based assays to test drug induced tissue damage, biomarkers and the immune response against damaged tissue. Our aim is to develop an in vitro platform that can model immunogenicity of drugs and robustly predict the human immune response to a greater extent. Compared to downstream analysis in liver and kidney in conventional toxicology such studies are very upstream in the drug pathway. This will bring a whole meaning to immunogenicity and make drug development more translational and safer. This platform applies to both small and large molecules and to all therapeutic areas including oncology. By generating more critical information on drug immune safety at preclinical stage we can avoid TeGenero-like scenarios and be better prepared rather than waiting for dangerous outcomes to happen in the clinical phase.

Introduction of a methoxymethyl side chain into PPD yielded the oxidative hair dye precursor 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance, and a significantly reduced sensitizing potency compared to the strong sensitizers p-phenylenediamine (PPD) and p-toluenediamine (PTD). Occupational exposure of hairdressers to PPD and PTD has been associated with the development of allergic contact dermatitis involving the hands. The exposure of hairdressers’ hands to ME-PPD was assessed following typical working conditions. Samples were obtained after hair dyeing treatments using a hand rinse method and HPLC analysis. Based on the exposure data a quantitative risk assessment (QRA) for the induction of contact sensitization was conducted. Daily hand exposure concentrations were derived by accounting for wet work, uneven exposure and inter-individual variability for professionals. Daily hand exposure was compared with the sensitization induction potency of ME-PPD defined as the No Expected Sensitization Induction Levels (NESIL).

First estimations for ME-PPD indicate that the hairdresser hand exposure level is approximately two orders of magnitude below its NESIL. Correspondingly, the risk of contact allergy induction for hairdressers to ME-PPD is significantly reduced compared to PPD and PTD, with hand exposure levels only 2.7 and 5.9 fold below the NESIL, respectively. In conclusion, the QRA indicates that induction risk for contact sensitization to ME-PPD is very low for hairdressers, because their occupational daily hand exposure to ME-PPD is approximately two orders of magnitude below the NESIL.

There are many examples of immunomodulatory compounds inducing a significant safety risk associated with cytokine storm responses. Many investigators have assessed in-vitro cellular assays for evaluation of this response but these do not always accurately reflect the true biologic mechanisms taking place. Importantly, as many new therapeutics are being developed that are human specific there is an increasing need for human cell based assays that help predict the safey risks associated with cytokine storm. This poster reviews some of the assay formats that have been tested in the field and reviews the disadvantages and advantages of the specific approaches. The reproducibility and predictive nature of these approaches will be discussed with the aim of showing how these approaches can be used to accurately predict the potential for cytokine storm for novel biologics.

Immunoregulatory pathways tip the scale for either activation of inflammatory responses or the mediation of suppressive effects. Upon chemical exposure, type and duration of the immunological responses decide on the direction of outcomes. For this, metabolic reprogramming is critical. Importantly, both physical and chemical characteristics of a chemical may influence the reaction cascades.

The interferon-gamma-induced metabolic pathways of tryptophan breakdown to kynurenine via indoleamine 2,3-dioxygenase (IDO-1), and neopterin formation via GTP-cyclohydrolase in human peripheral blood mononuclear cells (PBMC) can be used as a reliable readout to investigate immunomodulatory effects of compounds and materials.

Zinc oxide (ZnO) nanomaterials are used in technical and chemical industry, in pharmaceutical, food and cosmetic applications are frequent, due to the relatively low toxicity and biodegradability. Exposure of the healthy skin to ZnO and nanomaterials is generally considered as safe, but there are concerns regarding ZnO containing aerosols. In real life, the possibility for interferences with biological processes cannot be excluded.

ZnO bulk material and nanoparticles were analysed in this in vitro setting in a concentration range from 2.3 to 37.5 µg/ml. Nanoparticles showed higher cytotoxicity compared to the bulk material. The kynurenine to tryptophan ratio, a measure of IDO-1 activity, decreased with all materials dose-dependently in mitogen-stimulated cells. However, nanomaterial treatment of unstimulated cells increased IDO-1 activity, while this effect was less prominent with the bulk treatment. Neopterin concentrations showed the same trend.

Data indicate that within a certain concentration range, ZnO nanoparticles may have some activating effect on unstimulated PBMC, while in stimulated cells and with higher concentrations immunosuppressive effects prevail. A closer elucidation of these effects is clearly warranted.

The control of inflammation, which arises from complex biological responses to harmful stimuli, is an important determinant of both clinical outcomes and patient comfort. However, the side effects of many current therapies such as non-steroidal anti-inflammatory drugs mean that new safe treatments are required. We previously reported that 12.5 mg/ml hydroxytyrosol (HT) suppressed gene expression of the inducible nitric oxide (NO) synthase (iNOS) isoform, and NO production, in mouse peritoneal macrophages treated with lipopolysaccharide (LPS), where nuclear factor-κB (NF-kB) gene expression was not altered. The present study evaluated the anti-inflammatory effects of various concentrations of HT in LPS-induced RAW264.7 mouse macrophage cells. HT suppressed NF-kB signaling and downregulated LPS-mediated expression of iNOS, cyclooxygenase-2, and interleukin-1b at concentrations ≥ 12.5 mg/ml, resulting in reduced production of NO and prostaglandin E₂. Additionally, we aimed to determine whether HT suppresses COX-2-induced inflammation in a carrageenan-induced rat paw edema model. Additionally, we compared its activity with those of the selective COX-2 inhibitor, celecoxib, and a representative nonsteroidal anti-inflammatory drug (NSAID), indomethacin. HT, celecoxib, and indomethacin significantly suppressed swelling in carrageenan-injected rat paws. Although HT was less effective than celecoxib and indomethacin, it had a delayed onset of action, suggesting that the combined use of HT and celecoxib or indomethacin might yield a sustained anti-inflammatory effect. Moreover, we evaluated whether HT aggravates gastric damage, which is a typical adverse effect associated with NSAIDs, in an aspirin-induced gastric damage rat model. Unlike celecoxib and indomethacin, HT did not cause gastric damage when co-administered with aspirin. Our results indicate that HT displays potential as a new anti-inflammatory drug without gastric toxicity.

CuO nanoparticles (CuO NPs, 1-100 nm) are used in industrial and consumer products due to their antibacterial properties. The macrophage-NP interaction is highly important for the distribution, fate and toxicological profile of NPs.

Here we hypothesized that antibacterial metal-based NPs, including CuO NPs, that are already used in consumer products can be harmful to human cells. Since toxicity of CuO towards various cells may depend on their surface functionalization, four CuO NPs with primary sizes about 30 nm and different surface functionalizations (coatings) were studied: uncoated CuO, CuO functionalized with amino group (CuO-NH₂), CuO functionalized with carboxyl group (CuO-COOH) and CuO functionalized with polyethylene glycol (CuO-PEG). Escherichia coli MG1655 was selected to test antibacterial effects of differently functionalized CuO. Macrophages differentiated from THP-1 cells in vitro_,were selected for immunotoxicity testing. Differently from all the previous studies, antibacterial and immunotoxicity tests were performed in the same conditions: incubation at 37^◦ C in 10%-serum supplemented culture medium. Alamar blue assay was used as the toxicity endpoint in all cases. In parallel, we measured dissolution of CuO NPs in these test conditions and the toxicity of ionic Cu (as CuSO₄) as a control for solubility. Toxicity was expressed as EC₅₀ values adjusted to the Cu content.

All tested CuO NPs and ionic control CuSO₄ were toxic to bacterial cells. The antibacterial toxicity (24 h EC₅₀) varied from 19 mg Cu/l (CuO-PEG) to 42 mg Cu/l (uncoated CuO) and the EC₅₀ values increased in the order CuO-PEG < CuO-NH₂< CuO-COOH < CuSO₄ 50) varied from 15 mg Cu/l (CuO-NH₂) to >30 mg Cu/l (CuO-PEG) and the EC₅₀ values increased in the order CuO-NH₂< uncoated CuO < CuSO₄ < CuO-COOH < CuO-PEG. Thus, CuO-PEG could be the most suitable antibacterials as they were the most toxic to E. coli cells and not toxic to human immune cells in vitro. In contrast, CuO-NH₂ was significantly more toxic to THP-1 cells then to E. coli cells and is the least favorable as the antibacterial. This knowledge can be used for the synthesis of more efficient and safe antimicrobials. This work was supported by Estonian Research Council grants IUT23-5 and PUT1015.

Background: Mycotoxins are secondary metabolites and capable of causing life-threatening effects. Ergot alkaloids are mycotoxins which formed by Claviceps species. Among these alkaloids, ergotamine has been commonly prescribed for migraine headaches. Its mechanisms of toxicity nevertheless has not yet been completely understood. Among polyphenols, quercetin is mostly distributed in different fruits and vegetables.

Materials and Methods: In this study hepatocyte lysis and ROS formation have been investigated using accelerated cytotoxicity mechanism screening (ACMS).

Results: On the base of our results ergotamine induces ROS formation and lipid peroxidation in freshly isolated rat hepatocytes in dose dependent manner and these toxicity markers have been significantly prevented by quercetin.

Conclusion: It is concluded that the ROS formation play a key role in the ergotamine induced toxicity and quercetin could be considered as a supplement for who suffers from migraine.

Many pesticides including pyrethroid are subjected to metabolic biotransformation by hepatic cytochrome P450 (CYP) enzymes. CYP enzyme induction alters balance between detoxification and activation and in cases this may lead to enhanced toxicity. There are numerous examples where CYP enzymes catalyze metabolic activation of chemically inert agents to electrophiles. This work showed induction of CYP catalytic activities and mRNA levels by the pyrethroid lambda-cyhalothrin. Testosterone oxidative metabolism was determined in hepatic microsomes from control and treated rats. There are evidences that CYP3A1/2, CYP2A1, CYP2C11 and CYP2B1 isozymes catalyze testosterone oxidation at the 6β-, 7α-, 16α- and 16β-positions. Microsomal 6β-, 7α-, 16α- and 16β-testosterone hydroxylase activities were evaluated by conversion of testosterone to 6β-, 7α-, 16α- and 16β-hydroxytestosterone metabolites by HPLC. Quantitative real-time PCR assaysfor rat CYP1A1, CYP1A2, CYP2A1, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1 mRNA were also performed to analyze mRNA gene expressions. Oral exposure of rats to lambda-cyhalothrin resulted in a dose-dependent significant increase of hepatic microsomal testosterone 6β-, 7α- and 16β-hydroxylase activities. Lambda-cyhalothrin (8 mg/kg bw 6 days) produced significant increases in testosterone 6β-hydroxylase (68%,reflecting CYP3A1/2 activity), testosterone 7α-hydroxylase (52%, reflecting CYP2A1 activity) and testosterone 16β-hydroxylase (412%, reflecting CYP2B1 activity). CYP2E1 and CYP3A1/2 mRNA levels increased significantly (at 4 and 8 mg/kg bw 6 days). The major significant increase of mRNA levels was observed in CYP2B1 (1463% and 961%) and CYP2B2 (604% and 501%) in both treatment groups. It is concluded that lambda-cyhalothrin induces CYP enzymes which may enhanced the toxicity of these compounds. Work supported Projects Ref. S2013/ABI-2728, Comunidad de Madrid, and Ref. RTA2015-00010-C03-03, Ministerio de Economía, Industria y Competitividad, Spain.

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we document, for the first time, the generation of hydroxyl radicals (OH•) during the corrosion of Fe-based materials and their deleterious impacts on endothelial cells. The generation of OH• was documented by two independent acellular assays, terephtalic acid hydroxylation (fluorescence) and formation of spin trapping adducts (electron paramagnetic resonance spectroscopy). All tested Fe-based materials exhibited a strong potential to generate OH•, directly from the incomplete reduction of dissolved oxygen and/or via Fenton chemistry mediated by Fe ions. The reduction of these signals in presence of D-mannitol confirmed their specificity for OH•. Cellular responses were assessed in vitro on two human endothelial primary cells (HUVECs and HAOECs) using colorimetric and luminescence cytotoxicity assays. Cells were exposed directly to Fe powder, or to corrosion extracts. Only direct contact with Fe materials affected cell viability, consistent with the cytotoxic activity of short-lived OH• produced in direct contact with cells. To confirm the cellular impact of OH•, mRNA expression of oxidative stress response genes (heme oxygenase-1 (HO-1) and glutamate cysteine ligase modifier subunit (hGCLM) was also assessed. The expression of oxidative stress genes was dose-dependently increased 4h after direct exposure to the particles, not to extracts.

The demonstration of OH• production during corrosion and consequent oxidative stress on endothelial cells provides a new perspective on the biocompatibility of biodegradable Fe-based alloys, the future design of Fe-based vascular implants, and the protocols for assessing their biocompatibility in vitro.

Objective: Aluminum is a widely exposed neurotoxicant. However, the molecular mechanism underlying Aluminum toxicity remains to elucidate. In this study, expression profiling was performed using Illumina next generation sequencing to show the interfered hippocampus transcription induced by aluminum exposure. The ultimate goal was to find the key transcription changes in Aluminum-induced neurotoxicity.

Materials and Methods: Sub-chronic intraperitoneal injection of Aluminum-maltolate complex[AL(mal)₃] was performed for 3 months. cDNA libraries were constructed from six rats’ hippocampus (3 in control group and the other 3 in aluminum-exposed group). DNA-seq analysis of the two groups was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Differentially expressed genes (DEGs) were analyzed using DEseq2 method. Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was employed to analyze the function of DEGs.

Results: 96 up-regulated and 652 down-regulated DEGs were identified, comparing to the control group. GO analysis results showed that multiple functional genes are most significantly affected by aluminum exposure, including gial cell differentiation, neural transmission and vesicle trafficking. Furthermore, KEGG pathway analysis results revealed that these DEGs were clustered in several signal pathway, including ECM-receptor interaction; PI3K-Akt signaling pathway; Focal adhesion and cAMP signaling pathway.

Conclusion: Expression profiling identified that the genes involved in gial cell differentiation, cell adhesion and vesicle trafficking were candidate genes to be used in the research on aluminum induced neural toxicity.

(This work is supported by NSFC 81430078, 81372968)

Humans can be exposed at low concentrations to nephrotoxic agents with anthropogenic and natural origins such as uranium and fluoride. Nevertheless, there is a lack of knowledge of their mechanisms of nephrotoxicity. This study aims to compare these mechanisms in mouse to identify the cellular and molecular pathways of toxicity.

C57Bl6 mice are exposed by intraperitoneal injection of uranyl nitrate (UN) (0, 2, 4, 5 mg/kg) or sodium fluoride (NaF) (0, 2, 5, 7.5, 10 mg/kg) and euthanized 48h and 72h later. Renal phenotypic aspects and biological mechanisms are evaluated by urinary biochemistry, gene and protein expressions, enzyme activity, and histological analyses. Exposure to UN and NaF induces nephrotoxicity in a dose-dependent manner. 5 mg/kg of UN induces mild histopathological alterations and respectively 44 and 6-fold increase in gene expressions of nephrotoxicity markers KIM1 and osteopontin. In comparison, 10 mg/kg of NaF induces high nephrotoxicity with histopathological alterations scored as severe and late appearing parameters of toxicity whereas 7.5 mg/kg induces mild histopathological scoring and gene expressions of KIM1 and clusterin enhanced respectively by 70 and 4-fold compared to control. No signs of nephrotoxicity are observed below 5 mg/kg of NaF. Clusterin is respectively increased by 2.4 and 4.4-fold in urines after 7.5 mg/kg and 10 mg/kg injections of NaF. Apoptosis is evaluated through caspases 3/7 activity which is increased by 210% after UN treatment (5mg/kg) whereas NaF does not induce apoptosis. Inflammation is implied in UN and NaF acute nephrotoxicity as shown by gene and in situ overexpressions of ICAM and VCAM.

UN and NaF acute exposures resulted in a dose and time-dependent nephrotoxicity with a higher nephrotoxicity after 72h. Inflammation and apoptosis are both involved in UN or NaF toxicity. These observations allow us to identify the mechanisms that will be studied in a low-dose exposure protocol after a chronic exposure.

Epidemiological and experimental studies have shown that perinatal exposure to dioxin induces impaired cognitive function and behavioral abnormalities in humans and laboratory animals. Disruption of neuromorphology and expression of neurotransmitter receptor genes was observed in the brain of rodent offspring in utero and lactationally exposed to dioxin, suggesting impaired neural circuit structure and function by dioxin exposure. The aryl hydrocarbon receptor (AhR) that avidly binds dioxin is a ligand-activated transcription factor, and ligand-AhR complex induces various toxicities through activation of downstream signaling pathway. In addition, AhR plays important roles in the developing nervous system of invertebrates and vertebrates. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study was performed to examine AhR expression in the brain of C57BL/6J mice using western blot and in situ hybridization techniques. AhR protein was detected in the brain of 3-day-old mice although its expression level in the brain was significantly lower than that in the liver, lung, kidney, thymus, and spleen. Next, we examined AhR mRNA expression in the brain of embryonic and postnatal mice. On embryonic day 12.5, AhR mRNA was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebellum, and olfactory bulb of 3- and 14-day-old mice. In the hippocampus, mRNA expression in the CA1 and CA3 pyramidal cell layers was higher than stratum radiaum and oriens, suggesting that AhR is expressed in a subregion-specific manner. These results reveal temporal and spatial patterns of AhR expression in the mouse brain, which may contribute to understanding mechanisms of developmental neurotoxicity of AhR ligands, such as dioxin.

There are more than 6,000 chemical constituents in cigarette smoke (CS), many of them known toxicants or carcinogens. In vitro models, commonly used in toxicology, are often based on submerged cell cultures that cannot be exposed to smoke or aerosol directly. It is thus essential to develop adequate protocols to quantitatively trap smoke/aerosols in bio compatible solvent to allow testing them in submerged cell cultures.

With a rigorous Design of Experiment approach, we aimed to optimize the process of collecting the smoke from the 3R4F reference cigarette and the aerosol generated from the Tobacco Heating System (THS) 2.2 in phosphate-buffered saline (PBS) or in ethanol. In this study, the protocols to generate two different fractions, total particular matter (TPM) and aqueous extract (AE), were optimized for these two tobacco products to improve trapping efficiency, A detailed characterization of the chemical composition of each produced fraction was subsequently performed.

Trapping efficiencies for TPM and AE were determined by measuring nicotine concentration using a gas chromatography with a flame ionisation detector and the levels of eight carbonyls quantified by liquid chromatography with electrospray ionization mass spectrometry respectively . When comparing the extraction methods (shaking or syringe) from the Cambridge filter pad, nicotine concentration for THS 2.2 TPM fraction was slightly higher when shaking, while for 3R4F TPM, no difference was observed between the two extraction methods. In order to obtain the highest nicotine concentration per test item on one filter pad, we found that the smoke from six 3R4F cigarettes and the aerosol from four THS 2.2 test items were needed. We also found that for the AE preparation, collecting the aerosol before the puff volume-generating pump in an impinger filled with ice-cold PBS was important to obtain the highest carbonyl concentration per test item. Finally, the use of an impinger that contained glass pearls also improved the trapping efficiency for 3R4F AE smoke fraction.

In summary, we present optimized protocols for the collection of TPM and AE fractions from 3R4F and THS 2.2 tobacco products that may be leveraged in systems toxicology based in-vitro assessments.

The use of peroxisome proliferator-activated receptor-gamma agonists as anti-diabetic drugs has been limited due to serious adverse effects. PPAR-gamma targeting drugs such thiazolidinediones (TZDs) have been implicated with heart failure which has been linked to increased fluid retention. However, the exact underlying molecular mechanisms of PPAR-gamma mediated water resorption in the kidney are largely unkown. As part of an exploratory discovery safety strategy we seek to analyze whole transcriptome profiles upon drug exposure as a tool to decipher PPAR-gamma associated kidney-specific toxicity. Human induced pluripotent stem cell (hiPSC)-derived kidney organoids more closely recapitulate the complexity of kidney tissue in vitro than cell line models with regard to cellular composition and organization. As TZD-induced adverse outcome in vivo is manifested upon long-term treatment, an additional benefit of the 3D organoid model is that it allows for long-term culture and repeated dosing. Therefore, kidney organoids were the model of choice representing a meaningful model system to study TZD-mediated effects in vitro.

In an effort to cover acute and prolonged drug respone, TZD-treated kidney organoids are harvested after several exposure durations and processed for generation of sequencing libraries from mRNA, to obtain information from the coding transcriptome. Acquired data from high throughput whole transcriptome sequencing are subjected to bioinformatic preprocessing prior to integrated analysis.

Validation of kidney organoid model demonstrates suitable detection of drug-induced gene expression changes associated with kidney toxicity, water resorption and ion transport function. Quantification of gene expression data upon TZD exposure indicate the induction of known PPAR-gamma target genes as well as distinct transcriptional programs over time. We believe that in conjunction with future applications using samples of long-term treated animals, transcriptome analysis is a promising platform to discover novel mechanisms of toxicity and biomarkers. Ultimately, identification of safety biomarkers with regard to TZD-induced transcriptional changes will be crucial in order to overcome the lack of reliable in vitro methods to de-risk PPAR-gamma during early drug discovery.

A growing body of evidence suggests that air pollution can harm the brain, accelerating cognitive aging, and may even increase risk of Alzheimer’s disease and other forms of dementia. Neuronal apoptosis plays a crucial role in neurodegenerative diseases such as Alzheimer’s disease. Benzo[a]pyrene (B[a]P), a typical component of air pollution , can lead to neural cell apoptosis. However, little is known about the molecular mechanisms in B[a]P-induced neural cell apoptosis. In this study, Male SD rats were used to establish models of B[a]P-induced apoptosis, rats were randomly divided into five groups: the blank control group received no treatment and the others were received intraperitoneal injection of B[a]P (0, 1.0, 2.5, 6.25mg/kg.bw) for 1, 2 and 3 months respectively. The results of immunohistochemistry showed that B[a]P increased neural cell apoptosis and BPDE-DNA adducts in rat cortex . In the three dose groups, after 3 months treatment, B[a]P upregulated expressions of ATM, Chk2, Chk2thr68, p53, p53ser20 , PUMA and cleaved caspase3 proteins in a dose- and time-dependent manners in rat cortex . The findings suggested that B[a]P-induced cortex neurons apoptosis through DNA damage, in which ATM/Chk2/p53 pathway play an important role.

Background: Rechargeable Li-ion batteries (LIB) are increasingly used worldwide. They contain micrometric and low solubility particles consisting of toxicologically relevant elements. Their production and use has increased sharply in recent years, implying a potential for inhalation exposure in occupational settings. New proposed applications such as printable or spray-paintable batteries might also expose consumers. The health hazard of these materials is not documented. We performed the first study on the respiratory hazard of 3 leading LIB components (LiFePO₄ or LFP, Li₄Ti₅O₁₂ or LTO, and LiCoO₂ or LCO) and investigated their mechanisms of action.

Methods: Particles were characterizedphysico-chemically (composition, morphology, size distribution) and elemental bioaccessibility was documented at neutral and acidic pH. Lung inflammation, oxidative stress, fibrotic responses, and particle persistence were assessed in C57BL/6 and interleukin (IL) -1β deficient mice after oro-pharyngeal aspiration of LIB particles (0.5 or 2 mg) or crystalline silica (2 mg) used as reference. The implication of hypoxia-inducible factor (HIF)-1α in lung toxicity was assessed in vivo by using chetomin, an inhibitor of HIF-α activity.

Results: LIB compounds contain a significant fraction of respirable particles and their metallic elements are differently bioaccessible. Acute inflammatory lung responses (including IL-1β) and oxidative stress were recorded with the 3 LIB particles and silica, LCO being the most potent. Inflammation persisted 2 m after LFP, LCO and silica, in association with fibrosis in LCO and silica lungs. LIB particles persisted in the lung after 2 m and formation of ferruginous bodies was detected in LCO lungs. In vivo, only LCO and silica stabilized intracellular HIF-1α, a marker of Co ion activity, known to be implicated in inflammation and pro-fibrotic events. IL-1β deficiency or inhibiting HIF-1α activity significantly reduced in vivo lung inflammation induced by LCO.

Conclusions: We report here the varying respiratory toxicity of particles used in LIB, and conclude that they represent a respiratory hazard. LCO was at least as potent as crystalline silica to induce inflammatory and fibrotic responses. IL-1β and HIF-1α might represent key mediators of the elective lung toxicity of LCO particles.

Synthetic cathinones (SC) comprise a broad group of new psychoactive substances structurally related to cathinone, a naturally occurring stimulant found in the khat plant (Catha Edulis). The rapidly increasing use of SC, the second largest group of monitored new drugs, represents a major public health concern in view of several reports of intoxications and deaths. However, their toxicological data remains scarce. Pregnant and women of childbearing potential comprise particular risk groups, due to the potential onset of neurodevelopment disorders in the offspring.

This work aimed at evaluating the in vitro neurotoxicity of four commonly reported SC, as a first assessment of their effects on neuronal differentiation.

Briefly, different toxicological parameters (i.e. cell viability, mitochondrial integrity, energy metabolism) were evaluated in NG108-15 neuroblastoma cells exposed to increasing concentrations (1nM – 500μM) of Ethcathinone (Eth-Cat), 4-Chloroethcathinone (4-CEC), 4-chloro-alpha-pyrrolidinopropiophenone (4-Cl-α-PPP) or 4-fluoro-Pyrrolidinohexanophenone (4F-PHP). Neurite outgrowth was measured as the number of newly formed neurites per total number of cells, following differentiation with forskolin and retinoic acid, in the presence or absence of SC (single addition at day 0) at in vivo relevant concentrations (< 10μM).

4F-PHP showed the highest neurotoxicity, significantly decreasing metabolic activity (MTT reduction) above 1nM and was the only one inducing membrane integrity loss (LDH release) at 500μM. 4-Cl-α-PPP only decreased metabolic activity at the highest concentration tested, while neither Eth-Cat nor 4-CEC affected cell viability up to 500μM. Interestingly, only Eth-Cat induced the hyperpolarization of the mitochondrial membrane following exposure to 1 and 10μM and none of the SC significantly altered intracellular ATP levels up to 100μM. Moreover, none of the SC tested affected neurite outgrowth up to 10μM.

Overall, the tested SC showed different neurotoxicity profiles, suggesting the involvement of distinct toxicity mechanisms, which do not seem to affect neuronal differentiation at in vivo relevant concentrations.

Background: A parental prenatal high fat diet leads to an impaired metabolic phenotype in mouse offspring. The underlying mechanisms, however, are not yet fully understood. Therefore, this study investigated whether the impaired metabolic phenotype could have been programmed through altered gene expression and DNA methylation in promoter regions.

Methods: Both parent mice received either a high fat or a low fat diet starting from 6 weeks before gestation, continuing throughout pregnancy and lactation. At weaning, all offspring mice were switched to a high fat diet. Analyses were done on liver samples from male offspring at 12 and 28 weeks of age. Gene expression was assessed using microarray. Several differentially expressed genes (Cd163, Hmgcr, Aacs, Lpin1, Saa1, Orm2 and Il1r1) were subsequently selected for bisulfite pyrosequencing of the promotor region.

Results: Gene set enrichment analysis of microarray data revealed that metabolic pathways were indeed enriched as a result of the prenatal high fat diet. Bisulfite pyrosequencing of the promotor regions of Cd163, Hmgcr, Aacs, Lpin1, Saa1, Orm2 and Il1r1 indicated that DNA methylation levels were altered at several transcription factor binding sites.

Conclusion: These results suggest that an impaired metabolic phenotype in offspring mice due to prenatal high fat diet may be mediated by gene expression and DNA methylation.

Exposure to respirable silica dusts is associated to severe lung diseases, including silicosis and lung cancer, and is still a current source of concern for the heath of many workers and those using silica in nanotechnologies. Crystallinity has always been considered as the main feature of hazardous silica dusts. However, crystalline silicas are not all equally pathogenic, and some still unidentified physico-chemical differences between particles account for the variability of the silica hazard.

We recently synthesized a set of quartz crystals of respirable size, exposing as-grown, intact crystal faces. These particles showed a very low biological activity in a series of cellular tests relevant for the pathogenicity of quartz, indicating that the activity of quartz dust is not necessarily contingent to crystallinity. Mechanical grinding of these quartz crystals markedly increased their activity. By combining this finding with studies on a large set of both amorphous and crystalline silica particles, the surface spatial/energetic configuration of silanols emerged as a relevant characteristic accounting for damage to cellular membranes. We also correlated the ability of quartz particles to cause membranolysis with their capacity to activate the enzymatic machinery inflammasome and trigger a pro-inflammatory response, a step at the origin of silicosis and lung cancer. Our current hypothesis is that characterizing silanols through physico-chemical and in vitro analyses allows identifying the pathogenic activity of silica materials.

We are currently validating our hypothesis in vivo and developing a set of assays to identify and predict the respiratory hazard of silica particles based on the analysis of their surface silanol distribution. The results obtained on a panel of model quartz samples prepared ad hoc (i.e. particles with defined and designed physico-chemical properties) showed significant differences in their membranolytic and cytotoxic activity consistent with the hypothesis, confirming the relevance of silanols. Preliminary analyses via diffuse reflectance infrared spectroscopy (DRIFTS) indicated that this technique is promising to clearly reveal the silanol distribution of the silica particles.

Benzene is a widespread environmental and occupational pollutant. The major adverse health effect of benzene exposure is hematotoxicity. Hif-1a regulates the expression of genes encoding molecules that participate in cell proliferation. In the present study, we aimed to investigate the effects and molecular regulation of hif-1a on cell proliferation during benzene exposure. Male C57BL/6 mice were administered by subcutaneously injection with or without benzene for 2 weeks, hematotoxicity, cell proliferation and hif-1a protein level were evaluated. PTP4A3, a proliferation-related gene, was found by ChIP-Seq screening as a hif-1a target gene. Next, the hif-1a high expression mouse model (C57BL/6- hif-1a⁺) was constructed, the protein level of hif-1a and PTP4A3 were measured by western blot in WT and C57BL/6- hif-1a⁺ mouse. The bone marrow (BM) cell proliferation was also analyzed by BD FITC BrdU Flow Kit. Then the PTP4A3 inhibition K562 cell line was established by transfecting with the specific shRNA. Cells were exposed to 1,4-Benzoquinone (1,4-BQ) for 24 hours. The cell viability, proliferation, cell cycle and key protein levels in PI3K-ATK pathway were estimated. The results showed that Benzene exposure caused significant reduction in numbers of leukocytes, red blood cells, platelets and decreased hif-1a level in benzene exposed mice. Meanwhile, a significant decrease in the percentage of hematopoietic stem cells (HSCs) and an increase in the proliferation were observed in benzene exposed mice. Then we found that augmentation of hif-1a in C57BL/6 mouse significantly elevated the proliferation of BM cells and increased PTP4A3. In vitro, after treated with 1,4-BQ, PTP4A3 was significantly reduced in PTP4A3 inhibition and control cells. Compared with control cells at the same 1,4-BQ concentration, the cell viability and cell proliferation were significantly lower in PTP4A3 inhibition cells. Protein levels of PI3K and ATK were also dramatically decreased in cells with inhibited PTP4A3. In addition, G2/M phase found in the cells with inhibited PTP4A3 exposed to 1,4-BQ than those in control cells. In conclusion, the hif-1a regulates cell proliferation through PTP4A3 and PI3K-ATK pathway during benzene exposure. (Supported by National Natural Science Foundation of China 81573189, 81730087 and 81703265).

Introduction: Ethanol consumption is described to induce the expression of the inducible isoform of the enzyme nitric oxide synthase (iNOS). Purpose: Evaluate the role of iNOS in the renal toxicity induced by chronic ethanol consumption. Methods: Male C57BL/6J wild-type and iNOS-deficient (iNOS^-/-) mice were randomized into 4 groups: Control (WT); Ethanol (E-WT); iNOS (iNOS^-/-) and Ethanol iNOS^-/- (E-iNOS^-/-) (Ethics committee 14.1.847.53.0). The animals were treated with water or ethanol 20% (v/v) ad libitum for 10 weeks, and the temporal assessment of body weight and liquid and solid intake was determined. Systolic blood pressure (SBP) was measured. To evaluate the renal function, serum levels of urea, creatinine, K⁺ and Na⁺ ions were determined. The renal cortex was collected for the assessment of levels of: superoxide anion (O₂˙^-), hydrogen peroxide (H₂O₂), nitrate/nitrite (NOx), thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), oxidized glutathione (GSSG) and cytokines. Catalase (CAT), superoxide dismutase (SOD), metalloproteinases (MMP) and myeloperoxidase (MPO) activities, besides the Nox1, Nox2, Nox4, eNOS and SOD2 protein expression were evaluated. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s test (p<0.05). Results: Long-term ethanol consumption induced change in nutritional status and increased SBP, creatinine serum, IL-1β and GSH levels in WT and iNOS^-/- mice. Ethanol-treated animals showed high O₂˙^- levels and MPO activity, however these responses were lower in E-iNOS^-/-. High Nox4 expression, TNF-α and TBARS levels was noticed in E-WT mice. NOx levels were not modulated by ethanol, but the kidney of iNOS^-/- animals showed lower concentrations than WT. CAT, SOD, MMP-2 and MMP-9 activities, Nox1, Nox2, eNOS and SOD2 protein expression, GSSG, H₂O₂, IL-6, IL-10, urea, K⁺ e Na⁺ ion levels did not differ among the groups. Conclusion: Chronic ethanol consumption induces rise of O₂˙^-, TBARS and TNF-α levels, Nox4 protein expression and inflammatory cell infiltration in renal cortex, and these responses are mediated by iNOS. These results suggest a role for iNOS in renal toxicity induced by ethanol consumption. Financial support: FAPESP/CAPES

Evidence has been accumulated during the last three decades on the strong implication of several oxidants including hydroxyl radical, one-electron oxidants and singlet molecular oxygen [¹O₂] in the generation of hydroperoxides from several nucleobases, amino acids, and unsaturated lipid components. ¹O₂ is one of the major species responsible for the cytotoxic effects of photodynamic treatment and is being implicated in diseases such as porphyria and cataracts. In addition, ¹O₂ can trigger ultraviolet A-induced biological effects through activation of gene expression. Cell membrane contains polyunsaturated fatty acids, which are vulnerable to attack from many reactive species. The outcome of this process is lipid peroxidation and its primary products are lipid hydroperoxides. In addition, hydroperoxides can react with reactive nitrogen species leading to nitro or nitrosyl products. The aim of present work is to study the generation of ¹O₂ through the reaction of lipid hydroperoxides with nitronium tetrafluoroborate (NO₂BF₄). Ultraweak chemiluminescence arising from biomolecules oxidation has been attributed to the radiative deactivation of ¹O₂ and electronically excited triplet carbonyl products involving dioxetane intermediates. The approach used to unequivocally demonstrate the generation of ¹O₂ in these reactions is the use of the direct spectroscopic detection and characterization of ¹O₂ light emission. Characteristic light emission, corresponding to the singlet delta state monomolecular decay was observed.Chemiluminescence measurement of the monomol light emission in the near infrared region (λ= 1270 nm) has indicated that the reaction of organic and lipid hydroperoxides (cumene, t-butyl, oleic, linoleic and cholesterol hydroperoxides) with NO₂BF₄ generate ¹O₂.The use of sodium azide as a physical quencher of ¹O₂, associated with chemiluminescence measurements, contributed to identifying the generation of ¹O₂. Besides Russell mechanism, other possible mechanisms for ¹O₂ generation are under investigation.¹O₂ might be generated as a byproduct of lipid peroxidation, in conditions where reactive nitrogen species interact with lipid hydroperoxides. This might contribute to a better understanding of this complex event and physiological or physiopathological implications. Acknowledgments: FAPESP 2012/12663-1, CEPID 2013/07937-8, CNPq 301307/2013-0, CAPES NAP Redoxoma 2011.1.9352.1.8 and John Simon Guggenheim Memorial Foundation (P.D.M. Fellowship).

Globally, pollution problems are more severe in industrialized regions or in high density populated regions. The São Paulo Metropolitan Region is an example of a megalopolis with a unique set of emissions, solar flux and meteorological conditions that determinates its higher atmospheric pollution. Previously, we have shown the accurate quantification of 1,N²-propanodGuo in human urinary samples collected from residents of a polluted city (SP) and an unpolluted region showed significantly higher 1,N²-propanodGuo levels in the samples from SP donors than in samples from donors of the unpolluted region (Garcia et al. Chem. Res. Toxicol. 26, 1602-1604, 2013). This result provided evidence that elevated levels of 1,N²-propanodGuo in urinary samples may be correlated with urban air pollution. Here, we described a highly sensitive method involving HPLC/ESI-MS detection in SRM mode and employing isotope-labeled internal standards for the analysis of several DNA adducts (adductome) with aldehydes such as formaldehyde (1,N2-hydroxy-methyl-deoxyguanosine), acetaldehyde (1,N²-propano-2’-deoxyguanosine), crotonaldehyde, acrolein 1,N2-(α-Hydroxy) propano-2’-deoxyguanosine and 2,4-decadienal. The methodology was used to quantify the levels several DNA-aldehyde adducts in calf thymus DNA exposed to tobacco and cannabis smoke. In control DNA, 8-oxo-7,8-dihydro-2’-deoxyguanosine and 1,N2-hydroxy-methyl-2’-deoxyguanosine were detected. In the groups exposed to tobacco and cannabis smoke, all the analyzed adducts were detected. With the exception of the acetaldehyde adduct 1,N²-propano-2’-deoxyguanosine adduct, the groups exposed to tobacco smoke present higher levels of DNA adducts in relation to the group exposed to cannabis smoke. The method described herein can be used to study exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals after air pollution exposure. Supported by CEPID-Redoxoma (FAPESP: Proc. 2013/07937-8), NAP-Redoxoma (PRPUSP: Proc. 2011.1.9352.1.8), CNPq (301404/2016-0 and 301307/2013-0).

4-Fluoromethamphetamine (4-FMA) is a stimulant and entactogenic amphetamine, with alleged nootropic effects. Many drug users describe its subjective effects as similar to those of other amphetamines, but the potential health effects of recreational use of 4-FMA is hitherto unknown.

Because the liver plays a pivotal role in the metabolism and toxicity of amphetamines, we set out to evaluate the cytotoxic effects of 4-FMA using three complementary in vitro hepatocyte models.

Viability of human immortalised HepG2 and HepaRG cells, and primary rat hepatocytes (PRH) was evaluated by MTT reduction, after exposing cells for 24h to a concentration range of the drug that enable obtaining complete concentration vs. effect curves. 4-FMA was shown to induced concentration-dependent toxicity, primary rat hepatocytes being the most sensitive in vitro model (LC50 2.21 mM), followed by HepaRG and HepG2 (LC50 5.59 mM and 9.57 mM, respectively). Also, 4-FMA significantly impaired the glutathione and energetic storages in PRH, as well as disrupted redox homeostasis. Finally, 4-FMA activated the apoptosis common pathway, following activation of caspase-8 and caspase-9. The co-incubation of PRH with 4-FMA and cytochrome P450 specific inhibitors suggested that its toxicity is metabolism-dependent. CYP2D6 inhibition increased 4-FMA-induced cytotoxicity, while CYP2E1 inhibition decreased the cytotoxicity. These data suggest that 4-FMA may be metabolically activated by CYP2E1, while metabolism through CYP2D6 results in detoxification. CYP3A4 inhibition had no impact in the toxic effects of 4-FMA.

Our results show that 4-FMA is potentially hepatotoxic to its users. Since the metabolism of the drug is likely to greatly affect its toxicity, interindividual variability in susceptibility and potential for toxicologically relevant drug interactions are of concern.

Synthetic cathinones (β-Keto amphetamines; β-KA) abuse has been associated with renal injury. However, the exact mechanism behind the nephrotoxic effects of β-KA is not yet understood. This study aimed to investigate the role of autophagy in β-KA-induced nephrotoxicity in human kidney (HK-2) cells. To this purpose, HK-2 cells were exposed to two different concentrations of 3,4-dimethylmethcatinone (3,4-DMMC; 0.25 or 0.5 mM) and 3,4-methylenedioxymethcathinone (methylone; 2 or 4 mM) for 24 h. Both drugs prompted the formation of acidic vesicular organelles (evaluated by fluorescence microscopy of acridine orange-stained cells) and autophagosomes (evaluated using a CytoID Autophagy Detection Kit) in a concentration-dependent manner. Moreover, the autophagy inhibitors 3-methyladenine and wortmannin significantly potentiated cell death (evaluated by the reduction of the tetrazolium salt MTT), indicating that autophagy serves as a cell survival mechanism that protects β-KA exposed cells. We further examined the role of oxidative stress in autophagy induction and found that antioxidant treatment with N-acetylcysteine (NAC) or ascorbic acid significantly potentiated cell death induced by these cathinone derivatives, most likely due to inhibition of ROS-mediated autophagy and potentiation of apoptosis induced by β-KA. Further studies are ongoing to verify this theory.

Acknowledgements: This work was supported by national funds through FCT – Fundação para a Ciência e a Tecnologia, in the scope of FCT Project UID/Multi/04546/2013. A.M.A. thanks Fundação para a Ciência e Tecnologia (FCT), Portugal, for her PhD grant (SFRH/BD/107708/2015).

Oral clefts in any form (cleft lip and/or palate alone or associated with other head skeletal defects) represents one of the most frequent abnormality in humans (1:700 live births) showing increasing interest also because of their demonstrated multifactorial origin, involving both genetic and environmental risk factors. Environmental agents inducing cranio-facial malformations in experimental models include some pesticides, especially azole fungicides. The proposed common mode of action for azoles teratogenic effects is the inhibition of CYP26 enzymes, involved in the retinoic acid (RA) catabolism, with a consequent perturbation in endogenous RA levels in specific embryo segments. Risk assessment now focuses on combined exposures to molecules and, for those sharing the same mode of action, the use of relative potency factor (RPF) approach has been suggested. The aim of the work is to define RA-relative potency of four antifungal azoles by fitting postimplantation rat whole embryo culture (WEC) experimental data by using PROAST software analysis. The chemicals selected are all-trans retinoic acid (RA 0.025-1µM), cyproconazole (CYPRO 7.8-250µM), triadimefon (FON 6.25-125µM), flusilazole (FLUSI 1.56-125µM) and prochloraz (PCZ 6.25-50µM). Dose-related specific defects of the cranio-facial structures (reduced or fused branchial arches), were detected in rat embryos exposed in vitro to increasing concentration of RA or of the selected azoles. Collected dicotomic data were then modelled using PROAST 62.3 software in order to characterize the single dose-response curves. Curve parameter comparison showed that for all the used teratogenic agents steepness values were not dissimilar, so parallelism hypothesis and, by consequence, the hypothesis of a common mode of action shared between RA and the different tested azoles could not be rejected. Due to the azole RPFs versus RA, the potency ranking of the tested molecules resulting by the present work is RA>FLUSI>PCZ>CYPRO>FON. The obtained data suggest that WEC results could be a simple but predictive alternative method applicable to the novel hazard evaluation based on relative RPFs.

The study focuses on key events relating compound-induced mitochondrial perturbation to hepatotoxicity. The obtained data serve as input for the establishment of quantitative adverse outcome pathways (qAOPs) specific for mitochondrial toxicity. Ultimately, these qAOPs could contribute to the improvement of toxicity prediction and risk assessment of new and existing chemicals.

Analytical tools for assessment of mitochondrial functioning in HepG2 cells, including membrane potential, ROS formation, GSH expression and ATP production, were optimised for use in an automated real time confocal microscopy platform. The parameters were monitored along with measurements of cell functioning, including viability. In addition, gene and protein expression are studied using TempoSeq for transcriptomics and using BAC-GFP reporter technology for single cell real time analysis of candidate protein expression.

Time and concentration-resolved high content analysis of mitochondrial functionality and cell fate outcome were applied in a single exposure setting for mitochondrial toxicants including complex I, II and III inhibitors. Cluster analysis based on all mitochondrial functioning parameters demonstrated a clear separation of the different complex inhibitors. CI and CIII inhibitors reduced membrane potential with different potencies. CI inhibitors induced ROS formation and diminished ATP production at lower concentrations compared to CIII inhibitors. No effect was observed after CII inhibition in the applied concentration range. Cell viability after CI and CIII inhibition was reduced only when cells were switched from glucose- to galactose-containing medium, indicating that a switch to glycolysis represented an adaptive response to CI/III inhibition.

Besides compound characterisation, the data was used to develop a qAOP. A temporal relationship between parameters supported the AOP paradigm: for concentrations above the threshold value a reduction in membrane potential was observed along with a subsequent induction of an unfolded protein response and thereafter a loss of cell viability.

Ongoing experiments address the impact of repeat dose exposures and generate information on global transcriptional changes. The obtained data provides further insight into the chain of events triggered by mitochondrial toxicants. Altogether, this will support optimisation of the testing platform for assessment of mitochondrial toxicity.

Funded by EU Horizon2020 research grant agreement No 681002

The desire of boasting a healthy and fitbody leads many individuals to use ‘fat burners’ as a quick way to reduce body weight, under a misperception that such products endure a low health risk. An example of such substances is 2,4-dinitrophenol (2,4-DNP), well-known for its weight loss enhancer properties, but also for the occurrence of several related fatal cases among users. Although liver damage has been associated to the toxicity of 2,4-DNP, the underlying mechanisms remain to be established.

The aim of the present study was to evaluate the toxicity of 2,4-DNP in primary rat hepatocytes. In vitro assays were performed in primary cultures of rat isolated hepatocytes, obtained with a two-step collagenase perfusion. Our data showed that 2,4-DNP induced concentration-dependent toxicity in primary hepatocytes, at concentrations ranging from 0.55μM to 1 mM, at 37° C for 24 h, as assessed by three viability assays [neutral red uptake assay: EC₅₀ 98.05 μM; MTT reduction assay: EC₅₀ 96.82 μM; and lactate dehydrogenase leakage assay: EC₅₀ 174.83 μM]. A significant concentration-dependent increase of intracellular levels of reactive oxygen species (p<0.01, at ≥50 μM; Dunnett’s test) concurrent to a concentration-dependent decline in antioxidant defenses [decreased reduced glutathione (redGSH; p<0.01, at ≥100 μM; Dunnett’s test); decreased total glutathione (p<0.01, at ≥100 μM; Dunnett’s test); increased oxidized glutathione (GSSG; p<0.05, at ≥1 μM; Dunnett’s test); and decreased redGSH/GSSG (p<0.001, at ≥1 μM; Dunnett’s test)] were observed. Significant increase of mitochondrial membrane potential was observed at 10 μM and 50 μM (p<0.05; Holm-Sidak’s test), in accordance with the exacerbated synthesis of adenosine triphosphate (ATP). Energy-dependent activation of apoptosis was also confirmed by activation of pro-caspase-8 at all tested concentrations (p<0.05; Dunnett’s test) and by the increased activities of caspase-3 and -9, at concentrations higher than 10 μM (p<0.05; Dunnett’s test).

Abstract

Hepatic steatosis is a common liver disease that can lead to hepatotoxicity. Many drugs, including the anticonvulsant valproic acid (VPA), can induce hepatic steatosis as a side effect. Besides human clinical data, VPA and some of its structural analogues have shown to induce steatosis in rodents. Transcriptomics data derived from yeast cells suggest that VPA activates several stress response pathways, which are related to hepatotoxicity. This study aims to investigate stress response pathway activation by VPA and its structural analogues by using HepG2 BAC-GFP reporter cell lines in a high throughput screening platform. Activation of different stress responses were visualized by GFP tagged to target proteins SRXN1 (Nrf2-dependent oxidative stress response), P21 (p53-dependent DNA damage), BiP (unfolded protein response) and A20 (NFκB-dependent inflammatory signaling). Cells were treated with VPA and 18 structural analogous to generate dynamic concentration and time response datasets on pathway activation using high content confocal imaging. SRXN1 activation could discriminate between stimulation of VPA and structural in vivo steatosis positive analogues and in vivo negative analogues. P21 and A20 expression was also changed upon VPA exposure, where BiP did not show a response. Additionally, we observed no cytotoxic effects in our dose range. However, cells treated with valproic analogues that cause steatosis and co-stimulated with TNFα showed an increase in apoptosis, further supporting activation of common signaling pathways by the various toxic valproic analogues that drive onset of cytotoxicity. In conclusion, our current data support the application of a high content imaging stress response reporter platform to provide biological weight-of-evidence support for read across-based risk assessment strategies by providing mechanistic mode-of-action information.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002.

Smoking is a well-established risk factor for vascular diseases and vascular complication is a major outcome of diabetes. This study was performed to test the effect of cigarette smoke exposure on the progress of vascular impairment in diabetic condition. Seven-week old male Sprague-Dawley rats were divided into four groups consisting of each 10 rats; control group (C), cigarette smoke-exposed group (CS), diabetic group (D), and diabetic, cigarette smoke-exposed group (DCS). Diabetes was induced by a single, intraperitoneal injection of 60 mg/kg streptozotocin, which was confirmed by blood glucose level higher than 300 mg/dL. After 4 weeks, CS and DCS was exposed to cigarette smoke generated from 3R4F reference cigarettes at total particulate matter concentration of 200 microgram/L, 4 h/day, 5 day/week for 4 weeks. Filtered air was supplied instead of cigarette smoke for C and D. After treatment, thoracic aorta and blood plasma were analyzed for pathological changes by cigarette smoke. Medial area and thickness were decreased in aorta of diabetic rats, whereas internal diameter was not changed. However, cigarette smoke exposure had minimal effect on the structure of aorta. Collagen content was increased in diabetic aorta and such increase was further augmented by cigarette smoke exposure, although cigarette smoke did not affect collagen content in aorta of control rats. Cigarette smoke exposure caused endothelial nitric oxide synthase (eNOS) uncoupling only in diabetic aorta which was evidenced by an increase in monomer/dimer ratio. Malondialdehyde was increased and glutathione was decreased in the plasma of diabetic rats. However, such alterations of oxidative stress markers were not further changed by cigarette smoke exposure. Expression of NADPH oxidase (NOX) 2 and NOX4, representative reactive oxygen species-generating enzymes in aorta, were enhanced by cigarette smoke exposure in diabetic rats, not in control rats. The results of this study indicate that cigarette smoke aggravates vascular fibrosis and eNOS uncoupling in diabetes, which may accelerate the progress of diabetic macrovascular impairments such as loss of arterial elasticity and endothelial dysfunction.

2,4-D is a derivative of the chlorophenoxyacetic acid, it is a part of pesticides with herbicidal activity. In most cases, mercury is contained in environmental objects in the form of methylmercury; therefore, the heavy metal under investigation was used in the experiment in the form of methylmercury.

The purpose of the present studies was to establish the pathogenetic mechanism for potentiation of the combined effects of global environmental pollutants 2,4-D and methylmercury on warm-blooded organisms. The experiments were performed on albino rats in acute and subchronic experiments. Literature data on the toxicity of the derivatives of chlorophenoxyacetic acid and mercury were referred to when choosing research methods to determine the specific features of toxicodynamics of the studied substances.

A biological effect greater than additive was established for single and prolonged exposure to the combination of methylmercury and 2,4-D in the conducted experiments based on a number of changes in the studied parameters in experimental animals (the blocking of SH-groups, an increase in the activity of cytosolic alanine aminotransferase, aspartate aminotransferase, γ-gamma-glutamyltransferase enzymes, etc. in the serum, LPO processes, a decrease in the activity of antioxidant enzymes).

The leading pathogenetic links of the potentiation mechanism under the conditions of the combined action of methylmercury and 2,4-D herbicide on the warm-blooded organism with single and multiple exposures were revealed. The mechanism of potentiation of this combination is due to the blocking of SH-groups by mercury, and the process is strengthened by the additional blocking of sulfhydryl groups of protein molecules of enzymes by peroxides formed in the body during the biological oxidation of 2,4-D, against the background of a decrease in the activity of antioxidase enzymes. At the same time, the decrease in the activity of antioxidant enzymes occurs both due to the exposure to methylmercury directly and is also mediated by liver dysfunction. This is evidenced by an increase in the activity of alanine and aspartate aminotransferases, γ-glutamyltransferase in the blood serum, which, in turn, along with an increase in lipid peroxidation indicate damage to cellular and subcellular biomembranes.

Purpose: Many organochlorine compounds have been classified by the International Agency for Research on Cancer (1979) as carcinogenic in animals and also suspected to be carcinogenic in humans, hexachlorobenzene (HCB), is a typical member of this family. HCB has been found in feed in several regions of the world. In our study, we performed an in vitro evaluation of ROS generation and oxidative stress induced by HCB on the human intestinal Caco-2 cell line as a target cell type regarding to oral exposure.

Methods: Caco-2 cells were cultured in an atmosphere of 5% carbon dioxine and 95% air at 37°C. HCB was dissolved in absolute ethanol, for a stock solution of 2mM, and the same final concentration of the vehicle (0.1%) was used in the corresponding control. After 15 hours of seeding, cells were washed with serum free medium and then incubated with 4% FBS and treated with HCB (0.04 to 2000 nM). Treatments were repeated every 48 hours in all experimental designs for 14 days. Intracellular production of ROS was analyzed using the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (H₂-DCFDA) which characterized H₂O₂ production. Antioxidant enzymes activities like catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined in the cytosolic fraction.

Results: Our results showed that exposure of caco-2 cells to HCB elicited a dose-dependent induction of the intracellular production of H₂O₂ and the highest intensity of fluorescence being observed upon a 3 hours-period of incubation. Indeed, treatment of caco-2 cells with HCB induced an oxidative stress demonstrated by the increased activity of antioxidant enzymes including SOD, CAT and GPx. This induction was significantly observable at dose 4 nM and was more marked at the highest dose tested (2µM).

Normal cellular functions depend on the balance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms in the cell. In our study the cellular enzymatic antioxidants are unable to control the production of ROS, and an oxidative stress is occurs, leading to damaged cell functions.

The study demonstrates ROS-mediated organismal and sub-organismal injuries in Drosophila melanogaster following chronic organophosphate (acephate) exposure. Drosophila was reared on food supplemented with sub-lethal concentrations (1-6 µg/mL) of acephate (LC₅₀ 8.71 µg/mL). The treated adults demonstrated reduced longevity (~to half at 6 µg/mL exposure) along with reduced neuromuscular coordination and physical activities. Conspicuous developmental defects in compound eyes were confirmed through detection of apoptotic lesions in larval eye imaginal discs. Larval gut manifested tissue damage at various sites. Neural and fat cell viability was reduced by ~1.89 and ~3.38 fold at 6 µg/mL acephate treatment. Significant reduction in hemocyte viability confirmed the immunotoxic potential of acephate. Nearly 1-3fold increase in expression of various oxidative stress-markers (MDA, protein carbonyl contents, SOD, Catalase and HSP70) in treated larvae served as evidence of reactive oxygen species (ROS) production. Post-treatment increases in CYP450 and GST activities reflects ‘switch-on’ states of phase-I and phase-II detoxification mechanism. Genotoxic potential of Acephate was confirmed through alkaline single cell gel electrophoresis. Thus, findings of present study validate the fact that, besides traditional cholinesterase inhibition, chronic sub-lethal exposure to acephate potentially induces ROS-mediated toxic responses in non-target model organism Drosophila.

Propaquizafop is an herbicide with a long history of use. Chronic dietary exposure of rats led to liver tumors without genotoxicity, subchronic studies demonstrated dose-dependent increases in liver weights with hepatocellular hypertrophy, and two weeks administration induced liver CYP4A and peroxisomal enzymes. A rodent-specific mode of action (MOA) via activation of the peroxisome proliferator-activated receptor α (PPARα), increased liver peroxisomal activity, hypertrophy, liver enlargement, subsequent cell proliferation and finally adenoma formation was postulated by others based on the alignment of these findings with an established MOA for fibrates. Experience with PPARα-inducing pharmaceuticals indicates a lack of human relevance. In this study, the results of a MOA investigation following two weeks of dietary feeding in wildtype (WT) and PPARα-knockout (KO) rats are presented to confirm the dependency of key events following propaquizafop administration on PPARα. In WT animals dose-dependent liver weight increases (65-84%), liver CYP4A (20 fold) and acyl-CoA oxidase (10-15 fold) induction, hepatocellular hypertrophy and proliferation were observed, while in KO rats only a marginal increase in liver weight (24%) was observed without any effect on the other parameters confirming PPARα-dependency and sequelae comparable to a well-understood hepatocarcinogenic MOA in rodents. Based on an evaluation of the data according to the MOA-Human Relevance Framework, we conclude that liver tumors observed in rodents after long-term dietary administration of propaquizafop do not pose a relevant health risk to humans.

Caffeine is often used as an analgesic adjuvant combined with acetaminophen (APAP) in several prescription drugs and over-the-counter drugs. Apart from the therapeutic benefit of APAP-caffeine combination, possible risk of caffeine to potentiate APAP-induced liver injury (AILI) is of clinical concern. In animal experiments, it has been shown that caffeine both increase and decrease AILI, which might depend on the animal species used and the relative contents of hepatic cytochrome P450 enzymes (CYP). In the present study, we evaluated the effects of caffeine on AILI in male CD-1 mice, which are susceptible to AILI, in relation to changes in CYP isoforms contents and inflammation mediators. Overnight-fasted male CD-1 mice were given APAP intraperitoneally. Some mice were pretreated with phenobarbital or acetone to induce hepatic CYP2B/3A or CYP2E1, respectively. Caffeine was administered at various time points before or after the APAP injection. AILI was assessed by serum leakage of alanine aminotransferase (ALT). Glutathione (GSH) contents were determined in the liver homogenates. Hepatic TNF-α mRNA expression was assayed by real-time RT-PCR. Serum ALT leakage in male CD-1 mice treated with APAP (300 mg/kg) was attenuated by coadministration of caffeine. Caffeine administration 1 hour before APAP was also effective against AILI, but that 16 hour before or 1 hour after APAP did not. Phenobarbital and acetone potentiated AILI. The protective effect of caffeine against AILI was reduced in the mice pretreated with phenobarbital, whereas it was pronounced in acetone-pretreated mice, suggesting the importance of CYP2E1 in the protective effects of caffeine. Early depletion of hepatic GSH, which has been observed prior to initiation of AILI and an index of the rate of APAP activation into N-acetyl-p-benzoquinone imine, was slightly attenuated by caffeine, but the hepatic GSH levels in the mice treated with both APAP and caffeine were still much lower than control mice. On the other hand, caffeine suppressed basal TNF-α expression and early TNF-α induction by APAP. These results suggest that caffeine attenuates AILI via suppression of TNF-α, which has been implicated in the pathogenesis of AILI as an inflammatory mediator, in addition to the inhibition of CYP2E1.

Lipid accumulation and oxidative stress are major pathologic contributors to the development of hepatic steatosis. Treatment with molybdate reduces hepatic levels of lipids in diabetic rats. Potential activities of molybdate as an antioxidant have also been demonstrated in various animal models. In the present study, we evaluated the effects of sodium molybdate dihydrate (SM) on hepatic steatosis and associated disturbances in a widely used mouse model of the metabolic disease. Male C57Bl/6 mice at 10 weeks of age were fed a diet deficient in methionine and choline (MCD) and bottled water containing SM for four weeks. The SM treatment markedly attenuated MCD-induced accumulation of lipids, mainly triglycerides, in the liver. Lipid catabolic autophagic pathways were activated by SM in the MCD-fed mouse livers, as evidenced by a decreased level of p62 expression. MCD-induced oxidative damage, such as lipid and protein oxidation, was also alleviated by SM in the liver. However, the level of MCD-induced hepatocellular damage was not affected by SM. Taken together, these findings suggest that molybdate can be used in the treatment and prevention of hepatic steatosis without inducing adverse effects in the liver. To the best of our knowledge, this is the first experimental study to investigate the effects of molybdate in non-alcoholic fatty liver disease, and also the first that demonstrates molybdate-induced autophagy.

Rodent models are often used to study biochemical mechanisms of toxicity and to establish a mode of action (MOA) for adverse, chemical-induced effects. Phenobarbital (PB) has been shown to induce differential gene expression and CYP2B, CYP3A and UGT enzymes in the rodent liver leading to increased thyroid hormone clearance and subsequent stimulation of cell proliferation in the thyroid that eventually produces thyroid tumors after long-term exposures. This pathway has not been completely investigated in nonrodents. In this study, male Gottingen minipigs were orally administered 15 mg/kg/day PB for 6 days followed by investigation of phase I and II liver enzymes (mRNA expression and enzyme activities), circulating thyroid hormone levels and differential gene expression in the liver, in addition to standard toxicological evaluations. PB-administered minipigs had 46% increased absolute and 42% increased relative liver weights with mild, diffuse hepatocellular hypertrophy versus the control group. PB reduced plasma concentrations of T3 (by about 27% and 47%) and T4 (by about 15% and 20%) versus predose values at 24 hours and after 6 days of administration, respectively. PB induced a 2-fold increase in CYP3A429 and CYP4A24, 5-fold increase in CYP1A2 and around 11-fold increase in CYP2B22 compared to the control group, with increases in mRNA expression of these genes. A 1.6-fold increase in T4-specific UGT phase II enzyme activity was observed in the PB-administered group, and CYP2B22, CYP2A19, CYP2C42, CYP3A39 and CYP3A46 were among the top 20 upregulated genes. This study indicates that several of the early biochemical key events in the PB-induced (rodent) MOA are present in minipigs. It is concluded that the minipig is an interesting model for further study of the effects of xenobiotics on the liver-thyroid axis.

This work is focused on two mycotoxins: ochratoxin-A (OTA), produced by the fungi os the genus Aspergillus and Penicillium, and beauvericina (BEA) mainly produced by Fusarium. The cytotoxic effect in HepG2 cells exposed to OTA, BEA and OTA+BEA was performed by the MTT method at different concentration ranges (OTA 0.35-100 μM, BEA 0.003-25 μM, OTA+BEA 10:1 ratio) and at three exposure times (24, 48 and 72h); while lipid peroxidation (LPO) through the malondyaldehyde (MDA) production assay. The IC₅₀ values of individual mycotoxins were for BEA 12.5 ± 0.047 µM, 7 ± 0.055 μM and 5.5 ± 0.071 μM at 24, 48 and 72h, respectively; while for OTA 75±0.047 μM, 52.62±0.067 μM and 36±0.09 μM at 24, 48 and 72h, respectively. The IC₅₀ values decreased over the time; so increasing potential of toxicity; however, a greater toxic potential of BEA vs OTA is observed at all times tested in HepG2 cells. Combination OTA+BEA (10:1 ratio) at 24h no IC₅₀ was reached; while at 48h and 72h IC₅₀ values were 3.4±0.045 μM and 2.9±3.034 μM, respectively. The isobologram method in combination showed antagonistic effect at 24h, synergism effect at 48h and 72h at CI₂₅, CI₅₀ and CI₇₅; while additive effect was observed at CI₉₀. The results obtained in LPO assay showed that BEA and OTA increased the production of MDA from 2.1- to 3.6 fold and from 2- to 6.2-fold respect to control, respectively; while the combination of OTA+BEA (10:1 ratio) reached the highest increases of MDA production, which ranged from 3- to 4.6-fold respect to the control. In conclusion, simultaneous presence of mycotoxins in food and feed could increase toxic risk to human and animal health.

Acknowledgments: Spanish Ministry of Economy and Competitiveness (AGL2016-77610R).

Polyhexamethyleneguanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, was elucidated as a major cause of idiopathic pulmonary fibrosis that resulted in deaths of pregnant women and infants in South Korea. Fibrosis is triggered by recurrent damage in epithelial cells and is largely affected by epigenetic effects including microRNAs (miRNAs). In our previous study, epithelial-mesenchymal transition(EMT), one of the mechanisms in lung fibrogenesis, was observed, and mRNA-miRNA regulatory networks of PHMG-phosphate were investigated. The aim of this study was to elucidate the mechanism underlying PHMG-phosphate-induced EMT through miRNA. Microarray of miRNA profiling was conducted using Affymetrix GeneChip miRNA 4.0 array and was demonstrated by real-time qPCR. To show the specificity of PHMG-phosphate, miRNA expressions were compared with paraquat and transforming growth factor- β(TGF-β). In vivo study rats were repeatedly exposed to PHMG-phosphate 0.1 mg/kg 6 times for 4 weeks. As a result in A549 cells exposed to PHMG-phosphate 0.75 and 3 μg/ml for 24 and 48 hours, expression of miR-33b-3p was signicantly decreased and that of miR-6126 was increased. Moreover the specificity of miR-6126 up-regulation by PHMG-phosphate exposure was showed by comparison with paraquat and transforming growth factor- β(TGF-β). Otherwise miR-33b expressions were decreased similarly by exposure to all sorts of chemicals. Also expression of miR-6126 was markedly increased in lung of rats exposed to PHMG-phosphate repeatedly. To study the functions of miR-6126 at EMT caused by PHMG-phosphate, several proteins involved in EMT were assessed in A549 cells transfected with miR-6126 mimic or negative control mimic using western blot analysis. In conclusion, EMT was induced by miR-6126 up-regulation, so miR-6126 expression can be one of the markers for lung injury by PHMG-phosphate. For further understanding of the effects by miR-6126, more research on the targets of miR-6126 is needed.

Organophosphorus compounds (OPs) caused several effects and not all can be explained by the classical recognized target: acetylcholinesterase (AChE). A fraction of the membrane proteins with phenylvalerate esterase activity (PVase) is called or neuropathy target esterase (NTE) and is involved in the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN) for which adult chickens is the model animal for testing. We showed that several PVase enzymatic components could be discriminated by their inhibitory kinetic properties in the soluble fraction in chicken brain (Eα, Eβ, and Eγ) and four ChE activity components (C1, C2, C3, and C4). We demonstrated that the PVase activity of the Eα component showed cholinesterase (ChE) activity and it is a butyrylcholinesterase protein, while the other components have no ChE activity. C1 was identified with the Eα activity. We suggested that it might be related to the potentiation/promotion phenomenon of the OPIDN. Human butyrylcholinesterase (hBuChE) also shows PVase activity. Evidences are showed that both activities are associated to the same active center: (1) Both substrates (acethylthiocholine and phenyl valerate) compete in their activities. (2) Mipafox, iso-OMPA and PMSF inhibited with similar kinetic behavior. Further kinetic and structural research is under study. In the membrane brain fractions, four components (EPα, EPβ, EPγ and EPδ) of PVase have been discriminated by their inhibitory kinetic properties using irreversible inhibitors (paraoxon, mipafox, PMSF). Only EPα PVase activity were inhibited by acetylthiocholine and its cholinesterase activities by phenylvalerate. Four ChE activity enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in membrane fraction, on the basis of inhibitory kinetic with irreversible inhibitors (mipafox, paraoxon, PMSF and iso-OMPA). The ChE components CP1 and CP2 could be related to the PVase activity component EPα; This is resistant to PMSF and is highly sensitives to mipafox and paraoxon but is spontaneously reactivated when inhibited by paraoxon. The relation of PVases with ChEs need to be included in the molecular adverse pathways of OP delayed neurodegenerative toxicity under approaches with multi-target or multi-initiating molecular events.

Polyhexamethylene guanidine-phosphate (PHMG-phosphate) was widely used as a biocidal disinfectant to inhibit microbial growth in household humidifiers, which caused severe lung injury including idiopathic pulmonary fibrosis in hundreds of victims in South Korea. One of the mechanisms in lung fibrogenesis is epithelial-mesenchymal transition (EMT), a process in which epithelial cells gain a mesenchymal phenotype. Our previous study showed that PHMG-phosphate exposure caused EMT in human alveolar epithelial A549 cells in vitro. PI3K/Akt and Notch signaling have been shown to induce EMT after repressing transcription of the E-cadherin gene. To elucidate the mechanism of EMT induced by PHMG-phosphate, the activation of PI3K/Akt and Notch pathway by PHMG-phosphate was examined. The expressions of representative markers of PI3K/Akt and Notch pathway, p-Akt and NICD, after exposure to PHMG-phosphate in A549 cells were increased dose-dependently in Western blotting. The effect of PI3K/Akt and Notch pathway in EMT was confirmed using PI3K inhibitor, LY294002 and Notch inhibitor, DAPT. Moreover, the transcription factor expressions of down-stream targets of PI3K/Akt pathway, ZEB and β-catenin, and Notch pathway, Hes-1, were increased in A549 cells exposed to PHMG-phosphate. Our results demonstrate that PHMG-phosphate can trigger the activation of PI3K/Akt and Notch signaling pathway, which mediates EMT induced by PHMG-phosphate. To provide further understanding of the molecular effects induced by PHMG-phosphate, the mechanisms of Akt and Notch activation are required to be studied.

Sterigmatocystin (STC) is a polyketide mycotoxin with a bifuran ring structurally similar to aflatoxin. It is a precursor in aflatoxin biosynthesis in some Aspergillus species from the section Flavi and the final product in a number of Aspergilli from the section Versicolores. STC has been detected in various foodstuffs as well as indoor environments such as damp, moldy dwellings, carpets, and grain dust. In acute oral exposure it targets the liver and the kidneys. Its oral LD₅₀ in male rats is about 160 mg kg^-1 b.w. Similar to aflatoxin, STC's mechanism of action has been linked to the formation of exo-epoxide, which readily forms DNA adducts. Considering that aflatoxin toxicity has also been associated with oxidative stress, the aim of this study was to check whether the same is true for STC toxicity. With that in mind, we treated male Wistar rats with single oral STC doses of 1/4, 1/8, and 1/16 of LD₅₀ (40, 20, and 10 mg kg^-1 b.w., respectively) and measured superoxide dismutase (SOD) activity in plasma on a plate reader using a commercial kit. We also measured glutathione (GSH) and malondialdehyde (MDA) in plasma, kidney, and liver tissue using a spectrophotometer and HPLC, respectively. To assess oxidative DNA damage in the liver and kidneys we used comet tail intensity (i. e. DNA % in tail) obtained with the hOGG1-modified comet assay. STC did not affect GSH concentrations. SOD activity in plasma dropped significantly only in rats treated with 1/8 of LD₅₀ (9.26±0.26 vs. 10.08±0.82 U mL^-1 in control). All doses significantly increased MDA concentrations in plasma and kidney, but not in the liver. Oxidative DNA damage showed up in the liver and kidney cells, and was more pronounced in the kidneys, particularly at 1/8 of LD₅₀. Our findings suggest that acute oral exposure to STC causes greater oxidative stress in the kidneys than the liver. This work has been fully supported by the Croatian Science Foundation under the project MycotoxA (HRZZ-IP-09-2014-5982).

Mitochondrial dysfunction has been implicated in numerous drug induced adverse events, such as liver failure and cardiac toxicity. The potential of drugs to be mitochondrial toxicants can be determined by either comparing the increase in cytotoxicity of compounds in media containing galactose compared to glucose (Glu/Gal assay). In galactose conditions, cells are more reliant on oxidative phosphorylation and therefore more sensitive to mitochondrial disruption. Alternatively mitochondrial toxicants can be determined using a mitochondrial respiration assay which measures cellular oxygen consumption rate (OCR), reserve capacity (RC) and extracellular acidification rate (ECAR). A third approach utilises fluorescence dyes to measure changes in mitochondrial membrane potential (MMP) using high content imaging and compared to ATP depletion and cell loss. Alteration of MMP is strongly associated with mitochondrial toxicity. These approaches were analysed for the predictivity of compounds known to result in mitochondrial toxicity by different mechanisms of action.

Seventy compounds (known mitochondrial toxicants with varying mechanism of action and compounds with no mitochondrial effect) were screened through the Glu/Gal assay, the MMP and cytotoxicity assay, and the mitochondrial respiration assay, in HepG2 cells. Comparing the ECAR and OCR data with Glu/Gal data showed there were a number of toxins such as rosiglitazone, amiodarone and perhexiline which were not detected in the Glu/Gal assay, however were flagged as being positive for mitochondrial toxicity based upon the mitochondrial respiration assay. The MMP and cytotoxicity assay is higher throughput in comparison to the mitochondrial respiration assay with the benefit of better specificity (e.g. positive response to rosiglitazone) than the Glu/Gal assay and can provide some additional mechanistic information.

In summary, determining cellular OCR, reserve capacity and ECAR provides a more predictive, and sensitive measure of mitochondrial toxicity and in addition gives an understanding of potential mechanisms of action when compared to the Glu/Gal assay. The MMP and cytotoxicity assay provides a more sensitive alternative to the Glu/Gal assay where high throughput is required in early drug discovery.

Background: Combining potential drug originated from edible plant such as benzyl isothiocyanate (BITC) with antioxidant like caffeic acid (CA) could be more compatible to human body and produce synergistic effect to kill cancer. Our recent study demonstrates the trends and mechanisms of cytotoxicity effects of BITC, CA and the novel combination of BITC and CA to enhance the effectiveness of the cancer treatment.

Methods: Cytotoxicity effects of BITC, CA and their combination toward human breast adenocarcinoma (MCF-7) cells were evaluated by using MTT assay after 24 and 48 hours of treatments. We explored mechanisms of cytotoxicity effects by examining the protein expression of glutathione-S-transferase (GST)-pi and GST-alpha, p38, ERK 1/2, Nrf-2 and Bcl-2 by Western blot technique. Glutathione (GSH), reactive oxygen species (ROS) and caspase 3/7 level were measured fluorometrically.

Results: High dose of CA needed to cause the death of MCF-7 cells. Interestingly, we found that the combination of BITC and CA created the synergistic effect to kill MCF-7 cells in time and dose-dependent. BITC increased the ROS level and exploit it to induce MCF-7 cell death whereas the combination of CA and BITC decrease ROS level but effectively reduced MCF-7 cells viability. Coordination of p38 MAPK, ERK1/2, NRF-2, Bcl-2, caspase 3/7 and phase II of xenobiotic metabolism by the up-regulation of GS- pi and GST-alpha revealed the intracellular signaling to cause MCF-7 cells death.

Conclusion: Conclusively, CA boosted the efficiency of BITC to kill the cancer cells. The elevation of GST catalyzed the conjugation of GSH with BITC, CA and their combination, thus lowered the amount of GSH to create the imbalance of redox homeostasis and cause the death of cancer cells.

Acknowledgment: Ministry of Higher Education Malaysia and UMT for funding provided (Vot. 53131).

Drug-induced mitochondrial toxicity is a major issue for patients because such toxicity can trigger acute or chronic injury involving different organs or tissues including liver, heart, muscle, kidney and adipose tissue. We developed a medium-throughput in vitro screening platform to detect loss of mitochondrial function and integrity on isolated liver mitochondria [1] and HepaRG differentiated cells [2] investigating acute, indirect and chronic mitochondrial alterations for DILI prediction. For early detection of cardiotoxicity potential of lead compounds, we set up multiparametric assays on isolated rat heart mitochondria and terminally differentiated human iPSC-derived cardiomyocytes, Cor.4U cells. Both models were characterized in terms of mitochondrial (OxPHOS) activity and Cor.4U cells were shown to present highly functional mitochondria suitable for compound testing. Four reference drugs selected on their clinical-determined likelihood to induce (amiodarone, sulindac, tamoxifen) or not (erlotinib) mitochondrial damages in human heart were tested on both models. On isolated mitochondria their ability to induce swelling, transmembrane potential loss, inhibition of O₂ consumption either driven by complex I, complex II or fatty-acids beta-oxidation (FAO) was challenged. Amiodarone, sulindac and tamoxifen were found to directly inhibit mitochondrial respiratory chain while erlotinib did not alter rat heart mitochondria. In addition, Cor-4U cells were treated for 3 hours with compounds and subjected to O₂ consumption driven by Complex I or Complex II, mitochondrial transmembrane potential and mtROS production measurements. Results showed that amiodarone and tamoxifen presented strong mitochondrial toxicity in Cor.4U cells, while sulindac did not induce strong mitochondrial toxicity (only slight inhibition of CII-driven O₂ consumption). Surprisingly, erlotinib was shown to have an uncoupling-like effect that was not measured on isolated mitochondria. Thus, using a combination of both rat mitochondria and human cardiomyocytes models allows a good understanding of compounds mechanism of action. Overall, such assays help to early investigate the potential mitochondrial toxicity of new compounds, in addition to classical preclinical studies, to select safer drug candidates.

[1] Porceddu M et al (2012) Toxicol Sci. 129: 332-45.

[2] Porceddu et al (2018) Methods in Pharmacology and Toxicology, Eds Chen, M & Will Y.

Previous assessment of heavy and poorly refined petroleum substances demonstrate a strong association of developmental toxicity with levels and types of polyaromatic hydrocarbon (PAH) content. While suggestive evidence exists indicating a role for the aryl hydrocarbon receptor (AHR) in this toxicity, no direct experimental evidence exists. To assess the role of AHR in developmental toxicity of PAHs, a positive control PAH was sought. Although benzo[a]pyrene (BaP) is coal-derived, as it is only present at low ppb levels in most petroleum products, BaP is among the best characterized PAHs thus was identified as a reasonable candidate. Groups of five or six pregnant wild-type (WT) and AHR^-/- rats (Sprague-Dawley background) were exposed to 0 or 300 mg BaP/kg/d by single daily oral gavage from gestation day (GD) 6 through 20. Maternal body weight gain significantly decreased in BaP exposed WT and AHR^-/- dams from GD9 and GD12, respectively. Reduced weight gain resulted in final body weights 11% lower in BaP exposed WT dams and 18% lower in AHR^-/- dams. BaP exposure significantly increased total number of fetal resorptions, particularly early resorptions, in WT dams (56.4% of fetuses [55.0% early and 1.4% late] versus 3.7% in WT control), but did not significantly affect fetal resorptions in AHR^-/- dams (4.0% of fetuses versus 0.0% in AHR^-/- control). Pre-implantation losses were also increased in BaP exposed WT rats (16.7% compared to 2.4% in WT control, 0.0% in AHR^-/- control, and 1.7% in BaP exposed AHR^-/- rats). Consistent with increased resorptions, WT BaP exposure significantly decreased number of viable foetuses (43.6% versus 96.3% in control) and post-implantation loss (56.4% versus 3.7% in control), but not AHR^-/- dams. The results suggest 300 mg BaP/kg/d is an acceptable dose as a positive control for developmental toxicity in WT rats. Overall, this dose range finding experiment provides a first indication for a role of the AHR in PAH related developmental toxicity.

Among various nanomaterial silver nanoparticles (AgNPs) has been used widely for many application. However, with the rise in their applications the concerns of their safety are also increasing at alarming rate. Therefore, it is need of the hour to study their nanotoxicological aspect. Therefore, the present study was designed to evaluate the toxicity of mycosynthesised AgNPs. They were synthesised by using three species of Fusarium and a species of Phoma. Later on, they were characterised by UV-Vis Spectrophotometry to confirm their synthesis, Fourier Transform Infra-Red spectroscopy (FTIR) to confirm the protein capping on synthesised AgNPs, Zeta potential to know their stability, Nanosight LM20 to know their siez in working condition, X-ray diffraction (XRD) to confirm the silver as elemental composition and Transmission Electron Microscopy (TEM) to know their size and shape. The AgNPs, at different concentration, were then exposed to a specially designed Psueodomonas putida biosensor having lux gene. For eukaryotic studies, they were exposed to various cell lines including Caco-2, a colon cancer cell line, A549, a human alveolar cell line and IEC6, a rat intestinal epithelial cell line.

In result we found stable AgNPs to reduce the culturability of P. putida at the concentration range of 0.4 to 0.8 μg/ml. However, with the help of MTT assay, the AgNPs within size range of 5 to 25 nm were found to show the IC50 of 8 μg/ml against cell lines. The effect of AgNPs on interaction with cell membrane, formation of reactive oxygen species and fragmentation of DNA was tested by using LDH leakage assay, ROS assay and DNA fragmentation assay. The study revealed that AgNPs damages the cell membrane, promoting the accumulation of reactive oxygen species and damaging the nuclear material. Overall these mechanisms contribute to the toxicity of mycosynthesised AgNPs. This study is probably unique in performing the risk assessment and the mechanism of toxicity of mycosynthesised AgNPs on prokaryotic and eukaryotic model.

Question

Anethole is a natural constituent in many herbs and spices such as anise, star anise and fennel and belongs to the group of phenylpropenes. In natural products, anethole is usually present as a mixture of its trans- (1) and cis-configured (2) stereoisomers. Anethole or anethole containing plant components are commonly used as flavoring substances for foods, cosmetics and drugs, e.g. tea, alcoholic beverages like ouzo, or mouthwashes. Although 1 has been given GRAS status (generally recognized as safe) by FEMA (Flavor and Extract Manufacturers Association), recent data showed the DNA adduct formation of trans-anethole in fetal turkey livers. To get more insights in the toxicological properties of both, trans- and cis-anethole, we investigated the microsomal metabolism, mutagenicity and DNA reactivity of 1 and 2 and their main metabolites trans- (3) and cis-anethole epoxide (4).

Methods

The microsomal metabolism of 1 and 2 was investigated using liver microsomes of different species. Metabolites were characterized using HPLC-UV/Vis and HPLC-MS/MS. The mutagenicity of 1–4 was examined using the Ames fluctuation test with different Salmonella typhimurium strains. Nucleoside adducts were synthesized by the reaction of 3 or 4 with 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG).

Results

We were able to identify eleven metabolites of 1 and 2 from microsomal incubations. The threo- and erythro-configured dihydroxydihydrodiols, likely formed from 3 and 4 as precursors, as well as 3′-hydroxylated metabolites were identified as the main metabolites of 1 and 2. The Ames test showed no mutagenic effect for 1 and 2 in the tested concentrations (up to 1 mM), whereas 3 and 4 were found to be mutagenic at concentrations at or above 100 µM. The reaction of 3 and 4 with dA and dG resulted in the formation of adducts with a m/z of416 and 432, respectively.

Conclusion

Our results show that both anethole isomers, 1 and 2,are metabolizedmainly viaepoxidation of the side-chain. The intermediate formed reactive epoxides 3 and 4 may be responsible for the formation of DNA adducts detected in recent studies, since both epoxides react readily with dA and dG to form nucleoside adducts of the expected mass. Investigations to elucidate if trans- or cis-anethole forms these DNA adducts also in vitro and in vivo are currently ongoing in our group.

Tissues are a complex entity of different cells. Tissue heterogeneity increases variability in toxic responses, and makes it difficult to interpret adverse outcomes. A different intracellular signature, differentiation state and surrounding niche result in cell type-specific coping strategies. Within the complexity of multicellular tissues, stem cells often have a lower sensitivity to toxic compounds. Stem cells are characterized by a low metabolic state, a preference to maintain low oxygen levels and a well functioning repair system. Their presence in developing and carcinogenic tissues makes them an even more relevant toxicological target.

In this study, we focused on the coping strategies of pluripotent stem cells to chemically-induced damage in an in vivo situation, i.e. in the planarian Schmidtea mediterranea. Planarians are long known for their regenerative ability, which hinges on pluripotency. This renders the opportunity to study pluripotent stem cell responses while stem cells reside in their original niche. Cellular damage was induced via different toxicants: MMS, Cd and AgNPs. Pluripotent stem cells were able to recover from the induced cellular and DNA damage and successfully regenerated an entire organism. Depending on the microenvironment and needs of the organism, stem cell responses toggled between apoptosis and DNA repair, with p53 as a probable regulator of the stem cell’s self-renewal switch. Stem cell repair responses were further investigated by combining two top-down approaches, an in silico screening and a proteomic screening after chemical exposure. Underlying stem cell plasticity, we observed the involvement of several TSGs, either known or novel, who’s loss-of-function, in combination with chemical exposure, destabilized the stem cell compartment, triggering tumor formation. A knock down of matrix metalloproteinase B induced different types of protrusions, suggesting a control exerted by the ECM on the stem cell compartment. Although a further effort is needed to unravel how the integrated network of TSGs prevents stem cell transformation and tumorigenesis, our work pinpoints that planarian tumors originate from the ectopic, uncontrolled proliferation of the ordinary stem cells, and that TSGs and the stem cell niche have a central role in their transformation.

Although there have been major technological advances in how we study biological processes and systems in the last years, studying toxicity pathways still remains difficult, time-consuming and resource-intensive. High-content imaging of cells stained with multiplexed dyes, “Cell Painting”⁽¹⁾, has been successfully applied in drug screening⁽²⁾ of chemical compounds based on morphological changes they induce. However its application in the field of toxicity is still rather unexplored and comprises an exciting opportunity to unravel toxicity mechanisms and pathways.
We are investigating the usefulness of image-based morphology profiling toward understanding toxicology mechanisms and pathways, by studying a range of cell lines treated with different toxicants. To this end we implemented a cell painting screening assay, involving 4 - 6 fluorescent dyes, used to stain at least 6 - 8 subcellular components simultaneously, followed by subsequent imaging with an ImageXpress XLS high-content microscope. To analyse these compound-induced morphological changes we use a “CellProfiler”⁽³⁾ analysis pipeline to compare morphological profiles and classify compounds based on mechanisms.

Our initial experiments show promising results for mechanistic classification of toxicity mechanisms and we hypothesize that the method could be used for pathway enrichment. To this end we will strive towards automating the platform, and develop robust analysis pipelines to handle data/analysis to be able to study mechanisms on larger scale

⁽¹⁾ Bray, Mark-Anthony et al. 'Cell Painting, A High-Content Image-Based Assay For Morphological Profiling Using Multiplexed Fluorescent Dyes'. Nature Protocols 11.9 (2016): 1757-1774.

⁽²⁾ Simm, Jaak et al. “Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery”. Cell Chemical Biology, In press

⁽³⁾ Carpenter, Anne et al. “CellProfiler: image analysis software for identifying and quantifying cell phenotypes”. Genome Biology (2006) 7:R100

Exposure to neurotoxic metals lead (Pb), arsenic (As), and methylmercury (MeHg) occurs to mixtures and not to single contaminants. Metal interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of metal mixtures interactions on protein and cytotoxicity responses, we established an in vitro omics approach integrating bioinformatics, and statistics, to measure mixtures interactions at protein level in hippocampal cells. Multivariate statistical analysis showed the correlation of protein response between mixtures. Gene functional and pathway analyses of proteomic data confirms alterations related to mitochondrial dysfunction, oxidative stress, m-RNA splicing, and ubiquitin system dysfunction related neurodegeneration. Bioinformatics analysis of mixtures protein data are presented in comparative manner. Our results showed that, the significant differences of affected proteins in the mixtures. This study provides a novel proteomic approach for characterization neurotoxicity of metal mixtures in hippocampal cells.

Carbon nanotubes due to their structure and specific chemical, mechanical, thermal, optical and electrical properties have a great potential for applications and constitute an important group of nanomaterials. The aim of this study was to investigate the effect of selected carbon nanotubes on the surface properties of a phospholipid monolayer as the simplest model of biological membranes. Carbon nanotubes of various types, dimensions and modifications were chosen. Identification involved microscopic analysis using scanning electron microscopy (SEM), measurement of specific surface area (BET) and analysis of particle size distribution in liquid dispersion (DLS). The research was carried out using Langmuir-Wilhelmy balance (KSV, Finland). The phospholipid monolayer was prepared by applying 1 mg/ml solution of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Sigma-Aldrich, USA) on the surface of the aqueous subphase and evaporation of the solvent. The suspensions of carbon nanotubes at surface concentrations ranging from 10.3 ng/cm² to 205.1 ng/cm² were instilling on DPPC monolayer. The DPPC monolayer was isothermally compressed (37 °C) with constant velocity (1.25 cm²/s). It was found that carbon nanotubes cause (a) the shift of compression isotherms and compressibility curves towards higher values of surface pressure and surface area per one DPPC molecule, (b) the increase of maximum compressibility of DPPC monolayer and the corresponding surface pressure, (c) the reorganization of DPPC molecules at the air-liquid interface in gaseous phase (G), liquid-expanded phase (LE), liquid-condensed - liquid-expanded coexistence (I) and liquid-condensed phase (LC). The changes in surface properties of DPPC monolayer indicates a disruption of elasticity of the phospholipid membrane in the presence of the carbon nanotubes tested.

This paper has been based on the results of research task No. II.N.10 carried out within the scope of the fourth stage of the National Programme “Improvement of safety and working conditions” partly supported in 2017–2019 — within the scope of research and development — by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection – National Research Institute is the Programme’s main co-ordinator.

Nanoparticle-induced toxicological mechanisms have become one of the most studied topics in toxicology during the last few years. Because of their excellent antimicrobial activity, silver nanoparticles (AgNPs) are recognized as a promising nanomaterial with widespread applicability. However, the impact of AgNPs on biological systems, regarding their possible effects and fate in living cells are still limited. In order to assess AgNPs hazard and risk, reliable and reproducible screening approaches are needed to test the basic materials as well as nanoenabled products. In recent years, zebrafish was recommended as the nanomaterials toxicity screening animal model. The purpose of current study is to investigate the toxic effects and mechanism of AgNPs on zebrafish embryos. The results indicated zebrafish embryos exposed to AgNPs led to reduced survival ratio in a size-, dose-, and time dependent manner. Abnormal phenotypes (abnormal jaw, axis flexure, pericardial edema, and yolk sac edema) and developmental retardation can also be found. In addition, oxidative stress, ER stress and apoptosis are involved in the underlying toxic mechanisms. These results demonstrated that different size of the AgNPs can trigger different toxicity, in which the larger size cysteamine-modified AgNPs was more toxic than smaller size AgNPs.

Purpose

Graphene represent two-dimensional (2D) nanomaterial with unique physical and chemical properties. Unlike graphene oxide less is known about biological effects of other derivatives such as graphene platelets and sheets (GPs) which workers are exposed during manufacturing. These non-biodegradable materials could pose a risk especially for respiratory system after exposure by inhalation. It has been proved that GPs of a certain size can be delivered beyond the ciliated airways and deposit in alveoli. There are internalized mostly by alveoli macrophages or simply persists in intercellular spaces, where can physically interact with other cells. This may lead to inflammation, disruption off cell metabolism and tissue damage. This study represents preliminary data of possible interactions between two different commercially available GPs and selected monocyte/macrophage cell lines.

Methods

Dynamic light scattering method and Transmission Electron Microscopy (TEM) were used for characterization of GPs. THP-1 cells, THP-1 null cells, RAW BLUE cells and primary monocytes were used for experiments. The viability of cells was evaluated via WST-1 and LDH assays after 24, 48 and 72 hours of exposition. Localization of GPs was detected by TEM and confocal microscopy. Potential immune response was detected by activation of inflammasome NLRP3 test.

Results

TEM confirmed presence of both GPs in cytoplasm after 24 hours, but no particles were located in nucleus. First results have shown that both GPs caused neither acute cytotoxicity nor activation of inflammasome NLRP3 after 24 as well as 48 hours of exposition. However, levels of LDH which is released through damaged cell membrane increased with GPs concentration after 48 and 72 hours exposition of RAW BLUE cells. Interestingly, there was no increase of LDH after exposition of activated THP-1 cells and monocytes.

Supported by program PROGRES Q-09 and Q-10, ERDF/ESF projects NANOBIO CZ CZ.02.1.01/0.0/0.0/17_048/0007421 and FIT CZ.02.1.01/0.0/0.0/15_003/0000495, project CENATOX GAP503/12/G147.

The purpose: Silver nanoparticles (nanoAg) are used in a wide array of industry and biomedicine. The increasing applications of nanoAg may lead to necessitate a general health and environmental risk assessment of them. The toxicity of nanoparticles depends on their unusual features including growing reactivity and particle-to-cell contact (due to a large number of particles). Despite a few toxicological reports, in vitro and in vivo, neurotoxic effects of nanoAg are poorly known. The aim of the present study was to investigate the possible behavioural changes of rats after prolonged neonatal exposure (21 days) to small (10 nm) citrate-stabilized nanoAg at a dose of 0.2 mg/kg b.w. using a wide range of behavioural assessments.

Methods: Rat pups (14 days old) were administered intragastrically with nanoAg (0.2 mg/kg) or silver citrate (Ag⁺) for 21 days. Behavioural tests were performed twice – one day and one month after the discontinuation of substance administration. The measurement of locomotor activity (in actimeters), motor coordination (rota-rod test), memory performance (novel object recognition test), depression (forced swimming test) and anxiety-like behavior (elevated plus maze test), generally accepted in investigations of the central activity of new compounds, were made.

Results and conclusion: NanoAg exposure did not significantly influence locomotor activity and motor coordination of rats, both in the short and long term. Pro-depressive, anti-anxiety and pro-cognitive effects were observed after prolonged administration of nanoAg, but only one day after the discontinuation. These results were not shown one month after the discontinuation. Our data may suggest that nanoAg exerts a significant central action, together with low neurotoxicity as measured in behavioural animal tests.

The study was supported by the Statutory Activity provided by the Polish Ministry of Science and Higher Education for Medical University of Lublin (DS 23/2016).

Potassium octatitanate (POT) fibers are commonly used as alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, previous studies showed that inhaled POT fibers had little carcinogenic potential. Therefore, we conducted a short-term study in which we administered well-dispersed POT fibers to rats using transtracheal intrapulmonary spraying (TIPS). We found that POT fibers had greater biopersistence in the lung and pleural cavity and caused stronger tissue response than either anatase titanium dioxide nanoparticles (a-nTiO₂) or rutile titanium dioxide nanoparticles (r-nTiO₂), which have been evaluated as group 2B carcinogens. Consequently, we are conducting a 2-year study of POT fibers to investigate their carcinogenicity using MWCNT-7, a known carcinogen in rats, and inert r-nTiO₂ as reference materials. Male F344 rats were administered 0.25 and 0.5 mg POT fibers and r-nTiO₂ and 0.5 mg MWCNT every other day over a period of 15 days by TIPS. Rats were killed 3 weeks and 52 weeks from the start of experiment. In the lung, POT and MWCNT fibers induced significant increases in alveolar macrophage number, granuloma formation, reactive hyperplasia and thickening of the alveolar wall, pulmonary collagen deposition, and pulmonary 8-OHdG levels. In contrast, for both of the r-nTiO₂ treated groups, the tested parameters were not different from the controls. Both visceral and parietal pleura showed increase in thickness, PCNA labeling indices, and few areas of visceral pleural hyperplastic proliferation in the 0.5 mg POT and the MWCNT treated groups. The pleural cavity lavage showed significant increases in total protein levels in the 0.5 mg POT fiber group, approximately 4 fold higher than in MWCNT treated group. Also, numerous POT fibers but few MWCNT fibers were detected in the pleural cavity lavage cell pellet. In conclusion, POT fibers were persistent inside the lung and pleura and induced a similar or greater tissue response compared to MWCNT-7, indicating the possibility that inhaled POT fibers are carcinogenic in rats.

Financial support:

This work was supported by Health and Labour Sciences Research Grants of Japan, the 5^th Term Long-Range Research Initiative (2017) by Japan Chemical Industry Association, and Egyptian cultural affairs and missions sector.

Carbon-based nanoplatelets (CNP) represent a new class of 2-D nanostructures in multiple variants and with interesting functional properties (e.g. material enforcement and electrical conductivity). The hazard characterization of CNP is still incomplete. Commercial CNP candidates (ACS Material, USA) were selected, covering single layer (SLG) or multi layer graphene (MLG), carboxyl graphene, single layer graphene oxide, and graphite oxide. Technical soot (Printex 90) served as non-platelet reference. Sterility and endotoxin content of the CNP were analysed and the morphology (SEM pictures) and the specific surface area (BET method) were re-evaluated. As in vitro screening models both, primary rat alveolar macrophages (AM) and MRC-5 human lung fibroblast cells were analysed on membrane damage (LDH release) and metabolic activity (AlamarBlue test). Interestingly, the two SLG induced marked concentration-dependent membrane damage in AM after 24h of incubation, with a BMD₃₀ of 3.2 and 2.5 µg/cm², whereas no such effect was observed for MRC-5 cells. Some LDH release was also shown for single layer graphite oxide (BMD₃₀: 39.3 µg/cm²). The other materials were nearly inactive. Significant effects on metabolic activity were not observed. In AM, SLG additionally induced direct DNA damage and release of PGE₂ and TXB₂. In conclusion, SLG showed a (geno)toxic potential in vitro in AM, but not in lung fibroblasts. Based on the in vitro screening data and for validation, an SLG (highest) and an MLG (lowest toxic potential) were selected for in vivo investigations. In a dose range finding (DRF) test, with dosing by intratracheal instillation (0.02 and 0.2 mg/rat, each) SLG was confirmed as the stronger inflammogenic sample (bronchoalveolar lavage fluid - BALF analysis) inducing a significant recruitment of neutrophils and - surprisingly - of eosinophils. In a subsequent 4-week nose-only inhalation study with the same total doses (predicted by MPPD model) the pro-inflammatory effect of SLG was weaker and no eosinophils were detected in BALF. Again, SLG but not MLG exhibited some inflammogenic potential. The histopathological examination mirrored the results of the BALF analysis. Granulocyte infiltration and giant cells were observed only in the SLG high dose group. – Funding: FP7- ERA-NET SIINN - BMBF-PtJ

International awareness towards safe and sustainable nanostructured materials have conducted to the development of pioneering and fangled green and smart nanosolutions, and have boosted a remarkable expansion potential for multiple industry sectors. Yet, current uncertainty in the regulatory framework for the commercialization of these nano-sized structures, the barely available literature on their potential effects towards human health and environmental safety, and the scarce information on their specific properties have limited their use in the industry.

Our research group has screened the in vivo nanotoxicity of calcium carbonate, antimony tin oxide and titanium dioxide nanoparticles. All these engineered nano-sized structures are already commercially incorporated in materials developed in the industrial sector of plastics improving their barrier, mechanical and conductive properties. The above-mentioned nanoparticles were characterized for their biocompatibility via in vivo zebrafish embryo toxicity (ZET) protocol and the results were contrasted with the ones from zebrafish acute toxicity testing in order to assess the ability to substitute for the acute fish test in nano EHS (environmental health and safety) investigation [1].

Fertilized zebrafish eggs were monitored via continuous waterborne exposure to the different nanoparticles at the nominal concentrations of 0.01-100 mg.mL-1 for 80 hours postfertilization, permitting a fast-track screening of the embryonic lethality, developmental delay signals, phenotypical malformations, spontaneous movements and free-swimming patterns, heart rate and hatching. F0 parental zebrafish (i.e. adult fish) were further waterborne exposed to the same nanoparticles and at the same nominal concentrations range for 1 week.

Adult zebrafish acute toxicity testing results were accurately extended by in vivo ZET data that allowed for a sensitive and early warning signal of nanotoxicity. Inversely to titanium dioxide nanoparticles, calcium carbonate and antimony tin oxide nanoparticles did not cause significant (sub-)lethality to zebrafish. Yet, all nanoparticles tested induced-developmental cardiotoxicity.

References
[1] S. Lin, Y. Zhao, A.E. Nel, S.Lin, 2013. Zebrafish: an in vivo model for nano EHS studies. Small. 9(910):1608-18.

It is assumed that the key role in the cytotoxicity of nano-sized ZnO compared to other metallic nanoparticles is played exactly Zn²⁺ due to ion-shedding effect, which is capable in a high concentration to trigger an apoptosis. On the other hand, the shape of the nanomaterial can also contribute to these processes under interaction with the components of cell membrane.

The aim of work – to conduct a comparative analysis of the membrane effects of nano-sized ZnO in different shapes: rods (ZnO NRs) and spherical particles (ZnO NPs), after their interaction with human lymphocytes.

Materials and methods: Lymphocytes were isolated from heparinized peripheral blood of healthy donors. The morphology of the nanostructured ZnO was studied by scanning electron microscopy (SEM). PI test is used for cells viability assessment. Fluorescent labeling with TMA-DPH, laurdan, N-(1-pyrenyl)-maleimide, fluorescamine allow examining the conformation of cell membrane components.

Results and discussion. It was shown that incubation of isolated lymphocytes with 1– 100 μg/ml ZnO NRs and ZnO NPs during 20 and 40 h leads to a dose-dependent increase of count cells in a late apoptotic phase but the highest cytotoxity was revealed for ZnO NPs. Study of physical state of membrane lipids after exposure of lymphocytes to ZnO NRs revealed a decrease of lipid microviscosity of the hydrophilic membrane area but increase it in hydrophobic area. The effects of ZnO NPs on membrane lipid components were not significant. Investigations of the membrane proteins conformation state revealed rise of protein NH₂-groups level on the membrane surface only after lymphocytes exposure to ZnO NRs. At that time, level of the membrane protein SH-groups after action of ZnO NPs was increased, otherwise interaction of ZnO NRs with cells lead to oxidation of thiol groups. According to the results of SEM, the geometric size of the spherical ZnO NPs did not exceed 50 nm, the diameter of the ZnO NRs was 50-80 nm and the length did not exceed 500 nm. So, the obtained results of toxicological tests can be partly explained a possibility of spherical ZnO NPs to enter into cell while the more probable mechanisms for interaction with cell of NRs are electrostatic interaction or membrane puncturing.

The work is supported by the BRFFR Grant B17-128 and the EU Horizon 2020 programme under grant № 778157.

Increasing number of in-vitro acute toxicity studies suggest that amorphous silica nanoparticles (SiNPs) could induce adverse effects. However, different dispersion methods are being used to disperse SiNPs and their effect on size distribution/Aggregation (not agglomeration) were not properly addressed. We found that the acute toxicity of SiNPs was strongly influenced when dispersed using different methods. The main objective of this study was to investigate the adverse effects of repeated low dose exposure (estimated from OELs) in-vitro to SiNPs prepared by different dispersion methods. NM-200 (precipitated silica) was used in this study and two stock dispersions were prepared by ultra-sonication and vortexing respectively. Characterization of these suspensions by Transmission Electron Microscopy (TEM) and Dynamic Light scattering (DLS) indicated that only nanofractions were present in the sonicated suspension while a mixture of nano and micro (aggregated) particles were found in the vortexed suspension. Human bronchial epithelial cell line (16HBE14o-) and Human monocytic cell line (THP-1) were repeatedly exposed to low dose (0.76 µg/cm²) of these suspensions, three times a week for three weeks and the cell viability, cell number, total glutathione, IL-8, IL-6 and genotoxicity were evaluated at the end of third week. Sonicated suspensions strongly reduced the cell number/cell growth rate, induced strong increase in IL-8 and IL-6 only in HBE cells. No significant effect was noticed in THP-1 exposed to any of these suspensions. Our results suggest that the dispersion method strongly influenced the size distribution of SiNPs and therefore, acute and chronic in-vitro toxicity of SiNPs.

The application of nanomaterials has been rapidly increasing during the last years. Current studies show, that inhalational exposure to nanoparticles (NP) probably brings about negative toxic effects, but there is a critical lack of human studies, especially related to possible DNA alterations. In this study we analyzed twice (pre-shift and post-shift) a group of 20 persons, long-time working (17.8±10.0 years) in nanocomposites research, and 21 matched controls. Detail aerosol exposure monitoring during working shift in a sampling day (including processes as welding, smelting, machining) to assess the differences in exposure to a wide range of particulate matter (PM) including fractions <25 – 100 nm, and chemical analysis of nano-sized fraction was completed. The micronucleus assay combined with fluorescence in situ hybridization using Human Pan Centromeric Chromosome Paint was applied to recognize not only the frequency of total micronuclei (MN) in binucleated cells (BNC), but also other types of chromosomal damage (losses and breaks), including the centromere positive (CEN+) and centromere negative (CEN-) micronuclei. The monitoring data showed differences in the risk of exposure to NP related to individual working processes as well as differences in chemical composition of nano-fraction. Cytogenetic results demonstrated possible adaptation to long-time (years) exposure of NP (related to total frequency of MN), although this exposure may be responsible for DNA damage pattern changes (increase of chromosomal breaks from 36% to 48% – clastogenic effect). However, short-time (daily) exposure could be a reason for the increase of chromosomal losses (aneugenic effect) as well as breaks (clastogenic effect) in the exposed group, depending on the particular type of exposure. Supported by the Grant Agency of the Czech Republic #18-02079S and the Ministry of Education Youth and Sports Czech Republic #LO1508.

From the perspective of the immune system, nanomaterials (NMs) represent invading agents. Macrophages are immune cells residing in all organs and tissues as the first line of defense. Interactions of macrophages with NMs can determine the fate of NMs as well as their potential toxic effects. In the present study, we compared toxicity of four different types of NMs [NM-100 (TiO2, 110 nm), NM-110 (ZnO, 20 nm), NM-200 (SiO2, 150 nm) and NM-300K (Ag, 20 nm)], in THP-1 macrophage-like cells. Cells were incubated with non-cytotoxic concentrations (1-25 µg/ml) of NMs for 24 hours and microarray technology was used to analyze changes in whole-genome expression. Gene expression profiling revealed a substantially different molecular response following exposure to diverse NMs. While NM-100 did not exert any significant effect on gene expression profile, all other NMs triggered a pro-inflammatory response characterized by an activation of NF-κB transcription factor and induced expression of numerous chemokines and cytokines. NM-110 and NM-300K further affected processes such as p53 signaling, oxidative and replication stress as well as modulation of cell cycle and DNA repair. NM-300K, the most toxic nanomaterial, was also characterized by enhanced expresion of genes coding protesomal complex involved in degradation of diverse proteins. We suppose that genotoxicity of ZnO and Ag NMs leading to DNA damage in THP-1 macrophages is probably caused by the extensive intracellular dissolution of these NPs, as confirmed by TEM imaging. Supported by the Ministry of Youth, Education and Sports of the Czech Republic (LO1508).

E171 (Titanium dioxide) is an EC approved food additive (EC 1129/2011), authorized to be used as color in foodstuffs. It is widely used for its refractive properties (shiny coating, UV protection) in the food and pharmaceutical industries. It is intended and assumed to be present in bulk form. A nanofraction may be present.Dispersion is crucial step to characterize the particle properties of food additives as it allows to separate the primary particles. To better characterize titanium dioxide food additives (E171) their dispersion method was optimized.

A dispersion methodology based on the Guiot and Spalla approach was applied. It electrosterically stabilizes the (nano)materials, dispersed by sonication, using BSA at a pH determined by zeta potential measurement. Dispersion efficiency was examined by descriptive TEM and using a combination of TEM imaging and image analysis. The latter approach allows to assess the distribution of the particle properties (size, shape, surface structure) quantitatively. For both the pristine TiO₂ food additive E171 and the JRC TiO₂ representative test material zeta-potential measurement allowed to identify the conditions (pH) where a stable dispersion with a minimal level of agglomeration was observed. The stability of the dispersion was confirmed by descriptive TEM: preparing dispersions of TiO₂ through a pH adjustment provides a stable dispersion of single primary particles and small aggregates and agglomerates.

Under the optimized conditions, the minimal external dimension of the primary particles could be measured more precisely and accurately by a combination of EM imaging and image analysis than in metastable conditions, such that the materials could be better classified according to the EC definition of a nanomaterial.

There is strong evidence indicating that some inhaled particles can induce systemic effects. Translocation of particles from the airways or the lung is one of the possible ways for particles to induce systemic effects. Some nanomaterials have been proved to induce changes in the tight junctions of airway epithelial cells. In the present study we are using an automatized imager to evaluate changes in tight junctions and comparing with the results obtained by measuring the transepithelial electric resistance (TEER). We are using human airway epithelial cell line 16HBE as a model of epithelial barrier. To standardize the conditions of our cell cultures we seeded different cellular densities of 16HBE (8,000 to 33,000 cells) inside inserts with a surface of 0.3 cm². Once the cultures look confluent, we measured the TEER every day. After 5 to 7 days in culture, the higher density of cells did present a large increase in the TEER values, and these values remain high during 3-5 days. When exposing to NaHClO at concentrations from 0.0003 to 0.3% we observed drops in the TEER values. At the highest concentrations (0.3 and 0.03%) there was no recovery on the TEER values even after 24 h of exposure. With the lowest concentrations, after the TEER values get reduced in the first 2-5 h, there was a recovery after 24 h. Some particulate matter was used to evaluate the effect on the TEER values. TiO₂, PM₁₀ and PM_2.5 were tested with transient drops on TEER values at concentrations above 10 mg/cm². Using the same cellular densities as on inserts, we seeded 96 well plates (with a surface of 0.3 cm²/well). When we register an increase in the TEER values on the inserts, we treated the 96 well plates in the same way. To evaluate alterations in the tight junctions we did immunofluorescence staining for ZO-1 (tight junction protein) and evaluating changes in the tight junction patter using ImageXpress Micro equipment. With this method we are looking to automatize the evaluation of changes in tight junction patterns. Due to the variability on TEER values measurement, more efficient ways of evaluating changes in tight junctions is needed to evaluate and screen large amount of chemicals and particulate matter. Our preliminary results indicate that this approach is promising.

Nanoparticles (NPs) are colloidal systems with a size below 100 nm. They are present in some innovative drugs called 'nanomedicines' and in many food products. Among them, silica NPs are used as a lubricant in the production of tablets but also as an anti-caking agent in powdered foods (named additive E551). Some recent information has alarmed on a potential human health risk induced by the oral ingestion of the additive E551 composed of a high part of silica NPs. The lack of knowledges regarding their effects due to their unique physicochemical properties (size and specific surface area) requires a rigorous evaluation of the safety of these NPs, especially at the level of the intestinal barrier. Indeed, a modification of its permeability could have deleterious consequences for human health. The aim of the present work is to determine the effects of silica NPs and the corresponding food additive on the permeability of the intestinal barrier. For this purpose, silica NPs at different sizes and the additive E551 were added for 2h to Caco-2/HT29-MTX cells in co-culture. This in vitro model is recognized as a relevant model to mimic human intestinal barrier. The integrity of the intestinal barrier was evaluated through the measurement of transepithelial electrical resistance (TEER). In addition, paracellular transport was studied through the quantification of lucifer yellow, a membrane integrity marker. The level of reactive oxygen species (ROS) and different cytokines (IL-1α, IL-6, IL-8 and IL-10) was measured to evaluate the oxidative cellular stress and the inflammatory process. At non-cytotoxic concentrations, silica NPs dramatically alters the integrity of the intestinal barrier. This effect was not strictly inversely proportional of the NP size since the most deleterious effects were observed with the 30nm NPs. The enhanced paracellular transport could promote the passage into the body of potentially toxic molecules for humans. Moreover, the ROS production and the proinflammatory signaling pathway were increased after NP treatment. Taken altogether, these results suggested that the silica NPs can strongly alter the barrier function of the human intestine and interact with the epithelial cells by inducing an inflammatory response and an oxidative stress.

Double-walled carbon nanotubes (DWNTs) have been expected to be one of important materials in the fields of nanotechnology, because they have not only high mechanical strength and thermal stability but also unusual electronic and optical properties. But their safety for human health is poorly known. To evaluate immunotoxicity of DWNTs, effects of DWNTs on the pneumonia in respiratory syncytial virus (RSV)-infected mice were assessed.

We used DWNT-1 (1 μm in length), DWNT-7 (7 μm in length) and DWNT-15 (15 μm in length) in this study. The RSV infection test was performed as reported previously (Hashiguchi et al., 2015). Briefly, female (6 weeks old) BALB/c mice were exposed intranasally to DWNTs (0 - 0.25 mg/kg) on days 1, 3 and 5 before RSV infection under anesthesia. These mice were intranasally infected with 3.5 × 10⁵ PFU of RSV under anesthesia.

The levels of chemokine CCL5 (RANTES), a representative marker of pneumonia, in the bronchoalveolar lavage fluids (BALF) of RSV-infected mice were increased due to DWNT-1-exposure (0.025 mg/kg) compared with the control on day 5 post-infection. Histopathological analysis for lung tissues showed that the infiltration of mononuclear cells in alveolar wall was also increased due to DWNT-1-exposure compared with the control. Ingestion of the carbon tubes in macrophages was observed in the lung tissues of DWNT-1-exposed (0.25 mg/kg) RSV-infected mice. Effects of DWNT-7 and DWNT-15 on the pneumonia were not clear compared with DWNT-1.

Thus, exposure to DWNTs exacerbated the pneumonia in RSV-infected mice and might influence macrophages/monocytes in an early phase of RSV infection.

*Hashiguchi et al., 2015. Environ. Toxicol. Pharmacol. 39, 879-886.

The use of iron oxide nanoparticles (IONPs) as an environment remediation tool is based on their ability to adsorb metals and decrease their bioavailability. Within the Reground project, a humic acid coating was introduced on IONPs (ha-IONPs) to improve their reactivity and stability in the environment and could as well affect their toxicity. Cytotoxicity assays on human cell lines (A549 and Caco-2) and acute toxicity tests on soil and aquatic organisms (enchytraeids, plants and daphnids) were used to evaluate the toxicity of these particles. In addition, possible toxicological interactions between adsorbed zinc ions (Zn, as a model pollutant) and the ha-IONPs were evaluated.

ha-IONPs caused no toxicity effects on A549 and Caco-2 cells, except at rather high concentrations (above 100 mg/mL), in which assay artefacts induced by the NPs could not be totally excluded. In the ecotoxicological studies, ha-IONPs did not show any toxic effect to soil invertebrates (enchytraeids) and plants (Lactuca sativa), but caused immobilization to Daphnia magna (EC₅₀ > 900 mg/L).

The co-incubation of Zn ions with ha-IONPs led to slight changes in cellular toxicity when compared to the cellular response of the metals alone. Although the EC50 values did not change, the shape of the dose responses became shallower in the presence of ha-IONPs. This suggests a protective effect of the NPs at high metal concentrations and an enhanced toxicity at low concentrations. To further investigate the mechanisms leading to such increased toxicity, the mixture was also tested on 21-day differentiated Caco-2 cells, which are characterized by minimal nanoparticle uptake. In this model, Zn toxicity was much lower than in A549 cells, regardless of the presence of ha-IONPs. These results, together with additional evidence showing the precipitation of Zn salts in the assay medium, suggest that cell internalization as particles is a determinant event of metal toxicity.

In the Daphnia magna assays, co-exposure to Zn salts and ha-IONP, decreased the toxicity of the Zn salts (EC₅₀ increased from 0.013 mg Zn/L to 0.575 mg Zn /L), showing a protective effect of the particles. This could be due to the reduced bioavailability of the Zn ions due to settling in association with ha-IONPs.

Acknowledgments. Supported by the EU H2020 Project REGROUND, ID: 641768 (2015-2018).

Polymers are widely used as biomaterials due to their unique properties such as biocompatibility, easy design preparation and a variety of structures.Several methods have been developed to improve polymer design and it based conjugates provides many advantages, especially in clinical studies. There is limited number of studies on this subject. The aim of the study was characterized and determined of in vitro toxicities of newly synthesized L-glutamic acid-g-p(HEMA) nanoparticles. The characterization analyses showed that polymers have an average size of around 194.6 nm and zeta potential is a negative value which is -18 mV. SEM image was also supported these findings. The sample was scanned from 750 to 4000 cm^-1 for FT-IR spectroscopy and the characteristic peaks of stretching band were observed in spectrums. It was determined that the nanoparticle (12.5-100 µg/ml) has not been showed cytotoxic effect on healthy cell line (HEK 293, ATCC-CRL-1573) with WST-1 test after 24 and 48 hours of exposure. It has not hemolytic activity on rabbit erythrocytes even at the highest concentration (20-160 µg/ml). Also, Ames Test results showed that it was not caused genotoxic effects on Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains (even at 5000 µg/ml concentration). So, it has shown promising results for the future works.

[Background] Some studies showed reproductive and developmental toxicity of multi-wall carbon nanotube (MWCNT) in mice, but the mechanism remains unknown. We preliminary found that the strength of the toxicity was varied depending on pretreatments for MWCNT. We here conducted a comparative study using the MWCNT samples with different pretreatment. [Materials and Methods] Pregnant ICR mice were intratracheally instilled with three types of MWCNT samples: bulk (B-group), heat-treated (incubation 2-hr at 250 ^oC; H-group) and well-dispersed by Taquann method (T-group). Animals were repeatedly administered on gestational days 6, 9, 12 and 15 at the dose of 4 mg/kg/day, and then dissected on gestational day 17 for evaluation of reproductive parameters. Pulmonary toxicity of the dams was analyzed at gestational day 15 after three times instillation. [Results and Discussion] The body weight of dams in H-group significantly decreased at the day 17 comparing to vehicle control. Number of dead fetuses was increased in H-group, although the change was not significant. Body weight of fetuses was significantly decreased in B- and H- groups, and the mean value in H-group was lower than that in B-group. Frequency of visceral malformations of the fetuses was significantly high in B- and H-groups. In contrast, these parameters were not changed in T-group. Histological analysis of the lung parenchyma of dams revealed that the level of inflammation was high in order of H- > B- > T- group. LDH and total protein levels in lavage of the lung were increased in all exposed groups, but to a lesser degree in T-group. Further, expression of inflammation-related genes in the lung, such as ccl-2 and Il-6, was markedly upregulated in H-group. These data suggest that the developmental toxicity of MWCNT could be depending on the level of inflammation in the dam’s lung. [Acknowledgement] This study was supported by a Health and Labour Sciences Research Grant (H30-Kagaku-Shitei-004) from the MHLW, Japan.

VITO started to implement the ‘Safe Innovation’ concept in the H2020 NANOTUN3D project (Grant Agreement 685952; www.nanotun3d.eu/; 2015-2019) concerning the development of nano-modified alloys for additive manufacturing. It’s a way to incorporate Health, Safety, and Environmental (HSE) aspects already at an early stage of the innovation process in order to guarantee safety at the workplace, for consumers, and the environment. Identification of exposure scenarios, exposure modelling, and on-site measurements provided information on the relevant exposure routes. The human and ecological hazard assessment was performed for these routes and these results are shown for 7 NANOTUN3D test items, i.e. silicium carbide (SiC, 50 and 500 nm), titanium dioxide (TiO2, 50 nm), SiC@TiO2 core-shell nanoparticle (50 and 500nm), and yttrium oxide (Y2O3, 40 nm, Alfa Aesar and HWNANO).

Human toxicity screening consisted of acute exposure of in vitro cultured human cell models (airways: A549 alveolar epithelial cells type II), skin: SkinEthic^TM Reconstructed Human Epidermis (OECD Test Guideline (TG) 439), and eye: SkinEthic^TM Human Corneal Epithelium (SkinEthic^TM HCE, OECD TG 492)) and endpoint analysis of cytotoxicity and oxidative stress (A549 cells), and irritation responses (RHE, HCE). For eye irritation, the Bovine Corneal Opacity and Permeability test (OECD TG 437) was performed in a testing strategy together with SkinEthic^TM HCE. Ecological hazard identification for the aquatic environment was assessed. To simulate potential emissions to the aquatic compartment, leachable fractions (LF) were prepared. The acute aquatic toxicity of the LFs was measured (Algae growth inhibition (OECD TG 201) and Daphnia acute immobility (OECD TG 202)) for all compounds. Also the chronic ecotoxicity for selected compounds SiC 50 nm and Y2O3was measured (Algae NOEC/LOEC (OECD TG 201) and Daphnia reproduction (OECD TG 211)).

No human health hazard effects were observed for the NANOTUN3D compounds. Adverse effects were seen in the Algae growth inhibition test for TiO2 and Y2O3 (EC₅₀ value = 73% LF (730 mgeq/l); NOEC/LOEC = 250/500 mgeq/l). No adverse acute effects were seen in the Daphnia test. SiC 50 nm also had no inhibiting effect on Daphnia (reproduction test). Y2O3 showed chronic toxicity for Daphnia (NOEC/LOEC = 500/1000 mgeq/l).

Abstract

Introduction: Different usages of organophosphate (OP) poisons and availibility of these compounds for most peoples cause serious intentional and accidental toxicity. Hydroxyl group of serine in active site of acetylcholine estrase (AChE) enzyme inhibits by OPs and inactivat the enzyme. Treatment of OPs poisoning is not always successful. Use of synthetic polymers because of biocompatibility and biodegredetivity is very important. In this study we attempt to attach serine amino acid to the polyethylenglycol (PEG) as a novel antidote for Ops poisoning.

Methods: Serine and polymer were conjugated with reductive amination reaction. nanoparticles were purificated by ultrafilteration. Structure of conjugated compound were analyzed by ¹H NMR, ¹³C NMR, IR and DSC. Particle size of this compound determined by dynamic light scattering method. Finally, amount of blood hemolysis and its cytotoxicity effects on SKBR₃ cell line were calculated. Effectivness of NPs were evaluated in Albino poisoned mice. NPs at doses of (100, 200, 400 mg/kg) were administered (ip) 20 minutes after a single dose of diazinon (LD50 = 166 mg/kg). Atropine (20 mg/kg, ip) with Pralidoxime ( 20 mg/kg, ip) or alone as compared to the standard treatment were used. LD50 decreasing, cholinestrase reactivation enzymes in brain, RBC and serum and oxidative dammage in brain mitochondria were assessed in mice.

Results: According to NMR, IR and DSC data, conjugation of PEG to Serine was achieved succesfully. Peak 9.5 ppm in ¹HNMR spectrum of PEG-aldehyde which is attributed to the aldehyde hydrogen, does not exist in PEG-Serine spectrum and also appearance of peak 179.89 ppm in 13C-NMR of PEG-serine are indications of the product correct structure. Avarage particle size of nanomicelle was determined 192.4 nm. Amount of hemolytic activity of this NP was calculated 0.867 % and IC₅₀ was calculated 36 mg/ml. LD50 significantly decreased (25 %) by NPs when compared with only diazinon group. Cholinestrase enzymes activity, lipid peroxidation, protein carbonyl content, and mitochondrial function significantly improved by NPs when compared with diazinon group.

Conclusion: Synthetized conjugated compound is very biocompatible with none toxicity that can successfully decrease the acute toxicity of diazinon as a new combination therapy.

In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). The presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy. A higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. No global DNA methylation alteration on 5-methylcytosine (5-mC) or LINE-1 elements were observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites). Significant changes in sequence-specific methylation was observed also observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). The study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) in different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. Based on our results, CNT induced DNA methylation and expression changes could have significant impact on several critical cellular processes.

Nanomaterials, occurring naturally or engineered for many different purposes, have a wide range of applications due to have a small size and large surface area. Silver nanoparticles (AgNPs) are used as antimicrobial agent in various products such as textiles, cosmetics, medical tools, food packaging. This widespread use of this materials causing concern about the effects of human and environmental health. Moreover, to increase the antimicrobial activity, different groups are added to AgNPs. AgI, nano-sized a silver halide, is noticeable composite in this area. With the advances in nanotechnology, new materials are produced day by day, although the information on the potential toxicity of these materials is very limited. In this study, the effects of nano silver composite AgI (AgI NP) was investigated with the COMET assay (single cell gel electrophoresis) on Allium cepa root cells. A. cepa root cells were exposed to AgI NP during 18 hours. Five onion bulbs were used for each dose and three were randomly selected. 50 cells were counted for each dose and the genotoxic damage caused by the nanocomposite was assessed according to the tail length parameter. Distilled water and ethyl methane sulfonate were used as a negative and positive control, respectively. The statistical analysis was performed using SPSS software and One-Way ANOVA was used to evaluate the differences between the distilled water and the treated groups. The results obtained from this study showed statistically significant positive results at 0.05 mM and 0.1 mM doses of AgI NPs. The genotoxicity observed at the lowest dose of 0.01 mM and the highest dose of 0.5 mM was not statistically significant. This study revealed that AgI nanocomposite induced DNA damage on Allium cepa root cells.

Silver nanoparticles (AgNPs) are widely used due to their physico-chemical properties in nanotechnology, especially their antibacterial effect. Recent studies are progress to produce new silver composites to enhance the antibacterial activity of AgNPs. Since silver nano composites are new products, there is limited information about the effects on the environment and the living organisms during their production, use and disposal. Determination of the genotoxic potential of these AgNPs composites will enable for a safer use of AgNPs. In this study, micronucleus (MN) test, mitotic index (MI) and other chromosome aberrations (CA) tests were applied Allium cepa root meristematic cells in order to investigate the potential genotoxic effects of AgI NPs. The cells were exposed to four different doses (0.01, 0.05, 0.1 and 0.5 mM) of AgI NP during 18 hours. The CA test was evaluated according to different categories such as bridge, c-mitosis, polar deviation, binucleate, nuclear bud, laggard, early movement, and other deviations. The mutagenic effect of the NP tested was examined by the MN test. The cytotoxic effect was also determined by calculating the MI by the ratio between the number of dividing cells and the total of cells. In the tests, 3 repetitive 1000 cells (total 3000 cells) were evaluated.

Analysis of variance (ANOVA) and Dunnett’s t-test (p<0.05) was used for the statistical analysis of MI, MN, and CA data. Our results reveal that although the mitotic index did not statistically different from control group, chromosomal damages were significantly induced in all doses. On the other hand, incidence of MI was not statistically significant at any doses.

Silica nanoparticles (SiO₂ NPs) are easily produced in different sizes and surface modifications enabling their use in several industrial and biomedical applications, which increases the risk of human exposure to these nanoparticles. Because the toxicological profile of nanoparticles is dependent upon their physicochemical properties, the aim of this study was to investigate the influence of the physicochemical properties (size, surface capping and hydrophilicity/hydrophobicity) in the cyto- and genotoxicity of SiO₂ NPs in rat alveolar epithelial cells (RLE-6TN), a primary target during inhalation exposure.

To evaluate the cytotoxic potential of SiO₂ NPs, RLE-6TN cells were exposed for 24 h to different concentrations (0-56 µg/cm²) of differently capped (naked, phosphonate- and amino-modified), differently sized (7, 15 and 40 nm), and with different hydrophilicity/hydrophobicity nature variants dispersed in serum-free culture medium. Cytotoxicity of the SiO₂ NPs was assessed by determining the LDH release and WST-1 metabolization in RLE-6TN cells. To evaluate the genotoxic potential of the SiO2 NPs, RLE-6TN cells were exposed to subtoxic concentrations of the different variants tested (0-28 µg/cm²) and the potential DNA damage was assessed by the alkaline comet assay.

Exposure to hydrophilic SiO₂ NPs induced a concentration-dependent cytotoxicity in RLE-6TN cells, being the cytotoxic effect more marked in the cells exposed to the smallest SiO₂ NPs. Amino and phosphonate surface modification mitigated the cytotoxicity of the SiO₂ NPs as demonstrated by the lower LDH released levels and higher metabolization of WST-1 comparing with cells exposed to naked SiO₂ NPs with the same size. Exposure to hydrophobic SiO₂ NPs did not induce cytotoxicity on RLE-6TN cells. No DNA damage was observed in RLE-6TN cells after 24 h of exposure to all tested SiO₂ NPs.

Our data shows that size and hydrophilic/hydrophobic nature influence the cytotoxic profile of SiO₂ NPs. In turn, amino and phosphonate surface modification attenuates cytotoxicity of SiO₂ NPs on RLE-6TN and might constitute a strategy to increase biocompatibility of SiO₂ NPs. Nevertheless, further research such as the evaluation of other toxicity endpoints, should be conducted to support our findings.

This work is supported by Fundação para a Ciência e a Tecnologia through European project NanoToxClass (SIINN/0001/2013) and under the grants SFRH/BD/101060/2014, SFRH/BPD/96196/2013 and SFRH/BD/120646/2016.

Multiwalled carbon nanotubes (MWCNTs) represent tube-like structures with nano-sized diameters, made of multiple carbon layers. MWCNTs exhibit promising properties, including electrical/thermal conductivity and high tensile strength, thus practically pointing to a wide range of applications in the electronics and composite materials sectors. However, hazard characterization of MWCNTs, highly needed for work place and consumers’ safety, is still incomplete. Besides morphology, surface functiona­lization of MWCNTs may have marked impact on adverse biological activity. The ERA-NET SIINN project ICONS (“International Collaboration On Nanotube Safety”) thus aimed at mechanis­tically evaluating and ranking the pro-fibrotic and genotoxic potential of eight differentially purified (chemically or thermally) and functionalized (-COOH and -NH₂) MWCNT samples, made from one batch of industri­ally relevant Nanocyl NC7000 (tangled morphology). One focus of the subproject, carrried out by Fraunhofer ITEM (BMBF-funded: FKZ: 03XP0063), deals with the comparative in vitro (geno)toxicity testing of the eight samples, using primary human lung fibroblast MRC‑5 and primary human peritoneal mesothelial LP9 cells. MWCNTs were intially tested for sterility and endotoxin, followed by development of an ultrasound-based dispersion protocol. Scanning electron microscopy indicated presence of both free fibers and agglomerates, and turbidity measurements pointed to diffe­ren­tial sedi­men­tation and thus perhaps unequal in vitro doses. Subsequent in vitro testing revealed concentration-dependent membrane damage (LDH release), differential inhi­bition of proliferation (RICC, mitotic index), slight induction of micro­nuclei (1 µg/cm², 24 h), and loss of chromosomes in MRC-5 cells. In LP9 cells (5 µg/cm², 24 h), induction of LDH release, differential accumulation of MWCNTs around the cell nucleus and impairment of the cyto­skeleton (α-tubulin immunofluores­cence) were noted. But, no direct DNA damage (γH2A.X or 53BPI foci) was evident, and only sporadic γH2A.X panstaining occurred to account for induction of apoptosis or cellular senescence. Our results point so far to a rather mechanical than clastogenic adverse activity of the MWCNTs. Two candidate MWCNTs will be selected for a 28-day inhalation test in rats.

Cerium dioxide nanoparticles (CeO₂NPs) have the potential to impact disease outcome but have not been investigated for their effect on models of asthma and inflammatory lung disease. We therefore set out to examine the impact of CeO₂NPs on a house dust mite (HDM) allergen induced model of asthma in Balb/c mice. Repeated intranasal instillation of CeO₂NPs in the presence of HDM resulted in a type II inflammatory response, characterised by increased pulmonary mast cell markers, plasma IgE and airway goblet cell metaplasia. This was accompanied by increases in IL-4, CCL11, mucin levels and inflammatory regulators CLCA1 and SLC26A4 within the lung. Repeated HDM instillations followed by a single exposure to CeO₂NPs failed to produce changes in type II inflammation but did result in alterations in the neutrophil activation marker CD177. RNA-SEQ was used to explore early effects of CeO₂NPs and HDM exposure after 24hr exposure. Changes in serum amyloid A3 expression paralleled increased neutrophil levels within BAL fluid, while no changes in eosinophil or lymphocyte levels were observed. RNA-Seq was also used to investigate transcriptional responses within the lung after repeat CeO₂NP and HDM treatments. Changes in macrophage differentiation markers indicative of type II inflammation were prominent among a general inflammatory profile. Overall changes in gene expression were consistent with regulation of dendritic cells, macrophage functionality and interferon signalling as the potential key events for how CeO₂NPs may guide pulmonary responses to allergen towards type II inflammation. As this type of inflammatory response is present within asthmatic endotypes our findings may have implications for how occupational or incidental exposure to CeO₂NPs should be considered for those susceptible to disease.

Several ceramic industries have already incorporated within their production processes the manufacture of different types of ceramic nanoparticles, as well as the application of those nanomaterials on conventional products, which increases the risk of human exposure to these nanoparticles, particularly in occupational settings.

The aim of this study was to investigate the in vitro toxicity of ceramic nanoparticles (ZrO2, and CeO2 and Sb2O3•SnO2 NPs) in human alveolar epithelial cells, a primary target during inhalation exposure. The cells were grown for 7 days onto Transwell inserts in RPMI 1640 medium supplemented with Glutamax, 25 mM HEPES, 10% FBS, 50 U/mL penicillin and 50 µg/mL streptomycin. Cells were exposed to aerosolized nanoparticles for 2 and 4 h at air-liquid interface using a Vitrocell® exposure system and cytotoxicity assessed by the LDH release and WST-1 metabolization assays immediately after exposure or in the recovery time (24h). DNA damage was also assessed by the alkaline comet assay at 24 h after exposure. The percentage of DNA in the tail and the olive tail moment (OTM) were used as a measure of the amount of DNA damage.

A concentration-dependent increase in LDH release was observed after 2 and 4 h of exposure to all the tested aerosolized NPs comparing with cells exposed to clean air (exposure control). However, 24 after exposure, no differences in LDH release levels were observed among the exposed cells (exposure control and NPs aerosol-exposed cells) but those were significantly higher than the incubator control. A concentration-dependent decrease in cellular metabolic activity, as assessed by the WST-1 assay, was observed 24 h after exposure to all tested NPs aerosols. Regarding the DNA damage induced by exposure to the NPs aerosols, no significant increase in % tail intensity and OTM was detected comparing with control cells.

Under our experimental conditions, exposure to the NPs aerosols affected plasma membrane integrity and the metabolic activity of A549 cells. Further research should be conducted to get further insight into the mechanisms involved in the toxicity of the selected ceramic nanoparticles.

This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the CERASAFE project (SIINN/0004/2014). MJB and FB thank FCT for their PhD scholarships (SFRH/BD/120646/2016 and SFRH/BD/101060/2014).

The MEtabolomics standaRds Initiative in Toxicology (MERIT) is a European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) supported project to provide guidance on best practice, quality standards and the reporting of analytical and computational metabolomics methods used in regulatory toxicology. ECETOC relies on world-wide expert collaboration “to develop an agreed understanding on how the state of the science can be used to improve [chemical] risk assessment by developing novel tools”. Although there is strong interest in using metabolomics from industry and regulatory scientists, the lack of best practice guidelines is a concern that has hindered its use in regulation. Fourteen metabolomics scientists from academia, industry, regulatory and government agencies were recruited as the MERIT core team, with a much larger number acting as a wider consultative group. The primary objective of MERIT is to develop best practice guidelines and minimal reporting standards in the context of metabolomics in regulatory toxicology. Several case studies representing near-future applications of metabolomics - including for the discovery of molecular key events and biological read-across - are being used to ensure the relevance of the guidelines to regulatory toxicology. MERIT will publish its recommendations for best practice and reporting standards and is liaising with other groups, such as the NIH Metabolomics Quality Assurance and quality Control Consortium (mQACC) and the Metabolomics Society, to ensure harmonization of the recommendations.

The use of 0.1% DMSO (dimethyl sulfoxide) as solvent for in vitro assays is widespread and effects are often assumed to be negligible. Initially, DMSO was researched for medical applications although clinical trials were halted in 1965 due to adverse effects whereupon DMSO was labeled extremely toxic. Years later, upon reviewing new results, the FDA classified DMSO in the safest category. Thereafter, DMSO was used in applications such as cryopreservation or cell differentiation inducer, but mainly as a solvent. This study uses complete transcriptome and epigenome sequencing (RNA-seq & MeDIP-seq) to chart the effects of 0.1% DMSO on 3D cardiac and hepatic microtissues for the purpose of assessing whether DMSO induces bias in analyzing findings from toxicant-treated in vitro assays. RNA-seq detected over two thousand differentially expressed genes (DEGs; FDR >0.05) in both cardiac and hepatic tissue (compared to untreated microtissues). Pathway analysis of these DEGs identified hundreds significantly overrepresented pathways (FDR <0.05). Although the amount of DEGs per biological process differs between the tissues, similar processes are affected, indicating a consistent mode of action of DMSO. Since affected pathways displayed a majority of downregulated genes, processes of transcriptional regulation were investigated in detail, focusing on DNA methylation. Differentially methylated regions (FDR <0.05) found in both tissues indicate changes in the epigenetic landscape, suggesting that DMSO can interfere with regulatory systems in cardiac and hepatic tissues. While the field is evolving towards more sophisticated in vitro models, using 3D conformation and/or physiological-consistent low dose concentrations, omics technologies clearly demonstrate that the effect of DMSO on cell regulation has to be kept in mind when designing in vitro studies and interpreting the data. Consequently, the lowest possible dose of DMSO should be used as even low incubation concentrations may induce solvent-induced bias.

Smoking cessation has been reported as one of the most effective approaches to reducing the effects of cigarette smoke (CS). We used integrated omics analyses to investigate the impact of smoking cessation in murine liver and kidney, the primary organs involved in xenobiotic response. Female C57BL/6J mice were exposed to filtered room air (sham) or K3R4F reference CS for 6 months. Moreover, two additional groups as smoking cessation groups, mice were exposed to CS for 5 or 13 week, followed by filtered air up to 6 months. Livers and kidneys were subjected to transcriptomic and proteomic analysis. The number of differentially expressed genes (DEGs) was higher in livers than in kidneys in all exposure groups, suggesting a greater impact on liver. Notably, the number of DEGs decreased as the duration of the cessation period increased, in particular DEGs related to cell stress, oxidative stress, and inflammatory responses. Cessation periods reduced the numbers of DEGs in both organs, approaching the numbers in the sham group. Canonical pathway analysis predicted that CS inhalation would activate the aryl hydrocarbon receptor signaling pathway in the liver, showing a good correlation between gene expression and the abundance of proteins related to aryl hydrocarbon receptor signaling. These findings suggest that CS-inducible cell stress, especially in the liver, is mediated by aryl hydrocarbon receptor signaling. Moreover, this impact can decrease with the duration of smoking cessation.

The dynamic, time- and dose-dependent perturbation of intracellular pathways is an important mechanism underlying toxic responses in several species, in vivo and in vitro; the identification of such perturbations is essential to understand the mechanisms and to design effective predictive models. Despite the large amount of gene expression data generated for this purpose, certain limitations exist when using available analytical methods, including database annotation, arbitrary thresholds for statistical testing, as well as species- and model- specific responses. In this study, we sought to overcome these limitations by applying aQREN - POPH - Tipologia 4.1 - Formação Avançada', co-sponsored by Fundo Social Europeu (FSE) and by national funds of Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).

Aim. The global contamination from the released amounts of POPs into the environment has raised great concern by the adverse impacts in humans and several species of marine and terrestrial ecosystems. The objective of the current study was to assess the burden of POPs in wild terrestrial mammals.

Materials and Methods. A total of 16 hair samples were collected from wild animals (leopard cats, musk deers, wolf, amur hedgehog and raccoon dogs) that found dead due to traffic accident or by hunting of Primorsky Krai, Russia. The analysis of hair samples was focus on the determination of HCB (hexachlorobenzene), DDTs (opDDE, ppDDE and opDDD), PCB congeners (28, 52, 101, 118, 138, 153 and 180) and PAHs (acenaphylene, fluorene, anthracene, phenathrene and pyrene). The detection of pollutant was conducted by a one-step hair extraction method, using headspace SPME technique and analyzed by a simple GC-MS.

Results: The use of HSSPME in the current study provides low detection limits for most of the POPs. The majority of wild animal hair samples were found positive. More specific, the percentages of positive hair samples for HCB, opDDE and ppDDE were 93.8%, 100.0%, 93.8 %, respectively. In contrast, opDDD was detected only in the 18.8 % of the samples. DDTs’ detection levels ranged from 1.26 to 52.06 pg/mg. The most frequently detected PCB congeners were PCB28, PCB52, PCB101 and PCB138 (from 93.8 to 100.0%) in a concentration range from 0.73 to 31.34 pg/mg. PCB118, PCB153 and PCB180 was detected in the 18.8, 68.8 and 6.3 % of the samples, respectively. Fluorene and anthracene/phenathrene were detected in the 100% of the samples in a range from 6.53 to 23.56 pg/mg, while the acenaphylene and pyrene in the 87.5% and 75.0% of the samples providing levels below the LOQ values.

Conclusion. The highest mean concentrations levels of all tested pollutants were found for musk deer compared to the other species. For HCB, DDTs and PCBs the order of higher to lower mean detected concentrations was: musk deer> leopard cat> wolf> raccoon dog> amur hedgehog. To the best of our knowledge, the present study is the first that provide results for the contamination levels in hair samples by persistent organic pollutants in five different terrestrial mammals.

This work was supported by the Russian Science Foundation (project No. 18-14-00120)

The purpose

Cigarette smoking is considered a chronic medical disorder, as nicotine has a highly addictive potential.

In the last decades, studies were focused on the pharmacological effects of nicotine and its main metabolite cotinine.

Nicotine and cotinine have been suggested to be psychoactive in humans and animals, facilitating memory, cognition, and executive function.

Epidemiological studies indicate that the incidence of cigarette smoking is very high among depressed individuals.

Experimental animal studies revealed the antidepressant effects of acute or chronic nicotine treatments. In addition, nicotine and other nicotinic agonists have been demonstrated to improve performance on attention and memory tasks. In animal models, cotinine was demonstrated to have antipsychotic, anxiolytic, and antidepressant properties.

Both nicotine and cotinine are very similar structurally, but despite this structural similarity, cotinine shows distinctive effects from nicotine.

The objective of this study was to investigate comparatively the antidepressant and anxiolytic effects of cotinine and nicotine in mice.

Method

Nicotine 0.5 mg/kg and cotinine 5 mg/kg were administered orally to NMRI mice for 14 days. Pharmacological tests were performed after 1, 7 and 14 days. Antidepressant effect was evaluated using forced swimming test (FST) after acute administration and tail suspension test (TST) after 7 and 14 days. Plus suspended maze test was used for anxiolytic effect evaluation in the final day of the experiment. In order to eliminate false positive results, we also determined the mice motor activity at each test moment with the Ugo Basile activity cage.

Results

Immobilisation time in TST was shorter in cotinine group than the control after 7 days (24.7%; p<0.05), close to imipramine reference group (31.85%; p<0.05). After 14 days, this effect no longer occurs, probably due to a significant reduction in motor activity for cotinine group which could influence the immobility time values in TST. Time spent in open arms of the labyrinth was longer for nicotine group than the control (129.61%; p<0.05), which supports a consistent anxiolytic effect.

Conclusion

Both nicotine and cotinine are psychoactive in mice. Further studies are needed to understand the mechanisms for these effects.

Silver NPs (AgNPs) have emerged as an important class of nanomaterials, due to their unique optical, antimicrobial, electrical and physical properties, which enabled the use of these nanoparticles in various fields, including medical, food, health care, consumer, and industrial purposes. However, the widespread use of AgNPs raises unmet safety concerns, which may be related to their size and the coating agents used for its stabilization.

Neutrophils, as host defense cells, play a key role in recognizing, ingesting, digesting, and eliminating foreign agents, including nanoparticles. Despite the central role of neutrophils as key players in immune system, little is known about their interaction with AgNPs, with different coatings and sizes. In that sense, it was our aim to study the effect of three different sizes of citrate and polyvinylpyrrolidone (PVP) -AgNPs (5, 10 and 50 nm) in human neutrophils cell death and oxidative burst.

It was possible to conclude that the effect of AgNPs was size and coating-dependent.The citrate and 5 nm AgNPs were the most cytotoxic and potent inducers of oxidative burst, through theactivation of NADPH oxidase, via the action of PKC.

This work brings new insights about the interaction of AgNPs and human neutrophils which is essential for the safer use of these nanoparticles.

Acknowledgements

This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013, and “Programa Operacional Competitividade e Internacionalização” (COMPETE) (PTDC/QEQ-QAN/1742/2014 – POCI-01-0145-FEDER-016530), and under the framework of QREN (NORTE-01-0145-FEDER-000024). Tânia Soares acknowledges FCT the financial support for the PhD grant (SFRH/BD/128647/2017), in the ambit of 'QREN - POPH - Tipologia 4.1 - Formação Avançada', co-sponsored by Fundo Social Europeu (FSE) and by national funds of Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).

Isoparaffins are branched aliphatic hydrocarbons with a carbon skeleton length ranging from approximately C7 to C20. They are mainly used as solvents in textile cleaning products (dry-clean cloth) due to their low odor, low surface tension and excellent cleaning efficiency. Many of the isoparaffins are multi-constituent mixtures with variable and often unknown composition and have been defined as substances with unknown or variable composition (UVCB). Because of the compositional complexity, the assessments of their toxicity and environmental fate is complicated. Grouping these chemicals according to their carbon chain length and the aromatic content is suggested by recognized scientific and advisory bodies to be an ideal way to assess their toxicity. To this end and as part of ToxServices’ Full Materials Disclosure (ToxFMD®) program, we used the GreenScreen® hazard assessment tool to assess and compare the hazard properties of four isoparrafins solvents with low aromatic content (< 2%) that are used as solvents for commercial textile cleaning. The substances belong to three different categories (short carbon chain (C7-9), medium chain (C9-14) and long-chain (C14-C20)). Our results show that all four investigated isoparrafins, regardless of the carbon length chain, are associated with a high hazard for aspiration toxicity, a common concern for such type of solvents, as their measured kinematic viscosity parameters are below the GHS Guidance value for Category 1. Aspiration hazard was recently incorporated in the GreenScreen® methodology under systemic toxicity single exposure endpoint. In terms of environmental fate (persistence) and toxicity, the properties vary with the carbon chain length; substances with short chain (C7-C11) have low potential to persist, but are toxic to aquatic organisms. In contrast, substances with a carbon length greater than 11 are not readily biodegradable but are not expected to be toxic to aquatic organisms. All compounds have high potential for bioaccumulation. These results can be utilized to characterize hazards for other isoparaffin solvents with similar properties (carbon length/aromatic content) and provide informed choices for selecting less hazardous isoparaffin solvent alternatives.

The aim of the research was to choose non-toxic fragrance oils for use in an aroma-dispenser innovative device applied in a vehicle ventilation system. The levels of proinflammatory cytokines IL-6 and TNF-α and NF-κB activation level were determined in the human lung cells line A549 stimulated with selected essential oils.

Twenty commercial fragrance oils was used for the experiment in two concentrations: 25% and 12.5%. The A549 cell culture was stimulated with the studied fragrance oils according to the procedure. The A549 cell culture (ATCC CCL 185) was performed in Dulbecco's culture fluid (DMEM) supplemented with 10% inactivated calf serum, 100 μ/ml penicillin, 100 μg/ml streptomycin, and L-glutamine. The cell culture was adjusted to a density of 2x106 cells per ml and then incubated in 37°C until a single-layer culture was obtained.

The level of TNF-α and IL-6 cytokines was determined by immunoenzymatic ELISA using the R&D system kit and results were given in pg/ml. 96-well microplates were coated with anti-TNF or anti-IL-6 monoclonal antibodies, rinsed with buffer (0.05% Tween80 in PBS) and then blocked with PBS with 10% fetal bovine serum. Samples of 100 μl (supernatant of the cell culture) were applied to microplates. The reaction were stopped and the absorbance was measured at wavelength λ = 470 nm with a correction to λ = 570nm using the BioTek reader. The obtained results were referenced to the standard curve.

The diluted anti-NFκB p-65 primary antibody (Chemicon Internationatl Inc., USA), the biotinylated antibody (Novocastra Laboratories Ltd.) and the Novostain Super ABC Reagent solution (Novocastra Laboratories Ltd.) were applied to microscopic slides according to the procedure (using the cell culture). The DAB solution (0,1% DAB solution in 0,1 M Tris buffer containing 0,02% H2O2) (Novocastra Laboratories Ltd.) was used to stain microscopic slides, which allowed to determine the percentage of cells with positively stained nucleus and determine the level of NF-κB activity.

Studies have shown that only a few tested fragrance oils do not stimulate A549 cells for the production of proinflammatory cytokines such as TNF-α and IL-6 and do not affect NF-κB activation in the human lung cells line, so they are recommended for use in the innovative fragrance dispenser for vehicle interior refreshing.

The aim of this study was to investigate the influence of vitamin B1 (thiamine) on biochemical changes in the liver of Japanese quail (Coturnix japonica) treated with chlorpyrifos. The following parameters of oxidative stress were examined: concentration of malondialdehyde –MDA, activity of catalase–CAT, glutathione S-transferase–GST, myeloperoxidase–MPO, but also and activity of butyrylcholinesterase–BChE, and cyclooxygenase –COX and extracellular signal–regulated kinase –ERK (apoptotic regulators).

The study was conducted on eighty male Japanese quails (2 control groups and 6 experimental groups, n= 10), 3-4 weeks old. One control group was treated only with vitamin B1, while the second one received pure corn oil. CPF dissolved in corn oil was administered to three groups of quails by gavage for 7 consecutive days at doses of 1.5 mg/kg BW, 3 mg/kg BW and 6 mg/kg BW. Another three groups were treated with 10 mg/kg BW of vitamin B1 i.m. 30 min after CPF administration (in above mentioned doses) for 7 consecutive days.

Activity of BChE, CAT GST and MPO, and concentration of MDA were measured spectrophotometrically using a Cecil CE 2021 UV/VIS spectrophotometer. For detection of COX and ERK, we used Western blot analysis.

Our studie have shown that CPF significantly inhibited butyrylcholinesterase in the liver, while vitamin B1 increased activity of this enzyme in a dose dependent way. Also CPF has led to increase in the concentration of MDA, activity of CAT, GST and MPO, but after thiamine treatment there has been a decrease of these parameters. Increase of COX and ERK expression after CPF treatment demonstrates an increase of apoptotic vulnerability of cells exposed to CPF, while vitamin B1 caused a decrease in their expression.

Overall these results confirm that CPF causes oxidative stress and apoptosis, and also are giving new insights into thiamine research as an 'antistress vitamin'.

Introduction

The interest of plant extracts as phytopharmaceuticals has recently increased, due to their natural bioactivity, as antimicrobial and antioxidant effects. Consequently, the toxicity assessment of plant extracts is relevant to evaluate its potential use as ingredients for pharmaceutical products. Cistus ladanifer medicinal plant has been shown to be of interest in this field, due to the presence of phenolic compounds and compounds belonging to the terpene family in its composition. This species is traditionally used for dermal application due to putative cicatrization effect.

This study intends to assess the toxicity profile of Cistus ladanifer different extracts and their antioxidant bioactivity.

Methods

The antioxidant potential of 2 different aqueous extracts (hydrolates) from Cistus ladanifer (A and B), as well as an essential oil, were enrolled. The antioxidant effect was tested by measuring the extracts capacity to reduce the compound 2,2-diphenyl-1-picryl hydrazyl (DPPH). EC50 was calculated as the amount of sample able to reduce half of DPPH initial concentration, by comparison with a DPPH concentration calibration curve. The cytotoxicity was tested, cells of Skin fibroblasts (3T3) were treated with the hydrolates of Cistus ladanifer and the MTT assay was performed.

Results

The hydrolates did not show relevant antioxidant activity, since a reduction of at least 50% of DPPH initial solution was not observed. On the other hand, the essential oil presented a EC50 of 12.73 ul/ml. The fibroblasts presented cell viability above 50% after treatment with up to 10% of hydrolate A and 35% of hydrolate B.

Discussion/Conclusion
Cistus ladanifer essential oil showed to be antioxidant in contrast to both hydrolates tested that were found to have poor antioxidant activity. The results regarding the cytotoxic activity reveal that extracts of this plant are bioactive. Our results show that both the essential oil and hydrolate extracts from Cistus ladanifer may be interesting ingredients for pharmaceutical products considering the traditional use of this plant. The essential oil may be a good candidate as preservative and hydrolates may be a better choice for less reactive products development. More studies are needed to explore this approach and their use as pharmaceutical ingredients.

Indoor air pollution plays a major role with regard to public health. There are three established strategies to reduce indoor air pollution, namely source control, ventilation and mechanical air cleaners. Despite the existence of these established strategies, indoor air pollution is still one of the world’s largest environmental problems according to World Health Organization. Consequently, new methods or strategies to reduce indoor air pollution are continuously being sought. The purpose of this study was to investigate a novel technology that aims to construct an indoor self-cleaning ecosystem by using microorganisms, namely Bacillus species. Instead of applying conventional cleaning products in indoor spaces, microorganisms are nebulized on surfaces, objects and in the indoor air. The key element of the self-cleaning ecosystem is bioremediation, which is the use of living (micro)organisms for the cleaning up of contaminated soil, water, sediment or air. The pollutants might be metabolized to less hazardous metabolites. Scientific evidence concerning the effectiveness of this novel technology to reduce indoor air pollution is still lacking. Therefore, a crossover pilot study was carried out in an urban region in Flanders and lasted 3 weeks. The microorganisms were nebulized in one office building. Of this building, two offices were included in this study. One office of a well-matched neighbouring office building was included as control. The indoor air quality was assessed by tracking particulate matter, carbon dioxide, aldehydes, polycyclic aromatic hydrocarbons, volatile organic compounds, temperature, relative humidity and ventilation rate. Despite the complexity of the real-life study concerning the large amount of variables, the data obtained in this real-life pilot study indicates that constructing a self-cleaning ecosystem might help in reducing the indoor levels of acetaldehyde, particulate matter and total amount of hydrocarbons. The percentage decreases of acetaldehyde and particulate matter concentrations were respectively up to 1.5 and 4 times larger in the offices equipped with a self-cleaning ecosystem than in the control office (P<0.05).

Purpose: Kidney stone is one of the most prevalent diseases in globe. It can result in obstructing urinary tract which followed by renal colic and severe pain. The treatment considered for renal colic pain relief is to use nonsteroidal anti-inflammatory drugs or opioid analgesics and/or drug combinations which cause side effects and toxicity. In traditional medicine, camel thorn distillate (Alhagi persarum) is widely used to treat urinary stones. This study was carried out to speed up the kidney stones removal through concomitant use of camel thorn distillate and Rowatinex drug.

Case: The case was a 45-year-old woman with symptoms of flanks pain and kidney stone diagnosis. The sonograms of the patient showed that, there were two stones with 3.5mm and 3mm diameters in the left and right kidney calyx, respectively. There was also seen another stone with diameter of 3.5 mm on the front right distal urethral and ureterovesical junction. In the right kidney; there were hydro-urethra and nephritic grade I which was evident based on two diagnostic imagines.

The treatment protocol on this patient was started with Rowatinex capsule (two times daily) and 150 ml of camel thorn distillate which followed by a thirty- minute walk. This treatment was continued for 20 days.

Results: The sonograms taken one month after treatment showed; there was no trace of kidney stones. The concomitant use of camel thorn and Rowatinex can efficaciously help kidney stones in small sizes to be removed more appropriately and drug toxicity will be lowered.

Introduction

Capsaicin, the principal pungent ingredient of hot chili is wide used in various topic formulations to relieve diabetic neuropathic pain. However, this natural ingredient exhibits a very high cytotoxicity and finally induces apoptosis by ROS (Reactive Oxygen Species) production. The use of a drug delivery system for capsaicin could alleviate the toxicity of free capsaicin extracts and decrease the numeral side effects that free capsaicin administration triggers. In this context, the aim of our study was to investigate in vitro the potential of novel synthetized capsaicin alginate microcapsules to generate ROS and induce apoptosis of human dermal fibroblasts.

Experimental procedures

In this view, the bare alginate microcapsules and capsaicin loaded alginate microcapsules were washed with PBS supplemented with 5% antibiotic – antimicotic and then immersed in complete culture medium. During 24h, 4 extracts were collected at predefined time points and subsequently frozen until analysis. The potential of the novel alginate microencapsulation of capsaicin to induce ROS production was investigated by measuring the level of hydrogen peroxide in CCD – 1070Sk human dermal fibroblasts cell culture after treatment with the defrosted extracts. Moreover, the apoptotic status of the cells exposed to defrosted extracts was also investigated by flow cytometry.

Results

Our results show that the capsaicin free alginate microcapsules doesn`t induce ROS production or apoptosis. Furthermore, the capsaicin loaded alginate microcapsules treatment doesn`t determine an increase of the hydrogen peroxide levels, but caused the appearance of a very small population of apoptotic cells. However, the un encapsulated capsaicin displayed a statistically significant increase of ROS production, correlated with a dramatically considerable increase of the apoptotic cells.

Conclusions

In conclusion, the novel synthetized capsaicin alginate microcapsules represent a much safer approach for capsaicin therapeutic use and might be further included in different pharmacological formulations for topic administration in diabetic neuropathy.

Zinc is a micronutrient at low level but at high concentrations leads to environmental contamination and causes health problems. The aim of this study was to evaluate the effects of Zinc Chloride (ZnCl₂) exposure on cognition and locomotion behavior in adult zebrafish (Danio rerio) and correlate these findings with different tissue accumulation of Zn, overall brain AChE and ROS level, Cortisol and Metallothionine levels in the nervous system. Although low level heavy metal exposure poses a potential risk to the zebrafish larvae, little or no data about the effects of ZnCl₂ exposure on zebrafish adult fishes. We therefore, exposed adult zebrafishes for 4 days (0.5,1 and 1.5mg/L) displayed decreases locomotor behavior, such as average speed and time in upper zone and an increase speed of meandering. Interestingly, adult fishes exposed to ZnCl₂ for 4 days showed impaired frigid long-term memory in the passive avoidance test. Furthermore, zinc chloride treated fish showed memory deficit, increased ROS and AChE levels and decreased locomotor and swimming activities compared to control. A significantly positive correlation was observed between memory and AChE activity, as well as between locomotion and ROS production. These results indicate that acute exposure to ZnCl₂ in adults leads to anxiogenic effects, impaired memory and decrease aggressive behavior that might be associated with damage caused by this metal in the CNS, particularly in the cholinergic system.