Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach.

Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome.

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Combined Exposure to Multiple Mycotoxins: An Example of Using a Tiered Approach in a Mixture Risk Assessment.

Humans are exposed to mycotoxins on a regular basis. Exposure to a mixture of mycotoxins may, therefore, result in a combination of adverse effects, or trigger the same effects. This should be accounted for when assessing the combined risk of multiple mycotoxins. Here, we show the outcome of using different approaches in assessing the risks related to the combined exposure to mycotoxins. We performed a tiered approach using assessment groups with a common target organ (kidney, liver and haematologic system), or a common adverse effect (phenomenon) (reduced white blood cell count), to combine the exposure to mycotoxins. The combined exposure was calculated for the individuals in this assessment, using the Monte Carlo Risk Assessment (MCRA) tool. The risk related to this combined exposure was assessed using toxicological reference values, e.g., health based guidance values. We show that estimating the combined risk by adding the single compounds' risk distributions slightly overestimates the combined risk in the 95th percentile, as compared to combining the exposures at an individual level. We also show that relative potency factors can be used to refine the mixture risk assessment, as compared to ratios of toxicological reference values with different effect sizes and assessment factors.

Structure-toxicity relationships, toxicity mechanisms and health risk assessment of food-borne modified deoxynivalenol and zearalenone: A comprehensive review.

Mycotoxin, as one of the most common pollutants in foodstuffs, poses great threat to food security and human health. Specifically, deoxynivalenol (DON) and zearalenone (ZEN)-two mycotoxin contaminants with considerable toxicity widely existing in food products-have aroused broad public concerns. Adding to this picture, modified forms of DON and ZEN, have emerged as another potential environmental and health threat, owing to their higher re-transformation rate into parent mycotoxins inducing accumulation of mycotoxin in humans and animals. Given this, a better understanding of the toxicity of modified mycotoxins is urgently needed. Moreover, the lack of toxicity data means a proper risk assessment of modified mycotoxins remains challenging. To better evaluate the toxicity of modified DON and ZEN, we have reviewed the relationship between their structures and toxicities. The toxicity mechanisms behind modified DON and ZEN have also been discussed; briefly, these involve acute, subacute, chronic, and combined toxicities. In addition, this review also addresses the global occurrence of modified DON and ZEN, and summarizes novel methods-including in silico analysis and implementation of relative potency factors-for risk assessment of modified DON and ZEN. Finally, the health risk assessment of modified DON and ZEN has also been discussed comprehensively.

Urinary Metabolites of Organophosphate Pesticides among Pregnant Women Participating in the Japan Environment and Children's Study (JECS).

Organophosphate pesticides (OPPs) exhibit neurodevelopmental toxicity. To evaluate the effect of prenatal exposure to OPPs in the Japan Environment and Children's Study, a nationally representative birth cohort study, 4575 maternal urine samples were analysed for six OPP metabolites, i.e., dialkylphosphates (DAPs). This study aimed to investigate predictors of urinary DAPs using machine learning approaches and to assess the cumulative risk based on relative potency factors among Japanese pregnant women. The median creatinine-normalised urinary concentrations (interquartile ranges) of dimethylphosphate, dimethylthiophosphate and diethylphosphate, which had a detection rate of 50% or higher, were 3.53 (1.91-6.78), 4.09 (1.66-10.8) and 3.28 (1.88-5.98) µg/g-creatinine, respectively. Possible predictors of urinary DAP concentrations were the month of urine sampling, consumption of apple and maternal body mass index. When fenitrothion was used as an index chemical for cumulative risk assessment, 0.36% of participants exceeded the lower 95% confidence limit of the benchmark dose10.

RNA-protein correlation of liver toxicity markers in HepaRG cells.

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells.

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Combined hazard assessment of mycotoxins and their modified forms applying relative potency factors: Zearalenone and T2/HT2 toxin.

This paper describes a methodology for hazard assessment of groups of related substances for which toxicity data are insufficient, and which utilises, next to conventional toxicological assessments and mechanistic information, the derivation of relative toxicity potency factors (RPFs). Zearalenone (ZEN) and T-2 toxin (T2) and HT-2 toxin (HT2) and their modified forms have been used as examples. A tolerable daily intake (TDI) for ZEN of 0.25 µg/kg bw was established. In vitro and in vivo studies suggested that modified forms of ZEN act via the same mode of action as ZEN (oestrogenicity). Results from in vivo uterotrophic assays were used to establish RPFs, allowing inclusion the different modified forms in a group TDI with ZEN. A TDI for the sum of T2/HT2 of 0.02 µg/kg bw per day and an acute reference dose (ARfD) of 0.3 µg/kg bw for the sum of T2/HT2 was established. In vitro studies show that phase I metabolites of T2/HT2 act via a similar mode of action as their parent compounds, namely protein synthesis inhibition with immune- and haematotoxicity. The phase I metabolites as well as conjugates of T2/HT2 and their phase I metabolites can be included in a group TDI with T2/HT2 applying RPFs.

Selecting mixtures on the basis of dietary exposure and hazard data: application to pesticide exposure in the European population in relation to steatosis.

Populations are exposed to mixtures of pesticides through their diet on a daily basis. The question of which substances should be assessed together remains a major challenge due to the complexity of the mixtures. In addition, the associated risk is difficult to characterise. The EuroMix project (European Test and Risk Assessment Strategies for Mixtures) has developed a strategy for mixture risk assessment. In particular, it has proposed a methodology that combines exposures and hazard information to identify relevant mixtures of chemicals belonging to any cumulative assessment group (CAG) to which the European population is exposed via food. For the purposes of this study, food consumption and pesticide residue data in food and drinking water were obtained from national surveys in nine European countries. Mixtures of pesticides were identified by a sparse non-negative matrix underestimation (SNMU) applied to the specific liver steatosis effect in children from 11 to 15 years of age, and in adults from 18 to 64 years of age in nine European countries. Exposures and mixtures of 144 pesticides were evaluated through four different scenarios: (1) chronic exposure with a merged concentration dataset in the adult population, (2) chronic exposure with country-specific concentration datasets in the adult population, (3) acute exposure with a merged concentration dataset in the adult population, and (4) chronic exposure with a merged concentration dataset in the paediatric population. The relative potency factors of each substance were calculated to express their potency relative to flusilazole, which was chosen as the reference compound. The selection of mixtures and the evaluation of exposures for each country were carried out using the Monte Carlo Risk Assessment (MCRA) software. Concerning chronic exposure, one mixture explained the largest proportion of the total variance for each country, while in acute exposure, several mixtures were often involved. The results showed that there were 15 main pesticides in the mixtures, with a high contribution of imazalil and dithiocarbamate. Since the concentrations provided by the different countries were merged in the scenario using merged concentration data, differences between countries result from differences in food consumption behaviours. These results support the approach that using merged concentration data to estimate exposures in Europe seems to be realistic, as foods are traded across European borders. The originality of the proposed approach was to start from a CAG and to integrate information from combined exposures to identify a refined list of mixtures with fewer components. As this approach was sensitive to the input data and required significant resources, efforts should continue regarding data collection and harmonisation among the different aspects within the pesticides regulatory framework, and to develop methods to group substances and mixtures to characterise the risk.

Methylated PACs are more potent than their parent compounds: A study of aryl hydrocarbon receptor-mediated activity, degradability, and mixture interactions in the H4IIE-luc assay.

Twenty-six polycyclic aromatic compounds (PACs; including native polycyclic aromatic hydrocarbons [PAHs], hydroxylated PAHs, alkylated and oxygenated PAHs, and [alkylated] heterocyclic compounds) were investigated for their aryl hydrocarbon receptor (AhR)-mediated potencies in the H4IIE-luc bioassay. Potential degradabilities of PACs were investigated by use of various durations of exposure (24, 48, or 72 h), and various mixtures of PACs including PAHs, alkylated and oxygenated PAHs, and heterocyclic compounds were tested for their joint AhR-mediated potency. Additive behaviors of PACs in mixtures were studied by comparing observed mixture potencies with mixture potencies predicted by use of the concentration addition model. Methylated derivatives were more potent than their parent compounds in the H4IIE-luc assay. A time-dependent decrease in relative potency was observed for all AhR-active compounds, which may be indicative of in vitro biotransformation. Monomethylated compounds seemed to be more rapidly transformed than analogous unsubstituted compounds. In addition, the results showed that the predictive power of the concentration addition model increased with the number of compounds, suggesting additivity in multicomponent mixtures. Due to the greater potency of methylated derivatives and their ubiquitous occurrence, there is a need for further research on the toxicity and mixture behavior of these environmentally and toxicologically relevant compounds. Environ Toxicol Chem 2018;37:1409-1419. © 2018 SETAC.

Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines.

Pyrrolizidine alkaloids (PAs) are among the most potent natural toxins occurring in a broad spectrum of plant species from various families. Recently, findings of considerable contamination of teas/herbal infusions prepared from non-PA plants have been reported. These are obviously due to cross-contamination with minor amounts of PA plants and can affect both food and herbal medicines. Another source of human exposure is honey collected from PA plants. These findings illustrate the requirement for a comprehensive risk assessment of PAs, hampered by the enormous number of different PA congeners occurring in nature. Up to now, risk assessment is based on the carcinogenicity of certain PAs after chronic application to rats using the sum of detected PAs as dose metric. Because of the well-documented large structure-dependent differences between sub-groups of PA congeners with respect to their genotoxicity and (cyto)toxicity, however, this procedure is inadequate. Here we provide an overview of recent attempts to assess the risk of PA exposure and the available literature on the toxic effects and potencies of different congeners. Based on these considerations, we have derived interim Relative Potency (REP) factors for a number of abundant PAs suggesting a factor of 1.0 for cyclic di-esters and open-chain di-esters with 7S configuration, of 0.3 for mono-esters with 7S configuration, of 0.1 for open-chain di-esters with 7R configuration and of 0.01 for mono-esters with 7R configuration. For N-oxides we suggest to apply the REP factor of the corresponding PA. We are confident that the use of these values can provide a more scientific basis for PA risk assessment until a more detailed experimental analysis of the potencies of all relevant congeners can be carried out.