Substance-tailored testing strategies in toxicology : an in silico methodology based on QSAR modeling of toxicological thresholds and Monte Carlo simulations of toxicological testing

International audienceThe design of toxicological testing strategies aimed at identifying the toxic effects of chemicals without (or with a minimal) recourse to animal experimentation is an important issue for toxicological regulations and for industrial decision-making. This article describes an original approach which enables the design of substance-tailored testing strategies with a specified performance in terms of false-positive and false-negative rates. The outcome of toxicological testing is simulated in a different way than previously published articles on the topic. Indeed, toxicological outcomes are simulated not only as a function of the performance of toxicological tests but also as a function of the physico-chemical Properties of chemicals. The required inputs for Our approach are QSAR predictions for the LOAELs of the toxicological effect of interest and statistical distributions describing the relationship existing between in vivo LOAEL values and results from in vitro tests. Our methodology is able to correctly predict the performance of testing strategies designed to analyze the teratogenic effects of two chemicals: di(2-ethylhexyl)phthalate and Indomethacin. The proposed decision-support methodology can be adapted to any toxicological context as long as a Statistical Comparison between in vitro and in Vivo results is possible and QSAR models for the toxicological effect of interest can be developed

Predicting in vivo gene expression in macrophages after exposure to benzo(a)pyrene based on in vitro assays and toxicokinetic/toxicodynamic models

International audiencePredictive toxicology aims at developing methodologies to relate the results obtained from in vitro experiments to in vivo exposure. In the case of polycyclic aromatic hydrocarbons (PAHs), a substantial amount of knowledge on effects and modes of action has been recently obtained from in vitro studies of gene expression. In the current study, we built a physiologically based toxicokinetic (PBTK) model to relate in vivo and in vitro gene expression in case of exposure to benzo(a)pyrene (BaP), a referent PAH. This model was calibrated with two toxicokinetic datasets obtained on rats exposed either through intratracheal instillation or through intravenous administration and on an in vitro degradation study. A good agreement was obtained between the model's predictions and the concentrations measured in target organs, such as liver and lungs. Our model was able to relate correctly the gene expression for two genes targeted by PAHs, measured in vitro on primary human macrophages and in vivo in rat macrophages after exposure to BaP. Combining in vitro studies and PBTK modeling is promising for PAH risk assessment, especially for mixtures which are more efficiently studied in vitro than in vivo

Toxicité des perturbateurs endocriniens au cours du développement de la barrière hémato-testiculaire chez le rat

PARIS-BIUP (751062107) / SudocSudocFranceF

Perturbateurs de la fonction endocrinienne et santé : un point non exhaustif sur les connaissances

National audienc

Endocrine disruption and steroidogenesis : integrated evaluation from gonadal steroidogenic enzymes in vitro and in vivo to hormonal balance and fertility assessment in vivo

In the present study, we propose an integrated evaluation of the effects of some endocrine disruptors in vivo in male and female adult rats, and in vitro in several models. We focused our work on the last step of steroidogenesis and the aim was to determine to what extent in vitro models could be predictive of in vivo alterations. We have evaluated the selected chemicals for their ability to modulate the expression and activity of steroidogenic enzymes in vitro and in vivo, to disrupt hormonal balance, and to disrupt certain fertility parameters, during a sub-acute exposure. Gene expression profiles were assessed by RT-qPCR, in vitro and ex vivo aromatase activity was assessed by the method of tritiated water, blood and gonadal steroid concentrations were evaluated with LC-MS/MS and ELISA. Our results showed that all treatments induced disturbance of hormonal balance, which was related to gene expression disordersin some cases. For example, rats treated with atrazine presented high levels of estradiol and low levels of testosterone. This observation may be related to aromatase induction observed in testes and ovaries as well as in gonadal primary cultures and cell lines. A decrease of testosterone level is also observed in male rats treated with methoxychlor, which can be linked not only to aromatase induction, but also to HSD17B3down-regulation.Taken together, these results contribute to identify toxic endpoints as well as relevant biomarkers of endocrine disruption. Further studies should determine if, on the basis of these data, anew predictive tool could be developed for endocrine disrupting chemicals assessment

Effects of endocrine disruptors in adult rats: integrative evaluation from gonadal steroidogenic gene expression profiles to hormonal balance

Endocrine disruptors are chemicals that can alter functions of the endocrine system by several ways. Their risk assessment with regard to REACH legislation remains a challenge. In the present study, we propose an integrative evaluation for the effects of some endocrine disruptors on hormonal balance in male and female adult rats. We focused our work on the last step of steroidogenesis, where two enzymes are implicated: aromatase, which plays a central role by catalyzing the irreversible conversion of androgens to estrogens, and 17beta-hydroxysteroid dehydrogenase, which catalyses the conversion of inactive sexual hormones to active ones. We have evaluated the selected chemicals for their ability to i) modulate the expression and activity of steroidogenic enzymes in gonads, to ii) disrupt hormonal balance, and to iii) disrupt certain fertility parameters, during a sub-acute exposure. Gene expression profile was assessed by RT-qPCR, ex vivo aromatase activity was assessed by the method of tritiated water, blood and gonadal steroid concentrations were evaluated with LC-MS/MS. Our results showed that all treatments induced disturbance of hormonal balance, related or not with gene expression disorders. For example, rats treated with atrazine presented high levels of estradiol and low levels of testosterone. It may be related to aromatase induction observed in testes and ovaries as well as in gonadal primary cultures. A decrease of testosterone level is also observed in male rats treated with methoxychlor, which can be linked not only to aromatase induction, but also to HSD17B3 down-regulation. These results are part of a work aiming to evidence hormonal concentration modifications related to reproductive toxicology effects in in vitro models compared to in vivo model. Taken together and discussed with regards to extra-gonadal regulation, they contribute to provide information concerning the action of endocrine disruptors, which need to be considered when assessing the effects on human healt

Effects of endocrine disruptors on steroidogenic enzymes gene expression and on aromatase activity in two human cell lines

Endocrine disruptors are chemicals that can alter functions of the endocrine system by several ways. In the present study, we have evaluated the effects of several endocrine disruptors and of some of their metabolites on the last step of steroidogenesis in two cell line models. Two enzymes are implicated in this step: aromatase (CYP19A1), which plays a central role by catalyzing the irreversible conversion of androgens to estrogens, and 17beta-hydroxysteroid dehydrogenase (HSD17B), which catalyses the conversion of inactive sexual hormones to active ones in different steroidogenic organs. We have screened the selected chemicals for their ability to modulate the expression of steroidogenic enzymes and aromatase activity in the human choriocarcinoma JEG-3 cell line and in the human adrenocortical H295R cell line after both short (4 h) and long exposure (24 h). All chemicals were tested at concentrations that did not cause cytotoxicity after 24h of exposure, as tested by the MTT viability assay. Gene expression profile was assessed by real-time RT-PCR and aromatase activity was assessed by the method of tritiated water. Treatments of JEG-3 cells by atrazine, methoxychlor and the vinclozolin metabolite M2 resulted in an increase of CYP19A1 mRNA levels. In contrast, Bisphenol-A and the methoxychlor metabolite HPTE down-regulated the expression of CYP19A1 mRNA. Methoxychlor and HPTE decreased the amount of HSD17B1 mRNAs. In H295R cells, atrazine up-regulated CYP19A1 mRNA expression, HPTE increased HSD17B1 mRNA levels and methoxychlor increased HSD17B1 and HSD17B5 mRNA levels. Concerning the aromatase activity, treatment of H295R cells by atrazine induced aromatase activity. Furthermore, exposure of these cells to either bisphenol A or HPTE inhibited aromatase activity. In JEG-3 cells, which display higher basal aromatase expression than H295R cells, the pattern of aromatase activity regulation was similar but less pronounced. Taken together, these initial results contribute to a better characterization of the action of endocrine disrupting chemicals and their metabolites on the steroidogenic pathways, which needs to be considered when assessing their effects on human health

A comparison of two human cell lines and two rat gonadal cell primary cultures as in vitro screening tools for aromatase modulation

International audienceEnvironmental toxicants are a serious health concern, and numerous studies have been devoted to studying the effects of environmental Endocrine Disrupting Chemicals (EDCs). The balance between androgens and estrogens controls the function of many EDC-sensitive organs, and the aromatase enzyme plays a key role in maintaining this balance. In vitro studies have suggested that aromatase expression and activity is a promising biomarker for initial screenings of putative hormonal disrupting compounds. To further validate the aromatase biomarker, we tested several EDCs (atrazine, bisphenol A. methoxychlor, methoxychlor metabolite HPTE, vinclozolin, vinclozolin metabolite M2) in four different models (human cell lines H295R and JEG-3, rat primary cultures of granulosa and leydig cells). We evaluated the similarities/differences in the chemical impact on aromatase mRNA levels and enzymatic activity for the different species and cell types. Aromatase gene expression was assessed by q-RT-PCR, and enzymatic activity was assessed via a tritiated water method with either intact cells or isolated microsomes. The aromatase gene mRNA levels and cellular enzymatic activity varied between the four different models tested, which suggests that the EDC effect varies among different cell types. However, regulation of microsomal aromatase activity appeared to be conserved across all the species and cell types tested. These results suggest that several well characterized complementary cellular models are required to fully characterize the effects of putative EDCs and predict the in vivo effects

AhR-dependent induction of the NADPH oxidase component p47phox by benzo(a)pyrene in human macrophages

International audienc

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 phox expression leading to priming of human macrophage oxidative burst.

International audiencePolycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47(phox) in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47(phox) protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor alpha-naphtoflavone. These results indicated that BaP induced NCF1/p47(phox) expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases