F. Schmidt

Hepatotoxic effects of (tri)azole fungicides in a broad dose range

Abstract The toxicological relevance of effects observed at molecular stage, which occur at dose levels well below classical no-observed adverse effect levels is currently subject to controversial scientific debate. While the importance of molecular effects for the identification of a mode of action or an adverse outcome pathway is undisputed, their impact for other regulatory purposes remains uncertain. Here, we report the results of a 28-day rat-feeding study including three widely used hepatotoxic (tri)azole fungicides (cyproconazole, epoxiconazole and prochloraz) administered individually at five dose levels, ranging from slightly above the reference values to a clear toxic effect dose. Parameters analysed included pathology, histopathology, clinical chemistry and particularly effects on the molecular level. Since azole fungicides are considered to cause liver toxicity by a mechanism involving the constitutive androstane receptor (CAR), a known CAR activator (phenobarbital, PB) was administered to investigate potential similarities between triazoles and PB-mediated liver toxicity by pathway-focused gene expression analysis. Our results show an increase in liver weights and additionally histopathological changes (hepatocellular hypertrophy) for all substances at the top dose levels. The effects on liver weight were most pronounced for cyproconazole by which also the animals receiving the next lower dose were affected. In addition, vacuolisation of hepatocytes was observed at the top dose level. No such findings were obtained with any substance at lower doses to which consumers and operators might be exposed to. In contrast, the expression of sensitive marker genes (like some cytochrome-P-450 isoforms) was significantly affected also at the lower dose levels. While some of these changes, like the induction of genes related to fatty acid and phospholipid metabolism (e.g. Fasn, Fat/Cd36, Ppargc1a) or xenobiotic metabolism (Cyp1a1, Cyp2b1, Cyp3a2), could be associated with high dose effects like hepatocellular vacuolisation or hypertrophy, a histopathological correlate was lacking for others.

Application of omics data in regulatory toxicology: report of an international BfR expert workshop.

Abstract Advances in omics techniques and molecular toxicology are necessary to provide new perspectives for regulatory toxicology. By the application of modern molecular techniques, more mechanistic information should be gained to support standard toxicity studies and to contribute to a reduction and refinement of animal experiments required for certain regulatory purposes. The relevance and applicability of data obtained by omics methods to regulatory purposes such as grouping of chemicals, mode of action analysis or classification and labelling needs further improvement, defined validation and cautious expert judgment. Based on the results of an international expert workshop organized 2014 by the Federal Institute for Risk Assessment in Berlin, this paper is aimed to provide a critical overview of the regulatory relevance and reliability of omics methods, basic requirements on data quality and validation, as well as regulatory criteria to decide which effects observed by omics methods should be considered adverse or non-adverse. As a way forward, it was concluded that the inclusion of omics data can facilitate a more flexible approach for regulatory risk assessment and may help to reduce or refine animal testing.

Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice.

The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500xa0ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.

Combination effects of azole fungicides in male rats in a broad dose range

Two 28-day feeding studies were performed in male rats to investigate combination effects of azole fungicides in a broad dose range. Following separate administration of cyproconazole, epoxiconazole, prochloraz, propiconazole, and tebuconazole at five dose levels, the first three compounds were selected to be administered in two different mixtures at three dose levels including very low doses. Here we present the data obtained by clinical observations, pathology, histopathology, clinical chemistry and haematology. The liver was the common main target organ of all compounds and their mixtures. In addition, epoxiconazole exhibited an effect on the adrenals. Furthermore, food consumption and efficiency and body weight (gain) were affected. Adverse effects of the combinations were observed at dose levels at which the individual substances caused similar effects. No evidence of adverse effects was found at dose levels below the previously established NOAELs. Our findings indicate that the concept of dose additivity appears sufficiently protective for risk assessment of the fungicides examined. Besides toxicological testing, tissue residues of the azole compounds in liver, testis and kidney were determined revealing remarkable differences following administration of the single substances and of the mixtures.

Hepatotoxic combination effects of three azole fungicides in a broad dose range

Single active substances of pesticides are thoroughly examined for their toxicity before approval. In this context, the liver is frequently found to be the main target organ. Since consumers are generally exposed to multiple residues of different active substances via the diet, it is important to analyse combinations of active substances for potential mixture effects. For the (tri-)azoles, a group of agricultural fungicides and antifungal drugs, combination effects on the liver are likely because of a similar mode of action. Hepatotoxic effects of mixtures of two triazoles (cyproconazole and epoxiconazole) and an imidazole (prochloraz) were investigated in a 28-day feeding study in rats at three dose levels ranging from a typical toxicological reference value to a clear effect dose. Test parameters included organ weights, clinical chemistry, histopathology and morphometry. In addition, molecular parameters were investigated by means of pathway-focused gene expression arrays, quantitative real-time PCR and enzyme activity assays. Effects were compared to those caused by the individual substances as observed at the same dose levels in a previous study. Mixture effects were substantiated by increases in relative and absolute liver weights, histopathological findings and alterations in clinical chemistry parameters at the top dose level. On the molecular level also at lower dose levels, additive effects could be observed for the induction of several cytochrome P 450 enzymes (Cyp1a1, Cyp2b1, Cyp3a2), transporters (Abcb1a, Abcc3) and of genes encoding for enzymes involved in fatty acid or phospholipid metabolism (Ppargc1a, Sc4xa0mol). In most cases, treatment with mixtures caused a more pronounced effect as compared to the individual substances. However, the assumption of dose additivity was in general sufficiently conservative to cover mixture effects observed under the conditions of the present study.

Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells

Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.