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, Sc4 mol). 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.