ABCG2-pesticide interactions in the bovine mammary gland and pesticide-mediated induction of ABCG2 efflux activity by the arylhydrocarbon receptor

INTRODUCTION The ABC efflux transporter ABCG2 represents the main route for active secretion of various xenobiotics like drugs into human and dairy milk. Our recent studies showed that the pesticide prochloraz (PZ) induced ABCG2 activity in primary bovine mammary cells by the aryl hydrocarbon receptor (AhR). Pesticides are extensively used in agriculture and exposure of dairy animals occurs through contaminated feed. Despite the toxicological relevance of the mammary ABCG2 secretory activity, there is no detailed information on ABCG2-pesticide interactions in dairy cows. MATERIAL AND METHODS For this study, we used MDCKII cells stably expressing mammary bovine ABCG2 (bABCG2) that exhibits an AhR binding motif in its 50-untranslated region. Cells were incubated with selected pesticides. Chosen concentrations reflected the oneand ten-fold maximum residue levels (MRLs) in ruminant tissues. The bABCG2 promoter activity was examined by reporter assays. The mRNA levels of ABCG2, AhR and of the prototypical AhR target gene cytochrome P450 (CYP) 1A1 were detected by quantitative RT-PCR. The CYP1A1 catalytic activity was measured by using 7-ethoxyresorufin –deethylase (EROD) as a substrate. We assessed ABCG2 efflux activity by the Hoechst H33342 test. RESULTS Of all 14 tested substances, we detected ABCG2-pesticide interactions with one-fold MRL concentrations of chlorpyrifosmethyl (CPM), diflufenican (DF), glyphosate (GY), methiocarb (MT), PZ and thiacloprid (TC). This interaction was reflected by the competitive inhibition of bABCG2-mediated H33342 excretion through the tested pesticide. A combined incubation of CPM and MT resulted in a further increase of cellular H33342 levels. Pre-treatment of cells (72 h) with PZ, CPM and iprodion caused a dose-dependent increase in bABCG2 mRNA levels and EROD activity. The AhR antagonist CH 223191 significantly blocked the PZ-induced increase in bABCG2 mRNA amounts. Moreover, 10-fold MRL levels of these pesticides caused an increase in CYP1A1 but not AhR mRNA levels. In this concentration, PZ further induced bABCG2 promoter activity. CONCLUSIONS This study identified several pesticides as potential bABCG2 substrates. For some of them activation of AhR and AhRdepen-dent induction of bABCG2 gene expression was noticed. Hence, our results suggested that animal exposure with these feed contaminants may increase the risk of potential harmful residues of ABCG2 substrates in dairy milk like drugs and toxins.