Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University Leipzig, Leipzig, Germany.
Division of Veterinary Pharmacology and Toxicology, Department of Comparative Biomedicine and Food Science, University of Padua, Agripolis Legnaro (Padua), Italy.
PLoS One. 2020 Aug 7;15(8):e0237163. doi: 10.1371/journal.pone.0237163. eCollection 2020.
In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-Ο-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies.
在奶牛的乳腺中，ABCG2 转运蛋白将外源性物质主动分泌到牛奶中。当奶牛的饲料中残留有农药时，这种情况就会产生重大影响。最近的研究表明，杀菌剂丙环唑激活了芳烃受体（AhR）通路，增加了牛 ABCG2（bABCG2）基因的表达和外排活性。这可能会增加牛奶中 bABCG2 底物的积累，从而影响农药风险评估。因此，我们研究了欧洲常用的 13 种农药是否能诱导 AhR 和 bABCG2 的活性。将表达乳腺 bABCG2 的 MDCKII 细胞与农药孵育，时间长达 72 小时。为了反映体内情况，应用的农药浓度与牛脂肪或肌肉中允许的最大残留限量（MRL）相对应。通过 qPCR 和 7-乙氧基resorufin-O-脱乙基酶（EROD）测定分别测量 CYP1A mRNA 表达和酶活性来确定 AhR 的激活。使用 Hoechst 33342 积累测定法评估农药介导的 bABCG2 外排活性的增加。对于所有测定，已知的 AhR 激活农药丙环唑作为阳性对照，而非激活的托氯氟草甲酯作为阴性对照。在 10 倍 MRL 浓度下，氯吡硫磷、二甲吩草胺、碘苯腈、三唑磺隆和噻菌灵与载体对照相比，显著增加了 CYP1A1 mRNA 水平、CYP1A 活性和 bABCG2 外排活性。相比之下，乐果、二甲嘧酚、草甘膦、异菌脲、灭多威和噻虫啉对 AhR 介导的 CYP1A1 mRNA 水平、CYP1A 活性或 bABCG2 外排没有影响。总之，MDCKII-bABCG2 细胞模型被证明是一种识别 AhR 和 bABCG2 诱导性农药的合适工具。这提供了一种体外方法，可以减少在农药批准研究中所需的动物数量。
