Gräns Johanna, Johansson Junko, Michelová Marie, Wassmur Britt, Norström Elisabeth, Wallin Margareta, Celander Malin C
Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE 405 30 Gothenburg, Sweden.
Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE 405 30 Gothenburg, Sweden.
Aquat Toxicol. 2015 Jul;164:43-51. doi: 10.1016/j.aquatox.2015.04.016. Epub 2015 Apr 14.
The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist β-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7μM), whereas omeprazole had no effect on this activity (IC50=72μM). Exposure to 10μM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10μM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.
在亮体食蚊鱼肝癌（PLHC-1）细胞系中研究了细胞色素P450 1A（CYP1A）生物标志物反应，该细胞系是研究芳烃受体（AhR）-CYP1A信号传导的良好模型。将PLHC-1细胞暴露于典型的CYP1A诱导剂和AhR激动剂β-萘黄酮（BNF），并与不同的唑类联合使用。使用了两种咪唑（克霉唑和咪鲜胺）和两种苯并咪唑（诺考达唑和奥美拉唑）。暴露于克霉唑、咪鲜胺和诺考达唑后，在24小时和48小时时CYP1A介导的乙氧异吩唑酮-O-脱乙基酶（EROD）活性诱导了2至4倍，而暴露于奥美拉唑48小时对EROD活性没有影响。克霉唑、诺考达唑和咪鲜胺在PLHC-1细胞原位也作为EROD活性的抑制剂（IC50=1.3-7.7μM），而奥美拉唑对该活性没有影响（IC50=72μM）。与对照组相比，暴露于10μM咪鲜胺导致CYP1A mRNA诱导3倍，暴露于10μM诺考达唑导致24小时时CYP1A mRNA水平诱导16倍。在混合实验中，BNF与唑类克霉唑、咪鲜胺和诺考达唑之间对EROD活性的混合效应明显大于相加效应，其中诺考达唑的混合效应最强。诺考达唑的存在使对BNF的反应在CYP1A mRNA上增加至200倍，在EROD活性上增加至16倍，并将BNF暴露对EROD活性的影响延长24小时或更长时间。这表明作为CYP1A酶抑制剂和/或竞争底物的唑类在混合化学暴露情况下，可导致对依赖CYP1A代谢进行消除的化学物质暴露的敏感性增加。结果还表明，在受唑类污染的区域，EROD生物标志物反应可能会受到显著影响。在预先用诺考达唑以已知在这些细胞中24小时可拆解微管的浓度处理5或24小时的PLHC-1中，研究了EROD生物标志物对BNF暴露的反应性。用诺考达唑对PLHC-1细胞进行5或24小时的预处理对其对BNF暴露的反应性没有影响，这意味着在微管已拆解的细胞中EROD活性仍可被诱导。
