Endosulfan Induces CYP1A1 Expression Mediated through Aryl Hydrocarbon Receptor Signal Transduction by Protein Kinase C

Abstract

CYP1A1 is a phase I xenobiotic-metabolizing enzyme whose expression is mainly driven by AhR. Endosulfan is an organochlorine pesticide used agriculturally for a wide range of crops. In this study, we investigated the effect of endosulfan on CYP1A1 expression and regulation. Endosulfan significantly increased CYP1A1 enzyme activity as well as mRNA and protein levels. In addition, endosulfan markedly induced XRE transcriptional activity. CH-223191, an AhR antagonist, blocked the endosulfan-induced increase in CYP1A1 mRNA and protein expression. Moreover, endosulfan did not induce CYP1A1 gene expression in AhR-deficient mutant cells. Furthermore, endosulfan enhanced the phosphorylation of calcium calmodulin (CaM)-dependent protein kinase (CaMK) and protein kinase C (PKC). In conclusion, endosulfan-induced up-regulation of CYP1A1 is associated with AhR activation, which may be mediated by PKC-dependent pathways.

Keywords: Aryl hydrocarbon receptor; CYP1A1; Calcium; Endosulfan; Protein kinase C.

Fig. 1.. Effects of endosulfan on CYP1A1 activity. (A) Effects of endosulfan on cytotoxicity in Hepa-1c1c7 and Tao BpRcl cells. Cells were seeded into 96-well plates and treated with various concentrations of endosulfan (Endo) for 24 h. Cell viability was assessed using the MTT assay. (B) Effects of endosulfan on EROD activity in Hepa-1c1c7 cells. EROD activity was measured in cells treated with endosulfan or 3-MC for 18 h. Values represent the means from three independent experiments, each performed in triplicate. * p< 0.01, significantly different from the control, as determined by ANOVA by the Newman-Keuls test.

Fig. 2.. Effects of endosulfan on CYP1A1 transcriptional activity. (A) Effects of endosulfan on CYP1A1 mRNA expression in Hepa-1c1c7 cells. Cells were treated with endosulfan (Endo) or 3-MC for 6h. The cells were lysed, and total RNA was prepared to analyze CYP1A1 gene expression. PCR amplification of the housekeeping gene, β-actin, was performed for each sample. CYP1A1 mRNA expression was compared between treated and untreated cells at each time point by real-time PCR. Values represent the means from three independent experiments, each performed in triplicate. * p< 0.01, significantly different from the control, as determined by ANOVA by the Newman-Keuls test. (B) Effects of endosulfan on CYP1A1 promoter activity in Hepa-1c1c7 cells. Cells were transfected with pGL3-CYP1A1-Luc and subsequently treated with endosulfan or 3-MC for 18 h. Cells were then harvested and assayed for luciferase activity. Values represent the means from three independent experiments, each performed in triplicate. * p<0.01, significantly different from the control, as determined by ANOVA by the Newman-Keuls test. (C) Effects of endosulfan on CYP1A1 protein levels in Hepa-1c1c7 cells. Cells were treated with endosulfan or 3-MC for 24 h. The membrane was probed with a CYP1A1-specific antibody, and bands were visualized with ECL reagents. Each blot in this figure is representative of three independent experiments with similar results.

Fig. 3.. Effects of endosulfan on the CYP1A1 regulatory mechanism. (A) Effects of endosulfan on XRE promoter activity in Hepa-1c1c7 cells. Cells were transfected with the XRE-Luc construct and subsequently treated with endosulfan (Endo) or 3-MC for 18 h. Cells were then harvested and assayed for luciferase activity. Values represent the means from three independent experiments, each performed in triplicate. * p<0.01, significantly different from the control, as determined by ANOVA by the Newman-Keuls test. (B) Effects of an AhR antagonist on XRE transcriptional activity by endosulfan in Hepa-1c1c7 cells. Cells were transfected with XRE-Luc and treated with CH-223191 (CH), endosulfan or 3-MC for 18 h. Cells were then harvested and assayed for luciferase activity. Values represent the means from three independent experiments, each performed in triplicate. * p< 0.01, ** p < 0.01, and # p < 0.01, significantly different from the control, 3-MC, and endosulfan, as determined by ANOVA by the Newman-Keuls test. (C) Effects of an AhR antagonist on CYP1A1 protein levels by endosulfan in Hepa-1c1c7 cells. Cells were pretreated with CH-223191 for 30 min and then treated with endosulfan or 3-MC for 24 h. The membrane was probed with a CYP1A1-specific antibody, and bands were visualized with ECL reagents. (D) Effects of endosulfan on CYP1A1 mRNA levels in Tao BpRcl cells. Hepa-1c1c7 and Tao BpRcl cells were treated with endosulfan or 3-MC for 6 h. Values represent the means from three independent experiments, each performed in triplicate. * p< 0.01, significantly different from the control, as determined by ANOVA by the Newman-Keuls test.

Fig. 4.. Effects of endosulfan on the AhR upstream signaling pathway. (A) Effects of endosulfan on CaMK protein activation in Hepa-1c1c7 cells. Cells were treated with endosulfan (Endo) for 5min. Phosphorylated CaMKIa at threonine 177 (P-CaMKIα) was then analyzed by Western blotting, and β-actin expression was measured as a loading control. (B-C) Effects of endosulfan on PKC phosphorylation in Hepa-1c1c7 cells. Cells were treated with endosulfan for 10 min (B). Cells were pretreated with W7 and subsequently treated with endosulfan for 10 min (C). P-PKC was then analyzed by Western blotting, and β-actin expression was measured as a loading control. (D) Cells were pretreated with W7 or Gö 6983 (Gö) and treated with endosulfan for 24 h. CYP1A1 protein levels were then analyzed by Western blotting, and β-actin expression was measured as a loading control. Each blot in this figure is representative of three independent experiments with similar results.