Selective Disruption Of Cis-bifenthrin Enantiomers On Secretion Of Adrenocortical Hormones In H295R Cells

ObjectiveTo examine the disruption of pyrethroid pesticide cis-bifenthrin(cis-BF)on adrenocortical hormone secretion in H295R cells and the underlying mechanism at enantiomeric level.MethodsThe two enantiomers of cis-BF(1S-cis-BF and 1R-cis-BF)were separated by high performance liquid chromatography equipped with chiral chromatographic column.The quantitative analysis and determination of enantiomer configuration were carried out by gas chromatography and circular dichroism,respectively.H295R cells were used as the research model,and the cells were exposed to different concentrations(0.001,0.1,0.1,1,10μmol/L)of 1S-cis-BF/1R-cis-BF or the mixture of 1S-cis-BF/1R-cis-BF and 8-Br-cAMP(500 μmol/L)for 24 h.DMSO(0.1%,v/v)and 8-Br-cAMP(500 μmol/L)were used as the solvent control and positive control,respectively.The cell proliferation activity was detected via MTS assay to determine the exposure concentrations.The levels of cortisol and aldosterone in supernatant as well as the content of intracellular cAMP were measured by ELISA,and the expression levels of enzymes involved in steroid hormone synthesis(StAR,P450scc,3βHSD2 and CYP17)were analyzed by RT-qPCR method.ResultsThe two enantiomers of cis-BF were successfully separated by high performance liquid chromatography equipped with chromatographic column,and the purity of 1S-cis-BF and 1R-cis-BF was more than 99%.MTS results showed that high concentrations(1,10μmol/L)of 1 S-cis-BF/1R-cis-BF stimulated cell proliferation,and non-cytotoxic concentrations(0.001,0.01,0.1 μmol/L)of cis-BF enantiomers were selected for further exposure experiments.The results of hormone determination indicated that,compared with the solvent control group,exposure to 0.001,0.01,0.1μmol/L 1S-cis-BF/1R-cis-BF significantly decreased the level of basal cortisol secretion(P<0.05),and the inhibitory effects of 1S-cis-BF was 1.49,1.68,1.04 times higher than those of 1R-cis-BF,respectively Exposure to 0.001,0.1 μmol/L 1S-cis-BF and 0.1 μmol/L 1R-cis-BF significantly decreased the basal aldosterone secretion(P<0.05),and 1S-cis-BF showed 2.16-fold higher inhibitory effects on basal aldosterone secretion than 1R-cis-BF in the 0.1 μmol/L group,with significant enantimeric difference(P<0 05).Compared with the positive control group,the levels of cortisol and aldosterone induced by 8-Br-cAMP decreased with increasing concentrations of 1S-cis-BF,while 1R-cis-BF had no such inhibited effect,and no significant difference was found between the two enantiomer treatment groups.The results of RT-qPCR showed that,in comparison with the solvent control group,exposure to 0.1 μmol/L 1S-cis-BF significantly down-regulated the basal mRNA levels of StAR,P450scc,3 βHSD2 and CYP17(P<0 05),and exposure to 0.01,0.1 μmol/L 1R-cis-BF significantly down-regulated the basal mRNA levels of 3βHSD2(P<0.05).Moreover,in the 0.1 μmol/L group,the inhibitory effects of 1S-cis-BF on the basal mRNA levels of StAR,3βHSD2 and CYP17 was 2.23,2.04 and 5.00 times higher than those of 1R-cis-BF,respectively,with significant enantimeric difference.The inhibitory effects of 1S-cis-BF on 8-Br-cAMP-induced StAR,P450scc,3βHSD2 and CYP17 mRNA levels were increased with increasing exposure concentrations compared with the positive control group,and there was a statistic difference of 3βHSD2 and CYP17 transcription in 0.1 μmol/L group(P<0.05).However,1R-cis-BF slightly inhibited the induced expression of all genes by 8-Br-cAMP,but the difference was not statistically significant,and there was no significant enantiomeric difference in the transcription of all four genes.The data of cAMP revealed that,compared with the control group,the intracellular cAMP content in 0.1 μmol/L 1S-cis-BF group decreased by 8.10%(P<0.05),and exposure to 0.1 μmol/L 1R-cis-BF slightly decreased cAMP content,but the difference was not significant.Furthermore,1S-cis-BF exhibited 1.95-fold higher inhibitory effects on cAMP content than 1R-cis-BF in 0.1μmol/L group,with significant enantiomeric difference(P<0.05)ConclusionBoth of the two enantiomers of cis-BF could inhibit the secretion of adrenocortical hormone in H295R cells to varying degrees,and the inhibitory effects of 1S-cis-BF was stronger than those of 1R-cis-BF.Cis-BF enantiomers disrupted cAMP signal pathway and down-regulated the gene expression of downstream steroidogenesis-related enzymes,which leads to abnormal adrenocortical hormones secretion.