Background Chiral pesticides are pesticide compounds with chiral centers in their molecular structures, containing one or more pairs of enantiomers which are mirror images of each other. Cis-bifenthrin (cis-BF) is one of the most widely used chiral pyrethroid insecticides, containing two enantiomers:1S-cis-BF and 1R-cis-BF. Cis-BF is a potential endocrine disruptor. Previous studies have found that cis-BF exhibits an antagonistic effect on the receptors of glucocorticoids and corticosteroids secreted by hypothalamic-pituitariay-adrenal (HPA) axis, but limited evidence has been available concerning the enantioselective disruption effects of cis-BF on the hormone synthesis and secretion of HPA axis as well as the potential mechanisms.
Objective This experiment investigates the effects of enantiomers of chiral pesticide cis-BF on the secretion levels of adrenocortical hormones (cortisol and aldosterone) as well as the potential molecular mechanisms in human adrenocortical carcinoma (H295R) cells.
Methods Chiral resolution of cis-BF was carried out by high-performance liquid chromatography. The configuration of the individual enantiomers (1S-cis-BF/1R-cis-BF) was identified by circular dichroism, and their concentrations were analyzed by gas chromatography. H295R cells were cultured in vitro and exposed to different concentrations (0, 1×10-9, 1×10-8, 1×10-7, 1×10-6, and 1×10-5 mol/L) of 1S-cis-BF/1R-cis-BF, and exposure concentrations were determined by testing the cell viability via 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium assay. Then the cells were treated with non-cytotoxic concentrations (0, 1×10-9, 1×10-8, and 1×10-7 mol/L) of 1S-cisBF/1R-cis-BF for 24 h. The expression levels of genes encoding enzymes involved in adrenocortical hormone synthesis were determined by real-time fluorescence quantitative polymerase chain reaction (real-time PCR), and the level of intracellular cyclic adenosine monophosphate (cAMP) as well as the levels of cortisol and aldosterone in supernatant were measured by enzyme-linked immunosorbent assay (ELISA).
Results The enantiomers of cis-BF were successfully separated by high-performance liquid chromatography, and the purities of 1S-cisBF and 1R-cis-BF were 99.5% and 99.2% respectively. The results of MTS assay showed that the cell viability was elevated in the 1×10-6 and 1×10-5 mol/L groups (P < 0.05), but the 1×10-9, 1×10-8, and 1×10-7 mol/L 1S-cis-BF/1R-cis-BF had no significant effect on cell viability compared with the control group. The PCR data showed that, in comparison with the control group, the 1×10-7 mol/L 1S-cis-BF significantly down-regulated the mRNA levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD2), and 17α-hydroxylase (CYP17) (P < 0.05); the 1×10-8 and 1×10-7 mol/L 1R-cis-BF significantly down-regulated the 3βHSD2 mRNA levels (P < 0.05). Moreover, there were enantiomeric differences in the mRNA levels of StAR, 3βHSD2, and CYP17 in the 1×10-7 mol/L group with 2.23-, 2.04-, and 5.00-fold higher inhibitory effects of 1S-cis-BF than the effects of 1R-cis-BF (P < 0.05). The data of cAMP revealed a decrease of 8.10% in the intracellular cAMP content in the 1×10-7 mol/L 1S-cis-BF group compared with the control group (P < 0.05), while there was no significant difference between the 1R-cis-BF group and the control group. A significant enantiomeric difference was found in the cAMP content in the 1×10-7 mol/L group, with higher inhibitory effect of 1S-cis-BF than that of 1R-cis-BF (P < 0.05). The results of hormone determination indicated that the cortisol levels in all concentration (1×10-9, 1×10-8, and 1×10-7 mol/L) groups as well as the aldosterone levels in the 1S-cis-BF (1×10-9 and 1×10-7 mol/L) and the 1R-cis-BF (1×10-7 mol/L) groups were significantly decreased compared with the control group (P < 0.05). In the 1×10-8 mol/L group, 1S-cis-BF showed 1.68-fold higher inhibitory effect on cortisol level compared with 1R-cis-BF, and in the 1×10-7 mol/L groups, 1S-cis-BF showed 2.16-fold higher inhibitory effects on aldosterone levels than 1R-cis-BF, and the differences were significant (P < 0 05).
Conclusion The enantiomers of cis-BF could inhibit the secretion of cortisol and aldosterone in H295R cells via reducing cAMP level and down-regulating the gene expressions of cAMP-dependent enzymes related to steroidogenesis. Comparatively, the inhibitory effects of 1S-cis-BF is more potent than those of 1R-cis-BF.
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