王春蕾, 杨叶, 朱光平, 刘克澄, 姚春冀, 吴南翔. 顺式联苯菊酯对映体对H295R细胞肾上腺皮质激素分泌的选择性干扰效应[J]. 环境与职业医学, 2019, 36(10): 909-915. DOI: 10.13213/j.cnki.jeom.2019.19502
引用本文: 王春蕾, 杨叶, 朱光平, 刘克澄, 姚春冀, 吴南翔. 顺式联苯菊酯对映体对H295R细胞肾上腺皮质激素分泌的选择性干扰效应[J]. 环境与职业医学, 2019, 36(10): 909-915. DOI: 10.13213/j.cnki.jeom.2019.19502
WANG Chun-lei, YANG Ye, ZHU Guang-ping, LIU Ke-cheng, YAO Chun-ji, WU Nan-xiang. Selective disruption of cis-bifenthrin enantiomers on secretion of adrenocortical hormones in H295R cells[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 909-915. DOI: 10.13213/j.cnki.jeom.2019.19502
Citation: WANG Chun-lei, YANG Ye, ZHU Guang-ping, LIU Ke-cheng, YAO Chun-ji, WU Nan-xiang. Selective disruption of cis-bifenthrin enantiomers on secretion of adrenocortical hormones in H295R cells[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 909-915. DOI: 10.13213/j.cnki.jeom.2019.19502

顺式联苯菊酯对映体对H295R细胞肾上腺皮质激素分泌的选择性干扰效应

Selective disruption of cis-bifenthrin enantiomers on secretion of adrenocortical hormones in H295R cells

  • 摘要: 背景 手性农药是指分子结构中含有手性中心的农药化合物,含有一对或多对呈镜像关系的对映异构体(简称对映体)。顺式联苯菊酯(cis-bifenthrin,cis-BF)是当前应用较为广泛的一种手性拟除虫菊酯农药,含有1S-cis-BF和1R-cis-BF两个对映体。cis-BF是一种潜在的内分泌干扰物,有研究发现,cis-BF对下丘脑-垂体-肾上腺(HPA)轴分泌的糖皮质激素和盐皮质激素受体具有明显的拮抗效应,但关于cis-BF对映体对HPA轴激素合成分泌的选择性干扰效应及其机制仍不甚明了。

    目的 探讨手性农药cis-BF对映体对人肾上腺皮质癌(H295R)细胞肾上腺皮质激素(皮质醇和醛固酮)分泌水平的影响及其分子机制。

    方法 利用高效液相色谱仪对cis-BF进行手性拆分,用圆二色谱仪对单个对映体(1S-cisBF/1R-cis-BF)构型进行鉴定,用气相色谱对单个对映体浓度进行分析。体外培养H295R细胞,以不同浓度(0、1×10-9、1×10-8、1×10-7、1×10-6、1×10-5 mol/L)1S-cis-BF/1R-cis-BF染毒细胞,用单溶液噻唑兰(MTS)比色法检测细胞增殖活性,确定染毒浓度。以无细胞毒性浓度(0、1×10-9、1×10-8、1×10-7 mol/L)1S-cis-BF/1R-cis-BF处理细胞24 h,采用实时荧光定量-聚合酶链式反应(real-time PCR)检测肾上腺皮质激素合成相关酶基因的表达水平,用酶联免疫吸附实验(ELISA)测定细胞内环磷酸腺苷(cAMP)含量和细胞上清液中皮质醇、醛固酮水平。

    结果 利用高效液相色谱仪完成cis-BF对映体的拆分,两种对映体1S-cis-BF和1R-cis-BF纯度分别高达99.5%和99.2%。MTS实验结果显示,与对照组相比,1×10-6和1×10-5 mol/L浓度组细胞增殖活性升高(P < 0.05),而1×10-9、1×10-8和1×10-7 mol/L 1S-cis-BF/1R-cis-BF对H295R细胞增殖活性无明显影响。PCR结果显示,相对于对照组,1×10-7 mol/L 1S-cis-BF可下调类固醇合成急性调节蛋白(StAR)、细胞色素P450胆固醇侧链裂解酶(P450scc)、3β-羟基类固醇脱氢酶(3βHSD2)和17α-羟化酶(CYP17)的mRNA水平(P < 0.05);1×10-8、1×10-7 mol/L 1R-cis-BF可下调3βHSD2 mRNA水平(P < 0.05),且在1×10-7 mol/L浓度组StAR3βHSD2CYP17 mRNA水平具有对映体差异,1S-cis-BF的抑制作用分别是1R-cis-BF的2.23、2.04和5.00倍(P < 0.05)。cAMP测定结果显示,1×10-7 mol/L 1S-cis-BF染毒致细胞内cAMP含量相对于对照组下降了8.10%(P < 0.05),而1R-cis-BF与对照组差异无统计学意义;且1×10-7 mol/L浓度组cAMP含量有明显的对映体差异,1S-cis-BF的抑制作用高于1R-cis-BF(P < 0.05)。激素测定结果显示,与对照组相比,所有浓度组(1×10-9、1×10-8、1×10-7 mol/L)皮质醇分泌水平均明显降低(P < 0.05),1×10-9、1×10-7 mol/L 1S-cis-BF及1×10-7 mol/L 1R-cis-BF组醛固酮分泌水平明显降低(P < 0.05);其中在1×10-8 mol/L浓度组,S型对映体对皮质醇分泌水平的抑制作用是R型对映体的1.68倍,在1×10-7 mol/L浓度组,S型对映体对醛固酮分泌水平的抑制作用是R型对映体的2.16倍,差异均有统计学意义(P < 0.05)。

    结论 cis-BF对映体通过降低cAMP水平,下调cAMP依赖性类固醇激素合成酶mRNA的表达而抑制H295R细胞内皮质醇和醛固酮的分泌水平,且1S-cis-BF的干扰效应明显高于1R-cis-BF。

     

    Abstract: 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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