微囊藻毒素-LR对活性氧产生及细胞色素P450各亚型表达的影响
Effects of Microcystin-LR on Intracellular Reactive Oxygen Species Generation and mRNA Expression of Cytochrome P450 Isoforms
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摘要:
目的 探讨低剂量微囊藻毒素-LR(MCLR)诱导细胞内活性氧(ROS)产生的潜在来源。 方法 采用转染有机阴离子转运多肽OATP1B3的人胚肾细胞株HEK293-OATP1B3,设3个浓度的MCLR处理组(10、20、50 nmol/L)和未处理对照组(0.1%二甲基亚砜)。染毒4、24 h后,测定乳酸脱氢酶(LDH)漏出率,用荧光探针法检测细胞内ROS水平,采用实时荧光定量聚合酯链反应检测细胞色素P4501A1(CYP1A1)、1A2、2E1 mRNA的表达水平。 结果 低浓度(10~50 nmol/L)的MCLR作用HEK293-OATP1B3细胞4 h未产生明显细胞毒性;24 h后, LDH漏出率随着处理浓度的增加而增加。50 nmol/L MCLR处理细胞4 h后引起ROS水平明显升高(P<0.01)。同时, MCLR还上调了CYP2E1的mRNA表达水平(P<0.01);但未影响CYP1A1 mRNA的表达;对照组和MCLR处理组的CYP1A2 mRNA表达水平均非常低。 结论 低剂量MCLR可诱导细胞内ROS产生和上调CYP2E1 mRNA表达,提示CYP2E1可能是MCLR诱导ROS产生的一个潜在来源。 Abstract:Objective To study the sources responsible for the generation of reactive oxygen species (ROS) induced by lo w doses of microcystin-LR(MCLR). Methods Human embryonic kidney cells HEK293 stably expressing the organic anion transporting polypeptides 1B3 (HEK293-OATP1B3 cells) were treated with the solvent(0.1% dimethyl sulfoxide) and MCLR (10-50 nmol/L)for 4 or 24 h. The lactate dehydrogenase (LDH)release was measured and the levels of ROS were determined using fluorescent probes. The mRNA expressions of cytochrome P450 isoforms CYP1A1, 1A2 and 2E1 were determined by realtime polymerase chain reaction(PCR). Results MCLR did not increase LDH release in HEK293-OATP1B3 cells at low concentrations (10-50nmol/L)when incubated for 4h. However, significant LDH releases were observed when the cells were incubated with MCLR at 20 nmol/L and higher concentrations for 24 h (P < 0.05). Exposure to MCLR (50 nmol/L)for 4 h significantly increased in tracellular ROS level (P < 0.01). MCLR did not affect the expression of CYP1A1. In contrast, the expression level of CYP2E1 mRNA was significantly upregulated in MCLR-treated cells (P < 0.01). The expression level of CYP1A2 was extremely low in both control and MCLR-treated cells. Conclusion Low doses of MCLR induce the generation of ROS, and upregulate the expression of CYP2E1 mRNA, suggesting that CYP2E1 may be a potential source responsible for ROS generation by MCLR.
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