低剂量汞、铅和镉联合暴露致大鼠海马神经元细胞损伤的实验研究

Cellular damage of low-dose combined exposure to mercury, lead and cadmium on hippocampal neurons in rats

目的研究低剂量汞、铅和镉联合暴露对大鼠海马神经元的损伤。方法采用2×2×2析因设计将人神经母细胞瘤细胞按随机数字表法随机分为8组，染毒24h、48h和72h后，检测各组细胞活力值。将20只8周龄SD孕鼠按随机数字表法随机分配至4组（每组5只），分别为对照组（蒸馏水）、1倍3种重金属混合物暴露组（1×MM组，氯化汞、三水合醋酸铅、氯化镉含量分别为0．15、25．00、7．50mg／L），5倍3种重金属混合物暴露组（5×MM组，氯化汞、三水合醋酸铅、氯化镉含量分别为0．75、125．（30、37．50mg／L）和10倍3种重金属混合物暴露组（10×MM组，氯化汞、三水合醋酸铅、氯化镉含量分别为1．50、250．00、75．00mgL），饮水染毒至孕鼠分娩。分娩后，每组选择20只雄性仔鼠先经母乳染毒至断乳，调整染毒剂量后经饮水染毒至成年（出生后83d）处死。采集大鼠血样和海马组织，观察海马超微结构变化，并测定血中汞、铅、镉含量及海马神经元细胞凋亡率、活性氧和细胞内游离钙（[Ca2＋]，）荧光强度值。结果染毒48h和72h后，汞＋铅＋镉联合暴露（未观察到有害作用浓度，分别为1．0、0．5和0．1μmol／L）对细胞活力影响具有协同毒性（P值均〈0．05）。动物实验发现1×MM组较对照组比较变化不明显，5×MM组海马神经元开始出现线粒体减少、嵴和基质逐渐溶解，10×MM组加重出现明显的核内染色质聚集成团、线粒体大量减少且嵴与基质大量溶解。1×MM组、5×MM组、10×MM组大鼠血中汞、铅和镉含量均高于对照组，差异具有统计学意义（P值均〈0．001）。1×MM组海马神经元细胞凋亡率与对照组的差异无统计学意义，5×MM组和10xMM组细胞凋亡率均高于对照组，差异具有统计学意义（P值均〈0．001）。1×MM组海马神经元活性氧荧光强度值与对照组的差异无统计学意义，5×MM组和10×MM组活性氧荧光强度值均高于对照组，差异具有统计学意义（P值均〈0．05）。1×MM组海马神经元[Ca2＋]i荧光强度值与对照组的差异无统计学意义，5×MM组和10×MM组[Ca2＋]i荧光强度值均高于对照组，差异具有统计学意义（P值均〈0．001）。结论低剂量汞、铅和镉联合暴露可引起协同神经毒性损伤，可能与海马神经元凋亡、活性氧及[Ca2＋]i变化有关。

Objective To observe the cellular damage of low-dose combined exposure to Hg, Pb and Cd on hippocampal neurons in rat. Methods SH-SY5Y cells were randomly divided into 8 groups by 2×2×2 factorial design： control group, Pb exposure group, Hg exposure group, Pb＋Hg exposure group, Pb＋ Cd exposure group, Hg＋Cd exposure group and Pb＋Cd＋Hg exposure group. And the cell viabilities were measured. On this basis, an animal model was established. Twenty eight-week-old SD pregnant rats were randomly divided into four groups by random number table, and five in each group： the control group （distilled water）, 1-fold metal mixture exposure group （1 × MM, poisoning solution containing mercury chloride 0.15 mg/L, lead acetate trihydrate 25 mg/L, cadmium chloride 7.5 mg/L）, 5-fold metal mixture exposure group （5×MM, poisoning solution containing mercury chloride 0.75 mg/L, lead acetate trihydrate 125.00 mg/L, cadmium chloride 37.50 mg/L）, 10-fold metal mixture exposure group （10×MM, poisoning solution containing mercury chloride 1.50 mg/L, lead acetate trihydrate 250.00 mg/L, cadmium chloride 75.00 mg/L）. Pregnant rats drank water until delivery. Twenty male pups were selected and exposed to these metals through breast milk until weaned. The heavy metals dose of poisoning water was adjusted, and then the weaned rats were exposed to heavy metals via drinking poisoning water until adulthood （postnatal day 83）. The blood samples and brain hippocampus samples were collected to observe the uhrastructural changes of hippocampus, and to determine the levels of Hg, Pb and Cd in blood. In addition, apoptosis rate and fluorescence intensity of reactive oxygen species and intracellular free calcium concentration （[Ca2＋ ]i） in hippocampal neurons were measured. Results Cellular factorial design analysis showed that Hg＋Pb＋Cd （at no observed adverse effect level, 1.0, 0.5 and 0.1 μmol/L, respectively）had a interaction on cell viability after 48 or 72 hours of combined exposure （P〈0.05）. The results of ultrastructure showed that mitochondria decreased, ridges and matrixes gradually dissolved in rat hippocampal neurons of 5 × MM group; nuclear chromatin aggregated, more ridges and matrixes dissolved and the mitochondria also decreased in rat hippocampal neurons of 10×MM group. The concentration of Hg, Pb and Cd in the blood of 1×MM group, 5× MM group and 10× MM group were higher than those in the control group, and the differences were statistically significant （P〈0.001）. There was no significant difference in apoptosis rate between the 1 ×MM group and the control group. The apoptosis rate of 5×MM group and 10×MM group was higher than that in the control group, and the differences were statistically significant （P〈0.001）. There was no statistically significant difference in the fluorescence intensity of reactive oxygen species in hippocampal neurons of the 1 × MM group and the control group. The fluorescence intensity of reactive oxygen species in the 5 × MM group and the 10 × MM group was higher than that in the control group, the difference was statistically significant （P〈0.05）. There was no significant difference in the fluorescence intensity of [Ca2＋]i between the 1 ×MM group and the control group. The fluorescence intensity values of [Ca2＋]i in the 5×MM group and the 10×MM group were higher than the control group, the differences were statistically significant （P〈0.001）. Conclusion Low-level combined exposure to Hg, Pb, and Cd caused synergistic neurotoxic damage, and the process may be related to the changes of neuronal apoptosis, reactive oxide species, and [Ca2＋]i levels.