Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury.
Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury.
复制标题
兔肾近曲小管细胞的原代培养。
DOI：
10.1016/0041-008x(92)90201-3
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发表时间：
1992
影响因子：
3.8
通讯作者：
Kostyniak,PJ
中科院分区：
文献类型：
--
作者：
Aleo,MD;Taub,ML;Kostyniak,PJ
本研究进一步开发了兔肾近曲小管细胞（RPTC）的原代培养物作为体外模型，通过研究氯化汞（HgCl2）和氯化甲基汞（CH3HgCl）对RPTC的比较细胞毒性来研究化学诱导的毒性。暴露于 HgCl2 和 CH3HgCl 24 小时的 RPTC 汇合单层培养物在培养基浓度分别大于 25 和 2.5 μm 时表现出浓度依赖性细胞活力损失。活体染料排除是比乳酸脱氢酶活性、碱性磷酸酶活性、N-乙酰氨基葡萄糖苷酶活性和培养皿上剩余的蛋白质含量更敏感的细胞毒性指标。根据活体染料排除，暴露 24 小时后，HgCl2 对培养物中的近端小管细胞的毒性低于 CH3HgCl，无论细胞毒性是基于 LC50 值（34.2 μm HgCl2 与 6.1 μm CH3HgCl）还是总细胞汞摄取量（4.6 nmol Hg2+/105cells vs 1.25 nmol） CH3Hg+/105 细胞）。金属吸收程度和速率的差异也很明显。 Hg2+ 的最大细胞摄取发生在暴露后 6-24 小时内，并且不依赖于浓度，而 CH3Hg+ 的最大摄取发生在暴露后 3 小时内，并且依赖于浓度。两种汞在酸溶性和酸不溶性结合位点之间的细胞内分布也不同。在非细胞毒性浓度的 HgCl2 (0.04–5 μm) 下，细胞内 Hg2+ 与酸溶性结合位点的结合随着时间的推移而增加，从暴露 6 小时后的 15–30% 到暴露 72 小时后的 40–60%。然而，在亚细胞毒性 (25 μm) 和细胞毒性 (34.2 μm) 浓度下，Hg2+ 与酸溶性结合位点的结合在暴露后 6、12、24 和 72 小时内保持恒定在约 30-40%。相比之下，暴露于 0.039 至 6.1 μm CH3HgCl 6、12 和 24 小时后，只有 20% 的总细胞 CH3Hg+ 与酸溶性结合位点结合。暴露于 0.04–5 μm HgCl2 和 0.039–6.1 μm CH3HgCl 后，细胞总谷胱甘肽含量不受影响，但暴露于 25 和 34.2 μm HgCl2 后 6 小时耗尽。这些结果表明，在原代培养物中，CH3HgCl 是比 HgCl2 更有效的 RPTC 细胞毒剂。此外，与 Hg2+ 相比，CH3Hg+ 与酸溶性结合位点的结合较低，并且 CH3Hg+ 没有从酸不溶性结合位点重新分布到酸溶性结合位点，这似乎导致其对培养细胞具有更强的毒性。这项研究表明，原代培养中的兔 RPTC 是在细胞水平上研究化学诱导毒性的有用体外模型。
The present study further developed primary cultures of rabbit renal proximal tubule cells (RPTC) as an in vitro model to study chemical-induced toxicity by investigating the comparative cytotoxicity of mercuric chloride (HgCl2) and methyl mercury chloride (CH3HgCl) to RPTC. Confluent monolayer cultures of RPTC exposed to HgCl2and CH3HgCl for 24 hr exhibited a concentration-dependent loss in cell viability at culture medium concentrations greater than 25 and 2.5 μm, respectively. Vital dye exclusion was a more sensitive indicator of cytotoxicity than the amount of lactate dehydrogenase activity, alkaline phosphatase activity, N-acetylglucosaminidase activity, and protein content remaining on the culture dish. On the basis of vital dye exclusion, HgCl2was less toxic to proximal tubule cells in culture than CH3HgCl after 24 hr of exposure, whether cytotoxicity was based on LC50 values (34.2 μm HgCl2vs 6.1 μm CH3HgCl) or total cellular mercury uptake (4.6 nmol Hg2+/105cells vs 1.25 nmol CH3Hg+/105cells). Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg2+occurred within 6–24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CH3Hg+occurred within 3 hr of exposure and was concentration-dependent. The intracellular distribution of both mercurials between acid-soluble and acid-insoluble binding sites also differed. At noncytotoxic concentrations of HgCl2(0.04–5 μm), intracellular Hg2+bound increasingly to acid-soluble binding sites as a function of time, from 15–30% after 6 hr of exposure to 40–60% after 72 hr of exposure. However, at subcytotoxic (25 μm) and cytotoxic (34.2 μm) concentrations, Hg2+binding to acid-soluble binding sites remained constant at approximately 30–40% for 6, 12, 24, and 72 hr after exposure. In contrast, only 20% of total cellular CH3Hg+was bound to acid-soluble binding sites after exposure to 0.039 to 6.1 μm CH3HgCl for 6, 12, and 24 hr. Total cellular glutathione content was unaffected after exposure to 0.04–5 μm HgCl2and 0.039–6.1 μm CH3HgCl, but was depleted 6 hr after exposure to 25 and 34.2 μm HgCl2. These results indicate that CH3HgCl was a more potent cytotoxicant to RPTC in primary culture than HgCl2. Furthermore, compared to Hg2+, the low binding of CH3Hg+to acid-soluble binding sites and the absence of a redistribution of CH3Hg+from acid-insoluble to acid-soluble binding sites appeared to contribute to its more potent toxicity to cultured cells. This study demonstrated that rabbit RPTC in primary culture were a useful in vitro model for studying chemical-induced toxicity on a cellular level.