Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro.
Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro.
复制标题
DOI：
10.1016/j.taap.2013.04.008
复制
发表时间：
2013-08
影响因子：
3.8
通讯作者：
S. L. Ondovcik;T. J. Preston;G. McCallum;P. G. Wells
中科院分区：
文献类型：
--
作者：
S. L. Ondovcik;T. J. Preston;G. McCallum;P. G. Wells
急性暴露于高水平甲基汞（MeHg），或通过每日食用鱼类的慢性低水平饮食暴露于甲基汞，可导致成年和发育中的孕妇产生不利的神经系统影响。为了确定可变DNA修复能力的影响，以及活性氧（ROS）和氧化损伤DNA在毒性机制中的作用，我们使用转基因人胚胎肾（HEK） 293细胞，稳定表达人氧鸟嘌呤糖基酶1 （hOgg1）或其细菌同源物甲脒嘧啶糖基酶（Fpg），主要修复氧化损伤的8-氧-2 ' -脱氧鸟嘌呤（8-oxodG），以评估它们对MeHg的体外效应。Western blotting证实了hOgg1或Fpg在各自细胞系的核室和线粒体室中的表达。在0-10 μM MeHg急性（1-2小时）培养后，克隆性存活和细胞生长的浓度依赖性降低伴随着乳酸脱氢酶（LDH）释放、ROS形成、8-oxodG水平和无尿嘧啶/无嘧啶（AP）位点的浓度依赖性增加，与细胞毒性的发生一致。矛盾的是，在所有细胞和生化参数中，表达hOgg1-和pg的HEK 293细胞比空载体对照稳定转染的野生型细胞对MeHg更敏感，表现出克隆存活和细胞生长减少，LDH释放和DNA损伤增加。因此，由于缺乏修复有毒中间基位的下游过程的代偿性增强，碱基切除修复（BER）途径中特定组分的上调可能被证明是有害的。因此，DNA修复活性的个体间差异可能是环境引发的氧化性DNA损伤及其病理后果的重要危险因素。
Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess thein vitroeffects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences.