School of Public Health, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China.
Shijiazhuang Center for Disease Control and Prevention, Shijiazhuang, Hebei 050011, P.R. China.
Int J Mol Med. 2021 Jun;47(6). doi: 10.3892/ijmm.2021.4936. Epub 2021 Apr 28.
The toxicity of chloroacetamide herbicide in embryo development remains unclear. Acetochlor (AC) is a chloroacetamide that metabolizes into 2‑ethyl‑6‑methyl-2-chloroacetanilide (CMEPA) and 6‑ethyl‑o‑toluidine (MEA). The present study determined the potential effect of AC and its metabolites on embryo development. Both HepG2 cells and zebrafish embryos were exposed to AC, CMEPA and MEA in the presence or absence of co‑treatment with anti‑reactive oxygen species (ROS) reagent N‑acetylcysteine. The generation of ROS, levels of superoxide dismutase (SOD) and glutathione (GSH) in HepG2 cells and lactate dehydrogenase (LDH) leakage from HepG2 cells were investigated. The effects of AC, CMEPA and MEA on DNA breakage, MAPK/ERK pathway activity, viability and apoptosis of HepG2 cells were examined by comet assay, western blotting, MTT assay and flow cytometry, respectively. Levels of LDH, SOD and GSH in zebrafish embryos exposed to AC, CMEPA and MEA were measured. The hatching and survival rates of zebrafish embryos exposed to AC, CMEPA and MEA, were determined, and apoptosis of hatched fish was investigated using acridine orange staining. The present data showed AC, CMEPA and MEA induced generation of ROS and decreased levels of SOD and GSH in HepG2 cells, which in turn promoted DNA breakage and LDH leakage from cells, ultimately inhibiting cell viability and inducing apoptosis, as well as phosphorylation of JNK and P38. However, co‑treatment with N‑acetylcysteine alleviated the pro‑apoptosis effect of AC and its metabolites. Moreover, exposure to AC, CMEPA and MEA lead to toxicity of zebrafish embryos with decreased SOD and GSH and increased LDH levels and cell apoptosis, ultimately decreasing the hatching and survival rates of zebrafish, all of which was attenuated by treatment with N‑acetylcysteine. Therefore, AC and its metabolites (CMEPA and MEA) showed cytotoxicity and embryo development toxicity.
氯乙酰胺类除草剂的胚胎发育毒性尚不清楚。乙草胺 (AC) 是一种氯乙酰胺，可代谢为 2-乙基-6-甲基-2-氯乙酰胺 (CMEPA) 和 6-乙基-o-甲苯胺 (MEA)。本研究旨在确定 AC 及其代谢物对胚胎发育的潜在影响。在存在或不存在抗活性氧 (ROS) 试剂 N-乙酰半胱氨酸的情况下，将 HepG2 细胞和斑马鱼胚胎暴露于 AC、CMEPA 和 MEA 中。检测 HepG2 细胞中 ROS 的产生、超氧化物歧化酶 (SOD) 和谷胱甘肽 (GSH) 的水平以及 HepG2 细胞中乳酸脱氢酶 (LDH) 的渗漏。通过彗星试验、western blot、MTT 试验和流式细胞术分别检测 AC、CMEPA 和 MEA 对 HepG2 细胞 DNA 断裂、MAPK/ERK 通路活性、活力和凋亡的影响。测量暴露于 AC、CMEPA 和 MEA 的斑马鱼胚胎中 LDH、SOD 和 GSH 的水平。测定暴露于 AC、CMEPA 和 MEA 的斑马鱼胚胎的孵化率和存活率，并通过吖啶橙染色研究孵化鱼的凋亡。本研究数据表明，AC、CMEPA 和 MEA 诱导 HepG2 细胞中 ROS 的产生，降低 SOD 和 GSH 的水平，进而促进细胞 DNA 断裂和 LDH 渗漏，最终抑制细胞活力并诱导细胞凋亡，同时还激活 JNK 和 P38 的磷酸化。然而，用 N-乙酰半胱氨酸处理可减轻 AC 及其代谢物的促凋亡作用。此外，暴露于 AC、CMEPA 和 MEA 会导致斑马鱼胚胎毒性，表现为 SOD 和 GSH 水平降低，LDH 水平和细胞凋亡增加，最终导致斑马鱼孵化率和存活率降低，所有这些都可被 N-乙酰半胱氨酸处理所减轻。因此，AC 及其代谢物 (CMEPA 和 MEA) 表现出细胞毒性和胚胎发育毒性。
