The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B₁ (AFB₁) on human renal cells. Hek293 cells were exposed to AFB₁ (0–100 μM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC₅₀) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB₁ concentration was increased, with a half maximum inhibitory concentration (IC₅₀) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB₁-treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB₁ activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB₁ treated renal cells. The results suggest that AFB₁ induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

中文翻译：

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黄曲霉毒素B1致肾损伤的毒性及机制途径

该研究调查了黄曲霉毒素 B ₁ (AFB ₁ ) 对人肾细胞的毒性作用、分子机制和蛋白质组学评估。Hek293 细胞暴露于 AFB ₁ (0–100 μM) 24 小时。使用甲基噻唑四唑 (MTT) 测定评估了对细胞活力的影响，该测定也产生了半数最大抑制浓度 (IC ₅₀) 用于后续分析。通过定量丙二醛 (MDA) 和硝酸盐浓度来评估自由基的产生，而使用单细胞凝胶电泳 (SCGE) 测定和 DNA 凝胶电泳测定 DNA 断裂。使用乳酸脱氢酶 (LDH) 测定法确定对细胞膜的损伤。ATP、还原型谷胱甘肽 (GSH)、坏死、膜联蛋白 V 和半胱天冬酶活性的浓度通过发光测定法测量。使用蛋白质印迹和定量 PCR 来评估与细胞凋亡和氧化应激相关的蛋白质和基因的表达。MTT 测定显示，随着 AFB ₁浓度的增加，Hek293 细胞的细胞活力降低，最大抑制浓度的一半（IC ₅₀) 的 32.60 μM。降低的生存力对应于降低的 ATP 浓度。Hsp70 的上调表明在 AFB ₁处理的细胞中诱导了氧化应激。虽然这意味着自由基的产生增加，但伴随的抗氧化系统的上调表明防御机制的激活以防止细胞损伤。因此，如 LDH 释放和坏死减少所表明的那样，防止了与自由基形成增加相关的膜损伤。此外，细胞毒作用也很明显，因为 AFB ₁激活细胞凋亡的内在途径，相应地增加 DNA 片段化、p53 和 Bax 上调以及增加半胱天冬酶活性，但磷脂酰丝氨酸 (PS) 外化是细胞凋亡的主要标志，在 AFB ₁处理的肾细胞中没有发生。结果表明，AFB ₁通过肾细胞凋亡的内在途径诱导氧化应激导致细胞死亡。