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.
Context: Medicinal plants are a highly sought-after alternative to current pharmaceutical drugs because they can be locally cultivated, inexpensive and possess minimal adverse effects. Given that Terminalia phanerophlebia (TP) possesses many useful properties and plays a role in modulating lethal diseases, the cytotoxic effect should be evaluated before its application for therapeutic use. Aims: To investigate the oxidative effect and molecular mechanisms of TP on human embryonic kidney (HEK293) cells. Methods: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays were used to determine the cell viability whilst the thiobarbituric acid reactive species (TBARS) assay was used to detect lipid peroxidation. Endogenous antioxidants, catalase, superoxide dismutase, glutathione peroxidase, heat shock protein 70 and nuclear factor erythroid 2-related factor 2 (Nrf2), were used as oxidative stress markers and were detected via western blotting. Results: A decrease in cell viability with an IC50 of 1.36 mg/mL and ATP were noted. The concentration of malondialdehyde (MDA) increased significantly (p<0.005). Superoxide dismutase, Nrf2 and heat shock protein concentrations were increased. However, glutathione, glutathione peroxidase and catalase were depleted. Conclusions: The results obtained suggest that Terminalia phanerophlebia extract is toxicogenic and induced oxidative stress in HEK293 cells.
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