Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury

Abstract

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.

Keywords: aflatoxin B1 (AFB1); cell death; cytotoxicity; free radicals; mechanistic pathways; oxidative stress; proteomic assessment; renal cells.