Aflatoxin B1 modulates oxidative stress and apoptosis in human embryonic kidney cells.

Abstract

Introduction: Aflatoxin B1 (AFB1) is produced by filamentous fungal strains of Aspergillus flavus and Aspergillus parasiticus that infect field crops, therefore AFB1 is a frequent contaminant of dietary staples such as rice, maize and peanuts. Humans and animals are exposed to AFB1 through consumption of contaminated foods, predisposing them to various diseases. AFB1 is a potent hepatotoxin that has been classified by the International Agency of Research on cancer (IARC) as a group1 carcinogen. The carcinogenic effects of AFB1 have been attributed to the metabolism of this toxin to an epoxide that promotes the production of free radicals, mitochondrial toxicity and induction of cell death. With the increasing prevalence of kidney associated diseases in humans, and the AFB1-associated kidney toxicity observed in animals, this study investigated the cytotoxic effects/mechanism of AFB1 in human embryonic kidney (Hek293) cells. Methods: Hek293 cells were exposed to AFB1 (0-100μM) for 24hrs. 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. Results and discussion: 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 externalisation of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated Hek293 cells.

Conclusion: The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in Hek293 cells. Keywords : Aflatoxin B1 (AFB1), oxidative stress, apoptosis, Hek293 cells