STUDIES ON AFLATOXIN B1-INDUCED OXIDATIVE DAMAGE AND ITS SIGNIFICANCE

Abstract

Aflatoxin B1 (AFB1) is a potent hepatotoxin and hepatocarcinogen and it plays an important role in the etiology of human cancer. However, the mechanism(s) of AFB1 hepatocarcinogenesis has not been fully elucidated. In recent years, reactive oxygen species (ROS) and oxidative damage have been implicated in the process of carcinogenesis and many other diseases. Some preliminary evidence has suggested that ROS and oxidative damage many be involved in AFB1 cytotoxicity and genotoxicity. The main objective of the present study is thus to investigate AFB1 -induced oxidative damage and its significance in the cytotoxity, genotoxicity, and carcinogenicity of AFB1. The study was conducted by the following three steps: (i) studies on AFB1 -induced oxidative cellular damage (both in vivo and in vitro), and (ii) studies on AFB1 -induced oxidative DNA damage (both in vivo and in vitro) and (iii) studies on AFB1 –induced intracellular ROS formation (in vitro).

The induction of lipid peroxidaion by AFB1 was investigated by the determination of malondialdehyde (MDA) and conjugated dienes in rat liver after a single injecction of AFB1 (100 ?g/100 g body wt). The increase of MDA concentration was found to be AFB1 dose dependent. The pre-treatment of selenium (Se) and vitamin E (VE), both antioxidants and deferoxamine (DFO), a specific iron chelator, all significantly inhibited lipid peroxidation, as well as liver cell damage. AFB1 –induced lipid peroxidation, as well as liver cell damage. AFB1 –induced lipid peroxdation and cell injury were also investigated in cultured rat hepatocytes. A time-course and AFB1 –dose dependent changes of MDA concentration and LDH leakage were observed. On the other hand, it was found that AFB1 –induced lipid peroxidant and cell injury in cultured rat hepatocytes were effectively inhibited by superoxide dismutasee (SOD), catalase (CAT), DFO, or dimethyl sulfoxide (DMS), a hydorxyl radical scavenger).

8-Hydroxydeoxyguanosine (8-OHdG) formation is one of the main representative forms of oxidative DNA damage and there is substantial evidence showing that 8-OHdG is implicated in the process of carcinogenesis. In this part of the study, AFB1 –induced oxidative DNA damage was investigated by the determination of 8-OHdG in rat liver and after AFB1 administration by using HPLC-UV-ECD. A time course and dose-dependent changes of 8-OHdG concentration were found in both AFB1- treated rat liver and cultured rat hepacyptes. Morever, AFB1 –induced 8-OHdG formation was significantly inhibited by pre-treatment with Se and DFO in vivo, and SOD, CAT, DFO and DMSO in vitro. In contrast to other antioxidants, VE failed to inhibit , AFB1 –induced 8-OHdG formation in rat liver.

ROS production in , AFB1 –treated hepatocytes was examined using a flyurescent probe, 2’, 7’-dichlorofluorescin diacetuate (DCPH-DA). AFB1 significantly enhanced ROS production in cultured rat hepatocytes in a dose-dependent manner. CAT, DFO and DMSO were all able to inhibit the increase of ROS production in cultured hepatocytes.

Based on the overall results, it is concluded that AFB1 is able to induce oxidative cellular damage (lipid peroxidation) and oxidative DNA damage (8-OHdG formation) both in in vivo and in vitro, and increase ROS production hepatocytes. It is believed that AFB1 –induced oxidative damage plays and important role in AFB1 cytotoxicity, genotoxicity and carcinogenicity. The present study also suggests the feasibility of using antioxidants in the chemoprevention of AFB1 –related liver cancer in humans.