Genotoxicity Of Acrylamide In Human Hepatoma G2 (HepG2) Cells

Introduction:In April 2002, scientists in Sweden discovered that large amounts of acrylamide, a potent carcinogen is formed in foods rich in starch that have been heated at high temperatures. It may be formed through the Maillard reaction from amino acids and reducing sugars during drying and baking. This finding raises human health concerns about the risks of AA for the general population.Acrylamide is neurotoxic in humans and laboratory animals, and was classified as"probably carcinogenic to humans"(Group 2A carcinogen) by a working group of the International Agency for Research on Cancer. This might represent a potential threat to public health. But until now, epidemiological studies found no association between consumption of foods containing acrylamide and cancer risk. The relevance of current intake levels in terms of cancer risk still remains an issue under debate. With respect to AA genotoxicity data, AA could induce DNA damage in the PC Cl3 and FRTL5 rat thyroid cell lines, as well as in human lymphoblastoid TK6 cells in the comet assay. In the spermatid micronucleus test, positive result was observed in the rat meiotic cells. In addition to the tests in vitro, induction of structural chromosome aberrations, micronuclei or polyploidy was observed in various studies in mice treated in vivo (dose around 100 mg/kg bw).In the present study, we used human hepatoma G2 (HepG2) cells to investigate the genotoxicity of AA and possible mechanisms. HepG2 cells retain many characteristics of hepatocytes such as the activities of phase I and phase II enzymes and reflect the metabolism of xenobiotics in the human body better than other metabolically incompetent cells. HepG2 cellshave been shown to be a suitable system for genotoxicity testing. The liver is the initial site of acrylamide metabolism following oral or i.p. dosing, and acrylamide is found at high concentrations in the liver.Method: Genotoxicity of AA was assessed by micronucleus test (MNT) and comet assay. To further investigate the mechanism of genotoxicity of AA in HepG2 cells, we measured the intracellular generation of reactive oxygen species (ROS). We analyzed the oxidative DNA damage in AA-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG). To clarify if the AA-induced oxidative stress is via the depletion of glutathione (GSH), we treatedHepG2 cells with DL-buthionine sulphoximine (BSO) to deplete GSH and MTT assays was performed to determine the cytotoxicity of AA in GSH-depleted and normal control HepG2 cells.Result: The damage to DNA-strand of HepG2 cells significantly increased after exposure to 2.5~20mM AA for 1 h, in a dose-dependent manner. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 0.625~2.5mM AA for 24 h. ?After exposure to AA for 1 h, intracellular ROS significantly increased. The 8-OHdG was positive in the HepG2 cell treated with AA. With the help of the MTT method described above, we pretreated HepG2 cells with 40μM BSO to deplete GSH for 16 hours ,the cytotoxicity of AA in GSH-depleted significantly increased, in a dose-dependent manner.Conclusion: AA could be genotoxic to HepG2 cell. AA exerts genotoxic effects in HepG2 cells, probably through depletion of GSH,oxidative DNA damage induced by intracellular ROS and increase of 8-OHdG formation.