Genotoxic Effect And Nitrative DNA Damage In HepG2 Cells Exposed To Aristolochic Acid

Introduction: Aristolochic acid (AA), the basic active component of Aristolochia extracts, was the first nitro compound of the plants. Aristolochia plants were extensively distributed in tropical and subtropical zone. There were more than two hundred kinds of Aristolochia plants in the word, about forty varieties were in China. It was extensively used as a traditional herbal medicine to treat infections, dermatosis,tumor, edema, tussis, arthralgia and so on. Since Vanherweghen reported the first case with interstitial renal fibrosis rapidly progressing to end-stage renal disease after following a slimming regimen containing AA in Brussels, Belgium in 1993, AA elicited safety concerns about traditional Chinese herbal products that are known or suspected to contain AA throughout the word.In the 1980s, studies had found that AA is a mutagenic and carcinogenic extract from aristolochic herbs. International Agency for Research on Cancer (IARC) classified herbal remedies containing plant species of the genus Aristolochia as human carcinogens in 2002.It reported that AA was the direct mutagen to TA100, TA1537 and TA102 in Salomonella typhimurium. In sex-link recessive lethal in drosophila melanogaster, AA caused chromosome deletion in sperm cells and larvae of somatic cell gene rearrangements. The results from 6-Thioguanine guanine resistant mutations, sister chromatid exchange (SCE) chromosome aberration test were positive induced by AA in cultured mammalian cells. AA mixture was genotoxic to CHO cells, mouse bone-marrow cells, porcine epithelial cells, human lymphocytes and human-derived hepatoma (HepG2) cells.Many studies presented evidence that DNA damage induced by AA wasattributed to AA-DNA adducts in vivo and in vitro. Recently, nitrative DNA damage, caused by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivatives, such as peroxynitrite (ONOO-) and nitrogen dioxide, has been discussed in relation to the association between chronic inflammation, genotoxicity and carcinogenesis. The aim of present study is to explore whether AA exerts genotoxic effect on human HepG2 cells and whether nitrative DNA damage occurs in AA-treated cells.This study adopt HepG2 cells as a model to assess the genotoxicity and nitrative DNA damage in vitro because they retain many characteristics of hepatocytes such as the activities of phaseⅠandⅡenzymes that play a key role in the activation of genotoxic mutagens or carcinogens. It has been shown to be a suitable system for genotoxicity testing. Thus it may provide help to reveal the mechanism of its genotoxitity and carcinogenicity.Methods: HepG2 cells were used as test system. Cytotoxicity of AA was determined by MTT assay. DNA-strand breaks and frequencies of micronuclei (MN) in HepG2 cells were evaluated by single cell gel electrophoresis assay (SCGE) and micronuclei test (MNT), respectively. Intracellular nitrate were measured by 2, 3-diaminonaphthalene (DAN) assay. The level of 8-OHdG was investigated using immunocytochemical analysis.Results: After treatment with increasing concentrations of AA, the inhibition rates of growth of HepG2 cells increased in a dose-dependent manner. After exposure to AA for 24 and 48 h, the inhibitory concentration 50% (IC50) value was 132.2 and 62.2μM, respectively. The DNA-strand damage in HepG2 cells significantly increased after exposure to 25-200μM ?AA for 1 h, in a dose-dependent manner. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 12.5-50μM ?AA for 24 h ?After exposure to AA (50-200)μM for 1 h, intracellular nitrate significantly increased in a dose-dependent manner. The staining intensity of 8-OHdG increased after treatment with 50-200μM AA for 3 h.Conclusions: AA presentes cytoxicity on HepG2 cells and causes DNA and chromosome damage in comet assay and MNT in HepG2 cells. The increases in detection of NO and immunoperoxidase staining of 8-OHdG also occur in AA-treated cells at higher concentrations. Therefore, ourfindings support the assumption that AA exerts genotoxicity and causes nitrative DNA damage to HepG2 cells probably via NO and its derivatives, such as ONOO-.