3-monochloropropane-1, 2-diol Induces Oxidative DNA Damage In HEK 293 Cells

Introduction: Chloropropanols are a group of well-known food conta- minants.3-monochloropropane-1,2-diol (3-MCPD) has great pollution in food, so it is often as a representation and a poisioning reference of chloropro- panols. In the latter half of 1999, The flavoring material we exported to Europe was stopped selling because the content of 3-MCPD exceeded the limit of imported country, which made our country suffering serious econo- mic loss. 3-MCPD has been detected in several foods and food ingredients, including acid-hydrolysed vegetable protein(acid-HVP), soy sauces, malt products, roasted cereal, fermented sausages and health food. It has also been found in food package materials and drinking water .3-MCPD was considered as a carcinogen in rats, it can induce leydig cells, mammary and preputial gland tumor, benign kidney tumor, renal tubule carcinomas and renal tubule adenomas. It also can induce chronic progressive nephropathy. In the tests in vitro, Ames'assay demonstrated that 3-MCPD was mutagenic in Salmonella typhimurium in the absence of exogenous metabolic activation. The addition of S9 mix did not increase the mutagenic response. Positive results were also reported in yeast test systems, in the sister chromatid exchange(SCE)assay in mammalian cells and in the mouse lymphoma assay. The comet assay showed increased DNA-migration in Chinese hamster ovary-K1(CHO- K1) cells only at tested concentrations above 2.5mg/ml (22.6mM). Two in vivo assays, namely a bone marrow micronucleus and a rat liver Unscheduled DNA Synthesis (UDS) assay, provided negative results. Therefore the genotoxic activity of 3-MCPD observed in vitro was not expressed in vivo.In this study, we investigated DNA damage induced by 3-MCPD in HEK293 cells and compared the sensitivity of 3-MCPD-induced DNA damage in HEK293 cells and CHO-K1 cells and further explore the mechanisms of 3-MCPD-induced DNA damage in HEK293 cells.Methods: HEK293 cells and CHO- K1 cells were selected as test systems. We used the single cell gel electrophoresis assay (SCGE) to study 3-MCPD-induced DNA damage. Since the molecular mechanism may involve the generation of various reactive oxygen species (ROS), we measured the level of intracellular ROS by use of the 2, 7-dichloro- flu- orescein diacetate (DCFH-DA) assay. The level of the oxidative DNA damage was evaluated using immunoperoxidase staining for 8-hydroxy- deoxyguanosine (8-OHdG). We evaluated 3-MCPD-induced DNA damage after pre-treatment with antioxidant Hydroxytyrosol (HT) by comet assay.Results: The results showed that 3-MCPD at all tested concentrations (1.25 mM-10 mM) caused a significant increase of DNA strand breaks in HEK293 cells for 1h. The treatment with 3-MCPD in CHO-K1 cells did not cause DNA strand breaks at any tested concentrations(1.25 mM-5 mM).The level of intracelluar ROS was significantly increased when exposed to 10 mM 3-MCPD for 1h. The formation of 8-OHdG was significantly increased at the concentration of 3-MCPD (2.5 mM-5 mM) for 3h. A significantly concen- tration-dependent decline was observed in 3-MCPD (5 mM)-induced DNA migration after pre-treatment with HT(25μM-100μM)in HEK293 cells.Conclusion: 3-MCPD can induce DNA damage in HEK293 cells. HEK293 cells are more sensitive than CHO-K1 cells to the DNA-damaging effects of 3-MCPD. The DNA damage is probably induced by the increased level of intracellular ROS and formation of 8-OHdG, which cause oxidatively generated DNA damage and formation of DNA strand breaks.