Citreoviridin (CIT)-induced Genotoxicity And Possible Involvement Of Oxidative Stress In Human HepG2Cells

Introduction: Citreoviridin (CIT) is a toxic secondary metabolite mainlyproduced by Penicillium citreoviride. It widespreads in moldy cereal food such as rice,maize, grain and wheat. It has been found that citreoviride grows up so fast andproduces the highest toxin in rice production than other productions. The temperatureand PH around the environment are closed related with the growth and ability ofproducing toxin of citreoviride.The history of citreoviridin research began more than100years ago in Japan. In1891, Sakaki demonstrated that moldy, unpolished rice was fatal to experimentalanimals, with symptoms indicating paralysis of the central nervous system. In1920,Prof. I. Miyake and Dr. Takada first reported that Penicillium commune, which wasknown as a causal agent of "mossy diseased rice" was found to be toxic to experimentalanimals by feeding the moldy rice to rabbits and rats. Miyake and his co-workersdiscovered the first sample of yellow rice grains from Taiwan and domestic rice, fromwhich was isolated a species of Penicillium and later identified it with P. citreonigrum.The fungus produced a highly toxic metabolite, citreoviridin.CIT led to CNS disturbance associated with convulsions, tremors, respiratoryarrest and leg weakness through disturbed glycogen metabolism in brain. Researcherfound that the toxic effects of CIT resulted from respiratory and cardiovascular failures(apnea, delta EEG waves, sinus arrhythmia, hypotension) leading to central nervoussystem depression due to systemic hypoxia. CIT causes primary myocardial necrosis,the particles degeneration of myocardial cells and the aggregation and dissolve ofmyocardial fiber in rats. CIT can bond the mitochondria and then inhibit adenosinetriphosphate synthase. Studies suggest that CIT has close relation with yellow ricepoisoning in Japan, Keshan disease in China and beriberi in Brazil.CIT can induce DNA damage of human myocardial cells and SGC-790cells. However, the exact mechanism is still not clear. In this assay, we selected ametabolically competent human hepatoma line (HepG2) as test system to further detectthe CIT-induce DNA damage in vitro and hepatoma the role of oxidative stress in thisprocedure. HepG2retains, which roots in human embryonic cell tumor and is thewell-differentiated cells, not only lots of the functions of normal liver cells but also theactivities of several Phases I and II xenobiotic metabolizing enzymes. Therefore, it wasconsidered to be more suitable for reflecting the metabolism of xenobiotics in thehuman body better than other mammalian cell lines. Besides, another reason forselecting HepG2as test system would be that CIT is a lipophilic toxin and reaches thehigh concentration in the liver after absorption.The aim of this study was to assess whether CIT causes genotoxic effects inHepG2cells and to elucidate the underlying mechanism of CIT-induced DNA damagein HepG2cells. We hope the results could give some useful information for the safetyassessment to humans on CIT.Methods: HepG2cells were selected as test system. We operated the single cellgel electrophoresis assay (SCGE) to study the genotoxic effects of CIT. To elucidate theunderlying mechanism of CIT-induced genotoxicity in HepG2cells, the2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were used tomonitor the levels of ROS and glutathione (GSH). N-acetylcysteine (NAC), as anantioxidant, was used to modulate the level of ROS in HepG2cells and prevent theDNA damage induced by CIT in HepG2cells.8-hydroxyderoxyguanosine (8-OHdG),which is a reliable marker for oxidative DNA damage, was measured byimmunocytochemistry staining analysis. To further investigate the mechanism ofgenotoxicity of CIT in HepG2cells, we used acridine orange (AO) and rhodamine123to measure the changes of lysosomal membrane stability and mitochondrial membranePotential. The data were statistically analyzed by SPSS v11.5software.Results: In the SCGE, a dose-dependent increase of DNA migration was foundafter treatment with CIT. CIT at the concentrations of2.5-5μM causes a significantincrease of DNA damage in HepG2cells for treating60min. The formation ofintracellular ROS was significantly increased and intracellular GSH was decreased inthe cells treated with CIT2.5-5μM. DNA damage and increased level of ROS inducedby CIT was significantly reduced in cells pre-treated with NAC (10mM) for60min inthe SCGE assay and ROS test. CIT at dose1.25-5μM caused a significant oxidativedamage through8-OHdG formation in HepG2cells for3h. CIT significantly caused the change of lysosomal membrane stability in HepG2cells at concentrations from1.25μMto5μM. In addition, the change of mitochondrial membrane Potential induced by CITonly at dose5μM in the assay.Conclusion: The results suggest that CIT caused DNA strand breaks whichindicate that CIT induced genotoxic effects in HepG2cells. Significantly increasedlevels of ROS and decreased levels of GSH were observed in HepG2cells at higherconcentrations. Moreover, DNA damage and increased level of ROS induced by CITwas significantly reduced in cells pre-treated with NAC. NAC, as an antioxidant, playsa vital role in the defense against CIT-induced DNA damage. These results indicate thatCIT induced genotoxic effects in HepG2cells probably depend on oxidative stresspathway. It has been further tested by CIT-induced increase of8-OHdG formationconsidered as the maker of oxidative damage of DNA. In addition, CIT significantlycaused the change of lysosomal membrane stability and the change of mitochondrialmembrane potential in HepG2cells. In all, the data suggest that CIT exerts genotoxiceffects in HepG2cells, probably through the formation of ROS, depletion of GSH,increase of8-OHdG formation, the change of lysosomal membrane stability anddecrease of mitochondrial membrane potential.