Metabolic Activation And Genetic Toxicity Of Some Indirect Genetic Poison In Food

BackgroudV79-hCYP2E1-hSULT1A1, a genetically engineered Chinese hamster V79cell line expressing human CYP2E1and human sulfotransferase1A1(SULT1A1), demonstrates mutagenic response to promutagens requiring metabolic activation by either expressed enzyme.For the purpose of investigating the effect of either or both enzyme, it is highly necessary to establish a test model wherein either of the enzymes is specifically inhibited. We found that, specifically and completely, ABT and PCP are capable of prohibiting CYP2E1and SULT1A1dependent mutagenic response, respectively, which is a valuable test model of reliable value for investigating metabolic activation of genotoxicitys.There is a common point of the chemicals, which including NDMA,2-NP, NDEA, AA, benzene, phenol, that is, they are widely exist in the food and to have some general toxicity.Through the drug metabolizing enzymes, especially the drug metabolizing enzymes of liver, the metabolites will still have the genetic toxicity, and these chemicals are metabolized, often toxic not failed to eliminate, but activated by some liver drug enzyme, where the toxicity will be stronger than before, meanwhile, cost more lasting irreversible cell damage.That is, in vivo or in vitro studies, continuous catalysis can be affected by various enzymes of phase Ⅰ and phase Ⅱ reactions.This type of poison called indirect genotoxicity.These genetic poisons in the human body metabolic activation process, and how to cause these cells damage, that will be our focus.Micronucleus test is widely used as a biological indicator of chromosome breaks and polyploidy generated.Overall, the micronucleus from the chromosomal damage centromere fracture fragment, fracture or spindle fibers and free single or multiple chromosomes.In recent years, is a useful tool to predict the risk of cancer.The formation of micronuclei from free or broken chromosomes chromatin structure, regardless of the in vivo experiments or in vitro experiments have evidence that the formation of the number of chromosome breaks and multi-core formation in a dose-response relationship.The cause of the micronucleus of chromosome fragments of the fracture can not be the mitotic spindle full traction, where that is the formation of micronuclei, where still some are part micronucleus anaphase spindle fibers fracture, and also some are cost by the loss of whole chromosomes.There are are closely related between chromosome damage and the formation of micronucleus, and also the spindle damage and the formation of micronucleus.Through the micronucleus test can determine the extent of cell damage by, but can not determine the cell type of injury.Metabolic activation is to study these indirect genotoxicity of food lead to spindle damage, spindle structure mutations in mitosis the end of the cytoplasm can not be separated, resulting in polyploid formation.The cellular P-tubulin and γ-tubulin were stained with fluorescence immunoassay hybridization observed the expression of tubulin, to determine cell damage types, identify the type of toxic effect of indirect genotoxicity.Objective In order to preliminarily verified drug mechanism of action mechanism of the direct or indirect genetic toxic in the food environment, as well as CYP2E1and SULT1A1alone or in synergy, the mutagenic effect of these mutagens.In this study, the micronucleus test to calculate role of mutagen cell micronucleus rate and multi-core rate of change, a mutagen in the presence or absence of two enzymes, poison the changing role of features, combined with the micronucleus test results by the indirect immunofluorescence technique to observe the continuous catalytic mutagens by two enzymes, the structure of the cytoskeleton, especially the spindle and centromere damage, then according to the statistical analysis, to make these chemical mutagens to determine and inference the mechanism of cytotoxicity.Further more, lay a foundation for the further study.MethodsIn this study, we selectively separately or jointly blocked hCYP2E1and hSUL1A1these two enzymes of this cell line, so that it contact with a series of indirect genotoxicity micronucleus test and cell fluorescence immunoassay (IF), Statistics the difference about micronucleus and multinucleus amone different concentrations, observe the extent of the damage of the cytoskeleton structure and type of damage, To reflect the CYP2E1and hSUL1A1-of indirect genotoxicity what role in the intracellular metabolism, also, to understand enzyme and indirect genetic poison interaction is the interaction between how cell damage, as well as enzyme activation the indirect genotoxicity metabolism or the metabolic detoxification of cellular damage.ResultsResults are as follows: 1, In the present study, we further observed that the spontaneous Fmi and Fmu were also elevated, though in a smaller intensity.The accession of ABT and PCP did not affect cell micronucleus rate, but lower multinucleus rate.2, NDMA, in the concentrations of10-100uM, increased Fmi and Fmu simutaneously, with Fmi elevated marginally more than Fmu.NDMA-induced increases in Fmi and Fmu were completely inhibited by ABT.3,2-NP (in the concentrations ranging from1to5mM) caused concentration-dependent increase in both Fmu and Fmi, with the increase in Fmu being slightly more intensive than Fmi.2-NP-induced increases in Fmi and Fmu were blocked by PCP.4, NDEA caused concentration-dependent increase in both Fmu and Fmi, with the increase in Fmu being slightly more intensive than Fmi.The the multicore rate compared with control group was significantly different when ABT added and no inhibitors or substrates added.5, A A in the dose range of cell lines did not observe significant changes on frequencies of micronucleated or multinucleated after ABT added. Statistical significance was attained for comparison between1000μM (AA) group and the control when no inhibitors or substrates added.However, Statistical significance was attained for comparison only between300μM and the control after ethanol added into this system.Also, we found that Cells spindle fibers and centrioles damaged cells can lead to a significant increase in1500μM (AA) above, and above this dose can significantly interfere with cell centrioles.6, In the dose range, benzene did not cause significant changes in rate of micronuclei and multinucleated rate when ethanol added.However, we found that the frequency of micronucleated cells was increased significantly, but the ratio of multinucleated when PCP added into the system.Also, the frequency of micronucleated cells and multinucleated ones was increased significantly when PCP and ethanol both added.7, Phenol on the toxic effect of the cell lines shows a simple dose-response relationship. All dose groups in the dose range of frequencies of micronucleated were significantly increased.ConclusionsFrom all the article discussion, we can draw a conclusion that,1, When CYP2E1enzyme expressed, the genotoxicity of NDMA in V79-hCYP2E1-hSULT1A1cell line has been enhanced, Therefore, the bioactivating enzyme responsible for the effects of NDMA observed in the present study is human CYP2E1.2, We found that NDEA has chromosome breakage toxicity and aneuploidy toxicity both, suggesting SULT1A1and CYP2E1enzyme for the second of the NDEA metabolism, both with antagonism and synergy. When two enzymes exist at the same time, you can reduce the NDEA aneuploid toxicity, but at the same time or enhance the NDEA fragmentation of toxicity.3, The observed induction of micronuclei and multinuclei by2-NP in V79-hCYP2E1-hSULT1A1is dependent on human SULT1A1, but not human CYP2E1, which is in accordance to the previous report on2-NP-induced genotoxic response in human SULT1A1-expressing V79cells.4, On the toxic effects of V79-hCYP2El-hSULTlA1cell lines, AA in the dose range there is fragmentation and aneuploid toxicity, toxicity to aneuploid.SULT1A1Ⅱ and CYP2E1enzyme on metabolic activation of AA there is a synergistic effect. And under the second enzyme together, caused by AA aneuploid toxic not only on the damage of spindle fibers, also on the destruction of the Center particle synthesis and regulation, may appear on the large chromosome breakage.In the synergy of two enzymes, toxic effect of showing the complexity of diversity characteristics of AA.5, Within the dose range, CYP2E1enzyme activates the genetic toxicity of benzene on V79-hCYP2E1-hSULT1A1cell line, the genetic toxicity of main toxicity is aneuploid, larger doses may have fragmentation or other toxicity.When both of the CYP2E1and when SULT1A1, two enzymes on the presence of metabolic activation of antagonism.Under the second enzyme antagonistic, benzene does not appear within the dose range fragmentation and aneuploid toxicity.6, The type of toxic effect of phenol on this cell line are similar with the CYP2E1enzyme activated toxicity of benzene exposure on it.Micronucleus test of phenol, no study CYP2E1enzyme on metabolic activation of phenol metabolism, no study CYP2E1and SULT1A1, two enzymes have a synergistic or antagonistic on metabolism of phenol. Within the dose range, the toxicity of phenols is independent of CYP2E1, also does not rely on SULT1A1.