The Molecular Mechanisms Of Genotoxicity Of Quinoxalines

Quinoxalines had quinoxaline-1,4-dioxides basic structure and include carbadox, olaquindox, cyadox, mequindox and quinocetone. They were widly used for feed additive and growth promotion. Carbadox and olaquindox had been forbidden by European Commission because of their potential properties of canceration, aberration and mutation. Mequindox and quinocetone wer the new synthetic veterinary medicine in the quinoxaline-1,4-dioxides family. There were many reports about their potental genotoxicity but the mechanisms were not clear. It was thought that ROS played an important role in genotoxicity. But the resource and species of ROS were not clear, and whether were there some other mechanisms of DNA damage induced by quinoceton was also not clear. Therefore, this topic studied the DNA damage molecular mechanism from many aspects:free radical, DNA adduct topoisomerase activity and DNA replication and repair enzymes.1Screening the most toxic compound of genotoxicity, sensitivity cell and determining the appropriate toxic indicators:We used MTT and SCGE assay to select most toxic compound and sensitivity cell. Growth inhibition and DNA damage of HepG2cells induced by quinoxalines was worst. Quinocetone could cause the DNA strand break and cells growth inhibition seriously. Therefore, we used HepG2cell and quinocetone as the research materials to study the molecular mechanism of genotoxicity.2. Researching the resource, species of free radical and the relationship of DNA damage with free radical:We used fluorescent probe of superoxide anion to detect free radical species and gel electrophoresis methods to detect mtDNA damage to search the resource of free radical. Our result showed that quinocetone could induce HepG2cell producing persistent ROS releasing, more superoxide radical and little hydroxy radical. It could also be metabolized and generation superoxide radical and hydroxy radical with xanthine oxidase system. We found that the free radical scavenger SOD could reduce the DNA strand break induced by quinocetone. Quinocetone could cause the mtDNA gene COX1, COX3and ATP6mutation. Mutation of ATP6gene may affect the number of ATP and energy supplying.3Detecting topoisomerase activity:Nuclear extracted from HepG2cell treated by etoposide, quinocetone had markedly reduced topo II activity as judged by an inability to convert pBR322DNA from the catenated to the decatenated form, but the topoisomerase I was not. Quinocetone can also inhibit the reconnection ability of topoisomerase. We also showed that topo II becomes covalently bound to DNA. Quinocetone can also induce HepG2cell producing stable DNA-topo II complexes which palyed a role in DNA damage.4Detecting the DNA adducts:We used ultraviolet absorption spectrum and HPLC methods to detect the quinocetone bound to DNA. Our result showed that quinocetone could groove or electrostatic bound to DNA, not the covalent combination.5Researching the gene expression of DNA replication and repair enzymes treated by quinocetone used RT-PCR method. We find the expression of Gadd45, TopoII and PCNA were affected seriously. The Gadd45gene expression of multiple increased2folds after30μM expoure. The expression levels of TopoII gene were decreased over2folds seriously after30μM expoure.In a word, there were two aspects of primal action of DNA damage induced by quinocetone:First, quinocetone could be metabolized and generated superoxide radical and hydroxy radical which attacked DNA, lipid and reapair enzymes and led to DNA damage, lipid superoxide and repair activity decrease; Second, quinocetone could combine with DNA and inhibit topoisomerase II acticity. DNA damage could cause many kinds of consequences, such as ROS increase, DNA replication and repair decrease.