Mechanism Of Quinoxalines Mediated DNA Damage In Escherichia Coli

Quinoxaline compounds are a class of synthetic antibacterial agents, which are derivatives of quinoxaline-1,4-dioxides, and include carbadox, olaquindox, mequindox, quinocetone and cyadox. The first four of compounds are produced in the 20th century, and cyadox is a new member of QdNO drugs, attracting widespread attention because of its significant antibacterial growth-promoting effect, low toxicity and wide range of use. In vitro antibacterial test of cyadox has been carried out in our lab. The results indicate that cyadox does have high antibacterial effect and can damage bacterial DNA which has a similar mechanism with other drugs. But antibacterial mechanism of quinoxaline compounds how DNA is damaged has not been well revealed. The topic selects cyadox and olaquindox from quinoxalines represented for the study and E. coli which is sensitive to them were selected as objects of this study. This paper will study mechanism of drug action from two aspects of the direct role of cyadox and olaquindox on DNA and enzymes related to DNA synthesis and repair. Detect the direct effect of drugs on DNA damage in vivo and in vitro. And select some key enzymes which are related to DNA synthesis and repair processes:E. coli DNA Topoisomerase I, E. coli DNA Gyrase, E. coli DNA polymerase and E. coli DNA ligase. Investigate the mechanisms of quinoxaline damaging bacterial DNA in vitro with gel electrophoresis and fluorescence detection techniques.1. Determination of MIC and MBC of quinoxaline drugsIn this experiment, Escherichia coli CVCC2943 from chicken was selected to detect the sensitivity of cyadox, olaquindox, carbadox, mequindox under anaerobic and aerobic conditions respectively. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of quinoxalines in pathogenic E. coli CVCC2943 were performed by a broth macrodilution method. The MIC and MBC of Escherichia coli CVCC2943 against cyadox were determined to be 1μg/mL and 4μg/mL in anaerobic conditions, and to be 16μg/mL and 128μg/mL in aerobic conditions. Those of olaquindox were determined to be 4μg/mL and 16μg/mL in anaerobic conditions, and to be 16μg/mL and 128μg/mL in aerobic conditions. Those of carbadox were determined to be 0.25μg/mL and 1μg/mL in anaerobic conditions, and MIC of carbadox were 2μg/mL in aerobic conditions. Those of mequindox were determined to be 0.5μg/mL and 2μg/mL in anaerobic conditions, and to be 2μg/mL and 16μg/mL in aerobic conditions. As can be seen from the results cyadox had a good selection of the anaerobic activity like other quinoxaline drugs. Cyadox was a new drug of quinoxalines and low toxicity and low residue. Olaquindox was conventional quinoxaline drugs and the antibacterial mechanism of action was no reports. Combinding with previous studies on olaquindox in our laboratory, we chose cyadox and olaquindox as a representative to study quinoxaline-induced DNA damage in bacteria.2. Direct roles of cyadox and olaquindox on DNA damageDetect nucleic acid changes previous by isotopically labeled and electron microscopy on studies of quinoxalines antimicrobial mechanism of action. In this study, DNA damage was detected by gel electrophoresis, divided the effect of drugs on pBR322 DNA in vitro and observation damage of drugs on DNA after extracting plasmid DNA by bacterial incubated with the drug. The study was under aerobic and anaerobic in vitro, they were divided into the drug directly on DNA and drugs after metabolism with xanthine oxidase on DNA under anaerobic conditions. The results showed that cyadox and olaquindox can not break DNA without the presence of xanthine oxidase under either aerobic or anaerobic conditions. Cyadox and olaquindox can break DNA significantly with the presence of xanthine oxidase under anaerobic. The final concentration of DNA breakage was 8μg/mL. DNA slightly broke when incubated for 30min, and after one hour incubation, there are obviously DNA damage. The result showed cyadox and olaquindox could directly damaged DNA after metabolism in vitro. We first tranfered plasmid pBR322 DNA into bacteria, then incubated with drugs and extracted plasmid DNA. The results showed that plasmid DNA slightly broke when the concentration of cyadox was 4μg/mL. With the increase of drug concentration, the degree of degradation increased. Plasmid DNA obviously degradated when the concentration of cyadox was 16μg/mL. Plasmid DNA slightly broke when the concentration of olaquindox was 32μg/mL. With the increase of drug concentration, the extent of plasmid DNA damage had not changed too much.3. Impact cyadox and olaquindox on DNA synthesis and repair enzymesGel electrophoresis and intensity changes of fluorescence signal were used to detect impact of cyadox and olaquindox on enzymes activity, including E. coli DNA topoisomerase I, gyrase, polymerase and ligase. The first two enzymes took helicase of supercoiled DNA as activity indicator, and after two enzymes took kinetic parameter-reaction initial velocity calculated by the changes of fluorescence intensity as activity indicator. Detection of gyrase and topoisomerase was by supercoiled pBR322 DNA, the results showed enzymes can not helicase DNA substrate either the drug directly or after metabolism with them. We can conclude cyadox and olaquindox did not inhibit the activity of E. coli DNA gyrase and topoisomerase I, and the drugs did not block DNA synthesis by inhibiting the activity of two enzymes. For E. coli DNA polymerase and ligase, their activity was determined by initial velocity. The results showed cyadox and olaquindox reacted with DNA polymerase directly or after metabolism with it, and the initial velocity of the polymerization reaction catalyzed by polymerase significantly decreased. So we concluded cyadox and olaquindox could inhibit the activity of polymerase, and with the increase of drug concentration, the degree of inhibition increased. Inhibition can happen when drugs incubated for 30min. Both drugs could decrease the initial velocity of enzyme-catalyzed ligation reaction when they directly react with DNA ligase, but the initial velocity did not significantly change when the drugs after metabolism reacted with ligase. The result showed drugs prototype can inhibite ligase activity.Thus, we can conclude cyadox and olaquindox directly damage DNA after reducted metabolism with xanthine, xanthine oxidase reduction system. Another way of DNA damage was that drugs prototype directly inhibited the DNA polymerase and ligase activity, which were indispensable in DNA synthesis, repair and recombination. Thus DNA brought about damage. Cyadox and olaquindox after metabolism can also make influence the initial velocity of the polymerization reaction. Combinding with the impact of N1-deoxy cyadox on polymerase, it showed Nl-deoxy cyadox produced by cyadox metabolism inhibited polymerase activity, but N4-deoxy cyadox or dioxide cyadox can not. Cyadox and olaquindox after metabolism can not inhibit ligase activity.