The Genotoxicity Of Quinoxalines To Mammalian Cells

Quinoxalines, which have the structure of quinoxaline-1, 4-dioxides are syntheticantimicrobial agents, which have the characteristic for improving feed efficiency,promoting growth and increasing the rate of weight gain. For these reasons, they havebeen widely used as growth promoters in the most countries of the world. However,carbadox and olaquindox, which the first were developed and used, have been banned orstrictly limited to use due to their toxicities. Mequindox was synthesized by LanzhouInstitute of Animal and Veterinary Pharmaceutics sciences of CAAS in China, and usedas antibacterial agent. Quinocetone and Cyadox, which were new growth promoters ofthe quinoxalines family, had the characteristic of low toxicity, less side effect and obviousgrowth promotion in food-producing animals. Some researches showed that quinoxalineshad the mutagenic activity in germ and could induce base pair substitutions andframeshift mutations in germ. But, the mutagenicity of quinoxalines in mammalian cellsin vitro systems have few reported. In order to investigate the genotoxicity ofquinoxaline-1,4-dioxides in mammalian in vitro, five similar structural compounds:mequindox, carbadox, olaquindox, quinocetone and cyadox were chosen and tested theeffects of hgprt gene mutation in V79 cells and unscheduled DNA synthesis in Humanperipheral blood lymphocytes.Five quinoxalines were investigated for their capabilities of inducing HGPRT genemutation in V79 cells in the absence and presence of rats S9, which were PCB-induced asmetabolism activation systems. Every drug was divided four groups: differentconcentrations groups, negative control (DMEM, -S9与+S9), solvent control (DMSO,-S9与+S9), EMS(-S9) and B(a)P(+S9) control. Mequindox, carbadox, olaquindox andquinocetone had the same final concentration: 10μg/mL, 5μg/mL, 2.5μg/mL and1.25μg/mL, the final concentration of cyadox were 5μg/mL, 2.5μg/mL, 1.25μg/mL and0.625μg/mL, the negative control(DMEM) and the solvent control(DMSO) were 0.5%(V/V), the final concentration of positive control EMS was 1mg/mL and B(a)P was 2μg/mL. After V79 cells were treated for 4h, the cytotoxicity of quinoxalines wereevaluated in V79 cells line by using clone formation assay. Based on the results, wefurther observed the effects of quinoxalinees on hgprt locus mutation frequency. Theresults showed that the mutation frequency of the positive control EMS was more 67times than that of the solvent control in the absence of S9. B(a)P was more 32 times thanthat of the solvent control in the presence of S9. Both of them are typical positivecompounds. Mequindox, carbadox and olaquindox all induced gene mutation at the hgprtlocus in the absence and presence of S9-mix, and there were significant increase of hgprtgene mutation frequency with an approximately dose-effect relationship. However,quinocetone only produced a slight increasing of the mutant frequency at a concentrationof 10μg/mL, and cyadox failed to produce significant increase in the frequency of thehgprt locus mutation at concentration from 0.625 to 5μg/mL whether S9-mix was addedto the plates or not. The results suggest that mequindox, carbadox and olaquindox canlead to mutation of the hgprt locus in V79 cells, while quinocetone and cyadox can notinduce V79 gene mutation.To study the effects of quinoxalines on DNA damage and repair in Humanperipheral blood lymphocytes, unscheduled DNA synthesis test was used in the absenceand presence of S9-mix in vitro. Every drug was divided four groups: four differentconcentration groups, negative control (RPMI1640, -S9与+S9), solvent control (DMSO,-S9与+S9), positive control HN₂·HCl(-S9) and B(a)P (+S9). Mequindox, carbadox,olaquindox and quinocetone have the same final concentration:20μg/mL, 10μg/mL,5μg/mL and 2.5μg/mL, the final concentration of cyadox were 10μg/mL, 5μg/mL,2.5μg/mL and 1.25μg/mL, the negative control(RPMI1640) and the solventcontrol(DMSO) were 1%(V/V), the final concentration of positive control HN₂·HCl was20μg/mL and B(a)P was 25μg/mL. Human peripheral blood lymphocytes were treatedwith quinoxalines, hydroxyurea and ³H-TdR for 4h, then were collected on glass fiberfilters. The filters were dried and counted CPM in the Bckman liquid scintillator. Theresults showed that the CPM of HN₂·HCl and B(a)P groups had significant increase ofthat of the solvent control, which suggests that they are both typical positive compounds.Cyadox could not make the significance increase of unscheduled DNA synthesis inhuman peripheral blood lymphocytes at a concentration from 1.25 to 10μg/mL in theabsence and presence of S9-mix, suggest that it will not induce DNA damage. However,the CPM of mequindox, carbadox, olaquindox and quinocetone had significant difference from that of the solvent control. Quinocetone could produce the significance increase ofunscheduled DNA synthesis at a concentration from 10 to 20μg/mL, carbadox,olaquindox and mequindox make the similar effect at concentrations from 5 to 20μg/mL,and they were an approximately dose-effect relationship, the results suggested quincetone,olaquindox, carbadox and mequindox could cause the lymphocytes DNA damage inHuman peripheral blood.Based on the results, we found cyadox had no genotoxicity in mammalian cells;quinocetone at high concentration had slight genotoxicity; olaquindox, carbadox andmequindox had strong genotoxicity in mammalian cells.