The Study Of Genotoxicity And Carcinogenicity Of Quinoxalines

Quinoxalines is a class of broad-spectrum antimicrobial and growth-promoting compounds,which contains carbadox?CBX?,olaquindox?OLA?,mequindox?MEQ?,quinocetone?QCT?and cyadox?CYA?.With the widespread use of Qd NOs,the toxicity especially genotoxicity and carcinogenicity,has gradually attracted people's attention.Due to the potential genotoxicity and carcinogenicity,their use is prohibited or strictly restricted.Although the genotoxicity study has been carried out for many years,the focus of research is mainly on prototype of Qd NOs,which can't reveal the relationship between genotoxicity and metabolites of Qd NOs.In addition,the mechanism of genotoxicity of metabolites remains unclear.Pre-clinical toxicological studies of MEQ have shown that it has strong toxicity and genotoxicity.Therefore,MEQ are suspected to be carcinogenic.In this study,the genotoxicity of main N-O reduction metabolites of Qd NOs were studied according to the requirements of the“Test method for special toxicity of new veterinary drugs”,the Food and Drug Administration of the United States?FDA?and the Organization for Economic Cooperation and Development?OECD?.Furthermore,the carcinogenicity of MEQ and QCT were studied according to the guidelines of FDA and OECD and GB15193.1. Investigations into the genotoxicity of quinoxaline-di-N-oxides and their primary metabolitesIn this study,the genotoxicity of main N-O reductive metabolites of Qd NOs was studied by Ames test,V79 cell chromosome aberration test and mouse bone marrow cell micronucleus test.1.1 Ames testThe mutagenicity of metabolites of Qd NOs was tested in a reverse mutation assay using six S.typhimurium strains?TA97,TA98,TA100,TA102,TA1535,and TA1537?with and without S₉-mix.The highest concentration was 5 mg/plate for B-OLA,B-CBX and B-MEQ,2.5mg/plate for B-QCT,and 1.25mg/plate for N1-QCT and N1-MEQ,respectively.These results indicated that the metabolites of Qd NOs have no mutagenicity to S.typhimurium strains.1.2 Chromosomal aberration testThe metabolites of Qd NOs were administed to 3 different concentrations?67,33.5and 16.75?g/m L?.The results shown that there was induction of chromosome aberrations for B-CBX and B-MEQ in V79 cells without S₉at concentration of 67?g/m L.There was induction of chromosome aberrations for N1-MEQ in V79 cells with and without S₉at concentration?33.5?g/m L.N1-QCT,B-QCT and B-OLA didn't cause chromosome aberration in V79 cells with or without S₉at considered concentrations.1.3 In-vivo micronucleus assayThis test was performed to test the induction of micronuclei in polychromatic erythrocytes from femural bone marrow of Kunming mice resulting from exposure to metabolites of Qd NOs.Twenty groups of Kunming mouse?5 mice/sex/group?were selected.The results show that N1-MEQ caused a significant increase in the number of micronuclei at doses of 0.31 and 0.16g/kg b.w.in mice.A significant increase in the number of micronuclei was noted in male mice after treated with B-CBX and B-MEQ at5g/kg b.w.B-CBX significantly increased in the number of micronuclei at doses of 5 and2.5g/kg b.w.in female mice.N1-QCT,B-QCT and B-OLA can't cause micronuclei formation in mice at considered dose.The above results indicated that except for negative results in Ames test,N1-MEQ,B-MEQ and B-CBX gave positive in the other three genotoxicity tests.The genotoxicity order was same as prototype,namely N1-MEQ>B-MEQ>B-CBX.B-OLA,N1-QCT and N4-QCT gave negative in genotoxicity tests.2.Carcinogenicity study of MEQFour groups of SPF Kunming mice?50 mice/sex/group?and four groups of SPF Wistar rats?55 rats/sex/group?were fed diets containing MEQ?0,25,55 and 110 mg/kg?for 78 and 104 weeks,respectively.Regular sampling schedules are scheduled for 26,52and 78 weeks for mice,and 26,52,78 and 104 weeks for rats,respectively.During the experiment,clinical observation of rats was conducted and body weight was recorded.Hematology and serum biochemical analysis were performed at regular autopsy and end of exposure to MEQ.Gross autopsy and histopathological observation were carried out and the survival rate,organ weight,organ coefficient,incidence of tumors and benchmark dose were calculated.Hematology analysis showed that MEQ could cause significant changes in some parameters in mice,such as HGB,HCT,MCV,MCH,RDW,PCT and PDW,indicating that MEQ caused significant blood toxicity in mice.At week 52,78 and 104,the number of hematology indicators with significant changes in the MEQ groups increased gradually,suggesting that long-term exposure to MEQ could cause significant blood toxicity in rats,and this toxicity increased with the prolongation of exposure time.MEQ caused significant changes in serum biochemical indicators such as Na⁺,K⁺and BUN in mice.Pathological examination showed that kidneys and adrenal glands in the MEQ groups had obvious pathological damage,suggesting that MEQ had toxic effects on the kidneys and adrenal glands in mice and rats.In addition,the ALP,ALB,ALT and AST in mice were significantly decreased in MEQ groups,and the liver coefficients in MEQ groups in male mice at 52 weeks and female mice at 78 weeks were significantly decreased.At the 26th week,ALT and AST in M110 rat group were significantly increased.At week 52,ALT in M110 rat group was significantly higher than that in control group.At week 78,ALT and AST in M25 group and AST in M55 group were significantly increased.At week 104,the AST of rats in the MEQ groups was significantly increased.Pathological examination showed that the liver of mice at 52 and 78 weeks and rats at 52,78 and 104 weeks in the MEQ groups had obvious pathological damage.These observations suggested that liver is the main toxic target organ of MEQ.At the 26th week,the brain coefficient of female rats in M110 group was significantly increased.At week 52,the brain coefficient in M25 rat group and testicular coefficient in M110 rat group were significantly increased;and lung coefficient in M55and M110 rat groups was significantly decreased.At week 104,the coefficients of heart and lung of male rats in M25 group were significantly increased,while the coefficients of spleen and brain of female rats in MEQ groups were significantly reduced.At week 52,the heart coefficient of female mice in M55 group was significantly decreased;the testis and epididymis coefficient of M55 group and the brain coefficient of male mice in M110group was significantly increased.At week 78,the organ coefficients of lungs,uterus and ovaries of female mice in M25 group were significantly increased;the brain coefficient of female mice in M55 and M110 groups,and the spleen coefficient of male mice in M110group were significantly decreased.Pathological examination showed that the heart and brain in the MEQ groups had slight pathological changes,while lung,spleen,testis,uterus and ovary had obvious pathological changes.These results indicated that MEQ had toxic effects on heart,brain,spleen,lung,testis,uterus and ovary.In this study,the tumors of rats and mice in control group mainly occurred in late life,which accorded with the characteristics of spontaneous tumors.The first case of cancer appeared in M25 female rats at week 62,and the carcinogenic latency of MEQ was significantly earlier than that of the control group.The main types of tumors induced by MEQ in mice were liver cancer,splenic large granular lymphocytic leukemia,vascular cancer,lung adenoma,adrenal cortex tumor,uterine leiomyoma,breast fibroma,mammary adenoma,testicular Leydig cell tumor and melanoma.The main types of tumors induced by MEQ in rats were pituitary adenoma,myelodysplastic syndrome-leukemia,liver cancer,lung mesothelioma,lung adenocarcinoma,melanoma,uterine leiomyoma,adrenal cortex tumor,ovarian granuloma,breast fibroma,breast duct cancer,squamous cell carcinoma and testicular interstitial cell carcinoma.MEQ induced new types of tumors,such as thyroid C-cell carcinoma,adrenocortical tumors and testicular interstitial cell carcinoma in mice,ependymoma,ovarian granuloma and adrenocortical tumors in rats.Compared with the control group,the incidence of tumorigenic lesions increased significantly,suggesting that MEQ is carcinogenic in mice and rats.By using Bayesian BMD system software and dichotomous Hill model,the benchmark dose of MEQ was 0.0102 mg/kg when the BMR?benchmark dose response?was 5%.As MEQ is widely used in China,its carcinogenic risk to target animals and consumers should be paid attention to.3. Carcinogenicity study of QCTFour groups of SPF Wistar rats?50 rats/sex/group?were fed diets containing QCT?0,50,100 and 300 mg/kg?for 104 weeks.Regular sampling schedules are scheduled for 52,78 and 104 weeks.During the experiment,clinical observation of rats was conducted.Hematology and serum biochemical analysis were performed on rats at regular autopsy and at end of exposure to QCT.Gross autopsy and histopathological observation were carried out and the survival rate,organ weight,organ coefficient,incidence of tumors and benchmark dose were calculated.The survival rates of rats in control,Q50,Q100 and Q300 group were 60%,60%,54%,60%in females,and 52%,54%,58%and 60%in males,respectively.This results suggested that the survival rate of each dose group is more than 50%,which meets the relevant criteria of carcinogenic test.At week 52,AST,ALT and TG of females in QCT groups were significantly decreased,and CREA of females in Q100 and Q300 groups was significantly reduced.At week 78,TBA of females in QCT groups was significantly increased,and AST of females in Q50 group was significantly decreased.Histopathological examination showed that the liver in QCT groups had obvious pathological changes,indicating that liver was the main target organ of chronic toxicity of QCT.At week 52,the concentration of Na⁺in Q50 and Q300 groups was significantly reduced,and K⁺had an upward trend compared with the control group.Therefore,it is speculated that QCT may induce water-salt imbalance in vivo.Histopathological examination showed obvious pathological changes in kidneys and adrenal glands in the QCT groups,suggesting that the kidneys and adrenal glands were also the main target organs of the chronic toxicity of QCT.At week 52,the coefficients of uterus and lung in QCT groups were significantly increased.At week 78,the coefficients of heart and lung in Q50 group were significantly higher than those in control group;the coefficients of spleen and brain in Q100 group and the ovary coefficient in Q300 group were significantly increased,while the coefficients of testis and epididymis in Q300 group were significantly decreased.At week 104,the coefficients of heart and spleen in Q50 group and the heart coefficient in Q100 group was significantly increased,while the coefficients of lung,testis and epididymis in Q300group were significantly reduced.Pathological examination showed that the heart and brain in QCT groups had slight pathological changes,while the lung and spleen had obvious pathological changes.The main manifestations were aggregation of pulmonary macrophages and proliferation of splenic lymphoid follicles.The main pathological changes of reproductive organs in QCT groups were uterine cavity dilatation,ovarian vesicles,testicular atrophy,vas deferens cavity dilatation and testicular interstitial enlargement.These observations suggested that long-term exposure to QCT has toxic effects on the reproductive organs,heart,brain,spleen and lungs of rats.After feeding rats with QCT for 104 weeks,the incidence of tumors increased significantly.The main types of tumors are pituitary adenoma,liver cancer,spleen cancer,kidney cancer,lung adenoma,lung mesothelioma,uterine leiomyoma,ovarian clear cell carcinoma,breast fibroma,breast cancer,lymphoid tissue-like melanoma,adrenocortical tumor,testicular Leydig cell carcinoma and colon cancer.Compared with the control group,QCT induced the emergence of new types of tumors,such as renal cancer,ovarian clear cell carcinoma and adrenocortical tumors.In this study,the first case of cancer appeared in the Q100 female rat group at the fifty-fifth week,and carcinogenic latency of QCT was significantly earlier than that of control group.Therefore,QCT is carcinogenic in rats,which is consistent with the predicted carcinogenicity of QCT and its metabolites.By using Bayesian BMD system software and dichotomous Hill model,the benchmark dose of QCT was 0.0308 mg/kg when the BMR was 5%.In view of the recommended dosage of 50?75 mg/kg QCT to feed in the pig feed,there is a potential safety hazard in the process of clinical use of QCT.