| Veterinary drugs residues may not only affect the growth and normal physiological function of livestock and other animals but also may threaten human health though the bioaccumulation of the food chain. Studies have reported that the increased incidence of human cancer could associate with some environmental pollution, Veterinary drugs residues in animal food induced mutagenic and carcinogenic may be important. Quinoxaline drugs are a class of compounds with the basic structure of synthetic Quinoxaline-1,4-dioxide (QdNOs), they can inhibit against kinds of bacteria such as Staphylococcus aureus, Escherichia coli and Salmonella, and accelerate the animal growth. Both carbadox and olaquindox, two traditional QdNOs drugs, have shown mutagenicity and potential carcinogenicity in various test systems, so the Commission of the European Community has forbidden the use of carbadox and olaquindox as anima growth promoters in 1999. Quinocetone (QCT) is a new QdNOs and has been approved as an animal growth promoter in China since 2003 because of its higher effective and less toxic. However, based on available evidences from the literature, QCT may lead to bacterial and mammalian cell lines mutagenesis, and can cause DNA damage in HepG2 cell lines. But the studies on the QCT caused mutagenesis in vivo were limited, and the results of related studies were considerable controversy. Therefore, much research, used of sensitive laboratory animal species with carcinogenic chemicals, is necessary to evaluate accurately the genetic toxicity induced by QCT in vivo, which is important to establish reference norms and standards. Oxidative stress caused by many exogenous compounds is one of early molecular mechanisms of toxicity. The related studies showed that some QdNOs members, such as carbadox and qlaquindox, can lead to DNA damage in vitro and this injure was directly related to accumulation of ROS. With the same structure to some QdNOs, whether the oxidative stress was also occurred during the metabolic process and associated with oxidative DNA damage is largely unknown.Many studies confirmed that activation of stress response proteins is the early protective mechanism of the toxic effects induced by exogenous compounds. Heat shock protein (heat shock protein, HSP) family has become the focus in this field. Among the panel of potential candidate genes, heme oxygenase-1 (HO-1) seems to draw much attention with its potent anti-inflammatory, anti-oxidant, and anti-proliferative effects, close correlation with oxidative stress-related injury. Due to the function of multiple defenses against oxidative damage, good irritability and inducibility, HO-1 has become an important target gene to prevent oxidative damage. Some studies reported that a variety of dietary antioxidants can induce HO-1 expression and play antioxidant function. Chinese black tea (Pu-erh fermented tea), originally produced in the Yunnan province of China, is obtained by first parching crude green tea leaves and then fermenting them with microorganisms such as Aspergillus sp. Favorable evidence support the benefits of a diet rich in Pu-erh black tea and its associated bioactive components, such as antioxidation, hypocholesterolemia, and anti-obesity effects, and the main mechanism with these benefits may associate with its strong function by scavenging excess reactive oxygen species and reactive nitrogen substances. Suggesting that Pu-erh black tea may develop protective effect against oxidative DNA damage induced exogenous carcinogens and this could play an important role in anti-mutation effect. However, this hypothesis has not been approved in vivo study, and there is no related research on the regulation mechanism of HO-1 by Pu-erh black tea.Therefore, to further evaluate QCT-induced genetic toxicity and molecular toxicology mechanism in vivo, the aim of this study is to discuss the relationship among oxidative stress, inflammatory reaction, apoptosis and oxidative damage induced by QCT though acute or subchronic exposure, and the target function of HO-1 in exogenous carcinogens induced DNA damage. Besides, the protective effect and relative molecular mechanism of Pu-erh black tea extract are also evaluated as one part of this study. Part 1:Evaluation of genotoxicity induced by quinocetone in Balb/c mice though acute exposureObjectives:The aim of this study is to evaluate the genotoxicity of quinocetone though acute exposure in vivo, and discuss the relationship between genotoxicity and oxidative stress.Methods:Mice were divided randomly into total 10 groups (5 groups with females and males, respectively) of six animals with the same sex each and treated for acute QCT exposure as follows:(QCT groups) mice received the treatment comprised two successive administrations of QCT which was given as a solution in 0.5% sodium carboxymethylcellulose (Sinopharm Chemical Reagent Co., Ltd. P. R. China) at 24-h intervals by gavage for 12000 mg/kg·bw (QCT-H group),6000 mg/kg·bw (QCT-M group) and 3000 mg/kg-bw (QCT-L group), respectively; (Negative control group) normal control group received only the vehicle (0.5% sodium carboxymethylcellulose); (Positive control group) the mice received 40 mg/kg bw cyclophosphamidum injection. At the sampling time (between 3 and 6 h after the second dosing), all the animals were sacrificed. Giemsa staining method was used for micronucleus test of bone marrow polychromatic erythrocyte (PCE). The in vivo comet assay was used to evaluate the DNA damage in hepatic and nephric isolated cells. Besides, the ROS level, as well as GSH and T-AOC, and the activity of glutathione peroxidase, superoxide dismutase and catalase in liver and kidney were determined.Results:(1) The micronucleus rates of bone marrow PCE in 12000 mg/kg-bw of females and males were 9.2%o and 9.4%o, respectively. The differences between QCT groups and negative groups were statistically significantly. The 6000 and 3000 mg/kg-bw QCT groups can not lead to significant increase of micronucleus rate as compared to negative groups.(2) The DNA damage was observed in all the groups treated with single QCT, the OTM value in QCT-treated group were significantly higher than controls except the liver with dose of 3000 mg/kg-bw (P<0.01).(3) The ROS level in liver and kidney appeared a dose dependent increase during all QCT group, it was accompanied by decreased GSH and T-AOC level and repression of antioxidative enzymes activity, including GPx, SOD and CAT.Conclusion:(1) These data demonstrate that acute QCT exposure can develop a potential genotoxicity in mammalian.(2) The genotoxicity induced by QCT was mediated by oxidative stress though generating excessive ROS and inhibiting antioxidative systerm.Part 2:Evaluation of genotoxicity induced by QCT though subchronic exposure in SD ratsObjectives:The aim of this study was aimed to further determine the genotoxicity induced by QCT though subchronic exposure in SD rats.Methods:A total of 72 male SD rats were randomly divided into four groups as below:(1) high dose of QCT (QCT-H):2400 mg/kg/day; (2) middle dose of QCT (QCT-M):800 mg/kg/day; (2) low dose of QCT (QCT-L):50 mg/kg/day; (4) Control group:only received the vehicle. All the animals were sacrificed at end of one-month (n=8) and three-month (n=10), respectively. The serum ALT, AST, Cr and BUN levels, as well as the urinary 8-hydroxy 2-deoxyguanosine (8-OHdG) among all groups were determined and compared. Comet assay was used to evaluate the endocellular DNA damage in hepatic cell suspension. The ROS level, as well as GSH and MAD, and the activities of GPx, GST, GR, SOD and CAT were measured using commercial assay kits. The mRNA levels of related enzymes of OGG1-mediated base excision repair pathway, including Xrccl, PARP1 and PARP2, were analyzed by RT-PCR test. RT-PCR and western blot were used to detect inflammation and apoptosis related gene mRNA and protein expression levels, respectively. Besides, liver HO-1 protein expression was detected in 4 week and 13 week QCT exposure in SD rats.Results:(1) During the 13-week toxicological experiment, QCT-fed rats had significantly lower body weight and food consumption than control rats from the 1st week and 9th week of experiment, respectively (P<0.05). These trends were still evident throughout the rest of study period. Compared to the control group, QCT administration group in higher doses resulted in greater values for relative liver weight (P<0.01). Subchronic QCT administration resulted in a sustained elevation of ALT, AST, Cr and BUN levels. Hepatic histopathological examination showed that QCT caused some morphological abnormalcy in the liver characterized by physaliferous cells, inflammatory cell infiltration, slight fibration, cell necrosis and lysis.(2) The DNA damage was observed in all the higher doses groups treated with QCT, the OTM value in high dose of QCT group was significantly higher than controls (P<0.01), while the OTM value in QCT-L group was significantly lower than controls (P<0.01).(3) Increased hepatic cell ROS level, urinary 8-hydroxy 2-deoxyguanosine (8-OHdG) contents and lipid peroxidation level were observed in the 2400 mg/kg/day QCT groups. Inhibited antioxidant system, i.e. glutathione glutathione S-transferase, glutathione peroxidase and glutathione reductase was also observed in the liver homogenate of high dose of QCT groups (P<0.01 or P<0.05).(4) The OGG1 mRNA level in QCT-H group was almost 5.15 times higher than controls (P <0.01), this result accorded with the 8-OHdG results during the two groups. Besides, the mRNA levels of related enzymes of OGG1-mediated base excision repair pathway, including Xrcc1, PARP1 and PARP2, were also significantly increased in QCT-H group as compared to controls (P<0.01 or P<0.05).(5) High dose of QCT subchronic exposure can not only directly lead to increases NF-κB mRNA expression, but also enhance multiple inflammatory related genes expression, including iNOS, TNF-a and Cox-2 (P<0.05). One the other hand, the mRNA levels of some apoptosis related gene like p53 and casepase-3 were also increased significantly as compared to controls (P<0.05). The Bcl-xl mRNA level was decreased, but the difference was not statistically significantly (P>0.05). The changes of protein expression levels were parallel with the mRNA levels among the groups.(6) The dose dependent and time dependent decrease of HO-1 protein expression was observed in the QCT-treated groups. As compared to controls, the level of HO-1 protein was not significantly decreased in the liver of four week QCT exposure (P<0.05), while the difference was significant at the end of thirteen week (P>0.05).Conclusion:(1) QCT, which is a generator of ROS, induces DNA damage in hepatic cells tested by comet assay and activiates OGG1-mediated base excision repair pathway in SD rats, which lead to accumulation of DNA adduts. The results of this study demonstrate that QCT could induce potential genotoxicity after a 13-week subchronic exposure in vivo.(2) The endocellular high oxdatived stress accompanied by inhibition of Nrf-2/HO-1 pathway could be the main molecular mechanism of QCT-induced genotoxicity. HO-1 could be an important molecular target which mediated the genotoxicity induced by some exogenous toxicants.Part 3:Protective effects of Pu-erh black tea extract supplementation against quinocetone-induced genotoxicity through subchronic exposure in SD ratsObjectives:The present study was designed to investigate the protective effects and molecular mechanisms of Pu-erh black tea extract (BTE) in a rat model of QCT-induced genotoxicity.Methods:A total of 40 male SD rats were divided into five groups (eight animals for each group) and treated for 13-week as follows:(Control group) normal control group received only the basal diet; (QCT groups) SD rats were feed the diet with 2400 mg/kg/day QCT adjusted by daily food consumption; (QCT+BTE groups) QCT plus BTE groups received BTE once daily with doses of 1000 mg/kg/day (QCT+BTE-H group) and 500 mg/kg/day (QCT+BTE-L group); (BTE control group) single BTE administration with dose of 1000 mg/kg/day was selected as the BTE control group. Body weights and food consumption were measured weekly. All the animals were sacrificed at the 92-day. Serum from blood samples was collected for biochemical examination. The fresh liver was removed and weighted, then parts of liver in every group were placed in 10% neutral buffered formalin, sectioned and stained with hematoxylin and eosin (H&E) for histopathological examination. The DNAdamage in liver cell was evaluated by comet assay. The ROS level, as well as urinary 8-OHdG contents and lipid peroxidation level, was measured as the describtion in the second part. The mRNA levels of HO-1, iNOS, TNF-a, Cox-2, p53, Bcl-xl and casepase-3 were determined by RT-PCT. The protein expression levels of HO-1, Nrf-2, Keap-1, iNOS, NF-κB, Cox-2, Bcl-xl, casepase-3, ERK 42/44 and pERK 42/44 (Thr202/Tyr204) were measured by western blot. Results:(1) QCT-fed rats had significantly lower body weight and food consumption than control rats. Compared to the control group, QCT administration group resulted in greater values for relative liver weight and higher levels of serum ALT and AST (P<0.01 or P 0.05). Hepatic histopathological examination showed that QCT caused some morphological abnormalcy in the liver characterized by physaliferous cells, inflammatory cell infiltration, slight fibration, cell necrosis and lysis. BTE supplementation could effectively ameliorate QCT-induced hepatosis, including descreasing the serum ALT and AST levels, alleviating the pathological lesions in the liver.(2) BTE supplementation groups of both doses could significantly increase the DNA damage induced by QCT (P<0.01), the BTE itself can not lead to DNA damage in the SD rats though subchronic exposure.(3) BTE supplementation groups of both doses could increase the HO-1 and Nrf-2 mRNA and protein level in the liver as compared to the QCT-fed group. The Keap-1 protein level in the BTE control group was significantly higher than any other groups (P<0.01), but there was no significant difference among the four other groups.(4) The difference of ERK 42/44 protein in every group was not statistically significantly (P>0.05), but the phosphorylation of ERK 42/44 in BTE plus groups was higher than QCT-treated group.(5) The ROS level in hepatic cell, as well as the urinary 8-OHdG contents and lipid peroxidation level, was significantly increased in the 1000 mg/kg/day BTE supplementation group (P<0.01 or P<0.05). These were accompanied by modulation of antioxidative system for increasing the GSH level and activities of GPx, GST, GR, SOD and CAT in the liver homogenate.(6) As compared to QCT-fed group, BTE supplementation could alleviate the inflammatory response and cell apoptosis throgh inhibiting multiple inflammatory and apoptosis related genes and protein expression, including iNOS, TNF-a, Cox-2, p53 and casepase-3 and upregulating the mRNA and protein levels of Bcl-xl.Conclusion:(1) BTE supplementation can effectively protect the liver against QCT-induced DNA damage in SD rats.(2) BTE could upregulate the Nrf-2/HO-1 pathway via activation of ERK1/2 in liver through inhibiting oxidative stress, which plays a positive function of antimutagenic.
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