氧化应激介导的喹烯酮遗传毒性及普洱茶水提取液保护作用研究
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摘要
兽药残留不仅可能影响到畜禽等动物的生长发育和正常生理机能的维持,而且可能通过食物链的生物富集作用影响人类的健康。相关研究证实人类肿瘤发病率的升高与许多环境污染有关,来自动物食品的某些残留兽药的致突变和致癌作用可能是重要的原因之一。喹恶啉类药物(Quinoxalines)是人工合成的一类具有喹恶啉-1,4-二氧化物(quinoxaline-1,4-dioxides, QdNOs)基本结构的化合物,可抑制多种细菌的生长和繁殖,包括金黄色葡萄球菌、大肠杆菌和沙门氏菌等。卡巴氧和喹乙醇作为两种传统的QdNOs,体内和体外的大量研究发现这两种药物存在着潜在的致突变性和致癌性。正因为如此,欧洲共同体委员会(Commission of the European Community, CEC)于1999年将这两种化学物列为禁止使用的动物促生长剂。然而,作为一种新型的替代产品,喹烯酮(Quinocetone, QCT)与其他QdNOs也存在着相同的主体结构,在大量关注其药效的同时,喹烯酮药物使用的安全性也日益受到人们的关注。有限的资料显示,体外研究报道,QCT可能导致细菌和哺乳动物细胞株的致突变作用,并能引起HepG2细胞株的DNA损伤。但是目前关于QCT在体内引起致突变作用的研究很有限,且存在较大争议,因此选用对致癌性化学物较敏感的实验动物种群,并结合短期和长期实验,对于准确评价喹烯酮对哺乳动物的遗传毒性评价有重要的科学价值,并为制定相应的使用规范和标准有参考价值。氧化应激是许多外源化合物导致机体毒性作用的主要早期分子机制之一,有研究证明QdNOs的一些成员如卡巴氧和喹乙醇诱导的DNA损伤与在细胞内产生的ROS堆积直接相关。由于QCT与其他QdNOs一样具有相同的主体结构,其代谢过程中是否也能引起机体氧化应激,进而导致氧化性DNA损伤,目前还未见报道。
许多研究已经证实,应激性反应蛋白的激活是机体应对外源化合物引起毒性作用的早期保护性机制,其中热休克蛋白家族(heat shock protein, HSP)是当前研究的热点。在众多候选基因之中,血红素氧化酶-1(heme oxygenase-1, HO-1)因具有较强的抗炎、抗氧化和抗凋亡作用,因此与多种氧化应激相关性损伤密切相关。正是由于HO-1对氧化损伤的多重防御效应和良好的应激性、可诱导性,近年来它已经成为预防氧化损伤的重要靶基因,被广泛关注。大量研究发现,多种膳食抗氧化物能够诱导HO-1的表达并发挥抗氧化功能。普洱茶是生长在我国云贵高原特有的茶种,相关研究发现普洱茶提取液(BTE)具有较强的抗氧化作用,抗高脂血症和减肥功能,而发挥其功能作用的主要机制是能有有效清除体内过多的活性氧自由基和活性氮物质。提示普洱茶可能通过其强的抗氧化活性,降低外源性毒物引起机体的氧化性DNA损伤,进而发挥其抗突变作用,但是这一推断目前尚未在体内实验中证明,此外关于普洱茶对HO-1的调节作用目前还没有相关的研究。
因此,为进一步评价喹烯酮在体内的遗传毒性及其分子毒作用机制,本研究通过急性和亚慢性暴露动物染毒,探讨氧化应激与遗传毒性之间的关系,并研究HO-1在外源性化合物作用下在遗传毒性中的分子靶点作用。同时,通过普洱茶提取液补充实验,探索研究BTE对QCT诱导的遗传毒性的保护作用及其相关分子机制。
第一部分急性暴露下喹烯酮对Balb/c小鼠遗传毒性研究
目的：探讨QCT对Balb/c小鼠的致突变作用,并初步探讨QCT致突变作用与氧化应激的相互关系。
方法：雄性Balb/c小鼠(48只)及雌性小鼠(48只),同一性别小鼠随机分为以下8组：(1)阴性对照组(Control)：0.5%羧甲基纤维素钠水溶液灌胃；(2)喹烯酮高剂量组(QCT-H):12000 mg/kg·bw;(3)喹烯酮中剂量组(QCT-M):mg/kg-bw;(4)喹烯酮低剂量组(QCT-L):3000 mg/kg·bw;(5)阳性对照组(CY)：环磷酰胺40 mg/kg-bw腹腔注射。第二次灌胃4～6h颈椎脱臼处死动物,取胸骨骨髓涂片,Giemsa染色检查嗜多染红细胞(PCE)微核发生率。取新鲜肝、肾组织,制作单细胞悬液,碱性凝胶电泳实验评价肝肾分离细胞DNA损伤程度。制作肝、肾组织冰冻切片,DHE染色,荧光显微镜下观察荧光强度,并计算平均荧光密度。制备肝、肾组织线粒体匀浆,检测GSH水平和总抗氧化能力(T-AOC),以及GPx, SOD和CAT活力。
结果：(1)高剂量QCT组雌、雄性Balb/c小鼠骨髓细胞微核发生率分别为9.2‰和9.4‰,均显著性高于阴性对照组。中、低剂量QCT组小鼠微核发生率与阴性对照组相比无统计学差异。
(2)彗星实验结果显示急性QCT暴露均可引起Balb/c小鼠肝、肾分离细胞的DNA损伤。对于肝分离细胞,除了3000 mg/kg·bw QCT组,其余QCT剂量组OTM值显著性高于阴性对照组；对于肾分离细胞,所有QCT剂量组OTM均显著性高于阴性对照组。
(3)肝、肾细胞中ROS水平呈剂量依赖性升高,同时GSH水平和T-AOC水平在高剂量QCT组线粒体匀浆中显著性低于对照组,GPx, SOD和CAT活性也受到明显抑制。
结论：(1)喹烯酮急性暴露能够引起Balb/c小鼠骨髓PCE微核实验阳性和肝、肾分离细胞DNA损伤程度增加,提示喹烯酮对哺乳动物具有潜在的遗传毒性。
(2)喹烯酮急性暴露引起的遗传毒性跟其代谢中产生过多的ROS以及对抗氧化系统的抑制有直接关系。
第二部分：亚慢性暴露下喹烯酮对SD大鼠遗传毒性及分子机制研究
目的：以QCT在SD大鼠中的亚慢性暴露造模,以肝脏为主要的靶器官,进一步探讨QCT较长时期的暴露对大鼠的遗传毒性及其分子机制。
方法：成年雄性SD大鼠(160～180g)72只随机分为4组：喹烯酮高剂量组(QCT-H):2400 mg/kg/day;喹烯酮中剂量组(QCT-M):800 mg/kg/day;喹烯酮低剂量组(QCT-H):50 mg/kg/day;正常对照组(control):0 mg/kg/day。在喂养期间分别于第5 week(8只)和第14 week(10只)处死一批老鼠,分别检测血清ALT, AST, Cr和BUN水平和尿8-OHdG含量。制备13 week SD大鼠肝细胞悬液,彗星实验测定肝细胞DNA损伤程度。检测肝细胞ROS水平以及肝组织匀浆中GSH, MDA含量,GPx, GST, GR, SOD和CAT活性。RT-PCR法检测碱基切除修复相关基因OGG1, XRCC1, PARP1, PARP2和PARG mRNA水平。RT-PCR和western blot法分别检测炎症相关基因和凋亡相关基因mRNA和蛋白表达水平。同时,分别检测喹烯酮暴露4 week和13 week SD大鼠肝脏HO-1蛋白表达水平。
结果：(1) 2400 mg/kg/day喹烯酮暴露组SD大鼠体重显著性低于对照组,肝脏相对脏器重量显著性高于正常对照组。血清生化结果显示：QCT染毒大鼠血清ALT, AST水平呈剂量依赖型和时间依赖型升高,且高剂量QCT组大鼠血清ALT和AST水平均显著性高于对照组(P<0.05)。此外肝脏病理结果显示肝细胞内呈现大量空泡样改变,汇管区出现单核细胞侵润和结缔组织增生性改变,可见少量肝细胞坏死和凋亡现象。
(2)彗星结果显示高剂量QCT组大鼠肝脏分离细胞DNA损伤程度明显增加,其OTM和Tail DNA%值显著性高于对照组(P<0.01)。
(3)肝细胞内ROS,尿8-OHdG和肝组织匀浆MDA水平呈剂量依赖性升高,而抗氧化系统包括GSH水平,GPx, GST, GR, SOD和CAT活力呈剂量依赖性降低。其中高剂量QCT组各个指标与对照组相比,差异均具有显著性(P<0.05或P<0.01)。
(4)与对照组相比,QCT亚慢性暴露后OGG1 mRNA表达呈剂量依赖性升高,其中高剂量QCT组大鼠肝脏OGG1 mRNA升至对照组的5.15倍(P<0.01)。此外,在OGG1介导的碱基切除修复通路相关酶mRNA水平,如Xrcc1, PARP1和IPARP2 mRNA表达水平均显著性升高(P<0.05)。
(5)高剂量QCT的亚慢性暴露,可直接造成NF-κB mRNA表达的上升,同时也增加iNOS, TNF-a和Cox-2多个基因InRNA的表达水平(P<0.05)。高剂量QCT可造成p53 mRNA水平的显著性增高,同时casepase-3 mRNA水平显著性升高(P<0.05),以及对凋亡反应有一定抑制作用的Bcl-xl mRNA水平一定程度的下降,但是与对照组相比其差异没有显著性(P>0.05)。而各个基因蛋白表达水平与mRNA水平相一致。
(6)高剂量组HO-1蛋白水平呈剂量依赖性和时间依赖性降低,其中QCT染毒三个月SD大鼠肝脏中HO-1蛋白水平显著性低于对照组(P<0.05), Nrf-2mRNA水平和蛋白水平也显著性低于对照组(P<0.05),而Keap-1蛋白水平在各个组别之间没有显著性差异(P>0.05)。
结论：(1)QCT亚慢性染毒可造成肝细胞DNA损伤程度增加和OGG1修复系统活化,并产生大量的DNA加合物,进一步提示其具有潜在的遗传毒性。
(2)HO-1的表达抑制造成的细胞内高氧化应激可能是QCT诱导的遗传毒性的重要分子机制。
第三部分：普洱茶水提取液对喹烯酮诱导的遗传毒性的保护作用及其机制研究
目的：探讨普洱茶水提取液对喹烯酮亚慢性暴露所致遗传的保护作用及其机制
方法：成年雄性SD大鼠(160～180g)40只随机分为5组：喹烯酮染毒组(QCT)：2400mg/kg/day;普洱茶补充高剂量组(QCT+BTE-H):2400 mg/kg/day QCT+1000 mg/kg/dayBTE；普洱茶补充低剂量组(QCT+BTE-L):2400 mg/kg/day QCT+500 mg/kg/day BTE;正常对照组(control)正常进食；普洱茶对照组：1000 mg/kg/day BTE灌胃。所有SD大鼠喂养13周,每周记录体重和进食量,于13周末处死所有大鼠。分离血清检测ALT和AST水平,分离整个肝脏计算肝脏相对重量并做病理切片H＆E染色。彗星实验检测肝细胞DNA损伤程度,高效液相色谱电化学法检测尿液8-OHdG水平,冰冻切片检测肝细胞ROS水平,制作组织匀浆检测GSH和MDA含量,GPx, GST, GR, SOD和CAT活性。RT-PCR法检测HO-1, iNOS, TNF-a, Cox-2, p53, Bcl-xl和casepase-3 mRNA水平,western blot法检测HO-1, Nrf-2, Keap-1, iNOS, NF-κB, Cox-2, Bcl-xl casepase-3, ERK 42/44和pERK 42/44 (Thr202/Tyr204)蛋白表达水平。
结果：(1)喹烯酮染组SD大鼠体重,进食量显著性低于对照组,肝脏脏器系数,血清ALT和AST水平显著性高于对照组(P<0.05或P<0.01)。肝脏病理结果显示肝细胞内呈现大量空泡样改变,汇管区出现单核细胞侵润和结缔组织增生性改变,可见少量肝细胞坏死和凋亡现象。BTE补充组能在一定程度上改善QCT染毒造成的肝功能损伤,表现为降低血清ALT和AST水平,减轻QCT染毒诱导的肝脏病理损伤。
(2)各个剂量的BTE补充均能够降低QCT染毒所致的DNA损伤,而BTE本身对肝细胞DNA没有DNA损伤作用。
(3)各个剂量的BTE补充能提高肝脏中HO-1 mRNA和蛋白水平,并能提高Nrf-2蛋白水平。此外Keap-1蛋白在BTE对照组呈高表达状态,而其他各组间的差别不显著(P>0.05)。
(4)各个组别间ERK42/44蛋白表达水平没有明显差别(P>0.05),但是BTE补充能提高ERK42/44的磷酸化水平。
(5)BTE补充能有效降低QCT染毒引起的高ROS,8-OHdG和MDA状态,并能上调肝脏线粒体抗氧化系统,包括GSH含量,GPx, GST, GR, SOD和CAT活力。
(6)与QCT染毒组相比,BTE补充能够改善SD大鼠肝脏炎症反应和细胞凋亡。表现为降低iNOS, NF-κB, Cox-2, p53和casepase-3 mRNA和蛋白表达水平,提高Bcl-xlmRNA和蛋白表达水平。
结论：(1)BTE补充能改善QCT亚慢性染毒所致的DNA损伤。
(2)BTE通过提高ERK1/2的磷酸化水平,活化Nrf-2/HO-1通路,并抑制氧化应激,继而发挥抗突变作用。
本研究的创新之处
(1)本研究第一次证实喹烯酮药物能够对实验动物造成遗传毒性。
(2)本研究从DNA损伤和修复、氧化应激两方面探讨喹烯酮遗传毒性的分子毒理学机制。
(3)喹烯酮可能通过抑制HO-1的表达,造成肝脏细胞内高氧化应激状态而导致DNA损伤。进而证明HO-1可能是QCT导致SD大鼠遗传毒性的分子靶点。
(4)普洱茶作为一种抗氧化物质,可通过活化ERK1/2上调HO-1的表达,并抑制氧化应激,继而发挥抗DNA损伤作用。
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 exposure
Objectives: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 rats
Objectives: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 rats
Objectives: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.
许多研究已经证实,应激性反应蛋白的激活是机体应对外源化合物引起毒性作用的早期保护性机制,其中热休克蛋白家族(heat shock protein, HSP)是当前研究的热点。在众多候选基因之中,血红素氧化酶-1(heme oxygenase-1, HO-1)因具有较强的抗炎、抗氧化和抗凋亡作用,因此与多种氧化应激相关性损伤密切相关。正是由于HO-1对氧化损伤的多重防御效应和良好的应激性、可诱导性,近年来它已经成为预防氧化损伤的重要靶基因,被广泛关注。大量研究发现,多种膳食抗氧化物能够诱导HO-1的表达并发挥抗氧化功能。普洱茶是生长在我国云贵高原特有的茶种,相关研究发现普洱茶提取液(BTE)具有较强的抗氧化作用,抗高脂血症和减肥功能,而发挥其功能作用的主要机制是能有有效清除体内过多的活性氧自由基和活性氮物质。提示普洱茶可能通过其强的抗氧化活性,降低外源性毒物引起机体的氧化性DNA损伤,进而发挥其抗突变作用,但是这一推断目前尚未在体内实验中证明,此外关于普洱茶对HO-1的调节作用目前还没有相关的研究。
因此,为进一步评价喹烯酮在体内的遗传毒性及其分子毒作用机制,本研究通过急性和亚慢性暴露动物染毒,探讨氧化应激与遗传毒性之间的关系,并研究HO-1在外源性化合物作用下在遗传毒性中的分子靶点作用。同时,通过普洱茶提取液补充实验,探索研究BTE对QCT诱导的遗传毒性的保护作用及其相关分子机制。
第一部分急性暴露下喹烯酮对Balb/c小鼠遗传毒性研究
目的：探讨QCT对Balb/c小鼠的致突变作用,并初步探讨QCT致突变作用与氧化应激的相互关系。
方法：雄性Balb/c小鼠(48只)及雌性小鼠(48只),同一性别小鼠随机分为以下8组：(1)阴性对照组(Control)：0.5%羧甲基纤维素钠水溶液灌胃；(2)喹烯酮高剂量组(QCT-H):12000 mg/kg·bw;(3)喹烯酮中剂量组(QCT-M):mg/kg-bw;(4)喹烯酮低剂量组(QCT-L):3000 mg/kg·bw;(5)阳性对照组(CY)：环磷酰胺40 mg/kg-bw腹腔注射。第二次灌胃4～6h颈椎脱臼处死动物,取胸骨骨髓涂片,Giemsa染色检查嗜多染红细胞(PCE)微核发生率。取新鲜肝、肾组织,制作单细胞悬液,碱性凝胶电泳实验评价肝肾分离细胞DNA损伤程度。制作肝、肾组织冰冻切片,DHE染色,荧光显微镜下观察荧光强度,并计算平均荧光密度。制备肝、肾组织线粒体匀浆,检测GSH水平和总抗氧化能力(T-AOC),以及GPx, SOD和CAT活力。
结果：(1)高剂量QCT组雌、雄性Balb/c小鼠骨髓细胞微核发生率分别为9.2‰和9.4‰,均显著性高于阴性对照组。中、低剂量QCT组小鼠微核发生率与阴性对照组相比无统计学差异。
(2)彗星实验结果显示急性QCT暴露均可引起Balb/c小鼠肝、肾分离细胞的DNA损伤。对于肝分离细胞,除了3000 mg/kg·bw QCT组,其余QCT剂量组OTM值显著性高于阴性对照组；对于肾分离细胞,所有QCT剂量组OTM均显著性高于阴性对照组。
(3)肝、肾细胞中ROS水平呈剂量依赖性升高,同时GSH水平和T-AOC水平在高剂量QCT组线粒体匀浆中显著性低于对照组,GPx, SOD和CAT活性也受到明显抑制。
结论：(1)喹烯酮急性暴露能够引起Balb/c小鼠骨髓PCE微核实验阳性和肝、肾分离细胞DNA损伤程度增加,提示喹烯酮对哺乳动物具有潜在的遗传毒性。
(2)喹烯酮急性暴露引起的遗传毒性跟其代谢中产生过多的ROS以及对抗氧化系统的抑制有直接关系。
第二部分：亚慢性暴露下喹烯酮对SD大鼠遗传毒性及分子机制研究
目的：以QCT在SD大鼠中的亚慢性暴露造模,以肝脏为主要的靶器官,进一步探讨QCT较长时期的暴露对大鼠的遗传毒性及其分子机制。
方法：成年雄性SD大鼠(160～180g)72只随机分为4组：喹烯酮高剂量组(QCT-H):2400 mg/kg/day;喹烯酮中剂量组(QCT-M):800 mg/kg/day;喹烯酮低剂量组(QCT-H):50 mg/kg/day;正常对照组(control):0 mg/kg/day。在喂养期间分别于第5 week(8只)和第14 week(10只)处死一批老鼠,分别检测血清ALT, AST, Cr和BUN水平和尿8-OHdG含量。制备13 week SD大鼠肝细胞悬液,彗星实验测定肝细胞DNA损伤程度。检测肝细胞ROS水平以及肝组织匀浆中GSH, MDA含量,GPx, GST, GR, SOD和CAT活性。RT-PCR法检测碱基切除修复相关基因OGG1, XRCC1, PARP1, PARP2和PARG mRNA水平。RT-PCR和western blot法分别检测炎症相关基因和凋亡相关基因mRNA和蛋白表达水平。同时,分别检测喹烯酮暴露4 week和13 week SD大鼠肝脏HO-1蛋白表达水平。
结果：(1) 2400 mg/kg/day喹烯酮暴露组SD大鼠体重显著性低于对照组,肝脏相对脏器重量显著性高于正常对照组。血清生化结果显示：QCT染毒大鼠血清ALT, AST水平呈剂量依赖型和时间依赖型升高,且高剂量QCT组大鼠血清ALT和AST水平均显著性高于对照组(P<0.05)。此外肝脏病理结果显示肝细胞内呈现大量空泡样改变,汇管区出现单核细胞侵润和结缔组织增生性改变,可见少量肝细胞坏死和凋亡现象。
(2)彗星结果显示高剂量QCT组大鼠肝脏分离细胞DNA损伤程度明显增加,其OTM和Tail DNA%值显著性高于对照组(P<0.01)。
(3)肝细胞内ROS,尿8-OHdG和肝组织匀浆MDA水平呈剂量依赖性升高,而抗氧化系统包括GSH水平,GPx, GST, GR, SOD和CAT活力呈剂量依赖性降低。其中高剂量QCT组各个指标与对照组相比,差异均具有显著性(P<0.05或P<0.01)。
(4)与对照组相比,QCT亚慢性暴露后OGG1 mRNA表达呈剂量依赖性升高,其中高剂量QCT组大鼠肝脏OGG1 mRNA升至对照组的5.15倍(P<0.01)。此外,在OGG1介导的碱基切除修复通路相关酶mRNA水平,如Xrcc1, PARP1和IPARP2 mRNA表达水平均显著性升高(P<0.05)。
(5)高剂量QCT的亚慢性暴露,可直接造成NF-κB mRNA表达的上升,同时也增加iNOS, TNF-a和Cox-2多个基因InRNA的表达水平(P<0.05)。高剂量QCT可造成p53 mRNA水平的显著性增高,同时casepase-3 mRNA水平显著性升高(P<0.05),以及对凋亡反应有一定抑制作用的Bcl-xl mRNA水平一定程度的下降,但是与对照组相比其差异没有显著性(P>0.05)。而各个基因蛋白表达水平与mRNA水平相一致。
(6)高剂量组HO-1蛋白水平呈剂量依赖性和时间依赖性降低,其中QCT染毒三个月SD大鼠肝脏中HO-1蛋白水平显著性低于对照组(P<0.05), Nrf-2mRNA水平和蛋白水平也显著性低于对照组(P<0.05),而Keap-1蛋白水平在各个组别之间没有显著性差异(P>0.05)。
结论：(1)QCT亚慢性染毒可造成肝细胞DNA损伤程度增加和OGG1修复系统活化,并产生大量的DNA加合物,进一步提示其具有潜在的遗传毒性。
(2)HO-1的表达抑制造成的细胞内高氧化应激可能是QCT诱导的遗传毒性的重要分子机制。
第三部分：普洱茶水提取液对喹烯酮诱导的遗传毒性的保护作用及其机制研究
目的：探讨普洱茶水提取液对喹烯酮亚慢性暴露所致遗传的保护作用及其机制
方法：成年雄性SD大鼠(160～180g)40只随机分为5组：喹烯酮染毒组(QCT)：2400mg/kg/day;普洱茶补充高剂量组(QCT+BTE-H):2400 mg/kg/day QCT+1000 mg/kg/dayBTE；普洱茶补充低剂量组(QCT+BTE-L):2400 mg/kg/day QCT+500 mg/kg/day BTE;正常对照组(control)正常进食；普洱茶对照组：1000 mg/kg/day BTE灌胃。所有SD大鼠喂养13周,每周记录体重和进食量,于13周末处死所有大鼠。分离血清检测ALT和AST水平,分离整个肝脏计算肝脏相对重量并做病理切片H＆E染色。彗星实验检测肝细胞DNA损伤程度,高效液相色谱电化学法检测尿液8-OHdG水平,冰冻切片检测肝细胞ROS水平,制作组织匀浆检测GSH和MDA含量,GPx, GST, GR, SOD和CAT活性。RT-PCR法检测HO-1, iNOS, TNF-a, Cox-2, p53, Bcl-xl和casepase-3 mRNA水平,western blot法检测HO-1, Nrf-2, Keap-1, iNOS, NF-κB, Cox-2, Bcl-xl casepase-3, ERK 42/44和pERK 42/44 (Thr202/Tyr204)蛋白表达水平。
结果：(1)喹烯酮染组SD大鼠体重,进食量显著性低于对照组,肝脏脏器系数,血清ALT和AST水平显著性高于对照组(P<0.05或P<0.01)。肝脏病理结果显示肝细胞内呈现大量空泡样改变,汇管区出现单核细胞侵润和结缔组织增生性改变,可见少量肝细胞坏死和凋亡现象。BTE补充组能在一定程度上改善QCT染毒造成的肝功能损伤,表现为降低血清ALT和AST水平,减轻QCT染毒诱导的肝脏病理损伤。
(2)各个剂量的BTE补充均能够降低QCT染毒所致的DNA损伤,而BTE本身对肝细胞DNA没有DNA损伤作用。
(3)各个剂量的BTE补充能提高肝脏中HO-1 mRNA和蛋白水平,并能提高Nrf-2蛋白水平。此外Keap-1蛋白在BTE对照组呈高表达状态,而其他各组间的差别不显著(P>0.05)。
(4)各个组别间ERK42/44蛋白表达水平没有明显差别(P>0.05),但是BTE补充能提高ERK42/44的磷酸化水平。
(5)BTE补充能有效降低QCT染毒引起的高ROS,8-OHdG和MDA状态,并能上调肝脏线粒体抗氧化系统,包括GSH含量,GPx, GST, GR, SOD和CAT活力。
(6)与QCT染毒组相比,BTE补充能够改善SD大鼠肝脏炎症反应和细胞凋亡。表现为降低iNOS, NF-κB, Cox-2, p53和casepase-3 mRNA和蛋白表达水平,提高Bcl-xlmRNA和蛋白表达水平。
结论：(1)BTE补充能改善QCT亚慢性染毒所致的DNA损伤。
(2)BTE通过提高ERK1/2的磷酸化水平,活化Nrf-2/HO-1通路,并抑制氧化应激,继而发挥抗突变作用。
本研究的创新之处
(1)本研究第一次证实喹烯酮药物能够对实验动物造成遗传毒性。
(2)本研究从DNA损伤和修复、氧化应激两方面探讨喹烯酮遗传毒性的分子毒理学机制。
(3)喹烯酮可能通过抑制HO-1的表达,造成肝脏细胞内高氧化应激状态而导致DNA损伤。进而证明HO-1可能是QCT导致SD大鼠遗传毒性的分子靶点。
(4)普洱茶作为一种抗氧化物质,可通过活化ERK1/2上调HO-1的表达,并抑制氧化应激,继而发挥抗DNA损伤作用。
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 exposure
Objectives: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 rats
Objectives: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 rats
Objectives: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.
引文
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