马兜铃酸的肾毒性及DNA损伤机制研究
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摘要
自1964年以来，马兜铃属(Aristolochia)植物及其成分马兜铃酸(Aristolochic acid，AA)引起急性肾衰屡有报道。1993年比利时医生Vanherweghem报道了某诊所数例服用含马兜铃属植物减肥药者患慢性肾病。当时国际上称这种慢性肾损害为“中草药肾病”(Chinese Herbs Nephropathy，CHN)。此后多个国家和地区均有CHN的报道。在大量病因学研究基础上，我国学者提出“中草药肾病”的命名欠妥，应称之为马兜铃酸肾病(Aristolochic Acid Nephropathy，AAN)。据报道，含铁线莲状马兜铃的面粉可引起巴尔干肾病(Balkan Emdemic Nephropathy，BEN)，其临床表现和病理改变与CHN极为相似，该病年龄标化发病率女性高于男性。但含AA植物所致实验动物急性肾损害是否存在性别差异，作者尚未见国内外报道。目前对AAN机制研究表明，AA可致肾小管上皮细胞坏死、凋亡，小管上皮-肌成纤维细胞转分化，AA-DNA加合物形成等。对其DNA损伤的类型、生物学效应等方面尚需进一步研究。本研究采用整体动物试验系统，建立我国临床应用最广泛的关木通(Aristolochia manshuriensis Kom.,AMK)所致大鼠急性肾损害模型，研究其肾毒性及其性别差异，并从肾代谢酶性别二态性探讨其可能原因。利用体外试验研究AA所致DNA损伤及其代谢活化作用。为进一步探索AA肾毒性机制，阐明易感性提供毒理学依据。
方法：以传统方法制备关木通水煎剂，测定其AA含量。健康成年Wistar大鼠60只，雌雄各半，按体重随机分为3组：对照组，低剂量组(25g/kg)，高剂量组(45g/kg)。每日灌胃2次，连续给药5天。给药后第6天经腹主动脉采血处死大鼠。观测指标包括：体重，摄食量，肾脏系数及组织形态学观察，血尿素氮(BUN)、肌酐(SCr)含量，肾皮质微粒体细胞色素P450 1A1(CYP1A1)酶活性，并比较其性别差异。体外培养人胚肾293细胞系与转染了核苷酸切除修复基因的ERCC1-XPF-293细胞系，采用MTT比色法比较AA对两细胞系毒性差异。采用单细胞凝胶电泳(SCGE)试验研究AA对293
细胞致DNA链断裂作用。利用AA与牛胸腺DNA(CT一DNA)共孵育体系
研究AA致DNA交联作用，并在该体系中加入大鼠肝微粒体及特异性代谢
酶抑制剂研究了AA所致DNA交联的代谢活化作用。
结果:AMK每日经口给药459/kg(以AA计12 0 mg/kg)连续5天，
可引起大鼠明显的肾毒性。表现为体重降低，肾脏系数升高，BUN、SCr水
平升高，与对照组比较有显著性差别;组织形态学表现为肾小管上皮细胞水
肿、变性及坏死。两性间肾功能存在明显差异，雌性更敏感，其BUN、SCr
约为雄性3倍。肾皮质微粒体CYPIAI酶活性在对照组即存在明显性别差异，
未见AMK对该酶有明显的诱导或抑制作用，高剂量组大鼠5 Cr与CYPI Al
间存在正相关关系(r二0.5743，尸=O，0081)。MTT试验表明，AA对293与
ERCCI一XpF一293细胞系的半数抑制浓度(ICS。)分别为163.3和225.lp
g/mL，差异具有显著性。SCGE试验结果显示，AA在20 p g/mL即具有致DNA
链断裂作用。AA与CT一DNA共孵育不引起DNA交联，但经大鼠肝微粒体
代谢引起DNA交联，加入CYP IA抑制剂。一蔡黄酮(。一NY)降低DNA交联
率，而CYPZEI、ZD、3A抑制剂不改变其交联率。
结论:AMK每日经口给药459/kg(以AA计12 0 mg/kg)连续5天，
可引起大鼠明显的肾损害，雌性较雄性更敏感。AMK所致大鼠肾毒性性别
差异可能与肾CYP 1 Al酶活性有关。AA对体外培养肾细胞具有细胞毒作用，
部分是由于DNA损伤所致。AA具有致DNA链断裂作用，其代谢物具有致
DNA交联作用，CYP IA对AA所致DNA交联具有活化作用。
Introduction It have been widely reported that aristolochia species plants and its ingredient aristolochic acid(AA) would induce acute renal failure in clinical since 1964. In 1993 Belgian doctor Vanherweghem reported several chronic renal disease cases who followed the same slimming regimen containing aristolochia in certain clinic. It was designated "Chinese Herbs Nephropathy"(CHN) internationally at that time. To date it have been reported in many countries and regions. Based on a great deal of etiological research, scholars in our country alleged it should be named by "Aristolochic Acid Nephropathy"(AAN) rather than CHN. It is similar between CHN and Balkan Emdemic Nephropathy(BEN) induced by flour containing A. dematins on clinical and pathological grounds, and its incidence standardized by age is higher in female than that in male. But it is unknown whether it is the same case in animal model in home and abroad . Studies in mechanisms of AAN showed that AA induced renal tubular necrosis or apoptosis, t
ubular epithelial-myofibroblast transdifferentiation . AA-DNA adducts formation and so on . It needs further study to probe the types and biology effects of its DNA damage . In this study , acute nephro -toxicity animal model induced by Aristolochia manshuriensis Kom. (AMK) , the most used in our country , was established , in order to study its sex difference in nephrotoxicity , and explore its possible mechanism based on sex dimorphism of metabolic enzyme in renal cortex . In vitro test , determinations of the DNA damage induced by AA and its metabolic activation were performed . It would provide help to reveal the mechanism of AAN and clarity its susceptibility .
Methods Prepared AMK decoction with traditional method and determined its contents of AA . Sixty adult Wistar rats (female and male in half) were randomly divided into three groups: the control, low AMK (25 g/kg), high AMK(45 g/kg). Rats were treated orally twice a day for 5 consecutive days, and sacrificed with
collecting blood via abdomial aorta on day 6. The following indices were observed and determined: body weight, food intake, kidney index and histological examination, contents of blood urea nitrogen (BUN) and serum creatinine (SCr) , and activities of cytochrom P4501A1(CYP1A1) in renal cortex as well. Comparing the sex difference in above indices. In cultured human embryonic kidney cell lines 293 and ERCC1-XPF-293 transfected with nucleotide excision repair genes, cytotoxiciry difference between them induced by AA with MTT test was performed. With single cell gel electrophoresis(SCGE) assay the DNA strand-breaks induced by AA in 293 was observed. To probe the DNA cross -links induced by AA and its metabolic activation . co-incubatin system both AA and calf thymus DNA(CT-DNA), liver microsome of rat, and specific inhibitors of CYPs as well were used.
Results AMK(45 g/kg per day orally, the dose equivalent to AA 120 mg/kg for 5 consecutive days) caused noticeable nephrotoxicity in rats and characterized by body weight loss, increase of kidney index and contents of BUN and SCr, which have significance compared with the control. Histologically, edema,denaturation and necrosis in renal tubular epithelium were observed . There was remarkable sex difference in renal function between male and female , the latter was more sensitive than the former. The contents of BUN and SCr in female were about three times as degree as those in male. In control, there were markedly difference in activities of CYP1A1 in renal cortex, and no sign showed AMK could induce or inhibit its activitites. There were correlation between SCr and CYP1A1 ( r=0.5743 -P=0.0081) of rats in high AMK . MTT test showed there were significant difference in IC50 values between AA treated 293 and ERCC1-XPF-293 cells (163.3 and 225.1 n g/mL, respectively). SCGE assay showed AA above 20 u g/mL induced DNA strand-breaks. There was no DNA cross-links in co-incubation system involved CT-DNA and AA alone, whereas DNA cross-links were observed when added liver microsome of rat , whic
方法：以传统方法制备关木通水煎剂，测定其AA含量。健康成年Wistar大鼠60只，雌雄各半，按体重随机分为3组：对照组，低剂量组(25g/kg)，高剂量组(45g/kg)。每日灌胃2次，连续给药5天。给药后第6天经腹主动脉采血处死大鼠。观测指标包括：体重，摄食量，肾脏系数及组织形态学观察，血尿素氮(BUN)、肌酐(SCr)含量，肾皮质微粒体细胞色素P450 1A1(CYP1A1)酶活性，并比较其性别差异。体外培养人胚肾293细胞系与转染了核苷酸切除修复基因的ERCC1-XPF-293细胞系，采用MTT比色法比较AA对两细胞系毒性差异。采用单细胞凝胶电泳(SCGE)试验研究AA对293
细胞致DNA链断裂作用。利用AA与牛胸腺DNA(CT一DNA)共孵育体系
研究AA致DNA交联作用，并在该体系中加入大鼠肝微粒体及特异性代谢
酶抑制剂研究了AA所致DNA交联的代谢活化作用。
结果:AMK每日经口给药459/kg(以AA计12 0 mg/kg)连续5天，
可引起大鼠明显的肾毒性。表现为体重降低，肾脏系数升高，BUN、SCr水
平升高，与对照组比较有显著性差别;组织形态学表现为肾小管上皮细胞水
肿、变性及坏死。两性间肾功能存在明显差异，雌性更敏感，其BUN、SCr
约为雄性3倍。肾皮质微粒体CYPIAI酶活性在对照组即存在明显性别差异，
未见AMK对该酶有明显的诱导或抑制作用，高剂量组大鼠5 Cr与CYPI Al
间存在正相关关系(r二0.5743，尸=O，0081)。MTT试验表明，AA对293与
ERCCI一XpF一293细胞系的半数抑制浓度(ICS。)分别为163.3和225.lp
g/mL，差异具有显著性。SCGE试验结果显示，AA在20 p g/mL即具有致DNA
链断裂作用。AA与CT一DNA共孵育不引起DNA交联，但经大鼠肝微粒体
代谢引起DNA交联，加入CYP IA抑制剂。一蔡黄酮(。一NY)降低DNA交联
率，而CYPZEI、ZD、3A抑制剂不改变其交联率。
结论:AMK每日经口给药459/kg(以AA计12 0 mg/kg)连续5天，
可引起大鼠明显的肾损害，雌性较雄性更敏感。AMK所致大鼠肾毒性性别
差异可能与肾CYP 1 Al酶活性有关。AA对体外培养肾细胞具有细胞毒作用，
部分是由于DNA损伤所致。AA具有致DNA链断裂作用，其代谢物具有致
DNA交联作用，CYP IA对AA所致DNA交联具有活化作用。
Introduction It have been widely reported that aristolochia species plants and its ingredient aristolochic acid(AA) would induce acute renal failure in clinical since 1964. In 1993 Belgian doctor Vanherweghem reported several chronic renal disease cases who followed the same slimming regimen containing aristolochia in certain clinic. It was designated "Chinese Herbs Nephropathy"(CHN) internationally at that time. To date it have been reported in many countries and regions. Based on a great deal of etiological research, scholars in our country alleged it should be named by "Aristolochic Acid Nephropathy"(AAN) rather than CHN. It is similar between CHN and Balkan Emdemic Nephropathy(BEN) induced by flour containing A. dematins on clinical and pathological grounds, and its incidence standardized by age is higher in female than that in male. But it is unknown whether it is the same case in animal model in home and abroad . Studies in mechanisms of AAN showed that AA induced renal tubular necrosis or apoptosis, t
ubular epithelial-myofibroblast transdifferentiation . AA-DNA adducts formation and so on . It needs further study to probe the types and biology effects of its DNA damage . In this study , acute nephro -toxicity animal model induced by Aristolochia manshuriensis Kom. (AMK) , the most used in our country , was established , in order to study its sex difference in nephrotoxicity , and explore its possible mechanism based on sex dimorphism of metabolic enzyme in renal cortex . In vitro test , determinations of the DNA damage induced by AA and its metabolic activation were performed . It would provide help to reveal the mechanism of AAN and clarity its susceptibility .
Methods Prepared AMK decoction with traditional method and determined its contents of AA . Sixty adult Wistar rats (female and male in half) were randomly divided into three groups: the control, low AMK (25 g/kg), high AMK(45 g/kg). Rats were treated orally twice a day for 5 consecutive days, and sacrificed with
collecting blood via abdomial aorta on day 6. The following indices were observed and determined: body weight, food intake, kidney index and histological examination, contents of blood urea nitrogen (BUN) and serum creatinine (SCr) , and activities of cytochrom P4501A1(CYP1A1) in renal cortex as well. Comparing the sex difference in above indices. In cultured human embryonic kidney cell lines 293 and ERCC1-XPF-293 transfected with nucleotide excision repair genes, cytotoxiciry difference between them induced by AA with MTT test was performed. With single cell gel electrophoresis(SCGE) assay the DNA strand-breaks induced by AA in 293 was observed. To probe the DNA cross -links induced by AA and its metabolic activation . co-incubatin system both AA and calf thymus DNA(CT-DNA), liver microsome of rat, and specific inhibitors of CYPs as well were used.
Results AMK(45 g/kg per day orally, the dose equivalent to AA 120 mg/kg for 5 consecutive days) caused noticeable nephrotoxicity in rats and characterized by body weight loss, increase of kidney index and contents of BUN and SCr, which have significance compared with the control. Histologically, edema,denaturation and necrosis in renal tubular epithelium were observed . There was remarkable sex difference in renal function between male and female , the latter was more sensitive than the former. The contents of BUN and SCr in female were about three times as degree as those in male. In control, there were markedly difference in activities of CYP1A1 in renal cortex, and no sign showed AMK could induce or inhibit its activitites. There were correlation between SCr and CYP1A1 ( r=0.5743 -P=0.0081) of rats in high AMK . MTT test showed there were significant difference in IC50 values between AA treated 293 and ERCC1-XPF-293 cells (163.3 and 225.1 n g/mL, respectively). SCGE assay showed AA above 20 u g/mL induced DNA strand-breaks. There was no DNA cross-links in co-incubation system involved CT-DNA and AA alone, whereas DNA cross-links were observed when added liver microsome of rat , whic
引文
1．马红梅，张伯礼．关木通肾毒害及其防治．中草药，2001，32(4)：369-370．
2. Jackson L, Kofman S, Weiss A, et al. Aristolochic acid (NSC-50413): phase Ⅰ clinical study. Cancer Chemother Rep, 1964, 42(1):35-37.
3. Vanherweghem JL, Depierreux M, Tielemans C, et al. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs.Lancet, 1993, 341(8842): 387-391.
4. Vanherweghem LJ. Misuse of herbal remedies: the case of an outbreak of terminal renal failure in Belgium (Chinese herbs nephropathy).J Altern Complement Med, 1998, 4(1):9-13.
5. Cosyns JP. Aristolochic Acid and "Chinese Herbs Nephropathy": a review of the evidence to date. Drug Saf, 2003, 26 (1): 33-48.
6．郑法雷，张晓明，黄庆元，等．慢性马兜铃酸肾病动物模型的建立及其意义．中华医学杂志，2001，81(18)：1095-1100．
7. Castegnaro M.Endemic nephropathy and urinary tract tumors in the Balkans. Cancer Res, 1987, 47(13): 3608-3609.
8．谌贻璞，陈文．马兜铃酸肾病的研究进展．肾脏病与透析肾移植杂志，2002，11(1)：63-66．
9．尚明英，李军，胡波，等．中药关木通中总马兜铃酸的含量测定．中草药，2000，31(12)：899-900．
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35. Cosyns JP, Jadoul M, Squifflet JP, et al. Urothelial lesions in Chinese-herb nephropathy.Am J Kidney Dis, 1999, 33(6): 1011-1017.
2. Jackson L, Kofman S, Weiss A, et al. Aristolochic acid (NSC-50413): phase Ⅰ clinical study. Cancer Chemother Rep, 1964, 42(1):35-37.
3. Vanherweghem JL, Depierreux M, Tielemans C, et al. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs.Lancet, 1993, 341(8842): 387-391.
4. Vanherweghem LJ. Misuse of herbal remedies: the case of an outbreak of terminal renal failure in Belgium (Chinese herbs nephropathy).J Altern Complement Med, 1998, 4(1):9-13.
5. Cosyns JP. Aristolochic Acid and "Chinese Herbs Nephropathy": a review of the evidence to date. Drug Saf, 2003, 26 (1): 33-48.
6．郑法雷，张晓明，黄庆元，等．慢性马兜铃酸肾病动物模型的建立及其意义．中华医学杂志，2001，81(18)：1095-1100．
7. Castegnaro M.Endemic nephropathy and urinary tract tumors in the Balkans. Cancer Res, 1987, 47(13): 3608-3609.
8．谌贻璞，陈文．马兜铃酸肾病的研究进展．肾脏病与透析肾移植杂志，2002，11(1)：63-66．
9．尚明英，李军，胡波，等．中药关木通中总马兜铃酸的含量测定．中草药，2000，31(12)：899-900．
10．唐跃年，张顺国，李岚，等．肝微粒体的制备和细胞色素P450氧化酶活性测定．中国医院药学杂志，1998，18(12)：535-536．
11. Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binging. Anal Biochem, 1976,72:248-254.
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