Nephrotoxicity Induced By Aristolochic Acid And Mechanism Of Its DNA Damage

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, tubular 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 withcollecting 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...