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Food and Chemical Toxicology 44 (2006) 28–35 www.elsevier.com/locate/foodchemtox
Induction of urothelial proliferation in rats by aristolochic acid through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2 Horng-Rong Chang a,b, Jong-Da Lian a, Chia-Wen Lo c, Yun-Ching Chang c, Mon-Yuan Yang c, Chau-Jong Wang c,* a
c
Division of Nephrology, Department of Internal Medicine, Chung-Shan Medical University Hospital, Taichung 402, Taiwan b Institute of Medicine, Chung-Shan Medical University, Taichung 402, Taiwan Institute of Biochemistry and Biotechnology, Chung-Shan Medical University, No. 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan Received 21 September 2004; accepted 1 June 2005
Abstract Aristolochic acid (AA) has been implicated in urothelial carcinoma in humans. However, the mechanism by which AA induces this cancer has not been completely established. To evaluate the effects of AA on the urinary bladder of rats, a histopathological study of three-month intragastric feeding with mixture of AA (41% AA I, 56% AA II) was carried out. A total of 18 experimental rats were divided into three feeding regimens, with six rats in each group (group I, normal basal diet; groups II and III received intragastric 5 mg and 10 mg isolated AA mixture/kg/day for 5 days/week for 12 weeks). Dosage-dependent urothelial proliferation, but not carcinoma, was found in the urothelium of the bladder of the rats administered with AA mixture. Immunoprecipitation showed elevations of cyclin D1/cdk4 (increased induction by 1.57- and 1.95-fold in the groups II and III) and/ or cyclin E/cdk2 complex (increased induction by 1.46- and 1.62-fold in the groups II and III), which promote the increasing phosphorylation of Rb (increased induction by 1.75- and 2.07-fold in the groups II and III) and result in decrease of the Rb/ E2F complex (decreased expression by 0.65- and 0.24-fold in the groups II and III). Our results provide evidence to suggest that exposure to AA results in urothelial proliferation in rats through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Aristolochic acid; Cell cycle; Urothelial proliferation
1. Introduction Aristolochic acid (AA) is a nitrophenanthrene derivative isolated from all Aristolochia species and has been shown to be a genotoxic mutagen (Robisch et al., 1982;
*
Corresponding author. Tel.: +886 4 24730022x11670; fax: +886 4 23248167. E-mail address: [email protected] (C.-J. Wang). 0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.06.002
Schmeiser et al., 1986) and a potent carcinogen to both rats (Mengs et al., 1982; Schmeiser et al., 1990; Rossiello et al., 1993; Cosyns et al., 1998) and human (Cosyns et al., 1993; Nortier et al., 2000). AA I is the major component of the carcinogenic plant extract AA. Toxicological studies of AA have also given information on its nephrotoxicity (Jackson et al., 1964; Peters and Hedwall, 1963). Clinically, rapidly progressive renal fibrosis after weight-reducing regimen including Chinese herbs containing AA has been identified as Chinese-herb
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nephropathy (Vanherweghem et al., 1993; Vanhaelen et al., 1994). Several clinical presentations of AA-related nephropathy have been described from Spain, France, UK, and Taiwan (Pena et al., 1996; Stengel and Jones, 1998; Lord et al., 1999; Yang et al., 2000; Arlt et al., 2002). Previous studies have shown that the mutagenic and carcinogenic properties of AA are based on the formation of DNA adducts (Arlt et al., 2001). After reductive metabolic activation, both AAs react with DNA preferentially at the exocyclic amino groups of adenine and guanine (Pfau et al., 1990, 1991). The DNA adducts (e.g. 7-(deoxyadenosin-N6-yl)aristolactam I or II and 7(deoxyguanosin-N2-yl)aristolactam I or II) have been detected in kidney and ureter tissues of patients taking herbs containing AAs, several months or even years after cessation of the herbal consumption. The former was the most predominant DNA adduct in human and rat tissues (Pfau et al., 1990; Schmeiser et al., 1996). Because previous evidences have demonstrated that DNA adduct levels could associate with cell proliferation and tumor induction (Culp et al., 2000; Jackson et al., 2003) while little information exists regarding the cell signals of the carcinogenic characteristics of AA, we conducted this histopathological study using three-month intragastric feeding with AA to male Wistar rats to evaluate the effects of AA on the urothelium of rats. We aimed to evaluate the relationships between the histopathological changes of the urinary bladder and the cell cycle.
2. Material and method 2.1. Chemicals Tris–HCl, EDTA, EGTA, 2-mercaptoethanol, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), leupeptin, glycerol, AA mixture (AAM) (41% AA I, 56% AA II) and bromophenol blue were purchased from Sigma Chemical Co. (St. Louis, MO). SDS, bovine serum albumin (BSA), nonidet p-40, deoxycholic acid, sodium orthovanadate, and aprotinin were purchased from the Sigma Chemical Co. (St. Louis, MO). Protein assay kits were obtained from Bio-Rad Labs. (Hercules, CA). Polyclonal antibodies against cyclin A, and Rb were obtained from Transduction Lab (KY). Antibodies against cdk4 and E2F were obtained from Pharmingen (CA), and those against cyclin D1, cyclin E, and cdk2 were from Santa Cruz (CA). Phospho-Rb was from Cell Signaling Tech (MA). 2.2. Animals Male Wistar rats (200 ± 10 g body weight at four to five weeks old) were purchased from the National Sci-
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ence Council Animal Center, Taiwan. These animals were housed six per cage in an environmentally controlled animal room with a 12-h light, 12-h dark cycle maintained. Food (Purina Lab Chow) and drinking water were provided ad libitum. All animals used were handled according to the guidelines of the Institutional Animal Care and Use Committee of Chung-Shan Medical University (IACUC, CSMU) for the care and use of laboratory animals. A total of 18 experimental rats were divided into three feeding regimens, with six rats in each group. Rats in group I were fed only the basal diet (Purina Lab Chow) and an equivalent amount (4 ml/kg) of the solvent (distilled water), while groups II and III received intragastric 5 mg and 10 mg isolated AAM/kg per day for 5 days/week for 12 weeks. 2.3. Autopsy and histology At the end of experiments, all animals were sacrificed and a complete autopsy was performed immediately. The urinary bladders were removed from all the rats in all experimental and control groups and were examined histologically. Tissues were fixed in 10% buffered formalin, were processed for histological tests according to conventional methods, and stained with haematoxylin and eosin (H&E stain). Step sections (five sections per block of tissue) were prepared from the tissue. The morphology, number and locations of any lesions observed were noted. 2.4. Preparation of tissue extract and immunoblot analysis The tissues of the bladder were homogenized in a buffer containing (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 0.3 M sucrose, 50 lM 2mercaptoethanol, 0.1% Triton X-100, 2 mM PMSF, and 10 mg/ml leupeptin). The homogenates were then centrifuged at 12,000 rpm at 4 °C for 10 min, and the protein contents of the supernatants were determined with the coomassie blue total protein reagent (Kenlor Industries, Inc, USA) using bovine serum albumin as standard. For Western blot analysis, an equal volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue) was added to the samples, which contained 50 lg of tissue protein. The mixture was boiled for 5 min before being subjected to polyacrylamide gel electrophoresis. After being transferred into nitrocellulose membranes, the samples were immunoblotted with the indicated antibodies (against cyclin A, cyclin D1, cyclin E, cdk2, cdk4, Rb, phospho-Rb, E2F, and ß-actin as internal control). Relative protein expression levels were quantified by densitometric measurement of ECL
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reaction bands (AlphaImagerTM 2000, Alpha Innotech) and normalized with values of ß-actin. 2.5. Immunoprecipitation Cell lysates were prepared using lysis buffer containing 50 mM Tris, 5 mM EDTA, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 lg/ml aprotinin, 170 lg/ml leupeptin, and 100 lg/ml, pH 7.5 phenylsulfonyl fluoride. Five hundred micrograms of protein from cell lysates was pre-cleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using monoclonal anti-cdk2, -cdk4, and -E2F (Santa Cruz Biotech) antibodies. Immune complexes were harvested with protein A-sepharose, and immunoprecipitated proteins were analyzed by SDSPAGE. Immunodetection was carried out using monoclonal anti-cdk2, -cdk4, -E2F, and -cyclin D1; polyclonal anti-cyclin E, and -Rb antibodies.
3. Results 3.1. Effect of AAM on urothelium To demonstrate the impact of AAM on urinary bladder, the removed bladders of the 12 experimental rats and 6 controls were examined histologically. Although there was no carcinoma in any bladder tissues, an AAM-dose-dependent proliferation of the urothelium of the urinary bladders (Fig. 1) was found. These histological data indicate that AAM can induce urothelial proliferation in rats. 3.2. Effect of AAM on the expression of cyclins and cdks In mammalian cells, cyclins comprise an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle (Hunter and Pines, 1994). To investigate how AAM induces cell proliferation by possible activation of cyclins, the levels of cyclin A, cyclin D1, and cyclin E were measured. As shown in Fig. 2A, the expression of cyclins A, D1 and E increase in response to high doses of AAM treatment. Cyclin-dependent kinases (cdks) play a critical role in the commitment of a cell to proliferate (Vermeulen et al., 2003a,b). Cdks are activated by their association with regulatory subunits known as cyclins. To confirm that AAM induces possible activation of cdks in addition to activation of cyclins, the expressions of cdk2 and cdk4 were also checked. The western-blot data showed that the protein level of cdk2 and cdk4 change in response to high dose of AAM (Fig. 2A).
Fig. 1. Section of bladder urothelium from a normal rat (a) and those treated with aristolochic acid mixture 5 mg/kg (b) or 10 mg/kg (c) for 12 weeks, showing urothelial dose-dependent proliferation (single arrow and double arrows) (Hematoxylin and eosin, X100).
3.3. Effect of AAM on cyclin/cdk association Cell cycle transition from G1 to S requires the temporal activation of complexes of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 (Weinberg, 1995). To investigate how AAM induces cyclin and cdk activation and how it can promote cell-cycle progression, we used immunoprecipitation to ensure whether cyclin-cdk complexes were activated to promote cell cycles. As shown in Fig. 2B, cyclin D1-cdk4 and cyclin E-cdk2, which controls the progression through the G1-phase, increased
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Fig. 2. Three independent experiments for three rats in each group were conducted and showed the similar pattern of changes in the levels of cyclins A, D1, E, cdk 2 and cdk4 (A), in the levels of association of cyclin D1/cdk 4 and cyclin E/cdk 2 (B) and in increasing activation of Rb-p795 and decreasing expression of Rb/E2F complex in response to aristolochic acid mixture (C), a representative one is shown here. N, control; L, low dose AAM; H, high dose AAM; IP, immunoprecipitation.
induction by 1.57- and 1.46-fold in the low dose AAM group and increased induction by 1.95- and 1.62-fold in the high dose AAM group. This result implies that
activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes by AAM did promote cell-cycle transition from G1 to the S phase.
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3.4. Effect of AAM on the expression of Rb-Phosphorylation (pRb) Cyclin D1-cdk4 plays a major role in the initiation of the cell cycle, passage through the restriction point (G0), and entry into the S phase. The only known target of active cyclin D1-cdk4 is the retinoblastoma tumor-suppressor protein (pRb); however, other cyclin-dependent kinases, such as cyclin E-cdk2, and cyclin A-cdk2 have also been shown to phosphorylate pRb in the G1-phase and the G1/S transition of the cell cycle (Wang et al., 1994). Cyclin-cdk complexes will phosphorylate the retinoblastoma gene product (Rb), releasing E2F from its sequestration by Rb and allowing E2F to transactivate genes essential for the S phase (Vermeulen et al., 2003a,b). Demonstrating the AAM induced cyclin-cdkcomplexes could phosphorylate the Rb, we found that Rb phosphorylation was increased (Fig. 2C), with induction increased by 1.75- and 2.07-fold in the low dose and high dose AAM groups, respectively. At the same time, we also found that Rb/E2F association was markedly reduced (Fig. 2C) with expression decreased by 0.65and 0.24-fold in each low dose and high dose AAM group, respectively. These results demonstrate that AAM-induced cyclin-cdk complexes could phosphorylate Rb, thus allowing the release of E2F from Rb to promote cell-cycle transition from the G1 to the S phase.
4. Discussion AA has been implicated in nephrotoxicity and carcinogenesis in humans. The nephrotoxic effect was shown as early in Jackson et al. (1964) and was again noted in 1993 from Belgium because one group of young female patients taking a slimming regimen including Chinese herbs containing AA suffered from rapidly progressing interstitial renal fibrosis (Vanherweghem et al., 1993). Recently, animal models with chronic renal tubulointerstitial nephropathy induced by aristolochic acid were established (Zheng et al., 2001; Debelle et al., 2002). In 1994, two of these Belgian AAN patients were reported with urothelial cancer (Cosyns et al., 1994; Vanherweghem et al., 1995). Thereafter, an increasing number of urothelial carcinomas were reported (Cosyns et al., 1999). In 2000, Nortier et al. reported that the prevalence of urothelial carcinoma among these patients with end stage AAN is rather high, with 46% urothelial carcinoma (18 in 39 patients), 49% urothelial dysplasia (19 in 39 patients) and only 5% (2 in 39 patients) with normal urothelium. The cumulative dose of Aristolochia fangchi and the dose of AA were associated with a significantly higher risk of developing urothelial cancer (Nortier et al., 2000). Patients with a mean intake of 200 g Chinese herbs had a 50% higher risk of developing urothelial cancer.
In animals, AA has been shown to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis, urinary bladder, hepatic nodules, ear duct and small intestine (Schmeiser et al., 1990; Izumi et al., 2000). Recently, Cosyns et al. (1998) reported that AA induces tumors in the forestomach but without interstitial nephropathy in rats. They orally treated two groups of seven rats with either pure AA (10 mg/kg, 5 days/week, for three months) or herb powders (containing AA) mixed with fenfluramine. The animals in both groups developed multisystemic tumors and forestomach epidermoid carcinomas but no fibrosis of the renal interstitium. The feeding protocol of administration of AAM and the results of our study were similar to those of CosynsÕs study. We did not find deterioration of renal functions or interstitial nephropathy in any Wistar rat after a three-month oral AAM therapy. However, in the urothelium of urinary bladders, we found a phenomenon of dose-dependent urothelial proliferation. We, therefore, conducted this study focused on the impact of AAM on the cell cycle of urinary bladder tissues. Eukaryotic cells have precise and well-regulated mechanisms to control progression through the cell cycle (Vermeulen et al., 2003a,b). Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of cyclin (regulatory) and cyclin-dependent kinase (cdk; catalytic) subunits. The associations of cyclin D1 and cdk4, cyclin E and cdk2, as well as cyclin A and cdk2 have also been shown to phosphorylate rubidium (Rb) in the G0/G1 and the G1/S-phase transitions of the cell cycle (Vermeulen et al., 2003a,b). Upon phosphorylation, pRb releases and activates a number of proteins, such as the E2F family of transcription factors at the G1/S transition phase (Wang et al., 1994; Nevins et al., 1997), which in turn regulates the expression of several genes involved in DNA replication, such as dihydrofolate reductase, thymidine kinase, and DNA polymerase (Izumi et al., 2000). Regulation of G1 cyclin-cdk activity is also dependent on cdk inhibitory proteins (CKIs), which can bind and inactivate cyclin-cdk complexes (Hunter and Pines, 1994). Our immunoprecipitation showed the effect of AAM increasing the expression of cyclins, cdks, and the association of cyclins/cdks which promoted the activation of Rb, resulting in decrease of the Rb/E2F complex. The results provide evidence to suggest that AAM-induced urothelial proliferation of urinary bladder in rats was through cell cycle progression. The cell cycle progression was via the induction of cyclin D1/cdk 4 and/or cyclin E/ cdk 2 activity as well as the increasing phosphorylation of Rb which may act as possible procarcinogenic initiation. Recently, there have been many studies on the mutagenic and carcinogenic properties of AA, with most
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focusing on the formation of AA DNA adducts, activation of the H-ras oncogene and overexpression of the p53. In renal and ureteral tissue of AAN patients, three AA-specific DNA adducts including one major (dA– AAI) and two minor (dG–AAI and dA–AAII) were identified (Schmeiser et al., 1996; Nortier et al., 2000). The major adenine adduct of AAI, dA–AAI, is detectable at relatively high levels in native kidney tissue up to 89 months after the patients stopped taking the herbal medicine (Nortier et al., 2000). Furthermore, the dA– AAI adduct was also highly persistent in rat kidney (Bieler et al., 1997). Therefore, the AA–DNA adducts have been a suitable biomarker and have played a critical role in the carcinogenic process of AA. Another distinct molecular characteristic of AA-initiated carcinogenesis in rodents is the activation of the H-ras oncogene by a specific AT ! TA transversion mutation at the first adenine in codon 61 (CAA) (Schmeiser et al., 1990). The H-ras gene is activated at high frequency by an AT ! TA transversion mutation in codon 61 of DNA from AAI-induced tumors in rats (Schmeiser et al., 1990) and since both adenines in codon 61 (CAA) were shown to be AA–DNA binding sites (Arlt et al., 2000), this is another evidence suggesting a relevant role of dA–AAI adducts in AAN-related urothelial cancer. Furthermore, in AAN patients, urothelial carcinomas as well as urothelial atypia were associated with overexpression of p53 protein (Cosyns et al., 1994), suggesting that the p53 gene is also mutated in AANassociated urothelial cancer. The p53 gene is one of the most commonly mutated genes observed in human tumors and is mutated in >50% of all human cancers (Greenblatt et al., 1994; Hussain and Harris, 1998). These mutations can trigger tumorigenesis in humans in the same way as mutations in codon 61 of H-ras trigger tumorigenesis by AA in rodents. Although AA is associated with both carcinogenic and fibrogenic properties in humans, however in rats both properties are dissociated since only tumors and no fibrosis were induced (Cosyns et al., 1998). In 1998, Cosyns et al. treated rats with 10 mg AA/kg per day for 5 days/week for 3 months. At sacrifice at the third and eleventh month, animals in both groups had developed the expected tumors involving forestomach, small intestine, kidney, bladder, heart and prostate but not fibrosis of the renal interstitium. Our results also confirm CosynsÕs data that tumors of forestomach and no fibrogenesis of kidneys were observed in rats with AA induction. But, AAM causes only urothelial proliferation without urothelial cancer in our study, partly because we used mixture of AA and the dose of AA I was only 41% of that in CosynÕs study. Gatenby and Vincent (2003) recently described an evolutionary model of carcinogenesis in which carcinogenesis is considered to be an emergent phenomenon requiring a sequence of evolutionary steps as cellular proliferation follows
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successful adaptation to varying environmental constraints. In the initial development of preneoplastic lesions, cellular proliferation is controlled exclusively by interactions with other cells, the extracellular matrix, and soluble or insoluble growth factors so that gain of function mutations in oncogenes, loss of function mutations in tumor suppressor genes, and disruption of normal senescence pathways will permit clonal expansion. Our results were not sufficient to confirm that urothelial proliferation caused by the genotoxic agent of AAM is the preneoplastic lesion of urothelial carcinoma. However, the properties of loss of control of cellular proliferation in the urothelium might be a procarcinogenic phenomenon in tumorigenesis. The molecular mechanism by which AA induces urothelial carcinogenesis needs more studies to clarify it. Same as the absence of development of urothelial cancer in this study, the expression of p53 of urinary bladder tissues does not increase dose dependently, with 0.95-fold expression in the low dose AAM group and with 0.97-fold expression in the high dose AAM-group, comparing with the normal control group. The absence of the development of urothelial malignancy might because of the insufficient therapeutic dose or duration of AA I administration. For example, the increased expressions of cdk2 and cdk4 only exist in the high dose AAM group (Fig. 2A). Clinically, the cumulated dose of Aristolochia fangchi is associated with higher risk of developing urothelial cancer (Nortier et al., 2000). We infer that the therapeutic dose and duration of AA, particularly AA I, play important roles in developing urothelial cancer and in increasing expression of p53. Regarding the result of urothelial proliferation caused by AAM in our study, we did not examine that it was via genotoxic, non-genotoxic or both mechanisms. Although there is good evidence that AA is genotoxic through the formation of specific DNA adducts
Fig. 3. Proposed model for the induction of urothelial proliferation in rats caused by aristolochic acid mixture through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2.
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and genotoxicity has been found to be associated with cell proliferation and tumorigenesis (Culp et al., 2000; Jackson et al., 2003), the possibility of non-genotoxic mechanism could not be excluded whether the urothelial proliferation was secondary to injury. In this field, further study is needed to clarify this issue. In conclusion, our results suggest that exposure to AAM results in urothelial proliferation in rats caused by cell cycle progression via activation of cyclin D1/ cdk4 and cyclin E/cdk2 (Fig. 3).
Acknowledgement This work was supported by a grant from the Chung Shan Medical University Research Fund (CSMU91OM-B-045).
References Arlt, V.M., Wiessler, M., Schmeiser, H.H., 2000. Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. Carcinogenesis 21, 235–242. Arlt, V.M., Schmeiser, H.H., Pfeifer, G.P., 2001. Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. Carcinogenesis 22, 133– 140. Arlt, V.M., Stiborova, M., Schmeiser, H.H., 2002. Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17, 265–277. Bieler, C.A., Stiborova, M., Wiessler, M., Cosyns, J.P., van Ypersele, de S.C., Schmeiser, H.H., 1997. 32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis 18, 1063–1067. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Wese, F.X., van Ypersele, de S.C, 1993. Urothelial lesions in Chinese-herb nephropathy. American Journal of Kidney Diseases 33, 1011–1017. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Van Cangh, P.J., van Ypersele, de S.C, 1994. Urothelial malignancy in nephropathy due to Chinese herbs. Lancet 344, 188. Cosyns, J.P., Goebbels, R.M., Liberton, V., Schmeiser, H.H., Bieler, C.A., Bernard, A.M., 1998. Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats. Archives of Toxicology 72, 738– 743. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Wese, F.X., van Ypersele, de S.C., 1999. Urothelial lesions in Chinese-herb nephropathy. American Journal of Kidney Diseases 33, 1011–1017. Culp, S.J., Warbritton, A.R., Smith, B.A., Li, E.E., Beland, F.A., 2000. DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene. Carcinogenesis 21, 1433–1440. Debelle, F.D., Nortier, J.L., De Prez, E.G., Garbar, C.H., Vienne, A.R., Salmon, I.J., Deschodt-Lanckman, M.M., Vanherweghem, J.L., 2002. Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats. Journal of the American Society of Nephrology 13, 431–436. Gatenby, R.A., Vincent, T.L., 2003. An evolutionary model of carcinogenesis. Cancer Research 63, 6212–6220. Greenblatt, M.S., Bennett, W.P., Hollstein, M., Harris, C.C., 1994. Mutations in the p53 tumor suppressor gene: clues to cancer
etiology and molecular pathogenesis. Cancer Research 54, 4855– 4878. Hunter, T., Pines, J., 1994. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79, 573–582. Hussain, S.P., Harris, C.C., 1998. Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Research 58, 4023–4037. Izumi, M., Yokoi, M., Nishikawa, N.S., Miyazawa, H., Sugino, A., Yamagishi, M., Yamaguchi, M., Matsukage, A., Yatagai, F., Hanaoka, F., 2000. Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1. Biochimica et Biophysica Acta 24, 341–352. Jackson, L., Kofman, S., Weiss, A., Brodovsky, H., 1964. Aristolochic acid (NSC-50413): phase I clinical study. Cancer Chemotherapy Reports 42, 3–37. Jackson, P.E., OÕConnor, P.J., Cooper, D.P., Margison, G.P., Povey, A.C., 2003. Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumour yield in mice treated with 1,2-dimethylhydrazine. Carcinogenesis 24, 527–533. Lord, G.M., Tagore, R., Cook, T., Gower, P., Pusey, C.D., 1999. Nephropathy caused by Chinese herbs in the UK. Lancet 354, 481– 482. Mengs, U., Lang, W., Poch, J.A., 1982. The carcinogenic action of aristolochic acid in rats. Archives of Toxicology 51, 107– 119. Nevins, J.R., Leone, G., DeGregori, J., Jakoi, L., 1997. Role of the Rb/E2F pathway in cell growth control. Journal of Cellular Physiology 173, 233–236. Nortier, J.L., Martinez, M.C., Schmeiser, H.H., Arlt, V.M., Bieler, C.A., Petein, M., Depierreux, M.F., De Pauw, L., Abramowicz, D., Vereerstraeten, P., Vanherweghem, J.L., 2000. Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). The New England Journal of Medicine 342, 1686– 1692. Pena, J.M., Borras, M., Ramos, J., Montoliu, J., 1996. Rapidly progressive interstitial renal fibrosis due to a chronic intake of a herb (Aristolochia pistolochia) infusion. Nephrology, Dialysis, Transplantation 11, 1359–1360. Peters, G., Hedwall, P.R., 1963. Aristolochic acid intoxication: a new type of impairment of urinary concentrating ability. Archives Internationales de Pharmacodynamie et de Therapie 145, 334– 355. Pfau, W., Schmeiser, H.H., Wiessler, M., 1990. Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA. Carcinogenesis 11, 313–319. Pfau, W., Schmeiser, H.H., Wiessler, M., 1991. N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen. Chemical Research in Toxicology 4, 581– 586. Robisch, G., Schimmer, O., Goggelmann, W., 1982. Aristolochic acid is a direct mutagen in Salmonella typhimurium. Mutation Research 105, 201–204. Rossiello, M.R., Laconi, E., Rao, P.M., Rajalakshmi, S., Sarma, D.S., 1993. Induction of hepatic nodules in the rat by aristolochic acid. Cancer Letters 71, 83–87. Schmeiser, H.H., Pool, B.L., Wiessler, M., 1986. Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver. Carcinogenesis 7, 59–63. Schmeiser, H.H., Janssen, J.W., Lyons, J., Scherf, H.R., Pfau, W., Buchmann, A., Bartram, C.R., Wiessler, M., 1990. Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer Research 50, 5464–5469. Schmeiser, H.H., Bieler, C.A., Wiessler, M., van Ypersele, de S.C, Cosyns, J.P., 1996. Detection of DNA adducts formed by
H.-R. Chang et al. / Food and Chemical Toxicology 44 (2006) 28–35 aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. Cancer Research 56, 2025–2028. Stengel, B., Jones, E., 1998. End-stage renal insufficiency associated with Chinese herbal consumption in France. Nephrologie 19, 15– 20. Vanhaelen, M., Vanhaelen-Fastre, R., But, P., Vanherweghem, J.L., 1994. Identification of aristolochic acid in Chinese herbs. Lancet 343, 174. Vanherweghem, J.L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R., et al., 1993. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387– 391. Vanherweghem, J.L., Tielemans, C., Simon, J., Depierreux, M., 1995. Chinese herbs nephropathy and renal pelvic carcinoma. Nephrology, Dialysis, Transplantation 10, 270–273.
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Vermeulen, K., Van Bockstaele, D.R., Berneman, Z.N., 2003a. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Proliferation 36, 131–149. Vermeulen, K., Berneman, Z.N., Van Bockstaele, D.R., 2003b. Cell cycle and apoptosis. Cell Proliferation 36, 165–175. Wang, J.Y., Knudsen, E.S., Welch, P.J., 1994. The retinoblastoma tumor suppressor protein. Advances in Cancer Research 64, 25– 85. Weinberg, R.A., 1995. The retinoblastoma protein and cell cycle control. Cell 81, 323–330. Yang, C.S., Lin, C.H., Chang, S.H., Hsu, H.C., 2000. Rapidly progressive fibrosing interstitial nephritis associated with Chinese herbal drugs. American Journal of Kidney Diseases 35, 313– 318. Zheng, F., Zhang, X., Huang, Q., 2001. Establishment of model of aristolochic acid-induced chronic renal interstitial fibrosis in rats. Zhonghua Yi Xue Za Zhi 81, 1095–1100.
Induction of urothelial proliferation in rats by aristolochic acid through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2 Horng-Rong Chang a,b, Jong-Da Lian a, Chia-Wen Lo c, Yun-Ching Chang c, Mon-Yuan Yang c, Chau-Jong Wang c,* a
c
Division of Nephrology, Department of Internal Medicine, Chung-Shan Medical University Hospital, Taichung 402, Taiwan b Institute of Medicine, Chung-Shan Medical University, Taichung 402, Taiwan Institute of Biochemistry and Biotechnology, Chung-Shan Medical University, No. 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan Received 21 September 2004; accepted 1 June 2005
Abstract Aristolochic acid (AA) has been implicated in urothelial carcinoma in humans. However, the mechanism by which AA induces this cancer has not been completely established. To evaluate the effects of AA on the urinary bladder of rats, a histopathological study of three-month intragastric feeding with mixture of AA (41% AA I, 56% AA II) was carried out. A total of 18 experimental rats were divided into three feeding regimens, with six rats in each group (group I, normal basal diet; groups II and III received intragastric 5 mg and 10 mg isolated AA mixture/kg/day for 5 days/week for 12 weeks). Dosage-dependent urothelial proliferation, but not carcinoma, was found in the urothelium of the bladder of the rats administered with AA mixture. Immunoprecipitation showed elevations of cyclin D1/cdk4 (increased induction by 1.57- and 1.95-fold in the groups II and III) and/ or cyclin E/cdk2 complex (increased induction by 1.46- and 1.62-fold in the groups II and III), which promote the increasing phosphorylation of Rb (increased induction by 1.75- and 2.07-fold in the groups II and III) and result in decrease of the Rb/ E2F complex (decreased expression by 0.65- and 0.24-fold in the groups II and III). Our results provide evidence to suggest that exposure to AA results in urothelial proliferation in rats through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Aristolochic acid; Cell cycle; Urothelial proliferation
1. Introduction Aristolochic acid (AA) is a nitrophenanthrene derivative isolated from all Aristolochia species and has been shown to be a genotoxic mutagen (Robisch et al., 1982;
*
Corresponding author. Tel.: +886 4 24730022x11670; fax: +886 4 23248167. E-mail address: [email protected] (C.-J. Wang). 0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.06.002
Schmeiser et al., 1986) and a potent carcinogen to both rats (Mengs et al., 1982; Schmeiser et al., 1990; Rossiello et al., 1993; Cosyns et al., 1998) and human (Cosyns et al., 1993; Nortier et al., 2000). AA I is the major component of the carcinogenic plant extract AA. Toxicological studies of AA have also given information on its nephrotoxicity (Jackson et al., 1964; Peters and Hedwall, 1963). Clinically, rapidly progressive renal fibrosis after weight-reducing regimen including Chinese herbs containing AA has been identified as Chinese-herb
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nephropathy (Vanherweghem et al., 1993; Vanhaelen et al., 1994). Several clinical presentations of AA-related nephropathy have been described from Spain, France, UK, and Taiwan (Pena et al., 1996; Stengel and Jones, 1998; Lord et al., 1999; Yang et al., 2000; Arlt et al., 2002). Previous studies have shown that the mutagenic and carcinogenic properties of AA are based on the formation of DNA adducts (Arlt et al., 2001). After reductive metabolic activation, both AAs react with DNA preferentially at the exocyclic amino groups of adenine and guanine (Pfau et al., 1990, 1991). The DNA adducts (e.g. 7-(deoxyadenosin-N6-yl)aristolactam I or II and 7(deoxyguanosin-N2-yl)aristolactam I or II) have been detected in kidney and ureter tissues of patients taking herbs containing AAs, several months or even years after cessation of the herbal consumption. The former was the most predominant DNA adduct in human and rat tissues (Pfau et al., 1990; Schmeiser et al., 1996). Because previous evidences have demonstrated that DNA adduct levels could associate with cell proliferation and tumor induction (Culp et al., 2000; Jackson et al., 2003) while little information exists regarding the cell signals of the carcinogenic characteristics of AA, we conducted this histopathological study using three-month intragastric feeding with AA to male Wistar rats to evaluate the effects of AA on the urothelium of rats. We aimed to evaluate the relationships between the histopathological changes of the urinary bladder and the cell cycle.
2. Material and method 2.1. Chemicals Tris–HCl, EDTA, EGTA, 2-mercaptoethanol, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), leupeptin, glycerol, AA mixture (AAM) (41% AA I, 56% AA II) and bromophenol blue were purchased from Sigma Chemical Co. (St. Louis, MO). SDS, bovine serum albumin (BSA), nonidet p-40, deoxycholic acid, sodium orthovanadate, and aprotinin were purchased from the Sigma Chemical Co. (St. Louis, MO). Protein assay kits were obtained from Bio-Rad Labs. (Hercules, CA). Polyclonal antibodies against cyclin A, and Rb were obtained from Transduction Lab (KY). Antibodies against cdk4 and E2F were obtained from Pharmingen (CA), and those against cyclin D1, cyclin E, and cdk2 were from Santa Cruz (CA). Phospho-Rb was from Cell Signaling Tech (MA). 2.2. Animals Male Wistar rats (200 ± 10 g body weight at four to five weeks old) were purchased from the National Sci-
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ence Council Animal Center, Taiwan. These animals were housed six per cage in an environmentally controlled animal room with a 12-h light, 12-h dark cycle maintained. Food (Purina Lab Chow) and drinking water were provided ad libitum. All animals used were handled according to the guidelines of the Institutional Animal Care and Use Committee of Chung-Shan Medical University (IACUC, CSMU) for the care and use of laboratory animals. A total of 18 experimental rats were divided into three feeding regimens, with six rats in each group. Rats in group I were fed only the basal diet (Purina Lab Chow) and an equivalent amount (4 ml/kg) of the solvent (distilled water), while groups II and III received intragastric 5 mg and 10 mg isolated AAM/kg per day for 5 days/week for 12 weeks. 2.3. Autopsy and histology At the end of experiments, all animals were sacrificed and a complete autopsy was performed immediately. The urinary bladders were removed from all the rats in all experimental and control groups and were examined histologically. Tissues were fixed in 10% buffered formalin, were processed for histological tests according to conventional methods, and stained with haematoxylin and eosin (H&E stain). Step sections (five sections per block of tissue) were prepared from the tissue. The morphology, number and locations of any lesions observed were noted. 2.4. Preparation of tissue extract and immunoblot analysis The tissues of the bladder were homogenized in a buffer containing (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 0.3 M sucrose, 50 lM 2mercaptoethanol, 0.1% Triton X-100, 2 mM PMSF, and 10 mg/ml leupeptin). The homogenates were then centrifuged at 12,000 rpm at 4 °C for 10 min, and the protein contents of the supernatants were determined with the coomassie blue total protein reagent (Kenlor Industries, Inc, USA) using bovine serum albumin as standard. For Western blot analysis, an equal volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue) was added to the samples, which contained 50 lg of tissue protein. The mixture was boiled for 5 min before being subjected to polyacrylamide gel electrophoresis. After being transferred into nitrocellulose membranes, the samples were immunoblotted with the indicated antibodies (against cyclin A, cyclin D1, cyclin E, cdk2, cdk4, Rb, phospho-Rb, E2F, and ß-actin as internal control). Relative protein expression levels were quantified by densitometric measurement of ECL
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reaction bands (AlphaImagerTM 2000, Alpha Innotech) and normalized with values of ß-actin. 2.5. Immunoprecipitation Cell lysates were prepared using lysis buffer containing 50 mM Tris, 5 mM EDTA, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 lg/ml aprotinin, 170 lg/ml leupeptin, and 100 lg/ml, pH 7.5 phenylsulfonyl fluoride. Five hundred micrograms of protein from cell lysates was pre-cleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using monoclonal anti-cdk2, -cdk4, and -E2F (Santa Cruz Biotech) antibodies. Immune complexes were harvested with protein A-sepharose, and immunoprecipitated proteins were analyzed by SDSPAGE. Immunodetection was carried out using monoclonal anti-cdk2, -cdk4, -E2F, and -cyclin D1; polyclonal anti-cyclin E, and -Rb antibodies.
3. Results 3.1. Effect of AAM on urothelium To demonstrate the impact of AAM on urinary bladder, the removed bladders of the 12 experimental rats and 6 controls were examined histologically. Although there was no carcinoma in any bladder tissues, an AAM-dose-dependent proliferation of the urothelium of the urinary bladders (Fig. 1) was found. These histological data indicate that AAM can induce urothelial proliferation in rats. 3.2. Effect of AAM on the expression of cyclins and cdks In mammalian cells, cyclins comprise an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle (Hunter and Pines, 1994). To investigate how AAM induces cell proliferation by possible activation of cyclins, the levels of cyclin A, cyclin D1, and cyclin E were measured. As shown in Fig. 2A, the expression of cyclins A, D1 and E increase in response to high doses of AAM treatment. Cyclin-dependent kinases (cdks) play a critical role in the commitment of a cell to proliferate (Vermeulen et al., 2003a,b). Cdks are activated by their association with regulatory subunits known as cyclins. To confirm that AAM induces possible activation of cdks in addition to activation of cyclins, the expressions of cdk2 and cdk4 were also checked. The western-blot data showed that the protein level of cdk2 and cdk4 change in response to high dose of AAM (Fig. 2A).
Fig. 1. Section of bladder urothelium from a normal rat (a) and those treated with aristolochic acid mixture 5 mg/kg (b) or 10 mg/kg (c) for 12 weeks, showing urothelial dose-dependent proliferation (single arrow and double arrows) (Hematoxylin and eosin, X100).
3.3. Effect of AAM on cyclin/cdk association Cell cycle transition from G1 to S requires the temporal activation of complexes of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 (Weinberg, 1995). To investigate how AAM induces cyclin and cdk activation and how it can promote cell-cycle progression, we used immunoprecipitation to ensure whether cyclin-cdk complexes were activated to promote cell cycles. As shown in Fig. 2B, cyclin D1-cdk4 and cyclin E-cdk2, which controls the progression through the G1-phase, increased
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Fig. 2. Three independent experiments for three rats in each group were conducted and showed the similar pattern of changes in the levels of cyclins A, D1, E, cdk 2 and cdk4 (A), in the levels of association of cyclin D1/cdk 4 and cyclin E/cdk 2 (B) and in increasing activation of Rb-p795 and decreasing expression of Rb/E2F complex in response to aristolochic acid mixture (C), a representative one is shown here. N, control; L, low dose AAM; H, high dose AAM; IP, immunoprecipitation.
induction by 1.57- and 1.46-fold in the low dose AAM group and increased induction by 1.95- and 1.62-fold in the high dose AAM group. This result implies that
activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes by AAM did promote cell-cycle transition from G1 to the S phase.
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3.4. Effect of AAM on the expression of Rb-Phosphorylation (pRb) Cyclin D1-cdk4 plays a major role in the initiation of the cell cycle, passage through the restriction point (G0), and entry into the S phase. The only known target of active cyclin D1-cdk4 is the retinoblastoma tumor-suppressor protein (pRb); however, other cyclin-dependent kinases, such as cyclin E-cdk2, and cyclin A-cdk2 have also been shown to phosphorylate pRb in the G1-phase and the G1/S transition of the cell cycle (Wang et al., 1994). Cyclin-cdk complexes will phosphorylate the retinoblastoma gene product (Rb), releasing E2F from its sequestration by Rb and allowing E2F to transactivate genes essential for the S phase (Vermeulen et al., 2003a,b). Demonstrating the AAM induced cyclin-cdkcomplexes could phosphorylate the Rb, we found that Rb phosphorylation was increased (Fig. 2C), with induction increased by 1.75- and 2.07-fold in the low dose and high dose AAM groups, respectively. At the same time, we also found that Rb/E2F association was markedly reduced (Fig. 2C) with expression decreased by 0.65and 0.24-fold in each low dose and high dose AAM group, respectively. These results demonstrate that AAM-induced cyclin-cdk complexes could phosphorylate Rb, thus allowing the release of E2F from Rb to promote cell-cycle transition from the G1 to the S phase.
4. Discussion AA has been implicated in nephrotoxicity and carcinogenesis in humans. The nephrotoxic effect was shown as early in Jackson et al. (1964) and was again noted in 1993 from Belgium because one group of young female patients taking a slimming regimen including Chinese herbs containing AA suffered from rapidly progressing interstitial renal fibrosis (Vanherweghem et al., 1993). Recently, animal models with chronic renal tubulointerstitial nephropathy induced by aristolochic acid were established (Zheng et al., 2001; Debelle et al., 2002). In 1994, two of these Belgian AAN patients were reported with urothelial cancer (Cosyns et al., 1994; Vanherweghem et al., 1995). Thereafter, an increasing number of urothelial carcinomas were reported (Cosyns et al., 1999). In 2000, Nortier et al. reported that the prevalence of urothelial carcinoma among these patients with end stage AAN is rather high, with 46% urothelial carcinoma (18 in 39 patients), 49% urothelial dysplasia (19 in 39 patients) and only 5% (2 in 39 patients) with normal urothelium. The cumulative dose of Aristolochia fangchi and the dose of AA were associated with a significantly higher risk of developing urothelial cancer (Nortier et al., 2000). Patients with a mean intake of 200 g Chinese herbs had a 50% higher risk of developing urothelial cancer.
In animals, AA has been shown to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis, urinary bladder, hepatic nodules, ear duct and small intestine (Schmeiser et al., 1990; Izumi et al., 2000). Recently, Cosyns et al. (1998) reported that AA induces tumors in the forestomach but without interstitial nephropathy in rats. They orally treated two groups of seven rats with either pure AA (10 mg/kg, 5 days/week, for three months) or herb powders (containing AA) mixed with fenfluramine. The animals in both groups developed multisystemic tumors and forestomach epidermoid carcinomas but no fibrosis of the renal interstitium. The feeding protocol of administration of AAM and the results of our study were similar to those of CosynsÕs study. We did not find deterioration of renal functions or interstitial nephropathy in any Wistar rat after a three-month oral AAM therapy. However, in the urothelium of urinary bladders, we found a phenomenon of dose-dependent urothelial proliferation. We, therefore, conducted this study focused on the impact of AAM on the cell cycle of urinary bladder tissues. Eukaryotic cells have precise and well-regulated mechanisms to control progression through the cell cycle (Vermeulen et al., 2003a,b). Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of cyclin (regulatory) and cyclin-dependent kinase (cdk; catalytic) subunits. The associations of cyclin D1 and cdk4, cyclin E and cdk2, as well as cyclin A and cdk2 have also been shown to phosphorylate rubidium (Rb) in the G0/G1 and the G1/S-phase transitions of the cell cycle (Vermeulen et al., 2003a,b). Upon phosphorylation, pRb releases and activates a number of proteins, such as the E2F family of transcription factors at the G1/S transition phase (Wang et al., 1994; Nevins et al., 1997), which in turn regulates the expression of several genes involved in DNA replication, such as dihydrofolate reductase, thymidine kinase, and DNA polymerase (Izumi et al., 2000). Regulation of G1 cyclin-cdk activity is also dependent on cdk inhibitory proteins (CKIs), which can bind and inactivate cyclin-cdk complexes (Hunter and Pines, 1994). Our immunoprecipitation showed the effect of AAM increasing the expression of cyclins, cdks, and the association of cyclins/cdks which promoted the activation of Rb, resulting in decrease of the Rb/E2F complex. The results provide evidence to suggest that AAM-induced urothelial proliferation of urinary bladder in rats was through cell cycle progression. The cell cycle progression was via the induction of cyclin D1/cdk 4 and/or cyclin E/ cdk 2 activity as well as the increasing phosphorylation of Rb which may act as possible procarcinogenic initiation. Recently, there have been many studies on the mutagenic and carcinogenic properties of AA, with most
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focusing on the formation of AA DNA adducts, activation of the H-ras oncogene and overexpression of the p53. In renal and ureteral tissue of AAN patients, three AA-specific DNA adducts including one major (dA– AAI) and two minor (dG–AAI and dA–AAII) were identified (Schmeiser et al., 1996; Nortier et al., 2000). The major adenine adduct of AAI, dA–AAI, is detectable at relatively high levels in native kidney tissue up to 89 months after the patients stopped taking the herbal medicine (Nortier et al., 2000). Furthermore, the dA– AAI adduct was also highly persistent in rat kidney (Bieler et al., 1997). Therefore, the AA–DNA adducts have been a suitable biomarker and have played a critical role in the carcinogenic process of AA. Another distinct molecular characteristic of AA-initiated carcinogenesis in rodents is the activation of the H-ras oncogene by a specific AT ! TA transversion mutation at the first adenine in codon 61 (CAA) (Schmeiser et al., 1990). The H-ras gene is activated at high frequency by an AT ! TA transversion mutation in codon 61 of DNA from AAI-induced tumors in rats (Schmeiser et al., 1990) and since both adenines in codon 61 (CAA) were shown to be AA–DNA binding sites (Arlt et al., 2000), this is another evidence suggesting a relevant role of dA–AAI adducts in AAN-related urothelial cancer. Furthermore, in AAN patients, urothelial carcinomas as well as urothelial atypia were associated with overexpression of p53 protein (Cosyns et al., 1994), suggesting that the p53 gene is also mutated in AANassociated urothelial cancer. The p53 gene is one of the most commonly mutated genes observed in human tumors and is mutated in >50% of all human cancers (Greenblatt et al., 1994; Hussain and Harris, 1998). These mutations can trigger tumorigenesis in humans in the same way as mutations in codon 61 of H-ras trigger tumorigenesis by AA in rodents. Although AA is associated with both carcinogenic and fibrogenic properties in humans, however in rats both properties are dissociated since only tumors and no fibrosis were induced (Cosyns et al., 1998). In 1998, Cosyns et al. treated rats with 10 mg AA/kg per day for 5 days/week for 3 months. At sacrifice at the third and eleventh month, animals in both groups had developed the expected tumors involving forestomach, small intestine, kidney, bladder, heart and prostate but not fibrosis of the renal interstitium. Our results also confirm CosynsÕs data that tumors of forestomach and no fibrogenesis of kidneys were observed in rats with AA induction. But, AAM causes only urothelial proliferation without urothelial cancer in our study, partly because we used mixture of AA and the dose of AA I was only 41% of that in CosynÕs study. Gatenby and Vincent (2003) recently described an evolutionary model of carcinogenesis in which carcinogenesis is considered to be an emergent phenomenon requiring a sequence of evolutionary steps as cellular proliferation follows
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successful adaptation to varying environmental constraints. In the initial development of preneoplastic lesions, cellular proliferation is controlled exclusively by interactions with other cells, the extracellular matrix, and soluble or insoluble growth factors so that gain of function mutations in oncogenes, loss of function mutations in tumor suppressor genes, and disruption of normal senescence pathways will permit clonal expansion. Our results were not sufficient to confirm that urothelial proliferation caused by the genotoxic agent of AAM is the preneoplastic lesion of urothelial carcinoma. However, the properties of loss of control of cellular proliferation in the urothelium might be a procarcinogenic phenomenon in tumorigenesis. The molecular mechanism by which AA induces urothelial carcinogenesis needs more studies to clarify it. Same as the absence of development of urothelial cancer in this study, the expression of p53 of urinary bladder tissues does not increase dose dependently, with 0.95-fold expression in the low dose AAM group and with 0.97-fold expression in the high dose AAM-group, comparing with the normal control group. The absence of the development of urothelial malignancy might because of the insufficient therapeutic dose or duration of AA I administration. For example, the increased expressions of cdk2 and cdk4 only exist in the high dose AAM group (Fig. 2A). Clinically, the cumulated dose of Aristolochia fangchi is associated with higher risk of developing urothelial cancer (Nortier et al., 2000). We infer that the therapeutic dose and duration of AA, particularly AA I, play important roles in developing urothelial cancer and in increasing expression of p53. Regarding the result of urothelial proliferation caused by AAM in our study, we did not examine that it was via genotoxic, non-genotoxic or both mechanisms. Although there is good evidence that AA is genotoxic through the formation of specific DNA adducts
Fig. 3. Proposed model for the induction of urothelial proliferation in rats caused by aristolochic acid mixture through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2.
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and genotoxicity has been found to be associated with cell proliferation and tumorigenesis (Culp et al., 2000; Jackson et al., 2003), the possibility of non-genotoxic mechanism could not be excluded whether the urothelial proliferation was secondary to injury. In this field, further study is needed to clarify this issue. In conclusion, our results suggest that exposure to AAM results in urothelial proliferation in rats caused by cell cycle progression via activation of cyclin D1/ cdk4 and cyclin E/cdk2 (Fig. 3).
Acknowledgement This work was supported by a grant from the Chung Shan Medical University Research Fund (CSMU91OM-B-045).
References Arlt, V.M., Wiessler, M., Schmeiser, H.H., 2000. Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. Carcinogenesis 21, 235–242. Arlt, V.M., Schmeiser, H.H., Pfeifer, G.P., 2001. Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. Carcinogenesis 22, 133– 140. Arlt, V.M., Stiborova, M., Schmeiser, H.H., 2002. Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17, 265–277. Bieler, C.A., Stiborova, M., Wiessler, M., Cosyns, J.P., van Ypersele, de S.C., Schmeiser, H.H., 1997. 32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis 18, 1063–1067. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Wese, F.X., van Ypersele, de S.C, 1993. Urothelial lesions in Chinese-herb nephropathy. American Journal of Kidney Diseases 33, 1011–1017. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Van Cangh, P.J., van Ypersele, de S.C, 1994. Urothelial malignancy in nephropathy due to Chinese herbs. Lancet 344, 188. Cosyns, J.P., Goebbels, R.M., Liberton, V., Schmeiser, H.H., Bieler, C.A., Bernard, A.M., 1998. Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats. Archives of Toxicology 72, 738– 743. Cosyns, J.P., Jadoul, M., Squifflet, J.P., Wese, F.X., van Ypersele, de S.C., 1999. Urothelial lesions in Chinese-herb nephropathy. American Journal of Kidney Diseases 33, 1011–1017. Culp, S.J., Warbritton, A.R., Smith, B.A., Li, E.E., Beland, F.A., 2000. DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene. Carcinogenesis 21, 1433–1440. Debelle, F.D., Nortier, J.L., De Prez, E.G., Garbar, C.H., Vienne, A.R., Salmon, I.J., Deschodt-Lanckman, M.M., Vanherweghem, J.L., 2002. Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats. Journal of the American Society of Nephrology 13, 431–436. Gatenby, R.A., Vincent, T.L., 2003. An evolutionary model of carcinogenesis. Cancer Research 63, 6212–6220. Greenblatt, M.S., Bennett, W.P., Hollstein, M., Harris, C.C., 1994. Mutations in the p53 tumor suppressor gene: clues to cancer
etiology and molecular pathogenesis. Cancer Research 54, 4855– 4878. Hunter, T., Pines, J., 1994. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79, 573–582. Hussain, S.P., Harris, C.C., 1998. Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Research 58, 4023–4037. Izumi, M., Yokoi, M., Nishikawa, N.S., Miyazawa, H., Sugino, A., Yamagishi, M., Yamaguchi, M., Matsukage, A., Yatagai, F., Hanaoka, F., 2000. Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1. Biochimica et Biophysica Acta 24, 341–352. Jackson, L., Kofman, S., Weiss, A., Brodovsky, H., 1964. Aristolochic acid (NSC-50413): phase I clinical study. Cancer Chemotherapy Reports 42, 3–37. Jackson, P.E., OÕConnor, P.J., Cooper, D.P., Margison, G.P., Povey, A.C., 2003. Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumour yield in mice treated with 1,2-dimethylhydrazine. Carcinogenesis 24, 527–533. Lord, G.M., Tagore, R., Cook, T., Gower, P., Pusey, C.D., 1999. Nephropathy caused by Chinese herbs in the UK. Lancet 354, 481– 482. Mengs, U., Lang, W., Poch, J.A., 1982. The carcinogenic action of aristolochic acid in rats. Archives of Toxicology 51, 107– 119. Nevins, J.R., Leone, G., DeGregori, J., Jakoi, L., 1997. Role of the Rb/E2F pathway in cell growth control. Journal of Cellular Physiology 173, 233–236. Nortier, J.L., Martinez, M.C., Schmeiser, H.H., Arlt, V.M., Bieler, C.A., Petein, M., Depierreux, M.F., De Pauw, L., Abramowicz, D., Vereerstraeten, P., Vanherweghem, J.L., 2000. Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). The New England Journal of Medicine 342, 1686– 1692. Pena, J.M., Borras, M., Ramos, J., Montoliu, J., 1996. Rapidly progressive interstitial renal fibrosis due to a chronic intake of a herb (Aristolochia pistolochia) infusion. Nephrology, Dialysis, Transplantation 11, 1359–1360. Peters, G., Hedwall, P.R., 1963. Aristolochic acid intoxication: a new type of impairment of urinary concentrating ability. Archives Internationales de Pharmacodynamie et de Therapie 145, 334– 355. Pfau, W., Schmeiser, H.H., Wiessler, M., 1990. Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA. Carcinogenesis 11, 313–319. Pfau, W., Schmeiser, H.H., Wiessler, M., 1991. N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen. Chemical Research in Toxicology 4, 581– 586. Robisch, G., Schimmer, O., Goggelmann, W., 1982. Aristolochic acid is a direct mutagen in Salmonella typhimurium. Mutation Research 105, 201–204. Rossiello, M.R., Laconi, E., Rao, P.M., Rajalakshmi, S., Sarma, D.S., 1993. Induction of hepatic nodules in the rat by aristolochic acid. Cancer Letters 71, 83–87. Schmeiser, H.H., Pool, B.L., Wiessler, M., 1986. Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver. Carcinogenesis 7, 59–63. Schmeiser, H.H., Janssen, J.W., Lyons, J., Scherf, H.R., Pfau, W., Buchmann, A., Bartram, C.R., Wiessler, M., 1990. Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer Research 50, 5464–5469. Schmeiser, H.H., Bieler, C.A., Wiessler, M., van Ypersele, de S.C, Cosyns, J.P., 1996. Detection of DNA adducts formed by
H.-R. Chang et al. / Food and Chemical Toxicology 44 (2006) 28–35 aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. Cancer Research 56, 2025–2028. Stengel, B., Jones, E., 1998. End-stage renal insufficiency associated with Chinese herbal consumption in France. Nephrologie 19, 15– 20. Vanhaelen, M., Vanhaelen-Fastre, R., But, P., Vanherweghem, J.L., 1994. Identification of aristolochic acid in Chinese herbs. Lancet 343, 174. Vanherweghem, J.L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R., et al., 1993. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387– 391. Vanherweghem, J.L., Tielemans, C., Simon, J., Depierreux, M., 1995. Chinese herbs nephropathy and renal pelvic carcinoma. Nephrology, Dialysis, Transplantation 10, 270–273.
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Vermeulen, K., Van Bockstaele, D.R., Berneman, Z.N., 2003a. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Proliferation 36, 131–149. Vermeulen, K., Berneman, Z.N., Van Bockstaele, D.R., 2003b. Cell cycle and apoptosis. Cell Proliferation 36, 165–175. Wang, J.Y., Knudsen, E.S., Welch, P.J., 1994. The retinoblastoma tumor suppressor protein. Advances in Cancer Research 64, 25– 85. Weinberg, R.A., 1995. The retinoblastoma protein and cell cycle control. Cell 81, 323–330. Yang, C.S., Lin, C.H., Chang, S.H., Hsu, H.C., 2000. Rapidly progressive fibrosing interstitial nephritis associated with Chinese herbal drugs. American Journal of Kidney Diseases 35, 313– 318. Zheng, F., Zhang, X., Huang, Q., 2001. Establishment of model of aristolochic acid-induced chronic renal interstitial fibrosis in rats. Zhonghua Yi Xue Za Zhi 81, 1095–1100.