Cancer Letters

Cancer Letters

Volume 232, Issue 2, 8 February 2006, Pages 236-242
Cancer Letters

Chronic renal failure rats are highly sensitive to aristolochic acids, which are nephrotoxic and carcinogenic agents

https://doi.org/10.1016/j.canlet.2005.02.021Get rights and content

Abstract

Aristolochic acid (AA), a component of some Chinese herbal medicines, may cause Chinese Herbs Nephropathy (CHN) and multi-systemic tumors by the formation of AA–DNA adducts. In this study, we established an animal model to further characterize the mechanisms of AA-induced diseases. Our results indicated that AA significantly inhibited rat growth in terms of weight gain. By measuring the serum creatinine levels, AA resulted in considerable damage to the rat renal system, not only for those in which chronic renal failure (CRF) was induced but also for normal healthy rats. Mutation-specific polymerase chain reaction (PCR) and XbaI restriction fragment length polymorphism (RFLP) revealed the CAA→CTA transversion mutation at codon 61 of the H-ras proto-oncogene from the stomach tissues of CRF rats fed with AA, but not from other tissues of rats in the same experimental group. In addition, no such mutations were found in the tissues of CRF rats without AA treatment or healthy rats fed with AA. Our results strongly demonstrated that AA was in fact nephrotoxic and carcinogenic, especially to those CRF rats.

Introduction

Aristolochic acids (AA) extracted from genus Aristolochia, including 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) and 6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAII), are regarded as the principal components of herbal remedies to stimulate the defense mechanism against infections, to treat patients with arthritis, gout, as well as tumors [1], [2], [3], [4], [5]. However, in 1991, AA in slimming capsules was suspected of causing the so-called Chinese herb nephropathy (CHN), a progressive renal fibrosis that was diagnosed in a group of young Belgian women who had all followed the same slimming regimen [6]. Since then, multiple cases of CHN have been sporadically reported worldwide [4], [7], [8] and the potential nephrotoxicity of AA to rodents has also been ascertained in several laboratories [8], [9], [10], [11]. Furthermore, animals treated with AA seemed to be predisposed to develop various tumors [5], [12], [13], [14].

Recent studies have revealed the potential genotoxic mechanisms of AA in rodents, in which AA was metabolized through the nitroreduction pathway to turn into the mutagenic species aristolactam-nitriumion that might form covalent AA–DNA adducts along with deoxyadenosin or deoxyguanosin residues in DNA [15], [16]. Of these, the adenine adducts (dA–AAI and dA–AAII) have been detected not only in the tumors of rodents [15], [16], [17] but also in urethelial tissue of CHN patients [18]. To search for the tumorigenic mechanisms of AA, it has been suggested that the adenine adducts of AA–DNA might mistakenly be incorporated with dAMP during DNA synthesis, thereby leading to an A:T→T:A transversion mutation during the subsequent DNA replication [19], [20]. The A:T→T:A mutation has been identified in the activated H-ras proto-oncogene, specifically at its codon 61 (CAA→CTA), in the tumors of rodents treated with plant extracts containing AA [21], suggesting that the adenine adducts caused by the intake of AA components might bypass the eukaryotic DNA repair machinery.

Although there is increasing evidence demonstrating the nephrotoxicity and carcinogenicity of AA in rodents [13], [22], only a small percentage of participants who followed the similar slimming regimen offered by the same clinical center in Belgium have actually developed CHN or cancers [23]. It has been suggested that the composition difference of AA in the slimming pills due to the batch variation of production might lead to this clinical discrepancy [24], [25]. On the other hand, the impacts of AA administration may greatly depend on each individual physical history. Therefore, in this study, we characterized the outcomes of AA intake in rats some of which had undergone the 5/6 chronic renal failure (CRF) treatment. Our results demonstrated that AA caused rat renal failure and was tumorigenic by inducing the A:T→T:A transversion mutation at codon 61 of the H-ras gene thereby triggering oncogenic activity. The mutation incidence was heightened in the stomach tissue of rats with 5/6 CRF.

Section snippets

Chemicals

Aristolochic acid (AA) in the form of sodium salt was purchased from SIGMA (St Louis, MO, USA), and contained 58% AAI and 36% AAII.

Animal model

Eight-week-old wistar rats weighing ∼150±60 g were used in this study. Rats were divided into four groups as follows: group A (control group), normal rats (n=5) fed without AA; group B, normal rats (n=10) fed with AA; group C, 5/6 CRF (chronic renal failure) rats (n=10) fed without AA; and group D, 5/6 CRF rats (n=10) fed with AA. For the 5/6 CRF model (groups C and

Animal growth

We found that normal rats in group A without AA treatment gained considerably more weight than those in group B fed with AA (Fig. 1). 5/6 CRF treatment (group C) also reduced weight gain as shown in Fig. 1. The combination of 5/6 CRF treatment and AA supplement further slowed rat growth. In addition, the survival of animals was lower after administration of AA in 5/6 CRF-treated rats.

Serum creatinine

Elevated serum creatinine is generally considered as a major indicator for renal failure; therefore, both

Discussion

Aristolochic acids (AAs) have been detected in several commercially available Chinese herbal medicines [30] and are suspected to be associated with more than 200 cases of severe nephropathy or Chinese herb nephropathy (CHN) worldwide [4], [5], [7], [31], [32], [33]. The majority of CHN patients needed to undergo renal replacement therapy and some of them have developed cancer [4], [5], [31]. However, many other people who followed the same slimming regimen were not diagnosed with CHN [23]. Some

Acknowledgements

This work was supported by grants from Tunghai University, Taichung, ROC and Taichung Veterans General Hospital, Taichung, ROC to J.Y. Gu and C.L. Cheng.

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