Carcinogenic Aristolochic Acid Research Articles

Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene.

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m(2)) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C -->T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A --> T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.

Effects of dexfenfluramine on aristolochic acid nephrotoxicity in a rat model for Chinese-herb nephropathy

Chinese-herb nephropathy (CHN) is a progressive renal interstitial fibrosis initially reported after concomitant intake of an anorexigen, (dex)fenfluramine, and a Chinese herb ( Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acid (AA). We thus tested the possible enhancing effect of the active enantiomer dexfenfluramine (DXF) on AA nephrotoxicity in a rat model for CHN. Groups of 12 salt-depleted male Wistar rats received daily subcutaneous injections of 7 mg/kg body weight DXF (DXF group), 7 mg/kg body weight AA (AA group), a combination of the same doses of AA and DXF (AA+DXF group), or vehicle (control group) for up to 35 days. Six animals per group were killed on day 10 and the remaining six on day 35. Renal function was evaluated by determining serum creatinine and urinary leucine aminopeptidase activity. Histological evaluation of kidney samples was performed and tubulointerstitial injuries were semiquantified. The DXF group did not differ from controls for any parameter. Similarly elevated serum creatinine levels, decreased leucine aminopeptidase enzymuria, and renal lesions were observed in the AA and the AA+DXF groups after both 10 and 35 days. The formation of specific AA-DNA adducts in liver and renal tissue samples was assessed by the (32)P-postlabelling method. Specific AA-DNA adduct levels were significantly increased in kidney tissues from AA+DXF rats compared with AA rats. These functional and histological data suggest that DXF does not enhance AA nephrotoxicity in a rat model for CHN. Further investigations are needed to clarify the mechanism by which DXF may enhance AA-DNA adduct formation.

Aristolochic acid and 'Chinese herbs nephropathy': a review of the evidence to date.

Chinese herbs nephropathy (CHN) is a rapidly progressive interstitial nephropathy reported after the introduction of Chinese herbs in a slimming regimen followed by young Belgian women. It is characterised by early, severe anaemia, mild tubular proteinuria and initially normal arterial blood pressure in half of the patients. Renal histology shows unusual extensive, virtually hypocellular cortical interstitial fibrosis associated with tubular atrophy and global sclerosis of glomeruli decreasing from the outer to the inner cortex. Urothelial malignancy of the upper urinary tract develops subsequently in almost half of the patients. Suspicion that the disease was due to the recent introduction of Chinese herbs in the slimming regimen was reinforced by identification in the slimming pills of the nephrotoxic and carcinogenic aristolochic acid (AA) extracted from species of Aristolochia. This hypothesis was substantiated by the identification of premutagenic AA-DNA adducts in the kidney and ureteric tissues of CHN patients. Finally, induction of the clinical features (interstitial fibrosis and upper urothelial malignancy) typical of CHN in rodents given AA alone removed any doubt on the causal role of this phytotoxin in CHN, now better called aristolochic acid nephropathy (AAN). AAN is not restricted to the Belgian cases. Similar cases have been observed throughout the world, but AA is sometimes incriminated on the basis of the known content of AA in the herbs. The possibility remains that in some individuals in whom AA has not been demonstrated, other phytotoxins might be implicated. Biological and morphological features of AAN are strikingly similar to those reported in another fibrosing interstitial nephropathy of still unknown aetiology, Balkan endemic nephropathy (BEN). Interestingly, AA was incriminated as the cause of BEN many years ago, a hypothesis yet to be fully explored. The intake of AA and the presence of tissular AA-DNA adducts in patients with an unequivocal diagnosis of BEN remains to be demonstrated. The tragic phenomenon of CHN, recognised only 10 years ago, has been at the root of significant research and progress both in nephrology and oncology. It has provided a fascinating opportunity to understand the link between a fibrosing interstitial nephropathy and urothelial carcinoma. It allows the categorisation of interstitial nephritis on the basis of histological findings, of initiating toxic substances and of associated clinical features. Finally, it has led to the withdrawal in several countries of a previously unsuspected carcinogenic and nephrotoxic substance.

Renal interstitial fibrosis and urothelial carcinoma associated with the use of a Chinese herb ( Aristolochia fangchi)

A new renal disease called ‘Chinese-herb nephropathy’ (CHN) has been reported to occur in women who have ingested slimming pills containing powdered extracts of the Chinese herb Stephania tetrandra (ST) . Moderate to end-stage renal disease developed, requiring renal replacement therapy by dialysis or transplantation. Phytochemical analyses of the pills revealed the presence of aristolochic acids (AA) instead of tetrandrine, suggesting the substitution of ST (Han fang ji) by Aristolochia fangchi containing nephrotoxic and carcinogenic AA. A typical histological feature of CHN is a progressive interstitial fibrosis leading to a severe atrophy of the proximal tubules, as documented by the urinary excretion rates of markers of tubular integrity (reduction of neutral endopeptidase enzymuria and high levels of microproteinurias). Removal of the native kidneys and ureters in end-stage CHN patients provided a high prevalence of urothelial carcinoma (46%). Tissue samples contained AA-related DNA adducts, which are not only specific markers of prior exposure to AA but are also directly involved in tumorigenesis. Exposure to Aristolochia species (spp.) is associated with the development of renal interstitial fibrosis (CHN) and urothelial cancer in humans. Health professionals should be aware that in traditional Chinese medicine, Aristolochia spp. are considered interchangeable with certain other herbal ingredients and are also sometimes mistaken for ST, Akebia, Asarum, Clematis spp. and Cocculus spp. in herbal remedies.

Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy.

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of enzymes of rat renal and hepatic cytosolic fractions to activate AA to metabolites forming DNA adducts by the nuclease P1-enhanced version of the (32)P-postlabeling assay. Cytosolic fractions of both these organs generated AA-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI and 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II were generated from AAII by hepatic cytosol. Qualitatively the same AA-DNA adduct patterns were observed, although at lower levels, upon incubation of AAs with renal cytosol. To define the role of cytosolic reductases in the reductive activation of AA, we investigated the modulation of AA-DNA adduct formation by cofactors, specific inducers or selective inhibitors of the cytosolic reductases, DT-diaphorase, xanthine oxidase (XO) and aldehyde oxidase. The role of the enzymes in AA activation was also investigated by correlating the DT-diaphorase- and XO-dependent catalytic activities in cytosolic sample with the levels of AA-DNA adducts formed by the same cytosolic sample. On the basis of these studies, we attribute most of the cytosolic activation of AA to DT-diaphorase, although a role of cytosolic XO cannot be ruled out. With purified DT-diaphorase, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The binding orientation of AAI in the active site of DT-diaphorase was predicted by computer modeling based on published X-ray structures. The results presented here are the first report demonstrating a reductive activation of carcinogenic AAs by DT-diaphorase.

Progression rate of Chinese herb nephropathy: impact of Aristolochia fangchi ingested dose

Renal failure after ingestion of Chinese herbs between 1990 and 1992 was related to the replacement of Stephania tetrandra by Aristolochia fangchi (ST-AF), containing nephrotoxic and carcinogenic aristolochic acids. However, the relationship between ST-AF and renal failure is still a matter of debate. We therefore tested the impact of the ST-AF ingested dose on the progression of renal function deterioration. Analysis of medical charts and prescriptions between 1990 and 1992 was carried out to determine the presence of risk factors for kidney failure and the cumulative dose of pill components. Individual progression rate of renal impairment was studied by the time-course of the inverse of blood creatinine level (1/P(creat)). Patients were divided into an end-stage renal disease (ESRD) group (n=44) and a chronic renal failure (CRF) group (n=27) according to their P(creat) at the time of this study. The mean number of risk factors (+/-SD) was equally distributed within both groups (1.50+/-0.18 vs 1.59+/-0.17, P=0.74). Patients from the ESRD group ingested significantly higher cumulative doses of ST--AF (192+/-13.1 g vs 138+/- 16.3 g), Magnolia officinalis, (80.1+/-6.3 g vs 59.8+/-11.7 g), diethylpropion (14.7+/-1.4 g vs 10.0+/-1.4 g) and fenfluramine (14.1+/-1.6 g vs 8.7+/-1.3 g). In the ESRD group, some patients who had received steroids had a slower progression to ESRD than the others. In multiple regression analysis, ST-AF emerged as the only significant drug predicting the slope of the progression of renal failure. Moreover, hypothesizing a linear dose-response relationship, the risk of developing ESRD linearly increased with ST-AF doses. The relationship between the cumulative ST-AF dose and the renal failure progression rate confirms that regular ingestion of Aristolochia sp. extracts is causally involved in the onset of chronic interstitial nephropathy leading to ESRD.

Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats.

Chinese-herb nephropathy (CHN) is a rapidly progressive renal fibrosis associated with the intake of a Chinese herb (Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acids (AA). This study attempted to reproduce the main features of human CHN (renal failure, tubular atrophy, and interstitial fibrosis) in a rat model similar to that of cyclosporin-induced nephropathy. Salt-depleted male Wistar rats received daily subcutaneous injections of either 1 mg/kg body wt AA (low-dose AA group), 10 mg/kg body wt AA (high-dose AA group), or vehicle (control group) for 35 d. On days 10 and 35, assessment of renal function, measurements of urinary excretion of glucose, protein, and leucine aminopeptidase, and histologic analyses were performed (six rats euthanized/group). High-dose AA induced glucosuria, proteinuria, and elevated serum creatinine levels and reduced leucine aminopeptidase enzymuria on days 10 and 35, whereas low-dose AA had no significant effect. Tubular necrosis associated with lymphocytic infiltrates (day 10) and tubular atrophy surrounded by interstitial fibrosis (day 35) were the histologic findings for the high-dose AA-treated rats. In both AA groups, urothelial dysplasia was also observed, as well as fibrohistiocytic sarcoma at the injection site. A short-term model of AA-induced renal fibrosis was established in salt-depleted Wistar rats. These results support the role of AA in human CHN and provide a useful model for examination of the pathophysiologic pathways of renal fibrosis.

Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: evidence for reductive activation by cytochromes P450 1A1 and 1A2.

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Determining the capability of humans to metabolize AA and understanding, which human enzymes are involved in AA activation is important in the assessment of individual susceptibility. Using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we compared the ability of human, minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles reproducing those found in renal tissues from humans exposed to AA. Identical patterns of AA-DNA adducts were also observed when AA was activated by minipig and rat microsomes. Therefore, microsomes of both animals are suitable in vitro systems mimicking the enzymatic activation of AA in humans. To define the role of specific P450 enzymes and NADPH:P450 reductase in the activation of AA by human microsomes we investigated the modulation of AA-DNA adduct formation by specific inducers or selective inhibitors of P450s and cofactors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, beta-naphthoflavone, significantly stimulated the levels of AA-DNA adducts formed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such effect. Furthermore, only inhibitors of the P450 1A subfamily (alpha-naphthoflavone, furafylline) significantly decreased the amount of adducts formed by microsomes from humans, minipigs and rats. alpha-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower extent. On the basis of these results, we attribute most of the microsomal activation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase cannot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH:P450 reductase) and microsomes from baculovirus transfected insect cells expressing recombinant human P450 1A1/2 and NADPH:P450 reductase, the participation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductase in catalyzing the reductive activation of AA.

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase — 32P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin- N 6-yl)aristolactam I, 7-(deoxyguanosin- N 2-yl)aristolactam I and 7-(deoxyadenosin- N 6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin- N 2-yl)aristolactam II and 7-(deoxyadenosin- N 6-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (α-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.

Isolation of DT-Diaphorase [NAD(P)H Dehydrogenase (Quinone)] from Rat Liver Cytosol: Identification of New Enzyme Substrates, Carcinogenic Aristolochic Acids

Cytosolic fractions isolated from liver and kidney of rats treated with β-naphthoflavone, Sudan I, ellipticine, phenobarbital, ethanol, acetone and natural carcinogenic and nephrotoxic nitroaromatics, aristolochic acids, were tested for the activity of DT-diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2]. While the most efficient inducers of DT-diaphorase in liver were Sudan I, ellipticine and aristolochic acids, the highest increase in the DT-diaphorase activity in kidney was produced by aristolochic acids. No increase in the enzyme activity was determined after treatment of rats with acetone. DT-Diaphorase was isolated from liver cytosol of Sudan I-treated rats by the procedure consisting of fractionation with ammonium sulfate, gel permeation chromatography on a Sephadex G-150 column, affinity chromatography on an Affi-Gel Blue (Cibracron Blue Agarose) column and re-chromatography on Sephadex G-150. Rat DT-diaphorase catalyzed NAD(P)H-dependent reduction of menadione (vitamin K3), vitamin K1and 4-nitrosophenol as substrates. Moreover, we newly identified two carcinogenic nitroaromatic compounds, aristolochic acids, as other substrates of DT-diaphorase. A selective inhibitor of the human DT-diaphorase, dicoumarol, inhibited the catalytic activity of the rat purified enzyme.

Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase α

In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3′ end by 32P-postlabeling. These adducted templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase α. Both AAI-DNA adduct (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80–95%) at the nucleotide 3′ to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis ot the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase α three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion.

Cytochrome P-450 and Peroxidase Oxidize Detoxication Products of Carcinogenic Aristolochic Acids (Aristolactams) to Reactive Metabolites Binding to DNA in vitro

We report the analysis of DNA adducts formed from aristolactams I and II, which are the final metabolites derived from carcinogenic aristolochic acids in vivo, after their oxidation by microsomal cytochrome P-450 and horseradish peroxidase in vitro. DNA adducts were detected and quantified using the nuclease P1-enhanced variation of the 32P-postlabeling assay. Quantitative analysis revelead that the extent of modification of DNA by aristolactams activated by peroxidase was more than one order of magnitude higher than for activation by microsomal cytochrome P-450. Peroxidase catalyzes the formation of active oxygen in the presence of NADH, H2O2 and aristolactams. Aristolactams are also oxidized by mammalian peroxidase prostaglandin H synthase. The possible role of aristolactams in carcinogenesis induced by aristolochic acid is discussed.

Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid.

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.

Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver.

The rat liver 9000 g supernatant mediated metabolism of the carcinogenic aristolochic acid, which consists of aristolochic acid I (AAI) and aristolochic acid II (AAII), was investigated. Under anaerobic conditions the major metabolites were the corresponding aristolactams for both AAI and AAII. In contrast under aerobic conditions AAII was not detectably metabolized and the only metabolite found for AAI was the O-demethylated derivative aristolochic acid Ia (AAIa). The metabolites were identified by their u.v., mass and n.m.r. spectra and by comparison with reference standards. The mutagenic activities of the three metabolites were determined in Salmonella typhimurium strains TA1537 and TA 100. The aristolactams were mutagenic in both strains when a metabolizing system was present. These results indicate that AAI or AAII and their aristolactams exert their effect via a common reactive intermediate, probably the corresponding hydroxylamine. AAIa was only very weakly mutagenic and this metabolite may therefore not be regarded as a major mutagenic metabolite of AAI. These findings suggest that the acids are preferentially metabolized by two totally different pathways in vitro, namely an oxidative pathway for AAI and a reductive pathway for AAII.

Mutagenicity of the two main components of commercially available carcinogenic aristolochic acid in Salmonella typhimurium

One of the 2 main components of the commercially available carcinogenic aristolochic acid (AA) was isolated, the other was enriched. Three different aristolochic acid samples (AAI 99% pure; AAI 65% + AAII 35%; AAI 32% + AAII 68%) were assayed for mutagenic activity in Salmonella typhimurium TA1537, TA100 and TA100 NR with and without the addition of a metabolizing mixture. The two main components (AAI and AAII) were direct mutagens in Salmonella strains TA1537 and TA100 with almost equal mutagenic potency. In TA100 NR the aristolochic acid samples showed no or only a very low level of biological activity, indicating the necessity of nitroreduction for the bioactivation of the samples. These findings suggest that both AAI as well as AAII can be used in further studies to elucidate the metabolism of aristolochic acid.