Impact of Dietary Practices on DNA Adduct Formation by Aristolochic Acid I in Mice: Drinking Alkaline Water as a Risk Mitigation Strategy.

Volker M. ARLT,* Dusan FERLUGA, Marie STIBOROVA, Annie PFOHL-LESZKOWICZ, Mato VUKELIC, Stjepan CEOVIC, Heinz H. SCHMEISER and Jean-Pierre COSYNS Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, United Kingdom Institute of Pathology, University of Ljubljana, Ljubljana, Slovenia Department of Biochemistry, Charles University, Prague, The Czech Republic Ecole Nationale Superieure Agronomique de Toulouse, Laboratoire de Toxicologie et Securite Alimentaire, Auzeville Tolosane, France Department of Pathology, General Hospital, Slavonski Brod, Croatia Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany Department of Pathology, Universite Catholique de Louvain, Medical School, Brussels, Belgium

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.

Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804–2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

This PhD thesis aimed to provide additional evidence to demonstrate the potential of an integrated testing strategy using in vitro assays with physiologically based kinetic (PBK) modeling based-reverse dosimetry to predict in vivo toxicity without animal testing. Kidney toxicity was chosen as the toxicity endpoint and aristolochic acids (AAs) were selected as model chemicals. AAs are natural nephrotoxic, genotoxic and carcinogenic chemicals present in Aristolochia species. PBK models for rat, mouse and human were developed for aristolochic acid I (AAI) based on kinetic parameter values derived from in vitro incubations using relevant tissue fractions. Then, in vitro concentration-response curves for cytotoxicity of AAI were obtained in kidney cell lines and translated to in vivo dose-response curves for kidney toxicity using PBK modeling-based reverse dosimetry. The points of departure (PODs) obtained from these predicted in vivo dose-response curves generally fell within the range of PODs derived from in vivo literature data on kidney toxicity of AAI. The same PBK models were subsequently used to translate the in vitro concentration-response curves for AAI-DNA adduct formation to in vivo dose-response curves for kidney AAI-DNA adduct formation. The predicted in vivo AAI-DNA adduct formation in the rat, mouse and human kidney varied within an order of magnitude compared to the in vivo values reported in the literature. The PBK models were also used to predict the dose level that would be required in humans to obtain the level of DNA adducts in rats at the BMD10 (the benchmark dose causing a 10% extra risk above background level) value for AAI-induced tumor formation in the rat kidney. This analysis revealed that the dose level required to induce the level of DNA adduct formation that equals the DNA adduct level at the BMD10 were similar to AA doses estimated to be taken in Belgian patients that developed urinary tract cancer. Given that the exposure to AAI is often accompanied by the presence of AAII, in a next study the relative formation of DNA adducts by these two major AA congeners was investigated. The results revealed that the relative higher formation of AAI-DNA adducts as compared to AAII-DNA adducts observed in vitro was not reflected in vivo where the levels formed upon exposure to equal dose levels were relatively similar. PBK model based translation of the in vitro data to the in vivo situation revealed that PBK model based prediction of in vivo DNA adduct formation is feasible. However, predicted AAI-DNA adduct levels were higher than predicted AAII-DNA adduct levels, indicating that the difference between the in vitro and in vivo AAI-/AAII-DNA adduct ratios could only in part be explained by differences in in vivo kinetics of AAI compared to AAII. The discrepancy between the difference in DNA adduct formation of AAI and AAII in the in vitro and the in vivo situation is an issue that needs further investigation to also adequately predict the relative differences between the two AAs. In a final chapter this thesis aimed to investigate the possible risks associated with exposure to AAs based on AA levels measured in plant food supplements (PFS) and herbal products. This is of interest given the restrictions on the presence of AAs in food, installed in various countries including The Netherlands, after the incidences with induction of Aristolochic Acid Nephropathy upon use of herbal weight loss preparations that accidentally contained AAs. The risk assessment of PFS and herbal products containing AAs purchased via online markets revealed that consumers can still be exposed to AA-containing PFS and herbal products and that the corresponding levels of exposure raise concern especially for people who frequently use the products. Altogether, this thesis presented further support for the use of combined in vitro-PBK modeling based alternative tools for risk assessment and revealed the continued risks posed by AAs present in PFS and herbal products.

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.

To find out whether ochratoxin A (OTA), citrinin (CIT), aristolochic acids (AA) are etiologic agents of Balkan endemic nephropathy (BEN) or Chinese herbal nephrotoxicity, and associated urinary tract tumor (UTT), we have compared (i) in human kidney cell culture, the DNA adduct formation and persistence of OTA/CIT and AA adducts (ii) analyzed DNA adduct in several tumors from human kidney suspected to be exposed to either OTA and CIT, or AAs (iii) analyzed OTA, CIT, and AA in food. In kidney cell cultures, formation of specific OTA-DNA adduct and AA-DNA adduct were detected in the same range (around 10 adducts/10(9) nucleotides) and were time- and dose-dependent. After 2 days all disappeared. DNA adduct related to OTA and CIT are found in human kidney tissues from Balkans, France, and Belgium whereas no DNA adducts related to AA could be found in any tumors of BEN patients from Croatia, Bulgaria, or Serbia. No DNA adduct was found in kidney biopsy or necropsy of the French women suspected to be exposed to AA. OTA and CIT are more frequently found in rural area. AA was never detected. All these plead for implication of mycotoxins, especially OTA, in BEN and UTT.

Recently, chronic Aristolochia poisoning was found responsible for the aetiology of Balkan endemic nephropathy (BEN) in Croatia, Serbia, and Bosnia, and diet was the likely route of exposure to aristolochic acid (AA). BEN, often associated with an increased incidence of upper urinary tract carcinoma (UUC), also affects residents of certain rural villages in Romania. AA is a nephrotoxin and human carcinogen that forms DNA adducts after metabolic activation, which induce characteristic TP53 mutations in urothelial tumours. Here we present the first evidence linking AA exposure to UUC in residents of an endemic region in the Romanian Mehedinti County. DNA was extracted from kidney and tumour tissue of seven patients who underwent nephroureterectomy for UUC and resided in BEN villages (endemic group). Five patients with UUC from nonendemic villages served as controls. AA-DNA adducts (7-(deoxyadenosin-N(6) -yl)-aristolactam I), established biomarkers of AA exposure, were identified by (32)P-postlabelling in renal DNA of six patients from the endemic group and in one of the nonendemic group (adduct levels ranged from 0.3 to 6.5 adducts per 10(8) nucleotides). Additionally, an A to T transversion in TP53, a base substitution characteristic of AA mutagenic activity was found in urothelial tumour DNA of one patient from the endemic group. Our results provide a molecular link to the cause of urothelial tumours in BEN regions of Romania indicating that AA is the common aetiological agent for BEN across its numerous geographical foci.

Aristolochic acid (AA) is a plant alkaloid that causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases frequently associated with upper urothelial cancer (UUC). This review summarizes the significance of AA-derived DNA adducts in the aetiology of UUC leading to specific A:T to T:A transversion mutations (mutational signature) in AAN/BEN-associated tumours, which are otherwise rare in individuals with UCC not exposed to AA. Therefore, such DNA damage produced by AA-DNA adducts is one rare example of the direct association of exposure and cancer development (UUC) in humans, confirming that the covalent binding of carcinogens to DNA is causally related to tumourigenesis. Although aristolochic acid I (AAI), the major component of the natural plant extract AA, might directly cause interstitial nephropathy, enzymatic activation of AAI to reactive intermediates capable of binding to DNA is a necessary step leading to the formation of AA-DNA adducts and subsequently AA-induced malignant transformation. Therefore, AA-DNA adducts can not only be utilized as biomarkers for the assessment of AA exposure and markers of AA-induced UUC, but also be used for the mechanistic evaluation of its enzymatic activation and detoxification. Differences in AA metabolism might be one of the reasons for an individual’s susceptibility in the multi-step process of AA carcinogenesis and studying associations between activities and/or polymorphisms of the enzymes metabolising AA is an important determinant to identify individuals having a high risk of developing AA-mediated UUC.

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause another type of kidney fibrosis with malignant transformation of the urothelium, called Balkan Endemic Nephropathy (BEN). The compound predominantly responsible for the nephropathy and urothelial cancer of AA, is aristolochic acid I (AAI) which is a genotoxic mutagen after metabolic activation The activation pathway involves reduction of the nitro group to a cyclic N-acylnitrenium ion that can form covalent DNA adducts. These specific DNA adducts have been detected in experimental animals exposed to AAI, and in urothelial tissues from AAN patients. In rodent tumours induced by AAI, 7-(deoxyadenosin-N(6)-yl)aristolactam I was the most abundant DNA adduct formed and associated with activation of ras oncogenes through a characteristic transversion mutation. Such A:T-->T:A mutations have been identified in TP53 of urothelial tumour DNA of an AAN patient and in several patients suffering from BEN along with specific AA-DNA adducts. Understanding which enzymes are involved in AAI activation to species forming DNA adducts and/or detoxification to its O-demethylated metabolite aristolochic acid Ia (AAIa) is important in order to assess susceptibility to this carcinogen. A literature search. The most important human enzymes activating AAI by simple nitroreduction in vitro are hepatic and renal cytosolic NAD(P)H:quinone oxidoreductase, hepatic microsomal cytochrome P450 (CYP) 1A2 and renal microsomal NADPH:CYP reductase as well as cyclooxygenase which is highly expressed in urothelial tissue. However, the contribution of most of these enzymes to the development of AAN and BEN diseases is still unclear. Hepatic CYP enzymes were found to detoxify AAI to AAIa in mice, and thereby protect the kidney from injury. CYP enzymes of the 1A subfamily seem to play a major role in this process in mouse liver. Likewise, among human CYP enzymes, CYP1A1 and 1A2 were found to be the most efficient enzymes participating in AAI oxidation to AAIa in vitro. Nevertheless, which CYPs are the most important in this process in both animal models and in humans have not been entirely resolved as yet. In addition, the relative contribution of enzymes found to activate AAI to species responsible for induction of urothelial cancer in humans remains still to be resolved.

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

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN.

Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography-tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly.

Balkan endemic nephropathy (BEN) is found in certain rural areas of the Balkans and affects at least 25,000 inhabitants. Of the many hypotheses on BEN, the Aristolochia hypothesis has recently gained ground substantiated by the investigations on aristolochic acid nephropathy (AAN). On both clinical and morphological grounds, AAN is very similar to BEN. That exposure to aristolochic acid (AA) of individuals living in endemic areas through consumption of bread made with flour contaminated with seeds of Aristolochia clematitis is responsible for BEN is an old hypothesis, but one which is fully consistent with the unique epidemiologic features of BEN. Here, we propose an approach to investigate AA-induced mutagenesis in BEN that can provide molecular clues to the aetiology of its associated urothelial cancer. The molecular mechanism of AA-induced carcinogenesis demonstrates a strong association between DNA adduct formation, mutation pattern and tumour development. A clear link between urothelial tumours, p53 mutations and AA exposure should emerge as more tumour DNA from BEN patients from different endemic areas becomes available for mutation analysis. We predict that the observed p53 mutation spectrum will be dominated by AT --> TA transversion mutations as has already been demonstrated in the human p53 gene of immortalized cells after exposure to AAI and urothelial tumours from BEN patients in Croatia. Moreover, the detection of AA-specific DNA adducts in renal tissue of a number of BEN patients and individuals living in areas endemic for BEN in Croatia provides new evidence that chronic exposure to AA is a risk factor for BEN and its associated cancer.

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(−/−) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(−/−) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(−/−) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation.