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.

• Keywords
• Balkan endemic nephropathy, DNA adducts, NAD(P)H:quinone oxidoreductase 1, aristolochic acid I, aristolochic acid II, aristolochic acid nephropathy, aristolochic acid-mediated carcinogenesis, cytochrome P450, genotoxicity,
• MeSH
• DNA Adducts metabolism   MeSH
• DNA, Neoplasm metabolism   MeSH
• Carcinogenesis * chemically induced   metabolism   MeSH
• Carcinogens toxicity   MeSH
• Rats MeSH
• Aristolochic Acids toxicity   MeSH
• Rats, Wistar MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Adducts MeSH
• aristolochic acid B MeSH Browser
• aristolochic acid I MeSH Browser
• DNA, Neoplasm MeSH
• Carcinogens MeSH
• Aristolochic Acids MeSH

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.

• Keywords
• DNA adduct formation, aristolochic acid, carcinogenicity, mutagenesis, nephrotoxicity,
• MeSH
• DNA Adducts metabolism   MeSH
• Balkan Nephropathy etiology   metabolism   MeSH
• Biomarkers * MeSH
• Carcinogens chemistry   metabolism   MeSH
• Aristolochic Acids chemistry   metabolism   MeSH
• Humans MeSH
• Disease Susceptibility MeSH
• Cell Transformation, Neoplastic genetics   metabolism   MeSH
• Urologic Neoplasms etiology   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA Adducts MeSH
• Biomarkers * MeSH
• Carcinogens MeSH
• Aristolochic Acids MeSH

ABSTRACT: The herbal drug aristolochic acid, a natural mixture of 8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AAI) and 6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AAII), is derived from Aristolochia species and is the cause of two nephropathies. Ingestion of aristolochic acid is associated with the development of urothelial tumors linked with aristolochic acid nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. The O-demethylated metabolite of AAI, 8-hydroxyaristolochic acid (AAIa), is the detoxification product of AAI generated by its oxidative metabolism. Whereas the formation of AAIa from AAI by cytochrome P450 (CYP) enzymes has been found in vitro and in vivo, this metabolite has not been found from AAII as yet. Therefore, the present study has been designed to compare the amenability of AAI and AAII to oxidation; experimental and theoretical approaches were used for such a study. In the case of experimental approaches, the enzyme (CYP)-mediated formation of AAIa from both carcinogens was investigated using CYP enzymes present in subcellular microsomal fractions and recombinant CYP enzymes. We found that in contrast to AAI, AAII is oxidized only by several CYP enzymatic systems and their efficiency is much lower for oxidation of AAII than AAI. Using the theoretical approaches, such as flexible in silico docking methods and ab initio calculations, contribution to explanation of these differences was established. Indeed, the results found by both used approaches determined the reasons why AAI is better oxidized than AAII; the key factor causing the differences in AAI and AAII oxidation is their different amenability to chemical oxidation.

• Keywords
• Enzymes, High pressure liquid chromatography, Molecular modeling, Redox reactions,
• Publication type
• Journal Article MeSH

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.

• Keywords
• 3-Nitrobenzanthrone, Aristolochic acid nephropathy, Balkan endemic nephropathy, Carcinogen metabolism, DNA adducts, Sulfotransferase 1A1,
• MeSH
• DNA Adducts drug effects   genetics   MeSH
• Arylsulfotransferase genetics   MeSH
• Benz(a)Anthracenes toxicity   MeSH
• Cytosol drug effects   metabolism   MeSH
• Liver drug effects   metabolism   MeSH
• Carcinogens toxicity   MeSH
• Aristolochic Acids toxicity   MeSH
• Kidney drug effects   metabolism   MeSH
• Humans MeSH
• Multigene Family MeSH
• Mice, Knockout MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3-nitrobenzanthrone MeSH Browser
• DNA Adducts MeSH
• Arylsulfotransferase MeSH
• Benz(a)Anthracenes MeSH
• Carcinogens MeSH
• Aristolochic Acids MeSH
• SULT1A1 protein, human MeSH Browser
• SULT1A2 protein, human MeSH Browser

Balkan endemic nephropathy (BEN) is a unique, chronic renal disease frequently associated with upper urothelial cancer (UUC). It only affects residents of specific farming villages located along tributaries of the Danube River in Bosnia-Herzegovina, Croatia, Macedonia, Serbia, Bulgaria, and Romania where it is estimated that ~100,000 individuals are at risk of BEN, while ~25,000 have the disease. This review summarises current findings on the aetiology of BEN. Over the last 50 years, several hypotheses on the cause of BEN have been formulated, including mycotoxins, heavy metals, viruses, and trace-element insufficiencies. However, recent molecular epidemiological studies provide a strong case that chronic dietary exposure to aristolochic acid (AA) a principal component of Aristolochia clematitis which grows as a weed in the wheat fields of the endemic regions is the cause of BEN and associated UUC. One of the still enigmatic features of BEN that need to be resolved is why the prevalence of BEN is only 3-7 %. This suggests that individual genetic susceptibilities to AA exist in humans. In fact dietary ingestion of AA along with individual genetic susceptibility provides a scenario that plausibly can explain all the peculiarities of BEN such as geographical distribution and high risk of urothelial cancer. For the countries harbouring BEN implementing public health measures to avoid AA exposure is of the utmost importance because this seems to be the best way to eradicate this once mysterious disease to which the residents of BEN villages have been completely and utterly at mercy for so long.

• Keywords
• Aristolochic acid, Aristolochic acid nephropathy, Balkan endemic nephropathy, Disease aetiology, Environmental and genetic factors, Upper urothelial cancer,
• MeSH
• Aristolochia chemistry   growth & development   toxicity   MeSH
• Balkan Nephropathy chemically induced   epidemiology   physiopathology   prevention & control   MeSH
• Diet adverse effects   MeSH
• Endemic Diseases * MeSH
• Confounding Factors, Epidemiologic MeSH
• Carcinogens, Environmental analysis   toxicity   MeSH
• Food Contamination * prevention & control   MeSH
• Aristolochic Acids analysis   toxicity   MeSH
• Kidney drug effects   physiopathology   MeSH
• Drug Resistance MeSH
• Humans MeSH
• Evidence-Based Medicine * MeSH
• Flour adverse effects   analysis   MeSH
• Plant Weeds chemistry   growth & development   toxicity   MeSH
• Prevalence MeSH
• Triticum growth & development   MeSH
• Risk MeSH
• Seeds growth & development   MeSH
• Urologic Neoplasms chemically induced   epidemiology   physiopathology   prevention & control   MeSH
• Crops, Agricultural growth & development   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Geographicals
• Europe, Eastern epidemiology   MeSH
• Names of Substances
• aristolochic acid I MeSH Browser
• Carcinogens, Environmental MeSH
• Aristolochic Acids MeSH

UNLABELLED: Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H: quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using (32)P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.

• Keywords
• 1A2 and 1B1, DNA adduct formation, aristolochic acid I, aristolochic acid nephropathy, nitroreduction, site-directed mutagenesis of cytochromes P450 1A1,
• MeSH
• DNA Adducts metabolism   MeSH
• Aryl Hydrocarbon Hydroxylases genetics   metabolism   MeSH
• Cytochrome P-450 CYP1A1 MeSH
• Cytochrome P-450 CYP1A2 MeSH
• Cytochrome P-450 CYP1B1 MeSH
• Catalytic Domain genetics   MeSH
• Catalysis MeSH
• Aristolochic Acids metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Mutagenesis, Site-Directed MeSH
• Oxidation-Reduction MeSH
• Recombinant Proteins MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• aristolochic acid I MeSH Browser
• Aryl Hydrocarbon Hydroxylases MeSH
• Cytochrome P-450 CYP1A1 MeSH
• Cytochrome P-450 CYP1A2 MeSH
• Cytochrome P-450 CYP1B1 MeSH
• Aristolochic Acids MeSH
• Recombinant Proteins MeSH

UNLABELLED: This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human NAD(P)H: quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/2 enzymes was investigated with pure enzymes, enzymes present in subcellular cytosolic and microsomal fractions, selective inhibitors, and animal models (including knock-out and humanized animals). For the theoretical approaches, flexible in silico docking methods as well as ab initio calculations were employed. The results summarized in this review demonstrate that a combination of experimental and theoretical approaches is a useful tool to study the enzyme-mediated reaction mechanisms of 3-NBA and AAI reduction.

• MeSH
• Acetyltransferases metabolism   MeSH
• DNA Adducts chemistry   metabolism   MeSH
• Aryl Hydrocarbon Hydroxylases metabolism   MeSH
• Benz(a)Anthracenes chemistry   metabolism   MeSH
• Biocatalysis MeSH
• Enzymes metabolism   MeSH
• Aristolochic Acids chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular * MeSH
• NAD(P)H Dehydrogenase (Quinone) metabolism   MeSH
• Sulfotransferases metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• 3-nitrobenzanthrone MeSH Browser
• Acetyltransferases MeSH
• DNA Adducts MeSH
• aristolochic acid I MeSH Browser
• Aryl Hydrocarbon Hydroxylases MeSH
• Benz(a)Anthracenes MeSH
• Enzymes MeSH
• Aristolochic Acids MeSH
• NAD(P)H Dehydrogenase (Quinone) MeSH
• Sulfotransferases MeSH

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy, which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause a similar type of kidney fibrosis with malignant transformation of the urothelium, the Balkan endemic nephropathy. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of a susceptibility to this carcinogen. The most important human enzymes activating AA by simple nitroreduction in vitro are hepatic and renal cytosolic NAD(P)H:quinone oxidoreductase, hepatic microsomal cytochrome P450 1A2 and renal microsomal NADPH:cytcohrome P450 reductase, besides cyclooxygenase, which is highly expressed in urothelial tissue. Despite extensive research, contribution of most of these enzymes to the development of these diseases is still unknown. Hepatic cytochromes P450 were found to detoxicate AA in mice, and thereby protect the kidney from injury. However, which of cytochromes P450 are the most important in this process both in animal models and in humans have not been entirely resolved as yet. In addition, the relative contribution of enzymes found to activate AA to species responsible for induction of urothelial cancer in humans remains still to be resolved.

• Keywords
• Aristolochic acid, Aristolochic acid- and Balkan endemic-nephropathy, metabolism, renal injury, tumor induction,
• Publication type
• Journal Article MeSH