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.

• Klíčová slova
• 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
• adukty DNA metabolismus   MeSH
• DNA nádorová metabolismus   MeSH
• karcinogeneze * chemicky indukované   metabolismus   MeSH
• karcinogeny toxicita   MeSH
• krysa rodu Rattus MeSH
• kyseliny aristolochové toxicita   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adukty DNA MeSH
• aristolochic acid B MeSH Prohlížeč
• aristolochic acid I MeSH Prohlížeč
• DNA nádorová MeSH
• karcinogeny MeSH
• kyseliny aristolochové 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.

• Klíčová slova
• DNA adduct formation, aristolochic acid, carcinogenicity, mutagenesis, nephrotoxicity,
• MeSH
• adukty DNA metabolismus   MeSH
• balkánská nefropatie etiologie   metabolismus   MeSH
• biologické markery * MeSH
• karcinogeny chemie   metabolismus   MeSH
• kyseliny aristolochové chemie   metabolismus   MeSH
• lidé MeSH
• náchylnost k nemoci MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• urologické nádory etiologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• adukty DNA MeSH
• biologické markery * MeSH
• karcinogeny MeSH
• kyseliny aristolochové 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.

• Klíčová slova
• 3-Nitrobenzanthrone, Aristolochic acid nephropathy, Balkan endemic nephropathy, Carcinogen metabolism, DNA adducts, Sulfotransferase 1A1,
• MeSH
• adukty DNA účinky léků   genetika   MeSH
• arylsulfotransferasa genetika   MeSH
• benz(a)anthraceny toxicita   MeSH
• cytosol účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• karcinogeny toxicita   MeSH
• kyseliny aristolochové toxicita   MeSH
• ledviny účinky léků   metabolismus   MeSH
• lidé MeSH
• multigenová rodina MeSH
• myši knockoutované MeSH
• myši transgenní MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3-nitrobenzanthrone MeSH Prohlížeč
• adukty DNA MeSH
• arylsulfotransferasa MeSH
• benz(a)anthraceny MeSH
• karcinogeny MeSH
• kyseliny aristolochové MeSH
• SULT1A1 protein, human MeSH Prohlížeč
• SULT1A2 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• Aristolochic acid, Aristolochic acid nephropathy, Balkan endemic nephropathy, Disease aetiology, Environmental and genetic factors, Upper urothelial cancer,
• MeSH
• Aristolochia chemie   růst a vývoj   toxicita   MeSH
• balkánská nefropatie chemicky indukované   epidemiologie   patofyziologie   prevence a kontrola   MeSH
• dieta škodlivé účinky   MeSH
• endemické nemoci * MeSH
• faktory vyvracející (epidemiologie) MeSH
• karcinogeny životního prostředí analýza   toxicita   MeSH
• kontaminace potravin * prevence a kontrola   MeSH
• kyseliny aristolochové analýza   toxicita   MeSH
• ledviny účinky léků   patofyziologie   MeSH
• léková rezistence MeSH
• lidé MeSH
• medicína založená na důkazech * MeSH
• mouka škodlivé účinky   analýza   MeSH
• plevel chemie   růst a vývoj   toxicita   MeSH
• prevalence MeSH
• pšenice růst a vývoj   MeSH
• riziko MeSH
• semena rostlinná růst a vývoj   MeSH
• urologické nádory chemicky indukované   epidemiologie   patofyziologie   prevence a kontrola   MeSH
• zemědělské plodiny růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Geografické názvy
• východní Evropa epidemiologie   MeSH
• Názvy látek
• aristolochic acid I MeSH Prohlížeč
• karcinogeny životního prostředí MeSH
• kyseliny aristolochové MeSH

UNLABELLED: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.

• Klíčová slova
• Aristolochic acid I, Cytochromes P450 1A1 and 1A2, DNA adducts, Oxidative detoxification, Reductive activation,
• MeSH
• adukty DNA antagonisté a inhibitory   biosyntéza   MeSH
• cytochrom P-450 CYP1A1 biosyntéza   MeSH
• cytochrom P-450 CYP1A2 biosyntéza   MeSH
• enzymová indukce účinky léků   fyziologie   MeSH
• karcinogeny toxicita   MeSH
• krysa rodu Rattus MeSH
• kyseliny aristolochové toxicita   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• aristolochic acid I MeSH Prohlížeč
• CYP1A1 protein, human MeSH Prohlížeč
• CYP1A2 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A1 MeSH
• cytochrom P-450 CYP1A2 MeSH
• karcinogeny MeSH
• kyseliny aristolochové 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.

• Klíčová slova
• 1A2 and 1B1, DNA adduct formation, aristolochic acid I, aristolochic acid nephropathy, nitroreduction, site-directed mutagenesis of cytochromes P450 1A1,
• MeSH
• adukty DNA metabolismus   MeSH
• aromatické hydroxylasy genetika   metabolismus   MeSH
• cytochrom P-450 CYP1A1 MeSH
• cytochrom P-450 CYP1A2 MeSH
• cytochrom P450 CYP1B1 MeSH
• katalytická doména genetika   MeSH
• katalýza MeSH
• kyseliny aristolochové metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• mutageneze cílená MeSH
• oxidace-redukce MeSH
• rekombinantní proteiny MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• aristolochic acid I MeSH Prohlížeč
• aromatické hydroxylasy MeSH
• cytochrom P-450 CYP1A1 MeSH
• cytochrom P-450 CYP1A2 MeSH
• cytochrom P450 CYP1B1 MeSH
• kyseliny aristolochové MeSH
• rekombinantní proteiny MeSH

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

• Klíčová slova
• contribution of cytochromes P450 in detoxification of aristolochic acid I in human and rat livers, cytochrome P450-mediated detoxification of aristolochic acid I, molecular modeling, plant nephrotoxin and carcinogen aristolochic acid I,
• MeSH
• inhibitory cytochromu P450 farmakologie   MeSH
• jaterní mikrozomy účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• katalytická doména MeSH
• katalýza MeSH
• krysa rodu Rattus MeSH
• kyseliny aristolochové škodlivé účinky   chemie   metabolismus   MeSH
• lidé MeSH
• metabolická aktivace MeSH
• metabolická inaktivace MeSH
• metylace účinky léků   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• nemoci ledvin etiologie   metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• systém (enzymů) cytochromů P-450 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aristolochic acid I MeSH Prohlížeč
• inhibitory cytochromu P450 MeSH
• kyseliny aristolochové MeSH
• systém (enzymů) cytochromů P-450 MeSH