RESUMO
Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.
Assuntos
Anticorpos , Corantes Fluorescentes , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Imunofenotipagem , PolímerosRESUMO
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.
Assuntos
Ácidos Aristolóquicos/toxicidade , Carcinogênese , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , DNA de Neoplasias/metabolismo , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.
Assuntos
Ácidos Aristolóquicos/metabolismo , Animais , Ácidos Aristolóquicos/administração & dosagem , Ácidos Aristolóquicos/urina , Cromatografia Líquida de Alta Pressão , Teoria da Densidade Funcional , Injeções Intraperitoneais , Masculino , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.
Assuntos
Benzo(a)pireno/metabolismo , Citocromo-B(5) Redutase/metabolismo , Adutos de DNA/metabolismo , Hepatócitos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Camundongos , Camundongos Knockout , Microssomos Hepáticos/enzimologiaRESUMO
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.
RESUMO
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.
Assuntos
Ácidos Aristolóquicos/toxicidade , Arilsulfotransferase/genética , Benzo(a)Antracenos/toxicidade , Adutos de DNA/efeitos dos fármacos , Animais , Carcinógenos/toxicidade , Citosol/efeitos dos fármacos , Citosol/metabolismo , Adutos de DNA/genética , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Família MultigênicaRESUMO
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.
Assuntos
Ácidos Aristolóquicos/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Domínio Catalítico/genética , Mutação , Catálise , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP1B1 , Adutos de DNA/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
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.
Assuntos
Ácidos Aristolóquicos/toxicidade , Carcinógenos/toxicidade , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A2/biossíntese , Adutos de DNA/antagonistas & inibidores , Adutos de DNA/biossíntese , Animais , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVES: The term "endocrine disruptor" (ED) is used for compounds that mimic or antagonize the effects of endogenous hormones. Synthetic estrogen 17α-ethinylestradiol (EE2) and a human carcinogen benzo[a]pyrene (BaP) are assigned as exogenous endocrine disruptors and an estrogenic hormone estradiol is a natural endogenous disruptor. Here, the potency of these three disruptors administered to rats individually and in combination to induce expression of cytochrome P450 (CYP) enzymes involved in their own metabolism (CYP1A1, 2C and 3A) in vivo was investigated. METHODS: Changes in CYP protein expression after exposure of rats to BaP, EE2 or estradiol were analyzed by Western blotting. Using the HPLC method, CYP1A1, 2C and 3A specific activities in hepatic microsomes isolated from exposed rats were analyzed. RESULTS: Whereas exposure to BaP induces expression of CYP1A1 protein and its marker activity (Sudan I oxidation) in liver, kidney and lung of rats, no significant induction of this CYP and its enzyme activity was produced by EE2 and estradiol. Treatment of BaP in combination with EE2 and/or estradiol decreased the BaP-mediated CYP1A1 induction in liver of exposed rats. BaP also induces CYP2C11 protein in rat liver and kidney, but does not increase its enzyme activity measured as testosterone 16α-hydroxylation. The enzyme activity of another enzyme of the 2C subfamily, CYP2C6, diclofenac 4'-hydroxylation, is even decreased by BaP. The CYP2C11 protein expression and/or its activity are also increased in liver of rats treated with EE2 and estradiol, but its expression is significantly decreased in lung. The CYP2C6 activity is also elevated by treatment of rats with EE2 and estradiol administered individually as well as in their combination. Whereas only a slight increase in CYP3A protein expression was found by BaP in rat liver, its enzyme activity, testosterone 6ß-hydroxyalation, increased significantly in this organ. In contrast, no effect or even a decrease in CYP3A expression and its enzyme activity was produced by EE2 and estradiol in rats exposed to these compounds.
Assuntos
Benzo(a)pireno/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Disruptores Endócrinos/farmacologia , Estradiol/farmacologia , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Microssomos Hepáticos/metabolismo , Animais , Masculino , Ratos , Ratos WistarRESUMO
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.
Assuntos
Ácidos Aristolóquicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Ativação Metabólica , Animais , Ácidos Aristolóquicos/efeitos adversos , Ácidos Aristolóquicos/química , Catálise , Domínio Catalítico , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Humanos , Inativação Metabólica , Nefropatias/etiologia , Nefropatias/metabolismo , Fígado/efeitos dos fármacos , Masculino , Metilação/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Oxirredução/efeitos dos fármacos , Ligação Proteica , RatosRESUMO
Exposure to the plant nephrotoxin and carcinogen aristolochic acid (AA) leads to the development of AA nephropathy, Balkan endemic nephropathy (BEN) and upper urothelial carcinoma (UUC) in humans. Beside AA, exposure to ochratoxin A (OTA) was linked to BEN. Although OTA was rejected as a factor for BEN/UUC, there is still no information whether the development of AA-induced BEN/UUC is influenced by OTA exposure. Therefore, we studied the influence of OTA on the genotoxicity of AA (AA-DNA adduct formation) in vivo. AA-DNA adducts were formed in liver and kidney of rats treated with AA or AA combined with OTA, but no OTA-related DNA adducts were detectable in rats treated with OTA alone or OTA combined with AA. Compared to rats treated with AA alone, AA-DNA adduct levels were 5.4- and 1.6-fold higher in liver and kidney, respectively, of rats treated with AA combined with OTA. Although AA and OTA induced NAD(P)H: quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA-DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA-DNA adduct formation in liver and kidney of rats.
Assuntos
Ácidos Aristolóquicos/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/efeitos dos fármacos , Ocratoxinas/farmacologia , Animais , Ácidos Aristolóquicos/metabolismo , Carcinógenos/metabolismo , Inativação Metabólica/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredução , Ratos , Ratos WistarRESUMO
OBJECTIVES: Balkan endemic nephropathy (BEN) is a chronic progressive fibrosis associated with upper urothelial carcinoma (UUC). Aetiology of BEN is still not fully explained. Although carcinogenic aristolochic acid I (AAI) was proven as the major cause of BEN/UUC, this nephropathy is considered to be multifactorial. Hence, we investigated whether other factors considered as potential causes of BEN [a mycotoxin ochratoxin A (OTA), Cd, Pb, Se and As ions and organic compounds (i.e. phthalates) released from lignite deposits in BEN areas] can influence detoxication of AAI, whose concentrations are crucial for BEN development. METHODS: Oxidation of AAI to 8-hydroxyaristolochic acid I (AAIa) in the presence of Cd, Pb, Se, As ions, dibutylphthalate (DBP), butylbenzylphthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP) and OTA by rat liver microsomes was determined by HPLC. RESULTS: Only OTA, cadmium and selenium ions, and BBP inhibited AAI oxidation by rat liver microsomes. These compounds also inhibited activities of CYP1A1 and/or CYP2C6/11 catalysing AAI demethylation in rat livers. Therefore, these CYP inhibitions can be responsible for a decrease in AAIa formation. When the combined effects of these compounds were investigated, the most efficient inhibition was caused by OTA combined with BBP and selenium ions. CONCLUSION: The results show low effects of BBP, cadmium and selenium ions, and/or their combinations on AAI detoxication. No effects were produced by the other metal ions (Pb, As) and phthalates DBP and DEHP. This finding suggests that they do not influence AAI-mediated BEN development. In contrast, OTA might influence this process, by inhibition of AAI detoxication.
Assuntos
Ácidos Aristolóquicos/metabolismo , Nefropatia dos Bálcãs , Carcinógenos/metabolismo , Metais Pesados/farmacologia , Ocratoxinas/farmacologia , Oxirredução/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Animais , Arsênio/farmacologia , Cádmio/farmacologia , Cromatografia Líquida de Alta Pressão , Íons , Chumbo/farmacologia , Microssomos Hepáticos/metabolismo , Ratos , Selênio/farmacologiaRESUMO
Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H: quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.
Assuntos
Ácidos Aristolóquicos/farmacologia , Carcinógenos/farmacologia , Rim/enzimologia , Fígado/enzimologia , Pulmão/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Animais , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos WistarRESUMO
Aristolochic acid I (AAI) is the major toxic component of the plant extract AA, which leads to the development of nephropathy and urothelial cancer in human. Individual susceptibility to AAI-induced disease might reflect variability in enzymes that metabolise AAI. In vitro NAD(P)H: quinone oxidoreductase (NQO1) is the most potent enzyme that activates AAI by catalyzing formation of AAI-DNA adducts, which are found in kidneys of patients exposed to AAI. Inhibition of renal NQO1 activity by dicoumarol has been shown in mice. Here, we studied the influence of dicoumarol on metabolic activation of AAI in Wistar rats in vivo. In contrast to previous in vitro findings, dicoumarol did not inhibit AAI-DNA adduct formation in rats. Compared with rats treated with AAI alone, 11- and 5.4-fold higher AAI-DNA adduct levels were detected in liver and kidney, respectively, of rats pretreated with dicoumarol prior to exposure to AAI. Cytosols and microsomes isolated from liver and kidney of these rats were analysed for activity and protein levels of enzymes known to be involved in AAI metabolism. The combination of dicoumarol with AAI induced NQO1 protein level and activity in both organs. This was paralleled by an increase in AAI-DNA adduct levels found in ex vivo incubations with cytosols from rats pretreated with dicoumarol compared to cytosols from untreated rats. Microsomal ex vivo incubations showed a lower AAI detoxication to its oxidative metabolite, 8-hydroxyaristolochic acid (AAIa), although cytochrome P450 (CYP) 1A was practically unchanged. Because of these unexpected results, we examined CYP2C activity in microsomes and found that treatment of rats with dicoumarol alone and in combination with AAI inhibited CYP2C6/11 in liver. Therefore, these results indicate that CYP2C enzymes might contribute to AAI detoxication.
Assuntos
Ácidos Aristolóquicos/toxicidade , Carcinógenos/toxicidade , Dicumarol/farmacologia , Ativação Metabólica/efeitos dos fármacos , Animais , Ácidos Aristolóquicos/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2 , Citocromos/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Testes de Mutagenicidade , Mutagênicos/farmacocinética , Mutagênicos/toxicidade , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVES: Dicoumarol is known to act as an inhibitor of NAD(P)H: quinone oxidoreductase (NQO1). This cytosolic reductase significantly contributes to the genotoxicity of the nephrotoxic and carcinogenic alkaloid aristolochic acid I (AAI). Aristolochic acid causes aristolochic acid nephropathy (AAN), and Balkan endemic nephropathy (BEN), as well as associated urothelial malignancies. NQO1 is the most efficient enzyme responsible for the reductive bioactivation of AAI to species forming covalent AAI-DNA adducts. However, it is still not known how dicoumarol influences the NQO1-mediated reductive bioactivation of AAI. METHODS: AAI-DNA adduct formation was determined by 32P-postlabeling. Expression of NQO1 mRNA and NQO1 protein was determined by real-time polymerase chain reaction and Western blotting, respectively. RESULTS: In this study, dicoumarol inhibited AAI bioactivation to form AAI-DNA adducts mediated by rat and human NQO1 in vitro as expected. We however, demonstrated that dicoumarol acts as an inducer of NQO1 in kidney and lung of rats treated with this NQO1 inhibitor in vivo, both at protein and activity levels. This NQO1 induction increased the potency of kidney cytosol to bioactivate AAI and elevated AAI-DNA adduct levels were found in ex-vivo incubations of AAI with renal cytosols and DNA. NQO1 mRNA levels were induced in liver only by dicoumarol. CONCLUSION: Our results indicate a dual role of dicoumarol in NQO1-mediated genotoxicty of AAI. It acts both as an NQO1 inhibitor mainly in vitro and as an NQO1 inducer if administered to rats.
Assuntos
Ácidos Aristolóquicos/metabolismo , Dicumarol/farmacologia , Inibidores Enzimáticos/farmacologia , Quinona Redutases/metabolismo , Animais , Humanos , RatosRESUMO
Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
Assuntos
Ácidos Aristolóquicos/metabolismo , Carcinógenos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Fígado/enzimologia , Animais , Ácidos Aristolóquicos/toxicidade , Ácidos Aristolóquicos/urina , Carcinógenos/toxicidade , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Inibidores do Citocromo P-450 CYP1A2 , Citosol/enzimologia , Adutos de DNA/metabolismo , Remoção de Radical Alquila , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Inativação Metabólica , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microssomos Hepáticos/enzimologia , Oxirredução , Proteínas Recombinantes/metabolismo , Neoplasias Urológicas/induzido quimicamente , Urotélio/efeitos dos fármacosRESUMO
OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.
Assuntos
Ácidos Aristolóquicos/farmacocinética , Sistema Enzimático do Citocromo P-450/fisiologia , Fígado/efeitos dos fármacos , Animais , Biotransformação , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos/genética , Citocromos/metabolismo , Adutos de DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inativação Metabólica , Fígado/enzimologia , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Ratos , Ratos WistarRESUMO
Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.