Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk.
- MeSH
- adukty DNA účinky léků metabolismus MeSH
- barvicí látky metabolismus MeSH
- cytochrom P-450 CYP1A1 genetika metabolismus MeSH
- cytosol účinky léků enzymologie MeSH
- játra účinky léků enzymologie MeSH
- karcinogeny metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny účinky léků enzymologie MeSH
- messenger RNA genetika MeSH
- mikrozomy účinky léků enzymologie MeSH
- NAD(P)H dehydrogenasa (chinon) genetika metabolismus MeSH
- naftoly metabolismus MeSH
- plíce účinky léků enzymologie MeSH
- potkani Wistar MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
3-Nitrobenzanthrone (3-NBA), a carcinogenic air pollutant, was investigated for its ability to induce cytochrome P450 (CYP) 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1) in liver, kidney and lung of rats treated by intra-tracheal instillation. The organs used were from a previous study performed to determine the persistence of 3-NBA-derived DNA adducts in target and non-target tissues (Bieler et al., Carcinogenesis 28 (2007) 1117-1121, [22]). NQO1 is the enzyme reducing 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize a human metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), to yield the same reactive intermediate. 3-NBA and 3-ABA are both activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and from liver and kidney. Each compound generated the same five DNA adducts detectable by (32)P-postlabelling. When hepatic cytosols from rats treated with 0.2 or 2mg/kg body weight of 3-NBA were incubated with 3-NBA, DNA adduct formation was 3.2- and 8.6-fold higher, respectively, than in incubations with cytosols from control animals. Likewise, cytosols isolated from lungs and kidneys of rats exposed to 3-NBA more efficiently activated 3-NBA than those of control rats. This increase corresponded to an increase in protein levels and enzymatic activities of NQO1. Incubations of hepatic, pulmonary or renal microsomes of 3-NBA-treated rats with 3-ABA led to an 9.6-fold increase in DNA-adduct formation relative to controls. The highest induction in DNA-adduct levels was found in lung. The stimulation of DNA-adduct formation correlated with expression of CYP1A1/2 induced by the intra-tracheal instillation of 3-NBA. The results demonstrate that 3-NBA induces NQO1 and CYP1A1/2 in livers, lungs and kidneys of rats after intra-tracheal instillation, thereby enhancing its own genotoxic and carcinogenic potential.
- MeSH
- adukty DNA MeSH
- benz(a)anthraceny metabolismus farmakologie MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochrom P-450 CYP1A2 metabolismus MeSH
- cytosol účinky léků MeSH
- enzymová indukce účinky léků MeSH
- játra enzymologie MeSH
- karcinogeny farmakologie MeSH
- krysa rodu rattus MeSH
- látky znečišťující vzduch farmakologie MeSH
- ledviny enzymologie MeSH
- mikrozomy účinky léků MeSH
- NAD(P)H dehydrogenasa (chinon) metabolismus MeSH
- plíce enzymologie MeSH
- potkani Sprague-Dawley MeSH
- trachea MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. The target of this study was to investigate a role of CYP and peroxidase enzymes in ellipticine oxidative activation in rats, a suitable model mimicking the fate of ellipticine in humans, in details. The contribution of pulmonary and renal CYP- and peroxidase enzymes to ellipticine metabolic activation is investigated and compared with that found in the liver. METHODS: Ellipticine oxidation and DNA adduct formation in vitro were investigated using microsomes isolated from liver, lung and kidney of rats, either control (untreated) or treated i.p. with a single dose of 40 mg of ellipticine per kg of body weight. HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites. Inhibitors of CYPs and cyclooxygenase (prostaglandin H synthase, COX) were used to characterize the enzymes participating in ellipticine oxidative activation in rat liver, lung and kidney. Ellipticine-derived DNA adducts were detected by 32P-postlabeling. RESULTS: Using α-naphthoflavone, furafylline and ketoconazole, inhibitors of CYP1A, 1A2 and 3A, respectively, we found that the CYP1A and 3A enzymes play a major role in ellipticine activation to species forming DNA adducts in liver microsomes. Because of lower expression of these enzymes in lungs and kidneys, even after their induction by ellipticine, they play a minor role in ellipticine activation in these extrahepatic tissues. Arachidonic acid, a cofactor of COX, increased ellipticine activation in the microsomes of extrahepatic tissues. In addition, indomethacin, an inhibitor of COX, efficiently inhibited formation of ellipticine-derived DNA adduct in these microsomes. Based on these results, we attribute the higher activation of ellipticine in lung and kidney microsomes to COX than to CYP enzymes. CONCLUSION: The results demonstrate that whereas CYP enzymes of 1A and 3A subfamilies are the major enzymes activating ellipticine in rat livers, peroxidase COX plays a significant role in this process in lungs and kidneys.
- MeSH
- adukty DNA účinky léků MeSH
- antitumorózní látky metabolismus MeSH
- biotransformace účinky léků MeSH
- elipticiny metabolismus MeSH
- játra metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus MeSH
- mikrozomy účinky léků metabolismus MeSH
- modely u zvířat MeSH
- oxidace-redukce účinky léků MeSH
- peroxidasy fyziologie MeSH
- plíce metabolismus MeSH
- potkani Wistar MeSH
- systém (enzymů) cytochromů P-450 fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.
- MeSH
- antioxidancia farmakologie MeSH
- benzoflavony chemie metabolismus MeSH
- beta-naftoflavon chemie metabolismus MeSH
- časové faktory MeSH
- cytochromy b5 metabolismus MeSH
- cytochromy c metabolismus MeSH
- ferrikyanidy metabolismus MeSH
- flavonoidy chemie farmakologie MeSH
- fluorometrie MeSH
- králíci MeSH
- kyselina lipoová farmakologie MeSH
- mikrozomy účinky léků enzymologie metabolismus MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- oxaziny metabolismus MeSH
- oxid uhelnatý metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- spektrofotometrie MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
3-aminobenzanthrone (3-ABA) is the metabolite of the carcinogenic air pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was investigated for its ability to induce cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) in kidney and lung of rats, and for the influence of such induction on DNA adduct formation by 3-ABA and 3-NBA. NQO1 is the enzyme that reduces 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize 3-ABA to the same intermediate. When activated by cytosolic and and/or microsomal fractions isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney, both compounds generated the same DNA-adduct pattern, consisting of five adducts. When pulmonary cytosols isolated from rats that had been treated i.p. with 40 mg/kg bw of 3-ABA were incubated with 3-NBA, DNA adduct formation was up to 1.7-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. In contrast, no induction of NQO1 expression by 3-ABA treatment was found in the kidney. Incubations of 3-ABA with renal and pulmonary microsomes of 3-ABA-treated rats led to an increase of up to a 4.5-fold in DNA-adduct formation relative to controls. The stimulation of DNA-adduct formation correlated with a higher protein expression and activity of CYP1A1 induced by 3-ABA. These results show that by inducing lung and kidney CYP1A1 and NQO1, 3-ABA increases its own enzymatic activation as well as that of the environmental pollutant, 3-NBA, thereby enhancing the genotoxic and carcinogenic potential of both compounds.
- MeSH
- adukty DNA MeSH
- benz(a)anthraceny farmakologie metabolismus MeSH
- biotransformace MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- karcinogeny životního prostředí metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus účinky léků MeSH
- lidé MeSH
- mikrozomy enzymologie metabolismus účinky léků MeSH
- NAD(P)H dehydrogenasa (chinon) metabolismus MeSH
- plíce metabolismus účinky léků MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- fytoterapie MeSH
- kosterní svalová vlákna účinky léků MeSH
- krysa rodu rattus MeSH
- léčivé rostliny MeSH
- mikrozomy účinky léků MeSH
- myokard MeSH
- ochranné látky farmakologie MeSH
- scavengery volných radikálů farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- MeSH
- cytochromy metabolismus MeSH
- herbicidy farmakologie MeSH
- krysa rodu rattus MeSH
- mikrozomy metabolismus účinky léků MeSH
- mitochondrie metabolismus účinky léků MeSH
- mozek účinky léků ultrasonografie MeSH
- oxadiazoly farmakologie MeSH
- oxidoreduktasy antagonisté a inhibitory MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- MeSH
- borohydridy chemie metabolismus toxicita MeSH
- játra metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus účinky léků ultrastruktura MeSH
- mikrozomy účinky léků MeSH
- mozek metabolismus účinky léků MeSH
- sulfhydrylové sloučeniny chemie metabolismus toxicita MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- techniky in vitro MeSH
