RESUMO
Coffee consumption has been associated with a significant decrease in the risk of developing chronic diseases such as Parkinson disease, diabetes type-2 and several types of cancers (e.g. colon, liver). In the present study, a coffee-dependent induction of enzymes involved in xenobiotic detoxification processes was observed in rat liver and primary hepatocytes. In addition, coffee was found to induce the mRNA and protein expression of enzymes involved in cellular antioxidant defenses. These inductions were correlated with the activation of the Nrf2 transcription factor as shown using an ARE-reporter luciferase assay. The induction of detoxifying enzymes GSTs and AKR is compatible with a protection against both genotoxicity and cytotoxicity of aflatoxin B1 (AFB1). This hypothesis was confirmed in in vitro and ex vivo test systems, where coffee reduced both AFB1-DNA and protein adducts. Interestingly, coffee was also found to inhibit cytochrome CYP1A1/2, indicating that other mechanisms different from a stimulation of detoxification may also play a significant role in the chemoprotective effects of coffee. Further investigations in either human liver cell line and primary hepatocytes indicated that the chemoprotective effects of coffee against AFB1 genotoxicity are likely to be of relevance for humans. These data strongly suggest that coffee may protect against the adverse effects of AFB1. In addition, the coffee-mediated stimulation of the Nrf2-ARE pathway resulting in increased endogenous defense mechanisms against electrophilic but also oxidative insults further support that coffee may be associated with a protection against various types of chemical stresses.
Assuntos
Anticarcinógenos/farmacologia , Café/química , Neoplasias Hepáticas Experimentais/prevenção & controle , Fator 2 Relacionado a NF-E2/biossíntese , Aflatoxina B1/toxicidade , Animais , Antioxidantes/metabolismo , Western Blotting , Carcinógenos/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/enzimologia , Luciferases/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Elementos Reguladores de Transcrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To assess the effects of iron removal on cytochrome P450 2E1 activity and oxidative stress in dysmetabolic iron overload syndrome. METHODS: Forty-eight patients were randomized to phlebotomy therapy consisting of removal of 300-500 mL of blood every 14 days until serum ferritin levels dropped under 100 microg/L or to follow-up without phlebotomy therapy. Cytochrome P450 2E1 activity was measured at baseline and at the end of treatment by using the 6-hydroxychlorzoxazone/chlorzoxazone blood metabolic ratio, 2 h after the intake of 500 mg of chlorzoxazone. RESULTS: In the treatment group, a mean of 3.9 +/- 1.3 L of blood was removed and serum ferritin levels dropped from 715 +/- 397 to 74 +/- 34 microg/L. Variation of cytochrome P450 2E1 activity was not significantly different between the 2 groups (0.07 +/- 0.26 vs. 0.03 +/- 0.19, P = 0.36). In the treatment group, low-density lipoprotein cholesterol and vitamin E were lowered after treatment compared with control group (-0.15 +/- 0.51 vs. 0.24 +/- 0.58, P = 0.002 and -1.3 +/- 4.4 vs. 2.3 +/- 5.2, P = 0.03, respectively). Inversely, vitamin C was increased (0.5 +/- 3.5 vs. -1.8 +/- 3.9, P = 0.03). CONCLUSIONS: In dysmetabolic iron overload syndrome, reduction of iron stores does not significantly influence cytochrome P450 2E1 activity but is associated with a significant decrease of low-density lipoprotein cholesterol, suggesting that venesection therapy may be a suitable option in these patients.
Assuntos
Citocromo P-450 CYP2E1/metabolismo , Sobrecarga de Ferro/terapia , Estresse Oxidativo/fisiologia , Flebotomia/métodos , Ácido Ascórbico/sangue , Biomarcadores/sangue , LDL-Colesterol/sangue , Ferritinas/sangue , Humanos , Sobrecarga de Ferro/enzimologia , Sobrecarga de Ferro/fisiopatologia , Masculino , Malondialdeído/sangue , Estudos Prospectivos , Vitamina E/sangueRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are major carcinogenic environmental contaminants known to exert bone marrow toxicity and to induce leukemias, suggesting that these chemicals target hematopoietic stem cells. To investigate this hypothesis, we studied the effects of PAHs on cell proliferation and differentiation in human hematopoietic CD34+ cell cultures. Benzo(a)pyrene (BP), a prototypical PAH, was shown to markedly impair CD34+ cell expansion and to inhibit CD34+ cell differentiation into various hematological cell lineages, including erythroid, granulomacrophagic, and megakaryocytic lineages. This was associated with the induction of a caspase- and mitochondrion-related apoptosis process. CD34+ progenitor cells were found to exhibit functional expression of the aryl hydrocarbon receptor (AhR), and the use of the pure AhR antagonist 3'-methoxy-4'-nitroflavone partially counteracted the deleterious effects of BP in CD34+ cell cultures, underlining the involvement of AhR in BP toxicity. Additional events such as CYP1A1/1B1-dependent PAH metabolism and adduct formation were also required since 1) 2,3,7,8-tetrachlorodibenzo-p-dioxin, a very potent ligand of the AhR that is poorly metabolized and therefore does not generate reactive metabolites in contrast to PAHs, failed to affect CD34+ cell expansion; 2) the CYP1A1/1B1 inhibitor alpha-naphthoflavone blocked both BP adduct formation and BP toxicity; and 3) benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide, a highly reactive BP metabolite, exerted a marked toxicity toward CD34+ cell cultures. Overall, these data indicate that human hematopoietic CD34+ cells can bioactivate chemical carcinogens such as PAHs and, in this way, constitute targets for such carcinogenic environmental contaminants.
Assuntos
Antígenos CD34/metabolismo , Carcinógenos/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Apoptose/efeitos dos fármacos , Benzo(a)pireno/metabolismo , Biotransformação/efeitos dos fármacos , Western Blotting , Carcinógenos/metabolismo , Caspase 3 , Caspase 9 , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Poluentes Ambientais/toxicidade , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Técnicas de Patch-Clamp , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , RNA/biossíntese , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The metabolism of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated with human and rat liver microsomes, recombinant human cytochrome P450 1A2 (P450 1A2) expressed in Escherichia coli cells, and rat P450 1A2. Human liver microsomes and human P450 1A2 catalyzed the oxidation of the exocyclic amine group of MeIQx to form the genotoxic product 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx). Human P450 1A2 also catalyzed the oxidation of C(8)-methyl group of MeIQx to form 2-amino-(8-hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH(2)OH-IQx), 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carbaldehyde (IQx-8-CHO), and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH). Thus, chemically stable C(8)-oxidation products of MeIQx may be useful biomarkers of P450 1A2 activity in humans. Rat liver microsomes were 10-15-fold less active than the human counterpart at both N-oxidation and C(8)-oxidation of MeIQx when expressed as nanomoles of product formed per minute per nanomoles of P450 1A2. Differences in regioselective oxidation of MeIQx were also observed with human and rat liver microsomes and the respective P450 1A2 orthologs. In contrast to human liver microsomes and P450 1A2, rat liver microsomes and purified rat P4501A2 were unable to catalyze the oxidation of MeIQx to the carboxylic derivative IQx-8-COOH, an important detoxication product formed in humans. However, rat liver microsomes and rat P4501A2, but not human liver microsomes or human P450 1A2, extensively catalyzed ring oxidation at the C-5 position of MeIQx to form the detoxication product 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline (5-HO-MeIQx). There are important differences between human and rat P450 1A2, both in catalytic activities and oxidation pathways of MeIQx, that may affect the biological activity of this carcinogen and must be considered when assessing human health risk.
Assuntos
Citocromo P-450 CYP1A2/metabolismo , Microssomos Hepáticos/metabolismo , Mutagênicos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Quinoxalinas/metabolismo , Teofilina/análogos & derivados , Aminas/metabolismo , Animais , Biotransformação , Células Cultivadas , Inibidores do Citocromo P-450 CYP1A2 , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inativação Metabólica , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Mutagênicos/química , Mutagênicos/toxicidade , Quinoxalinas/química , Quinoxalinas/toxicidade , Ratos , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Teofilina/farmacologiaRESUMO
Metabolic pathways of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) remain incompletely characterized in humans. In this study, the metabolism of MeIQx was investigated in primary human hepatocytes. Six metabolites were characterized by UV and mass spectroscopy. Novel metabolites were additionally characterized by 1H NMR spectroscopy. The carcinogenic metabolite, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline, which is formed by cytochrome P450 1A2 (P450 1A2), was found to be transformed into the N(2)-glucuronide conjugate, N(2)-(beta-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx), thought to be formed exclusively by the intestinal flora, were also identified. A novel metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), and it was the predominant metabolite formed in hepatocytes exposed to MeIQx at levels approaching human exposure. IQx-8-COOH formation is catalyzed by P450 1A2. This metabolite is a detoxication product and does not induce umuC gene expression in Salmonella typhimurium strain NM2009. IQx-8-COOH is also the principal oxidation product of MeIQx excreted in human urine [Turesky, R., et al. (1998) Chem. Res. Toxicol. 11, 217-225]. Thus, P450 1A2 is involved in both the metabolic activation and detoxication of this procarcinogen in humans. Analogous metabolism experiments were conducted with hepatocytes of untreated rats and rats pretreated with the P450 inducer 3-methylcholanthrene. Unlike human hepatocytes, the rat cell preparations did not produce IQx-8-COOH but catalyzed the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline as a major P450-mediated detoxication product. In conclusion, our results provide evidence of a novel MeIQx metabolism pathway in humans through P450 1A2-mediated C(8)-oxidation of MeIQx to form IQx-8-COOH. This biotransformation pathway has not been detected in experimental animal species. Considerable interspecies differences exist in the metabolism of MeIQx by P450s, which may affect the biological activity of this mutagen and must be considered when assessing human health risk.
Assuntos
Citocromo P-450 CYP1A2/metabolismo , Hepatócitos/metabolismo , Quinoxalinas/metabolismo , Animais , Biotransformação , Separação Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Inibidores do Citocromo P-450 CYP1A2 , Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Humanos , Técnicas In Vitro , Inativação Metabólica , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mutagênicos/toxicidade , Quinoxalinas/química , Quinoxalinas/toxicidade , Ratos , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2. F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine. They did not display either decreased cellular PAH accumulation or increased PAH efflux. In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity. Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells. These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Carcinógenos/toxicidade , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Benzopirenos/toxicidade , Células Cultivadas , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Adutos de DNA/análise , Adutos de DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Metilcolantreno/toxicidade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima , Vincristina/farmacologiaAssuntos
Citocromo P-450 CYP1A2/metabolismo , Mutagênicos/metabolismo , Quinoxalinas/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Estrutura Molecular , Mutagênicos/química , Mutagênicos/farmacologia , Oxirredução , Quinoxalinas/química , Quinoxalinas/farmacologia , Células Tumorais CultivadasRESUMO
Recent studies have demonstrated that two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1, 2-dithiole-3-thione (D3T), and sulforaphane (SF), an isothiocyanate, are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes (P450s) involved in xenobiotic metabolism. We examined P450 inhibition by the two compounds and compared two OPZ metabolites (OPZ M(3) and M(8)) and D3T using human P450s expressed in Escherichia coli membranes. OPZ was a more potent inhibitor than D3T or SF, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 approximately 1B1 > 2E1. OPZ M(3) also inhibited P450s 1A2, 1A1, 1B1, and 3A4 but not more effectively than OPZ. OPZ M(8) was not inhibitory. OPZ behaved as a competitive inhibitor of P450 1A2, with a K(i) of 1.5 microM. Incubation of P450 1A2 with OPZ and NADPH led to a first-order loss of the P450 spectrum, and the loss was not blocked by glutathione. No such time-dependent loss of P450 was seen with P450 1A2 and D3T, P450 1A2 and OPZ M(3), P450 1A2 and SF, P450 3A4 and OPZ, P450 3A4 and D3T, P450 2E1 and OPZ, or P450 2E1 and D3T. The time- and concentration-dependent loss of P450 1A2 activity in the presence of OPZ was characterized with a K(i) of 9 microM and a k(inactivation) of 0.19 min(-)(1). The activation of 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline (MeIQ) in an E. coli lac-based mutagenicity tester system containing functional human P450 1A2 was inhibited by OPZ (IC(50) < 1 microM) but not appreciably by 40 microM D3T. Our results indicate that OPZ is a competitive and mechanism-based inhibitor of P450 1A2, and the extent of this inhibition is significantly greater than that of other chemoprotective chemicals with P450 1A2 or other human P450s.
Assuntos
Anticarcinógenos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Pirazinas/farmacologia , Tiocianatos/farmacologia , Tionas/farmacologia , Tiofenos/farmacologia , Inibidores do Citocromo P-450 CYP1A2 , Humanos , Isotiocianatos , Quinolinas/metabolismo , Quinolinas/toxicidade , SulfóxidosRESUMO
Etheno (epsilon) and related DNA adducts are formed from the reaction of certain bifunctional electrophiles with DNA. Our interest has been focused on oxiranes substituted with leaving groups, e.g. 2-chlorooxirane, the epoxide derived from the carcinogen vinyl chloride. The chemical mechanisms of the formation of the major etheno products derived from adenine, cytosine and guanine have been elucidated by nuclear magnetic resonance analysis and 13C-labelled precursors. The amounts of all major etheno adducts have been quantified in DNA treated with 2-chlorooxirane by coupled high-performance liquid chromatography of nucleoside and base products. 1,N2-epsilon-Gua, its formally hydrated but stable hemiaminal HO-ethanoGua (5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine) and 1,N2-ethanoGua have all been inserted at a single site in oligonucleotides. All three of these bases block polymerases, cause misincorporations and produce some mutations in bacteria. The patterns of blockage and substitution vary among polymerases. In nucleotide excision repair-deficient Escherichia coli, 1,N2-epsilon-Gua yielded a calculated 16% mutation frequency (base-pair substitutions) when the results were corrected for strand usage. 1,N2-epsilon-Gua was also examined in Chinese hamster ovary cells with a stable integration system; the mutants are more complex than observed in bacteria and include rearrangements, deletions and base-pair substitutions other than at the adduct site.
Assuntos
Adutos de DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA/efeitos dos fármacos , Adutos de DNA/química , Relação Dose-Resposta a Droga , Escherichia coli/genética , Óxido de Etileno/análogos & derivados , Óxido de Etileno/química , Óxido de Etileno/toxicidade , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Cinética , Modelos Químicos , Dados de Sequência Molecular , MutaçãoRESUMO
The effect of Escherichia coli lipopolysaccharide (LPS), a classic inducer of the acute-phase response, has been analyzed on both constitutive and oltipraz (a chemoprotective agent)-inducible messenger RNAs (mRNAs) and enzyme activities of major cytochromes P450 (CYPs) and glutathione transferases (rGSTs) in rat liver. At the dose administered (1 mg/kg) and the time studied (6 and 24 hours), endotoxin had no effect on the expression of either CYPs and GSTs with the exception of CYP1A2, which was reduced at both mRNA and activity levels. A strong increase of rGSTA1/2, rGSTM1, rGSTP1, CYP1A2, CYP2B1/2, and CYP2E1 was observed after 3 days of treatment with oltipraz (0.075%, wt/wt). Oltipraz induction of these enzymes (with the exception of CYP2E1) was found to be suppressed at both mRNA, protein, and activity levels during the acute-phase response to endotoxin. Moreover, it is shown that oltipraz induction of CYP1A2 and CYP2B1/2 and its suppression by E. coli LPS occurred at a transcriptional level. These data support the idea that the chemoprotective effect of oltipraz is altered in the course of inflammation and that adaptation in chemoprotective strategies should be considered in certain physiopathologic situations.
Assuntos
Anticarcinógenos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Endotoxinas/farmacologia , Glutationa Transferase/metabolismo , Fígado/metabolismo , Pirazinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática/efeitos dos fármacos , Glutationa Transferase/genética , Lipopolissacarídeos/farmacologia , Fígado/enzimologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tionas , TiofenosRESUMO
Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.
Assuntos
Aflatoxina B1/farmacocinética , Carcinógenos/farmacocinética , Animais , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , DNA/efeitos dos fármacos , Epóxido Hidrolases/metabolismo , Humanos , Inativação MetabólicaRESUMO
Rat hepatocytes cocultured with rat liver epithelial cells (RLEC) were used to investigate the influence of all-trans retinoic acid (RA) on the regulation of apolipoproteins (Apo) A-I and A-II gene expression, the major protein constituent of high-density lipoproteins. In contrast to rat hepatocytes in conventional primary culture, Apo A-I and Apo A-II gene expression remained high and stable for several days in parenchymal cells in coculture. Treatment of cocultured rat hepatocytes with RA resulted in a specific decrease in Apo A-I mRNA levels whereas no marked difference in Apo A-II mRNA levels was observed. Such a negative effect of RA was already detected as early as 2 days of treatment and was effective for the entire experimental period (6 days). As controls, RARbeta mRNA levels increased whereas those of GAPDH mRNA were not affected by the RA treatment. The decrease in Apo A-I mRNA levels was associated with lower amounts of Apo A-I secreted in the culture medium within day 1 of treatment. This effect required active transcription and protein synthesis. These results show that, contrary to primary pure hepatocyte cultures and hepatoma cell lines, cocultures of rat hepatocytes reproduce the in vivo results suggesting that only well differentiated hepatocytes may correctly respond to RA. Furthermore, they demonstrate that RA can directly act on hepatocytes and differently affect Apo A-I and Apo A-II gene expression.
Assuntos
Apolipoproteína A-I/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Tretinoína/farmacologia , Albuminas/efeitos dos fármacos , Albuminas/metabolismo , Animais , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The isothiocyanate sulforaphane (SF) is thought to be a potential chemoprotective agent. Its effects on Phase I and Phase II enzymes of carcinogen metabolism in primary cultures of rat and human hepatocytes have been investigated. Northern blot analyses of rat hepatocytes showed a dose-dependent induction of mRNAs for rat glutathione S-transferases (rGSTs) A1/A2 and P1 but not M1. This was associated with enhanced levels of not only rGSTA1, A2, A4, A5, and P1 but also of rGSTs M1 and M2. On the other hand, the enzyme activities in rat hepatocytes associated with cytochromes P-450 (CYPs) 1A1 and 2B1/2, namely ethoxyresorufin-O-deethylase and pentoxyresorufin-O-dealkylase, respectively, were decreased in a dose-dependent manner. In SF-treated human hepatocytes, hGSTA1/2 but not hGSTM1 mRNAs were induced, and the expression of CYP1A2 was unaffected, whereas the expression of CYP3A4, the major CYP in human liver, was markedly decreased at both mRNA and activity levels. These observations demonstrate that in intact human and rat hepatocytes, SF may both induce a number of GSTs and cause enzyme inhibition of some but not all CYPs and, in the case of CYP3A4, inhibit both its enzyme activity and its expression.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Glutationa Transferase/efeitos dos fármacos , Isoenzimas/efeitos dos fármacos , Fígado/enzimologia , Tiocianatos/farmacologia , Adulto , Animais , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2B1/antagonistas & inibidores , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Indução Enzimática/efeitos dos fármacos , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isotiocianatos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Sulfóxidos , Fatores de TempoRESUMO
The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.
Assuntos
Anticarcinógenos/farmacologia , Citocromo P-450 CYP1A1/efeitos dos fármacos , Citocromo P-450 CYP2B1/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pirazinas/farmacologia , Aflatoxina B1/metabolismo , Animais , Cafeína/metabolismo , Células Cultivadas , Fígado/enzimologia , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Tionas , TiofenosRESUMO
Primary cultures of human and rat hepatocytes are widely used in pharmacotoxicological research. This model presents the advantages of retaining liver function for at least a few days, expressing both phase 1 and phase 2 enzymes, and responding to inducers. Recently we made use of primary hepatocytes to investigate the effects of chemoprotective agents on drug-metabolizing enzyme expression and activities. The treatment of rat and human hepatocytes with two chemoprotective agents, oltipraz (a synthetic derivative of 1,2-dithiole-3-thione) and sulforaphane (an isothiocyanate found in broccoli), clearly demonstrated that both of these compounds are inducers of glutathione transferases and transient inhibitors of cytochrome P450, suggesting that these two compounds could exert their chemoprotective effects by both reducing the formation of reactive metabolites of chemicals and enhancing their inactivation.
Assuntos
Testes de Carcinogenicidade , Células Cultivadas , Fígado/citologia , Testes de Toxicidade , Animais , Anticarcinógenos/farmacologia , Humanos , Isotiocianatos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Pirazinas/farmacologia , Ratos , Sulfóxidos , Tiocianatos/farmacologia , Tionas , TiofenosRESUMO
1,N2-Ethenoguanine (1,N2-epsilon-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5'-CAGTGGGTG*TCCGAATTGA-3', were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H2(18)O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3'-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-epsilon-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, -1 and -2 base frame shifts were detected with both 1,N2-epsilon-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in k(cat) for all dNTP incorporations compared to the normal G x dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-epsilon-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Guanina/análogos & derivados , Imidazóis/metabolismo , Purinas/metabolismo , Transcrição Gênica , Carcinógenos/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Desoxirribonucleotídeos/química , Guanina/química , Guanina/metabolismo , Imidazóis/química , Mutagênese , Oligodesoxirribonucleotídeos/biossíntese , Purinas/químicaRESUMO
Recently, we used human hepatocytes in primary culture to study the effects of inducers of glutathione-S-transferases (GSTs) in the expectation that information obtained can be used to predict the value of particular inducers for use in the chemoprevention of cancer and other toxicities. However, in vitro human studies cannot readily be confirmed by studies in vivo. This problem does not arise in experimental animals. In the current studies, the response of male rat hepatocytes in primary culture to the following inducers of GST isoenzymes has been determined: 3-methylcholanthrene (MC); phenobarbital (PB); 1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz (OPZ), and the results have been compared with induction obtained in livers of MC- and OPZ-treated rats. Each type of inducer was found to elicit a different response. In vitro, phenobarbital increased messenger RNA (mRNA) levels of subunits 1b and 3 after 12 and 72 hours, respectively; MC had a rapid effect on GST alpha class mRNAs (bringing about increase after only 2 hours of treatment), increased subunit 7 mRNA slightly, and had no effect on mu class mRNAs; dithiolethiones induced both subunit lb and 7 mRNAs after 4 hours and, to a much lower extent, subunit 3 mRNA after 72 hours. In vivo, MC induced significantly both subunit lb and 7 mRNAs whereas OPZ increased significantly subunits lb, 3 and 7 mRNA levels, and to a lower extent those of subunit 2, after 3 days and beyond to at least 5 days of treatment. Results obtained in mRNA studies were confirmed by high-pressure liquid chromatography (HPLC) analysis of GST subunits. HPLC also showed an induction of subunit 10 at the protein level of which the mRNA was not analyzed. Our results show that rat hepatocytes in primary culture prove to be a good model for the effect of inducers on both the expression of GST mRNA and protein levels in the rat liver in vivo. The demonstration of this good correlation in the rat with respect to increases gives support for the use of human hepatocytes for predictive studies of chemoprotection in human pharmacology.
Assuntos
Glutationa Transferase/biossíntese , Fígado/enzimologia , Animais , Células Cultivadas , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/análise , Glutationa Transferase/genética , Fígado/citologia , Masculino , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Pirazinas/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Tionas , TiofenosAssuntos
Aflatoxina B1/metabolismo , Carcinógenos/metabolismo , Fígado/metabolismo , Anticarcinógenos/farmacologia , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Transferase/biossíntese , Humanos , Fígado/citologia , Metilcolantreno/farmacologia , Pirazinas/farmacologia , Tionas , TiofenosRESUMO
Dithiolethiones are thought to act as potent chemoprotective agents against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by inducing glutathione S-transferases (GSTs). To determine whether these antioxidants can be similarly effective in human beings, we have investigated metabolism of AFB1, in primary human hepatocytes with or without pretreatment by oltipraz (OPZ), a synthetic derivative of the natural 1,2-dithiole-3-thione. Aflatoxin M1 (AFM1), glutathione conjugates of AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cultures derived from eight human liver donors. Parenchymal cells obtained from the three GST M1-positive livers metabolized AFB1 to AFM1 and to AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no AFBSGs were formed in the GST M1-null cells. Pretreatment of the cells with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and CYP3A4, respectively, caused a significant increase in AFB1 metabolism. Although OPZ induced GST A2, and to a lesser extent GST A1 and GST M1, it decreased formation of AFM1 and AFBSG, which involves CYP1A2 and CYP3A4. Inhibition by OPZ of AFB1 metabolism by reducing CYP1A2 and CYP3A4 was also demonstrated by decreased activity of their monooxygenase activities toward ethoxyresorufin and nifedipine, respectively. The significant inhibition by OPZ of human recombinant yeast CYP1A2 and CYP3A4 was also shown. These results demonstrate that AFBSG can be formed by GST M1-positive human hepatocytes only, and suggest that chemoprotection with OPZ is due to an inhibition of activation of AFB1, in addition to a GST-dependent inactivation of the carcinogenic exo-epoxide.