RESUMEN
beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.
Asunto(s)
Proteínas Portadoras/metabolismo , Citosol/química , Hígado/química , Receptores de Hidrocarburo de Aril/agonistas , beta-naftoflavona/metabolismo , Animales , Benzo(a)pireno/farmacología , Benzoflavonas/farmacología , Benzofuranos/farmacología , Unión Competitiva , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Citocromo P-450 CYP1A1/metabolismo , Citosol/efectos de los fármacos , Femenino , Glicina N-Metiltransferasa , Cinética , Hígado/citología , Hígado/efectos de los fármacos , Metilcolantreno/farmacología , Metiltransferasas/metabolismo , Peso Molecular , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Hidrocarburo de Aril/metabolismo , beta-naftoflavona/farmacologíaRESUMEN
The potential of an aryl hydrocarbon receptor (AhR) agonist, beta-naphthoflavone (beta-NF), to modulate ovarian hormone responses in the uterus and liver of 50-day-old Sprague-Dawley rats was examined. Treatment with beta-NF at 40 mg/kg of body weight consisted of 3 or 9 intraperitoneal injections in corn oil administered to ovariectomized (OVX) and sham-treated (SH) rats on day 5 through 7 or 1 through 9 after surgery performed on day 42 or 40 of age, respectively. Treatment of SH rats with either dose regimen of beta-NF effected a decrease (approximately 80%) in the uterine peroxidase activity, which was similar to that effected by ovariectomy (> 93%). By contrast, treatment of rats with alpha-naphthoflavone, an AhR antagonist, did not decrease the peroxidase activity. After the 9-dose treatment with beta-NF, decreases (approximately 70%) in hepatic estrogen receptor (ER) levels in both SH and OVX rats exceeded those effected by ovariectomy (30%). However, treatment with beta-NF partially prevented the ovariectomy-effected increase (approximately 1.5-fold) in body weight gain, decrease (approximately 67%) in uterine weight, and increase (3-fold) in uterine ER level. In both SH and OVX rats, treatment with beta-NF increased (1.7-fold) uterine progesterone receptor (PR) levels, which were unaffected by ovariectomy. Thus, the results suggest that the effect to of treatment with beta-NF is both mimicking and counteracting the effects of estrogen. Since beta-NF itself or upon conversion to metabolites by liver microsomes was shown herein not to be a ligand for uterine ER and PR, the aforementioned effects of beta-NF resembled those of certain halogenated polycyclic hydrocarbons, and thus may be mediated via AhR.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hígado/efectos de los fármacos , Útero/efectos de los fármacos , beta-naftoflavona/farmacología , Animales , Benzoflavonas/farmacología , Citocromo P-450 CYP1A1/metabolismo , Estradiol/farmacología , Femenino , Hormonas/fisiología , Hígado/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Peroxidasa/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/metabolismo , Útero/metabolismoRESUMEN
The effects of the route of benzo[a]pyrene administration on sister chromatid exchange (SCE) and B[a]P diol epoxide (B[a]PDE)-DNA adducts formation in bone marrow cells of Ah responsive (C57BL/6; B6) and Ah non-responsive (DBA/2; D2) mice were determined. Animals were treated intraperitoneally (i.p.), intragastrically (i.g.) or topically with two 100 mg/kg doses of benzo[a]pyrene 24 h apart and killed 96 h after the first treatment. Significant increase in the frequencies of SCE and the level of B[a]PDE-DNA adducts as measured by synchronous fluorescence spectrophotometry were detected in D2 mice as compared to B6 mice. The route of administration had little effect on SCE levels in bone marrow cells in D2 mice. In B6 mice higher levels of SCE were observed following i.p. administration as compared to i.g. or topical administration. In both strains the highest level of B[a]PDE-DNA adducts was formed after i.p. administration of B[a]P. We conclude that the i.p. route of B[a]P administration is the most effective in inducing SCE and B[a]P-DNA adducts formation. SCE induction does not correlate linearly with the amount of B[a]PDE-DNA adducts formed in these cells after administration of the above dose of B[a]P.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Benzo(a)pireno/administración & dosificación , Médula Ósea/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Aductos de ADN/metabolismo , ADN/metabolismo , Mutágenos/administración & dosificación , Intercambio de Cromátides Hermanas/efectos de los fármacos , Animales , Benzo(a)pireno/metabolismo , Médula Ósea/efectos de los fármacos , Citocromo P-450 CYP1A1/efectos de los fármacos , Vías de Administración de Medicamentos , Inducción Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Mutágenos/metabolismoAsunto(s)
Benzo(a)pireno/toxicidad , Aductos de ADN , Daño del ADN , ADN/efectos de los fármacos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Bazo/efectos de los fármacos , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/análisis , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Animales , Benzo(a)pireno/análisis , Benzo(a)pireno/metabolismo , Carcinógenos Ambientales/análisis , Carcinógenos Ambientales/metabolismo , ADN/análisis , ADN/metabolismo , Inyecciones Intraperitoneales , Riñón/química , Riñón/metabolismo , Hígado/química , Hígado/metabolismo , Pulmón/química , Pulmón/metabolismo , Masculino , Ratas , Ratas Wistar , Espectrometría de Fluorescencia , Bazo/química , Bazo/metabolismoRESUMEN
The techniques applicable for DNA adducts identification and quantitation are critically reviewed. The review also presents the data concerning an analysis of DNA adducts resulted from occupational exposure, tobacco smoking, diet and chemotherapy of tumors.
Asunto(s)
ADN de Neoplasias/análisis , Neoplasias/genética , Animales , Dieta , Humanos , Epidemiología Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/epidemiología , Enfermedades Profesionales/epidemiología , Enfermedades Profesionales/genética , Factores de Riesgo , Fumar/efectos adversosRESUMEN
Synchronous fluorescence spectrophotometry (SFS) developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)--DNA adducts was used to measure the formation and disappearance of DNA adducts in the lung, liver, kidney, spleen and small intestine of genetically responsive C57BL/10 (B10) and nonresponsive DBA/2 (D2) mice. After single stomach intubation of 100 mg/kg of benzo[a]pyrene (B[a]P) in both strains, binding of BPDE to DNA reached a peak 48 h after treatment. However, the levels of binding in the lung, liver, kidney and spleen were higher in D2 than in B10 mice. In contrast to this, in the small intestine the higher level of BPDE binding was found in B10 mice and reached its maximum 24 h earlier. Thereafter a very rapid drop in the level of BPDE--DNA adducts to a value of approximately 50% after 48 h was observed in this tissue. In the other tissues of the B10 mice the rate of adducts removal was slower, but by 14 days after treatment 90-100% of adducts were removed. In the D2 mice up to the 4th day after treatment the rates of removal of the BPDE--DNA adducts were similar to that of the B10 mice. Thereafter the level of bound hydrocarbon decreased at a slower rate. During the whole period after B[a]P treatment distinct differences between organs in the amount of BPDE--DNA adducts were observed. In D2 mice the highest level of binding was found in the spleen followed by the lung, kidney, liver and small intestine. In B10 mice the highest level of binding was observed in the DNA of small intestine. The data suggest that the decreased rate of B[a]P metabolism in D2 mice may be at least in some tissues the reason of higher binding of BPDE--DNA adducts in comparison with B10 mice.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/análisis , Benzo(a)pireno/metabolismo , Aductos de ADN , ADN/análisis , Animales , Benzo(a)pireno/farmacocinética , Carcinógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Espectrometría de Fluorescencia , Distribución TisularRESUMEN
The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.
Asunto(s)
Benzo(a)pireno/toxicidad , Receptores de Droga/genética , Intercambio de Cromátides Hermanas/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etilnitrosourea/efectos adversos , Linfocitos/efectos de los fármacos , Masculino , Metilcolantreno/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Pruebas de Mutagenicidad/métodos , Receptores de Hidrocarburo de Aril , Bazo/efectos de los fármacosRESUMEN
Monoclonal antibodies (MAb 1-7-1) directed against the isoenzymes of rat liver cytochrome P-450 induced by methylcholanthrene, inhibited benzo(a)pyrene hydroxylase less strongly at low (0.04 mM) than at high (2 mM) NADH concentration. Inhibition of NADPH dependent hydroxylation was the same irrespective of NADPH concentrations and corresponded to that observed at high NADH concentration. The same was also the h.p.l.c. pattern of the reaction products of benzo(a)pyrene hydroxylation supported by high concentration of both coenzymes. It is postulated that different cytochrome P-450 isoenzymes participate in benzo(a)pyrene hydroxylation, whereas the second one acts at high concentration of both NADH and NADPH.
Asunto(s)
Benzopirenos/metabolismo , Sistema Enzimático del Citocromo P-450/análisis , Microsomas Hepáticos/metabolismo , NADP/metabolismo , NAD/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Benzopireno Hidroxilasa/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/inmunología , Hidroxilación , Isoenzimas/análisis , Isoenzimas/inmunología , Masculino , Metilcolantreno/farmacología , Ratones , Ratones Endogámicos C57BLRESUMEN
The current research on utilization of monoclonal antibodies for isolation of cytochrome P-450 forms, substrate specificity determination, localization in tissues and contribution in DNA adduct formation is reviewed in this paper. Usefulness of monoclonal antibodies directed against rodent cytochrome P-450 isoenzymes to human investigations is emphasized.
Asunto(s)
Aminoácidos/genética , Anticuerpos Monoclonales , Sistema Enzimático del Citocromo P-450/genética , Isoenzimas/genética , Secuencia de Aminoácidos/genética , Aminoácidos/química , Animales , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Genes/genética , Humanos , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Conejos , RatasRESUMEN
Benzo(a)pyrene (BP) metabolism was studied in cultured fibroblasts from healthy donor. Cells were cultured in Eagle medium supplemented with 10% fetal calf serum, 2 mM 1-glutamine, 100 U/ml penicillin and 100 micrograms/ml streptomycin. Cultures were grown at 37 degrees C to confluence in 45 cm2 culture flasks containing 15 ml medium and refed with fresh medium 24 h prior to treatment with BP. Cells were treated for 24 h with [G-3H]BP diluted with BP to give a final concentration 10 microM and a specific activity of 0.3 Ci/mmol. The metabolites were extracted by ethyl acetate, dried with Na2SO4, evaporated with nitrogen and injected into high pressure liquid chromatograph. The column (LiChrosorb RP 18) was eluted with a linear gradient of 60% methanol in water to 100% methanol in 45 min at a flow rate 0.8 ml/min. Radioactivity of fractions was measured. Following metabolites were identified: 3-hydroxy-BP (23.7%), 9-hydroxy-BP (17.0%), quinones (22.9%), 7,8-dihydroxy-BP (6.1%), 9,10-dihydroxy-BP and other derivatives (30.3%). 7,8-benzoflavone--an inhibitor of BP hydroxylase and epoxide hydrase, strongly inhibited the metabolism of BP in human fibroblasts, changing the proportions in the amounts of the metabolites. The ratio of phenols to the other metabolites increased twice under the influence of 7,8-benzoflavone. This suggests that 7,8-benzoflavone has the stronger inhibitory effect on epoxide and diol formation in comparison with BP hydroxylation.
Asunto(s)
Benzo(a)pireno/metabolismo , Benzoflavonas/farmacología , Benzo(a)pireno/antagonistas & inhibidores , Benzo(a)pireno/química , Benzopirenos/química , Cromatografía Líquida de Alta Presión/métodos , Medios de Cultivo , Depresión Química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Técnicas In VitroRESUMEN
In this study, hepatic microsomes from 5,6-benzoflavone induced C57BL/10 mice were used. To inhibit monooxygenase activities, the monoclonal antibody MAb 1-7-1 recognizing two isoenzymes of methylcholanthrene-induced cytochrome P-450 was applied. Microsomes were incubated with tritium labeled benzo(a)pyrene [G-3H]BP for 10 min at 37 degrees C. The incubation mixture contained: 50 mM potassium phosphate buffer, pH 7.25; 30 mM KCl; 3 mM MgCl2; 2 mM NADPH; 80 microM [G-3H]BP (specific activity 50 mCi/mmol); and monoclonal antibody MAb 1-7-1 or ascites fluid (NBS) containing nonspecific IgG as a control. The ratio of antibody protein/microsomal protein was 2:5. BP metabolites were extracted from incubation mixtures by ethyl acetate. The organic layer was dried over sodium sulfate, and evaporated under a stream of nitrogen. To separate BP metabolites HPLC technology was used. The column was eluted with methanol gradient (60-100%) for 45 minutes. The radio-activity of collected samples was determined using liquid scintillation counter. Differential inhibitory effects of MAb 1-7-1 on BP-metabolites formation were found, e.g. 7,8-diol was inhibited by 86.1% and quinones by 62.5%. The predominant metabolite, 3-OH-BP, was inhibited by 80.4%. Moreover, it was found that MAb 1-7-1 inhibition of benzo(a)pyrene hydroxylase activity (by 75.8%, as measured by fluorescent technique) was very similar to the inhibition of 3-OH-BP along with 9-OH-BP formation (as measured by HPLC).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Benzo(a)pireno/metabolismo , Sistema Enzimático del Citocromo P-450/inmunología , Microsomas Hepáticos/metabolismo , Animales , Medios de Cultivo , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Técnicas In Vitro , Masculino , Metilcolantreno , Ratones , Ratones Endogámicos C57BLRESUMEN
The biphasic kinetics of hydroxylation of benzo(a)pyrene (B(a)P) dependent on the concentration of either NADPH or NADH has been observed in DBA/2 (AhdAhd) but not in C57BL/6 (AhbAhb) beta-naphthoflavone-treated mice. On the other hand, in nontreated mice, this biphasic kinetics has been observed in both strains of mice. This shows that characteristics of biphasic kinetics differentiate Ahb from Ahd mice only after treatment with methylcholanthrene-type of inducers. The discussed biphasic kinetics was more regular and distinct in the NADH-dependent reaction as compared with NADPH-dependent hydroxylation of B(a)P. It is suggested that the NADH-specific pathway of electron transport to cytochrome P-450 is necessary for the occurrence of this effect both in NADH- and NADPH supported reaction.