RESUMEN
Oxidative stress is known to play a key role in estrogen-induced breast cancer. This study assessed the chemopreventive activity of the naturally occurring γ-tocopherol-rich mixture of tocopherols (γ-TmT) in early stages of estrogen-induced mammary hyperplasia in ACI rats. ACI rats provide an established model of rodent mammary carcinogenesis due to their high sensitivity to estrogen. Female rats were implanted with 9 mg of 17ß-estradiol (E2) in silastic tubings and fed with control or 0.3% γ-TmT diet for 1, 3, 7, and 14 d. γ-TmT increased the levels of tocopherols and their metabolites in the serum and mammary glands of the rats. Histological analysis revealed mammary hyperplasia in the E2 treated rats fed with control or γ-TmT diet. γ-TmT decreased the levels of E2-induced nitrosative and oxidative stress markers, nitrotyrosine, and 8-oxo-dG, respectively, in the hyperplastic mammary tissues. 8-Isoprostane, a marker of oxidative stress in the serum, was also reduced by γ-TmT. Noticeably, γ-TmT stimulated Nrf2-dependent antioxidant response in the mammary glands of E2 treated rats, evident from the induced mRNA levels of Nrf2 and its downstream antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. Therefore, inhibition of nitrosative/oxidative stress through induction of antioxidant response is the primary effect of γ-TmT in early stages of E2-induced mammary hyperplasia. Due to its cytoprotective activity, γ-TmT could be a potential natural agent for the chemoprevention of estrogen-induced breast cancer.
Asunto(s)
Antioxidantes/uso terapéutico , Enfermedades de la Mama/dietoterapia , Suplementos Dietéticos , Glándulas Mamarias Animales/patología , Estrés Oxidativo/efectos de los fármacos , Tocoferoles/uso terapéutico , Animales , Enfermedades de la Mama/inducido químicamente , Enfermedades de la Mama/metabolismo , Enfermedades de la Mama/patología , Suplementos Dietéticos/análisis , Estrógenos , Femenino , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/dietoterapia , Hiperplasia/metabolismo , Hiperplasia/patología , Glándulas Mamarias Animales/metabolismo , Factor 2 Relacionado con NF-E2/genética , ARN Mensajero/genética , Ratas , Ratas Endogámicas ACI , Tirosina/análogos & derivados , Tirosina/análisis , Regulación hacia ArribaRESUMEN
Phase II enzymes, including Ugts, Sults, and Gsts, are critical for the disposition and detoxification of endo- and xenobiotics. In this study, the mRNA and protein expression of major phase II enzymes, as well as key regulatory transcription factors, were quantified in livers of time-matched pregnant and virgin control C57BL/6 mice on gestation days (GD) 7, 11, 14, 17, and postnatal days (PND) 1, 15, and 30. Compared with virgin controls, the mRNA expression of Ugt1a1, 1a6, 1a9, 2a3, 2b1, 2b34, and 2b35 decreased 40 to 80% in pregnant dams. Protein expression of Ugt1a6 also decreased and corresponded with reduced in vitro glucuronidation of bisphenol A in S9 fractions from livers of pregnant mice. Similar to Ugts levels, Gsta1 and a4 mRNAs were reduced in pregnant dams in mid to late gestation; however no change in protein expression was observed. Conversely, Sult1a1, 2a1/2, and 3a1 mRNAs increased 100 to 500% at various time points in pregnant and lactating mice and corresponded with enhanced in vitro sulfation of acetaminophen in liver S9 fractions. Coinciding with maximal decreases in Ugts as well as increases in Sults, the expression of transcription factors CAR, PPARα, and PXR and their target genes were downregulated, whereas ERα mRNA was upregulated. Collectively, these data demonstrate altered regulation of hepatic phase II metabolism in mice during pregnancy and suggest that CAR, PPARα, PXR, and ERα signaling pathways may be candidate signaling pathways responsible for these changes.
Asunto(s)
Hígado/metabolismo , Acetaminofén/metabolismo , Animales , Compuestos de Bencidrilo/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Femenino , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/metabolismo , Técnicas In Vitro , Inactivación Metabólica , Masculino , Ratones , Ratones Endogámicos C57BL , Fenoles/metabolismo , Embarazo , ARN/biosíntesis , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Estrógenos/biosíntesis , Espectrofotometría Ultravioleta , Fracciones Subcelulares/metabolismo , Sulfatos/metabolismo , Sulfotransferasas/metabolismoRESUMEN
Previous clinical and epidemiological studies of vitamin E have used primarily α-tocopherol for the prevention of cancer. However, γ-tocopherol has demonstrated greater anti-inflammatory and anti-tumor activity than α-tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ-tocopherol (γ-TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17ß-estradiol (E2 ) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ-TmT for 2 or 10 wk. Serum E2 levels were significantly reduced by the treatment with 0.5% γ-TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8-isoprostane, were suppressed by γ-TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2 -treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2), and estrogen receptor α (ERα), while there was an increase in cleaved-caspase 3, peroxisome proliferator-activated receptor γ (PPARγ), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in γ-TmT-treated rats. In addition, treatment with γ-TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERß and PPARγ were increased. In conclusion, γ-TmT was shown to suppress inflammatory markers, inhibit E2 -induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ-TmT may be a promising agent for human breast cancer prevention.
Asunto(s)
Proliferación Celular , Dieta , Receptor alfa de Estrógeno/metabolismo , Neoplasias Mamarias Experimentales/prevención & control , Factor 2 Relacionado con NF-E2/metabolismo , PPAR gamma/metabolismo , Tocoferoles/administración & dosificación , Animales , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Estradiol/sangre , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Hiperplasia/metabolismo , Hiperplasia/patología , Hiperplasia/prevención & control , Técnicas para Inmunoenzimas , Mediadores de Inflamación/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Microsomas Hepáticos/metabolismo , Factor 2 Relacionado con NF-E2/genética , PPAR gamma/genética , ARN Mensajero/genética , Ratas , Ratas Endogámicas ACI , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tocoferoles/sangreRESUMEN
Genistein is a natural phytoestrogen of the soybean, and bisphenol A (BPA) is a synthetic chemical used in the production of polycarbonate plastics. Both genistein and BPA disrupt the endocrine system in vivo and in vitro. Growing concerns of altered xenobiotic metabolism due to concomitant exposures from soy milk in BPA-laden baby bottles has warranted the investigation of the glucuronidation rate of genistein in the absence and presence (25 µM) of BPA by human liver microsomes (HLM) and rat liver microsomes (RLM). HLM yield V(max) values of 0.93 ± 0.10 nmol · min(-1) · mg(-1) and 0.62 ± 0.05 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively. K(m) values for genistein glucuronidation by HLM in the absence and presence of BPA are 15.1 ± 7.9 µM and 21.5 ± 7.7 µM, respectively, resulting in a K(i) value of 58.7 µM for BPA. Significantly reduced V(max) and unchanged K(m) in the presence of BPA in HLM are suggestive of noncompetitive inhibition. In RLM, the presence of BPA resulted in a K(i) of 35.7 µM, an insignificant change in V(max) (2.91 ± 0.26 nmol · min(-1) · mg(-1) and 3.05 ± 0.41 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively), and an increase in apparent K(m) (49.4 ± 14 µM with no BPA and 84.0 ± 28 µM with BPA), indicative of competitive inhibition. These findings are significant because they suggest that BPA is capable of inhibiting the glucuronidation of genistein in vitro, and that the type of inhibition is different between HLM and RLM.
Asunto(s)
Genisteína/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Fenoles/farmacología , Animales , Compuestos de Bencidrilo , Propuestas de Licitación , Femenino , Genisteína/antagonistas & inhibidores , Glucurónidos/antagonistas & inhibidores , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Inactivación Metabólica , Cinética , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Xenobióticos/metabolismo , Xenobióticos/farmacocinéticaRESUMEN
BACKGROUND: Type-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites. RESULTS: Transporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice. CONCLUSION: A severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.
RESUMEN
Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. In transmembrane segment (TM) 12, residues Tyr-490 and dileucine Leu-503/Leu-504 were identified to be critical for hOAT1 function. Substitution of Tyr-490 with alanine led to a dramatic reduction in protein expression of hOAT1 and its transport activity. The contribution of the side chain of Tyr-490 to transport activity was then evaluated by replacing this residue with Trp or Phe. Substitution of Tyr-490 with Trp or Phe partially or fully recovered the protein expression of hOAT1 and its transport activity, respectively, that were lost by substitution of Tyr-490 with alanine, suggesting that the aromatic ring and the size of the side chain of Tyr-490 are critical for hOAT1 expression and function. Studies with protease inhibitors and pulse-chase labeling further showed that the loss of expression of hOAT1 and its transport activity by replacing Tyr-490 with alanine resulted from accelerated degradation of the transporter, whereas its maturation efficiency was not affected. In contrast to Tyr-490, substitution of Leu-503/Leu-504 with alanine also resulted in complete loss of protein expression of hOAT1 and its transport activity. However, such loss of protein expression could not be prevented by treating mutant-expressing cells with protease inhibitors. Pulse-chase experiments showed that the mutant transporter (L503/L504A) was trapped in the endoplasmic reticulum without conversion into mature form of the transporter. Our results are the first to highlight the central role of TM 12 in maintaining the stability and in promoting the maturation efficiency of hOAT1.
Asunto(s)
Proteína 1 de Transporte de Anión Orgánico/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Humanos , Células LLC-PK1 , Mutagénesis Sitio-Dirigida , Proteínas Mutantes , Proteína 1 de Transporte de Anión Orgánico/biosíntesis , Proteína 1 de Transporte de Anión Orgánico/genética , Proteína 1 de Transporte de Anión Orgánico/fisiología , Porcinos , TransfecciónRESUMEN
Chemical carcinogenesis studies are powerful tools to obtain information on potential mechanisms of chemical factors for malignancies. In this study Western blot analyses, using monoclonal antibodies specific for three different cytochrome P450 (CYP) isozymes (CYP1A1, CYP1A2 and CYP2B), were employed to examine the effect(s) of 3-methylcholanthrene and/or pristane (2,6,10,14-tetramethylpentadecane) on the basal and inducible levels of expression of CYP proteins within Copenhagen rat tissues. Pristane exposure led to tissue specific differences in the CYP isozymes expressed and elicited increased CYP protein expression over 3-methylcholanthrene induced levels in microsomes isolated from liver, Peyer's Patches, and thymus. Within the context of the chemical carcinogenesis model employed in this study, these observations correlated with the induction of B-cell malignancies by low doses of 3-methylcholanthrene and of thymic lymphomas by a high 3-methylcholanthrene dose. The data suggest that pristane treatment affects CYP isozyme expression. This pristane-mediated effect clearly could be a contributing factor in the chemical carcinogenesis of the previously observed lymphoid malignancies, and a possible basis for the tumor enhancing effects of pristane.
Asunto(s)
Carcinógenos/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Ganglios Linfáticos Agregados/efectos de los fármacos , Terpenos/toxicidad , Timo/efectos de los fármacos , Animales , Benzo(a)Antracenos/farmacología , Femenino , Hígado/efectos de los fármacos , Hígado/enzimología , Metilcolantreno , Microsomas/efectos de los fármacos , Microsomas/enzimología , Ganglios Linfáticos Agregados/enzimología , Ratas , Ratas Endogámicas , Timo/enzimologíaRESUMEN
We systematically characterized the oxidative metabolites of 17beta-estradiol and estrone formed by 15 human cytochrome P450 (CYP) isoforms. CYP1A1 had high activity for 17beta-estradiol 2-hydroxylation, followed by 15alpha-, 6alpha-, 4-, and 7alpha-hydroxylation. However, when estrone was the substrate, CYP1A1 formed more 4-hydroxyestrone than 15alpha- or 6alpha-hydroxyestrone, with 2-hydroxyestrone as the major metabolite. CYP1A2 had the highest activity for the 2-hydroxylation of both 17beta-estradiol and estrone, although it also had considerable activity for their 4-hydroxylation (9-13% of 2-hydroxylation). CYP1B1 mainly catalyzed the formation of catechol estrogens, with 4-hydroxyestrogens predominant. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2D6 each showed a varying degree of low catalytic activity for estrogen 2-hydroxylation, whereas CYP2C18 and CYP2E1 did not show any detectable estrogen-hydroxylating activity. CYP3A4 had strong activity for the formation of 2-hydroxyestradiol, followed by 4-hydroxyestradiol and an unknown polar metabolite, and small amounts of 16alpha- and 16beta-hydroxyestrogens were also formed. The ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone with CYP3A4 was 0.22 or 0.51, respectively. CYP3A5 had similar catalytic activity for the formation of 2- and 4- hydroxyestrogens. Notably, CYP3A5 had an unusually high ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone (0.53 or 1.26, respectively). CYP3A4 and 3A5 also catalyzed the formation of nonpolar estrogen metabolite peaks (chromatographically less polar than estrone). CYP3A7 had a distinct catalytic activity for the 16alpha-hydroxylation of estrone, but not 17beta-estradiol. CYP4A11 had little catalytic activity for the metabolism of 17beta-estradiol and estrone. In conclusion, many human CYP isoforms are involved in the oxidative metabolism of 17beta-estradiol and estrone, with a varying degree of catalytic activity and distinct regioselectivity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Estradiol/metabolismo , Estrona/metabolismo , Isoenzimas/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Catálisis , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Expresión Génica , Humanos , Hidroxilación , NADP/farmacología , Oxidación-Reducción , Especificidad por Sustrato , TransfecciónRESUMEN
OBJECTIVE: Our objective was to characterize the oxidative metabolism of estradiol by human term placenta and its modulation by cigarette smoking. METHODS: Placental microsomes were prepared from term placentas obtained from 13 cigarette smokers (20 to 30 cigarettes per day until the time of delivery) and 13 control subjects who were nonsmokers. Estrogen metabolism was studied by incubation of 250 nmol/L [(3)H]estradiol with placental microsomes and NADPH, and the estrogen metabolites were determined by HPLC and gas chromatography-mass spectrometry. RESULTS: 2-Hydroxyestradiol was the major hydroxyestrogen detected, followed by 6alpha-hydroxyestradiol. Small amounts of several other hydroxyestrogen metabolites (4-hydroxyestradiol, 6beta-hydroxyestradiol, 7alpha-hydroxyestradiol, and 16alpha-hydroxyestradiol) were also detected. Large amounts of estrone plus small amounts of 2-hydroxyestrone and unidentified nonpolar metabolites were formed. Cigarette smoking stimulated the placental hydroxylation of benzo[a ]pyrene by about 16-fold. Cigarette smoking had little or no effect on the overall rate of placental estradiol metabolism or on the formation of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, or 16alpha-hydroxyestradiol. However, placental formation of 4-hydroxyestradiol and 7alpha-hydroxyestradiol was increased 38% (P =.08) and 150% (P =.05), respectively, in cigarette smokers. The formation of 6alpha-hydroxyestradiol was decreased 33% (P =.04). Metabolic formation of 15alpha-hydroxyestradiol was observed during incubations of estradiol with placental microsomes from 11 of the 13 cigarette smokers, but this metabolite was not detected during incubations with placental microsomes from any of the 13 nonsmokers. Analysis of data from all 26 placentas showed that the 15alpha-hydroxylation of estradiol was highly correlated with benzo[a ]pyrene hydroxylation (r = 0.93; P <.001). CONCLUSIONS: Many hydroxylated estradiol metabolites were formed by placental microsomes from cigarette smokers and nonsmokers. 15alpha-Hydroxylation of estradiol was markedly stimulated in the placentas of cigarette smokers.
Asunto(s)
Estradiol/análogos & derivados , Estradiol/metabolismo , Placenta/metabolismo , Fumar/metabolismo , Adulto , Estrógenos de Catecol , Femenino , Humanos , Hidroxilación , Microsomas/metabolismo , NADP/metabolismo , Placentación , Embarazo , Fumar/fisiopatologíaRESUMEN
Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO?) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO? formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450. Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO(.-) formation. HO(.-) formation in liver microsomes was inhibited by the HO(.-) radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO(.-). Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.
Asunto(s)
Radical Hidroxilo/metabolismo , Microsomas Hepáticos/enzimología , Ácidos Ftálicos/metabolismo , Agua/química , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Hidroxilación , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1alpha,25(OH)(2)D(3), the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1alpha,25(OH)(2)D(3) in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using (a) an N-methyl-N-nitrosourea-induced estrogen receptor (ER)-positive mammary tumor model and (b) an MCF10DCIS.com xenograft model of ER-negative mammary tumors. Among vitamin D analogues we tested, Gemini 0072 [1alpha,25-dihydroxy-20S-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] and Gemini 0097 [1alpha,25-dihydroxy-20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMU-induced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.
Asunto(s)
Calcitriol/análogos & derivados , Carcinoma/prevención & control , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Mamarias Experimentales/prevención & control , Receptores de Estrógenos/genética , Animales , Calcitriol/efectos adversos , Calcitriol/farmacología , Calcitriol/uso terapéutico , Carcinoma/inducido químicamente , Carcinoma/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Femenino , Humanos , Hipercalcemia/epidemiología , Hipercalcemia/etiología , Hipercalcemia/prevención & control , Incidencia , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/genética , Metilnitrosourea , Ratones , Ratones SCID , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16alpha-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 >> 3A18 > 3A9 approximately = 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively.
Asunto(s)
Anticuerpos/aislamiento & purificación , Citocromo P-450 CYP3A/biosíntesis , Carbonitrilo de Pregnenolona/farmacología , Sustancias Protectoras/farmacología , Caracteres Sexuales , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Citocromo P-450 CYP3A/inmunología , Inducción Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas In Vitro , Isoenzimas/biosíntesis , Isoenzimas/inmunología , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Conejos , Ratas , Ratas Sprague-Dawley , Testosterona/metabolismoRESUMEN
It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-kappaB activation by lipopolysaccharide and tumor necrosis factor-alpha plays a pivotal role in the suppression of cyp3a4 through interactions of NF-kappaB with the PXR.retinoid X receptor (RXR) complex. Inhibition of NF-kappaB by NF-kappaB-specific suppressor SRIkappaBalpha reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-alpha. Furthermore, we showed that NF-kappaB p65 disrupted the association of the PXR.RXRalpha complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-kappaB p65 directly interacted with the DNA-binding domain of RXRalpha and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR.RXRalpha complex. This mechanism of suppression by NF-kappaB activation may be extended to other nuclear receptor-regulated systems where RXRalpha is a dimerization partner.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Inflamación/fisiopatología , Lipopolisacáridos/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Citocromo P-450 CYP3A , Dimerización , Regulación hacia Abajo/inmunología , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/inmunología , Humanos , Inactivación Metabólica/fisiología , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/química , Receptores de Esteroides/química , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Human cytochrome P450 2A13 (CYP2A13) is highly efficient in the metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and another potent carcinogen, aflatoxin B1 (AFB1). Although previous studies demonstrated that CYP2A13 mRNA is predominantly expressed in human respiratory tissues, expression of CYP2A13 protein in these tissues and the involved cell types have not been determined because of the lack of CYP2A13-specific antibodies. To explore the toxicological and physiological function of CYP2A13, it is important to understand the tissue/cellular distribution of CYP2A13 protein. In this study, we generated a peptide-specific antibody against human CYP2A13 and demonstrated by immunoblot analysis that this antibody does not cross-react with heterologously expressed human CYP2A6 and mouse CYP2A5 proteins, both sharing a high degree of amino acid sequence similarity with CYP2A13. Nor does the antibody cross-react with heterologously expressed human CYP3A4, CYP2S1, or any of the cytochrome P450 enzymes present in the human liver microsomes. Using this highly specific antibody for immunohistochemical staining, we detected a high level of CYP2A13 protein expression in the epithelial cells of human bronchus and trachea, but a rare distribution in the alveolar cells. There was little expression of CYP2A13 protein in different types of lung cancers. In consideration of the high efficiency of CYP2A13 in NNK metabolic activation, our result is consistent with the reported observations that most smoking-related human lung cancers are bronchogenic and supports that CYP2A13-catalyzed in situ activation may play a critical role in human lung carcinogenesis related to NNK and AFB1 exposure.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Bronquios/enzimología , Neoplasias Pulmonares/enzimología , Alveolos Pulmonares/enzimología , Tráquea/enzimología , Animales , Anticuerpos/inmunología , Hidrocarburo de Aril Hidroxilasas/inmunología , Humanos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Isoenzimas/inmunología , Isoenzimas/metabolismo , Neoplasias Pulmonares/patología , Ratones , Microsomas Hepáticos/enzimología , Péptidos/inmunologíaRESUMEN
An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.
Asunto(s)
Glucuronosiltransferasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bilirrubina/química , Western Blotting , Cromatografía Líquida de Alta Presión , Femenino , Glucurónidos/química , Humanos , Isoenzimas/química , Hígado/química , Macaca mulatta , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Enterolactone (ENL) and enterodiol (END) are mammalian lignans derived from the plant lignans matairesionol (MAT), secoisolariciresinol (SECO), and other dietary precursors. ENL was found to undergo extensive glucuronidation with rhesus liver microsomes to form O-glucuronides at both phenolic hydroxy groups. In addition to glucuronidation, ENL was found to be a good substrate for oxidative metabolism. The major products had a m/z of 313 or 295 by LC-MS analysis in negative ion mode and were determined to be products of monohydroxylation of ENL. The m/z 295 products were the result of a dehydration of the m/z 313 in the MS source. Addition of N-acetylcysteine (NAC) to the NADPH incubations resulted in a decrease of at least 2 major monohydroxylated products and the formation of a major and several minor new products with a m/z of 474. The major adduct was isolated, purified for NMR, and confirmed to be the NAC adduct of the ENL catechol. Incubations of ENL with liver microsomes containing UDPGA, NADPH, and NAC resulted in the formation of ENL-glucuronides; no NAC adducts were detected by LC-MS. Incubations of ENL with human and rhesus hepatocytes resulted in several metabolites. The major metabolites in hepatocytes were the glucuronic acid conjugates; minor amounts of the sulfate conjugate(s) and monohydroxylated products were also detected by LC-MS. Glutathione or other thiol adducts were not detected in hepatocytes. Conclusion. The high efficiency and specificity for the glucuronidation of ENL decrease its potential toxicity via CYP450 bioactivation.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacocinética , Lignanos/farmacocinética , Acetilcisteína/administración & dosificación , Animales , Cromatografía Líquida de Alta Presión , Femenino , Hígado/metabolismo , Macaca mulatta , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Oxidación-ReducciónRESUMEN
Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.
Asunto(s)
Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Línea Celular , Cumarinas/química , Cumarinas/metabolismo , Estradiol/análogos & derivados , Estradiol/metabolismo , Femenino , Ácido Glucurónico/metabolismo , Glucuronosiltransferasa/aislamiento & purificación , Humanos , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Riñón/citología , Riñón/enzimología , Cinética , Macaca mulatta , Microsomas/metabolismo , Técnicas de Sonda Molecular , Datos de Secuencia Molecular , Narcóticos/química , Narcóticos/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
In this study we describe the mapping of epitopes on CYP3A4/5 recognized by a panel of monoclonal antibodies (MAbs). CYP3A4 and CYP3A5 cDNAs were cloned in GST expression vectors and the fusion proteins were subjected to Western blot. Eight MAbs reacted with the full-length GST-3A4 fusion protein as well as baculovirus cDNA-expressed CYP3A4, while six of these reacted with baculovirus cDNA-expressed CYP3A5. Five (MAb 347, 351, 352, 354, and 357) out of 8 MAbs were inhibitory in a metabolic assay using quinine as substrate. MAbs 352, 354, and 357 brought about a moderate inhibition of quinine metabolism (60-70%) while MAb 347 inhibited quinine 3- hydroxylation in human liver microsomes (n=6) by more than 70%. MAb 347 was a potent inhibitor of baculovirus-expressed CYP3A5-catalyzed metabolism of quinine (95%) at =0.20 mg IgG/nmol P450 but only moderately inhibited CYP3A4 at much higher ratios of MAb to P450. This MAb was mapped to a region of 283 to 504 amino acids on CYP3A4 protein and to an identical region on CYP3A5 protein. The region that was identified on the CYP3A5 construct was further validated based on the ability of the construct harboring the epitope to reverse the inhibition of hydroxylation of quinine by MAb 347. Our experiments clearly demonstrate that a spatial antigenic determinant is responsible for the inhibitory potency of MAb 347.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Sistema Enzimático del Citocromo P-450/química , Quinidina/análogos & derivados , Animales , Baculoviridae/metabolismo , Western Blotting , Citocromo P-450 CYP3A , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Epítopos , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Modelos Genéticos , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Conformación Proteica , Estructura Terciaria de Proteína , Quinidina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADNRESUMEN
Chemotherapy and androgen ablation therapy are only temporarily effective against prostate cancer, and current studies are ongoing to test agents that target proteins responsible for autocrine and paracrine stimulated growth. Given limitations of current laboratory models to test the effect of these agents on cell growth and protein targets, we developed a coculture model that can distinguish paracrine stimulated growth and effects on proteins. We found that LNCaP prostate cancer cells and an immortalized rat prostate cell line transfected to overexpress the antiapoptotic resistance protein Bcl-2 were stimulated to grow (>2-fold increase, p < 0.01) through autocrine effects from additional cells in an upper chamber of our system. Using a proteomic approach with a two-dimensional differential in gel electrophoresis method to increase fidelity, four proteins were found to increase after autocrine induced growth stimulation. These proteins were all identified by mass spectrometry as enzymes in the glycolytic pathway, validating the ability of this system to detect both clonogenic growth and the effect on proteins. These data, therefore, demonstrate a novel coculture model for further study of agents that target proteins in pathways of paracrine or autocrine stimulated cell growth.