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1.
Mol Carcinog ; 54(9): 916-25, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24782330

RESUMO

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.


Assuntos
Antioxidantes/uso terapêutico , Doenças Mamárias/dietoterapia , Suplementos Nutricionais , Glândulas Mamárias Animais/patologia , Estresse Oxidativo/efeitos dos fármacos , Tocoferóis/uso terapêutico , Animais , Doenças Mamárias/induzido quimicamente , Doenças Mamárias/metabolismo , Doenças Mamárias/patologia , Suplementos Nutricionais/análise , Estrogênios , Feminino , Humanos , Hiperplasia/induzido quimicamente , Hiperplasia/dietoterapia , Hiperplasia/metabolismo , Hiperplasia/patologia , Glândulas Mamárias Animais/metabolismo , Fator 2 Relacionado a NF-E2/genética , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos ACI , Tirosina/análogos & derivados , Tirosina/análise , Regulação para Cima
2.
J Pharmacol Exp Ther ; 344(1): 244-52, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23055538

RESUMO

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.


Assuntos
Fígado/metabolismo , Acetaminofen/metabolismo , Animais , Compostos Benzidrílicos/metabolismo , Western Blotting , Cromatografia Líquida de Alta Pressão , Feminino , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Glutationa Transferase/metabolismo , Técnicas In Vitro , Inativação Metabólica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenóis/metabolismo , Gravidez , RNA/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/biossíntese , Espectrofotometria Ultravioleta , Frações Subcelulares/metabolismo , Sulfatos/metabolismo , Sulfotransferases/metabolismo
3.
Mol Carcinog ; 52(7): 514-25, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22389237

RESUMO

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.


Assuntos
Proliferação de Células , Dieta , Receptor alfa de Estrogênio/metabolismo , Neoplasias Mamárias Experimentais/prevenção & controle , Fator 2 Relacionado a NF-E2/metabolismo , PPAR gama/metabolismo , Tocoferóis/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Estradiol/sangue , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Hiperplasia/metabolismo , Hiperplasia/patologia , Hiperplasia/prevenção & controle , Técnicas Imunoenzimáticas , Mediadores da Inflamação/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Microssomos Hepáticos/metabolismo , Fator 2 Relacionado a NF-E2/genética , PPAR gama/genética , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos ACI , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tocoferóis/sangue
4.
Drug Metab Dispos ; 40(3): 481-5, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22146138

RESUMO

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.


Assuntos
Genisteína/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Fenóis/farmacologia , Animais , Compostos Benzidrílicos , Proposta de Concorrência , Feminino , Genisteína/antagonistas & inibidores , Glucuronídeos/antagonistas & inibidores , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Inativação Metabólica , Cinética , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Xenobióticos/metabolismo , Xenobióticos/farmacocinética
5.
Comp Hepatol ; 11(1): 1, 2012 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-22524730

RESUMO

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.

6.
J Pharmacol Exp Ther ; 332(2): 650-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19892921

RESUMO

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.


Assuntos
Proteína 1 Transportadora de Ânions Orgânicos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Células LLC-PK1 , Mutagênese Sítio-Dirigida , Proteínas Mutantes , Proteína 1 Transportadora de Ânions Orgânicos/biossíntese , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/fisiologia , Suínos , Transfecção
7.
Int J Environ Res Public Health ; 2(1): 138-46, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16708425

RESUMO

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.


Assuntos
Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Nódulos Linfáticos Agregados/efeitos dos fármacos , Terpenos/toxicidade , Timo/efeitos dos fármacos , Animais , Benzo(a)Antracenos/farmacologia , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Metilcolantreno , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Nódulos Linfáticos Agregados/enzimologia , Ratos , Ratos Endogâmicos , Timo/enzimologia
8.
Endocrinology ; 144(8): 3382-98, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12865317

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Estradiol/metabolismo , Estrona/metabolismo , Isoenzimas/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica , Humanos , Hidroxilação , NADP/farmacologia , Oxirredução , Especificidade por Substrato , Transfecção
9.
Clin Pharmacol Ther ; 71(5): 311-24, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12011817

RESUMO

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.


Assuntos
Estradiol/análogos & derivados , Estradiol/metabolismo , Placenta/metabolismo , Fumar/metabolismo , Adulto , Estrogênios de Catecol , Feminino , Humanos , Hidroxilação , Microssomos/metabolismo , NADP/metabolismo , Placentação , Gravidez , Fumar/fisiopatologia
10.
Biochem Pharmacol ; 68(4): 747-52, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15276082

RESUMO

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.


Assuntos
Radical Hidroxila/metabolismo , Microssomos Hepáticos/enzimologia , Ácidos Ftálicos/metabolismo , Água/química , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Hidroxilação , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Solubilidade
11.
Cancer Prev Res (Phila) ; 1(6): 476-84, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19138995

RESUMO

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.


Assuntos
Calcitriol/análogos & derivados , Carcinoma/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/prevenção & controle , Receptores de Estrogênio/genética , Animais , Calcitriol/efeitos adversos , Calcitriol/farmacologia , Calcitriol/uso terapêutico , Carcinoma/induzido quimicamente , Carcinoma/genética , Transformação Celular Neoplásica/genética , Células Cultivadas , Feminino , Humanos , Hipercalcemia/epidemiologia , Hipercalcemia/etiologia , Hipercalcemia/prevenção & controle , Incidência , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Metilnitrosoureia , Camundongos , Camundongos SCID , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Arch Biochem Biophys ; 446(2): 101-10, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16448623

RESUMO

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.


Assuntos
Anticorpos/isolamento & purificação , Citocromo P-450 CYP3A/biossíntese , Carbonitrila de Pregnenolona/farmacologia , Substâncias Protetoras/farmacologia , Caracteres Sexuais , Animais , Anticorpos Monoclonais/isolamento & purificação , Citocromo P-450 CYP3A/imunologia , Indução Enzimática , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Isoenzimas/biossíntese , Isoenzimas/imunologia , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Coelhos , Ratos , Ratos Sprague-Dawley , Testosterona/metabolismo
13.
J Biol Chem ; 281(26): 17882-9, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16608838

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Inflamação/fisiopatologia , Lipopolissacarídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Citocromo P-450 CYP3A , Dimerização , Regulação para Baixo/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/imunologia , Humanos , Inativação Metabólica/fisiologia , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/química , Receptores de Esteroides/química , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismo , Ativação Transcricional/fisiologia
14.
Drug Metab Dispos ; 34(10): 1672-6, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16815959

RESUMO

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.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Brônquios/enzimologia , Neoplasias Pulmonares/enzimologia , Alvéolos Pulmonares/enzimologia , Traqueia/enzimologia , Animais , Anticorpos/imunologia , Hidrocarboneto de Aril Hidroxilases/imunologia , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Isoenzimas/imunologia , Isoenzimas/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Microssomos Hepáticos/enzimologia , Peptídeos/imunologia
15.
Arch Biochem Biophys ; 402(2): 289-95, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12051676

RESUMO

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.


Assuntos
Glucuronosiltransferase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bilirrubina/química , Western Blotting , Cromatografia Líquida de Alta Pressão , Feminino , Glucuronídeos/química , Humanos , Isoenzimas/química , Fígado/química , Macaca mulatta , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
16.
Arch Biochem Biophys ; 426(1): 55-62, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15130782

RESUMO

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.


Assuntos
Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cumarínicos/química , Cumarínicos/metabolismo , Estradiol/análogos & derivados , Estradiol/metabolismo , Feminino , Ácido Glucurônico/metabolismo , Glucuronosiltransferase/isolamento & purificação , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Rim/citologia , Rim/enzimologia , Cinética , Macaca mulatta , Microssomos/metabolismo , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Entorpecentes/química , Entorpecentes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
17.
Arch Biochem Biophys ; 429(2): 244-51, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15313229

RESUMO

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.


Assuntos
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacocinética , Lignanas/farmacocinética , Acetilcisteína/administração & dosagem , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Fígado/metabolismo , Macaca mulatta , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução
18.
Arch Biochem Biophys ; 414(2): 244-54, 2003 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12781776

RESUMO

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

Assuntos
Anticorpos Monoclonais/metabolismo , Sistema Enzimático do Citocromo P-450/química , Quinidina/análogos & derivados , Animais , Baculoviridae/metabolismo , Western Blotting , Citocromo P-450 CYP3A , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Epitopos , Escherichia coli/metabolismo , Glutationa Transferase/metabolismo , Humanos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Modelos Genéticos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Estrutura Terciária de Proteína , Quinidina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA
19.
Proteomics ; 4(10): 3268-75, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15378687

RESUMO

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.


Assuntos
Neoplasias da Próstata/patologia , Proteômica/métodos , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Espectrometria de Massas , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transfecção
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