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1.
Int Immunopharmacol ; 12(4): 651-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22310326

RESUMO

Arsenite is critical pharmacologically as a treatment for advanced stage blood cancer. However, environmental exposure to arsenic results in multiple diseases. Previous studies have shown that arsenic decreases expression of CYP3A, a critical drug metabolizing enzyme in human and rat liver. In addition, acute and chronic arsenic exposure in liver stimulates an inflammatory response. Our work has shown that arsenite decreases nuclear levels of RXRα the nuclear receptor that, as a heterodimer partner with PXR, transactivates the CYP3A gene. These results suggest that arsenite decreases transcription of CYP3A by decreasing RXRα. The present report shows that exposure to 5 µM arsenite decreased the activity of a rat CYP3A promoter luciferase reporter in HepG2 cells. The activity of a RARE-luciferase reporter, that is transactivated by the retinoic acid receptor (RAR)/RXRα, was also decreased. Previous studies have shown that arsenic in the concentration range of 2-5 µM affects CYP3A mRNA. When rifampicin-treated primary human hepatocyte cultures were exposed to arsenite concentrations as low as 50 nM, CYP3A mRNA was decreased. Treatment of primary human hepatocytes with the proteasome inhibitor MG132 increased RXRα suggesting the involvement of the proteasome pathway in regulation of RXRα. Finally, arsenic induces a pro-inflammatory response in liver. Surprisingly, we show that in hepatocytes arsenite decreases expression of two inflammatory mediators, TNF and VEGF, an effect that is not predicted from suppression of RXRα activity.


Assuntos
Arsenitos/toxicidade , Citocromo P-450 CYP3A/genética , Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Receptor X Retinoide alfa/metabolismo , Adulto , Células Cultivadas , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genética
2.
Drug Metab Rev ; 43(1): 1-26, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20860521

RESUMO

Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.


Assuntos
Apoenzimas/química , Apoenzimas/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/química , Heme/metabolismo , Animais , Apoenzimas/genética , Sistema Enzimático do Citocromo P-450/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estabilidade Enzimática , Heme/genética , Fígado/metabolismo , Estrutura Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional
3.
Neurosci Lett ; 461(3): 302-5, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19539709

RESUMO

Heme-a, is the heme prosthetic group of cytochrome c oxidase (COX), the terminal complex of the mitochondrial electron transport chain. We measured heme-a levels in postmortem brain tissue from nine patients diagnosed with dementia: Alzheimer's disease (AD) was the primary diagnosis in five, AD/diffuse Lewy body disease (DLBD) was diagnosed in two, DLBD was diagnosed in one, and DLBD (severe)/AD (mild) was diagnosed in one. Eight non-demented patients who died from non-neurological causes served as controls. When the primary diagnosis was AD (AD and AD/DLBD), levels of cerebral heme-a were increased almost two-fold on a protein basis compared to controls (p<0.001). Using perfused and non-perfused rats we showed that measured levels of cerebral heme-a were unaffected by the presence of blood in brain tissue. In mice we showed that levels of cerebral heme-a were unaffected by 24h of storage at 4 degrees C prior to freezing. These animal studies suggest that increased levels of cerebral heme-a in AD were not due to blood in postmortem brain or variation in postmortem interval.


Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Idoso , Animais , Biomarcadores/metabolismo , Lobo Frontal/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Subunidades Proteicas/metabolismo , Ratos , Ratos Endogâmicos F344
4.
Hepatology ; 46(6): 1927-834, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17854053

RESUMO

UNLABELLED: Porphyria cutanea tarda is a liver disease characterized by elevated hepatic iron and excessive production of uroporphyrin (URO). Phlebotomy is an effective treatment that probably acts by reducing hepatic iron. Here we used Hfe(-/-) mice to compare the effects on hepatic URO accumulation of two different methods of hepatic iron depletion: iron chelation using deferiprone (L1) versus iron-deficient diets. Hfe(-/-) mice in a 129S6/SvEvTac background were fed 5-aminolevulinic acid (ALA), which results in hepatic URO accumulation, and increasing doses of L1 in the drinking water. Hepatic URO accumulation was completely prevented at low L1 doses, which partially depleted hepatic nonheme iron. By histological assessment, the decrease in hepatic URO accumulation was associated with greater depletion of iron from hepatocytes than from Kupffer cells. The L1 treatment had no effect on levels of hepatic cytochrome P4501A2 (CYP1A2). L1 also effectively decreased hepatic URO accumulation in C57BL/6 Hfe(-/-) mice treated with ALA and a CYP1A2 inducer. ALA-treated mice maintained on defined iron-deficient diets, rather than chow diets, did not develop uroporphyria, even when the animals were iron-supplemented either directly in the diet or by iron dextran injection. CONCLUSION: The results suggest that dietary factors other than iron are involved in the development of uroporphyria and that a modest depletion of hepatocyte iron by L1 is sufficient to prevent URO accumulation.


Assuntos
Quelantes de Ferro/uso terapêutico , Deficiências de Ferro , Porfiria Cutânea Tardia/dietoterapia , Porfiria Cutânea Tardia/tratamento farmacológico , Piridonas/uso terapêutico , Animais , Deferiprona , Modelos Animais de Doenças , Fígado/química , Masculino , Camundongos , Uroporfirinas/análise
5.
Toxicol Appl Pharmacol ; 221(2): 235-42, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17466352

RESUMO

Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the high affinity form of the receptor (AHRb(1)), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(-/-) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(-/-) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(-/-) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Hexaclorobenzeno/farmacologia , Porfirias/induzido quimicamente , Receptores de Hidrocarboneto Arílico/metabolismo , Uroporfirinas/metabolismo , Animais , Feminino , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores Sexuais
6.
Drug Metab Dispos ; 35(7): 1223-31, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17392391

RESUMO

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Etanol/toxicidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Pentanóis/toxicidade , Acetaminofen , Alanina Transaminase/sangue , Animais , Benzoquinonas/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/deficiência , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Modelos Animais de Doenças , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucuronídeos/metabolismo , Glutationa/metabolismo , Hidroxilação , Iminas/metabolismo , Fígado/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Knockout , Índice de Gravidade de Doença , Testosterona/metabolismo , Troleandomicina/farmacologia
7.
Hepatology ; 45(1): 187-94, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17187429

RESUMO

UNLABELLED: Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. CONCLUSION: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.


Assuntos
Deficiência de Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Ferro/farmacologia , Porfiria Cutânea Tardia/metabolismo , Uroporfirinas/metabolismo , Ácido Aminolevulínico , Animais , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Deficiência de Ácido Ascórbico/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Suplementos Nutricionais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ferro/metabolismo , Complexo Ferro-Dextran/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Bifenilos Policlorados , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/tratamento farmacológico
8.
Am J Physiol Gastrointest Liver Physiol ; 290(6): G1269-79, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16439473

RESUMO

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-alpha. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-alpha. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity.


Assuntos
Acetaminofen/efeitos adversos , Etanol/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptor 4 Toll-Like/metabolismo , Analgésicos não Narcóticos/efeitos adversos , Animais , Sinergismo Farmacológico , Fígado Gorduroso/patologia , Feminino , Fígado/patologia , Camundongos
9.
Drug Metab Dispos ; 33(12): 1827-36, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16141365

RESUMO

The pregnane X receptor (PXR) is a transcriptional regulator of xenobiotic metabolizing enzymes, including cytochrome P450 3A (CYP3A), and transporters. Pretreatment of mice and rats with inducers of CYP3A increases acetaminophen (APAP) hepatotoxicity. In untreated mice, the amount of hepatic CYP3A11 mRNA is 4-fold greater in PXR(-/-) mice compared to wild-type mice (Guo et al., 2003), a finding anticipated to increase APAP hepatotoxicity in PXR(-/-) mice. We investigated APAP hepatotoxicity in wild-type and PXR(-/-) mice in a C57BL/6 background, with APAP administered by gavage. Despite a 2.5-fold higher level of total hepatic CYP3A protein and a 3.6-fold higher level of CYP3A activity compared to wild-type mice, PXR(-/-) mice were less sensitive to APAP hepatotoxicity. Hepatic levels of CYP2E1 were identical in the two mouse lines, but hepatic CYP1A2 levels were 3-fold greater in wild-type mice compared to PXR(-/-) mice. Caffeine, an inhibitor of CYP1A2 activity and an enhancer of CYP3A activity, decreased APAP hepatotoxicity in wild-type mice. APAP uptake was 1.5-fold greater in wild-type mice compared to PXR(-/-) mice. No significant differences in the formation of APAP glucuronide and sulfate-conjugated metabolites were observed between wild-type and PXR(-/-) mice. Glutathione levels were similar in the two mouse lines and were transiently decreased to similar amounts after APAP administration. Our finding that APAP hepatotoxicity was decreased in PXR(-/-) mice indicates that PXR is an important modulator of APAP hepatotoxicity, through positive modulation of constitutive CYP1A2 expression and possibly through increased APAP absorption.


Assuntos
Acetaminofen/toxicidade , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Acetaminofen/metabolismo , Animais , Benzoquinonas/metabolismo , Transporte Biológico , Cafeína/farmacologia , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP2E1/análise , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/análise , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Iminas/metabolismo , Absorção Intestinal , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Transcrição Gênica/efeitos dos fármacos
10.
Arch Biochem Biophys ; 439(1): 1-11, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15946643

RESUMO

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.


Assuntos
Ácido Aminolevulínico/metabolismo , Coproporfirinas/biossíntese , Fármacos Gastrointestinais/farmacologia , Glucagon/farmacologia , Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Uroporfirinas/biossíntese , Ácido Aminolevulínico/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Hepatócitos/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia
11.
Biochem Biophys Res Commun ; 333(4): 1211-7, 2005 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-15979568

RESUMO

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.


Assuntos
Arsenitos/administração & dosagem , Hidrocarboneto de Aril Hidroxilases/metabolismo , Calpaína/metabolismo , Glutationa/metabolismo , Heme/metabolismo , Hepatócitos/metabolismo , Animais , Células Cultivadas , Citocromo P-450 CYP3A , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Toxicol Appl Pharmacol ; 209(2): 174-82, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-15907335

RESUMO

Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 microM arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.


Assuntos
Arsenitos/toxicidade , Hidrocarboneto de Aril Hidroxilases/biossíntese , Fígado/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP3A , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Immunoblotting , Fígado/enzimologia , Fígado/metabolismo , Masculino , Polirribossomos/enzimologia , Polirribossomos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Drug Metab Dispos ; 33(7): 993-1003, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15833926

RESUMO

Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (P450) levels by arsenic. P450s are involved in the oxidative metabolism and elimination of numerous toxic chemicals. CYP3A4, a major P450 in humans, is involved in the metabolism of half of all currently used drugs. Acute exposure to arsenite decreases the induction of CYP1A1/2 proteins and activities in cultured human hepatocytes, as well as CYP3A23 in cultured rat hepatocytes. Here, in primary cultures of human hepatocytes, we assessed the effects of acute arsenite exposure on CYP3A4 and several transcription factors involved in CYP3A4 expression. The concentrations of arsenite used in these studies were nontoxic to the hepatocytes and failed to elicit an oxidative response. Treatment with arsenite in the presence of CYP3A4 inducers, rifampicin (Rif) or phenobarbital, caused major decreases in CYP3A4 mRNA, protein, and activity. In addition, the levels of CYP3A4 in untreated cells were decreased following arsenite treatment. Transcription of the CYP3A4 gene is primarily regulated by heterodimers of the retinoid X receptor alpha (RXRalpha) and the pregnane X receptor (PXR). We found that arsenite failed to affect expression of PXR or the transcription factor Sp1, yet caused a significant decrease in PXR responsiveness to Rif. Arsenite caused a large decrease in nuclear RXRalpha protein and, to a lesser extent, RXRalpha mRNA. These results suggest that arsenite inhibits both untreated and induced CYP3A4 transcription in primary human hepatocytes by decreasing the activity of PXR, as well as expression of the nuclear receptor RXRalpha.


Assuntos
Arsenitos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Hepatócitos/efeitos dos fármacos , Receptor X Retinoide alfa/antagonistas & inibidores , Adolescente , Adulto , Idoso , Sequência de Bases , Células Cultivadas , Criança , Pré-Escolar , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , Feminino , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética
14.
Hepatology ; 40(4): 942-50, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15382179

RESUMO

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.


Assuntos
Consumo de Bebidas Alcoólicas/genética , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Porfiria Cutânea Tardia/genética , 5-Aminolevulinato Sintetase/metabolismo , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Citocromo P-450 CYP1A2/metabolismo , Proteína da Hemocromatose , Ferro/metabolismo , Complexo Ferro-Dextran/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Porfiria Cutânea Tardia/etiologia , Porfiria Cutânea Tardia/metabolismo , Uroporfirinas/metabolismo
15.
Drug Metab Dispos ; 32(7): 681-4, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15205381

RESUMO

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.


Assuntos
Citocromo P-450 CYP2E1/metabolismo , Microssomos Hepáticos/metabolismo , Nitrofenóis/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2E1/biossíntese , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A , Indução Enzimática , Etanol/farmacologia , Feminino , Hidroxilação , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Microssomos Hepáticos/enzimologia , Oxirredutases N-Desmetilantes/biossíntese , Oxirredutases N-Desmetilantes/metabolismo , Pentanóis/farmacologia , Fatores Sexuais
16.
J Biochem Biophys Methods ; 59(2): 195-200, 2004 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-15163531

RESUMO

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.


Assuntos
Técnicas de Química Analítica/métodos , Ferro/análise , Animais , Heme , Ferro/química , Ferro/metabolismo , Camundongos , Fenantrolinas/química , Sensibilidade e Especificidade
17.
Toxicol Appl Pharmacol ; 196(3): 410-21, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15094312

RESUMO

To study liver toxicity and uroporphyrin (URO) accumulation and urinary excretion, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand for the aryl hydrocarbon receptor (AHR), is often used as the prototype. In this study, we asked the question how important is the role of CYP1A1 in causing TCDD toxicity. Using a single large intraperitoneal dose of TCDD (200 microg/kg) and following the response over an 8-week period, we found this dose: (a) was lethal in less than 4 weeks to Cyp1a1(+/+) males but not to Cyp1a1(-/-) males or to females of either genotype; (b) caused a wasting syndrome in Cyp1a1(+/+) but not Cyp1a1(-/-) mice; (c) resulted in thymic atrophy, regardless of gender or genotype; (d) decreased spleen size and caused leukocytopenia in males but not females of either genotype; (e) caused hepatocyte hypertrophy in Cyp1a1(+/+) more so than in Cyp1a1(-/-) mice; (f) increased intrahepatocyte lipids and total liver fat content in Cyp1a1(+/+) more than Cyp1a1(-/-) males and females; and (g) caused uroporphyria in Cyp1a1(+/+) males much more than Cyp1a1(+/+) females, or in Cyp1a1(-/-) mice. Contrary to Cyp1a2(-/-) knockout mice that exhibited 15 times less accumulation of TCDD in liver than Cyp1a1/1a2(+/+) wild-type mice, Cyp1a1(-/-) mice did not show this altered TCDD distribution-indicating that CYP1A2 but not CYP1A1 is the major hepatic TCDD-binding "sink". Our data demonstrate that CYP1A1 contributes to high-dose TCDD-induced toxicity, uroporphyria, and lethality.


Assuntos
Citocromo P-450 CYP1A1/fisiologia , Fígado/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Teratogênicos/toxicidade , Uroporfirinas/metabolismo , Síndrome de Emaciação/induzido quimicamente , Animais , Citocromo P-450 CYP1A1/genética , Feminino , Genótipo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Dibenzodioxinas Policloradas/antagonistas & inibidores , Dibenzodioxinas Policloradas/farmacocinética , Distribuição Tecidual , Síndrome de Emaciação/metabolismo
18.
J Biol Chem ; 278(50): 50771-80, 2003 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-14512429

RESUMO

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Heme/química , Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Western Blotting , Divisão Celular , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Ferroquelatase/química , Modelos Químicos , Oxigênio/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Tempo , Transativadores/química , Fatores de Transcrição
19.
Biochem Pharmacol ; 65(4): 545-50, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12566081

RESUMO

In experimental animals, CYP1A2 is absolutely required for the development of uroporphyria induced by treatment with polyhalogenated aromatic compounds or other compounds. Although the role of this CYP in clinical uroporphyria, porphyria cutanea tarda (PCT), is not clear, Cyp1a2(-/-) mice are resistant to the development of uroporphyria. Here, we compared the abilities of human and mouse CYP1A2 expressed in mouse hepatoma Hepa-1 cells to: (i) catalyze CYP1A2-dependent methoxyresorufin demethylase (MROD), and (ii) support uroporphyrin (URO) accumulation. Both CYP1A2 orthologs were expressed at similar levels as indicated by immunodetectable CYP1A2 proteins and MROD activities. URO accumulation was increased in cultures expressing either ortholog when supplemented with 5-aminolevulinic acid, the porphyrin precursor. Cells expressing mouse CYP1A2 produced more URO than cells expressing human CYP1A2. The results indicate that human CYP1A2 can support URO accumulation in hepatoma cells and thus may play a role in human PCT.


Assuntos
Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A2/fisiologia , Porfiria Cutânea Tardia/enzimologia , Uroporfirinas/metabolismo , Ácido Aminolevulínico/farmacologia , Animais , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Immunoblotting , Camundongos , Oxirredutases/metabolismo , Porfiria Cutânea Tardia/metabolismo , Transfecção , Células Tumorais Cultivadas
20.
Hepatology ; 37(2): 351-8, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12540785

RESUMO

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.


Assuntos
Etanol/farmacologia , Proteínas de Membrana/deficiência , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/metabolismo , Uroporfirinas/urina , 5-Aminolevulinato Sintetase/metabolismo , Animais , Citocromo P-450 CYP1A2/metabolismo , Modelos Animais de Doenças , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Ferro/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout/genética , Distribuição Tecidual
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