RESUMO
Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed by addition of dithiothreitol, Zn2+ or chelators of Fe2+ and Fe3+. Addition of FeSO4, ferric citrate, or ferritin to homogenates of control liver had no effect of activity of ALA dehydrase. The decrease in activity of ALA dehydrase, caused by iron-dextran, was mirrored by a reciprocal increase in ALA synthase. Iron-dextran potentiated the induction of ALA synthase by allylisopropylacetamide. However, this potentiation could be dissociated from the decrease in ALA dehydrase caused by iron loading.
Assuntos
Dextranos/farmacologia , Heme/biossíntese , Ferro/farmacologia , Fígado/enzimologia , Sintase do Porfobilinogênio/antagonistas & inibidores , 5-Aminolevulinato Sintetase/metabolismo , Alilisopropilacetamida/farmacologia , Ácido Aminolevulínico/metabolismo , Animais , Sinergismo Farmacológico , Masculino , Protoporfirinas/biossíntese , Ratos , Ratos Endogâmicos , Uroporfirinogênio Descarboxilase/metabolismoRESUMO
Cytochromes P-450IIB1 and P-450IIB2 were recently shown to be inducible in rat hepatocyte cultures maintained on a reconstituted extracellular tumor matrix (Matrigel) as indicated by increases in P-450IIB1 and -IIB2 mRNAs and immunoreactive proteins (J. Cell. Physiol., 134: 309-323, 1988). Here we show that treatment of cultured rat hepatocytes with phenobarbital and other compounds known to induce P-450IIB1/2 in vivo increased spectral cytochrome P-450, immunoreactive proteins, and benzyloxy- and pentoxy-resorufin dealkylases, activities known to be specific for cytochrome P-450IIB1/2. These increases were observed when cells were cultured on either Matrigel or collagen matrix in Williams E medium. Cytochrome P-450III was also increased by phenobarbital and dexamethasone on either matrix. Propoxycoumarin depropylase activity, which has been proposed as a specific activity catalyzed by cytochrome P-450III, was increased 3-4-fold more by treatment with 3-methylcholanthrene than by phenobarbital or dexamethasone. The activity catalyzed by P-450III could be distinguished from that catalyzed by other P-450 forms using the specific inhibitor triacetyloleandomycin. Benzoyloxyresorufin dealkylase was also increased in these cells by treatment with 2,4,5,2',4',5'-hexachlorobiphenyl, glutethimide, or mephenytoin. Treatment with phenobarbital or 2-allyl-2-isopropylacetamide slightly induced 5-aminolevulinate synthase activity. 5-Aminolevulinate synthase activity was slightly increased in cells treated with phenobarbital or 2-allyl-2-isopropylacetamide. Succinyl acetone also induced 5-aminolevulinate synthase activity and, in combination with either of the other two drugs, synergistically increased the enzyme activity regardless of whether cells were cultured on collagen or Matrigel. These results indicate that with simple and economical enzyme assays for holocytochrome P-450 and 5-aminolevulinate synthase, the rat hepatocyte culture system can be used for studies of the interrelationships between phenobarbital induction of cytochrome P-450 and heme metabolism.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Fígado/enzimologia , Animais , Células Cultivadas , Dexametasona/farmacologia , Indução Enzimática , Cinética , Fígado/efeitos dos fármacos , Masculino , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.
Assuntos
Sistema Enzimático do Citocromo P-450/análise , Fígado/efeitos dos fármacos , Mutagênicos , Quinolinas/toxicidade , Animais , Células Cultivadas , Embrião de Galinha , Citocromo P-450 CYP1A1 , Fígado/enzimologia , Masculino , Oxirredutases/análise , Ratos , Ratos Endogâmicos F344 , Uroporfirinogênios/metabolismoRESUMO
Cytochromes of the a-, b-, c- and d-type become reduced when intact cells of Hemophilus parainfluenzae have become anaerobic following respiration with substrates such as formate or succinate, as shown previously (J. Biol. Chem. (1970) 254, 5096-5100). In the presence of formate after depletion of O2, there is an unusual two-step time course of reduction of the membrane-bound cytochrome c. The proportion of the cytochrome c which is reduced during the second stage is oxidizable by either nitrate or H2O2 and is reduced again when the nitrate or H2O2 have been depleted. We conclude that the observed two-stage reduction of cytochrome c results from the presence of an oxidant, probably H2O2, produced by reaction of formate dehydrogenase with O2. This was shown by the effects of cyanide, catalase and O2. In addition, no evidence for the production of the oxidant is seen when succinate is the substrate oxidized. Although measurements of absorption spectra indicated only one species of cytochrome c, kinetic evidence is presented for some separation of the cytochrome c into more than one electron transport pathway.
Assuntos
Grupo dos Citocromos c/metabolismo , Haemophilus/metabolismo , Peróxido de Hidrogênio/farmacologia , Membranas/metabolismo , Nitratos/farmacologia , Consumo de Oxigênio , Sítios de Ligação , Cianetos/farmacologia , Citocromos/metabolismo , Formiatos/metabolismo , Congelamento , Oxirredução , Succinatos/metabolismoRESUMO
PURPOSE: To evaluate the efficacy of three hormonal manipulations in the palliation of chemoresistant ovarian cancer, and to analyze the results in the light of other clinical trials. PATIENTS AND METHODS: Three sequential phase II trials were performed in patients with refractory epithelial ovarian carcinoma, using high-dose megestrol acetate (800 mg/d for 30 days, then 400 mg/d), high-dose tamoxifen (80 mg/d for 30 days, then 40 mg/d), and aminoglutethimide (1 g/d plus tapering doses of hydrocortisone). Results were compared with those described in the world literature from trials of the same or similar agents. RESULTS: No responses were seen among 30 assessable patients treated with megestrol acetate, and most (but not all) similar trials have reported low response rates. Five responses (17%) were seen among 29 patients treated with tamoxifen. Two responses exceeded 5 years in duration. No responses were seen among 15 patients treated with aminoglutethimide. CONCLUSION: Antiestrogen therapy may offer the possibility of useful and, occasionally, long-term palliation of refractory epithelial ovarian carcinoma, with little toxicity. There may be a trend toward a dose-response effect, which represents a suitable topic for a future prospective trial.
Assuntos
Aminoglutetimida/uso terapêutico , Megestrol/análogos & derivados , Neoplasias Ovarianas/tratamento farmacológico , Cuidados Paliativos , Tamoxifeno/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Carcinoma/tratamento farmacológico , Resistência a Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Hidrocortisona/uso terapêutico , Megestrol/uso terapêutico , Acetato de MegestrolRESUMO
Uroporphyrinogen oxidation by hepatic microsomes from chick embryos or mice pretreated with methylcholanthrene was increased by addition of iron-EDTA. This increase was partially prevented by catalase, mannitol, ketoconazole and piperonyl butoxide, whereas only ketoconazole and piperonyl butoxide inhibited the oxidation in the presence and absence of iron-EDTA. These data suggest that the oxidations of uroporphyrinogen in the presence and absence of added iron occur by different mechanisms.
Assuntos
Ácido Edético/farmacologia , Compostos Férricos/farmacologia , Quelantes de Ferro/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Porfirinogênios/metabolismo , Uroporfirinogênios/metabolismo , Animais , Catalase/metabolismo , Embrião de Galinha , Cetoconazol/farmacologia , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxirredução , Butóxido de Piperonila/farmacologia , Bifenilos Policlorados/farmacologia , Superóxido Dismutase/metabolismoRESUMO
Uroporphyrinogen decarboxylase activity was investigated in cultures of chick embryo liver by two different methods: (1) analysis of porphyrin composition following incubation of intact cells with delta-aminolevulinic acid; and (2) a more conventional direct enzymic assay of cell homogenates. Activity was detectibly decreased following exposure of cells to 100 ng/ml 2,4,5,3',4'-pentabromobiphenyl using the first method, but not the second. This decrease in activity was reversed by homogenizing the cells treated with 100 ng/ml pentabromobiphenyl. It is concluded that the direct homogenate assay of the enzyme may miss or underestimate decreases in its in vivo activity.
Assuntos
Compostos de Bifenilo/farmacologia , Carboxiliases/metabolismo , Fígado/enzimologia , Bifenil Polibromatos/farmacologia , Uroporfirinogênio Descarboxilase/metabolismo , Ácido Aminolevulínico/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Fígado/efeitos dos fármacos , Porfirinas/metabolismoRESUMO
Seizures may occur in acute intermittent porphyria or other hepatic porphyrias. Management is difficult, because barbiturates and hydantoins exacerbate the porphyric state. We studied one patient with major motor seizures and acute intermittent porphyria. The seizure disorder was exacerbated by phenytoin and did not respond to a high-carbohydrate diet or to intravenous hematin. Clonazepam was ineffective in treating the seizures and, in high doses, seemed to exacerbate the porphyria. Both clonazepam and valproate were porphyrinogenic in experimental test systems. Because both drugs may exacerbate the acute hepatic porphyrias, bromide remains the drug of choice to treat these seizures.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Benzodiazepinonas/uso terapêutico , Clonazepam/uso terapêutico , Hepatopatias/tratamento farmacológico , Porfirias/tratamento farmacológico , Convulsões/tratamento farmacológico , Ácido Valproico/uso terapêutico , Adulto , Ácido Aminolevulínico/metabolismo , Animais , Embrião de Galinha , Sistema Enzimático do Citocromo P-450/metabolismo , Hemina/farmacologia , Humanos , Masculino , Porfobilinogênio/metabolismo , RatosRESUMO
This study investigated the effects of estrogens on the induction of cytochrome P-450 by polycyclic aromatic hydrocarbons in primary cultures of chick embryo hepatocytes. Exposure to polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene led to 2- to 3-fold increases of cytochrome P-450. The amount of cytochrome P-450 induced by 3-methylcholanthrene was increased 40-50% when the synthetic estrogen, 17 alpha-ethynylestradiol, was also present. The rate of decay of cytochrome P-450 in the presence of cycloheximide as measured spectrophotometrically was similar in cells previously treated with either 3-methylcholanthrene or 3-methylcholanthrene plus 17 alpha-ethynylestradiol, suggesting that 17 alpha-ethynylestradiol did not affect the stability of the 3-methylcholanthrene-induced cytochrome P-450. In contrast, 17 alpha-ethynylestradiol did not potentiate the induction of cytochrome P-450 by phenobarbital-like inducers, such as 2-propyl-2-isopropylacetamide, as indicated by a lack of increase in both the content of cytochrome P-450 and benzphetamine demethylase activity. The naturally occurring estrogens, 17 beta-estradiol and estrone, and the synthetic estrogen, diethylstilbestrol, did not affect cytochrome P-450 induction by 3-methylcholanthrene, suggesting that the effect of 17 alpha-ethynylestradiol was not mediated via the estrogen receptor. We investigated whether the amount of cytochrome P-450 increased in the presence of 17 alpha-ethynylestradiol was the same or different from that induced by 3-methylcholanthrene. Treatment with 17 alpha-ethynylestradiol alone resulted in a small increase in ethoxyresorufin deethylase activity. The enzymatic activities of 7-ethoxyresorufin and aryl hydrocarbon hydroxylase, when expressed per cytochrome P-450 content, were identical in microsomes from cells treated with either 3-methylcholanthrene or the combination of 3-methylcholanthrene and 17 alpha-ethynylestradiol. The data suggest that the additional cytochrome P-450 induced by the combination of 17 alpha-ethynylestradiol and 3-methylcholanthrene was the same isozyme as that induced by 3-methylcholanthrene alone.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Etinilestradiol/farmacologia , Metilcolantreno/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/análise , Células Cultivadas , Embrião de Galinha , Citocromo P-450 CYP1A1 , Sistema Enzimático do Citocromo P-450/análise , Dexametasona/farmacologia , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Fígado/metabolismo , Metilcolantreno/metabolismo , Oxirredutases/análise , Porfirias/induzido quimicamente , Receptores de Hidrocarboneto Arílico , Receptores de Droga/metabolismoRESUMO
In this study, using a combination of immunological and enzymatic characterizations, we compared the forms of cytochrome P-450 induced by ethanol and glutethimide in primary cultures of chicken embryo hepatocytes. Recently we purified a cytochrome P-450 of 50K molecular weight from chicken embryo liver using glutethimide as a prototypic inducer. Antibodies to both this chicken cytochrome P-450 and to rabbit cytochrome P-450 form 3a from the IIE subfamily detected microsomal proteins of 50K induced by either ethanol or glutethimide in cultured chick embryo hepatocytes, indicating the antigenic homology of these subfamilies of cytochromes P-450 among different animal species. However, the antibody to glutethimide-induced chick cytochrome P-450 of 50K inhibited p-nitrophenol hydroxylase and benzphetamine demethylase activities 85-90% in microsomes from both ethanol- and glutethimide-treated cells, indicating similar epitopes whose integrity is required for catalytic activity. In contrast, antibodies to rabbit cytochrome P-450 form 3a had little to no effect on these same microsomal activities. Both ethanol and glutethimide induced microsomal p-nitrophenol and aniline hydroxylase activities in cultured chick embryo hepatocytes. In microsomes from ethanol-treated cells, the turnover of p-nitrophenol per cytochrome P-450 was 2-fold greater than that induced by glutethimide treatment, suggesting that ethanol is inducing a form of cytochrome P-450 that has greater catalytic activity with this substrate than glutethimide-induced forms. Thus, in cultured chick embryo hepatocytes, ethanol may induce cytochromes P-450 from both the IIB and IIE subfamilies.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Etanol/farmacologia , Glutetimida/farmacologia , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Cinética , Peso Molecular , Oxirredutases N-Desmetilantes/metabolismo , Especificidade por SubstratoRESUMO
Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.
Assuntos
5-Aminolevulinato Sintetase/antagonistas & inibidores , Cobalto/metabolismo , Fígado/metabolismo , Porfirinas/metabolismo , Protoporfirinas/metabolismo , Animais , Embrião de Galinha , Cobalto/farmacologia , Dicarbetoxi-Di-Hidrocolidina/farmacologia , Ferroquelatase/análise , Técnicas In VitroRESUMO
The amount of cytochrome P-450 was increased to different extents after treatment of cultured chick embryo hepatocytes with n-propanol, isopropanol, n-butanol, or isobutanol. These increases were associated with increases in benzphetamine demethylase activity, a cytochrome P-450-catalyzed oxidation, and glucuronidation of phenol red, catalyzed by UDP-glucuronyl transferase. The responses were similar to those obtained with ethanol or propylisopropylacetamide, which the phenobarbital-like inducers. Pretreatment of cells with cycloheximide prevented the increases in both cytochrome P-450 and glucuronidation of phenol red, indicating that protein synthesis was required for these responses.
Assuntos
Álcoois/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Alilisopropilacetamida/análogos & derivados , Alilisopropilacetamida/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Cicloeximida/farmacologia , Oxigenases de Função Mista/metabolismo , Fenolsulfonaftaleína/farmacologiaRESUMO
Exposure of cultured chick embryo hepatocytes to ethanol, isobutanol, or isopentanol, the predominant alcohols present in commercial alcoholic beverages, resulted in increased metabolism of aminopyrine or biphenyl by the intact cells. The increases correlated with induction of cytochrome P-450. Hydroxylation of biphenyl at the 4-position was preferentially increased in cells pretreated with either the alcohols or propylisopropylacetamide, a barbiturate-like inducer of cytochrome P-450. In contrast, exposure of the cells to 3,4,3',4'-tetrachlorobiphenyl, a planar polycyclic aromatic hydrocarbon inducer of P-450, resulted in preferential increased hydroxylation at the 2- and 3-positions of biphenyl.
Assuntos
Álcoois/farmacologia , Aminopirina/metabolismo , Compostos de Bifenilo/metabolismo , Fígado/metabolismo , Alilisopropilacetamida/análogos & derivados , Alilisopropilacetamida/farmacologia , Animais , Butanóis/farmacologia , Células Cultivadas , Embrião de Galinha , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Etanol/farmacologia , Fígado/enzimologia , Pentanóis/farmacologia , Bifenilos Policlorados/farmacologiaRESUMO
Cellular glutathione concentrations in primary cultures of chick embryo hepatocytes were 15.3 +/- 5.3 nmoles/mg protein (mean +/- S.D.) and remained stable for up to 3 days in culture. The presence of insulin was not essential for the maintenance of glutathione concentrations. Induction of cytochrome P-450 by phenobarbital-like inducers (2-propyl-2-isopropylacetamide, 2-allyl-2-isopropylacetamide, and 2,4,5,2',4',5'-hexabromobiphenyl) was accompanied by 2- to 3-fold increases in glutathione concentrations and by increased glucuronidation of phenol red. The 3-methylcholanthrene-like inducers of cytochrome P-450 (beta-naphthoflavone and 3,4,3',4'-tetrachlorobiphenyl) did not have these effects. Glutathione was rapidly depleted to 15-30% of control levels in hepatocytes treated with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthase. No toxicity was observed with glutathione depletion. Glutathione depletion did not affect the ability of 2-propyl-2-isopropylacetamide to induce cytochrome P-450, glucuronidation of phenol red, or delta-aminolevulinate synthase.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Glutationa/análise , Fígado/análise , Alilisopropilacetamida/análogos & derivados , Alilisopropilacetamida/farmacologia , Animais , Butionina Sulfoximina , Células Cultivadas , Embrião de Galinha , Indução Enzimática , Glucuronatos/metabolismo , Insulina/farmacologia , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , Metilcolantreno/farmacologia , Fenolsulfonaftaleína/metabolismoRESUMO
In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.
Assuntos
Bilirrubina/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Icterícia/metabolismo , Animais , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/deficiência , Citocromo P-450 CYP1A2/genética , Modelos Animais de Doenças , Complexo Ferro-Dextran/farmacologia , Icterícia/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Bifenilos Policlorados/farmacologia , Dibenzodioxinas Policloradas/toxicidade , Ratos , Ratos Gunn , Ratos Wistar , Teratogênicos/toxicidadeRESUMO
Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Etanol/administração & dosagem , Pentanóis/administração & dosagem , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Interações Medicamentosas , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Necrose , Ratos , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Previous work has implicated CYP1A2 in experimental uroporphyria caused by polyhalogenated aromatic compounds, and in uroporphyria caused by iron and 5-aminolevulinate (ALA) in the absence of inducers of CYP1A2. Here we examined whether the different susceptibilities of SWR and C57BL/6 strains of mice to uroporphyria in the absence of inducers of CYP1A2 are related to different levels of CYP1A2. Enzymological assays (ethoxy- and methoxyresorufin dealkylases, and uroporphyrinogen oxidation) and immunoblots indicated that there was about twice the amount of hepatic CYP1A2 in SWR mice compared with C57BL/6 mice. Immunohistochemistry revealed that CYP1A2 was located centrilobularly in the liver, and the staining was more intense in SWR mice than in C57BL/6 mice. Hepatic non-heme iron was about double in SWR compared with C57BL/6 mice. In SWR mice given iron dextran, hepatic iron was 1.7-fold that of C57BL/6 mice given iron dextran. SWR mice administered ALA in the drinking water accumulated much less hepatic protoporphyrin than did C57BL/6 mice. To confirm the importance of small increases in CYP1A2, C57BL/6 mice were given a low dose of 3-methylcholanthrene (MC) (15 mg/kg), as well as iron and ALA. There was about a 5- to 6-fold increase in hepatic uroporphyrin accumulation after 32 days on ALA compared with animals not given MC. In these animals, CYP1A2 was increased by 10-fold at 2 days, but returned to basal levels by 14 days. We conclude that small and transient differences in CYP1A2 may be important in the development of uroporphyria.
Assuntos
Ácido Aminolevulínico/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Ferro/farmacologia , Fígado/enzimologia , Uroporfirinas/urina , Animais , Citocromo P-450 CYP1A2/análise , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie , Uroporfirinas/metabolismoRESUMO
Ethanol and isopentanol are the predominant alcohols in alcoholic beverages. We have reported previously that pretreatment of rats with a liquid diet containing 6.3% ethanol plus 0.5% isopentanol for 7 days results in a synergistic increase in acetaminophen hepatotoxicity, compared with rats treated with either alcohol alone. Here, we investigated the role of CYP3A in acetaminophen hepatotoxicity associated with the combined alcohol treatment. Triacetyloleandomycin, a specific inhibitor of CYP3A, protected rats pretreated with ethanol along with isopentanol from acetaminophen hepatotoxicity. At both 0.25 and 0.5 g acetaminophen/kg, triacetyloleandomycin partially prevented elevations in serum levels of alanine aminotransferase. At 0.25 g acetaminophen/kg, triacetyloleandomycin completely protected 6 of 8 rats from histologically observed liver damage, and partially protected the remaining 2 rats. At 0.5 g acetaminophen/kg, triacetyloleandomycin decreased histologically observed liver damage in 7 of 15 rats. In rats pretreated with ethanol plus isopentanol, CYP3A, measured immunohistochemically, was decreased by acetaminophen treatment. This effect was prevented by triacetyloleandomycin. These results suggest that CYP3A has a major role in acetaminophen hepatotoxicity in animals administered the combined alcohol treatment. We also found that exposure to ethanol along with 0.1% isopentanol for only 3 days resulted in maximal increases in acetaminophen hepatotoxicity by the combined alcohol treatment, suggesting that short-term consumption of alcoholic beverages rich in isopentanol may be a risk for developing liver damage from acetaminophen.
Assuntos
Acetaminofen/toxicidade , Hidrocarboneto de Aril Hidroxilases , Doença Hepática Induzida por Substâncias e Drogas , Etanol/farmacologia , Pentanóis/farmacologia , Troleandomicina/farmacologia , Analgésicos não Narcóticos/toxicidade , Animais , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Etanol/administração & dosagem , Hepatopatias/enzimologia , Hepatopatias/patologia , Hepatopatias/prevenção & controle , Masculino , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/metabolismo , Pentanóis/administração & dosagem , Substâncias Protetoras/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
Cultured chick hepatocytes were used to investigate the hepatotoxicity of methotrexate alone and in combination with paracetamol. Treatment with methotrexate alone at concentrations as high as 1 mg/ml resulted in no toxicity in cultured chick hepatocytes, as indicated by no detachment of cells and no effect on protein synthesis or on release of the intracellular enzyme lactate dehydrogenase. However, treatment with methotrexate alone resulted in a 30% decrease in reduced glutathione levels. Combined treatment with methotrexate and paracetamol was toxic, but only in cells preinduced for cytochrome P450 1A by treatment with beta-naphthoflavone. Under these conditions, methotrexate lowered the threshold concentration of paracetamol at which toxicity was observed. This methotrexate-mediated increase in paracetamol toxicity was associated with decreased formation of the glucuronide, sulfate and thiol metabolites of paracetamol and with increased covalent binding of radiolabeled paracetamol to macromolecules. In cells pretreated with beta-naphthoflavone, additional treatment with either methotrexate or buthionine sulfoximine, an inhibitor of glutathione synthesis, together with paracetamol, was associated with decreased restoration of glutathione levels. These results suggest that methotrexate increased paracetamol toxicity by decreasing the amount of glutathione available for conjugation with reactive metabolites of paracetamol.
Assuntos
Acetaminofen/toxicidade , Fígado/efeitos dos fármacos , Metotrexato/toxicidade , Acetaminofen/metabolismo , Animais , Benzoflavonas/farmacologia , Butionina Sulfoximina , Células Cultivadas , Embrião de Galinha , Sistema Enzimático do Citocromo P-450/metabolismo , Sinergismo Farmacológico , Indução Enzimática/fisiologia , Glutationa/metabolismo , Fígado/citologia , Fígado/metabolismo , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , beta-NaftoflavonaRESUMO
The formation of zinc protoporphyrin in response to lead or iron depletion has previously been investigated in erythroid systems. Because of its possible metabolic role in non-erythroid tissue, we investigated the formation of zinc protoporphyrin in cultured hepatocytes. The effects of lead and inhibitors of ferrochelatase, the iron insertion step of heme synthesis, on the conversion of 5-aminolevulinic acid to zinc protoporphyrin, protoporphyrin and heme were compared in rat and chick embryo hepatocyte cultures. In rat cultures, zinc protoporphyrin was synthesized enzymatically by ferrochelatase, since N-methylmesoporphyrin, an inhibitor of ferrochelatase. caused 40% or greater decreases in both heme and zinc protoporphyrin accumulation and markedly stimulated protoporphyrin accumulation. In addition, chelation of ferrous iron with 2,2'-dipyridyl decreased heme accumulation by 50%, but increased ZPP accumulation by 200%. Zinc protoporphyrin formation in chick embryo hepatocytes required the addition of zinc as well as 5-aminolevulinic acid and apparently was non-enzymatic, since it was not inhibited by N-methylmesoporphyrin nor increased by iron chelation. In the presence of 5-aminolevulinic acid, lead had no effect on zinc protoporphyrin, protoporphyrin or heme accumulation in chick hepatocytes, but decreased all three in rat hepatocytes, with the decrease in protoporphyrin being far greater than that of zinc protoporphyrin or heme. These findings indicate that, in contrast to the effect of lead in erythroid tissue, it did not specifically increase zinc protoporphyrin accumulation or alter iron availability in cultured hepatocytes.