RESUMO
Fluorescein-isothiocyanate dextran (FITC-dextran), a dye confined to the vascular space, was infused via the hepatic artery and portal vein into perfused livers from fed rats treated with diethylnitrosamine for 4 to 5 months. Fluorescence due to FITC-dextran was detected with fiberoptic microlight guides placed on surface nodules of about 5 mm in diameter. Nodules were categorized into groups with normal and compromised microcirculation based on their fluorescence following infusion of FITC-dextran. Similar results were obtained when nodules were classified based on reflectance of trypan blue. Despite compromised microcirculation, ATP and ADP levels as well as ATP/ADP ratios were comparable in both groups of nodules; however, AMP was elevated in FITC-dextran-negative nodules (i.e., those with compromised microcirculation). Nodules with compromised microcirculation also contained higher glucose and lactate levels than nodules that were well perfused; however, glycogen was five times lower than in FITC-dextran-positive nodules. Fasting reduced ATP/ADP ratios in poorly perfused nodules in comparison to well-perfused nodules. In perfused livers from fed rats where glycogen was high, however, ATP/ADP ratios and rates of ATP depletion during ischemia were the same in well-perfused and poorly perfused nodules. Products of glycogen breakdown (e.g., glucose and lactate) were elevated in nodules from livers of fed but not fasted rats. The results indicate that alteration of perfusion of hepatic nodules does not change ATP levels nor the capacity of nodules to utilize high energy phosphate during anoxia. Thus, near normal energy status is maintained from glycogen metabolism in poorly perfused nodules via glycolysis. Since basal ATP content and utilization is comparable in well and poorly perfused nodules, compromised energy status is unlikely to explain selection of nodules that regress to near normal hepatocytes.
Assuntos
Nucleotídeos de Adenina/metabolismo , Metabolismo dos Carboidratos , Fluoresceína-5-Isotiocianato/análogos & derivados , Circulação Hepática , Neoplasias Hepáticas Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Dextranos , Dietilnitrosamina , Fluoresceínas , Corantes Fluorescentes , Glucose/metabolismo , Lactatos/metabolismo , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Masculino , Microcirculação/fisiopatologia , Lesões Pré-Cancerosas/irrigação sanguínea , Lesões Pré-Cancerosas/induzido quimicamente , Ratos , Ratos Endogâmicos F344 , Espectrometria de FluorescênciaRESUMO
The biotransformation of selenite to dimethylselenide was studied in an oxygenated hepatocyte model system. The concentrations of selenite used were 20-100 microM. A lag period of one hour or more, during which no net formation of selenide could be detected characterized the system. The maximal rate of volatilization was recorded during the second hour and was 0.13 nmoles/10(6) cells/min with 50 microM selenite. The rate then declined and volatilization eventually ceased. Two-thirds of the added amount of Se was lost within 4 hr. Oxidation of glutathione (GSH) by cumene hydroperoxide delayed volatilization. An inhibitor of gluconeogenesis, p-tert-butylbenzoic acid (3 microM) prevented volatilization. There were indications that GSSG reductase dependent metabolism was the only major metabolic pathway in hepatocytes under the conditions studied. During the lag period Se accumulated in cells, but was subsequently partially released during volatilization. The accumulation of Se was paralleled by an increase in oxygen uptake. The above mentioned inhibitors of volatilization prolonged the phase of accumulation. With 50 microM selenite the rate of accumulation was 0.06 nmoles/10(6) cells/min and maximally 30-35% of the added dose was retained in the cells. The results are compatible with the assumption that Se mainly accumulated as Se-glutathione complexes. The possibility that such complexes autooxidized and entered futile redox cycles during the lag period is discussed.
Assuntos
Fígado/metabolismo , Selênio/metabolismo , Animais , Biotransformação , Glucose/farmacologia , Glutationa/análise , Glutationa Redutase/análise , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Ácido Selenioso , VolatilizaçãoRESUMO
To study the mechanism of centrilobular damage developing in the centrilobular region after high doses of acetaminophen (APAP), its metabolism and toxicity were compared in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. Contrary to earlier reports, based on perfusions, no evidence for a periportal dominance of APAP sulfation could be observed. Glucuronidation, the dominant pathway of conjugation at high (5 mM) APAP concentration, was faster in perivenous cells. During primary culture, prolonged exposure (> or = 24 hr) to 5 mM APAP damaged perivenous cells, with a higher P450 2E1 level than periportal cells. When cells were isolated from ethanol-pretreated rats, to induce P450 2E1 levels specifically in the perivenous region, perivenous hepatocytes exhibited enhanced APAP vulnerability and extensive glutathione depletion. In contrast, corresponding periportal cells retained good viability. Isoniazid, an inhibitor of cytochrome P450 2E1, protected cells against APAP toxicity and prevented glutathione depletion. Induction of P450 2E1 also caused a 3-fold increase in the covalent binding of reactive intermediates from [14C]APAP, and this increase was mainly confined to perivenous cells. These results indicate that in rat liver there is only slight perivenous zonation of APAP conjugation and suggest that zone-specific APAP activation, mediated by the regional expression of ethanol-inducible cytochrome P450 2E1, is responsible for the characteristic centrilobular liver damage elicited by APAP.
Assuntos
Acetaminofen/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Acetaminofen/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Etanol , Glutationa/metabolismo , Fígado/irrigação sanguínea , Circulação Hepática , Masculino , Oxirredutases N-Desmetilantes/biossíntese , Ratos , Ratos WistarRESUMO
Chloroform and selenite toxicity have been studied in isolated hepatocytes. Two different toxic mechanisms, which lead to cellular lysis after distinct lag periods, are compared. Chloroform toxicity can be divided into 2 phases, a first phase characterized by chloroform metabolism and a second phase characterized by lipid peroxidation. GSH depletion during the first phase is claimed to be a prerequisite for lipid peroxidation in the second phase. Selenite metabolism leads to cofactor depletion as well. Selenite reduction via a GSH reductase dependent pathway exhausts the cells of NADPH and this effect can be related to cellular lysis.
Assuntos
Clorofórmio/metabolismo , Fígado/metabolismo , Selênio/metabolismo , Animais , Células Cultivadas , Clorofórmio/toxicidade , Glutationa/metabolismo , Ratos , Selênio/toxicidadeRESUMO
Isolated hepatocytes incubated with selenite (30-100 microM) exhibited changes in the glutathione redox system as shown by an increase in O2 consumption, oxidation of glutathione and loss of NADPH. Selenite (50 microM) raised O2 consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 microM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl2 and KCN prevented the increase in O2 uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O2 consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O2 consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O2 in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 microM) there were indications that O2 reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 microM) it appeared that O2 reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite.
Assuntos
Benzoatos , Fígado/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Selênio/toxicidade , Animais , Benzoatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Glutationa/metabolismo , Técnicas In Vitro , Fígado/metabolismo , Masculino , NADP/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Ácido Selenioso , Selênio/metabolismoRESUMO
The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl4. In contrast, the mere presence of low concentrations (25-50 microM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.
Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Fígado/efeitos dos fármacos , Oxirredutases N-Desmetilantes/efeitos dos fármacos , Silimarina/farmacologia , 1-Propanol/farmacologia , Animais , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Glutationa/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Masculino , Oxirredutases N-Desmetilantes/metabolismo , Peróxidos/farmacologia , Propanóis , Ratos , Ratos Wistar , terc-Butil HidroperóxidoRESUMO
The effects of the hepatoprotective compound silymarin on hepatocytes in primary culture were studied. Exposure of cells in primary culture (both conventional cells and perivenous or periportal cells isolated by the digitonin-collagenase perfusion technique) to high concentrations of silymarin surprisingly demonstrated that silymarin per se was cytotoxic. Incubation of cells for 18 hr with silymarin at concentrations exceeding 25 mum abruptly increased cell damage, whereas viability decreased in a more linear fashion with increasing concentrations of its major constituent, silybin. Morphologically, cell cultures exposed to silymarin concentrations lower than 20 mum appeared normal, but at higher concentrations intercellular contacts were lost; cells appeared granulated and took up eosin. Silymarin and silybin at these doses were also found to prevent cell attachment. The mechanism responsible for this effect at relatively low concentrations of silymarin during prolonged exposure in the primary cell culture system is not clear at present. The effects of low doses on cell attachment to the matrix suggest an action on the cell membrane and/or on the cytoskeleton.
RESUMO
The flavonoid silymarin and its main active component silybin have been used in the treatment of toxic liver diseases. In order to evaluate the hepatoprotective potency of both these compounds, their effects on the viability, lipid peroxidation and reduced glutathione (GSH) depletion induced by allyl alcohol (AA) and tert-butyl hydroperoxide (t-BuOOH) in suspensions of isolated hepatocytes were investigated. Cells were preincubated for 30 min with silymarin and silybin before the addition of AA or t-BuOOH. Samples were taken after 1-2 hr of incubation. Cell death, after 2 hr of incubation with 0.2 mm AA, was prevented by 0.01 mm silymarin; however, 2 mm silybin was required to give comparable protection. The presence of silymarin reduced AA-induced lipid peroxidation by more than 90%, whereas silybin was much less effective. The near-complete depletion of intracellular GSH by AA was restored in a dose-dependent manner by silymarin, but silybin did not have this effect. Protection by silymarin against the toxic effects of t-BuOOH was less pronounced than that against AA. In conclusion, silymarin was much more effective than silybin in preventing the toxic effects induced by two pro-oxidant toxins, AA and t-BuOOH.
Assuntos
Arsênio/farmacologia , Arsenitos , Bile/efeitos dos fármacos , Glutationa/metabolismo , Animais , Bile/metabolismo , Glutationa/análogos & derivados , Dissulfeto de Glutationa , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Viability of isolated hepatocytes was not significantly dependent on oxygen at oxygen partial pressures (Po2) from 70 to 0.3 mmHg. The critical Po2 for induction of hypoxic cell death was close to 0.1 mmHg and was distinct from the value at which mitochondrial function becomes impaired (2-5 mmHg). Hypoxic damage in hepatocytes from fasted rats occurred within 1 h but was delayed by the addition of fructose, which increased rates of lactate formation from about 1 to 12 nmol.10(6) cells-1.min-1. Hepatocytes from fed rats maintained viability for almost 180 min of anaerobic incubation and then rapidly became damaged. Addition of fructose prevented hypoxic cell damage also in these hepatocytes. Rates of lactate formation were 11-15 nmol.10(6) cells-1.min-1 and were increased two- to threefold by fructose. The rapid initial degradation of glycogen and the release of glucose were delayed with fructose, which also could have contributed to sustained glycolytic rates. Further, under conditions in which lactate production was high, e.g., in the fed state, there was also a significantly better preservation of cellular ATP levels.
Assuntos
Glicólise , Fígado/citologia , Oxigênio/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular , Jejum , Alimentos , Frutose/farmacologia , Glucose/metabolismo , Glicogênio/metabolismo , Cinética , Lactatos/metabolismo , Ácido Láctico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Oxigênio/administração & dosagem , Ratos , Ratos EndogâmicosRESUMO
The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isolated from the periportal or perivenous region by digitonin-collagenase perfusion. Exposure of hepatocytes in culture to NDMA (0.5 or 5 mM) for up to 18 hrs caused little damage, but after 42 hr loss of cell viability became evident, and the extent of cell death was higher in perivenous cells than in periportal cells. Pretreatment of rats with ethanol caused a dramatically enhanced cell damage in perivenous cells (80%) compared to periportal cells (45%). This ethanol pretreatment caused a several-fold induction of cytochrome P450 2E1, as determined both with Western blot and as NDMA demethylase activity, and the effect was observed almost exclusively in perivenous cells. Isoniazid, an inhibitor of cytochrome P450 2E1, completely protected against NDMA toxicity. Glutathione dependent cytoprotective mechanisms and lipid peroxidation did not appear to be critical in NDMA toxicity, as evidence by lack of potentiation of toxicity by buthionine sulfoximine, an inhibitor of glutathione synthesis, and by the absence of increased lipid peroxidation. Instead, the higher expression of cytochrome P450 2E1 in the perivenous cells seems to be the main determinant for the regiospecific toxicity of NDMA, and, consequently, probably also for the associated genotoxicity.
Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Dimetilnitrosamina/toxicidade , Etanol/farmacologia , Fígado/enzimologia , Oxirredutases N-Desmetilantes/efeitos dos fármacos , Animais , Biotransformação , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Dimetilnitrosamina/farmacocinética , Glutationa/metabolismo , Peroxidação de Lipídeos , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , Masculino , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/fisiologia , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Tiobarbitúricos/metabolismo , Fatores de TempoRESUMO
Cellular lysis in freshly isolated hepatocytes incubated with varying concentrations of selenite could be related to the reductive metabolism of selenite. A decrease in intracellular GSH levels was observed concomitant with an increased rate of accumulation of oxidized glutathione in the incubation medium. Pretreatment of hepatocytes with an inhibitor of GSSG-reductase (1,3-bis(2-chloroethyl)-1-nitrosourea), prior to the addition of 50 microM selenite, resulted in substantially lower GSH-levels. The rate of GSSG reductase-catalyzed metabolism of selenite (50 microM) could be estimated to approximately 7 nmoles reduced/h per 10(6) cells. The results indicate that this was the major metabolic pathway for toxic concentrations of selenite in isolated hepatocytes. Furthermore, selenite considerably decreased cellular NADPH levels. In hepatocytes isolated from starved rats, the presence of alanine and glucose in the incubation medium protected against selenite-mediated cellular lysis. These observations suggest that an insufficient NADPH generation could be critical for selenite reduction and toxicity in isolated hepatocytes.
Assuntos
Glutationa Redutase/metabolismo , Fígado/metabolismo , Selênio/toxicidade , Animais , Permeabilidade da Membrana Celular , Glutationa/metabolismo , Técnicas In Vitro , Fígado/citologia , Masculino , Metionina/farmacologia , NADP/metabolismo , Ratos , Ratos Endogâmicos , Ácido Selenioso , Selênio/metabolismoRESUMO
Incubation of isolated rat intestinal epithelial cells with selenite resulted in a rapid decrease of intracellular glutathione (GSH) and about 5 nmol of Se (added as 75Se labelled selenite, 50 microM) was accumulated per 10(6) cells during 20 min. Addition of exogenous GSH enhanced cellular accumulation of Se, while the gamma-glutamyl transferase inhibitor, serine-borate, decreased uptake. The added exogenous GSH was rapidly consumed (oxidized) by selenite. Depletion of GSH by diethylmaleate (DEM) inhibited selenite uptake both in in situ isolated intestinal loop, everted gut sacs and in isolated intestinal cells. The findings indicate that both intracellular and extracellular GSH, and gamma-glutamyltransferase, play a role in the absorption of selenite in the intestine.
Assuntos
Glutationa/metabolismo , Absorção Intestinal , Intestino Delgado/metabolismo , Selênio/metabolismo , Animais , Interações Medicamentosas , Células Epiteliais , Epitélio/metabolismo , Técnicas In Vitro , Intestino Delgado/citologia , Líquido Intracelular/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Ácido Selenioso , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
Reoxygenation following hypoxia enhanced loss of viability of isolated hepatocytes compared to cells maintained under hypoxic conditions. Cell damage due to reoxygenation was not dependent on the conversion of xanthine dehydrogenase to xanthine oxidase which occurred at a time when almost all the hepatocytes had lost their viability. The effect of reoxygenation was critically linked to the duration of the hypoxic period. During the hypoxic period degradation of endogenous glycogen may have provided sufficient substrate for glycolysis to contribute to maintenance of cell integrity by preservation of adenine nucleotides.
Assuntos
Fígado/patologia , Aerobiose , Anaerobiose , Animais , Sobrevivência Celular , Células Cultivadas , Hipóxia/patologia , Fígado/citologia , Fígado/enzimologia , Masculino , Ratos , Ratos Endogâmicos , Xantina Oxidase/metabolismoRESUMO
Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected. Rates of glucose and lactate production from 4 mM fructose were around 100 and 75 mumol/g/h, respectively. Rates of fructose uptake were around 190 mumol/g/h when 4 mM fructose was infused. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, decreased glucose formation from fructose maximally by 20% suggesting that a fraction of the lactate formed from fructose is used for glucose synthesis. A good correlation (r = 0.92) between extra oxygen consumed and glucose produced from fructose was observed. At low fructose concentrations (less than 0.5 mM), the extra oxygen uptake was much greater than could be accounted for by glucose synthesis possibly reflecting fructose 1-phosphate accumulation. Furthermore, fructose diminished ATP/ADP ratios from about 4.0 to 2.0 in periportal and pericentral regions of the liver lobule indicating that the initial phosphorylation of fructose via fructokinase occurs in both regions of the liver lobule. Basal rates of oxygen uptake measured with miniature oxygen electrodes were 2- to 3-fold higher in periportal than in pericentral regions of the liver lobule during perfusions in the anterograde direction. Infusion of fructose increased oxygen uptake by 65 mumol/g/h in periportal areas but had no effect in pericentral regions of the liver lobule indicating higher local rates of gluconeogenesis in hepatocytes located around the portal vein. When perfusion was in the retrograde direction, however, glucose was synthesized nearly exclusively from fructose in upstream, pericentral regions. Thus, gluconeogenesis from fructose is confined to oxygen-rich upstream regions of the liver lobule in the perfused liver.
Assuntos
Frutose/metabolismo , Gluconeogênese , Fígado/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas In Vitro , Cinética , Fígado/anatomia & histologia , Circulação Hepática , Masculino , Consumo de Oxigênio , Perfusão , Ratos , Ratos EndogâmicosRESUMO
The effects of glutathione depletion in isolated hepatocytes have been studied. A list of compounds which depleted glutathione and induced lipid peroxidation and cell lysis is given. The effects of halogenated acetamides were studied in more detail and results of studies on the interaction of iodoacetamide with cellular constituents are presented. A single metabolite of iodoacetamide, tentatively identified as the glutathione conjugate, was excreted from the cells while less than one percent of the "parent compound" was retained, tightly bound to macromolecules. This bound component could not be associated with the cellular damage. Methionine, cysteine and alpha-tocopherol, as wellas paracetamol and ethylmorphine, were found to prevent lipid peroxidation and lysis. It is concluded that GSH deficiency per se can lead to lipid peroxidation and that this reaction caused the observed hepatocellular lysis.
Assuntos
Glutationa/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Peróxidos/metabolismo , Acetamidas/farmacologia , Animais , Células Cultivadas , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Maleatos/farmacologia , Proteínas/metabolismo , RatosRESUMO
The protective effect of N-acetylcysteine against the toxicity of paracetamol, acrolein, and paraquat was investigated using isolated hepatocytes as the experimental system. N-acetylcysteine protects against paracetamol toxicity by acting as a precursor for intracellular glutathione. N-acetylcysteine protects against acrolein toxicity by providing a source of sulfhydryl groups, and is effective without prior conversion. Paraquat toxicity can be decreased by coincubating the cells with N-acetylcysteine, but the mechanism for the protective effect is not as clear in this instance. It is probable that N-acetylcysteine protects against paraquat toxicity by helping to maintain intracellular glutathione levels.
Assuntos
Acetaminofen/toxicidade , Acetilcisteína/farmacologia , Acroleína/toxicidade , Aldeídos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Paraquat/toxicidade , Acetona/farmacologia , Acetilcisteína/uso terapêutico , Aminoácidos/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Glutationa/metabolismo , Fígado/citologia , Masculino , Maleatos/farmacologia , Fenobarbital/farmacologia , Ratos , Ratos EndogâmicosRESUMO
4-Methylumbelliferyl sulfate was used to characterize sulfatase activity in periportal and pericentral regions of the liver lobule in the perfused rat liver. Following infusion of 1.5 mM of this organic sulfatester, free 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were formed at rates of 13 and 9 mumoles/g/h, respectively, in livers from fasted, phenobarbital-treated rats. 5-Pregnen-3 beta-ol, 20-one sulfate inhibited hydrolysis and metabolite production completely, whereas perfusion with nitrogen-saturated perfusate or FCCP decreased total metabolite formation by only 30%. 4-Methylumbelliferone formed from the hydrolysis of 4-methylumbelliferyl sulfate was monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule. Detection of the desulfated product was always greater in the downstream region, i.e., infusion of 4-methylumbelliferyl sulfate produced a higher fluorescence signal in pericentral areas when perfusion was in the anterograde direction, while periportal areas demonstrated higher activity during perfusion in the retrograde direction. Perfusion with nitrogen-saturated perfusate abolished these differences. Taken together, these data suggest that uptake of organic sulfateesters is partially energy dependent, follows the hepatic oxygen gradient inversely, and is a major rate determinant for sulfatase activity in the liver.
Assuntos
Himecromona/metabolismo , Fígado/metabolismo , Umbeliferonas/metabolismo , Animais , Feminino , Tecnologia de Fibra Óptica , Hidrólise , Himecromona/análogos & derivados , Técnicas In Vitro , Perfusão , Ratos , Ratos Endogâmicos , Espectrometria de FluorescênciaRESUMO
The hydrolysis of 4-methylumbelliferyl sulfate by liver sulfatases to free fluorescent 4-methylumbelliferone and the subsequent formation of the glucuronide conjugate were studied in the isolated perfused rat liver. In livers from fed, phenobarbital-treated rats, 4-methylumbelliferyl sulfate (0.25-1.5 mM) was hydrolyzed rapidly to free 4-methylumbelliferone at maximal rates of about 5 mumol/g/hr. A major fraction of the free 4-methylumbelliferone formed was converted to the glucuronide at maximal rates around 20 mumol/g/hr. Similar rates of hydrolysis were observed in livers from fasted, phenobarbital-treated or normal rats, although the ratio of glucuronide to free product was decreased markedly by fasting. In liver homogenates, however, rates of organic sulfate hydrolysis exceeded those observed in the perfused liver by at least 2-fold, suggesting that 4-methylumbelliferyl sulfate content is an important determinant of rates of hydrolysis in the perfused liver. There was a good correlation (r = 0.91) between rates of product formation and fluorescence of 4-methylumbelliferone detected from the liver surface with fiber optic light guides. Fluorescence of 4-methylumbelliferone produced from hydrolysis of 4-methylumbelliferyl sulfate was also monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule for the estimation of local rates of product formation. When perfusions were in the anterograde direction, desulfation of 4-methylumbelliferyl sulfate was about 50% higher in pericentral (28.8 +/- 9.3 mumol/g/hr) than in periportal (18.2 +/- 2.7 mumol/g/hr) areas. Furthermore, 4-methylumbelliferyl sulfate content determined in microdissected samples was 1.5- to 2-fold higher in pericentral than in periportal regions of the liver lobule but the activity of 4-methylumbelliferyl sulfate sulfatase was identical in both zones of the liver lobule. We conclude, therefore, that the local substrate content is an important determinant of rates of 4-methylumbelliferyl sulfate hydrolysis in sublobular zones of the liver.
Assuntos
Himecromona/metabolismo , Fígado/metabolismo , Sulfatases/metabolismo , Umbeliferonas/metabolismo , Animais , Jejum , Feminino , Hidrólise , Himecromona/análogos & derivados , Fígado/anatomia & histologia , Consumo de Oxigênio , Perfusão , Fenobarbital/farmacologia , RatosRESUMO
Viability, glycolytic capacity and energy metabolism under anaerobic conditions were studied in the hepatoma cell lines HTC, FU5 and HepG2 and in rat and human hepatocytes using glucose and fructose as glycolytic precursors. During 6 hr of anaerobic incubation without additional substrate, viability decreased rapidly in FU5 and HTC cells, whereas viability of HepG2 cells was not significantly affected. In all tumor cells, 10 mmol/L glucose prevented hypoxic cell injury almost completely. Lactate formation from glucose was about five times higher than in hepatocytes under these circumstances. ATP content of the tumor cells remained almost constant under anaerobic conditions in the presence of glucose. Ten millimoles per liter of fructose diminished glycolysis in the hepatoma cells compared with glucose, ranging from 87% reduction in HTC cells to 43% reduction in HepG2 cells. Accordingly, ATP content decreased rapidly in the FU5 and slowly in the HepG2 cells. Viability was strongly diminished in the HTC and FU5 cells in the presence of fructose, whereas in the HepG2 cells no effect of fructose on viability was detectable. In contrast to the hepatoma cells, rat and human hepatocytes exhibited higher rates of anaerobic glycolysis in the presence of fructose and thus were able to maintain their viability under these conditions. These differences in glycolytic capacity, energy metabolism and hypoxia tolerance of hepatoma cells compared with hepatocytes may be used for the treatment of liver cancer by isolated liver perfusion and ex situ revision of the organ.