Irene Anundi

Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes

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.

The effectiveness of N-acetylcysteine in isolated hepatocytes, against the toxicity of paracetamol, acrolein, and paraquat

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 glutathinone levels.

Hepatoprotective mechanism of silymarin: No evidence for involvement of cytochrome P450 2E1

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.

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity.

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.

Involvement of glutathione reductase in selenite metabolism and toxicity, studied in isolated rat hepatocytes

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 μM selenite, resulted in substantially lower GSH-levels. The rate of GSSG reductase-catalyzed metabolism of selenite (50 μM) could be estimated to approximately 7 nmoles reduced/h per 106 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.

GSH release in bile as influenced by arsenite

Biliary release of glutathione disulfide (GSSG) and glutathione-S-conjugates by the isolated perfused liver under different metabolic conditions has been studied by several investigators [l-3]. Sies et al. [ 1,3] have shown that efflux of GSSG occurs during oxidation of drugs such as hexobarbital and ethylmorphine as well as during hydroperoxide metabolism. In the isolated liver there is also a transport of reduced glutathione (GSH) into the perfusate space [4]. However, it has also been reported that relatively high concentrations of GSH may be excreted in the bile produced in situ ]5a,bl. The present study, performed mainly with isolated perfused rat liver, concerns the release of both reduced and oxidized glutathione in bile after arsenite exposure. It has previously been shown that arsenic is eliminated via this route [6] and compounds such as selenite are known to interact with arsenite in a way that increases their respective elimination in the bile [7,8]. Furthermore, the form of As, e.g., if complexed to small molecular weight compounds, or the mechanism(s) involved in its biliary excretion, have not been described. Similar studies have indicated an involvement of GSH in the excretion of zinc and other metals [9,10] in rat bile in situ.

Selenite biotransformation to volatile metabolites in an isolated hepatocyte model system

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.

Metabolism of toxic substances in isolated hepatocytes.

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.

Chloroacetamide hepatotoxicity: Hydropic degeneration and lipid peroxidation

Abstract Rats were treated with chloroacetamide (75 mg/kg) to rapidly deplete hepatic glutathione (GSH) and induce lipid peroxidation. Three to six hours after administration of chloroacetamide, midzonal and peripheral lesions developed in the liver parenchyma, and during this period an enhancement of lipid peroxidation [measured as thiobarbituric acid (TBA)-reacting material in liver homogenate] was seen. Reversible morphological changes, notably hydropic degeneration, were also seen at 24 and 48 hr. After 1 week no sign of regenerative growth in the liver was observed. TBA values remained high for up to 24 hr and then decreased to normal levels at 48 hr. The GSH content rapidly decreased; less than 10% remained after 1 hr and thereafter a progressive increase was seen with values slightly higher than normal observed at 48 and 72 hr. Rats fasted overnight were more susceptible to chloroacetamide toxicity and midzonal necrosis and higher TBA values were observed in those rats. The morphological changes were also more severe in rats treated with a higher dose (112.5 mg/kg) of chloroacetamide. The results indicate that hepatic GSH depletion leads to lipid peroxidation in vivo . They further suggest that lipid peroxidation can be significantly increased without causing permanent damage to the hepatocytes.

LIPID PEROXIDATION INDUCED BY GLUTATHIONE DEPLETION

Publisher Summary This chapter discusses the lipid peroxidation induced by glutathione (GSH) depletion. GSH is a cofactor for enzymatic reduction of peroxides and may have a critical role in protection against lipid peroxidation. The chapter describes a study in which, isolated hepatocytes were treated with electrophiles that conjugate GSH to study the effect of GSH depletion. In this study, it was found that diethylmaleate and water-soluble halogenated acetamides readily induced lipid peroxidation. It could also be shown that cellular lysis was related to lipid peroxidation while covalent binding to macromolecules could be ruled out as a lysing mechanism. Substrates for cytochrome P-450, such as acetaminophen, inhibited lipid peroxidation and prevented cellular lysis indicating a role for the endoplasmic reticulum (ER) electron transport system in the toxicity of GSH-depleting agents. The chapter also focuses on the integrity of GSH-depleted cells before detectable lipid peroxidation and the in vivo effect of hepatic GSH depletion.