Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated hepatocytes

The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

ATP-generating glycolytic substrates prevent N-nitrosofenfluramine-induced cytotoxicity in isolated rat hepatocytes

The relationship between cytotoxicity induced by N-nitrosofenfluramine and mitochondrial or glycolytic adenosine triphosphate (ATP) synthesis-dependent intracellular bioenergetics was studied in isolated rat hepatocytes. The supplementation of fructose, an ATP-generating glycolytic substrate, to hepatocyte suspensions prevented N-nitrosofenfluramine-induced cell injury accompanied by the formation of cell blebs, abrupt loss of intracellular ATP and reduced glutathione and mitochondrial membrane potential (DeltaPsi), and the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation, during a 2h incubation period. Fructose (1-20mM) resulted in concentration-dependent protection against the cytotoxicity of N-nitrosofenfluramine at a concentration of 0.6mM, a low toxic dose. Pretreatment with xylitol, another glycolytic substrate, at concentration of 15mM also prevented the cytotoxicity caused by the nitroso compound, but neither glucose nor sucrose exhibited protective effects. In addition, fructose inhibited N-nitrosofenfluramine (0.5 and 0.6mM)-induced DNA damage, as evaluated in the comet assay, indicating that nuclei as well as mitochondria are target sites of the compound. These results indicate that (a) the onset of N-nitrosofenfluramine-induced cytotoxicity in rat hepatocytes is linked to mitochondrial failure, and that (b) the insufficient supply of ATP in turn limits the activities of all energy-requiring reactions and consequently leads to acute cell death.     

Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes

The biotransformation and cytotoxic effects of hydroxychavicol (HC; 1-allyl-3,4-dihydroxybenzene), which is a catecholic component in piper betel leaf and a major intermediary metabolite of safrole in rats and humans, was studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to HC caused not only concentration (0.25-1.0 mM)- and time (0-3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. At a concentration of 1 mM, the cytotoxic effects of safrole were less than those of HC. The loss of mitochondrial membrane potential and generation of oxygen radical species assayed using 2′,7′-dichlorodihydrofluoresein diacetate (DCFH-DA) in hepatocytes treated with HC were greater than those with safrole. HC at a weakly toxic level (0.25 and/or 0.50 mM) was metabolized to monoglucuronide, monosulfate, and monoglutathione conjugates, which were identified by mass spectra and/or ¹H nuclear magnetic resonance spectra. The amounts of sulfate rather than glucuronide or glutathione conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In hepatocytes pretreated with either diethyl maleate or salicylamide, HC-induced cytotoxicity was enhanced, accompanied by a decrease in the formation of these conjugates and by the inhibition of HC loss. Taken collectively, our results indicate that (a) mitochondria are target organelles for HC, which elicits cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage and subsequently lipid peroxidation through oxidative stress at a later stage; (b) the onset of cytotoxicity depends on the initial and residual concentrations of HC rather than those of its metabolites; (c) the toxicity of HC is greater than that of safrole, suggesting the participation of a catecholic intermediate in safrole cytotoxicity in rat hepatocytes.     

Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems

Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.     

Peroxidase-catalyzed oxidation of eugenol: formation of a cytotoxic metabolite(s)

The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells.     

Mechanisms of Phenytoin‐Induced Toxicity in Freshly Isolated Rat Hepatocytes and the Protective Effects of Taurine and/or Melatonin

Phenytoin is a widely used antiepileptic drug. However, hepatotoxicity is one of its adverse effects reported in some patients. The mechanism(s) by which phenytoin causes hepatotoxicity is not clear yet. This study was designed to evaluate the cytotoxic mechanism(s) of phenytoin toward rat hepatocytes (whose cytochrome P450 enzymes had been induced by Phenobarbital). Furthermore, the effect of taurine and/or melatonin on this toxicity was investigated. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and mitochondrial depolarization were monitored as toxicity markers. Results showed that phenytoin caused an elevation in ROS formation, depletion of intracellular reduced glutathione, increase in cellular oxidized glutathione, enhancement of LPO, and mitochondrial damage. Taurine (1 mM) and/or melatonin (1 mM) administration decreased the intensity of cellular injury caused by phenytoin. This study suggests the protective role of taurine and/or melatonin against phenytoin‐induced cellular damage probably through their reactive radical scavenging properties and their effects on mitochondria.     

Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes.

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mM cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mM fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.     

Mitochondrial dysfunction and biotransformation of β-carboline alkaloids,harmine and harmaline,on isolated rat hepatocytes

The cytotoxic effects and biotransformation of harmine and harmaline, which are known β-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0–0.50 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5 mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these β-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25 mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50 μM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.     

Induction of apoptosis by eugenol in human breast cancer cells

In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol.     

A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis

The depletion of cell calcium from isolated rat hepatocytes results in stimulated lipid peroxidation, loss of intracellular and mitochondrial GSH (reduced glutathione), and enhancement of both efflux and oxidation of GSH. These events are followed by cell injury and enhance the susceptibility of the cells to toxic chemicals. It is shown herein that an initial event in the generation of such injury is the depletion of cellular alpha-tocopherol. alpha-Tocopheryl succinate addition (25 microM) to the calcium-depleted cells markedly elevated the alpha-tocopherol content of the cells, inhibited the associated lipid peroxidation, and maintained intracellular GSH levels without affecting its efflux or redox status. This resulted in an enhanced formation of total glutathione after a 5-h incubation, which correlated with the alpha-tocopherol content of the cells, and was greater than that expected by a direct sparing action of vitamin E. Inhibition of hepatocyte glutathione biosynthesis by buthionine sulfoximine (0.5 mM) eliminated the enhancement of GSH formation by vitamin E. Analysis of endogenous and 35S-labelled precursors of glutathione biosynthesis by high-performance liquid chromatography demonstrated that the depletion of cellular alpha-tocopherol resulted in the efflux of glutathione precursors. It is concluded that cell injury associated with alpha-tocopherol depletion is partly the result of the efflux of glutathione precursors, and hence diminished biosynthesis and intracellular levels of GSH. These losses and resultant cell injury are preventable by maintenance of cellular alpha-tocopherol levels.     

Biosynthesis of chondroitin sulphate by a Golgi-apparatus-enriched preparation from cultures of mouse mastocytoma cells.

Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.     

Alteration of glutathione level in human melanoma cells: effect of N-acetyl-L-cysteine and its analogues

The effects of N-acetyl-L-cysteine (L-NAC), N,N-diacetyl-L-cystine (oxidized form of L-NAC) and N-acetyl-D-cysteine on the intracellular glutathione (GSH) level and their toxicity were investigated in the human melanoma cell culture IGR1. L-NAC applied in 3 mM concentration for 24 hr decreased; when applied for 48 hr it did not alter the intracellular GSH level. Treatment with 1 mM L-NAC for 24 hr had no effect on cellular glutathione, whereas the same concentration applied for 48 hr resulted in an increase in the level of GSH. Both concentrations also induced cell injury as determined by protein assay and trypan blue staining. N,N-diacetyl-L-cystine (0.5 and 1.5 mM, 24 hr) induced a decrease in cellular glutathione content without any apparent cell toxicity. D-NAC (1 and 3 mM, 24 hr) did not influence the GSH level of the melanoma cells; however, it had toxic effects resulting in cell loss.     

Role for an episulfonium ion in S-(2-chloroethyl)-DL-cysteine-induced cytotoxicity and its reaction with glutathione

The cysteine S conjugate of 1,2-dichloroethane, S-(2-chloroethyl)-DL-cysteine (CEC), is hepatotoxic, nephrotoxic, and mutagenic. To determine the cellular and chemical mechanisms involved in CEC-induced toxicity and to assess the role of an episulfonium ion, the effect of CEC on the viability of isolated rat hepatocytes was studied. CEC addition resulted in both a time- and concentration-dependent loss of cell viability. Depletion of intracellular glutathione concentrations (greater than 70%) and inhibition of microsomal Ca2+ transport and Ca2+-ATPase activity preceded the loss of cell viability, and initiation of lipid peroxidation paralleled the loss of viability. The depletion of glutathione concentrations was partially attributable to a reaction between glutathione and CEC to form S-[2-(DL-cysteinyl)ethyl]glutathione, which was identified by NMR and mass spectrometry. N-Acetyl-L-cysteine, vitamin E, and N,N'-diphenyl-p-phenylenediamine protected against the loss of cell viability. N,N'-Diphenyl-p-phenylenediamine inhibited CEC-initiated lipid peroxidation but did not protect against cell death at 4 h, indicating that lipid peroxidation was not the cause of cell death. The analogues S-ethyl-L-cysteine, S-(3-chloropropyl)-DL-cysteine, and S-(2-hydroxyethyl)-L-cysteine, which cannot form an episulfonium ion, were not cytotoxic, thus demonstrating a role for an episulfonium ion in the cytotoxicity associated with exposure to CEC and, possibly, 1,2-dichloroethane. These results show that an alteration in Ca2+ homeostasis and the generation of an electrophilic intermediate may be involved in the mechanism of cell death.     

Effects of 4-hydroxynonenal on isolated hepatocytes. Studies on chemiluminescence response, alkane production and glutathione status.

The effect of 4-hydroxy-2,3-trans-nonenal, a diffusible product of lipid peroxidation, on isolated hepatocytes was evaluated with two non-invasive techniques measuring low-level chemiluminescence and alkane evolution. Oxygen-induced low-level chemiluminescence and ethane and n-pentane formation by hepatocytes is enhanced over 7-fold in the presence of 4-hydroxynonenal (2 mM). Glutathione-depleted hepatocytes show a higher increase than controls in both low-level chemiluminescence and alkane formation upon supplementation with 4-hydroxynonenal. The effects on both parameters are diminished by vitamin E pretreatment of rats and are absent under anaerobiosis. At variance with chemiluminescence and alkane formation, 4-hydroxynonenal does not elicit a concomitant increase in malonaldehyde or diene-conjugate formation. Addition of 4-hydroxynonenal to a suspension of hepatocytes causes a rapid loss of cellular glutathione in the form of a glutathione conjugate with the alkenal as observed with high-pressure liquid-chromatographic analysis. The reaction between glutathione and 4-hydroxynonenal proceeds also spontaneously in vitro at 1:1 stoichiometry. The cellular effects of 4-hydroxynonenal evaluated by low-level chemiluminescence and alkane formation are independent of the formation of a glutathione conjugate and seem to rely on the remaining not-bound 4-hydroxynonenal. The sensitivity of 4-hydroxynonenal-enhanced chemiluminescence and alkane formation to free-radical quenchers suggests the participation of a free-radical propagation process.     

Mechanism of metalloid-induced death in Leishmania spp.: role of iron, reactive oxygen species, Ca2+, and glutathione

There is growing evidence that metalloid-induced cell death in protozoan parasites is due to oxidative injury; however, the biochemical changes related to this event are not fully understood. Leishmania spp. demonstrated cross-resistance to two related metalloids, arsenic and antimony, and both metalloids induced cell death accompanied by cell shrinkage and DNA fragmentation that was preceded by an increase in reactive oxygen species. Both drugs caused mitochondrial dysfunction in terms of loss of membrane potential and a drop in ATP levels. Arsenic treatment resulted in an elevation of intracellular Ca2+ levels that did not occur with antimony exposure. Cellular glutathione level was reduced after antimony treatment but arsenic did not affect glutathione. Inhibition of Ca2+ influx during arsenic treatment reduced cell death, whereas supplementation of glutathione during antimony treatment rescued cell loss. Under iron-depleted conditions, the cytotoxic effects of arsenic and antimony did not occur and cell survival increased; in contrast, the presence of excess iron resulted in higher cell death. Therefore, this study provides a new possibility that iron can potentiate parasite death induced by metalloids like arsenic and antimony. In addition, an important observation is that the two similar metalloids produce toxicity by very different mechanisms.     

Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated.

When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea).

Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI.

NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 μM extensive bleb formation was followed by loss of cell membrane integrity and cell death.

To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.     

Mechanisms of Trazodone‐Induced Cytotoxicity and the Protective Effects of Melatonin and/or Taurine toward Freshly Isolated Rat Hepatocytes

It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC₅₀ of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone‐induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone‐induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:457‐462, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21509     

Resistance to TGF-beta-induced apoptosis in regenerating hepatocytes

Treatment with transforming growth factor beta (TGF-beta) of hepatocytes from two different proliferative conditions, such as fetal development and adult liver regeneration, shows that regenerating cells respond to this cytokine in terms of growth inhibition, but are less sensitive than the fetal ones to the apoptosis induced by this factor. Regenerating TGF-beta treated cells show higher cell viability and lower percentage of apoptotic cells than the fetal treated ones. Furthermore, TGF-beta treated regenerating hepatocytes maintain a well-preserved parenchyma-like organization. Treatment with TGF-beta induces the loss of mitochondrial transmembrane potential in fetal but not in regenerating hepatocytes and activation of caspase-3 is lower in regenerating than in fetal cells. Regenerating hepatocytes show higher intracellular levels of some antiapoptotic proteins, such as Bcl-x(L) and c-IAP-1 and, interestingly, they present higher intracellular glutathione levels, which might provide of mechanisms to avoid potential dangerous effects of the oxidative stress-mediated apoptosis induced by TGF-beta. In fact, treatment with BSO (a glutathione synthesis inhibitor) restores the response of regenerating hepatocytes to TGF-beta in terms of cell death. In conclusion, increased levels of Bcl-x(L) and cIAP-1 and higher intracellular glutathione levels could confer resistance to the apoptosis induced by TGF-beta during liver regeneration.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.