Hepatic injury induces contrasting response in liver and kidney to chemicals that are metabolically activated: role of male sex hormone

Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase activities significantly only in male mice. Increases in Cyp2e1 and Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.     

4-hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells

An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.     

Potentiation of the antimalarial action of chloroquine in rodent malaria by drugs known to reduce cellular glutathione levels

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by 4-aminoquinolines. As a result FP accumulates in the membrane fraction and associates with enzymes of infected cells in parallel with parasite killing. Free FP is degraded by reduced glutathione (GSH). This degradation is inhibited by chloroquine (CQ) and amodiaquine (AQ) but not by quinine (Q) or mefloquine (MQ). Increased GSH levels in Plasmodium falciparum-infected cells confer resistance to CQ and vice versa, and sensitize CQ-resistant Plasmodium berghei by inhibiting the synthesis of glutathione. Some drugs are known to reduce GSH in body tissues when used in excess, either due to their pro-oxidant activity or their ability to form conjugates with GSH. We show that acetaminophen, indomethacin and disulfiram were able to potentiate the antimalarial action of sub-curative doses of CQ and AQ in P. berghei- or Plasmodium vinckei petteri-infected mice, but not that of Q and MQ. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. Although these results imply that alteration in GSH are involved, measurement of total glutathione either in uninfected or P. berghei-infected mice, treated with these drugs did not reveal major changes. In conclusion, experimental evidences provided in this study suggest that some off the counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.     

Acetaminophen alters microsomal ryanodine Ca2+ channel in HepG2 cells overexpressing CYP2E1

Acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding of drug metabolites to liver proteins. Acetaminophen metabolites have been shown to affect rat liver microsomal Ca2+ stores, but the mechanism is not well understood. The aim of the current work was to find out if the metabolism of acetaminophen by CYP2E1 affects ryanodine-sensitive Ca2+ stores in the endoplasmic reticulum of transduced HepG2 cells. Five millimoles acetaminophen decreased proliferation of CYP2E1-overexpressing HepG2 cells, increased cytosolic Ca2+ levels and produced significant cytotoxicity, while only little, mostly anti-proliferative effects were found in HepG2 cells lacking CYP2E1. CYP2E1 inhibitor-4-methylpyrazole decreased drug cytotoxicity in transduced cells and normalized elevated Ca2+ levels. Acetaminophen cytotoxicity was significantly higher in CYP2E1 expressing cells with depleted glutathione. In the cells engineered to overexpress CYP2E1, an increased [3H]ryanodine affinity (by 45%) and increased ligand maximal binding to ryanodine receptors (by 64%) was observed, most probably due to increased association rate of [3H]ryanodine. Ca2+ loading was decreased by about 53% in microsomal fractions isolated from transduced cells treated with acetaminophen and by 92% in glutathione depleted transfected cells treated with the drug. Ca2+/Mg2+-ATPase activity was unchanged in all microsomal fractions. Such effects were not observed in cells lacking CYP2E1. Our results confirm significant role of CYP2E1 in metabolic activation of acetaminophen and indicate that ryanodine receptors located in the liver endoplasmic reticulum are sensitive targets for acetaminophen metabolites.     

The role of oxidative stress in zearalenone-mediated toxicity in Hep G2 cells: oxidative DNA damage, gluthatione depletion and stress proteins induction

Zearalenone (ZEN) is a fusarial mycotoxin with several adverse effects in laboratory and domestic animals including mainly estrogenicity. While most ZEN toxic effects have been quite well investigated, little is known regarding its mechanism of toxicity. Our previous investigations have shown the involvement of cytotoxicity, inhibition of macromolecules synthesis as well as genotoxicity. However, there are no available data regarding the involvement of the oxidative stress pathway in ZEN toxicity. In this context, the aim of this study was to find out whether ZEN induces oxidative cell damage. Using human hepatocytes Hep G2 cells, ZEN-induced stress response is monitored at several levels in these cells. ZEN mediated induction of oxidative DNA damage (comet assay using the repair enzymes), modulation of gluthatione (GSH), cytotoxicity (growth inhibition) and the oxidative stress responsive gene Hsp 70 and Hsp 90 were investigated with respect to concentration and time dependency. Hep G2 cells respond to ZEN exposure by loss of cell viability, induction of oxidative DNA damage, GSH depletion and Hsp 70 and Hsp 90 induction already at concentrations, which are not yet cytotoxic. The perturbation of the oxidative status was further confirmed by the significant reduction of the induced oxidative DNA damage as well as stress protein induction when cells were pre-treated with Vitamin E prior to exposure to ZEN. Our study clearly demonstrates that oxidative damage is likely to be evoked as one of the main pathway of ZEN toxicity. This oxidative damage may therefore be an initiating event and contribute, at least in part, to the mechanism of ZEN different genotoxic and cytotoxic effects.     

Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.     

Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (>500 microM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 microM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.     

Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine

Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects.     

Increased mitochondrial stress and modulation of mitochondrial respiratory enzyme activities in acetaminophen-induced toxicity in mouse macrophage cells

Overdose of acetaminophen (APAP) causes tissue injury particularly in the liver. However, the precise mechanism of APAP toxicity is not clear. Glutathione (GSH) depletion and oxidative stress are believed to be the main cause of APAP toxicity. The role of macrophages in APAP-induced tissue injury is controversial. Using mouse macrophage J774.2 cells, we recently demonstrated that like in animal models, APAP reduces GSH pool and alters GSH metabolism by increasing the production of reactive oxygen species (ROS). In the present study, we show that APAP-induced cytotoxicity and apoptosis in macrophages are associated with increased mitochondrial metabolic and oxidative stress, alterations in the mitochondrial membrane potential and activities of the respiratory enzyme complexes. APAP treatment also altered ROS/NO production and inhibited the expression of COX-2 and iNOS in LPS-stimulated macrophages. Electron microscopic studies also confirmed morphological changes associated with apoptosis at the lower dose of APAP, while at the higher dose late apoptosis/necrotic changes were visible. These results suggest that mitochondrial metabolic and oxidative stress are the main causes of cytotoxicity and cell death in APAP treated macrophages. The study may have long term implications to better understand the role of macrophages in the toxicology and pharmacology of APAP.     

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 microM) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.     

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6 h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3 h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24 h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.     

Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: close structural connectivity between glutathione conjugation activity and non-substrate ligand binding

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.     

Evaluation of glutathione deficiency in rat livers by microarray analysis

Hepatic glutathione content was measured and gene expression data were obtained using an Affymetrix RG U34 array after treatment with tap water containing 20mM l-buthionine (S, R)-sulfoximine (BSO) to male F344 rats for four consecutive days. Both Spearman's and Pearson's correlation coefficients were calculated between the glutathione content and the mRNA content level obtained from the microarray analysis individually. Sixty-nine gene probes, which were statistically significant (Spearman's correlation, P < 0.05) and showed a Pearson's correlation coefficients (Pearson's r) less than -0.8 between mRNA content and hepatic glutathione content, were identified as glutathione deficiency-correlated probes. By comparing the hepatic gene expression profiles between BSO- and butylated hydroxyanisole (BHA)-treated rats, 14 probes of genes that showed an increase in the corresponding gene mRNA levels only after the BSO treatment were thought to be good indicators of glutathione deficiency. A principal component analysis successfully illustrated the time-course of hepatic gene expression after the treatment with acetaminophen, phenobarbital and clofibrate, and the expression profiles were thought to reflect the changes in hepatic glutathione levels. The identified gene probes in the present study would be useful as markers for assessing hepatocellular glutathione deficiency, or oxidative stress level, based on microarray data.     

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N(G)-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48+/-6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23+/-5%, while, SNAP or DETA-NONO increased viability to 66+/-8 or 71+/-6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.     

Structural requirements for the flavonoid-mediated modulation of glutathione S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells

The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1 activity in MCF7 pMTG5 cells can be inhibited by some flavonoids. Especially galangin was able to inhibit almost all cellular GSTP1-1 activity upon exposure of the cells to a concentration of 25microM. Other flavonoids like kaempferol, eriodictyol and quercetin showed a moderate GSTP1-1 inhibitory potential. For GSTP1-1 inhibition, no specific structural requirements necessary for potent inhibition could be defined. Most flavonoids appeared to be potent GS-X transport inhibitors with IC(50) values ranging between 0.8 and 8microM. Luteolin and quercetin were the strongest inhibitors with IC(50) values of 0.8 and 1.3microM, respectively. Flavonoids without a C2-C3 double bond like eriodictyol, taxifolin and catechin did not inhibit GS-X pump activity. The results of this study demonstrate that the structural features necessary for high potency GS-X pump inhibition by flavonoids are (1) the presence of hydroxyl groups, especially two of them generating the 3',4'-catechol moiety; and (2) a planar molecule due to the presence of a C2-C3 double bond. Other factors, like lipophilicity and the total number of hydroxyl groups do not seem to be dominating the flavonoid-mediated GS-X pump inhibition. To identify the GS-X pump responsible for the DNP-SG efflux in MCF7 cells, the effects of three characteristic flavonoids quercetin, flavone and taxifolin on MRP1 and MRP2 activity were studied using transfected MDCKII cells. All three flavonoids as well as the typical MRP inhibitor (MK571) affected MRP1-mediated transport activity in a similar way as observed in the MCF7 cells. In addition, the most potent GS-X pump inhibitor in the MCF7 cells, quercetin, did not affect MRP2-mediated transport activity. These observations clearly indicate that the GS-X pump activity in the MCF7 cells is likely to be the result of flavonoid-mediated inhibition of MRP1 and not MRP2. Altogether, the present study reveals that a major site for flavonoid interaction with GSH-dependent toxicokinetics is the GS-X pump MRP1 rather than the conjugating GSTP1-1 activity itself. Of the flavonoids shown to be most active especially quercetin is frequently marketed in functional food supplements. Given the physiological levels expected to be reached upon supplement intake, the IC(50) values of the present study point at possible flavonoid-drug and/or flavonoid-xenobiotic interactions especially regarding transport processes involved in toxicokinetics.     

Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium

Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3-4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca(2+)-containing Krebs-Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams' E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h. Also included was the bicarbonate-free HEPES buffer that does not require carbogen gassing, and is therefore handled more easily. The results clearly demonstrated that the type of incubation medium profoundly affected the functionality of the suspended hepatocytes, changing their sensitivity and response to exogenous damaging effects. While HEPES buffer and Williams' E medium offered the lowest background of spontaneous cell death, bicarbonate-based buffers and media seemed more suitable for obtaining both phases I and II biotransformation. Williams' E medium ensured a constant glutathione content of the cells and a lower level of oxidative stress.     

Cyclosporine A-induced apoptosis in renal tubular cells is related to oxidative damage and mitochondrial fission

Cyclosporine A (CsA) nephrotoxicity has been linked to reactive oxygen species (ROS) production in renal cells. We have demonstrated that the antioxidant Vitamin E (Vit E) abolished renal toxicity in vivo and in vitro models. As one of the main sources of intracellular ROS are mitochondria, we studied the effects of CsA on several mitochondrial functions in LLC-PK1 cells.     

Yeast Saccharomyces cerevisiae devoid of Cu, Zn-superoxide dismutase as a cellular model to study acrylamide toxicity

Acrylamide is known as a cytotoxic and genotoxic component of starch-containing heat-processed food. We demonstrate that yeast Saccharomyces cerevisiae may be used as a cellular model to examine the biochemical mechanisms of acrylamide toxicity. We found that acrylamide causes impairment of growth of the yeast deficient in Cu, Zn-superoxide dismutase (Δsod1) in a concentration-dependent manner. This growth inhibitory effect is not due to cell death but to decreased cell vitality and proliferative capacity. Treatment of the Δsod1 yeast with acrylamide induced generation of increased reactive oxygen species and depletion of glutathione. The toxicity of acrylamide for yeast cells may be abolished by antioxidants (ascorbate, cysteine, N-acetylcysteine, glutathione and dithiothreitol) or lowering oxygen content in the atmosphere.     

A role for glutathione,independent of oxidative stress,in the developmental toxicity of methanol

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6 h later in embryonic ROS formation, measured by 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2′-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.