The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. A marked decrease in mitochondrial membrane potential and inhibition of mitochondrial respiration that was accompanied by reactive oxygen species formation occurred when isolated rat hepatocytes were incubated with low concentrations of HCHO in a dose-dependent manner. Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner. At higher HCHO concentrations, lipid peroxidation ensued followed by cell death. Cytotoxicity studies were conducted in which isolated hepatocytes exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes. There was a marked increase in HCHO cytotoxicity when either alcohol dehydrogenase or aldehyde dehydrogenase was inhibited. Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion of GSH markedly increased hepatocyte susceptibility to HCHO. In each case, cytotoxicity was dose-dependent and corresponded with a decrease in hepatocyte HCHO metabolism and increased lipid peroxidation. Antioxidants and iron chelators protected against HCHO cytotoxicity. Cytotoxicity was also prevented, when cyclosporine or carnitine was added to prevent the opening of the mitochondrial permeability transition pore which further suggests that HCHO targets the mitochondria. Thus, HCHO-metabolising gene polymorphisms would be expected to have toxicological consequences on an individual's susceptibility to HCHO toxicity and carcinogenesis.     

Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: carbonyl-metabolizing enzymes versus oxidative stress

Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.     

Differences in glyoxal and methylglyoxal metabolism determine cellular susceptibility to protein carbonylation and cytotoxicity

Chronic hyperglycemia in diabetic patients often leads to chronic side effects associated with protein glycation and the formation of reactive carbonyl species, such as methylglyoxal (MGO) and glyoxal (GO). We have shown that both MGO and GO carbonylated bovine serum albumin (BSA) in vitro to the same degree and stability. The carbonylated BSA formed initially could be a reversible Schiff base as the UV absorbance formed after the addition of 2,4-dinitrophenylhydrazine was decreased when sodium borohydride was added. MGO and GO also carbonylated hepatocyte protein rapidly with similar dose and time dependence. In contrast to BSA carbonylation, the amount of carbonylated proteins in hepatocytes decreased over time, much more rapidly for hepatocytes treated with MGO than with GO. This could be attributed to the rapid hepatocyte metabolism of MGO with glyoxalase I, the predominant detoxification enzyme for MGO. Protein carbonylation and the associated toxicity caused by GO and MGO were studied in the following hepatocyte models: (1) control hepatocytes, (2) glutathione (GSH)-depleted hepatocytes, (3) mitochondrial aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, (4) hepatocyte inflammation model, and (5) catalase-inhibited hepatocyte model. Carbonylation and cytotoxicity caused by MGO or GO was markedly increased in GSH-depleted hepatocytes as compared to control hepatocytes. Hepatocytes exposed to non-toxic concentrations of H(2)O(2) or hepatocytes treated with catalase inhibitors also showed a marked increase in GO-caused cytotoxicity and protein carbonylation, whereas there were only minor increases with MGO. The GO effect was attributed to potential radical formation and the inhibition effect of H(2)O(2) on aldehyde dehydrogenase, a major GO metabolising enzyme. GO-caused cytotoxicity and protein carbonylation were also increased with ALDH2-inhibited hepatocytes whereas such an increase was only observed with MGO in GSH-depleted hepatocytes.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

S-adenosylmethionine exerts a protective effect against thioacetamide-induced injury in primary cultures of rat hepatocytes

S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.     

A decrease in S-adenosyl-L-methionine potentiates arachidonic acid cytotoxicity in primary rat hepatocytes enriched in CYP2E1

Previous studies show that treatment with a polyunsaturated fatty acid, arachidonic acid (AA), or high concentrations of cycloleucine, an inhibitor of methionine adenosyltransferase (MAT), which lowers levels of S-adenosyl-L-methionine (SAM), increased toxicity in hepatocytes from pyrazole-treated rats which expressed high levels of cytochrome P450 2E1 (CYP2E1). In this study, I used concentrations of cycloleucine or AA, which by themselves do not produce any toxicity, to evaluate whether a decrease in SAM sensitizes hepatocytes to AA toxicity, especially in hepatocytes enriched in CYP2E1. Levels of SAM were lower by 50% in hepatocytes from pyrazole- compared to saline-treated rats. Cycloleucine treatment caused a 50% decline in SAM levels with both hepatocyte preparations and SAM levels were lowest in the pyrazole-treated hepatocytes. The combination of cycloleucine plus AA produced some toxicity and apoptosis in hepatocytes from saline-treated rats but increased toxicity and apoptosis was found in the hepatocytes from pyrazole-treated rats. Cytotoxicity could be prevented by incubation with SAM, the antioxidant trolox, and the mitochondrial permeability transition inhibitor trifluoperazine. The enhanced cytotoxicity could also be protected by treating rats with chlormethiazole, a specific inhibitor of CYP2E1, thus validating the role of CYP2E1. Cycloleucine plus AA treatment elevated production of reactive oxygen species (ROS) and lipid peroxidation to greater extents with the hepatocytes from pyrazole-treated rats than that from the saline-treated rats. I hypothesize that increased production of ROS by hepatocytes enriched in CYP2E1 potentiates AA-induced lipid peroxidation and toxicity when hepatoprotective levels of SAM are lowered. Such interactions, e.g. induction of CYP2E1, decline in SAM and polyunsaturated fatty acid-induced lipid peroxidation, may contribute to alcohol-induced liver injury.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390-401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H(2)O(2) [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414-419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

Peroxidase catalysed formation of cytotoxic prooxidant phenothiazine free radicals at physiological pH

The antipsychotic phenothiazines may have other therapeutic applications because of their ability to kill bacteria, plasmids and tumor cells. They are also known to undergo a peroxidase-catalysed oxidation to form cation radicals that are stable at acid pH, but are not detected at a neutral pH. The objective of this project was to determine whether phenothiazine cation radical metabolites could cause oxidative stress at a neutral pH resulting in cytotoxicity. At a neutral pH, catalytic amounts of phenothiazines were found to be oxidised by a peroxidase/H2O2 system and also caused ascorbate, GSH and NADH cooxidation. NADH and GSH co-oxidation was accompanied by oxygen uptake and was increased by the addition of catalytic amounts of superoxide dismutase, indicating that the superoxide radical was formed. The phenothazines were different from other peroxidase substrates in that the NADH, ascorbate or GSH cooxidation was faster at pH 6.0 than pH 7.4, thereby partly reflecting the cation radical stability. The order of catalytic effectiveness found was promazine > chlorpromazine > trifluoperazine. Peroxidase/H2O2 also markedly increased phenothiazine cytotoxicity towards isolated rat hepatocytes at nontoxic phenothiazine concentrations. At both pH 6.0 and 7.4, the same order of phenothiazine catalytic effectiveness was observed as seen in the co-oxidation experiments. Cytotoxicity to hepatocytes could be attributed to oxidative stress as most hepatocyte glutathione oxidation and lipid peroxidation preceded phenothiazine induced cytotoxicity and that cytotoxicity was prevented by the antioxidant butylated hydroxyanisole. This hepatocyte/peroxidase/H2O2 system could be a useful model for studying drug induced idiosyncratic hepatic injury enhanced by inflammation.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390–401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H₂O₂ [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414–419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Effect of benzophenones from Hypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes

Five benzophenones and a xanthone, isolated from Hypericum annulatum Moris, were investigated for their protective effect against carbon tetrachloride toxicity in isolated rat hepatocytes. The benzophenones and the xanthone gentisein were administered alone (100 microM) and in combination with CCl4 (86 microM). CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (*CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. The levels of thiobarbituric acid reactive substances (TBARS) were assayed as an index of lipid peroxidation (LPO). Lactate dehydrogenase (LDH) leakage, cell viability and reduced glutathione (GSH) depletion were used as signs of cytotoxicity. CCl4 significantly decreased hepatocyte viability, GSH level and increased TBARS level and LDH leakage as compared to the control. Our data indicate that 2,3',5',6-tetrahydroxy-4-methoxybenzophenone, 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone and 2-O-alpha-L-3'-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone showed weaker toxic effects compared to CCl4 and in combination showed statistically significant protection against the toxic agent.     

Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+)

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Quinone toxicity in hepatocytes: studies on mitochondrial Ca2+ release induced by benzoquinone derivatives

Hepatocyte cytotoxicity caused by substituted benzoquinones was associated with increased cytosolic Ca2+ concentration. p-Benzoquinone-induced hepatotoxicity was enhanced when the hepatocytes were loaded with Ca2+ by preincubation with ATP. A similar order of potency of the substituted benzoquinones in releasing Ca2+ from isolated mitochondria and inducing hepatocyte cytotoxicity was found; in decreasing order, this was 2-Br-, unsubstituted-, 2-CH3-, 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5-(CH3)2-, 2,3,5-(CH3)3-, and 2,3,5,6-(CH3)4-benzoquinones (duroquinone). The cellular products of quinone metabolism, hydroquinones and glutathione conjugates, did not cause mitochondrial Ca2+ release. Benzoquinone-induced mitochondrial Ca2+ release was preceded by GSH conjugate formation and NAD(P)H oxidation but followed by mitochondrial swelling. With duroquinone, a slow GSH and NADPH oxidation preceded Ca2+ release, but GSH oxidation did not occur with Se-deficient mitochondria lacking glutathione peroxidase activity. Cyanide-insensitive respiration was also observed with duroquinone but not with benzoquinone, suggesting that duroquinone undergoes redox cycling. GSH was depleted by both arylation and oxidation with 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5(CH3)2-, and 2,3,5-(CH3)3-benzoquinones. Benzoquinone concentrations that totally depleted GSH did not cause Ca2+ release until intramitochondrial NAD(P)H was oxidized. Ca2+ release was also prevented when NAD(P)H generation was stimulated by the presence of isocitrate or 3-hydroxybutyrate. This suggests that mitochondrial Ca2+ release is associated with NAD(P)H oxidation catalyzed by NADH dehydrogenase with benzoquinone or by the glutathione peroxidase-glutathione reductase system with duroquinone.     

Contrasting role of Na(+) ions in modulating Cu(+2) or Cd(+2) induced hepatocyte toxicity

Previously we showed that hepatocyte lysis induced by Cu(+2)/Cd(+2) could be partly attributed to membrane lipid peroxidation induced by Cu(+2) or mitochondrial toxicity induced by Cd(+2) [5]. Changes in Na(+) and Ca(+2) homeostasis induced when Cu(+2) was incubated with hepatocytes markedly differed from that induced by Cd(+2). Na(+) omission from the media or addition of the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride markedly increased Cu(+2) cytotoxicity even though Cu(+2) did not increase hepatocyte Na(+) when the media contained Na(+). Intracellular Ca(+2) levels however were markedly increased when the hepatocytes were incubated with Cu(+2) in a Na(+) free media and removing media Ca(+2) with EGTA also prevented Cu(+2) induced hepatocyte cytotoxicity. This suggests that intracellular Ca(+2) accumulation contributes to Cu(+2) induced cytotoxicity and a Na(+)-dependent Ca(+2) transporter is involved in controlling excessive Ca(+2) accumulation caused by Cu(+2). The omission of Cl(-) from the media or addition of glycine, a Cl(-) channel blocker also enhanced Cu induced cytotoxicity. By contrast Cd(+2) induced cytotoxicity was prevented by Na(+) omission from the media or by the addition of 5-(N,N-dimethyl)-amiloride. Furthermore the omission of Cl(-) from the media or addition of glycine also prevented Cd(+2) induced hepatocyte toxicity. A hypotonic media also increased Cd(+2) but not Cu(+2) induced hepatocyte cytotoxicity. This suggests that Cd(+2) but not Cu(+2) cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. The increased osmotic load caused by the uncontrolled accumulation of intracellular Na(+) in Cd(+2) treated hepatocytes likely resulted from the activation of Na(+)/H(+) exchanger and the Na(+)/HCO(3)(-) cotransporter by the acidosis and ATP depletion caused by mitochondrial toxicity.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

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.     

Glutathione mediated reductive activation and mitochondrial dysfunction play key roles in lithium induced oxidative stress and cytotoxicity in liver

Lithium preparations are commonly used drug in treating mental disorders and bipolar diseases, but metal's cytotoxic mechanisms have not yet been completely understood. In this study, we investigated the cytotoxic mechanisms of lithium in freshly isolated rat hepatocytes. Lithium cytotoxicity were associated with reactive oxygen species (ROS) formation and collapse of mitochondrial membrane potential and cytochrome c release into the hepatocyte cytosol. All of the mentioned lithium-induced cytotoxicity markers were significantly (P?相似文献   

The effects of demethylsterigmatocystin and sterigmatin on ATP synthesis system in mitochondria: A comparison with sterigmatocystin

The effects of demethylsterigmatocystin and its structural isomer, sterigmatin, on oxidative phosphorylation of mitochondria were studied by means of isolated rat liver mitochondria in comparison with sterigmatocystin. Both compounds were found to uncouple the oxidative phosphorylation in mitochondria, causing marked decreases in RC index and P/O ratio. Sterigmatin, in which dihydrobisfuran ring and xanthone nucleus are combined in a linear manner, was evidently more toxic to mitochondrial functions than demethylsterigmatocystin which was an angular molecular shape. Though their uncoupling concentrations were rather high for assessing theirin vivo toxicity, a good correspond was observed between their orders of potencies in the toxicity to mitochondrial functions and in the cytotoxicity to rat hepatocytes. The demethylation of sterigmatocystin resulted in an obvious decrease of uncoupling activity.     

Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Acrolein is an air pollutant from cigarette smoking and other pollutions and also a by-product of lipid peroxidation. Studies have demonstrated that acrolein causes cytotoxicity and genotoxicity, including liver damage and death of hepatocytes. However, the toxic effects and the underlying mechanisms of acrolein on mitochondria, especially, on liver mitochondria, have not been well studied. In the present study, we investigated the toxic effects and mechanisms of acrolein on mitochondria isolated from rat liver by examining mitochondrial respiration, dehydrogenases, complex I, II, III, IV and V, permeability transition, and protein oxidation. Acrolein incubation (10-1000 microM, or 0.02-2 micromol/mg protein) with mitochondria caused dose-dependent inhibition of NADH- and succinate-linked mitochondrial respiration chain, change of mitochondrial permeability transition, increase in protein carbonyls, and selective enzyme inhibition of mitochondrial complex I, II, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, but no effects on mitochondrial complex III, IV, V and malate dehydrogenase. These results suggest that acrolein is a mitochondrial toxin and that mitochondrial dysfunction caused by acrolein may play an important role in acrolein toxicity such as hepatotoxicity and also smoking-related diseases.