On the role of calcium in chemical toxicity.
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Animals↗
Biomedical subjects
S Orrenius
Publications and source records attributed to S Orrenius.
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.
Animals↗
The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system.
Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+-activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+-dependent, non-lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.
Adenosine Triphosphate↗
On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine.
Incubation of isolated rat hepatocytes with either morphine or ethylmorphine resulted in glutathione (GSH) depletion followed by loss of cell viability. Pretreatment of cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase did not markedly affect the rates of GSH depletion seen in untreated cells. In contrast, hexobarbital stimulated H2O2 production in isolated liver microsomes, incubated aerobically with NADPH, whereas the effects of morphine and ethylmorphine on microsomal H2O2 production were minimal. Finally, incubation of hepatocytes with radioactively labeled morphine resulted in formation of 2 glutathione conjugates, one of which was tentatively identified as formyl glutathione. We conclude that GSH consumption during the metabolism of morphine or ethylmorphine by hepatocytes is due mainly to formation of glutathione conjugates.
Animals↗
Quinone toxicity in hepatocytes without oxidative stress.
The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.
Alkylating Agents↗
Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system.
Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.
Animals↗
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.
Alkylation↗
Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: studies with extracellular adenosine 5'-triphosphate.
The incubation of isolated rat hepatocytes with extracellular adenosine 5'-triphosphate (ATP) resulted in an inhibition of Ca2+ efflux. The ATP-induced Ca2+ accumulation as determined by the increase in phosphorylase a activity and the Ca2+-sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis-[carboxymethyl]aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca2+, suggesting that the increased free Ca2+ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[ 2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H- imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca2+-calmodulin process may be involved in the initiation of cytotoxicity.
Adenosine Triphosphate↗
tert-Butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation.
Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide-induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.
Animals↗
Role of thiols in protection against biological reactive intermediates.
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Adenosine Triphosphate↗
Cellular defense mechanisms against toxic substances.
Recent studies of cellular defense mechanisms against toxic substances are reviewed with particular emphasis on the critical functions of reduced glutathione. Studies of the metabolism of paracetamol and of the redox active quinone menadione in isolated rat hepatocytes, are summarized in order to illustrate how multiple defense mechanisms are involved in the protection of the cell against the toxicity of these agents. Cytotoxicity with both agents occurs only after the cellular defense mechanisms have become exhausted.
Animals↗
The mechanism of pentachlorobutadienyl-glutathione nephrotoxicity studied with isolated rat renal epithelial cells.
Isolated renal epithelial cells were used to study the mechanism of toxicity of pentachlorobutadienyl-glutathione (PCBG), a nephrotoxic glutathione conjugate of hexachlorobutadiene. The cytotoxicity of PCBG displayed a very steep dose-response relationship; at 10 microM PCBG no toxicity was observed whereas 25, 50, and 100 microM PCBG all resulted in a similar degree of toxicity. In all cases, loss of cell viability was observed only after a 30-min lag period and reached a plateau of 50 to 60% nonviable cells between 90 and 100 min. Toxic doses of PCBG also resulted in the depletion of cellular thiols. Blocking PCBG metabolism by inhibition of gamma-glutamyl transpeptidase [1-gamma-L-glutamyl-2-(2-carboxyphenyl)hydrazine (anthglutin), 2 mM] or renal cysteine conjugate beta-lyase (aminooxyacetic acid, 0.5 mM) resulted in complete protection against PCBG-induced cell damage. Exposure of isolated renal epithelial cells to 100 microM PCBG resulted in the rapid formation of plasma membrane blebs which appeared to be associated with a loss of Ca2+ from the mitochondrial compartment and an elevation of cytosolic Ca2+ concentration as measured by Quin-2. PCBG treatment also resulted in the inhibition of cell respiration and a marked depletion of cellular ATP content, indicating additional mitochondrial effects of the toxin. Our results support a role for renal cysteine conjugate beta-lyase in the metabolic activation of PCBG and suggest that PCBG-induced renal cell injury may be the result of selective effects on mitochondrial function.
Adenosine Triphosphate↗
Menadione (2-methyl-1,4-naphthoquinone)-induced Ca2+ release from rat-liver mitochondria is caused by NAD(P)H oxidation.
Incubation of rat-liver mitochondria with menadione in the presence of succinate and rotenone resulted in rapid glutathione and NAD(P)H oxidation followed by Ca2+ release and mitochondrial swelling. Ca2+ release, NAD(P)H oxidation and mitochondrial swelling, were also observed in mitochondria from selenium-deficient rats. Glutathione was only slowly oxidized, suggesting that glutathione oxidation, and subsequent NAD(P)H oxidation via the glutathione peroxidase-glutathione reductase system were not required for Ca2+ release by menadione. Isocitrate prevented and reversed Ca2+ release dose-dependently but dicoumarol had no effect indicating that NADH-ubiquinone oxidoreductase and not DT-diaphorase was responsible for NAD(P)H oxidation. Superoxide anion radical was formed by cyanide-resistant respiration, suggesting that menadione undergoes a one-electron reduction to an autoxidizable semiquinone radical by NADH-ubiquinone oxidoreductase. The inability of menadione to oxidize glutathione in selenium-deficient mitochondria indicates that the metabolism of the superoxide dismutation product, H2O2, by glutathione peroxidase was probably responsible for the glutathione oxidation in selenium-replete mitochondria.
Animals↗
Effects of oxidative stress caused by hyperoxia and diquat. A study in isolated hepatocytes.
The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated. The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyl)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below approximately 6 nmol/10(6) cells. The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant alpha-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxyl radical scavenger alpha-keto-gamma-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation. The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase - glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H2O2 and lipid hydroperoxides.
Animals↗
Studies of Ca2+-mediated toxicity in hepatocytes.
Molecular mechanisms involved in the development of toxic cell injury have attracted increased interest in recent years. In particular, the possible existence of a final common pathway in toxic cell killing has been addressed in a number of studies. Recent work from our laboratory suggests that a disruption of intracellular Ca2+ homeostasis may represent a common step in the development of toxic damage to hepatocytes, and that activation of Ca2+-dependent, neutral proteases by a sustained increase in cytosolic free Ca2+ concentration represents one mechanism by which toxic agents can kill hepatocytes.
Animals↗
The formation of both a mono- and a bis-substituted glutathione conjugate of hexachlorobutadiene by isolated hepatocytes and following in vivo administration to the rat.
The nephrotoxicity of hexachloro-1,3-butadiene (HCBD) appears to depend on the initial formation of a glutathione (GSH) conjugate in the liver. In the present study we have examined the hepatic metabolism of HCBD using isolated hepatocytes and following in vivo administration. Exposure of isolated hepatocytes to HCBD resulted in a dose-dependent depletion of GSH. HPLC analysis of the incubation medium demonstrated the formation of two products. When isolated hepatocytes containing [3H]GSH were exposed to [14C]HCBD, coincident elution of 3H and 14C corresponding to the previously recognized HPLC peaks was observed. Both products were sensitive to treatment with gamma-glutamyl transpeptidase (gamma-GT), providing additional support for their identification as GSH conjugates. The ratio of 3H to 14C in the two peaks indicated the formation of both a mono- and a bis-substituted GSH conjugate of HCBD. The identification of the mono- and bis-GSH conjugates was further confirmed by the preparation of synthetic standards which displayed retention times by HPLC identical to the biological products. The production of the total and individual GSH conjugates displayed both dose and time dependence. The production of the total as well as the ratio of mono- to bis-conjugate was found to depend on the availability of GSH. At low HCBD exposure levels the bis-substituted conjugate accounted for more than 20% of the total conjugate produced by isolated hepatocytes. This value decreased at higher HCBD concentrations. Analysis of bile collected from rats following intraportal administration of [14C]HCBD revealed the presence of both the mono- and bis-substituted GSH conjugates of HCBD as well as additional 14C-containing metabolites. The results of the present study clearly demonstrate the production of both a mono- and a bis-substituted GSH conjugate of HCBD. The potential importance of this finding in terms of the nephrotoxicity of HCBD is discussed.
Animals↗
Oxidative stress studied in intact mammalian cells.
Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.
Animals↗
The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca2+.
The effects of acetaminophen and its major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), have been investigated in hepatocytes isolated from 3-methylcholanthrene-pretreated and -untreated rats, respectively. The two compounds produced qualitatively similar changes although the quinone imine was toxic with shorter incubations periods and at lower doses. Both agents caused an elevation of cytosolic Ca2+, assessed by phosphorylase a activity, which was accompanied by the concomitant appearance of plasma membrane blebs. A loss of mitochondrial Ca2+ was also observed. This disruption of Ca2+ homeostasis always preceded cell death. Studies with NAPQI showed that low doses were able to cause complete Ca2+ release from isolated liver mitochondria which was accompanied by pyridine nucleotide oxidation and preceded membrane damage. NAPQI also produced a rapid, dose-dependent depletion of both cytosolic and mitochondrial reduced glutathione as well as a loss of protein-bound SH groups. This loss of protein thiols may have been responsible for the observed inhibition of the high-affinity Ca2+-ATPase activity of the plasma membrane fraction isolated from NAPQI-treated cells. In addition, NAPQI inhibited microsomal Ca2+ uptake which would further contribute to the elevation in cytosolic Ca2+. Our results suggest that acetaminophen and N-acetyl-p-benzoquinone imine exert their cytotoxic effects via a disruption of Ca2+ homeostasis secondary to the depletion of soluble and protein-bound thiols. This mechanism may prove to be of general applicability to a variety of hepatotoxins.
Acetaminophen↗