An evaluation of the role of calcium in cell injury.

It has been proposed that a number of chemical-induced cell injuries result from disruption of the ability of the cell to control calcium. Many of the techniques used to develop this theory have relied on indirect measurements of intracellular calcium. The advent of digital imaging fluorescence microscopy has allowed a more direct examination of the relationship between calcium and cell damage. Results indicate that cytosolic calcium does not play a central role in the initiation of oxidative injury in a number of cell types. Changes in calcium homeostasis occur well after the appearance of other indications of cell injury. However, recent studies indicate that a mitochondrial lesion occurs relatively early in the time course of oxidative cell injury. Calcium may play a role in the development of this lesion.

Oxidative stress in cultured hepatocytes exposed to acetaminophen.

The effect of acetaminophen (APAP) exposure on the formation of oxidized glutathione (GSSG) was investigated in cultured mouse hepatocytes to determine if oxidative damage is involved in the toxicity of this drug. Incubations of hepatocytes for 24 hr with 1 mM APAP produced a time-dependent loss of cell viability which was preceded by depletion of reduced glutathione (GSH) and an increase in GSSG formation. Pretreatment with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) (0.1 mM) for 30 min, which irreversibly inhibited glutathione reductase (GSSG-Rd) activity, increased the extent of GSSG formation produced by APAP exposure and potentiated its cell killing. Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for 1 hr to chelate ferric iron decreased GSSG formation and cell killing produced by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation as determined by the formation of the APAP-GSH conjugate or the covalent binding of APAP metabolites to cellular protein. Hence, increasing the susceptibility of hepatocytes to an oxidative stress with BCNU increased both the formation of GSSG and cell killing produced by APAP. Conversely, decreasing their susceptibility to an oxidative stress by chelating iron with DFO decreased GSSG formation and cell injury. It follows that APAP toxicity involves oxidative processes that occur early in the poisoning process and are a major factor contributing to injury in these cells.

Protection from oxidative damage in mouse liver cells.

Paracetamol toxicity in mouse hepatocytes involved oxidative stress initiated by the formation of NAPQI. This oxidative component of paracetamol injury is associated with the latter stages of the poisoning process. Ebselen, a drug with GSH-peroxidase activity, was effective in ameliorating these oxidative events.

Level of cytosolic free calcium during acetaminophen toxicity in mouse hepatocytes.

It has been suggested that elevated cytosolic free calcium plays a key role in acetaminophen-induced cell death. The present study has examined the effect of a toxic concentration of acetaminophen on cytosolic free calcium in single mouse hepatocytes, using the dye fura-2 and video imaging fluorescence microscopy. Cytosolic free calcium was calculated from the ratio of emitted fluorescence at greater than 475 nm produced by excitation at 340 and 380 nm, using a double-intensified silicon target camera and digital image processing. In the presence of 5 mM acetaminophen, cell death did not occur for 2 hr, but the toxic lesion that ultimately killed the cells occurred as early as 1 hr. If cytosolic free calcium plays an important role in these toxic events, it would be expected to increase during this period. However, during a 2-hr exposure, cytosolic free calcium concentration in cells exposed to acetaminophen was not different from control. In hepatocytes incubated for longer than 2 hr, the calcium concentration increased shortly before loss of cell viability (i.e., as a late event), consistent with an influx of calcium through a damaged cell membrane. This late increase in calcium occurred well after the appearance of cell surface blebs. The data suggest that there is no sustained change in cytosolic free calcium in acetaminophen injury either before or during the time when irreversible toxic events occur in hepatocytes.

Postnatal mice have low susceptibility to paracetamol toxicity.

The hepatotoxicity of paracetamol in mice of 2, 3, 8-10, 24-26, 32-34, and 52-54 wk of age was determined by lethality data, histopathologic examination of the liver, and appearance of glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase activities in the plasma over an 8-h exposure period. At a dose of 300 mg/kg, there was evidence of hepatocytic necrosis and transaminase leakage in the 32- to 34- and 52- to 54-wk-old mice, but lethality was only recorded in the oldest age group. At 500 mg/kg, paracetamol produced 30% lethality in 3-wk-old mice and between 50 and 90% lethality in the adult age groups. There was histologic evidence of hepatocytic necrosis at all of these ages and its extent increased with age. Similarly, there were increases in plasma transaminases in each of these age groups. However, in 2-wk-old mice there was no lethality, no hepatocytic necrosis, and no increase in plasma transaminases. The lack of susceptibility of 2-wk-old mice to paracetamol toxicity was not due to immaturity of the cytochrome P-450 enzymes responsible for metabolism of paracetamol to its reactive metabolite (N-acetyl-p-benzoquinone imine). In fact, the activity of this enzyme pathway in 2-wk-old mice was greater than that in adults. The partial clearance of the glutathione-derived metabolites of paracetamol after a nontoxic (50 mg/kg) dose was 80% greater in 2-wk-old mice than in 8- to 10-wk-old mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The killing of cultured hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI) as a model of the cytotoxicity of acetaminophen.

The killing of isolated hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI), the major metabolite of the oxidation of the hepatotoxin acetaminophen, has been studied previously as a model of liver cell injury by the parent compound. Such studies assume that the toxicity of acetaminophen is mediated by NAPQI and that treatment with exogenous NAPQI reproduces the action of the endogenously produced product. The present study tested these assumptions by comparing under identical conditions the toxicity of acetaminophen and NAPQI. The killing of hepatocytes by acetaminophen was mediated by oxidative injury. Thus, it depended on a cellular source of ferric iron; was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase; and was sensitive to antioxidants. By contrast, the cytotoxicity of NAPQI was not prevented by chelation of ferric iron; was unaffected by BCNU; and was insensitive to antioxidants. Thus, the killing of cultured hepatocytes by NAPQI occurs by a mechanism different from that of acetaminophen. The killing by NAPQI was preceded by a collapse of the mitochondrial membrane potential and a depletion of ATP. Monensin potentiated the cell killing, and extracellular acidosis prevented it. These manipulations are characteristic of the toxicity of mitochondrial poisons, and are without effect on the depletion of ATP and the loss of mitochondrial energization. Thus, mitochondrial de-energization by a mechanism unrelated to oxidative stress is a likely basis of the cell killing by NAPQI. It is concluded that treatment of cultured hepatocytes with NAPQI does not model the cytotoxicity of acetaminophen in these cells.

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes.

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.

Cytosolic free magnesium, ATP and blebbing during chemical hypoxia in cultured rat hepatocytes.

Cytosolic free Mg2+ concentration was determined in 1-day cultured rat hepatocytes using Multiparameter Digitized Video Microscopy (MDVM) of the fluorescent probe, mag-fura-2. Chemical hypoxia with KCN (5 mM) and iodoacetate (1 mM), a model which mimics the ATP depletion and reductive stress of hypoxia, caused a rapid increase of free Mg2+ from 1.1 +/- 0.2 to 1.6 +/- 0.2 mM within 4 min. Concurrently, numerous small plasma membrane blebs formed and ATP levels dropped from 13.24 to 1.32 nmol/10(6) cells. Removal of KCN and iodoacetate resulted in recovery of ATP to 60-70% of pre-exposure levels, a concomitant decrease in cytosolic free Mg2+ back toward basal levels, and reversal of blebbing (bleb resorption). These results indicate that changes of cytosolic free Mg2+ inversely reflect changes of ATP in a model of hypoxia and reoxygenation. Bleb formation and resorption were dependent on the fall and rise of ATP.

Mitochondrial dysfunction in paracetamol hepatotoxicity: in vitro studies in isolated mouse hepatocytes.

The effect of paracetamol intoxication on mitochondrial function was studied in isolated mouse hepatocytes. Inhibition of cellular respiration as well as a lowering of cellular ATP contents and ATP/ADP ratios was associated with exposure to toxic concentrations of paracetamol. Significantly, inhibition of 3-hydroxybutyrate- and lactate/pyruvate-supported respiration, as well as the reduction in cellular ATP levels and ATP/ADP ratios, preceded the appearance of plasma membrane damage, as assessed by LDH leakage. N-Acetylcysteine reduced the extent of plasma membrane damage induced by paracetamol and protected against the impairment of cellular respiration. This suggests that respiratory dysfunction was a consequence of the oxidation of paracetamol to its reactive metabolite within the liver cell. These findings indicate that paracetamol toxicity results in an impairment of mitochondrial function which precedes the loss of plasma membrane integrity.

Postnatal development of enzyme activities associated with protection against oxidative stress in the mouse.

A number of enzyme systems are important in the protection of cells from chemical-induced oxidative damage. Little is known of the relative importance of these enzymes during postnatal development and its is possible that changes in their activity during this period may alter the susceptibility to toxic agents. This study investigated the activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, gamma-glutamyl-cysteine synthetase and glutathione synthetase in the liver, lung and kidney of postnatal and adult mice. The first 3 postnatal weeks are characterized by marked changes in the activities of enzymes that protect against oxidative stress (glutathione peroxidase/reductase, catalase and superoxide dismutase). Overall, the activity of these enzymes suggests that the mouse has a higher level of protection against peroxides at various stages during this period but lower capacity to detoxify superoxide anions. The activities of the glutathione-synthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) were significantly lower in the kidney of the postnatal mice, but the liver and lung had levels similar to those in the adult. Glutathione turnover in the liver of 2-week-old mice was not different from that in adults. The results indicate a complex pattern of development in the activities of detoxification enzyme systems during postnatal development.

A role for the glutathione peroxidase/reductase enzyme system in the protection from paracetamol toxicity in isolated mouse hepatocytes.

The role of the glutathione peroxidase/reductase (GSH-Px/GSSG-Rd) enzyme system in protection from paracetamol toxicity was investigated in isolated mouse hepatocytes in primary culture. The effect of inhibitors of these enzymes on the toxicity of paracetamol and on t-butylhydroperoxide (t-BOOH), used as a positive control, was determined. 1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GSSG-Rd, and goldthioglucose (GTG) used to inhibit GSH-Px. Both these inhibitors increased cell membrane damage in response to oxidative stress initiated by t-BOOH. However, they also increased the susceptibility of hepatocytes to paracetamol toxicity, indicating that a component of paracetamol's toxic effect involves formation of species that are detoxified by the GSH-Px/GSSG-Rd enzymes. To further examine the role of these enzymes, age-related differences in their activity were exploited. Hepatocytes from two-week-old mice were less susceptible to both t-BOOH and paracetamol toxicity than were those from adult mice. This corresponds to higher activity of cytosolic GSH-Px/GSSG-Rd in this age group. However, after inhibition of GSSG-Rd with BCNU, hepatocytes from these postnatal mice were more susceptible to paracetamol toxicity. This suggests that the higher activity of GSH-Px/GSSG-Rd in hepatocytes from two-week-old mice is responsible for their reduced susceptibility to paracetamol toxicity. The data indicate that the GSH-Px/GSSG-Rd enzymes contribute to protection from paracetamol toxicity and suggest that formation of peroxides contributes to this drug's hepatotoxic effects.

Paracetamol-induced stimulation of glycogenolysis in isolated mouse hepatocytes is not directly associated with cell death.

Paracetamol intoxication in vivo is known to be accompanied by depletion of hepatic glycogen stores. We have demonstrated a dose-dependent stimulation of glycogenolysis by paracetamol in glycogen-rich hepatocytes isolated from the mouse. Concentrations of paracetamol that produced plasma membrane damage were also found to activate glycogen phosphorylase a and deplete cellular glycogen contents. However, paracetamol-mediated stimulation of glycogenolysis could be dissociated from the events associated with paracetamol-induced cell killing. Both N-acetylcysteine and 2,4-dichloro-6-phenylphenoxyethylamine markedly reduced the extent of hepatocellular plasma membrane damage induced by paracetamol, yet neither agent prevented the activation of phosphorylase a nor the depletion of glycogen. These findings suggest that the hepatic glycogen depletion that accompanies paracetamol intoxication in vivo is due, at least in part, to a direct effect of the drug on the liver.

Comparison of the susceptibility of hepatocytes from postnatal and adult mice to hepatotoxins.

Age-related changes of susceptibility to hepatotoxicity induced by four hepatotoxic compounds were investigated using an isolated mouse hepatocyte model. Hepatocytes isolated from 2-week-old mice and adult mice (8-10 weeks old) were exposed to different concentrations (including toxic concentrations) of paracetamol, furosemide, iodoacetic acid and t-butylhydroperoxide for incubation times up to 24 hr. Cell damage was assessed by leakage of lactate dehydrogenase. Analysis of variance indicated that the hepatocytes from the 2-week-old mice were less susceptible to the toxic effects of all four hepatotoxins. The activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase were determined in both hepatocytes and whole liver from the two age groups. While catalase was significantly greater in adults, glutathione peroxidase, glutathione reductase and superoxide dismutase were all higher in the 2-week-old mice. Since these three enzymes are involved with protection against oxidative stress, it is likely that the higher activity in hepatocytes from 2-week-old mice is responsible for the reduced susceptibility to damage induced by the four hepatotoxins.

Effect of acetaminophen hepatotoxicity on hepatic mitochondrial and microsomal calcium contents in mice.

The effect of toxic doses of acetaminophen on hepatic intracellular calcium compartmentation were studied in mice. No effects on the calcium contents of the mitochondria, microsomes or cytosol were observed 4 h after the administration of 175 and 375 mg/kg acetaminophen when compared to saline-treated controls. However, doses of 500 and 750 mg/kg of acetaminophen increased mitochondrial calcium contents at this time. Also, the 750 mg/kg dose caused marked alterations in the calcium contents of microsomal and cytosolic compartments. The time-course of the onset of these effects was examined using a 500 mg/kg dose. No changes in either mitochondrial, microsomal or cytosolic calcium contents were observed in the livers of mice treated with acetaminophen compared to saline-treated controls at either 1 or 2 h after dose administration. However, at 3, 4 and 24 h after acetaminophen, mitochondrial and cytosolic calcium contents were significantly increased above control values. The increases in mitochondrial and cytosolic calcium contents observed in the acetaminophen-intoxicated mouse liver appear to occur at the same time as the appearance of plasma membrane damage, as measured by sorbitol dehydrogenase leakage. The data suggest that a perturbation in hepatic calcium compartmentation is not an early event in acetaminophen-induced hepatotoxicity in the mouse.

Isolation of hepatocytes from postnatal mice.

A method for the isolation of hepatocytes from postnatal (1- to 3-week-old) mice has been developed. Cell isolation was carried out by retrograde perfusion of the liver with a collagenase-containing bicarbonate buffer. Viable cells were separated by selective adsorption onto collagen membranes. Cell viability was assessed by measuring ATP and glutathione content, lactate:pyruvate ratio, and the rate of protein synthesis. Comparisons of these parameters were made with those in cells isolated from adult mice and with values in whole liver. These hepatocytes were capable of metabolizing acetaminophen to its known hepatic conjugates and were susceptible to acetaminophen toxicity. This procedure for isolating mouse hepatocytes should be useful for the study of hepatic drug metabolism and its relationship to toxicity in the postnatal mouse.

Comparison of the protective effects of N-acetylcysteine, 2-mercaptopropionylglycine and dithiothreitol against acetaminophen toxicity in mouse hepatocytes.

The effects of N-acetylcysteine (NAC), 2-mercaptopropionylglycine (MPG) and dithiothreitol (DTT) on the metabolism and toxicity of acetaminophen (APAP) were examined in isolated mouse hepatocytes maintained in primary culture on collagen-coated dishes. Both NAC and MPG increased the formation of the glutathione and sulfate conjugates of APAP and decreased the covalent binding of the APAP reactive metabolite to cellular protein. DTT did not increase APAP metabolism but did decrease covalent binding. NAC, MPG and DTT decreased plasma membrane damage, as measured by leakage of lactate dehydrogenase from hepatocytes, during a 4-h incubation in 5.0 mM APAP. NAC, MPG and DTT also reduced the APAP-induced fall in glutathione levels in these cells. In other experiments, hepatocytes were exposed to 5.0 mM APAP for 1 h and then incubated during a post-exposure period in APAP-free medium. Damage increased during this post-exposure incubation. Addition of DTT, but not NAC or MPG, after APAP exposure protected the hepatocytes from plasma membrane damage during the post-exposure period. These results indicate that NAC and MPG exert their protective effects by their action on the reactive metabolite of APAP. As well as its effect in reducing the formation of the reactive metabolite, DTT has a potent protective effect against the toxic processes initiated by the APAP reactive metabolite.

Age-related toxicity of paracetamol in mouse hepatocytes.

Hepatocytes from postnatal and adult mice were isolated by perfusion of the liver with a collagenase-containing bicarbonate buffer. These were allowed to attach to collagen-coated tissue culture dishes and were then examined for their susceptibility to paracetamol toxicity. After an 8 hr incubation in either 0.1 or 1.0 mM paracetamol, the extent of lactate dehydrogenase leakage and depletion of glutathione were similar in hepatocytes from young (1-, 2- and 3-week-old) mice when compared to adult mice. The covalent binding of [14C]-paracetamol to protein was greater in the hepatocytes from young mice. The results indicate that while the amount of reactive metabolites free to react with cellular constituents is greater in hepatocytes from young mice, the amount of damage produced was not different than that found in those from adults.

The effectiveness of antioxidants in reducing paracetamol-induced damage subsequent to paracetamol activation.

Paracetamol toxicity was studied in isolated mouse hepatocytes. This drug produced a concentration- and time-dependent loss of cell viability. Exposure to 5 mM paracetamol produced a rapid fall in intracellular reduced glutathione (GSH) followed by a decrease in plasma membrane integrity. The antioxidant, diphenyl-p-phenylenediamine (DPPD) had no effect on either the loss of GSH, the binding of 14C-paracetamol metabolites to protein or the loss of plasma membrane integrity when hepatocytes were incubated in 5 mM paracetamol. When hepatocytes were exposed to paracetamol for 1 hr they showed a loss of plasma membrane integrity during the subsequent 7 hr incubation. DPPD, alpha-tocopherol and promethazine reduced this effect when added after the paracetamol exposure. It appears that paracetamol exposure initiates toxic events subsequent to the generation of the reactive metabolite but prior to cell death and that the progression of these events can be interrupted by compounds with antioxidant properties.

Studies on the fate of the glutathione and cysteine conjugates of acetaminophen in mice.

Studies were conducted in mice to examine the origin and fate of the amino acid-containing conjugates of acetaminophen (APAP). Collection of bile containing [14C]APAP metabolites (mainly the glutathione conjugate) in common duct-cannulated mice given a 250 mg/kg oral dose of the drug reduced by greater than 70% the urinary excretion of the cysteine and mercapturic acid conjugates of APAP. This confirmed previous reports which indicated that these urinary metabolites originated from the glutathione conjugate excreted in bile. The urinary excretion of cysteine and mercapturic acid conjugates was not altered, however, by ligation of the common bile duct in mice given APAP. Thus, biliary excretion of the glutathione conjugate is not obligatory for the appearance of cysteine and mercapturic acid conjugates in urine. Intravenous administration of purified glutathione conjugate to mice having a bile-duct cannula indicated that this conjugate did not appear in bile but appeared in urine primarily in the form of the cysteine conjugate. An identical pattern of excretion was observed after an iv dose of the purified cysteine conjugate of APAP to bile duct-cannulated mice. These results indicated that, if the glutathione conjugate leaves the liver via the blood, it is rapidly converted to the cysteine conjugate which is eliminated in urine. This conversion takes place at multiple sites in the body and evidence is presented to implicate both intestine and kidney in the process. The appearance of a small amount of glutathione conjugate in urine (16%) after an iv dose of the cysteine conjugate indicates that formation of the glutathione of APAP can occur by a route that does not involve direct conjugation of reactive metabolites of the drug with glutathione.