William J. Racz

Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity

Morphological changes in mitochondria are observed early in the course of acetaminophen (AA)-induced hepatotoxicity, and mitochondrial dysfunction has been observed both in vivo and in vitro following exposure to AA. This study examined the early effects of AA exposure in vivo on mitochondrial respiration and evaluated the effectiveness ofN-acetyl-L-cysteine (NAC) in protecting against respiratory dysfunction. Mitochondria were isolated from the livers of fasted, male CD-1 mice 0, 0.5, 1, 1.5 or 2 h after administration of a hepatotoxic dose of AA (750 mg/kg). Glutamate- and succinate-supported mitochondrial respiration were subsequently assessed by polarographic measurement of state 3 (ADP-stimulated) and state 4 (resting) rates of oxygen consumption and determination of the corresponding respiratory control ratios (RCR: state 3/state 4) and ADP:O ratios. Hepatotoxicity was assessed histologically and by measuring plasma alanine aminotransferase (ALT) activity. The earliest sign of mitochondrial dysfunction observed in this study was a significant decrease in the ADP:O ratio for the oxidation of glutamate 1 h post-dosing. At 1.5 and 2 h post-dosing the RCRs for both glutamate- and succinate-supported respiration were significantly decreased. All of the respiratory parameters measured in this study were significantly decreased, with the exception of succinate-supported state 4 respiration which was significantly increased, 2 h after AA administration. Thus, inhibition of mitochondrial respiration preceded overt hepatic necrosis, indicated by an elevation of ALT activity, which was not observed until 3 and 4 h post-dosing. In addition, mitochondrial respiratory dysfunction correlated with morphological alterations. Inhibition of mitochondrial respiration therefore appears to be an early event in the course of AA-induced hepatotoxicity. Cotreatment with NAC (1200 mg/kg) completely prevented the AA-induced impairment of mitochondrial respiration and the development of histopathologic damage. The protection afforded by NAC in these experiments indicates thatN-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of AA, is responsible for the observed inhibitory effects, and suggests that mitochondrial dysfunction makes an important, if not essential, contribution to the development of AA-induced hepatotoxicity.

Effects of N-acetylcysteine on metabolism, covalent binding, and toxicity of acetaminophen in isolated mouse hepatocytes☆☆☆

The effects of N-acetylcysteine (NAC) on the toxicity, conjugate formation, and covalent binding of acetaminophen (pHAA) and its presumed toxic metabolite were studied in suspensions of isolated mouse hepatocytes. Preincubation of liver cells with NAC prior to the addition of pHAA resulted in enhanced protection compared to the concurrent addition of pHAA and NAC, thus indicating a time lag between availability of NAC and exertion of a protective effect. Furthermore, a protective concentration of NAC caused a large increase in the proportion of pHAA plus metabolites found as the glutathione (GSH) conjugate and a decrease in covalent binding of radiolabeled pHAA metabolite to proteins. Thus, it appears that NAC protects against pHAA toxicity by increasing the availability of intracellular GSH.

The role of mitochondrial injury in bromobenzene and furosemide induced hepatotoxicity

Bromobenzene (BB) and furosemide (FS) are two hepatotoxicants whose bioactivation to reactive intermediates is crucial to the development of liver injury. However, the events which lead to hepatocellular toxicity following metabolite formation and covalent binding to cellular macromolecules remain unknown. The present study was undertaken to investigate the effect of administered BB and FS on mitochondrial total glutathione (GSH+GSSG, henceforth referred to as glutathione) content and respiratory function as potential initiating mechanisms of the hepatotoxicity of these compounds in the mouse. Bromobenzene (2 g/kg i.p.) significantly decreased mitochondrial glutathione to 48% of control at 3 h post administration, and to 41% at 4 h. This decrease in mitochondrial glutathione was subsequent to a significant decrease in cytosolic glutathione to 64 and 28% of control at 1 and 2 h, respectively. Oxygen consumption supported by complex I (glutamate-supported) of the respiratory chain was not inhibited by BB until 4 h, where state 3 (active) respiration was reduced to 16% of control. This resulted in a decreased respiratory control ratio (RCR) for complex I-supported respiration. Complex II (succinate)-supported state 3 and state 4 respiration were unaffected by BB until 4 h, at which time they were reduced to 57 and 48% of control, respectively. However, the similar reductions in state 3 and state 4 respiratory rates did not alter the corresponding RCR for complex II. Overt hepatic injury was detected at 4 h, with plasma alanine aminotransferase (ALT) activity increasing significantly at this time point. In contrast to the effects of BB, FS administration (400 mg/kg i.p.) did not alter mitochondrial or cytosolic glutathione, and had no effect on respiration supported by complex I or II for up to 5 h following dosing. However, ALT activity was significantly increased 5 h following FS administration. These results suggest that inhibition of mitochondrial respiratory function coinciding with a decrease in mitochondrial glutathione content may be crucial to the initiation of BB-induced hepatotoxicity, while such events are not required for the initiation of FS-induced hepatotoxicity.

Acetaminophen-induced hypothermia, hepatic congestion, and modification by N-acetylcysteine in mice

Abstract Acetaminophen-induced hypothermia and hepatic congestion, their modification by N -acetylcysteine (NAC) and their relationship to hepatotoxicity were studied in Swiss white mice. Acetaminophen (125–750 mg/kg) and NAC (1200 mg/kg) were administered orally and animals killed at various times up to 9 hr. Body temperature declined before overt liver injury and the associated congestion, thereby indicating that hypothermia was centrally mediated. The magnitude of hepatic congestion was sufficient to cause marked liver enlargement. This phenomenon was a possible cause of observed hypovolemia which may in turn have contributed to early mortality. Hypothermia and/or related CNS effects may also have contributed to the early mortality. Coadministration of NAC with acetaminophen prevented the hepatotoxicity, but only partially protected against the hypothermia. When administered 3 hr after acetaminophen, NAC immediately halted the development of congestion and hepatotoxicity, but reversal of the hypothermia was manifested only after several hours. The consequences of congestion and liver enlargement on the expression of biochemical variables such as covalent binding are discussed. Our results indicate that acetaminophen-induced hypothermia and hepatotoxicity develop separately in mice, and that an appreciation of both events is important in understanding the action of antidotal compounds.

Increased acetaminophen-induced hepatotoxicity after chronic ethanol consumption in mice

The effect of chronic ethanol consumption on acetaminophen (200, 400, and 600 mg/kg) toxicity was determined by maintaining mice for 10 days on diets consisting of chow and one of the following drinking solutions: 10% ethanol + 10% sucrose, 8% sucrose, or tap water. Toxicity as manifested by mortality, liver enlargement, and liver congestion was greatest in the ethanol-treated group. We suggest that the greater mortality was a result of the increased liver congestion and consequent hypovolemia. Despite the increased levels of cytochrome(s) P-450, covalent binding of [3H]acetaminophen reactive metabolite(s) to liver protein was not higher in ethanol-treated animals. This can be explained by the higher initial glutathione concentration and/or ability to replenish glutathione in the ethanol-treated group. We suggest that the enhancement of acetaminophen toxicity by ethanol is the result of an effect of ethanol on hepatocyte membranes which renders the cells more susceptible to toxic injury.

Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes

Isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen (AA) for 1.5 h to initiate glutathione (GSH) and protein thiol (PSH) depletion and cell injury. Cells were subsequently washed to remove non-covalently bound AA and resuspended in medium containing N-acetylcysteine (NAC, 2.0 mM) or dithiothreitol (DTT, 1.5 mM). The effects of these agents on the replenishment of GSH and total PSH content were related to the development of cytotoxicity. When cells exposed to AA were resuspended in medium containing NAC or DTT, both agents replenished GSH and total PSH content to levels observed in untreated cells but only DTT was able to attenuate cytotoxicity. Addition of the GSH synthesis inhibitor, buthionine sulfoximine (BSO, 1.0 mM, 1.5 h), to cells in incubation medium containing AA, enhanced GSH and total PSH depletion and potentiated cytotoxicity. Resuspension of these cells in medium containing NAC did not alter the potentiating effects of BSO; GSH and PSH levels were not replenished and no cytoprotective effects were observed. However, when cells exposed to AA and BSO were resuspended in medium containing DTT, PSH content was replenished but GSH levels were not restored. In addition, DTT was able to delay the development of cytotoxicity. It appears that DTT, unlike NAC, has a GSH-independent mechanism of PSH replenishment. These observations suggest that while replenishment of GSH and total PSH content does not result in cytoprotection, the regeneration of critical PSH by DTT may play an important role in the maintenance of proper cell structure and/or function.

Amiodarone-induced disruption of hamster lung and liver mitochondrial function : lack of association with thiobarbituric acid-reactive substance production

Amiodarone (AM) is an efficacious antidysrhythmic agent that is limited clinically by numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT) due to the potential for mortality. Mitochondrial alterations and free radicals have been implicated in the etiology of AM-induced toxicities, including AIPT. Isolated hamster lung and liver mitochondria were assessed for AM-induced effects on respiration, membrane potential, and lipid peroxidation. AM (50-400 microM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 microM) resulting in a subsequent inhibition. This biphasic effect of AM (200 microM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50-400 microM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 microM AM decreased lung mitochondrial membrane potential (p < 0.05), while a concentration-dependent (50-200 microM) decrease of membrane potential was observed with liver mitochondria exposed to AM (p < 0.05). Formation of thiobarbituric acid-reactive substances (TBARS) was not altered by AM (50-400 microM) in incubations lasting up to 1 h. These results indicate that lipid peroxidation, as indicated by levels of TBARS, does not play a role in AM-induced alterations in mitochondrial respiration and membrane potential.

Effects of dietary vitamin E supplementation on pulmonary morphology and collagen deposition in amiodarone- and vehicle-treated hamsters.

Amiodarone (AM) is a potent antidysrhythmic agent that is limited in clinical use by its adverse effects, including potentially life-threatening AM-induced pulmonary toxicity (AIPT). The present study tested the ability of dietary supplementation with vitamin E (500 IU d,1-alpha-tocopherol acetate/kg chow) to protect against pulmonary damage following intratracheal administration of AM (1.83 micromol) to the male golden Syrian hamster. At 21 days post-dosing, animals treated with AM had increased lung hydroxyproline content and histological disease index values compared to control (P < 0.05), which were indicative of fibrosis. Dietary vitamin E supplementation for 6 weeks resulted in a 234% increase in lung vitamin E content at the time of AM dosing, and maintenance on the diet prevented AM-induced elevation of hydroxyproline content and disease index 21 days post-dosing. Dietary vitamin E supplementation also decreased hydroxyproline content and disease index values in hamsters treated intratracheally with distilled water, the AM vehicle. These results demonstrate a protective role for vitamin E in an in vivo model of AIPT, and suggest that this antioxidant may have non-specific antifibrotic effects in the lung.

Direct mitochondrial dysfunction precedes reactive oxygen species production in amiodarone-induced toxicity in human peripheral lung epithelial HPL1A cells

Amiodarone (AM), a drug used in the treatment of cardiac dysrrhythmias, can produce severe pulmonary adverse effects, including fibrosis. Although the pathogenesis of AM-induced pulmonary toxicity (AIPT) is not clearly understood, several hypotheses have been advanced, including increased inflammatory mediator release, mitochondrial dysfunction, and free-radical formation. The hypothesis that AM induces formation of reactive oxygen species (ROS) was tested in an in vitro model relevant for AIPT. Human peripheral lung epithelial HPL1A cells, as surrogates for target cells in AIPT, were susceptible to the toxicity of AM and N-desethylamiodarone (DEA), a major AM metabolite. Longer incubations (> or =6 h) of HPL1A cells with 100 microM AM significantly increased ROS formation. In contrast, shorter incubations (2 h) of HPL1A cells with AM resulted in mitochondrial dysfunction and cytoplasmic cytochrome c translocation. Preexposure of HPL1A cells to ubiquinone and alpha-tocopherol was more effective than that with Trolox C or 5,5-dimethylpyrolidine N-oxide (DMPO) at preventing AM cytotoxicity. These data suggest that mitochondrial dysfunction, rather than ROS overproduction, represents an early event in AM-induced toxicity in peripheral lung epithelial cells that may be relevant for triggering AIPT, and antioxidants that target mitochondria may potentially have beneficial effects in AIPT.

Acetaminophen-induced hypothermia in mice: Evidence for a central action of the parent compound

Pretreatment of mice with phenobarbital, an inducer of oxidative drug metabolism, had no effect on the early hypothermic effect of a toxic dose of acetaminophen, while pretreatment with metyrapone, SKF-525A, or piperonyl butoxide (inhibitors of mixed-function oxidase) enhanced the hypothermia. In mice treated with acetaminophen alone, brain parent drug levels correlated with the degree of hypothermia, while liver drug levels did not. Also, intracerebroventricular injection of acetaminophen resulted in significant hypothermia within 20 min. These results indicate that the early hypothermia caused by acetaminophen in mice is due to the parent drug, not to its toxic reactive metabolite, and that the effect is mediated centrally. The observation that piperonyl butoxide and SKF-525A themselves caused significant hypothermia indicates that the use of these compounds should be avoided when body temperature is being followed in drug metabolism experiments.