Acetaminophen inhibits NF-kappaB activation by interfering with the oxidant signal in murine Hepa 1-6 cells.

A toxic dose of acetaminophen (APAP) reduces the activity of NF-kappaB in mouse liver. NF-kappaB inactivation may be important for APAP toxicity, as this transcription factor can play a central role in maintaining hepatic viability. We recently reported that APAP likewise inhibits serum growth factor activation of NF-kappaB in a mouse hepatoma cell line (Hepa 1-6 cells). Here we present evidence that APAP's antioxidant activity may be involved in this NF-kappaB inhibition in Hepa 1-6 cells. Like the antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC), APAP was found to suppress the H(2)O(2)-induced oxidation of an intracellular reactive oxygen species probe (dihydrodichlorofluorescein) in Hepa 1-6 cells. Treatment of Hepa 1-6 cells with H(2)O(2) was sufficient for NF-kappaB activation and IkappaBalpha degradation, and APAP was able to block both of these events. The APAP inhibition of NF-kappaB activation by serum growth factors may also be due to APAP's antioxidant activity, as the antioxidants NAC and PDTC likewise inhibit this activation. The potential role of NF-kappaB and oxidant-based growth factor signal transduction in APAP toxicity is discussed.

Modulation of serum growth factor signal transduction in Hepa 1-6 cells by acetaminophen: an inhibition of c-myc expression, NF-kappaB activation, and Raf-1 kinase activity.

Acetaminophen (APAP) is a widely used analgesic and antipyretic that can lead to severe liver damage when taken at excessive doses. APAP toxicity results when cytochrome P450-generated APAP metabolites trigger an oxidative stress and covalently modify target proteins. APAP has also been reported to inhibit cells from completing S-phase through a cytochrome P450-independent mechanism, raising the possibility that APAP may directly suppress liver regeneration and repair. Here we show that APAP also inhibits entrance of Hepa 1-6 cells into the cell cycle by blocking a number of events associated with the G0-G1 transition. We have found that APAP inhibits serum growth factor activation of c-myc expression, NF-kappaB DNA binding, and Raf kinase. Therefore, the ability of APAP to inhibit passage of cells through both G1 and S phases might interfere with organ regeneration and thus exacerbate acute liver damage caused by APAP.

Inhibition of protein phosphatase activity and changes in protein phosphorylation following acetaminophen exposure in cultured mouse hepatocytes.

Protein phosphorylation was determined in cultured mouse hepatocytes exposed to an hepatotoxic concentration of acetaminophen (APAP) for selected times up to 12 h. Cultures were radiolabled with 32P-orthophosphoric acid and the cell extracts were analyzed by 2D gel electrophoresis and autoradiography. APAP exposure selectively increased the phosphorylation state of proteins of molecular weight 22, 25, 28, and 59 kDa and decreased the phosphorylation of a 26-kDa protein. Evidence is presented that these changes (1) are dependent on cytochrome P-450 activation of APAP; (2) occur well before enzyme leakage in this in vitro model; (3) are not likely attributed to GSH depletion alone; (4) are in part mimicked by okadaic acid, calyculin A, and cantharidic acid, three structurally distinct inhibitors of protein phosphatases 1 and 2A; and (5) are paralleled by a decline in protein phosphatase activity. The physiological consequences of protein phosphatase inactivation could be significant in APAP overdose since these enzymes are involved in the dephosphorylation of regulatory proteins that control many cell functions. This study also provides the first evidence for disruption in signal transduction pathways as a response to or component of APAP-induced hepatic injury.

Selective protein covalent binding and target organ toxicity.

Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the tartgeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity.

Selective protein arylation and acetaminophen-induced hepatotoxicity.

More than 20 years have passed since the early reports of acute hepatotoxicity with APAP overdose. During that period investigative research to discover the "mechanism" underlying the toxicity has been conducted in many species and strains of intact animals as well as in a variety of in vitro and culture systems. Such work has clarified the primary role of biotransformation and the protective role of GSH. Understanding the former provides explanations for the toxic interactions which may occur with alcohol or other xenobiotics, while understanding of the latter led to the development of antidotes for the treatment of acute poisoning. Acetaminophen (APAP)-induced hepatotoxicity: roles for protein arylation. Initiating events in toxicity require biotransformation of APAP to NAPQI followed by arylation of several important proteins with subsequent alteration of protein structure and function. The immediate consequence of the alterations is detectable in several organelles and these may represent multiple initiating events which are depicted as acting in concert to cause cell injury (large arrowheads). Arylation of cytosolic 58-ABP with subsequent translocation to the nucleus is depicted as a possible signaling mechanism for determining outcome at the cell or organ level (within dotted boundary). For simplicity NAPQI's potentials for oxidizing protein sulfhydryls and direct binding to DNA have been omitted. Significant light has also been shed on the biochemical and cellular events which accompany APAP-induced hepatotoxicity. However, such studies have not identified a unique mechanism of toxicity that is universally accepted. The recent identification of several protein targets which become arylated during toxicity--along with the findings that arylation of some of those target proteins results in loss of protein function--demonstrates that covalent binding does, indeed, have biological consequences and is not merely an indicator of the fleeting presence of reactive electrophiles. These observations further suggest that multiple independent insults to the cell may be involved in toxicity. it is now apparent that the concept of a multistage process that involves both initiation and progression events is appropriate for APAP toxicity, and it is unlikely that a unique initiating event will ever be identified. In light of recent findings it is more likely that a number of such cellular events occur very early after toxic overdosage, and that they collectively set in motion and perpetuate the biochemical, cellular, and molecular processes which will determine outcome. The importance of 58-ABP arylation with early, apparently selective, translocation to the nucleus remains to be elucidated. To date there is nothing to suggest that this represents an initiating event in toxicity. rather it is plausible that the translocation may play a role in signaling electrophile presence and in calling for cellular defense against electrophile insult. This is reflected in the hypothetical model presented in Fig. 3. Critical experimental testing of this model will advance our understanding of the cellular and molecular responses to toxic electrophile insult.

Identification of a 54-kDa mitochondrial acetaminophen-binding protein as aldehyde dehydrogenase.

The covalent binding of acetaminophen (APAP) to mitochondrial proteins has been postulated to alter the function of the organelle and contribute to the development of the hepatotoxicity upon APAP overdose. To identify the arylated proteins CD-1 mice were administered 600 mg/kg APAP and Western blots of mitochondrial proteins collected 4 hr after dosing were probed with anti-APAP antibodies. Five proteins of approximately 75, 60, 54, 44, and 33 kDa were detected on 1-D gels. Immunostaining of the 54-kDa protein was most intense. Mitochondria were subsequently fractionated into inner and outer membrane, matrix, and intermembrane space using digitonin, sonication, and differential centrifugation. The 54-kDa target was most highly enriched in the inner membrane fraction. On 2-D gels this 54-kDa band was resolved into three arylated proteins with pIs of 6.4, 6.6, and 7.1. The pI 7.1 protein was excised from 55 2-D gels, and, after tryptic digestion, the two best-resolved peptides were sequenced and found to be 100% identical to mitochondrial aldehyde dehydrogenase. Coincident with APAP covalent binding the specific activity of the enzyme decreased; by the time of maximal covalent binding at 4 hr after APAP, the activity was 60% of control. Since the enzyme is an abundant mitochondrial dehydrogenase, its decreased activity may contribute to the impaired mitochondrial function observed after APAP administration.

Protein arylation precedes acetaminophen toxicity in a dynamic organ slice culture of mouse kidney.

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

Protection by clofibrate against acetaminophen hepatotoxicity in male CD-1 mice is associated with an early increase in biliary concentration of acetaminophen-glutathione adducts.

Repeated treatment with clofibrate (CFB) significantly increased hepatic glutathione (GSH) content and also diminished acetaminophen's (APAP) selective protein arylation, GSH depletion, and severity of hepatocellular necrosis. The present work was conducted to evaluate the role of elevated GSH and APAP detoxifying pathways in the amelioration of APAP's toxicity by CFB. Male CD-1 mice received 500 mg CFB/kg, i.p., daily for 10 days. Controls were given corn oil vehicle. They were challenged with 700 mg APAP/kg in 50% propylene glycol/water after an overnight fast. Results indicate that CFB pretreatment had no effect on 24-hr urinary excretion of APAP-glucuronide, sulfate, or GSH-derived conjugates; however, there was 50% less unchanged APAP excreted in urine of CFB-pretreated mice. CFB also did not alter microsomal UDP-glucuronyl transferase activity toward APAP in vitro. However, elimination of APAP from plasma and liver was much greater in CFB-pretreated mice. This was accompanied by elevated biliary APAP-GSH content in CFB-pretreated mice at 2 hr after APAP dosing with diminished levels in bile at 12 hr. The CFB-induced increase in biliary excretion of APAP-GSH may mediate the protection against APAP-induced hepatotoxicity.

Evidence suggesting the 58-kDa acetaminophen binding protein is a preferential target for acetaminophen electrophile.

Acetaminophen is an analgesic and antipyretic which causes liver toxicity in humans and experimental animals with overdose. Acetaminophen (APAP) covalent binding to a cytosolic protein of approximately 58 kDa (58-ABP) has been associated with target organ toxicity. Since hepatic content of 58-ABP varies, studies were conducted to determine if this influences APAP binding to other target proteins. In the liver, the amount of 58-ABP varied with individual male CD-1 mice, but in kidneys of the same mice there was no such variability in 58-ABP content. All male A/J mice tested had comparatively little detectable 58-ABP in liver cytosol. Similarly, female CD-1 mice had low 58-ABP content compared to males; however, administration of testosterone propionate to females significantly increased 58-ABP content in liver cytosol. At 4 hr after challenge of mice from the above-described groups with 600 mg APAP/kg, cytosolic covalent binding to proteins was determined by Western blot analysis with anti-APAP antibody. The Western blots were then stripped of antibody and blocking agents and reprobed with antibody prepared against purified 58-ABP (anti-58-ABP). In the liver, the level of APAP bound to the 58-ABP target corresponded with 58-ABP content. In cases where 58-ABP was poorly expressed, APAP adducts to other protein targets were more prominently detected. In the kidneys of the male CD-1 mice 58-ABP arylation by APAP varied little among animals, reflecting the relatively consistent levels of renal 58-ABP. These data suggest that binding to the 58-ABP may spare other potential targets of APAP electrophile attach and support a role of the 58-ABP as a preferred target of APAP electrophile in cytosol.

Acetaminophen-arylated proteins are detected in hepatic subcellular fractions and numerous extra-hepatic tissues in CD-1 and C57B1/6J mice.

To identify acetaminophen (APAP)-bound proteins in addition to the major 44 and 58 kDa APAP-binding proteins (Bartolone et al., 1992, Toxicol. Appl. Pharmacol. 113. 19-9; Pumford et al., 1992, Biochem. Biophys. Res. Commun. 182, 1348-1355; Bulera et al., 1995, Toxicol, Appl. Pharmacol. 134, 313-320), we investigated subcellular localization of liver proteins and tissue distribution of proteins arylated by a hepatotoxic dose of APAP in CD-1 and C57B1/6J mice. Western blot analysis with affinity-purified, anti-APAP antibodies allowed the detection of covalently bound proteins in liver mitochondria, nuclei, membrane, cytosol, and microsomes. Enzyme market assays revealed that subcellular fractions were 90-98% pure. The lack of contamination from other isolated subcellular fractions indicates that covalently bound proteins were specific to the particular subcellular fraction. APAP-arylated proteins with molecular weights similar to those detected in the liver were found in cytosolic fractions from kidney, lung, pancreas, heart, skeletal muscle, and stomach. The presence of arylated proteins in extra-hepatic organs suggests that other organs may be susceptible to APAP toxicity and may contain critical protein targets that are important in APAP toxicity. In contrast, covalently bound proteins were not detected in cytosols isolated from spleen, small intestine, brain, and testis. The characterization of the APAP-arylated proteins identified in this study will aid in elucidating the mechanism of APAP-induced toxicity.

Sex- and age-dependent acetaminophen hepato- and nephrotoxicity in Sprague-Dawley rats: role of tissue accumulation, nonprotein sulfhydryl depletion, and covalent binding.

Acetaminophen (APAP) produces sex-dependent nephrotoxicity and hepatotoxicity in young adult Sprague-Dawley (SD) rats and age-dependent toxicity in male rats. There is no information regarding the susceptibility of aging female SD rats to APAP toxicity. Therefore, the present studies were designed to determine if sex-dependent differences in APAP toxicity persist in aging rats and to elucidate factors contributing to sex- and age-dependent APAP hepatotoxicity and nephrotoxicity. Young adult (3 months old) and aging (18 months old) male and female rats were killed from 2 through 24 hr after receiving APAP (0-1250 mg/kg, ip) containing [ring-14C]APAP. Trunk blood was collected for determination of blood urea nitrogen (BUN) concentration, serum alanine aminotransferase (ALT) activity, and plasma APAP concentration; urine was collected for determination of glucose and protein excretion; and liver and kidneys were removed for determination of tissue glutathione (GSH) concentration, APAP concentration, and covalent binding. APAP at 1250 mg/kg induced nephrotoxicity (as indicated by elevations in BUN concentration) in 3-month-old females but not males, whereas APAP induced hepatotoxicity (as indicated by elevations in serum ALT activity) in 3-month-old males but not females. Sex differences in APAP toxicity were no longer apparent in 18-month-old rats. APAP at 750 mg/kg ip produced liver and kidney damage in 18-month-old but not 3-month-old male and female rats. No consistent sex- or age-dependent differences in serum, hepatic, and renal APAP concentrations were observed that would account for differences in APAP toxicity. No sex- or age-dependent differences in tissue GSH depletion or covalent binding of radiolabel from APAP in livers or kidneys were observed following APAP administration. Utilizing an affinity-purified polyclonal antibody raised against APAP, arylated proteins with electrophoretic mobility similar to those observed in mice were prominent in rat livers following APAP administration to 3- and 18-month-old rats of both sexes. In contrast, no arylated proteins were detected in any rat kidneys following APAP administration. Absence of immunochemically detectable proteins in rat kidney following APAP administration is in direct contrast to observations in mice and supports the hypothesis that mechanisms of APAP hepatotoxicity and nephrotoxicity in rats and mice are distinctly different. In conclusion, sex differences in APAP toxicity are observed only in young adult (3-month-old) rats and sex differences are organ-specific with males more susceptible to hepatotoxicity and females more susceptible to nephrotoxicity. Aging rats are more susceptible to APAP-induced damage to both the liver and the kidney than are 3-month-old rats but sex differences are no longer apparent in 18-month-old rats. The mechanisms contributing to sex- and age-dependent differences in APAP toxicity cannot be attributed to differences in tissue APAP concentrations, GSH depletion, or covalent binding.

Protection against acetaminophen hepatotoxicity by a single dose of clofibrate: effects on selective protein arylation and glutathione depletion.

Previous reports demonstrated that repeated administration of peroxisome proliferators protects against acetaminophen (APAP) hepatotoxicity in mice. This protection was associated with a decrease in APAP's selective protein arylation and glutathione depletion. This study was conducted to determine if a single dose of clofibrate (CFB), rather than repeated doses, would similarly prevent APAP toxicity. CD-1 male mice received a single dose of 500 mg CFB/kg and controls were given corn oil 24 hr prior to APAP challenge. After an 18-hr fast, mice were challenged with 800 mg APAP/kg (in 50% propylene glycol) and killed at 4 or 12 hr. Other mice similarly pretreated were killed without APAP challenge. The results showed that pretreatment with a single CFB dose significantly decreased APAP-induced hepatotoxicity. At 12 hr after APAP plasma sorbitol dehydrogenase activity and the severity of hepatocellular necrosis were decreased in CFB pretreated mice. Surprisingly, no differences in hepatic nonprotein sulfhydryl (NPSH) depletion or selective arylation of target proteins in cytosol were observed at 4 hr after APAP challenge. Neither did a single dose of CFB significantly alter hepatic NPSH content prior to APAP challenge. These results indicate that protection against APAP hepatotoxicity by CFB does not require repeated administration, and the absence of significant alterations in APAP's selective protein arylation or glutathione depletion suggests that the protection against APAP hepatotoxicity after a single treatment with CFB may differ mechanistically from the protection observed after repeated CFB dosing.

Acetaminophen nephrotoxicity in the CD-1 mouse. II. Protection by probenecid and AT-125 without diminution of renal covalent binding.

Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr fasted, 3-month-old male CD-1 mice results in necrosis of the convoluted renal proximal tubules with a corresponding elevation of plasma urea nitrogen (BUN). Administration of the gamma-glutamyl transpeptidase inhibitor, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydroxy- 5-isoxazoleacetic acid (AT-125) (50 mg/kg, ip), to mice 30 min before APAP significantly diminished the APAP-induced histopathologic damage and BUN elevation. Administration of the organic-anion transport inhibitor, probenecid (150 mg/kg, ip), 30 min before APAP challenge also protected against the APAP-induced elevation of BUN and detectable histopathologic changes. By contrast, pretreatment of mice with the cysteine conjugate beta-lyase inhibitor, (aminooxy)acetic acid (100 mg/kg, ip), 1 hr before APAP did not alter nephrotoxicity. None of the pretreatments altered the APAP-induced elevation of plasma sorbitol dehydrogenase activity, nor were there any detectable changes in liver histopathology after APAP challenge. Despite the protective effects of both probenecid and AT-125 against nephrotoxicity, they did not affect either the level of immunochemically detectable covalent binding to protein or the depletion of renal glutathione at 4 hr after APAP. Thus, the protection appears independent of effects on renal APAP uptake or activation and indirectly suggests that an APAP-glutathione conjugate may contribute to the observed nephrotoxicity.

Evidence for common binding of acetaminophen and bromobenzene to the 58-kDa acetaminophen-binding protein.

Acetaminophen (APAP) toxicity has been closely associated with covalent binding to a cytosolic protein of approximately 58 kDa (58-ABP). To determine if metabolites of other toxicants might also selectively target this protein, studies were conducted with bromobenzene (BrB). Mice were given phenobarbital (80 mg/kg/d x 4 d) and were killed 4 h after challenge with 800 mg BrB/kg. Liver cytosols from BrB-treated or naive mice were incubated with an APAP activating system. Cytosolic fractions were analyzed for APAP binding by Western blotting with anti-APAP antibody. Binding to 58-ABP was selectively decreased in liver cytosol from BrB-treated mice while binding to other targets was minimally affected. Western blotting of the same samples with anti-58-ABP antisera showed that this decrease in binding did not result from diminished 58-ABP content. HPLC analysis of APAP-N-acetyl cysteine conjugate formation in vitro indicates that APAP activation was not altered in the incubates with cytosol from BrB-treated mice. These results suggest that the 58-ABP may be a common target for electrophiles in reactive intermediate toxicity.

Identification of the mouse liver 44-kDa acetaminophen-binding protein as a subunit of glutamine synthetase.

Identification of proteins that covalently bind acetaminophen (APAP) is essential for a clearer understanding of the hepatotoxicity that results after an APAP overdose. Birge et al. (1991) have reported that in our mouse model system a membrane-associated 44-kDa acetaminophen-binding protein is the earliest target detected immunochemically following administration of an hepatoxic dose of APAP. To identify this 44-kDa protein, liver microsomes from mice administered 600 mg APAP/kg were extracted with Triton X-114 and the resulting aqueous fraction was adsorbed to DEAE-cellulose. Further purification of the DEAE eluate by reverse-phase HPLC and by two-dimensional (2D) gel electrophoresis indicated that four proteins of approximately 44 kDa with pIs ranging from 6.7-7.0 were targeted by APAP. The most highly arylated of these 44-kDa isoforms (pI 7.0) was excised from 2D gels, digested with trypsin, and the resulting peptides were separated by reverse-phase HPLC. The two best resolved peptides were sequenced and 14 of the 15 amino acids detected were found to be identical to subunits of mouse liver glutamine synthetase (EC 6.3.1.2). Purification of glutamine synthetase from APAP-treated mice confirmed that the enzyme is indeed targeted by APAP in vivo. Since the intracellular activity of glutamine synthetase is significantly decreased after the administration of APAP to hepatocytes in culture, it is likely that the arylation of this enzyme may be involved in the ensuing hepatotoxicity.

A comparative study of mouse liver proteins arylated by reactive metabolites of acetaminophen and its nonhepatotoxic regioisomer, 3'-hydroxyacetanilide.

Acetaminophen (4'-hydroxyacetanilide), a widely used analgesic/antipyretic drug, is hepatotoxic in large doses, whereas the m-hydroxy isomer of acetaminophen, 3'-hydroxyacetanilide, is not hepatotoxic. Both are oxidized by mouse liver cytochromes P-450 to reactive metabolites that bind covalently to hepatic proteins. Because previous studies have shown that peak levels of liver protein adducts formed after the administration of each of these compounds to mice are nearly equivalent, and because liver protein adduct formation correlates with hepatotoxicity caused by acetaminophen in mice, we investigated the abundance and patterns of protein adducts formed by acetaminophen and its regioisomer for significant differences. Hepatotoxic doses of acetaminophen to mice significantly altered the abundances of several liver proteins 2 h after dosing as revealed by densitometric analysis of two-dimensional electrophoretic patterns of these proteins. The same analysis after the administration to mice of 3'-hydroxyacetanilide indicated that this nonhepatotoxic regioisomer of acetaminophen caused several similar changes in protein patterns, but also revealed some significant differences. Binding of radiolabeled acetaminophen and 3'-hydroxyacetanilide to hepatic proteins corroborated and extended these results. Two hours after the administration of 14C-labeled analogs of these two compounds to mice, at a time when their extent of total covalent binding to hepatic proteins is approximately equivalent, there are many similarities but also some differences in selectivity of proteins that are adducted, as revealed by both one-dimensional and two-dimensional gel electrophoresis followed by phosphorimage analysis of radiolabel bound to protein bands. Moreover, protein adducts formed from 3'-hydroxyacetanilide were found to be less stable than those formed from acetaminophen under the conditions of electrophoretic analysis. Furthermore, a comparison of radiodetection and immunodetection of protein adducts formed from acetaminophen with an antibody specific for acetaminophen protein adducts indicates that the antibody detects most of the same proteins that are radiolabeled and that the relative quantitative contribution of various adducts to the overall covalent binding of acetaminophen to proteins is approximately the same by both methods. Thus, 3'-hydroxyacetanilide should prove to be a useful tool to aid in the discrimination of hepatic acetaminophen protein adducts that may be critical or noncritical to survival of hepatocytes.

Gender-related differences in susceptibility to acetaminophen-induced protein arylation and nephrotoxicity in the CD-1 mouse.

Acetaminophen (APAP) is a commonly used analgesic and antipyretic agent which, in high doses, causes liver and kidney necrosis in man and animals. Damage in both target organs is greatly dependent upon biotransformation. However, in the CD1 mouse only males exhibit cytochrome P450-dependent nephrotoxicity and selective protein covalent binding. The lack of renal toxicity in female mice may reflect the androgen dependence of renal CYP2E1. To study this, female mice were pretreated with testosterone propionate and then challenged 6 days later with APAP. Groups of control males and females were similarly challenged with APAP for comparison. All groups exhibited hepatotoxicity after APAP with similar glutathione (GSH) depletion, covalent binding, centrilobular necrosis, and elevation of plasma sorbitol dehydrogenase activity. By contrast, APAP-induced nephrotoxicity occurred only in males and in the females pretreated with testosterone. No nephrotoxicity was evident in APAP-challenged control females. The selective pattern of hepatic and renal protein arylation previously reported for male mice was similarly observed in testosterone-pretreated female mice. Western blot analysis of microsomes showed that testosterone increased renal CYP2E1 levels without altering hepatic CYP2E1. Testosterone pretreatment, in vivo, also resulted in increased activation of APAP in vitro in kidney microsomes with no effect on the in vitro activation of APAP in liver microsomes. These data suggest that APAP-mediated GSH depletion, covalent binding, and toxicity in the kidneys of testosterone-pretreated females results from increased APAP activation by the testosterone-induced renal CYP2E1. This further suggests that renal, rather than hepatic, biotransformation of APAP to a toxic electrophile is central to APAP-induced nephrotoxicity in the mouse.

Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: correspondence of covalent binding with toxicity.

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-microns paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity.

Peroxisome proliferators have been shown to diminish acetaminophen (APAP) hepatotoxicity (Biochem. Pharmacol. 43, 1395, 1992). To investigate the mechanistic basis for this protection CD-1 male mice were given corn oil or 500 mg clofibrate (CFB)/kg, ip, daily for 10 days. They were then fasted overnight and either killed without challenge or at 4 or 12 hr after challenge with 800 mg APAP/kg (in 50% propylene glycol). At 12 hr, hepatotoxicity was evidenced by elevated plasma sorbitol dehydrogenase and histopathology in corn oil but not in CFB-pretreated mice. At 4 hr after APAP treatment, hepatic glutathione (GSH) depletion and selective arylation of the major APAP target proteins were both greatly diminished by CFB pretreatment. Western blot analysis with the anti-58 antibody of liver cytosol from unchallenged mice showed no apparent changes in the levels of the 58-kDa major APAP target protein with CFB treatment. These findings suggest that protection could be the result of diminished net availability of generated electrophile. In vitro, measurements indicated that the specific activity in microsomes for APAP oxidation by cytochrome P450 was not changed by CFB treatment; whereas GSH S-transferase activity in cytosol was decreased by 25%. Pretreatment with CFB also produced a significant elevation in hepatic GSH. These studies indicate that protection by CFB might result from increased availability of hepatic GSH which could trap APAP electrophile nonenzymatically, thereby decreasing covalent binding and preventing toxicity.