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S D Cohen

Publications and source records attributed to S D Cohen.

At least 19 recordsLinked to original sources
PubMed2000

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.

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PubMed1999

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.

Acetaminophen↗
PubMed1999

Metallothionein-I/II knockout mice are sensitive to acetaminophen-induced hepatotoxicity.

The purpose of this study was to examine whether intracellular metallothionein (MT) protects against acetaminophen hepatotoxicity. MT-I/II knockout (MT-null) and control mice were given acetaminophen (150-500 mg/kg i.p.), and liver injury was assessed 24 h later. MT-null mice were more susceptible than controls to acetaminophen-induced lethality and hepatotoxicity, as evidenced by elevated serum enzyme activities and histopathology. Zinc pretreatment, a method of MT induction, protected against acetaminophen hepatotoxicity in control mice, but not in MT-null mice. The susceptibility of MT-null mice to acetaminophen hepatotoxicity was not due to the increased acetaminophen bioactivation, as cytochrome P-450 enzymes, and acetaminophen-reactive metabolites in bile and urine were not increased in MT-null mice. Western blots of liver cytosol indicated that acetaminophen covalent binding at 4 h increased with acetaminophen dose, but there was no consistent difference between control and MT-null mice. Acetaminophen injection depleted cellular glutathione similarly in both control and MT-null mice, but produced more lipid peroxidation in MT-null mice, as evidenced by the abundance of thiobarbiturate-reactive substances, and by immunohistochemical localization of 4-hydroxynonenal and malondialdehyde protein adducts. MT-null hepatocytes were more susceptible than control cells to oxidative stress and cytotoxicity produced by N-acetylbenzoquinoneimine, a reactive metabolite of acetaminophen, as determined by oxidation of 2', 7'-dichlorofluorescin diacetate and lactate dehydrogenase leakage. In summary, this study demonstrated that MT deficiency renders animals more vulnerable to acetaminophen-induced hepatotoxicity. The increased sensitivity does not appear to be due to increased acetaminophen activation, glutathione depletion, or covalent binding, but appears to be associated with the antioxidant role of MT.

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PubMed1998

Acetaminophen hepatotoxicity in tumor necrosis factor/lymphotoxin-alpha gene knockout mice.

Recent evidence suggests that macrophages and/or other nonparenchymal cells may release important mediators contributing to the hepatic necrosis induced by high doses of acetaminophen (APAP). The nature and causative role of these mediators has remained elusive, however. To investigate the role of the proinflammatory cytokine, tumor necrosis factor (TNF) in the initiation and early propagation of APAP-induced liver injury, we have used mice deficient in both TNF and the closely related lymphotoxin-alpha (LT-alpha). Male TNF/LT-alpha knockout mice and C57BL/6 wild-type mice were treated with a hepatotoxic dose of APAP (400 mg/kg, intraperitoneally), and the development of liver injury was monitored over 8 hours. Both genotypes exhibited similar basal activities of hepatic cytochrome P450 2E1 and 1A2. After APAP administration, both the rate of glutathione consumption and the extent of subsequent selective protein binding did not differ significantly in the knockout and wild-type mice. The TNF/LT-alpha-deficient mice developed severe centrilobular necrosis and exhibited highly increased levels of serum alanine aminotransferase and aspartate aminotransferase, the extent of which was not significantly different from that in wild-type mice. In C57BL/6 mice exposed to APAP, no increases in hepatic transcripts of TNF or LT-alpha were found by reverse transcription-polymerase chain reaction, nor was immunoreactive serum TNF detected by enzyme-linked immunosorbent assay over 8 hours posttreatment. These data indicate that, in the absence of the genes encoding for TNF and LT-alpha, APAP bioactivation was not altered and mice still developed severe hepatic necrosis. Thus, TNF is unlikely to be a key mediator in the early pathogenesis of APAP-induced hepatotoxicity.

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PubMed1998

Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice.

Acetaminophen (APAP) hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), that is capable of binding to cellular macromolecules. At least two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated in this reaction in mice. To test the combined roles of CYP1A2 and CYP2E1 in an intact animal model, a double-null mouse line lacking functional expression of CYP1A2 and CYP2E1 was produced by cross-breeding Cyp1a2-/- mice with Cyp2e1-/- mice. Animals deficient in the expression of both P450s developed normally and exhibited no obvious phenotypic abnormalities. Comparison of the dose-response to APAP (200-1200 mg/kg) indicated that double-null animals were highly resistant to APAP-induced toxicity whereas the wild-type animals were sensitive. Administration of 600 to 800 mg/kg of this drug to male wild-type animals resulted in increased plasma concentrations of liver enzymes (alanine aminotransferase, sorbitol dehydrogenase), lipidosis, hepatic necrosis, and renal tubular necrosis. In contrast, when APAP of equivalent or higher dose was administered to the double-null mice, plasma levels of liver enzymes and liver histopathology were normal. However, administration of 1200 mg of APAP/kg to the double-null mice resulted in infrequent liver lipidosis and mild kidney lesions. Consistent with the protection from hepatotoxicity, the expected depletion of hepatic glutathione (GSH) content was significantly retarded and APAP covalent binding to hepatic cytosolic proteins was not detectable in the double-null mice. Likewise, in vitro activation of APAP by liver microsomes from the double-null mice was approximately one tenth of that in microsomes from wild-type mice. Thus, the protection against APAP toxicity afforded by deletion of both CYP2E1 and CYP1A2 likely reflects greatly diminished production of the toxic electrophile, NAPQI.

Acetaminophen↗
PubMed1998

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.

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PubMed1997

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.

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PubMed1996

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.

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PubMed1996

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.

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PubMed1996

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.

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PubMed1996

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.

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PubMed1996

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.

Acetaminophen↗
PubMed1996

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.

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PubMed1996

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.

Acetaminophen↗
PubMed1996

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.

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PubMed1995

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.

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PubMed1995

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.

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