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 andhepatotoxicity in young adult Sprague-Dawley (SD) rats and age-dependenttoxicity in male rats. There is no information re garding thesusceptibility of aging female SD rats to APAP toxicity. Therefore,the present studies were designed to determine if sex-dependentdifferences in APAP toxicity persist in aging rats and to elucidatefactors contributing to sex- and age-dependent APAP hepatotoxicityand nephrotoxicity. Young adult (3 months old) and aging (18months old) male and female rats were killed from 2 through24 hr after receiving APAP (0–1250 mg/kg, ip) containing[ring-¹⁴C]APAP. Trunk blood was collected for determinationof blood urea nitrogen (BUN) concentration, serum alanine aminotransferase(ALT) activity, and plasma APAP concentration; urine was collectedfor determination of glucose and protein excretion; and liverand 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 elevationsin BUN concentration) in 3-month-old females but not males,whereas APAP induced hepatotoxicity (as indicated by elevationsin serum ALT activity) in 3-month-old males but not females.Sex differences in APAP toxicity were no longer apparent in18-month-old rats. APAP at 750 mg/kg ip produced liver and kidneydamage 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 accountfor differences in APAI toxicity. No sex- or age-dependent differencesin tissue GSH depletion or covalent binding of radiolabel fromAPAP in livers or kidneys were observed following APAP administration.Utilizing an affinity-purified polyclonal antibody raised againstAPAP, arylated proteins with electrophoretic mobility similarto those observed in mice were prominent in rat livers followingAPAP administration to 3- and 18-month-old rats of both sexes.In contrast, no arylated proteins were detected in any rat kidneysfollowing APAP administration. Absence of immunochemically detectableproteins in rat kidney following APAP administration is in directcontrast to observations in mice and supports the hypothesisthat mechanisms of APAP hepatotoxicity and nephrotoxicity inrats and mice are distinctly different. In conclusion, sex differencesin APAP toxicity are observed only in young adult (3-month-old)rats and sex differences are organ-specific with males moresusceptible to hepatotoxicity and females more susceptible tonephrotoxicity. Aging rats are more susceptible to APAP-induceddamage to both the liver and the kidney than are 3-month-oldrats but sex differences are no longer apparent in 18-month-oldrats. The mechanisms contributing to sex- and age-dependentdifferences in APAP toxicity cannot be attributed to differencesin tissue APAP concentrations, GSH depletion, or covalent binding.     

Increased hepatotoxicity of acetaminophen in Hsp70i knockout mice

The effect of the inducible forms of 70 kDa heat shock protein (Hsp70i) on acetaminophen (APAP) hepatotoxicity was assessed in an Hsp70i knockout mouse model. Absence of the Hsp70i protein in liver was verified by monitoring Hsp levels in knockout and control mice after heat stress (41.5 degrees C water bath immersion for 30 min). Hsp70i knockout mice were more susceptible to APAP-induced hepatotoxicity than controls, as indicated by elevated serum alanine aminotransferase activities 24 and 48 h after the APAP dose. Increased APAP hepatotoxicity in knockout mice was verified by morphological evaluation of liver sections. The difference in toxic response to APAP between knockout and control strain mice could not be attributed to differences in APAP bioactivation, assessed by measurement of CYP2E1 and glutathione S-transferase activities, hepatic nonprotein sulfhydryl content, or covalent binding of reactive APAP metabolites to proteins. Pretreatment with transient hyperthermia to produce a general upregulation of Hsps resulted in decreased APAP hepatotoxicity in both the knockout and control strains. Among thermally-pretreated mice, hepatotoxicity of APAP was greater in the knockouts compared with the control strain. These observations suggest that increased Hsp70i expression in response to APAP acts to limit the extent of tissue injury. Results further suggest that other factors related to heat stress can also contribute to protection against APAP toxicity.     

Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.     

Resistance to acetaminophen-induced hepatotoxicity in glutathione S-transferase Mu 1-null mice

We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3β and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.     

Role of pharmacokinetics and metabolism in the enhanced susceptibility of middle-aged male Sprague-Dawley rats to acetaminophen nephrotoxicity

Middle-aged male Sprague-Dawley (SD) rats (9-12 months) are more susceptible to acetaminophen (APAP)-induced nephrotoxicity than are young (2-3 months) adult males. The present studies were designed to evaluate the role of pharmacokinetics and renal and hepatic metabolism of APAP in age-dependent nephrotoxicity. Following 750 mg/kg APAP, ip, a nephrotoxic dosage in 12-month-old but not 3-month-old rats, renal cortical APAP concentrations were significantly greater in 12-month-old compared with 3-month-old SD rats at 3, 4, and 6 hr after treatment. Renal medullary APAP concentrations in 12 month-old rats were significantly greater than in 3-month-old rats at 2, 3, and 5 hr after treatment. Serum APAP concentrations were significantly elevated in 12-month-old compared with 3-month-old rats from 2 through 5 hr after APAP (750 mg/kg ip). However, APAP tissue/serum concentration ratios were similar in 3- and 12-month-old rats, indicating that differences in tissue concentration were secondary to increased serum concentrations in older rats. Conjugated APAP metabolites in blood were similar in 3- and 12-month-olds during the initial 2-3 hr after 750 mg/kg APAP, ip, but began to accumulate in 12-month-old but not 3-month-old rats within 6-8 hr after APAP administration, perhaps secondary to declining renal function. After 500 mg/kg APAP, iv, blood APAP concentrations were markedly elevated in 12-month-old compared with 3-month-old rats during the entire course of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonalcoholic steatohepatitic (NASH) mice are protected from higher hepatotoxicity of acetaminophen upon induction of PPARalpha with clofibrate

The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARalpha via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. (14)C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by (3)H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARalpha was tested. PPARalpha was downregulated in NASH. To investigate whether downregulation of PPARalpha in NASH is the critical mechanism of compromised liver tissue repair, PPARalpha was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARalpha expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity.     

Evidence against deacetylation and for cytochrome P450-mediated activation in acetaminophen-induced nephrotoxicity in the CD-1 mouse

Acetaminophen (APAP) administration (600 mg/kg, po) results in proximal tubular necrosis in 18-hr fasted, 3-month-old male CD-1 mice. This study was undertaken to determine if deacetylation of APAP to p-aminophenol (PAP) is a prerequisite to nephrotoxicity in the mouse, as it is in the Fischer rat. Administration of either APAP or PAP to mice resulted in significant elevations of plasma urea nitrogen and marked proximal tubular necrosis at 12 hr after dosing. Prior inhibition of APAP deacetylation by the carboxylesterase inhibitors bis(p-nitrophenyl) phosphate or tri-o-tolyl-phosphate did not alter APAP hepatotoxicity or nephrotoxicity. By contrast, pretreatment with the MFO inhibitor piperonyl butoxide decreased APAP nephrotoxicity but not that of PAP. Immunochemical analysis of kidneys from APAP-treated mice demonstrated covalently bound APAP but no binding was detected after mice were treated with a nephrotoxic dose of PAP. Since the antibody used has been characterized as being directed primarily against the N-acetyl moiety of bound APAP metabolite and since it did not react with kidney proteins of mice given a nephrotoxic dose of PAP, it is unlikely that APAP deacetylation preceded binding or that acetylation of bound PAP occurred. Taken together, these findings indicate that in the CD-1 mouse, APAP-induced nephrotoxicity differs from that previously described for the Fischer rat and likely involves cytochrome P450-dependent activation and subsequent covalent binding of a metabolite without prior deacetylation.     

Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity

The biliary excretion of acetaminophen (APAP) is reduced in transport deficient (TR⁻) hyperbilirubinemic rats lacking the multidrug resistance-associated protein 2 (Mrp2). This mutant strain of Wistar rats has impaired biliary excretion of organic anions and increased hepatic glutathione. The rational for this study was to determine if there is an altered risk for liver damage by APAP in the absence of Mrp2. Therefore, the susceptibility of TR⁻ rats to APAP hepatotoxicity was investigated. Male Wistar and TR⁻ rats were fasted overnight before APAP treatment (1 g/kg). Hepatotoxicity was assessed 24 h later by plasma sorbitol dehydrogenase activity and histopathology. In other studies, TR⁻ rats received buthionine sulfoximine before APAP to reduce hepatic glutathione to values similar to those in Wistar rats. mRNA expression of APAP metabolizing enzymes was also measured in naïve animals. Wistar rats treated with APAP showed significant elevations in plasma sorbitol dehydrogenase activity, while no increases in enzyme activity were observed in TR⁻ rats. Histopathology was in agreement. Hepatic non-protein sulfhydryls were significantly lower in Wistar rats receiving APAP than in TR⁻ rats. TR⁻ rats treated with buthionine sulfoximine and APAP showed dramatic increases in hepatotoxicity. TR⁻ rats had increased mRNA expression of several APAP metabolizing enzymes. Mrp2 expression not only is important in biliary excretion, but also influences the toxic potential of reactive intermediates by controlling intrahepatic GSH and possibly drug metabolism.     

Immunohistochemical Localization of Acetaminophen-Bound Liver Proteins

A sensitive immunofluorescence assay was developed for localizingacetaminophen (APAP) protein adducts in liver sections fromtreated mice. Affinity-purified anti-APAP antibodies, when appliedto liver sections from mice given 600 mg APAP/kg, po, were preferentiallylocalized in cells of the centrilobular region. At 30 min afterdosing, covalently bound APAP was detected only in those cellsmost proximal to the central vein. Thereafter, binding spreadthroughout the centrilobular zone. However, by 8 hr the overallintensity of staining decreased and binding appeared more diffuse.Western blot analysis of electrophoretically resolved proteinsfrom similarly treated mice revealed a corresponding temporalarylation of cytosolic proteins by APAP and indicated that thefluorescence detected at 30 min was associated with arylationof protein(s) of 44 kDa. The findings demonstrate the sensitivityand utility of immunohistochemical techniques in the study ofcovalently bound toxicants and emphasizes the temporal linkbetween selective protein arylation in individually targettedcells to the development of APAP hepatotoxicity.     

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.     

Acetaminophen metabolism does not contribute to gender difference in its hepatotoxicity in mouse.

Gender is an important factor in pharmacokinetics and pharmacodynamics. In the current study, gender difference in acetaminophen (APAP)-induced hepatotoxicity has been examined. Male and female mice were injected with a toxic dose of APAP (500 mg/kg, ip). Female mice were resistant to the hepatotoxic effects of APAP, depicted by serum alanine aminotransferase and sorbital dehydrogenase activities and histological analysis. Basal hepatic reduced glutathione (GSH) levels were lower in females than in males, suggesting that basal GSH level may not be a factor in determining the gender difference of APAP hepatotoxicity. APAP metabolism was slower in females than males, revealed by lower levels of glucuronidation and sulfation and higher amounts of free APAP in the livers of female mice. Lower basal Cyp1a2 mRNA levels and lower expression of Cyp1a2 and Cyp3a11 mRNAs after APAP dosing were also observed in females compared with males. However, there was no gender difference in N-acetyl-p-benzoquinone imine covalent binding 2 h after APAP administration, suggesting similar APAP bioactivation between genders. Moreover, liver Gst pi mRNA levels were significantly lower in females than males. This finding is consistent with a previous report, which showed that Gst pi knockout mice are protected from APAP-induced liver toxicity. In conclusion, gender difference of APAP-induced hepatotoxicity is not likely due to APAP metabolism. Perhaps, it is in part due to gender-dependent Gst pi expression. However, the mechanism underlying the association between reduction in Gst pi expression and hepatoprotective effect against APAP toxicity remains to be further explored.     

Intrinsic susceptibility of the kidney to acetaminophen toxicity in middle-aged rats

Acetaminophen (APAP)-induced nephrotoxicity is age-dependent in male Sprague-Dawley (SD) rats: middle-aged (9-12 months old) rats exhibit nephrotoxicity at lower dosages of APAP than do young adults (2-3 months old). The present study was designed to test the hypothesis that the intrinsic susceptibility of renal tissue to APAP toxicity is increased in middle-aged rats. APAP toxicity was evaluated in renal slices from naive 3- and 12-month-old male SD rats incubated with 0-50 mM APAP for 2-8 h. Renal slice glutathione (GSH) and APAP concentrations were determined; renal function was assessed by organic anion (para-aminohippurate, PAH) and cation (tetraethylammonium, TEA) accumulation; and cell viability was assessed by lactate dehydrogenase (LDH) leakage. At each concentration of APAP tested, accumulation of APAP by renal slices was similar in 3- and 12-month-olds. APAP toxicity in renal slices from both 3- and 12-month-old rats was characterized by concentration-dependent increases in LDH leakage. In contrast to APAP nephrotoxicity in vivo, APAP toxicity in renal slices was accompanied by decreased accumulation of PAH and TEA. Additionally, APAP produced marked reductions in renal slice GSH content in a concentration-dependent manner: however, in contrast to APAP nephrotoxicity in vivo, APAP-induced GSH depletion in vitro did not precede cytotoxicity. No consistent age-dependent differences in the time- and concentration-response curves for APAP nephrotoxicity were observed. These data suggest that APAP cytotoxicity in vitro is not increased in 12-month-old rats. However, since the pattern (and mechanisms) of APAP cytotoxicity in vitro appears to be different from that observed in vivo, extrapolation of in vitro cytotoxicity to in vivo nephrotoxicity is limited. Therefore, age differences in intrinsic susceptibility of the intact kidney cannot be excluded as a mechanism contributing to enhanced APAP nephrotoxicity in middle-aged rats.     

Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice.

The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.     

Examination of the differential hepatotoxicity of diallyl phthalate in rats and mice

In this study we confirmed that diallyl phthalate (DAP) is more hepatotoxic to rats than to mice, and we demonstrated the same species difference in toxicity for allyl alcohol (AA). The data suggest that the toxicity of DAP probably results from AA cleaved from DAP. To determine if the species difference in susceptibility to hepatotoxicity resulted from differences in the disposition and metabolism of DAP, Fischer-344 rats and B6C3F1 mice were given [14C]DAP, 1, 10, or 100 mg/kg po or 10 mg/kg iv, and placed in metabolism cages for 24 hr. In rats, 25-30% of the DAP was excreted as CO2, and 50-70% appeared in the urine within 24 hr. In mice, 6-12% of the DAP was excreted as CO2, and 80-90% was excreted in the urine within 24 hr. Monoallyl phthalate (MAP), allyl alcohol, 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite (PM) were found in the urine of rats and mice dosed with DAP. The polar metabolite was present in the urine of rats dosed with DAP or AA, indicating that the compound is a metabolite of AA. There was no difference between the species in the quantity of AA excreted, but mice excreted more MAP (39 vs 33%), HPMA (28 vs 17%), and PM (20 vs 8%) than rats. Because DAP is metabolized to AA, a potent periportal hepatotoxicant, and because the mouse produced more HPMA than rats, we postulate that the differential hepatotoxicity of DAP is related to the extent of glutathione conjugation with allyl alcohol or acrolein (the active metabolite of AA).     

Peroxisome proliferator-activated receptor alpha-null mice lack resistance to acetaminophen hepatotoxicity following clofibrate exposure.

The purpose of this study was to investigate whether activation of the nuclear receptor PPARalpha is needed for protection from acetaminophen (APAP) hepatotoxicity produced by repeated administration of the peroxisome proliferator clofibrate (CFB). Female wild-type and PPARalpha-null mice received corn oil vehicle or 500 mg CFB/kg, ip, daily for 10 days. They were then fasted overnight (18 h) and either killed at 4 or 24 h after challenge with 400 mg APAP/kg. Controls received 50% propylene glycol vehicle only. In this model of CFB hepatoprotection, liver injury was assessed by measuring plasma sorbitol dehydrogenase activity and by histopathology at 24 h after APAP challenge. Significant hepatocellular necrosis was evident in both corn oil-pretreated PPARalpha-null and wild-type mice at 24 h after APAP challenge. In agreement with previous studies, CFB-pretreated wild-type mice showed marked protection against APAP toxicity. In contrast, CFB did not provide protection against APAP hepatotoxicity in the PPARalpha-null mice. Similarly, at 4 h after APAP challenge, hepatic glutathione depletion and selective arylation of cytosolic proteins were reduced significantly in CFB-pretreated wild-type mice, but not in PPARalpha-null mice. The lack of changes in APAP binding and NPSH depletion in CFB-pretreated, PPARalpha-null mice is consistent with the presence of significant liver injury at 24 h in this treatment group. These findings demonstrate that the protection against APAP hepatotoxicity by peroxisome proliferator treatment is mediated by the activation of PPARalpha.     

Prevention of acetaminophen-induced hepatotoxicity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) has previously been shown to protect against acetaminophen (APAP)-induced hepatotoxicity, but the mechanism of this effect was not clear. Treatment of mice with 1 mg/kg DMSO 4 h before 250 mg/kg APAP resulted in significantly less hepatotoxicity than with APAP alone, as measured by serum glutamic pyruvic transaminase (SGPT) content 24 h after APAP. Protection was also evident when 1 ml/kg DMSO was given 4, but not 8 h after 250 mg/kg APAP. The APAP-induced depletion of liver glutathione was prevented in mice pretreated with DMSO, although DMSO alone had no effect on liver glutathione levels. The hepatic concentration of cytochrome P-450 (P450) 4 h after treatment of mice with 1 ml/kg DMSO, was significantly decreased compared to saline-treated animals. However, while this DMSO pretreatment significantly decreased the activity of cytochrome P-450-linked aminopyrine-N-demethylase, it increased the activity of aniline hydroxylase. Covalent binding of [14C]APAP to hepatic protein in vivo was significantly decreased in mice pretreated with DMSO. Covalent binding of [14C]APAP to hepatic microsomal protein in vitro was not significantly altered after in vivo treatment with DMSO. However, the presence of DMSO in the in vitro incubation mixture significantly decreased covalent binding of [14C]APAP in a dose-dependent manner compared to microsomal fractions from untreated, saline-treated or DMSO pretreated animals. These data suggest that the DMSO-induced alterations in cytochrome P-450 content and activity may not be the cause of the observed protective action of this chemical. The ability to competitively inhibit APAP bioactivation or to directly scavenge free radicals produced during APAP metabolism, including the activated species which covalently binds to protein, may account for the hepatoprotection afforded by DMSO.     

Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice

Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.     

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 CD-1 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.     

Short-term treatment with alcohols causes hepatic steatosis and enhances acetaminophen hepatotoxicity in Cyp2e1(-/-) mice

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.