The contribution of oxidation and deacetylation to acetaminophen nephrotoxicity in female Sprague-Dawley rats

Young adult female rats are more susceptible to acetaminophen (APAP) induced nephrotoxicity than are male rats. The purpose of the present study was to assess the contribution of oxidation and deacetylation to the expression of APAP nephrotoxicity. Male and female rats received APAP (1100 mg kg⁻¹ i.p.) alone or following pretreatment with 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochromes P450, or tri-o-tolylphosphate (TOTP), an irreversible carboxyesterase inhibitor. Rats were sacrificed 6 or 24 h following administration of 1100 mg APAP kg⁻¹ containing [ring-¹⁴C]APAP. Blood urea nitrogen (BUN) concentration was used as an index of nephrotoxicity. Renal and hepatic non-protein sulfhydryl (NPSH) contents and covalent binding of radiolabel derived from APAP were determined 6 h following APAP administration. Pretreating female rats with ABT, TOTP, or both compounds prevented the APAP-induced elevation in BUN concentration at 24 h. Pretreatment with ABT or ABT plus TOTP prevented APAP-induced depletion of both hepatic and renal NPSH content at 6 h in female rats. In male rats, APAP treatment did not significantly affect hepatic NPSH content. However, renal NPSH content in males was significantly decreased following APAP treatment and the decrease was prevented when rats were pretreated with ABT or ABT plus TOTP. Covalent binding of radiolabel derived from APAP was significantly greater in female kidney as compared to male kidney. Further, covalent binding in female kidney was significantly decreased when rats were pretreated with ABT, TOTP or both. These data suggest that both oxidative metabolism and deacetylation may contribute to APAP-induced nephrotoxicity in rats.     

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.     

Taurine protects acetaminophen-induced oxidative damage in mice kidney through APAP urinary excretion and CYP2E1 inactivation

Acute exposure of acetaminophen (APAP), a widely used analgesic and antipyretic drug, causes severe renal damage and no specific agent has been reported so far that plays any beneficial role in this organ pathophysiology. In the present study, the protective role of taurine on APAP-induced nephrotoxicity was investigated in mice. In order to induce acute nephrotoxicity, APAP was administered at a single dose of 2 g/kg body weight orally to male adult albino mice of Swiss strain. APAP exposure for 24 h significantly increased plasma level of blood urea nitrogen (BUN), creatinine, uric acid, TNF-α, NO production, urinary γ-glutamyl transpeptidase (γ-GT) activity, total urinary protein and urinary glucose level accompanied by a decrease in Na⁺–K⁺–ATPase activity. Moreover, APAP administration significantly increased MDA, protein carbonylation, GSSG level, intracellular ROS production and cytochrome P450 enzyme (CYPP450) activity. The same exposure decreased GSH level, ferric reducing/antioxidant power (FRAP) as well as the activities of antioxidant enzymes indicating that APAP-induced renal damage was mediated through oxidative stress. Besides, APAP exposure significantly reduced mitochondrial membrane potential and induced up-regulation of CYP2E1 in renal tissues although JNK did not play any significant role in this APAP-induced renal pathophysiology. Caspase 9/3 immunoblot and DNA fragmentation analyses showed that APAP-induced renal cell damage was mostly necrotic in nature, although some apoptosis also occurred simultaneously. Taurine treatment both pre and post (150 mg/kg body weight for 3 days, orally) to APAP exposure, however, significantly reduced APAP-induced nephrotoxicity through its antioxidant properties, urinary excretion of APAP and suppression of CYP2E1. Results suggest that taurine might be a potential therapeutic candidate against APAP-induced acute nephrotoxicity.     

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.     

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity in CD-1 mice: I. Enhancement of acetaminophen nephrotoxicity by acetaminophen-cysteine

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Recent studies suggest a contributory role for glutathione (GSH)-derived conjugates of APAP in the development of nephrotoxicity. Inhibitors of either gamma-glutamyl transpeptidase (gamma-GT) or the probenecid-sensitive organic anion transporter ameliorate APAP-induced nephrotoxicity but not hepatotoxicity in mice and inhibition of gamma-GT similarly protected rats from APAP nephrotoxicity. Protection against APAP nephrotoxicity by disruption of these GSH conjugate transport and metabolism pathways suggests that GSH conjugates are involved. APAP-induced renal injury may involve the acetaminophen-glutathione (APAP-GSH) conjugate or a metabolite derived from APAP-GSH. Acetaminophen-cysteine (APAP-CYS) is a likely candidate for involvement in APAP nephrotoxicity because it is both a product of the gamma-GT pathway and a probable substrate for the organic anion transporter. The present experiments demonstrated that APAP-CYS treatment alone depleted renal but not hepatic glutathione (GSH) in a dose-responsive manner. This depletion of renal GSH may predispose the kidney to APAP nephrotoxicity by diminishing GSH-mediated detoxification mechanisms. Indeed, pretreatment of male CD-1 mice with APAP-CYS before challenge with a threshold toxic dose of APAP resulted in significant enhancement of APAP-induced nephrotoxicity. This was evidenced by histopathology and plasma blood urea nitrogen (BUN) levels at 24 h after APAP challenge. APAP alone was minimally nephrotoxic and APAP-CYS alone produced no detectable injury. By contrast, APAP-CYS pretreatment did not alter the liver injury induced by APAP challenge. These data are consistent with there being a selective, contributory role for APAP-GSH-derived metabolites in APAP-induced renal injury that may involve renal-selective GSH depletion.     

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the gamma-glutamyl cycle

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid gamma-glutamyl acceptor substrates for gamma-glutamyl transpeptidase (gamma-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the gamma-glutamyl cycle through interaction with gamma-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a gamma-glutamyl acceptor for both murine and bovine renal gamma-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a gamma-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the gamma-glutamyl cycle.     

Role of nitric oxide and reduced glutathione in the protective effects of aminoguanidine, gadolinium chloride and oleanolic acid against acetaminophen-induced hepatic and renal damage

The potential protective role of aminoguanidine (AG), gadolinium chloride (GdCl(3)) and oleanolic acid (OA) in acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity was investigated in rats. Pretreatment of rats with AG (50mg/kg) orally, GdCl(3) (10mg/kg) intramuscularly or OA (25mg/kg) intramuscularly protected markedly against hepatotoxicity and nephrotoxicity induced by an acute oral toxic dose of APAP (2.5g/kg) as assessed by biochemical measurements and by histopathological examination. None of AG-, GdCl(3)- or OA-pretreated animals died by the acute toxic dose of APAP. Concomitantly, pretreatment of rats with these agents suppressed the profound elevation of nitric oxide (NO) production and obvious reduction of intracellular reduced glutathione (GSH) levels in liver and kidney induced by the acute toxic dose of APAP. Similarly, daily treatment of rats with a smaller dose of AG (10mg/kg), GdCl(3) (3mg/kg) or OA (5mg/kg) concurrently with a smaller toxic dose of APAP (750mg/kg) for 1 week protected against APAP-induced hepatotoxicity and nephrotoxicity. This treatment also completely prevented APAP-induced mortality and markedly inhibited APAP-induced NO overproduction as well as hepatic and renal intracellular GSH levels reduction. These results provide evidence that inhibition of NO overproduction and consequently maintenance of intracellular GSH levels may play a pivotal role in the protective effects of AG, GdCl(3) and OA against APAP-induced hepatic and renal damages.     

Role of receptor interacting protein (RIP)1 on apoptosis-inducing factor-mediated necroptosis during acetaminophen-evoked acute liver failure in mice

Acetaminophen (APAP) overdose induces apoptosis-inducing factor (AIF)-dependent necroptosis, but the mechanism remains obscure. The present study investigated the role of receptor interacting protein (RIP)1, a critical mediator of necroptosis, on AIF-dependent necroptosis during APAP-induced acute liver failure. Mice were intraperitoneally injected with APAP (300 mg/kg). As expected, hepatic RIP1 was activated as early as 1 h after APAP, which is earlier than APAP-induced hepatic RIP3 upregulation. APAP-evoked RIP1 activation is associated with hepatic glutathione (GSH) depletion. Either pretreatment or post-treatment with Nec-1, a selective inhibitor of RIP1, significantly alleviated APAP-induced acute liver failure. Moreover, Nec-1 improved the survival and prevented APAP-induced necroptosis, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that Nec-1 significantly inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. In addition, Nec-1 blocked APAP-induced translocation of AIF from the mitochondria to the nucleus. Of interest, no changes were induced by Nec-1 on hepatic CYP2E1 expression. In addition, Nec-1 had little effect on APAP-induced hepatic GSH depletion at early stage. Taken together, these results suggest that RIP1 is involved in APAP-induced necroptosis. Nec-1 is an effective antidote for APAP-induced acute liver failure.     

Acetaminophen nephrotoxicity in the rat. I. Strain differences in nephrotoxicity and metabolism

Acetaminophen (APAP) produced renal necrosis restricted to the straight segment of the proximal tubule in Fischer 344 (F344) rats but not in Sprague-Dawley (SD) rats. APAP-induced renal functional changes (elevation in blood urea nitrogen and reduction in the accumulation of p-aminohippurate by renal cortical slices) also correlated with strain-dependent histopathological changes. Such strain differences have been attributed to differences in renal P-450 activation of APAP or the deacetylation of APAP to the nephrotoxic metabolite, p-aminophenol (PAP). Kidneys from F344 rats displayed greater concentrations of P-450 and greater ethoxycoumarin-o-deethylase activity than kidneys from SD rats. However, covalent binding of [ring-14C]APAP to renal and hepatic microsomal protein in vitro was similar for both SD and F344 rats. Deacetylation of APAP to PAP was similar in renal and hepatic homogenates from SD and F344 rats. Furthermore, isolated kidneys from SD and F344 rats perfused with APAP excreted PAP at similar rates. PAP excretion, over a 24-hr period following APAP administration, was greater in F344 rats than in SD rats only at the highest dose (900 mg/kg) of APAP. Thus, strain differences in APAP-induced nephrotoxicity apparently cannot be attributed to differences in P-450 activation of APAP or in deacetylation to the nephrotoxic metabolite, PAP.     

The potential protective role of alpha-lipoic acid against acetaminophen-induced hepatic and renal damage

The potential protective role of alpha-lipoic acid (alpha-LA) in acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity was investigated in rats. Pretreatment of rats with alpha-LA (100mg/kg) orally protected markedly against hepatotoxicity and nephrotoxicity induced by an acute oral toxic dose of APAP (2.5 g/kg) as assessed by biochemical measurements and by histopathological examination. None of alpha-LA pretreated animals died by the acute toxic dose of APAP. Concomitantly, APAP-induced profound elevation of nitric oxide (NO) production and oxidative stress, as evidenced by increasing of lipid peroxidation level, reducing of glutathione peroxidase (GSH-Px) activity and depleting of intracellular reduced glutathione (GSH) level in liver and kidney, were suppressed by pretreatment with alpha-LA. Similarly, daily treatment of rats with a smaller dose of alpha-LA (25mg/kg) concurrently with a smaller toxic dose of APAP (750 mg/kg) for 1 week protected against APAP-induced hepatotoxicity and nephrotoxicity. This treatment also completely prevented APAP-induced mortality and markedly inhibited APAP-induced NO overproduction and oxidative stress in hepatic and renal tissues. These results provide evidence that inhibition of NO overproduction and maintenance of intracellular antioxidant status may play a pivotal role in the protective effects of alpha-LA against APAP-induced hepatic and renal damage.     

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on extrahepatic sulfhydryl levels in mice treated with acetaminophen

The cysteine (Cys) precursor 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA) has been shown to protect against acetaminophen (APAP)-induced hepatic GSH, GSSG, and Cys depletion and hepatic necrosis. The aim of this study was to determine the effects of PTCA on the concentrations of sulfhydryl compounds in extrahepatic tissues, including renal cortex, whole blood, and brain, in C57BL/6 mice treated with hepatotoxic doses of APAP. PTCA (1-5 mmol/kg, i.p.) was administered 30 min after the administration of APAP at a dose (800 mg/kg; 5.29 mmol/kg, i.p.) that depleted hepatic GSH and Cys at 4 hr by 95 and 86%, respectively. Tissue concentrations of GSH and Cys were determined by HPLC. At 4 hr following APAP administration, renal cortical GSH and Cys concentrations were decreased to 64 and 39%, respectively, of vehicle-treated control values, and blood concentrations were decreased to 87 and 30%, respectively, of vehicle controls. Brain GSH and Cys were not depleted by APAP. PTCA at 5 mmol/kg (i) attenuated the APAP-induced depletion of GSH and Cys at 4 hr in renal cortex (78 and 65%, respectively, of vehicle controls), (ii) prevented APAP-induced Cys depletion in blood (670% of vehicle controls) with no effect on GSH concentration (94% of vehicle controls), and (iii) increased GSH and Cys concentrations in brain (119 and 411%, respectively, of vehicle controls). The results demonstrate a high degree of tissue selectivity in the APAP-induced depletion of GSH and Cys, and in the effectiveness of PTCA in maintaining and even elevating sulfhydryl levels in extrahepatic tissues of APAP-treated mice.     

Glycyrrhetinic acid prevents acetaminophen-induced acute liver injury via the inhibition of CYP2E1 expression and HMGB1-TLR4 signal activation in mice

Acetaminophen (APAP) is a widely used antipyretic and analgesic drug, which is safe and effective at the therapeutic dose. Unfortunately, excessive dosage of APAP could cause severe liver injury due to lack of effective therapy. Successful therapeutic strategies are urgently requested in clinic. Glycyrrhetinic acid (GA), derived from a traditional medicine licorice, has been shown to exert anti-inflammatory and antioxidant actions. In this study, the effect and the underlying mechanism of GA on APAP-induced hepatotoxicity were explored. Our results showed that pretreatment with GA significantly reduced serum ALT and AST activities, alleviated hepatic pathological damages with hepatocellular apoptosis, down-regulated expression of CYP2E1 mRNA and protein, increased GSH levels, and reduced reactive oxygen species (ROS) productions in the liver of APAP-exposed mice. Furthermore, GA obviously inhibited APAP-induced HMGB1-TLR4 signal activation, as evaluated by reduced hepatic HMGB1 release, p-IRAK1, p-MAPK and p-IκB expression as well as the productions of TNF-α and IL-1β. In addition, GA attenuated hepatic neutrophils recruitment and macrophages infiltration caused by APAP. These findings reflected that GA could alleviate APAP-induced hepatotoxicity, the possible mechanism is associated with down-regulation of CYP2E1 expression and deactivation of HMGB1-TLR4 signal pathway.     

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.     

Role of mouse CYP2E1 in the O-hydroxylation of p-nitrophenol: comparison of activities in hepatic microsomes from Cyp2e1(-/-) and wild-type mice.

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.     

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.     

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.     

Type 1 diabetic mice are protected from acetaminophen hepatotoxicity.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.     

Sex difference in the nephrotoxicity of thiabendazole in mice depleted of glutathione by treatment with DL-buthionine sulphoximine.

In ICR mice depleted of glutathione (GSH) by treatment with DL-buthionine sulphoximine (BSO), males were much more susceptible to thiabendazole (TBZ) nephrotoxicity than females. The nephrotoxicity was indicated by increases in relative kidney weight and serum urea nitrogen (SUN) concentration and by a decrease in renal GSH concentration at 24 hr after TBZ administration. The susceptibility of males to TBZ-induced nephrotoxicity was completely eliminated by pretreatment with oestradiol (OD). Castration of male mice also reduced, though not completely, their susceptibility to TBZ nephrotoxicity. In females pretreated with testosterone (TS), the nephrotoxic effect of TBZ was increased to an extent comparable with that in males.