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.     

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.     

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.     

The development of acetaminophen-induced nephrotoxicity in male Fischer 344 rats of different ages

Male Fischer 344 rats classified as young (2–4 months), middle-aged (12–15 months) and aged (22–25 months) were administered 600 mg/kg acetaminophen (APAP) IP. Rats were killed 6 and 12 h after dosing, and renal damage evaluated by blood urea nitrogen (BUN) levels and histopathology. In addition, plasma levels of APAP and its sulfate and glucuronide conjugates were determined after 6 h. There was no evidence of renal damage in any age group 6 h after APAP. While no nephrotoxicity was present in young animals after 12 h, BUN was elevated 94% and 214% in middle-aged and aged rats, respectively, compared to young animals. At 12 h, APAP-induced renal lesions were more severe in aged rats compared to middle-aged animals. APAP-induced renal damage, as judged by BUN and histopathology, was not altered in young or middle-aged rats following unilateral nephrectomy.Six hours after APAP, both the middle-aged and aged animals had significantly higher plasma levels of APAP and APAP glucuronide compared to young rats. There were similar amounts of the sulfate conjugate in the plasma of each age group. This suggests pharmacokinetic differences could contribute to the age-related increased susceptibility of male Fischer 344 rats to APAP-induced nephrotoxicity.     

The shark bile salt 5 beta-scymnol abates acetaminophen toxicity, but not covalent binding

Acetaminophen (APAP) toxicity involves both arylative and oxidative mechanisms. The shark bile salt, 5 beta-scymnol (5beta-S), has been demonstrated to act as an antioxidant and free radical scavenger in vitro. To determine if 5beta-S protects against either APAP-induced hepatic or renal toxicity, 3-4-month-old male Swiss Laca mice were given APAP (500 mg/kg), and 5beta-S (100 mg/kg) was given at 0 and 2 h after APAP. Plasma SDH at 12 h after APAP alone was 1630 U/l and BUN was 19 mg/dl versus 20 U/l and 10 mg/dl, respectively, in controls. Either simultaneous or 2 h delayed treatment with 5beta-S significantly decreased the APAP-induced SDH increase while only the simultaneous pretreatment prevented the BUN elevation. 5beta-S alone did not increase liver glutathione content. Western analysis of APAP covalent binding using anti-APAP antibodies indicated the 5beta-S did not alter protein arylation either qualitatively or quantitatively. These results suggest that 5beta-S treatment did not impair APAP activation and are consistent with 5beta-S protection that likely results from its antioxidant activity.     

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.     

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.     

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.     

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.     

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.     

Strain differences in acetaminophen nephrotoxicity in rats: role of pharmacokinetics

Strain differences in susceptibility of rats to acetaminophen (APAP)-induced nephrotoxicity have been previously reported. Young adult male Fischer-344 (F-344) rats are susceptible whereas weight-matched Sprague-Dawley (SD) rats are not susceptible to APAP nephrotoxicity. The present study was designed to evaluate the role of pharmacokinetics in strain-dependent APAP nephrotoxicity. Age-matched (2-month-old) male F-344 and SD rats received 250-750 mg APAP/kg, i.v., or 0-1000 mg APAP/kg, i.p. Pharmacokinetic variables were evaluated following i.v. APAP and 24 h urinary excretion of APAP and major metabolites was determined following both i.v. and i.p. administration of APAP. Following i.p. administration, nephrotoxicity was observed only in F-344 rats following 1000 mg APAP/kg; SD rats were not susceptible to APAP-induced nephrotoxicity. In contrast, nephrotoxicity did not occur in either F-344 or SD rats administered APAP i.v. Pharmacokinetic variables (volume of distribution, apparent systemic clearance, and apparent terminal half-life) of APAP were similar in F-344 and SD rats. No striking differences in the pattern of specific urinary metabolites were observed between F-344 and SD rats treated with i.p. or i.v. APAP. Thus, strain differences in APAP-induced nephrotoxicity do not appear to be due to differences in pharmacokinetics or major pathways of APAP metabolism.     

Potentiation of acrylate ester toxicity by prior treatment with the carboxylesterase inhibitor triorthotolyl phosphate (TOTP)

The ability of the carboxylesterase inhibitor TOTP to modify the metabolism, mortality, and sulfhydryl depletion resulting from inhalation of acrylate esters was investigated in male rats. Methyl acrylate and ethyl acrylate were enzymatically hydrolyzed by plasma and by homogenates of rat liver, lung, and kidney, with highest activity found in liver homogenates. Hydrolysis of methyl and ethyl acrylate by tissue homogenates from rats treated with TOTP was inhibited in a dose-dependent manner. Pretreatment of rats with 125 mg/kg of TOTP potentiated the lethal action of inhaled methyl acrylate and ethyl acrylate. In the 72-hr period following termination of a 4-hr exposure to 500, 750, or 1000 ppm methyl acrylate the respective mortality rates in TOTP pretreated rats were 83, 100, and 100% compared with 0, 17, and 67% mortality in corn oil-pretreated rats. The acrylate esters reacted with glutathione in vitro and decreased tissue nonprotein sulfhydryl (NPSH) in vivo. The depletion of NPSH by inhaled acrylate esters was most pronounced in lung compared with that in liver, kidney, or blood. TOTP pretreatment significantly enhanced acrylate ester-induced decreases in tissue NPSH concentrations. In rats exposed for 4 hr to 135, 370, 490, or 720 ppm methyl acrylate, lung NPSH was reduced 34, 55, 69, and 83%, respectively, in TOTP-pretreated rats versus 0, 26, 27, and 52%, respectively, in corn oil-pretreated rats. Kidney NPSH following exposure to acrylate esters was significantly altered only in TOTP-pretreated rats. These studies demonstrate that carboxylesterases are important in the detoxification of methyl acrylate and ethyl acrylate and that exposure to inhibitors of carboxylesterases may potentiate the adverse effects of these esters.     

Protein Arylation Precedes Acetaminophen Toxicity in a Dynamic Organ Slice Culture of Mouse Kidney

Acetaminophen (APAP) is an analgesic and antipyretic agent whichmay cause hepatotoxicity and nephrotoxicity with overdose inman and laboratory animals. In vivo studies suggest that insitu activation of APAP contributes to the development of nephrotoxicity.Associated with target organ toxicity is selective arylationof proteins, with a 58-kDa acetaminophen binding protein (58-ABP)being the most prominent cytosolic target. In this study a mousekidney slice model was developed to further evaluate the contributionof in situ activation of APAP to the development of nephrotoxicityand to determine the selectivity of protein arylation. Precisioncut kidney slices from male CD-1 mice were incubated with selectedconcentrations of APAP (0–25 mM) for 2 to 24 hr. APAPcaused a dose- and time-dependent decrease in nonprotein sulfhy-dryls(NPSH), ATP content, and K⁺ retention. Preceding toxicity wasarylation of cytosolic proteins, the most prominent one beingthe 58-ABP. The association of 58-ABP arylation with APAP toxicityin this mouse kidney slice model is consistent with earlier,in vivo results and demonstrates the importance of in situ activationof APAP for the development of nephrotoxicity. Precision cutrenal slices and dynamic organ culture are a good model forfurther mechanistic studies of APAP-induced renal toxicity.     

A nutrient mixture prevents acetaminophen hepatic and renal toxicity in ICR mice

Acetaminophen (APAP) overdose is often fatal, leading to fulminant hepatic and renal tubular necrosis in humans and animals. We studied the effect of a nutrient mixture (NM) containing, among other nutrients, lysine, proline, ascorbic acid, N-acetyl cysteine, and green tea extract, which has previously been demonstrated to exhibit a broad spectrum of therapeutic properties on APAP-induced hepatic and renal damage in ICR (Imprinting Control Region) mice. Seven-week-old male ICR mice were divided into four groups (A-D) of five animals each. Groups A and C mice were fed a regular diet for 2 weeks, while groups B and D mice were supplemented with 0.5% NM (w/w) during that period. Groups A and B received saline i.p., while groups C and D received APAP (600 mg/kg) i.p. All animals were killed 24 h after APAP administration, serum was collected to assess the liver and kidney functions, and the livers and kidneys were excised for histology. Mean serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, BUN (Blood Urea Nitrogen), creatinine, and BUN/creatinine ratios were comparable in groups A and B, increased markedly in group C and significantly lower in group D compared with group C. APAP caused significant centrilobular necrosis and glomerular damage in unsupplemented animals, while NM prevented these alterations. The results indicate that NM has potential to protect against APAP-induced liver and kidney damage.     

Rhein protects against acetaminophen-induced hepatic and renal toxicity

This study investigated the possible protective effects and mechanism of rhein on Acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity in rats. Treatment of rats with APAP resulted in severe liver and kidney injuries, as demonstrated by drastic elevation of serum glutamate-pyruvate transaminase (GPT), glutamate-oxaloacetic transaminase (GOT), total bilirubin (TBIL), creatinine (CREA), urea nitrogen (UREA) levels and typical histopathological changes including necrosis, phlogocyte infiltration and fatty degeneration in liver, tubules epithelium swelling and severe vacuolar degeneration in kidney. APAP caused oxidative stress, as evidenced by increased reactive oxygen species (ROS) production, nitric oxide (NO) and malondiadehyde (MDA) levels, together with depleted glutathione (GSH) concentration in the liver and kidney of rats. However, rhein can attenuate APAP-induced hepatotoxicity and nephrotoxicity in a dose-dependent manner. Our results showed that GPT, GOT, UREA and CREA levels and ROS production were reduced dramatically, NO, MDA, GSH contents were restored remarkedly by rhein administration, as compared to the APAP alone treated rats. Moreover, the histopathological damage of liver and kidney were also significantly ameliorated by rhein treatment. These findings suggested that the protective effects of rhein against APAP-induced liver and kidney injuries might result from the amelioration of APAP-induced oxidative stress.     

Acetaminophen nephrotoxicity in the rat. Renal metabolic activation in vitro

High doses of acetaminophen (APAP) result in hepatic centrilobular and renal cortical necrosis in man and the F344 rat. Hepatic necrosis is considered to be due to the generation of an arylating intermediate via a microsomal cytochrome P-450 dependent system. Renal microsomes also metabolize APAP to an arylating intermediate via a P-450 dependent mechanism. Thus, at least part of the renal damage from APAP may be due to a biochemical mechanism similar to that in liver. Additionally, APAP is deacetylated to p-aminophenol (PAP) in renal and hepatic cytosol and microsomes. Previous results demonstrated that PAP may be activated in renal microsomes via an NADPH-independent mechanism. Therefore, significant metabolic activation of APAP in the kidney may occur subsequent to deacetylation. Covalent binding of [ring-14C]APAP to renal subcellular fractions was used to substantiate this hypothesis. Under appropriate incubation conditions, enzymatic NADPH-independent covalent binding of [ring-14C]APAP could be demonstrated in renal microsomes but not in 100,000g supernatant fractions. Combination of these subcellular fractions resulted in greater covalent binding of [ring-14C]APAP than in the individual subcellular fractions alone. Addition of glutathione, bis(p-nitrophenyl)phosphate (a deacetylase inhibitor), or PAP inhibited this covalent binding. In contrast, NADPH-independent covalent binding of [ring-14C]APAP could not be demonstrated in any combination of hepatic subcellular fractions. Experiments comparing [ring-14C]APAP and [acetyl-14C]APAP covalent binding to renal 10,000g supernatant fractions indicate that the compound which binds to renal macromolecules is derived from PAP. Thus, these results are consistent with the hypothesis that APAP can be metabolically activated in the kidney after deacetylation to PAP.     

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.     

Renal damage, metabolism and covalent binding following administration of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) to male Fischer 344 rats

In vivo metabolism, nephrotoxicity and covalent binding to proteins were evaluated in male Fischer 344 rats that received [2,3-¹⁴C]-N-(3,5-dichlorophenyl)succinimide (¹⁴C-NDPS). Some animals were pretreated with the enzyme inducer phenobarbital (PB, 80 mg/kg per day, for 3 days, i.p. in saline) prior to receiving a non-nephrotoxic dose of ¹⁴C-NDPS (0.2 mmol/kg, i.p. in corn oil). Other rats were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT, 100 mg/kg, 1 h prior to NDPS, i.p. in saline) before administration of a non-toxic or a toxic dose (0.2 or 0.6 mmol/kg, respectively, i.p. in corn oil) of ¹⁴C-NDPS. Non-pretreated animals received either dose of ¹⁴C-NDPS, but did not receive PB or ABT. All rats were sacrificed 6 h after administration of ¹⁴C-NDPS. Nephrotoxicity was monitored by measuring urine volume, urine protein concentrations, blood urea nitrogen levels, and kidney weights. The NDPS metabolic profile in tissue, blood, and urine was analyzed by HPLC. Covalent binding of ¹⁴C-NDPS-derived radioactivity to tissue proteins was also measured. Compared with non-pretreated rats, PB-pretreatment potentiated the toxicity of the non-toxic dose of ¹⁴C-NDPS. In contrast, ABT-pretreatment protected the rats against NDPS nephrotoxicity. The amount of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA), an oxidative, nephrotoxic metabolite of NDPS, was elevated in kidney homogenates and urine by PB-pretreatment (0.2 mmol/mg NDPS). ABT pretreatment inhibited NDPS metabolism at both doses. Covalent binding of ¹⁴C-NDPS (0.2 mmol/kg)-derived radioactivity to renal and plasma proteins was higher in the PB-pretreated rats than in the non-pretreated animals. In contrast, ABT-pretreatment partially inhibited covalent binding at both doses of ¹⁴C-NDPS. Our results suggest that there is a relationship between oxidative metabolism of NDPS, covalent binding of an NDPS metabolite to renal proteins, and NDPS-induced nephrotoxicity in rats.     

The nitric oxide donor,V-PYRRO/NO,protects against acetaminophen-induced nephrotoxicity in mice

The nitric oxide (NO) donor, O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), is metabolized by P450 enzymes to release NO in the liver and possibly other tissues. V-PYRRO/NO has been shown to be hepatoprotective, but little is known about its effect in the kidney, another organ rich in P450s. Thus, mice were given V-PYRRO/NO (0.4-5.4 mg/ml, 8 microl/h) before and/or after a nephrotoxic dose of acetaminophen (APAP; 600 mg/kg, i.p.) to examine its nephroprotective effects. V-PYRRO/NO administration significantly reduced APAP-induced nephrotoxicity in a dose- and time-dependent manner, as evidenced by mitigation of increased blood urea nitrogen levels and by amelioration of renal pathology, specifically interstitial congestion, proximal tubular cell degeneration and necrosis. The best protection was observed at the highest dose (5.4 mg/ml) and with V-PYRRO/NO pretreatment (4-16 h). Implanting V-PYRRO/NO pumps simultaneously with APAP also attenuated APAP nephrotoxicity. The protection is probably not due to a decreased APAP toxication metabolism, as similar depletion of renal glutathione levels was observed regardless of V-PYRRO/NO treatment. APAP-induced renal lipid peroxidation was reduced by V-PYRRO/NO, as determined by the concentrations of hydroxynonenals and malondialdehyde. In summary, this study demonstrates that the NO donor V-PYRRO/NO is effective in blocking APAP-induced nephrotoxicity in mice. The protection is probably due to multiple mechanisms involving attenuation of APAP-induced congestion and lipid peroxidation in the kidney.     

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.