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.     

Acetaminophen and p-Aminophenol Nephrotoxicity in Aging Male Sprague-Dawley and Fischer 344 Rats

Acetaminophen and p-Aminophenol Nephrotoxicity in Aging MaleSprague-Dawley and Fischer 344 rats. TARLOFF, J. B., GOLDSTEIN.R. S., MORGAN, D. G., AND HOOK, J. B. (1989). Fundam Appl Toxicol12, 78–91. Strain differences in susceptibility of ratsto acetaminophen (APAP)-induced nephrotoxicity have been reportedpreviously. Young adult male Fischer 344 (F344) rats are susceptible,whereas weight-matched Sprague-Dawley (SD) rats are not susceptibleto APAP nephrotoxicity. Susceptibility to APAP nephrotoxicityis also age dependent, at least in F344 rats. Middle-aged (12–15months old) male F344 rats are more susceptible to APAP-inducednephrotoxicity than are young adult (2–4 months old) males.APAP nephrotoxicity in aging SD rats has not been evaluated.The present studies were designed to define strain differencesin the nephrotoxicity of APAP and p-aminophenol (PAP), a nephrotoxicmetabolite of APAP, using 2-, 3-, and 9-to 12-month-old F344and SD rats. At 2 months of age, F344, but not SD, rats weresusceptible to APAP-induced nephrotoxicity. However, at 3 monthsof age, strain differences were less marked, as susceptibilityto APAP nephrotoxicity appeared to increase between 2 and 3months of age only in SD rats. By 9–12 months of age,susceptibility to APAP nephrotoxicity was comparable in F344and SD rats. No age- or strain-related differences were observedin the excretory pattern of urinary APAP and metabolites thatmight explain the increased susceptibility of aging rats toAPAP nephrotoxicity. Strain differences in age-matched ratswere not marked for PAP-induced nephrotoxicity. Susceptibilityof both 3-and 12-month- old F344 and SD rats to PAP-inducednephrotoxicity was greater compared to strain-matched 2-month-oldrats. In both F344 and SD rats, PAP nephrotoxicity increasedonly modestly between 3 and 12 months of age, indicating thatincreased susceptibility to PAP probably does not play a majorrole in the age-dependent increase in APAP nephrotoxicity. Thus,strain differences in APAP nephrotoxicity decrease with advancingage. The mechanisms mediating the increased susceptibility toAPAP nephrotoxicity in middle-aged rats are not known but mayrelate, at least in part, to age-dependent differences in pharmacokineties.The present study highlights the importance of considering theage of rats when evaluating drug toxicity. Even in young adultrats, subtle maturational changes in drug metabolism and/ordisposition may occur, making toxicological evaluation in weight-matchedrats of different strains and ages inappropriate.     

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.     

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.     

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.     

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.     

The role of glutathione in p-aminophenol-induced nephrotoxicity in the mouse.

p-Aminophenol (PAP) produces nephrotoxicity in rats through a mechanism presumably involving oxidation and conjugation with glutathione (GSH). Recently it was found that PAP also causes nephrotoxicity in mice as evidenced by elevated blood urea nitrogen (BUN) and serum creatinine levels. The objective of this study was to further investigate the mechanism and elucidate the role of GSH in PAP-induced nephrotoxicity in the mouse. Male C57BL/6 mice injected i.p. with various doses of PAP were sacrificed at 12 hr for measurement of BUN and serum creatinine levels and determination of the extent of renal cortical nonprotein sulfhydryl (NPSH) and GSH depletion. PAP depleted renal cortical NPSH content in a dose- and time-dependent manner. Depletion of NPSH in mouse kidney did not occur at PAP doses below 600 mg/kg. Buthionine sulfoximine, an inhibitor of GSH synthesis, decreased nephrotoxicity. Ascorbate, a reducing agent, prevented PAP-induced nephrotoxicity and attenuated renal cortical NPSH depletion. However, acivicin and aminooxyacetic acid, inhibitors of gamma-glutamyltranspeptidase and beta-lyase, respectively, did not prevent toxicity in the mouse. Piperonyl butoxide, an inhibitor of cytochrome P-450 enzymes, enhanced nephrotoxicity and renal cysteine depletion but not GSH depletion. The results suggest that PAP-induced nephrotoxicity in the mouse may involve oxidation and formation of a GSH conjugate.     

Nephrotoxicity of p-aminophenol,a metabolite of acetaminophen,in the Fischer 344 rat

Acetaminophen (APAP) produces proximal tubular necrosis in the Fischer 344 rat. APAP is deacetylated to p-aminophenol (PAP) in the hamster, and PAP has been reported to be a potent specific cortical nephrotoxicant in the rat. However, the role of PAP in APAP nephrotoxicity has not been defined. Therefore, it was of interest to quantify PAP formation after APAP administration and to correlate PAP formation with renal injury produced by APAP in the Fischer 344 rat. Urinary PAP excretion, measured as an index of PAP formation, increased with increasing doses of APAP. In addition, APAP was metabolized to PAP in isolated perfused kidneys. PAP at doses as low as 100 mg/kg produced significant renal toxicity (elevation in blood urea nitrogen and reduction in accumulation of p-aminohippurate by thin renal cortical slices). Ortho- and meta-aminophenol were not nephrotoxic. Pretreatment with polybrominated biphenyls or β-naphthoflavone, inducers of mixed function oxidases, protected against nephrotoxicity of PAP, possibly as a result of enhanced hepatic biotransformation of the parent compound. These studies indicate that PAP is a potent, selective nephrotoxicant that can be generated from APAP by the kidney and may be responsible for the renal necrosis subsequent to APAP administration.     

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.     

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.     

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)     

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.     

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.     

Gender differences in the potentiation of N-(3,5-dichlorophenyl)succinimide metabolite nephrotoxicity by phenobarbital

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute nephrotoxicity characterized as polyuric renal failure with proximal tubular necrosis. Phenobarbital pretreatment potentiates NDPS and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, a nephrotoxic metabolite of NDPS) nephrotoxicity in male rats. The purpose of this study was to determine the ability of phenobarbital pretreatment to potentiate (1) NDHS nephrotoxicity in female rats and (2) N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, a nephrotoxic metabolite of NDHS) nephrotoxicity in male and female rats. Age-matched male and female Fischer 344 rats (4/group) were pretreated intraperitoneally (ip) with phenobarbital (75 mg/d, 3 d). At 24 h after the last injection of phenobarbital, an ip injection of NDHS (0.025 mmol/kg), 2-NDHSA (0.025 mmol/kg, females; 0.05 mmol/kg, males), or vehicle was given and renal function was monitored at 24 and 48 h post NDPS metabolite or vehicle. Additional groups received the NDPS metabolite or vehicle only and were also monitored for 48 h. In a separate experiment, male Fischer 344 rats were pretreated with piperonyl butoxide (PIBX, 1360 mg/kg) or the PIBX vehicle. 2-NDHSA (0.1 mmol/kg) or vehicle was administered (ip) 30 min after PIBX, and renal function was monitored for 24 h. Phenobarbital markedly potentiated 2-NDHSA nephrotoxicity in male rats as evidenced by increased kidney weight, increased blood urea nitrogen (BUN) concentration, and decreased tetraethylammonium (TEA) accumulation by renal cortical slices. PIBX had no effect on 2-NDHSA nephrotoxicity. Phenobarbital pretreatment did not markedly enhance the nephrotoxic potential of NDHS or 2-NDHSA in female rats. These results indicate that phenobarbital exhibits differential potentiation of NDPS metabolite nephrotoxicity in male and female rats and that the potentiation of 2-NDHSA nephrotoxicity observed in males is not due to cytochrome P-450-mediated oxidative biotransformation.     

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.     

Effects of biliary cannulation and buthionine sulphoximine pretreatment on the nephrotoxicity of para-aminophenol in the Fischer 344 rat

The effects of a glutathione depletor, buthionine sulphoximine (BSO) and biliary cannulation on the nephrotoxicity ofp-aminophenol (PAP) have been investigated in the F344 rat. Preatment with BSO completely protected against the nephrotoxicity of a 50 mg/kg dose of PAP, assessed by clinical chemistry, renal histopathology, and¹ H-NMR urinalysis. Biliary cannulation partially protects against nephrotoxicity induced by 100 mg/kg PAP. These data suggest that the nephrotoxicity of PAP may be due in part to the formation of a proximate toxic metabolite in the liver which is excreted in the bile, subsequently reabsorbed and transported via the systemic circulation to the kidney where the toxic effects occur.     

The Role of Glutathione INp-Aminophenol-Induced Nephrotoxicity in the Mouse

ABSTRACT

p-Aminophenol (PAP) produces nephrotoxicity in rats through a mechanism presumably involving oxidation and conjugation with glutathione (GSH). Recently it was found that PAP also causes nephrotoxicity in mice as evidenced by elevated blood urea nitrogen (BUN) and serum creatinine levels. The objective of this study was to further investigate the mechanism and elucidate the role of GSH in PAP-induced nephrotoxicity in the mouse. Male C57BL/6 mice injected ip with various doses of PAP were sacrificed at 12 hr for measurement of BUN and serum creatinine levels and determination of the extent of renal cortical nonprotein sulfhydryl (NPSH) and GSH depletion. PAP depleted renal cortical NPSH content in a dose- and time-dependent manner. Depletion of NPSH in mouse kidney did not occur at PAP doses below 600 mg/kg. Buthionine sulfoximine, an inhibitor of GSH synthesis, decreased nephrotoxicity. Ascorbate, a reducing agent, prevented PAP-induced nephrotoxicity and attenuated renal cortical NPSH depletion. However, acivicin and aminooxyacetic acid, inhibitors of y-glutamyltranspeptidase and β-lyase, respectively, did not prevent toxicity in the mouse. Piperonyl butoxide, an inhibitor of cytochrome P-450 enzymes, enhanced nephrotoxicity and renal cysteine depletion but not GSH depletion. The results suggest that PAP-induced nephrotoxicity in the mouse may involve oxidation and formation of a GSH conjugate.     

D-amino-acid oxidase is involved in D-serine-induced nephrotoxicity

D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-amino-acid oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-amino-acid oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-amino-acid oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-amino-acid oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-amino-acid oxidase is responsible for the D-serine-induced nephrotoxicity.     

Acute N-(3,4,5-trichlorophenyl)succinimide-induced nephrotoxicity in Sprague-Dawley and Fischer-344 rats

Previous studies have shown that the experimental agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) produces acute nephrotoxicity via a reactive intermediate in Sprague-Dawley and Fischer-344 rats. The purpose of this study was to examine if an arene oxide intermediate is a toxic metabolite contributing to NDPS-induced nephropathy in rats. N-(3,4,5-Trichlorophenyl)succinimide (NTPS) was prepared to prevent arene oxide formation of NDPS, and its nephrotoxic potential was determined in Sprague-Dawley and Fischer-344 rats. Rats were administered a single intraperitoneal injection of NTPS (0.4 or 1.0 mmol/kg) or sesame oil (2.5 ml/kg), and renal function was monitored at 24 and 48 h. NTPS (0.4 or 1.0 mmol/kg) administration produced diuresis, proteinuria, glucosuria, hematuria, decreased accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA), and increased blood urea nitrogen (BUN) and kidney weight in both strains. Extensive proximal tubular necrosis was observed in both strains of rat. The magnitude of these effects was similar to those previously reported for NDPS-induced nephrotoxicity in Sprague-Dawley and Fischer-344 rats. It was concluded that an arene oxide metabolite does not contribute to the nephrotoxic potential of NDPS. The results of the present study indicate that lipophilic character alone is not a good predictor of the nephrotoxic potential for NDPS and NTPS.