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.     

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.     

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.     

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.     

Effects of age and dose on disposition and metabolism of salicylic acid in male Fischer 344 rats

Salicylic acid (SAL)-induced nephrotoxicity has been reported to be greater in older rats. To examine age- and dose-related changes in disposition and metabolism, male Fischer 344 rats aged 3, 12, and 25 months were administered single doses of 14C-SAL at 5,50, and 500 mg/kg po. At 5 mg 14C-SAL/kg, urinary excretion of 14C-SAL derived radioactivity (RA) followed first-order kinetics and was complete by 24 hr in 3- and 25-month-old rats, but not until 48 hr in 12-month-old rats. The percentage of administered 14C-SAL excreted as the oxidative metabolites 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-diOH), unmetabolized SAL, or salicyl ester glucuronide (SA-AG) was unchanged with age. The percentage excreted as the ether glucuronide (SA-PC) was significantly decreased in 25-month-old rats, while the percentage excreted as the glycine conjugate, salicyluric acid (SUA) was significantly increased in 12- and 25-month-old rats. At 50 mg SAL/kg, urinary elimination shifted toward zero-order kinetics and was not complete until 48 hr in all age groups. The percentage of an administered dose of 14C-SAL found in urine as 2,3- and 2,5-diOH and SA-AG increased significantly in all age groups, while the percentage excreted as SUA decreased significantly. Twelve- and 25-month-old rats excreted a significantly greater percentage of the total dose as 2,3- and 2,5-diOH than 3-month-old rats at this dose. No SA-PG was detected at this dose in any age group. At 500 mg SAL/kg, mortality was observed in both 3- and 25-month-old rats and excretion of SAL-derived RA in urine was incomplete at 48 hr. However, data indicated a further shift in biotransformation toward increased production of oxidative metabolites and a decrease in SUA production. No significant overall differences were observed between 3- and 25-month-old rats in plasma levels of 14C-SAL following iv administration of 5 and 50 mg SAL/kg. However, elimination half-life (t1/2) was significantly increased in 25-month-old rats at 5 mg SAL/kg vs. 3-month-old rats. These results indicate that the age-related increase in acute nephrotoxicity of SAL may result from increased production of oxidative metabolites in older rats at higher doses of SAL.     

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.     

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.     

Nephrotoxicity of hexachloro-1:3-butadiene in the rat: The effect of age,sex, and strain

Adult male rats are less susceptible to hexachloro-1:3-butadiene-induced nephrotoxicity than adult female and young male rats. A single dose of 50 mg/kg hexachloro-1:3-butadiene (HCBD) ip in adult Alderley Park (Wistar-derived) females produced marked renal tubular necrosis and an increased plasma urea by 24 hr. Young male rats were also more susceptible to HCBD-induced nephrotoxicity; a dose of 25 mg/kg produced marked tubular necrosis and an increased plasma urea in 21-day-old rats, while a dose of 200 mg/kg was required to produce a similar response in adult males. p-Aminohippurate accumulation by thin slices of renal cortex from rats treated 24 hr previously with HCBD was reduced in adult but not in young male rats. HCBD was more toxic to young male rats (21 and 29 days old, LD50 57 and 96 mg/kg. respectively) than to adult males (7 weeks old, LD50 360 mg/kg). HCBD administration produced a more marked nephrotoxicity in 21-day-old rats treated with phenobarbital in their drinking water for 7 days than in rats of the same age not treated with phenobarbital. Associated with the increased susceptibility of female rats, renal nonprotein sulfhydryl content (NP-SH) was decreased in female but not in male rats 4 hr after HCBD administration. This decrease suggests conjugation of HCBD by the female rat kidney. The sex and age differences observed in nephrotoxicity due to HCBD are probably related to differences in hepatic and renal enzymes responsible for the detoxification and/or activation of HCBD. Fischer 344 rats were slightly more susceptible and Long Evans rats slightly less susceptible than the Alderley Park strain to HCBD-induced nephrotoxicity, although the differences were not as marked as those seen with age and sex.     

Cephaloridine nephrotoxicity in streptozotocin induced diabetic Fischer 344 (F344) rats

The purpose of this study was to determine if cephaloridine nephrotoxicity is attenuated in streptozotocin (STZ)-induced diabetic rats. Fischer 344 (F344) rats (205-250 g) were given a single injection (i.p.) of STZ (27-35 mg/kg) or citrate buffer. The nephrotoxicity of (750 mg/kg) cephaloridine (i.p.) was then compared with normoglycemic and 14-day diabetic rats. Increased blood urea nitrogen (BUN) levels as well as diminished renal cortical slice accumulation of tetraethylammonium (TEA) and lactate-stimulated p-aminohippurate (PAH) were measured (P less than 0.05) in normoglycemic rats 48 h after cephaloridine administration. Cephaloridine failed to alter BUN levels and organic ion accumulation in diabetic rats. Diabetes did not totally protect against cephaloridine toxicity since kidney weights were elevated in normoglycemic and diabetic rats 48 h after administration of 750 mg/kg cephaloridine. A series of experiments also measured BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA 24, 48 and 72 h after (1500 mg/kg) cephaloridine administration. Cephaloridine increased (P less than 0.05) kidney wt and decreased PAH and TEA uptake (P less than 0.05) in the normoglycemic group at 24-72 h. No change in kidney wt, PAH or TEA uptake was observed in the diabetic rats. These data indicate diabetes reduces cephaloridine nephrotoxicity.     

Role of leukotrienes in N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity in male Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500mg/kg, ip), an inhibitor of LTA(4) synthesis, 1h before NDPS (0.4mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD(4) receptor antagonist LY171883 (100mg/kg, po) was administered 0.5h before and again 6h after NDHS (0.1mmol/kg, ip) or 2-NDHSA (0.1mmol/kg, ip) or vehicle. Renal function was monitored for 48h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.     

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.     

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.     

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.     

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.     

Renal glutathione depletion and nephrotoxicity of cadmium-metallothionein in rats

Renal glutathione (GSH) concentrations were reduced approximately 80% at 4 hr after a single injection of buthionine sulfoxime (BSO) (4 mmol/kg body wt) and remained reduced for at least 16 hr in male rats. Following BSO injection, rats were injected with a nephrotoxic dose of cadmium-metallothionein (Cd-MT) (0.3 mg Cd as Cd-MT/kg body wt) and killed 1, 4, or 12 hr later. Damage to the kidney was assessed histologically and by measurement of p-aminohippuric acid (PAH) uptake into renal cortical slices. Although the renal accumulation of Cd following Cd-MT injection was significantly lower in BSO-pretreated rats as compared to nonpretreated rats, the damage to kidney was more severe. At 4 and 12 hr, both Cd-MT-induced inhibition of PAH uptake and morphological damage were significantly increased in BSO-pretreated rats. In certain experiments, the induction of renal intracellular MT synthesis by zinc pretreatment slightly decreased the renal toxicity of Cd-MT in the BSO-treated rats. The results demonstrate that although GSH depletion decreases the renal accumulation of Cd in rats injected with Cd-MT, the nephrotoxicity of Cd-MT is increased. Preinduction of MT in the kidney can only partially overcome this increase in toxicity. Therefore both GSH and intracellular MT levels can influence the renal toxicity of injected Cd-MT.     

Lack of Correlation between para-Aminophenol Toxicity in Vivo and in Vitro in Female Sprague--Dawley Rats

The present study was designed to test the hypothesis that para-aminophenol(PAP) nephrotoxicity is due to autooxidation. We compared renalfunctional responses following PAP administration to femaleSprague-Dawley rats and following incubation of renal proximaltubules with PAP. The concentrations of PAP selected for invitro incubations produced cytotoxicity (for example, a decreasein oxygen consumption or adenine nucleotide concentration) inrat renal epithelial cells or rabbit proximal tubule suspensions.In rats, PAP (300 mg/kg ip) caused proximal tubular necrosiswithin 24 hr. Changes in renal function 24 hr following PAPadministration included increased kidney weight and blood ureanitrogen concentration and decreased renal glutathione (GSH)content and adenine nucleotide concentrations. PAP did not causehepatic damage. Within 2–4 hr following PAP administration,renal GSH content and adenine nucleotide concentrations weresignificantly decreased. In renal cortical slices prepared fromPAP-treated rats, oxygen consumption and accumulation of organicions (para-aminohippurate and tetraethylammonium) were significantlydecreased compared with renal cortical slices prepared fromcontrol rats. In liver, GSH content was significantly decreasedfrom 1 to 4 hr following PAP administration. In contrast tothe effects of PAP in vivo, renal proximal tubules showed littleevidence of injury when incubated with 0.1 or 0.5 mM PAP forup to 4 hr in the presence or absence of amino acids in theincubation medium. When tubules were incubated with 1 mM PAPfor 4 hr in the presence of amino acids, GSH content, AMP concentration,and TEA uptake were significantly decreased. When amino acidswere removed from the incubation medium, 1 mM PAP caused decreasesin oxygen consumption and ATP concentration after 4 hr of incubation.Functional changes observed during incubation with PAP in vitrowere not consistent with functional changes observed in vivo.The discrepancy between PAP toxicity in vivo and in vitro suggeststhat autooxidation is unlikely to be responsible for PAP nephrotoxicityand that nephrotoxicity in vivo is primarily mediated by extrarenalbioactivation. Further, depletion of hepatic GSH content priorto changes in renal function suggests that PAP or a PAP metabolitemay conjugate with hepatic GSH. These observations suggest thatPAP nephrotoxicity may be mediated by PAP-GSH conjugates ratherthan autooxidation of PAP in the kidney.     

Early onset of cisplatin-induced nephrotoxicity in streptozotocin-diabetic rats treated with insulin

Mechanism of protection against cisplatin nephrotoxicity in streptozotocin-diabetic rats is unclear but is associated with decreased renal platinum accumulation. This study was designed to determine whether normalization of hyperglycaemia by insulin treatment to six week streptozotocin-diabetic rats reversed protection against cisplatin nephrotoxicity. Male Sprague-Dawley rats divided into 3 groups (n=10/group) (1) non-diabetic (2) untreated streptozotocin-diabetic and (3) insulin-treated streptozotocin-diabetic groups were rendered diabetic using streptozotocin (65 mg/kg body weight, intravenous). At the end of 6 weeks, Group 3 animals were treated with insulin (subcutaneously) for 21 days to normalize glucose. After 21 days of insulin treatment, the mean +/- S.D. plasma glucose (mg%) in Group 3 animals at 144.8 +/- 22.03, was significantly lower than Group 2 animals (412 +/- 24.69) and comparable to age-matched non-diabetic (Group 1) animals. Blood urea nitrogen at 24 hr after intraperitoneal administration of cisplatin (5 mg/kg body weight) increased by a factor 2.5 in Group 3 compared to 1.1 and 1.3 in Group 1 and Group 2 animals respectively. In the same animals, at 96 hr the blood area nitrogen increased by a factor of 3.2 and 2.9 in Group 1 and Group 3 respectively compared to 1.14 for Group 2 animals. Renal platinum levels in Group 1, Group 2 and Group 3 after 96 hr after cisplatin administration were 6.92 +/- 0.83, 3.46 +/- 0.77 & 6.20 +/- 0.64 (microg/g wet weight of tissue) respectively. Results indicate that 21 day insulin treatment to streptozotocin-diabetic animal reverses protection against cisplatin toxicity. Moreover, insulin treatment increased the susceptibility of streptozotocin-diabetic rats to cisplatin-induced renal toxicity.     

A comparative study of the hepatotoxicity of 1-fluoropentane and 1-fluorohexane

Phenobarbitone pretreatment potentiated hepatocyte lesions in male rats 24 hr after treatment with 1-fluoropentane (3.5 mg/kg body weight) and 1-fluorohexane (0.17 mg/kg body weight). Serum levels of the enzymes ornithine carbamyltransferase, glutamic-pyruvic transaminase and gamma-glutamyltranspeptidase were significantly elevated by the test compounds with the peak effect occurring 24-72 hr after a single ip administration. Significant elevation of hepatocyte triglyceride content and mitochondrial calcium and citrate levels were demonstrated 24 and 48 hr after a single ip injection of 1-fluoropentane or 1-fluorohexane, respectively.     

Functional and structural alterations of the rat kidney induced by the naturally occurring organonitrile 2S-1-cyano-2-hydroxy-3,4-epithiobutane

The organonitriles, 2S-1-Cyano-2-hydroxy-3,4-epithiobutane (erythro and threo) (CHEB), isolated from the seed of Crambe abyssinica were administered by gavage to male Fischer-344 rats. Rats given 50 mg/kg/day were killed at 24, 48, and 72 hr. The rats given 100 mg/kg/day were killed at 48 and 72 hr. Serum urea nitrogen and creatinine were increased by 48 hr and further elevated by 72 hr. Glomerular filtration rate (GFR) of the 50 mg/kg CHEB rats was elevated at 24 hr but fell to subnormal values by 72 hr. The GFR of the 100-mg/kg group was decreased at 48 and 72 hr. Urine output of the 50-mg/kg group increased continuously through 72 hr, while urine output of the 100-mg/kg group was increased to a lesser degree. Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity (nmol/hr/mg creatinine) was significantly elevated in both groups by 48 hr, and further increased by 72 hr. Twenty-four hours after administration of 50 mg/kg, renal proximal tubular epithelial cells of some rats had fine cytoplasmic vacuolation. At 48 and 72 hr, necrosis and coarse vacuolation of proximal tubular epithelial cells occurred in both dose groups. The necrosis was most severe at the apexes of the medullary rays and the coarse vacuolation extended deeply toward the outer stripe of the outer zone of the medulla. Higher doses and/or longer times of CHEB administration resulted in a more extensive lesion distribution. It is concluded that CHEB induces nephrotoxicity in rats characterized by nonoliguric, acute renal failure, and morphological lesions preferentially involving the pars recta of the proximal tubules.     

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.