Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity.

Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells. Both Alamar blue and MTT are reduced by mitochondrial enzymes. We observed enhanced fluorescence of Alamar blue when kidney epithelial cells were co-incubated with hepatic post-mitochondrial supernatant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepatic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products. Livers from female Sprague-Dawley rats were used to prepare S9 fraction, and mitochondrial, microsomal and cytosolic fractions. Fractions containing 1 or 5 mg protein/ml were incubated with Alamar blue or MTT for up to 4 h. Fluorescence (Alamar blue) or absorbance (MTT) were determined and expressed as differences between treated wells and controls. Hepatic fractions (S9, mitochondria, microsomes and cytosol) caused concentration- and time-dependent increases in Alamar blue fluorescence and MTT absorbance. Reduction of Alamar blue and MTT by hepatic S9 fraction was abolished by heating. Reduction of Alamar blue by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or cytosol. Reduction of MTT by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or microsomes. These data indicate that mitochondrial, cytosolic and microsomal enzymes reduce Alamar blue and MTT. Therefore, caution should be exercised in ascribing decreases in viability as due solely to mitochondrial damage when using either of these dyes.

Metabolism of para-aminophenol by rat hepatocytes.

Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

Effect of antioxidants on para-aminophenol-induced toxicity in LLC-PK1 cells.

The present studies were designed to investigate the susceptibility of LLC-PK1 cells to cytotoxicity induced by para-aminophenol (PAP) and the ability of antioxidants to prevent PAP-induced cytotoxicity. LLC-PK1 cells were incubated for 4 h with varying concentrations of PAP (0-0.2 mM). Incubation was continued for 20 h and viability was monitored at 24 h after initial exposure to PAP. For coincubation experiments, cells were incubated for 4 h with various antioxidants [including ascorbate, glutathione (GSH), butylated hydroxytoluene (BHT), beta-nicotinamide adenine dinucleotide (NADH), or beta-nicotinamide adenine dinucleotide phosphate (NADPH)] in the absence or presence of 0.1 mM PAP. For preincubation experiments, cells were incubated for 1 h with ascorbate, GSH or NADPH. Antioxidants were removed and cells were exposed to 0 or 0.1 mM PAP for 4 h. Viability was determined 24 h following PAP exposure. LLC-PK1 cells displayed a steep concentration-response relationship for PAP; 0.1 mM PAP caused approximately 50% loss of viability. Coincubation with ascorbate, GSH and NADPH was without effect on cell viability in the absence of PAP and attenuated PAP-induced losses in viability. In contrast, NADH was ineffective in preventing PAP-induced cytotoxicity. BHT alone produced a significant loss of cell viability and was ineffective in preventing PAP cytotoxicity. Inability of NADH to prevent PAP-induced cytotoxicity was related to rapid degradation of NADH in aqueous solution. Preincubation of cells with ascorbate or GSH but not NADPH was associated with attenuation of PAP-induced cytotoxicity. These data suggest that (1) PAP is cytotoxic to LLC-PK1 cells, (2) a portion of PAP cytotoxicity is due to nonenzymatic oxidation that occurs in the incubation medium, and (3) a portion of PAP cytotoxicity is due to enzymatic or nonenzymatic oxidation that occurs within cells.

Comparative disposition of the nephrotoxicant N-(3, 5-dichlorophenyl)succinimide and the non-nephrotoxicant N-(3, 5-difluorophenyl)succinimide in Fischer 344 rats.

Disposition of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) was compared with that of a nontoxic analog, N-(3, 5-difluorophenyl)succinimide (DFPS). Male Fischer 344 rats were administered 0.2 or 0.6 mmol/kg [14C]NDPS or [14C]DFPS (i.p. in corn oil). Plasma concentrations were determined from blood samples obtained through the carotid artery. Urine samples were analyzed for metabolite content by HPLC. Rats were sacrificed at 3 h (DFPS) or 6 h (NDPS) and tissue radiolabel content and covalent binding were determined. [14C]NDPS-derived plasma radioactivity levels were 6- to 21-fold higher and peaked later than those from [14C]DFPS. Six hours after dosing, NDPS was 40% eliminated in the urine compared with approximately 90% for DFPS. By 48 h, only 67% of the NDPS dose was eliminated in urine. In contrast, DFPS excretion was virtually complete within 24 h. NDPS underwent oxidative metabolism to a slightly greater extent than DFPS. Distribution of [14C]NDPS-derived radioactivity into the kidneys was 3- to 6-fold higher than that into the liver or heart, and was more extensive than with [14C]DFPS. NDPS also covalently bound to plasma, renal, and hepatic proteins to a greater extent than DFPS. In summary, NDPS achieves higher tissue and plasma concentrations, covalently binds to a greater extent, and is eliminated more slowly than DFPS. Differences in the lipid solubility of NDPS metabolites and DFPS metabolites may help explain these results. The overall greater tissue exposure of NDPS and its metabolites may contribute to differential toxicity of these analogs.

Coincubation of rat renal proximal tubules with hepatic subcellular fractions potentiates the effects of para-aminophenol.

Treatment of rats with para-aminophenol (PAP) (300 mg/kg ip) produced decreases in renal nonprotein sulfhydryl (NPSH) content, oxygen consumption, and adenine nucleotide concentrations 2-4 hr following administration. In contrast, incubation of rat renal tubules with up to 1 mm PAP for 4 hr produced inconsistent changes in renal tubules. This discrepancy suggested that extrarenal metabolism of PAP may be involved in PAP bioactivation and nephrotoxicity. We designed the present studies to test the hypothesis that hepatic metabolism of PAP potentiates the effects of PAP on renal tubules. Incubation of renal tubules with 0.5 mm PAP and 10 mg protein from hepatic postmitochondrial supernatant (S9 fraction) in the absence of glutathione (GSH) for 4 hr did not alter renal oxygen consumption or adenine nucleotide metabolite concentrations. We observed no changes when we incubated tubules with 0.5 mm PAP and 1 mm GSH in the absence of hepatic S9 fraction. However, incubation of renal tubules with 0.5 mm PAP, 1 mm GSH, and 10 mg hepatic S9 protein for 4 hr significantly decreased renal oxygen consumption and adenosine triphosphate and total nucleotide concentrations. These data suggest that the effects of PAP in renal tubules may be potentiated by enzymatic metabolism of PAP, possibly involving oxidation and GSH conjugation. From experiments using hepatic microsomes or cytosol instead of S9 fraction, we found that changes were produced when we incubated tubules with PAP in the presence of hepatic microsomes, but not cytosol. These data suggest that hepatic microsomal metabolism of PAP may contribute to the production of changes in renal tubules in vitro. PAP-induced changes in renal proximal tubules were prevented when we included a beta-nicotinamide adenine dinucleotide phosphate (NADPH) generating system in the incubation medium. The protective effect of NADPH persisted when microsomes were inactivated by incubation with 1-aminobenzotriazole, a cytochrome P450 inhibitor. These data suggest that cytochrome P450-dependent oxidation is not involved in the production or prevention of PAP-induced changes in renal tubules. The enzyme(s) responsible for PAP bioactivation and the mechanism(s) by which NADPH protects renal tubules from PAP-induced decrements in oxygen consumption and adenine nucleotide concentrations are currently unclear.

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.

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 renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

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 and hepatotoxicity in young adult Sprague-Dawley (SD) rats and age-dependent toxicity in male rats. There is no information regarding the susceptibility of aging female SD rats to APAP toxicity. Therefore, the present studies were designed to determine if sex-dependent differences in APAP toxicity persist in aging rats and to elucidate factors contributing to sex- and age-dependent APAP hepatotoxicity and nephrotoxicity. Young adult (3 months old) and aging (18 months old) male and female rats were killed from 2 through 24 hr after receiving APAP (0-1250 mg/kg, ip) containing [ring-14C]APAP. Trunk blood was collected for determination of blood urea nitrogen (BUN) concentration, serum alanine aminotransferase (ALT) activity, and plasma APAP concentration; urine was collected for determination of glucose and protein excretion; and liver and 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 elevations in BUN concentration) in 3-month-old females but not males, whereas APAP induced hepatotoxicity (as indicated by elevations in serum ALT activity) in 3-month-old males but not females. Sex differences in APAP toxicity were no longer apparent in 18-month-old rats. APAP at 750 mg/kg ip produced liver and kidney damage 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 account for differences in APAP toxicity. No sex- or age-dependent differences in tissue GSH depletion or covalent binding of radiolabel from APAP in livers or kidneys were observed following APAP administration. Utilizing an affinity-purified polyclonal antibody raised against APAP, arylated proteins with electrophoretic mobility similar to those observed in mice were prominent in rat livers following APAP administration to 3- and 18-month-old rats of both sexes. In contrast, no arylated proteins were detected in any rat kidneys following APAP administration. Absence of immunochemically detectable proteins in rat kidney following APAP administration is in direct contrast to observations in mice and supports the hypothesis that mechanisms of APAP hepatotoxicity and nephrotoxicity in rats and mice are distinctly different. In conclusion, sex differences in APAP toxicity are observed only in young adult (3-month-old) rats and sex differences are organ-specific with males more susceptible to hepatotoxicity and females more susceptible to nephrotoxicity. Aging rats are more susceptible to APAP-induced damage to both the liver and the kidney than are 3-month-old rats but sex differences are no longer apparent in 18-month-old rats. The mechanisms contributing to sex- and age-dependent differences in APAP toxicity cannot be attributed to differences in tissue APAP concentrations, GSH depletion, or covalent binding.

Contribution of oxidation and deacetylation to the bioactivation of acetaminophen in vitro in liver and kidney from male and female Sprague-Dawley rats.

Young adult female Sprague-Dawley (SD) rats are more susceptible to acetaminophen (APAP)-induced nephrotoxicity than are age-matched male SD rats. Mechanisms contributing to sex-dependent APAP nephrotoxicity may involve differences in APAP bioactivation via cytochrome P450-dependent metabolism to N-acetyl-p-benzoquinoneimine and/or deacetylation to para-amino-phenol. APAP bioactivation by oxidation and deacetylation was assessed by examining the effects of 1-aminobenzotriazole (ABT), a suicide substrate inhibitor of cytochrome P450, and bis-(para-nitrophenyl) phosphate (BNPP), a reversible carboxyesterase inhibitor, on covalent binding of APAP-derived radiolabel. Hepatic and renal S9 fractions prepared from naive male and female rats were incubated with [14C-ring]-APAP in the presence and absence of NADPH. There were no sex-related differences in covalent binding of APAP-derived radiolabel in hepatic or renal S9 fractions from male and female rats. In both sexes, incubation of hepatic or renal S9 fractions with 10 mM ABT significantly reduced covalent binding of APAP-derived radiolabel as compared with covalent binding in the absence of ABT. In contrast, incubation of renal and hepatic S9 fractions with 10 mM BNPP did not alter covalent binding of radiolabel derived from APAP in either males or females. Thus, at least in vitro, differences in bioactivation of APAP in liver and kidney from male and female SD rats do not seem to contribute to sex-dependent APAP toxicity. Carboxyesterases inhibited by BNPP do not seem to contribute to covalent binding of APAP-derived radiolabel in vitro in either liver or kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Aminobenzotriazole-induced destruction of hepatic and renal cytochromes P450 in male Sprague-Dawley rats.

1-Aminobenzotriazole (ABT) is a suicide substrate of both hepatic and pulmonary cytochromes P450. The present studies were designed to compare the effects of ABT on hepatic and renal metabolism. Hepatic and renal microsomes and cytosol were prepared from male Sprague-Dawley rats following ABT pretreatment (0-100 mg/kg ip) for various times. Administration of 100 mg ABT/kg produced profound reductions in P450 content in both liver and kidney within 2 hr; loss of P450 in both tissues persisted for at least 48 hours. ABT-induced destruction of P450 was dose-dependent. Maximal destruction of about 80% of total hepatic P450 occurred at dosages of ABT equal to or greater than 10 mg/kg. Maximal destruction of about 80% of total renal P450 occurred at dosages of ABT equal to or greater than 50 mg/kg. In vitro, ABT rapidly and efficiently destroyed P450 in both hepatic and renal microsomes prepared from naive male Sprague-Dawley rats. Incubation of hepatic or renal microsomes in vitro with ABT produced detectable destruction of P450 within 5 min. Maximal destruction of P450 occurred within 10 min in both hepatic and renal microsomes during in vitro incubation with ABT. ABT-induced destruction of P450 in vitro was concentration-dependent. For hepatic microsomes, maximal destruction of about 70% of P450 required concentrations of ABT equal to or greater than 10 mM. For renal microsomes, maximal destruction of about 80% of P450 required concentrations of ABT equal to or greater than 10 mM. In both liver and kidney, only P450 content and P450-dependent activities were significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic and renal conjugation (Phase II) enzyme activities in young adult, middle-aged, and senescent male Sprague-Dawley rats.

Acetaminophen (APAP)-induced nephrotoxicity is age dependent in male Sprague-Dawley rats: nephrotoxicity occurs at lower dosages of APAP in 12- to 14-month olds compared with 2- to 3-month olds. The mechanisms responsible for enhanced nephrotoxicity in 12-month-old Sprague-Dawley rats are not entirely clear, but may be related to age-dependent differences in APAP metabolism in liver and/or kidney. Major pathways of hepatic APAP metabolism include sulfation and glucuronidation; glutathione conjugation represents a pathway for detoxification of reactive oxidative APAP metabolites. The present studies were designed to quantify in vitro activity of three Phase II enzyme activities: glutathione S-transferase using 1-chloro-2,4-dinitrobenzene as substrate, UDP-glucuronyl transferase using APAP as substrate, and sulfotransferase using APAP as substrate, in subcellular fractions of liver and kidney of 3-, 12-, 18-, and 30-month-old naive male Sprague-Dawley rats. In liver, glutathione S-transferase, UDP glucuronyl transferase, and sulfotransferase activities were not significantly different in rats from 3 through 30 months of age. Renal UDP glucuronyl transferase and sulfotransferase activities were similar in rats from 3 through 30 months of age. In contrast, renal glutathione S-transferase activity was characterized by a lower Km in 12- and 30-month olds when compared with 3-month olds. These data suggest that the reduced total systemic clearance of APAP in 12-month-old male Sprague-Dawley rats previously observed cannot be attributed to age-dependent differences in hepatic APAP metabolism. In addition, it is unlikely that differences in renal APAP metabolism contribute to age-dependent APAP nephrotoxicity.

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.

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)

Acetaminophen and p-aminophenol nephrotoxicity in aging male Sprague-Dawley and Fischer 344 rats.

Strain differences in susceptibility of rats to acetaminophen (APAP)-induced nephrotoxicity have been reported previously. Young adult male Fischer 344 (F344) rats are susceptible, whereas weight-matched Sprague-Dawley (SD) rats are not susceptible to APAP nephrotoxicity. Susceptibility to APAP nephrotoxicity is also age dependent, at least in F344 rats. Middle-aged (12-15 months old) male F344 rats are more susceptible to APAP-induced nephrotoxicity 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 differences in the nephrotoxicity of APAP and p-aminophenol (PAP), a nephrotoxic metabolite of APAP, using 2-, 3-, and 9- to 12-month-old F344 and SD rats. At 2 months of age, F344, but not SD, rats were susceptible to APAP-induced nephrotoxicity. However, at 3 months of age, strain differences were less marked, as susceptibility to APAP nephrotoxicity appeared to increase between 2 and 3 months of age only in SD rats. By 9-12 months of age, susceptibility to APAP nephrotoxicity was comparable in F344 and SD rats. No age- or strain-related differences were observed in the excretory pattern of urinary APAP and metabolites that might explain the increased susceptibility of aging rats to APAP nephrotoxicity. Strain differences in age-matched rats were not marked for PAP-induced nephrotoxicity. Susceptibility of both 3- and 12-month-old F344 and SD rats to PAP-induced nephrotoxicity was greater compared to strain-matched 2-month-old rats. In both F344 and SD rats, PAP nephrotoxicity increased only modestly between 3 and 12 months of age, indicating that increased susceptibility to PAP probably does not play a major role in the age-dependent increase in APAP nephrotoxicity. Thus, strain differences in APAP nephrotoxicity decrease with advancing age. The mechanisms mediating the increased susceptibility to APAP nephrotoxicity in middle-aged rats are not known but may relate, at least in part, to age-dependent differences in pharmacokinetics. The present study highlights the importance of considering the age of rats when evaluating drug toxicity. Even in young adult rats, subtle maturational changes in drug metabolism and/or disposition may occur, making toxicological evaluation in weight-matched rats of different strains and ages inappropriate.

Age-related nephropathy in laboratory rats.

Chronic progressive nephropathy is a spontaneous disease common among aging laboratory rats, often making it difficult to distinguish age-related from drug-related effects in chronic toxicity studies. Morphological changes of the kidney that occur with age include thickening of glomerular and proximal tubular basement membranes, mesangial proliferation, fusion of foot processes, and, ultimately, glomerular sclerosis. Proteinuria (specifically, albuminuria) is the most striking characteristic change in renal function of aging rats and, generally, correlates well with the severity of age-related glomerular pathology. Changes in tubular functions also may occur with aging but have not been investigated sufficiently. The pathogenesis of chronic progressive nephropathy is not known; however, hemodynamic adaptations after ad libitum consumption of protein-rich diets may be a contributing factor. High-protein diets increase glomerular pressures and flows, perhaps facilitating excretion of metabolic end products. These hemodynamic adaptations may impair the permselective properties of the glomerulus, leading to: enhanced accumulation of macromolecules in the mesangium, progressive mesangial expansion, and, ultimately, glomerular sclerosis. Indeed, decreasing total food or protein intake retards or prevents the progression of age-related nephropathy. Inasmuch as chronic toxicity studies are complicated by a high incidence of spontaneous nephropathy, implementation of a restricted dietary regimen may improve detection of drug-induced toxicity.

Biochemical mechanisms of cephaloridine nephrotoxicity.

Large doses of the cephalosporin antibiotic, cephaloridine, produce acute proximal tubular necrosis in humans and in laboratory animals. Cephaloridine is actively transported into the proximal tubular cell by an organic anion transport system while transport across the lumenal membrane into tubular fluid appears restricted. High intracellular concentrations of cephaloridine are attained in the proximal tubular cell which are critical to the development of nephrotoxicity. There is substantial evidence indicating that oxidative stress plays a major role in cephaloridine nephrotoxicity. Cephaloridine depletes reduced glutathione, increases oxidized glutathione and induces lipid peroxidation in renal cortical tissue. The molecular mechanisms mediating cephaloridine-induced oxidative stress are not well understood. Inhibition in gluconeogenesis is a relatively early biochemical effect of cephaloridine and is independent of lipid peroxidation. Furthermore, cephaloridine inhibits gluconeogenesis in both target (kidney) and non-target (liver) organs of cephaloridine toxicity. Since glucose is not a major fuel of proximal tubular cells, it is unlikely that cephaloridine-induced tubular necrosis is mediated by the effects of this drug on glucose synthesis.

Active tetraethylammonium uptake across the basolateral membrane of rabbit proximal tubule.

Tetraethylammonium (TEA) uptake was measured in isolated, nonperfused rabbit S2 proximal tubule segments. The TEA cell-to-bath concentration ratio in bicarbonate-Ringer bathing medium was 10-fold higher than that predicted by passive equilibration according to the basolateral electrochemical potential, indicating that TEA uptake is an active process as reported by previous investigators. Removing bicarbonate and CO2 reduced TEA uptake to 22% of control. When bicarbonate-CO2 was replaced by HEPES-O2 or butyrate-O2, TEA uptake was unaltered, but uptake was inhibited when the major buffer anion was omitted. In the presence of 10(-4) M ouabain and bicarbonate-CO2, the TEA cell-to-bath concentration ratio was reduced to 20% of control. TEA uptake in the absence of bicarbonate and CO2 was unaltered by the addition of 10(-4) M ouabain. TEA uptake was inhibited when the bathing medium contained 0 mM K+ or 2 mM Ba2+. These data 1) demonstrate that active basolateral TEA uptake is dependent on medium buffer capacity and 2) support the concept that a portion of TEA uptake occurs via a passive equilibration pathway.

The pentobarbital anesthetized dog: an animal model for assessing chemically induced changes in renal function and ultrastructure.

An experimental procedure was devised using the pentobarbital-anesthetized dog that could be used for the comprehensive evaluation of the renal effects of chemicals. After IV or renal arterial administration of 0.9% saline solution (vehicle), 12 renal function determinants were continuously monitored for periods of 2 and 6 hours. At the completion of the 2 or 6 hours of study, the kidneys of a number of dogs (usually between 1 and 7) in each vehicle-treated group were subjected to a modification of the intravascular perfusion-of-fixative technique to evaluate the ultrastructural status of the outer cortical, inner cortical, and outer medullary tissue. The remaining dogs (at least 3) in each vehicle-treated group were given a nonnephrotoxic, but maximally effective, diuretic dose of ethacrynic acid, which enabled an assessment of the functional integrity of the thick ascending limb of Henle's loop. Renal function and glomerular and tubular ultrastructure remained stable in the pentobarbital-anesthetized dog for up to 6 hours after administration of vehicle. Sustained infusion of inulin (included in the procedure to estimate glomerular filtration rate) throughout the duration of the experiments, and pentobarbital anesthesia of various durations did not alter the morphologic status of the canine nephron. The procedure used for the renal perfusion of fixative circumvented any manipulation of the kidneys before fixation and allowed for the acquisition of normal (unaltered) appearing tissue from all areas of the kidneys. The responses of pentobarbital-anesthetized dogs to ethacrynic acid administration were similar when given 2 and 6 hours after the vehicle administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of alkylating agents on canine renal function: specifically designed synthetic maleimides.

Maleimidohippurates and maleimidobenzoates were synthesized that possess a carboxy group for active uptake into renal proximal tubular cells and a reactive maleimide moiety to covalently bond with proximal tubular components. The reactivity of the maleimide moiety in each series was progressively reduced by substitution of methyl groups in place of the vinyl hydrogens. In contrast to N-ethylmaleimide (NEM), the resulting maleimidohippurates and maleimidobenzoates did not possess significant diuretic activity in the dog following renal arterial administration. However, as predicted, the nephrotoxicity of the maleimidohippurates paralleled their in vitro alkylating ability and was quite specifically located in the proximal portion of the canine renal tubule.