Investigations of the lactate minimum test.

We evaluated: the agreement between lactate minimum and maximal lactate steady state (MLSS) cycling powers (study 1); whether rates of change of blood lactate concentration during the lactate minimum test reflect that of constant power exercise (study 2); whether the lactate minimum power is influenced by the muscle groups used to elevate blood lactate concentration (study 3). Study 1: 32 subjects performed a lactate minimum test comprising a lactate elevation phase, recovery phase, and incremental phase (five 4 min stages); MLSS was subsequently determined. Study 2: 8 subjects performed a lactate minimum test and five 22 min constant power tests at the incremental phase exercise intensities. Study 3: 10 subjects performed two identical lactate minimum tests, except during the second test the lactate elevation phase comprised arm-cranking. Lactate minimum and MLSS powers demonstrated good agreement (mean bias+/-95% limits of agreement: 2+/-22 W). Rates of change of blood lactate concentration during each incremental phase stage and corresponding constant power test did not correlate. Lactate minimum power was lowered when arm-cranking was used during the lactate elevation phase (157+/-29 vs. 168+/-21 W; p<0.05). The lactate elevation phase modifies blood lactate concentration responses during the incremental phase, thus good agreement between lactate minimum and MLSS powers seems fortuitous.

Relationship between VO(2max) and repeated sprint ability using non-motorised treadmill ergometry.

AIM: The aim of this study was to investigate the relationship between maximal oxygen uptake (Vo(2max)) and repeated sprint ability (RSA) using non-motorised treadmill ergometry. METHODS: Ten male participants (mean [standard deviation] VO(2max): 57.5 [2.1] mL x kg(-1).min(-1)) completed a RSA test (10, 6-s sprints with 34-s recovery) on a non-motorised treadmill. Oxygen consumption (VO(2)) and heart rate (HR) were measured during the RSA test and the decrement of performance fatigue (%(d)) was calculated for the performance indices mean maximal speed (MxSp) and average power output (AvPO). RESULTS: There were significant relationships between VO(2max) and the %(d) MxSp (r=-0.75, P<0.05) and VO(2max) and the %(d) AvPO (r=-0.69, P<0.05). There were also significant relationships between VO(2max) and HR recovery (r=0.56, P<0.05) and VO(2max) and VO(2) recovery (r=0.7, P<0.01). However, while no significant relationships were reported between HR recovery and %(d) MxSp (r= 0.45, P>0.05) and HR recovery and %(d) AvPO (r=-0.52, P>0.05), significant relationships were observed between VO(2) recovery and %(d) MxSp (r=-0.75, P<0.05) and between VO(2) recovery and %(d) AvPO (r=-0.77, P<0.05). CONCLUSION: The findings of this study suggest that VO(2max) may be an important factor determining RSA during repeated, high-intensity running-based exercise, such as field hockey, rugby and soccer.

Role of stereochemistry in N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity.

The nephrotoxicity induced by the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is mediated through oxidative metabolites of NDPS. Oxidation of the succinimide ring in NDPS yields the nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and its hydrolysis product N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The oxidation of NDPS on the succinimide ring also introduces an asymmetric carbon atom into these NDPS metabolites, so that R- and S- enantiomers of NDHS and 2-NDHSA are possible. The purpose of this study was to begin to explore the importance of the stereochemical orientation at the asymmetric carbon atom for the nephrotoxicity induced by NDPS metabolites. Male Fischer 344 rats were administered a single intraperitoneal (ip) injection of R-(+)- or S-(-)-2-NDHSA (0.05, 0.1 or 2.0 mmol/kg) or vehicle, and renal function was monitored for 48 h. R-2-NDHSA (0.1 mmol/kg) administration had little effect on renal function. R-2-NDHSA (0.2 mmol/kg) treatment induced mild diuresis on day 1, increased proteinuria, and a small increase in blood urea nitrogen (BUN) concentration, but no change in kidney weight or glucosuria. S-2-NDHSA (0.1 mmol/kg) induced marked nephrotoxicity as evidenced by diuresis on both post-treatment days, increased proteinuria, glucosuria, and increased kidney weight and BUN concentration. No evidence of hepatotoxicity was obtained in any treated group. Thus, the S-isomer of 2-NDHSA is a more potent nephrotoxicant than the R-isomer, and stereochemistry may play a role in NDPS metabolite-induced 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.

Diversity in medical school: perceptions of first-year students at four southeastern U.S. medical schools.

PURPOSE: To assess students' perceptions of the extent of diversity in their classes, the role of diversity in their first-year curriculum, and their predictions of the amount of diversity in their future patient populations. METHOD: In 1998, students at four southeastern U.S. medical schools that had distinct demographics and differing institutional missions completed a questionnaire on diversity at the end of the first year. In the instrument, diversity was defined according to nine population characteristics: age, sex, race, ethnic background, physical disability, religious affiliation, sexual orientation, socioeconomic status, and rural background (growing up in a community of less than 5,000). Responses were compared according to students' institution, sex, and race. RESULTS: Questionnaires were returned by 349 of 474 students (74%). Students at the school with the most diverse first-year class placed the greatest value on the contributions of diversity to the learning environment. Women students placed more value on the inclusion of diversity issues in the curriculum than did men students, and they placed greater value on understanding diversity issues in their future medical practices than did men. Compared with Asian American, Hispanic, and white students, African American students were the least likely to think that the curriculum contained adequate information about diversity. CONCLUSIONS: The results indicate that perceptions of diversity were influenced by the students' own demographic characteristics and those of their medical school. The more diverse the class, the more comfortable the students were with diversity and the more they valued its contribution to their medical education.

Sodium sulfate potentiates N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity in the Fischer 344 rat.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity as its major toxicity in rats. Previous studies have shown that NDPS induces nephrotoxicity following oxidation of the succinimide ring to form N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and the hydrolysis product of NDHS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). Our recent work found that sodium sulfate potentiated NDPS nephrotoxicity, suggesting that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. The purpose of this study was to determine if sodium sulfate also potentiated the nephrotoxicity of the two nephrotoxic metabolites of NDPS and further to see if sodium sulfate potentiated NDHS and 2-NDHSA nephrotoxicity to the same degree. Male Fischer 344 rats (4-16 rats/group) received an intraperitoneal (ip) injection of sodium sulfate (10 mg/kg) 20 min before a non-nephrotoxic dose (0.05 mmol/kg, ip) of NDHS or 2-NDHSA, or vehicle (12.5% dimethyl sulfoxide in sesame oil). Renal function was then monitored over 48 h. Sodium sulfate pretreatment potentiated the renal effects of a non-nephrotoxic dose of NDHS and 2-NDHSA to induce nephrotoxicity. Nephrotoxicity was characterized by diuresis, increased proteinuria, elevated blood urea nitrogen (BUN) concentration, increased kidney weight and proximal tubular necrosis. Differences in the potentiation of NDHS and 2-NDHSA nephrotoxicity by sodium sulfate were also observed as NDHS nephrotoxicity was potentiated to a lesser degree than 2-NDHSA-induced nephrotoxicity. These results support the likelihood that one or more sulfate conjugate(s) of NDPS metabolites contribute to NDPS nephrotoxicity.

Nephrotoxic potential of N-(3,5-dichloro-4-fluorophenyl)succinimide in Fischer 344 rats: comparison with N-(3,4,5-trichlorophenyl)succinimide.

Numerous structure-nephrotoxicity relationship studies from our laboratory have demonstrated that N-(3,5-dichlorophenyl)succinimide (NDPS) is one of the most potent nephrotoxicants among the N-arylsuccinimides. The purpose of this study was to extend our previous structure-nephrotoxicity relationship studies by examining the effect of addition of a fluoro verses a chloro group at the 4-phenyl position in NDPS. Male Fischer 344 rats (four rats/group) received a single intraperitoneal (i.p.) injection of N-(3,5-dichloro-4-fluorophenyl)succinimide (NDCFPS) or N-(3,4,5-trichlorophenyl)succinimide (NTCPS)(0.4 or 0.8 mmol/kg) or vehicle, and renal function monitored at 24 and 48 h. NDCFPS did not induce significant nephrotoxicity at either dose tested. In contrast, NTCPS (0.4 or 0.8 mmol/kg) induced marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, elevated kidney weight and increased blood urea nitrogen (BUN) concentration. NTCPS also induced marked proximal tubular necrosis at both doses tested. Neither NDCFPS nor NTCPS induced hepatotoxicity at either dose tested. The results of these experiments indicate that addition of a fluoro group at the 4-position on the phenyl ring of NDPS produces a nonnephrotoxicant NDPS derivative (NDCFPS), while addition of a chloro group at this site produces an NDPS derivative with similar nephrotoxic potential to NDPS. The mechanism for this differential effect between 4-halophenyl substitution is unclear, but may result from increased hydrolysis of the succinimide ring and/or increased clearance of N-arylsuccinimide metabolites when a fluoro group is added to the 4-position of the phenyl ring.

Effect of glucuronidation substrates/inhibitors on N-(3,5-dichlorophenyl)succinimide nephrotoxicity in Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an acute nephrotoxicant in rats. Our previous studies have strongly suggested that glucuronide conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. In this study, effects of substrates and/or inhibitors of primarily glucuronidation on NDPS nephrotoxicity were examined to explore further the role of glucuronidation in NDPS nephrotoxicity. Male Fischer 344 rats (4-6/group) were administered one of the following intraperitoneal (i.p.) pretreatments (dose, pretreatment time) prior to NDPS (0.4 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg): (1) no pretreatment; (2) borneol (900 mg/kg, 30 min); (3) eugenol (500 mg/kg per day, 3 days); (4) clofibric acid (400 mg/kg, 15 min before (1/2 dose) and 3 h after (1/2 dose)), or (5) valproic acid, sodium salt (1.0 mmol/kg, 15 min). Following NDPS or NDPS vehicle administration, renal function was monitored at 24 and 48 h. Pretreatment with borneol or eugenol, substrates for ether glucuronidation and sulfation (mainly glucuronidation), afforded complete protection against NDPS nephrotoxicity. Substrates for acyl glucuronidation, clofibric acid or valproic acid, mildly reduced or had little effect on NDPS nephrotoxicity, respectively. These results suggest that ether glucuronide conjugates of NDPS metabolites, rather than acyl glucuronide conjugates, may be the primary ultimate nephrotoxicant species mediating NDPS nephrotoxicity.

The role of glucuronidation in N-(3,5-dichlorophenyl)succinimide (NDPS) nephrotoxicity: nephrotoxic potential of NDPS and NDPS metabolites in Gunn, Wistar, and Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an acute nephrotoxicant in rats. Although the mechanism of NDPS nephrotoxicity is not clear, our previous studies have strongly suggested that glucuronide conjugation of NDPS metabolite(s) is an important biotransformation reaction leading to the ultimate nephrotoxicant metabolite(s) mediating NDPS nephrotoxicity. In this study, the nephrotoxic potential of NDPS and its nephrotoxicant metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), was examined in Gunn rats, which contain a genetic deficiency in bilirubin uridine diphosphate-glucuronosyltransferase (UDPGT), to explore further the role of glucuronidation in NDPS nephrotoxicity. The nephrotoxic potential of NDPS, NDHS, and NDHSA was also examined in Wistar rats, the parent strain for Gunn rats and which generally have normal UDPGT activity. Comparisons were then made with the nephrotoxicity induced by these compounds in Fischer 344 (F344) rats. Age-matched male F344, homozygous (j/j) Gunn, and Wistar rats were used. Rats (four to eight rats/group) of each strain were administered NDPS (0.4 mmol/kg ip), NDHS (0.1 or 0.2 mmol/kg ip), NDHSA (0.1 mmol/kg ip), or vehicle, and renal effects were monitored functionally and morphologically for 48 h. NDPS and its nephrotoxicant metabolites, NDHS and NDHSA, were much weaker nephrotoxicants in Gunn rats than in F344 rats, while Wistar rats were susceptible to the nephrotoxicity induced by NDPS, NDHS, or NDHSA. These results suggest that the lack of NDPS nephrotoxicity observed in Gunn rats is due to the deficiency in UDPGT in this strain rather than the parent Wistar strain being inherently nonresponsive to NDPS nephrotoxicity. Therefore, it appears that glucuronide metabolite(s) of NDHS and/or NDHSA contribute(s) to NDPS nephrotoxicity, although the exact nature of the nephrotoxicant glucuronide metabolite(s) of NDPS remains to be determined.

Analysis of gabapentin in serum and plasma by solid-phase extraction and gas chromatography-mass spectrometry for therapeutic drug monitoring.

A simple method for the determination of gabapentin (Neurontin) is described. The method uses solid-phase extraction by disk column and derivatization followed by gas chromatographic-mass spectrometric analysis. The single-step derivatization with MTBSTFA produces a t-BDMS derivative of both the carboxylic and amine moieties of the molecule. Each step of the procedure was optimized to assure reliable performance of the method. The assay limit of detection was 0.1 microg/mL with a linear range from 1.0 to 35 microg/mL. Within-run (n = 3) and between-run (n = 40) coefficients of variation were less than 8.2 and 15.9%, respectively. The method has proven reliable in routine production for more than a year, producing clean chromatography with unique ion fragments, consistent ion mass ratios, and no interferences. Statistical analysis of the gabapentin concentrations measured in 1020 random specimens over a 2-month period showed a mean concentration of 6.07 microg/mL with a standard deviation of 5.28.

Gender differences in acute N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) nephrotoxicity in Fischer 344 rats.

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide that induces nephrotoxicity as its major toxicity. NDPS is also a more potent nephrotoxicant in female than in male rats. The purpose of this study was to examine the nephrotoxic potential of the two NDPS metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) in age-matched male and female Fischer 344 rats to determine if gender differences exist for the nephrotoxicity induced by the two NDPS metabolites. Rats (4 per group) were administered a single intraperitoneal (ip) injection of NDHS or 2-NDHSA (0.025 or 0.05 mmol/kg) or vehicle, and renal function was monitored for 48 h. Neither compound induced significant nephrotoxicity in male rats at the doses tested. However, in female rats both metabolites induced marked nephrotoxicity at the 0.05 mmol/kg dose level, and treatment with 0.025 mmol/kg 2-NDHSA induced some changes in renal function (transient diuresis, transient proteinuria, decreased organic ion accumulation). Little effect on renal function was induced in females by treatment with 0.025 mmol/kg NDHS. At toxic levels in female rats, the renal lesions were located primarily in the S2 and S3 segments of the proximal tubule. These results indicate that, like the parent compound, gender differences exist in the nephrotoxic potential of NDHS and 2-NDHSA. The results also suggest that in females, as in males, NDPS nephrotoxicity is mediated via NDHS and/or 2-NDHSA. However, it is not clear if the ultimate nephrotoxicant species following NDPS exposure is different in males and females or if the same ultimate nephrotoxicant species is produced in both species but handled differently by male and female kidneys. Thus, further studies are needed to determine the exact nature of the ultimate nephrotoxicant species and the mechanisms of the observed gender differences.

Effects of sodium sulfate on acute N-(3,5-dichlorophenyl)succinimide (NDPS) nephrotoxicity in the Fischer 344 rat.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure in rats. Results of previous studies have suggested that NDPS may induce nephrotoxicity via conjugates of NDPS metabolites. Thus, the purpose of this study was to examine if administered sodium sulfate could alter NDPS nephrotoxicity. Male Fischer 344 rats (four rats per group) were administered a single intraperitoneal (i.p.) injection of sodium sulfate (0.035, 0.07, 0.35 or 3.5 mmol/kg) or sodium chloride (7.0 mmol/kg) 20 min before NDPS (0.2, 0.4 or 0.8 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg) and renal function monitored at 24 and 48 h. High dose sodium sulfate (3.5 mmol/kg) markedly attenuated NDPS nephrotoxicity, while sodium chloride had no effect on NDPS-induced renal effects. NDPS nephrotoxicity was also attenuated by a pretreatment dose of 0.35 mmol/kg sodium sulfate, while 0.07 mmol/kg sodium sulfate pretreatment potentiated NDPS 0.2 mmol/kg to produce nephrotoxicity without markedly attenuating NDPS 0.4 mmol/kg to induce renal effects. A dose of 0.035 mmol/kg sodium sulfate did not potentiate NDPS 0.2 mmol/kg to induce nephrotoxicity. These results suggest that sulfate conjugates of NDPS metabolites might contribute to NDPS nephrotoxicity.

4-Amino-2,6-dichlorophenol nephrotoxicity in the Fischer 344 rat: protection by ascorbic acid, AT-125, and aminooxyacetic acid.

A halogenated derivative of 4-aminophenol, 4-amino-2, 6-dichlorophenol (ADCP), is a potent nephrotoxicant and a weak hepatotoxicant in Fischer 344 rats. Although the mechanism of ADCP nephrotoxicity is unknown, ADCP could undergo oxidation to a reactive intermediate, such as a 4-amino-2,6-dichlorophenoxy radical or 2,6-dichloro-1,4-benzoquinoneimine, which can generate additional free radicals and/or covalently bind to cellular proteins. The toxic process might also be mediated by glutathione (GSH) conjugates of ADCP, as suggested for the mechanism of 4-aminophenol nephrotoxicity. In this study, the effects of modulators of oxidation and GSH conjugation-related metabolism or transport on ADCP-induced nephrotoxicity were examined. In one set of experiments, male Fischer 344 rats (four/group) were intraperitoneally (ip) administered ADCP (0.38 mmol/kg) only or coadministered an antioxidant, ascorbic acid (1.14 mmol/kg, ip) with ADCP. Administration of ascorbic acid markedly reduced both functional nephrotoxicity and morphological changes induced by ADCP. Administration of a gamma-glutamyltransferase (GGT) inhibitor, l-(alphaS, 5S)-alpha-amino-3-chloro-4,5-dihydroxy-5-isoxazoleacetic acid (10 mg/kg, ip), or a cysteine conjugate beta-lyase inhibitor, aminooxyacetic acid (0.5 mmol/kg, ip), 1 hr before ADCP (0.38 mmol/kg) challenge partially protected rats against ADCP nephrotoxicity. In contrast, administration of an organic anion transport inhibitor, probenecid (140 mg/kg, ip), 30 min before ADCP had little effect on ADCP nephrotoxicity. The GSH depletor, buthionine sulfoximine (890 mg/kg, ip), was given 2 hr prior to ADCP and only minimal protection was noted. In addition, the nonprotein sulfhydryl (NPSH) contents in renal cortex and liver were determined at 2 hr following the administration of ADCP only or ascorbic acid/ADCP. Ascorbic acid afforded complete prevention of the depletion of NPSH in the kidney and liver caused by ADCP administration and also prevented the elevation of renal glutathione disulfide content induced by ADCP. The results indicate that oxidation of ADCP appears to be essential to ADCP nephrotoxicity and that GSH or GSH-derived conjugates of ADCP may be partly responsible for the nephrotoxic effects of ADCP via a GGT-mediated mechanism.

3,4-Dicholoroaniline acute toxicity in male Fischer 344 rats.

The aromatic amine, 3,4-dichloroaniline (DCA) is an important intermediate in the chemical production of agricultural chemicals. A previous study had shown that nephrotoxicity was apparent 48 h after injection of 3,4-DCA. The purpose of this study was to examine the potential for 3,4-DCA to be toxic to the kidney, liver and urinary bladder 24 h after acute administration. Male Fischer 344 (F344) rats were injected (intraperitoneal (i.p.)) with 0.4, 0.8 or 1.0 mmol/kg 3,4-DCA hydrochloride (HCl) salt (2.5 ml/kg, 25% ethanol). Nephrotoxicity was apparent within 24 h in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group and was characterized by elevated (P < 0.05) blood urea nitrogen (BUN) and kidney weight. Renal cortical slice accumulation ofp-aminohippurate (PAH) was also decreased in the 0.8 and 1.0 mmol/kg 3,4-DCA treated group relative to pair fed controls (PFC). Cellular changes were noted in the liver and bladder 24 h after 3,4-DCA administration. Plasma alanine transaminase (ALT) activity was elevated (P < 0.05) above PFC values 24 h after treatment with 0.8 or 1.0 mmol/kg indicating liver damage was apparent within 24 h. Morphological damage was apparent along the centrilobular region. Hematuria was observed in the 0.8 and 1.0 mmol/kg 3,4-DCA treated groups. Infiltration of erythrocytes and polymorphonuclear leukocytes was apparent within the urinary bladder upon examination by light microscopy. These results indicated that 3,4-DCA was toxic within 24 h and that the target tissues were the kidney, liver and urinary bladder. In vitro studies were conducted to compare the toxicity of two forms of 3,4-DCA, the free base and hydrochloride salt to determine whether chemical form contributes to renal cortical slice toxicity. Lactate dehydrogenase (LDH) release was elevated above control by 120 min exposure to 2 mM 3,4-DCA free base or hydrochloride salt. Pyruvate directed gluconeogenesis in renal slices was decreased relative to control by 0.5 mM 3,4-DCA free base and hydrochloride salt. The results from the in vitro studies indicates that the chemical form did not modify in vitro renal cortical slice toxicity.

Nephrotoxicity of N-(3-bromophenyl)-2-hydroxysuccinimide: role of halogen groups in the nephrotoxic potential of N-(halophenyl) succinimides.

Among N-(halophenyl)succinimides. N-(3,5-dichlorophenyl)succinimide (NDPS) is a potent nephrotoxicant as well as an agricultural fungicide. Although two chloride groups on the phenyl ring are essential to induce optimal nephrotoxicity, the role of halogen groups in NDPS nephrotoxicity is not clear. In this study, N-(3-bromophenyl)-2-hydroxysuccinimide (NBPHS) was prepared as a monohalophenyl derivative of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), an oxidative and nephrotoxicant metabolite of NDPS. The nephrotoxic potential of NBPHS was evaluated in vivo and in vitro to determine the role of halogen groups in N-(halophenyl)succinimide nephrotoxicity. Male Fischer 344 rats (four/group) were administered a single intraperitoneal (i.p.) injection of NBPHS (0.1, 0.4 or 0.8 mmol/kg) or vehicle (25% dimethyl sulfoxide in sesame oil) and renal function monitored for 48 h. Administration of NBPHS (0.8 mmol/kg) induced nephrotoxicity, while very mild changes or no changes in renal function were observed following administration of 0.4 mmol/kg or 0.1 mmol/kg of NBPHS, respectively. Nephrotoxicity induced by NBPHS (0.8 mmol/kg) was characterized by diuresis, transiently increased proteinuria, glucosuria and hematuria elevated kidney weight and reduced tetraethylammonium (TEA) uptake by renal cortical slices, and was not as marked as nephrotoxicity induced by NDHS (0.1 mmol/kg) or NDPS (0.4 mmol/kg). In the in vitro studies the effects of NBPHS on organic ion accumulation, pyruvate-stimulated gluconeogenesis, and lactate dehydrogenase (LDH) release were measured using renal cortical slices. NBPHS decreased p-aminohippurate (PAH) and TEA accumulation at NBPHS bath concentrations of 0.05 mM and 0.5 mM and 0.5 mM or greater, respectively. Renal gluconeogenesis was inhibited by NBPHS at 1 mM bath concentration, while LDH leakage was not increased at NBPHS bath concentrations up to 1 mM. The results demonstrate that NBPHS is a mild nephrotoxicant in vivo and in vitro, but does not have cytotoxic effects to renal tissues at the concentrations tested. From these results, it appears that halogen groups are essential to the nephrotoxic potential of N-(halophenyl)-2-hydroxysuccinimides or N-(halophenyl)succinimides and play an important role in the mechanism of NDPS nephrotoxicity following NDHS formation.

Nephrotoxicity of 4-amino-2-chlorophenol and 2-amino-4-chlorophenol in the Fischer 344 rat.

Aminophenols and halogenated anilines induce nephrotoxicity and mild hepatotoxicity in rats. In this study, the in vivo and in vitro nephrotoxic potential of 4-amino-2-chlorophenol and 2-amino-4-chlorophenol, monochlorinated aminophenols and potential metabolites of 3-chloroaniline, was evaluated. Hepatotoxicity of both compounds was also examined in vivo. Male Fischer 344 rats (four/group) were administered 4-amino-2-chlorophenol hydrochloride (0.4, 0.8 or 1.0 mmol/kg), 2-amino-4-chlorophenol hydrochloride (0.4, 0.8 or 1.2 mmol/kg) or vehicle intraperitoneally (i.p.) and renal and hepatic function monitored for 48 h. Administration of 4-amino-2-chlorophenol (0.8 mmol/kg) induced nephrotoxicity, while only minor changes in kidney function were observed following administration of 0.4 mmol/kg of 4-amino-2-chlorophenol or 0.8 mmol/kg of 2-amino-4-chlorophenol. Increasing the dose of 4-amino-2-chlorophenol to 1.0 mmol/kg or 2-amino-4-chlorophenol to 1.2 mmol/kg resulted in lethality. Nephrotoxicity induced by 4-amino-2-chlorophenol was characterized by diuresis, increased proteinuria, glucosuria, hematuria, elevated blood urea nitrogen (BUN) concentration and kidney weight, and marked proximal tubular damage, while 2-amino-4-chlorophenol induced milder effects on renal function and transient oliguria instead of diuresis. No hepatotoxicity was observed with either compound at any dose tested. In the in vitro studies, the direct effects of 4-amino-2-chlorophenol or 2-amino-4-chlorophenol on organic ion accumulation, pyruvate-stimulated gluconeogenesis and lactate dehydrogenase (LDH) leakage were determined using renal cortical slices. 4-Amino-2-chlorophenol and 2-amino-4-chlorophenol were almost equally effective in inhibiting organic anion or cation uptake and gluconeogenesis or increasing LDH leakage, although small differences in the minimum effective concentrations were present (minimum effective concentration, 0.01-0.5 mM range). These results demonstrate that 4-amino-2-chlorophenol is a more potent nephrotoxicant than 2-amino-4-chlorophenol in vivo. The results also indicate that the addition of a chloride group to aminophenols enhances renal toxicity.

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol.

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.

3,5-Dichloroaniline toxicity in Fischer 344 rats pretreated with inhibitors and inducers of cytochrome P450.

3,5-Dichloroaniline (3,5-DCA), a derivative needed in the manufacture of dyes, pesticides and industrial compounds has been reported to induce renal damage. This study investigated whether pretreatment with inducers or inhibitors of P450 altered 3,5-DCA toxicity. P450 levels were induced in male Fischer 344 rats (4-12/group) by pretreatment (i.p.) with phenobarbital (PB, 75 mg/kg/day for 3 days), beta-naphthoflavone (BNF, 100 mg/kg/day for 4 days) or pyridine (PYR, 100 mg/kg/day for 4 days). P450 activity was inhibited by pretreatment with piperonyl butoxide (PiBx) 30 min prior to injection of 3,5-DCA. Upon completion of a designated pretreatment regimen, 0.4 or 0.8 mmol/kg 3,5-DCA was injected into F344 rats. Pair-fed controls were injected with 25% ethanol solution or physiological saline (2.5 ml/kg). The renal changes monitored at 24 and 48 h following treatment with 0.8 mmol/kg 3,5-DCA were characterized by increased blood urea nitrogen (BUN) level and decreased renal cortical slice accumulation of p-aminohippurate (PAH). Plasma alanine transaminase activity (ALT/GPT) was increased 24 h after injection of 0.8 mmol/kg 3,5-DCA while liver wt. was unchanged. PB or PYR pretreatment did not alter the renal or hepatic effects of 3,5-DCA while BNF pretreatment slightly reduced toxicity. In contrast, PiBx pretreatment increased the renal and hepatic changes associated with 3,5-DCA. The results with PiBx suggest that either the parent compound possesses some direct cytotoxicity or that a toxic metabolite was generated through a biotransformation pathway not inhibited by PiBx.

Role of chloride groups in the nephrotoxic potential of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, an oxidative metabolite of N-(3,5-dichlorophenyl)succinimide.

Although the addition of chloride groups to the phenyl ring of N-phenylsuccinimide (NPS) is known to enhance the nephrotoxic potential of NPS, the mechanism of this enhancement is unknown. One chlorinated NPS derivative, N-(3,5-dichlorophenyl)succinimide (NDPS), is a potent nephrotoxicant which induces marked proximal tubular necrosis at i.p. doses of 0.4 mmol/kg or greater. The purpose of this study was to compare the nephrotoxic potential of 2-hydroxy-N-phenylsuccinimide (HNPS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), an oxidative and nephrotoxicant metabolite of NDPS, to determine the importance of the chloride groups for the nephrotoxic potential of NDHS. Male Fischer 344 rats (4/group) were administered a single i.p. injection of HNPS (1.0 or 1.5 mmol/kg), NDHS (0.1 mmol/kg) or vehicle (25% dimethyl sulfoxide in sesame oil), and renal function measured at 24 and 48 h. HNPS was a nonnephrotoxicant at both doses tested, while NDHS induced marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, elevated blood urea nitrogen (BUN) concentration and kidney weight, decreased organic ion accumulation by renal cortical slices and proximal tubular necrosis. In vitro, HNPS reduced p-aminohippurate (PAH) and tetraethylammonium (TEA) accumulation beginning at HNPS bath concentrations of 0.05 and 0.5 mM, respectively. The results of this study indicate that although HNPS has direct effects on renal function in vitro, HNPS is not a nephrotoxicant in vivo at doses up to 15 times the minimal nephrotoxicant dose of NDHS. Therefore, the chloro groups present on NDHS play an essential role in the nephrotoxic potential of NDHS and contribute to aspects of the nephrotoxic mechanism of NDPS beyond NDPS oxidation to form NDHS.