N-(3,5-dichlorophenyl)-2-hydroxysuccinimide Research Articles

Nephrotoxicity induced by the R- and S-enantiomers of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and their sulfate conjugates in male Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity characterized as polyuric renal failure and mediated via metabolites arising from oxidation of the succinimide ring. Recent findings have suggested that the stereochemical nature of NDPS metabolites may be an important factor in NDPS metabolite-induced nephrotoxicity. The purpose of the present study was to determine the role of stereochemistry in the in vivo nephrotoxicity induced by R-(+)- and S-(−)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide ( R- and S-NDHS) and the in vitro nephrotoxicity induced by their enantiomeric sulfate conjugates, R-(−)- and S-(+)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide- O-sulfate ( R- and S-NSC). Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) an enantiomer of NDHS (0.05, 0.1 or 0.2 mmol/kg) or vehicle, and renal function monitored for 48 h. R-NDHS (0.1 or 0.2 mmol/kg) had little effect on renal function. In contrast, S-NDHS (0.1 mmol/kg) induced marked nephrotoxicity. The nephrotoxic potential of R- and S-NSC (0.5, 0.75 or 1.0 mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3–4 × 10 6 cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1 h incubation period. The LDH release observed in these studies was similar between R- and S-NSC. These results indicate that stereochemistry is an important factor for NDPS metabolite nephrotoxicity and that the role of stereochemistry, at least for NSC, occurs at extra-renal sites.

Effect of gender on phase I and phase II metabolism of N‐(3,5‐dichlorophenyl)succinimide (NDPS)

NDPS is a model compound for examining chemically-induced kidney damage. The parent compound and its direct oxidative metabolite, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), induce nephrotoxicity in rats. Toxicity of NDPS is thought to require hepatic formation of NDHS followed by conjugation to reactive phase II metabolites. Further studies have shown that female rats are more susceptible to toxicity than males. In this study, the relationship between turnover and gender was examined. Metabolism was studied by incubating rat liver microsomes with the appropriate substrate and cofactor (oxidation: NDPS, NADPH; glucuronidation: NDHS, UDPGA) for time periods between 15 and 120 min. Samples were analyzed by HPLC with UV detection or HPLC-ESI-MS, and relative metabolite formation was determined. Results indicate that females are capable of producing the same oxidative metabolites and glucuronide conjugates that were previously found in males. A quantitative difference in the formation of oxidative metabolites was not observed. However, NDHS glucuronidation was significantly higher in male microsomes at incubation periods of 60 min or less. Whether this is relevant to the differential susceptibility of the genders to NDPS nephrotoxicity remains to be determined. Other phase II pathways, such as sulfation, may also vary between female and male rats. Supported by PHS grant ES012499.

Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats: Evidence for Bioactivation through Alcohol-O-glucuronidation and O-Sulfation

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS), was shown to be nephrotoxic in rats. Previous studies have indicated that the metabolism of NDPS contributes to its mechanism of toxicity and both phase I and phase II pathways may be involved. In the current report, we investigated the detailed biotransformation of [(14)C]NDPS in rats using HPLC-ESI-MS. The chemical reactivity of the phase II NDPS metabolites was also evaluated. In vivo studies were conducted by administering [(14)C]NDPS to male Fischer 344 rats. Urine, tissue (liver and kidney), and plasma samples were analyzed. The mechanism of formation and chemical reactivity of the glucuronide and sulfate metabolites of NDPS were investigated in vitro using liver subcellular preparations. Major in vivo metabolites of NDPS were identified as the oxidative [N-(3,5-dichlorophenyl)-2- and 3-hydroxysuccinamic acid, 2-/3-NDHSA] and hydrolytic products [N-(3,5-dichlorophenyl)succinamic acid]. N-Acetylcysteine and cysteine (with intramolecular aminolysis) conjugates were also detected in rat urine and fecal extracts, respectively, suggesting the formation of reactive intermediate(s) in the metabolism of NDPS. Small amounts of the alcohol-O-glucuronide and O-sulfate of 2-/3-NDHSA were detected in rat urine, plasma, and tissue homogenates. The formation of these phase II metabolites was found to be mediated through the initial conjugation of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) followed by hydrolysis. As compared to NDHS, NDHS-O-sulfate is approximately 500-fold more reactive toward GSH conjugation. In rat liver S9, fortifying phase II cofactors (UDPGA or PAPS) in incubation mixtures with NDHS also significantly increased the amount of GSH adducts produced. Results of this research demonstrate that phase II metabolites of NDPS were produced in rats. The formation of the alkyl alcohol-O-glucuronide and O-sulfate conjugates represents bioactivation pathways in the metabolism of NDPS that could potentially contribute to its mechanism of nephrotoxicity.

EFFECT OF THREE N -ACETYLAMINO ACIDS ON N -(3,5-DICHLOROPHENYL)SUCCINIMIDE (NDPS) AND NDPS METABOLITE NEPHROTOXICITY IN FISCHER 344 RATS

The agricultural fungicide N -(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in mammals characterized as polyuric renal failure and proximal tubular necrosis. Recent studies have suggested that NDPS-induced nephrotoxicity may be mediated by metabolites arising from the nephrotoxic NDPS metabolites N -(3,5-dichlorophenyl)2-hydroxysuccinimide (NDHS) and/or N -(3,5-dichlorophenyl)-2-succinamic acid (2-NDHSA). The purpose of this study was to examine the effects of N -acetylcysteine (NAC), a nucleophilic agent, and two nonnucleophilic N -acetylamino acids, N -acetylserine (NAS) and N -acetylalanine (NAA), on NDPS and NDPS metabolite-induced nephrotoxicity. Male Fischer 344 rats (4-8/group) were administered intraperitoneally (ip) an N -acetylamino acid (1 mmol/kg) 2 h before an ip injection of NDPS (0.4 mmol/kg), NDHS (0.1 mmol/kg), 2-NDHSA (0.1 mmol/kg), or vehicle. Renal function was then monitored at 24 and 48 h. NAC pretreatment markedly attenuated NDPS-, NDHS-, and 2-NDHSA-mediated nephrotoxicity. The nonnucleophilic N -acetylamino acids (NAS, NAA) only partly reduced NDPS and NDHS nephrotoxicity, and they had little effect on 2-NDHSA nephrotoxicity. These results suggest that reactive NDPS metabolites may be formed from NDHS and 2-NDHSA and that nucleophilic substrates (e.g., NAC) may offer protection from NDPS-induced nephrotoxicity. However, mechanisms other than chemical neutralization of reactive NDPS metabolites may also be contributing to the attenuation of NDPS nephrotoxicity, since nonnucleophilic N -acetylamino acids (e.g., NAA) also provided some protection against NDPS and NDHS nephrotoxicity.

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.

In vitro nephrotoxicity induced by N-(3,5-dichlorophenyl)succinimide (NDPS) metabolites in isolated renal cortical cells from male and female Fischer 344 rats: evidence for a nephrotoxic sulfate conjugate metabolite

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in vivo that is characterized as acute polyuric renal failure and proximal tubular necrosis. However, earlier in vitro studies have failed to reproduce the in vivo nephrotoxicity seen with NDPS or its nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The purpose of this study was to examine the nephrotoxic potential of NDPS, its known non-conjugated metabolites, the O-sulfate conjugate of NDHS (NSC), and the putative metabolite N-(3,5-dichlorophenyl)maleimide (NDPM) and its hydrolysis product N-(3,5-dichlorophenyl)maleamic acid (NDPMA) using freshly isolated renal cortical cells (IRCC). IRCC were obtained from untreated male or female Fischer 344 rats following collagenase perfusion of the kidneys. Cells (∼4 million per ml) ( N=4) were incubated with up to 1.0 mM NDPS or an NDPS metabolite or vehicle for up to 120 min. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release into the medium. Only NSC (>0.5 mM) and NDPM (≥0.5 mM) exposure increased LDH release from IRCC. NSC 1.0 mM or NDPM 0.5 mM increased LDH release from IRCC within 15–30 min of exposure. NDPS or the remaining NDPS metabolites did not increase LDH release at bath concentrations of 1.0 mM for exposures of 120 min. IRCC from male and female rats responded similarly to the toxic effects of NDPS and its metabolites. These results demonstrate that sulfate conjugates of NDPS metabolites can be fast acting nephrotoxicants and could contribute to NDPS nephrotoxicity in vivo. These results also suggest that the kidney probably accumulates toxic sulfate conjugates of NDPS metabolites rather than forming the conjugates. In addition, mechanisms responsible for gender differences in nephrotoxicity seen with NDPS and NDPS metabolites in vivo either occur prior to renal accumulation of sulfate conjugates and/or represent biochemical/physiological differences between the genders.

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.

The Role of Glucuronidation inN-(3,5-Dichlorophenyl)succinimide (NDPS) Nephrotoxicity: Nephrotoxic Potential of NDPS and NDPS Metabolites in Gunn, Wistar, and Fischer 344 Rats

The agricultural fungicideN-(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) andN-(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.

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 agematched 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.

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 lactatc 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 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.

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 a 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 ?M, 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 NDHS and contribute to aspects of the nephrotoxic mechanism of NDPS beyond NDPS oxidation to form NDHS.

Effect of dimethyl sulfoxide on N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity

Dimethyl sulfoxide (DMSO) is frequently used as a solvent to assist in dissolving compounds which are not readily soluble in other injection vehicles. The purpose of this study was to determine the suitability of DMSO as a vehicle for administering the nephrotoxicant, N-(3,5-dichlorophenyl)succinimide, (NDPS) and two nephrotoxicant NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA). Male Fischer 344 rats (4/group) were administered a single intraperitoneal injection of NDPS (0.4 or 0.8 mmol/kg), NDHS (0.1 or 0.2 mmol/kg), or NDHSA (0.1 or 0.2 mmol/kg) dissolved in 25% DMSO in sesame oil or 100% sesame oil (2.5 ml/kg), while control rats received vehicle only. Renal function was then monitored at 24 and 48 h. Including DMSO in the vehicle markedly attenuated NDPS 0.4 mmol/kg-induced nephrotoxicity and reduced NDPS 0.8 mmol/kg-induced renal effects. Thus, the magnitude of the attenuating effect of DMSO depended in part on the nephrotoxicant dose of NDPS. In addition, NDHS nephrotoxicity was not altered by DMSO and only slight effects on NDHSA nephrotoxicity were observed. These results suggest that DMSO is capable of attenuating NDPS nephrotoxicity, and that the primary mechanism of this interaction might be due to an inhibition of the biotransformation of NDPS to NDHS.

Effect of microsomal enzyme modulators on N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS)-induced nephrotoxicity in the Fischer 344 rat

We have previously reported that phenobarbital (PB) pretreatment enhances and piperonyl butoxide (PIBX) pretreatment or cobalt chloride (CoCl 2) pretreatment decreases the nephrotoxicity induced by the model nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) in the Fischer 344 rat. The objective of this study was to determine the effect of a microsomal enzyme inducer (PB) or microsomal enzyme inhibitor (PIBX or CoCl 2) on a single intraperitoneal (i.p.) injection of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.05, 0.1 or 0.2 mmol/kg), a nephrotoxicant metabolite of NDPS, or vehicle (sesame oil, 2.5 ml/kg). Renal function was monitored at 24 and 48 h post-NDHS for PB pretreated rats and at 24 h only for PIBX and CoCl 2 pretreated rats, due to lethality at 48 h in PIBX pretreated rats. PB pretreatment potentiated the renal toxicity induced by a non-toxic dose of NDHS (0.05 mmol/kg), inducing diuresis and elevated proteinuria, hematuria, glucosuria, blood urea nitrogen (BUN) concentration and kidney weight. PB pretreatment also enhanced some monitored renal effects of a toxic dose (0.1 mmol/kg) of NDHS, including reduced organic ion transport by renal cortical slices. PIBX and CoCl 2 pretreatments did not markedly affect the increased kidney weight, proteinuria, glucosuria, BUN concentration or altered organic ion transport induced by NDHS (0.2 mmol/kg) treatment. We conclude that PB potentiates NDHS-induced nephrotoxicity via a mechanism not influenced by CoCl 2 or PIBX.

Effect of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide on renal function and hemodynamics in the anesthetized rat

The experimental agricultural fungicide N-dichlorophenyl)succinimide (NDPS) is a nephrotoxicant in the Fischer 344 rat. NDPS induces nephrotoxicity via metabolic bioactivation to one or more metabolites. Both N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), oxidative metabolites of NDPS, are more potent nephrotoxicants than the parent compound. Preliminary studies in our laboratiry indicate that altered renal hemodynamics may contribute to the mechanism of NDPS-induced nephrotoxicity. However, it is not known if NDPS affects renal hemodynamics prior to or after altering tubular function. In this study, male Fischer 344 rats (375–300 g) were anesthetized with urethane (1.5 g/kg, i.p) and prepared for renal function experiments. Renal blood flow (RBF), glomerular filtration rate (GFR), urine flow rate (V) and fraction of GFR excreted as urine (V/GFR) were determined during eight 30 min intervals following NDHS (0.2 or 0.4 mmol/kg, i.p.) or vehivle (sesame oil, 2.5 ml/kg, i.p.) treatment. NDHS (0.2 or 0.4 mmol/kg) decreased GFR while urine flow rate and V/GFR were increased compared to vehicle-treated controls. These alterations in renal function were evident by 2 h post NDHS (0.4 mmol/kg) and by 3 h post-NDHS (0.2 mmol/kg) treatment. RBF of rats receiving NDHS (0.2 or 0.4 mmol/kg) tended to decrease post-NDHS treatment; however, this decrease was not significant. Results of this study indicate that NDHS (0.2 or 0.4 mmol/kg) initially alters renal function by reducing the tubular reabsorption of glomerular filtrate prior to a reduction of GFR and RBF.

Effect of buthionine sulfoximine on N-(3,5-dichlorophenyl)-2- hydroxysuccinimide and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid nephrotoxicity

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide which induces acute tubular necrosis as its primary toxicity. Two NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA) previously have been shown to be more potent nephrotoxicants than NDPS. In addition, buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, was found to attenuate NDPS-induced nephrotoxicity. The purpose of this study was to examine the effects of BSO pretreatment on NDHS- and NDHSA-induced nephrotoxicity. Male Fischer-344 rats (4 rats/group) were administered intraperitoneally (i.p.) BSO (890 mg/kg) 2 h before NDHS or NDHSA (0.1 or 0.2 mmol/kg i.p.) or vehicle (sesame oil 2.5 ml/kg), and renal function monitored at 24-h intervals for 48 h. BSO pretreatment markedly attenuated NDHSA (0.1 or 0.2 mmol/kg)-induced effects on the renal functional parameters monitored. BSO pretreatment also markedly reduced NDHS (0.1 mmol/kg)-induced renal effects. However, NDHS (0.2 mmol/kg) nephrotoxicity was attenuated to a lesser extent than NDHS (0.1 mmol/kg) nephropathy. These results indicate that glutathione is an important mediator of NDPS metabolite nephrotoxicity and suggests that BSO did not attenuate NDPS nephropathy by inhibiting NDPS biotransformation to NDHS or NDHSA.

Effect of calcium antagonism by nifedipine and chlorpromazine on acute N-(3,5-dichlotophenyl)succinimide-induced nephrotoxicity in Fischer 344 rats

The nephrotoxicity induced by a wide variety of chemical compounds can be attenuated by agents which modify calcium ion (Ca 2+) movement across membranes or calcium-dependent processes. The purpose of this study was to examine the ability of nifedipine, a calcium channel blocking drug, and chlorpromazine (CPZ), an antagonist of many calcium-dependent processes, to attenuate the nephrotoxicity induced by the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) or its metabolite N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS). Male Fischer 344 rats (4 rats per group) were pretreated intraperitoneally (i.p.) with nifedipine (0.25 or 0.50 mg/kg), CPZ (1.0 or 5.0 mg/kg) or vehicle 1 h before NDPS (0.4 mmol/kg), NDHS (0.1 mmol/kg) or vehicle (sesame oil, 2.5 ml/kg). In separate experiments, rats were pretreated with nifedipine (0.25 or 0.50 mg/kg/day, i.p.) starting 2 days before NDPS or NDPS vehicle and continuing throughout the experiment. Renal function was monitored at 24 and 48 h. Nifedipine (single or multiple treatments) and CPZ (1.0 mg/kg) were ineffective in substantially altering NDPS (0.4 mmol/kg)-induced nephrotoxicity. However, CPZ (5.0 mg/kg) markedly attenuated all aspects of NDPS-induced nephropathy. Also, CPZ (5.0 mg/kg) partially protected against NDHS (0.1 mmol/kg)-induced renal effects. These results demonstrate the inability of the calcium channel blocker nifedipine to attenuate NDPS nephrotoxicity. Attenuation of NDPS nephrotoxicity by CPZ could suggest that CPZ is antagonizing calcium influx into renal tissue and/or renal intracellular calcium-dependent processes to modify the renal response to NDPS. However, the inability of CPZ to markedly attenuate NDHS nephrotoxicity could indicate that CPZ protected against NDPS nephrotoxicity by inhibiting biotransformation of the parent compound to its toxic chemical species.

N(3,5-dichlorophenyl)succinimide nephrotoxicity: evidence against the formation of nephrotoxic glutathione or cysteine conjugates

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity via one or more metabolites. Previous studies suggested that glutathione is important for mediating NDPS-induced nephropathy. The purpose of this study was to examine the possibility that a glutathione or cysteine conjugate of NDPS or an NDPS metabolite might be the penultimate or ultimate nephrotoxic species. In one set of experiments, male Fischer 344 rats were administered intraperitoneally (i.p.) NDPS (0.4 or 1.0 mmol/kg) 1 h after pretreatment with the gamma glutamyltranspeptidase inhibitor AT-125 (acivicin) (10 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. In general, AT-125 pretreatment had few effects on NDPS-induced nephropathy. In a second set of experiments, rats were treated i.p. or orally (p.o.) with a putative glutathione ( S-(2-( N-(3,5-dichlorophenyl)succinimidyl)glutathione (NDPSG)), a cysteine ( S-(2-( N-3,5-dichlorophenyl)succinimudyl)cysteine (NDPSC) (as the methyl ester) or ( N- acetylcysteine ( S-(2-( N-(3,5- dichlorophenyl) succinimidyl)- N- acetylcysteine ( NDPSN)) conjugate of NDPS (0.2, 0.4 or 1.0 mmol/kg) or vehicle and renal function was monitored at 24 and 48 h. An intramolecular cyclization product of NDPSC, 5-carbomethoxy-2-( N-(3,5-dichlorophenyl)carbomyl-methyl) -1,4-thiazene-3-one (NDCTO) was also examined for nephrotoxic potential. None of the compounds produced toxicologically important changes in renal function or morphology. The in vitro ability of the conjugates to alter organic ion accumulation by cortical slices was also examined. All of the conjugates tested caused a reduction in p-aminohippurate (PAH) accumulation at a conjugate bath concentration of 10 −4 M, but none of the conjugates reduced tetraethylammonium (TEA) uptake. In a third experiment, the ability of the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid (AOAA) (0.5 mmol/kg, i.p.) to alter the nephrotoxicity induced by two NDPS metabolites, N-(3,5-dichlorophenyl) -2-hydroxysuccinimide (NDHS) of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA) (0.2 mmol/kg, i.p.), was examined. AOAA pretreatment had no effect on NDHS- or NDHSA-induced nephrotoxicity. These results do not support a role for a glutathione or cysteine conjugate of NDPS or an NDPS metabolite as being the penultimate or ultimate nephrotoxic species.

Effect of autacoid modulation on N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide which has been shown to induce acute tubular necrosis. The purpose of the present study was to determine if creatinine clearance was altered early in the development of NDPS nephrotoxicity. This study also examined the effect of autacoid modulation on the renal effects induced by NDPS and two metabolites of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA). In one set of experiments, male Fischer 344 rats (4 rats/group) were administered a single intraperitoneal (i.p.) injection of NDPS (1.0 mmol/kg) or vehicle and creatinine clearance was determined at 3 and 6 h post-treatment. NDPS administration resulted in a marked decrease in creatinine clearance at both time points. In a second set of experiments, rats (4–8 rats/group) were pretreated with the cyclooxygenase inhibitor indomethacin (3.0 or 5.0 mg/kg, i.p.) or the thromboxane synthase inhibitor dazmegrel (20 mg/kg, i.p.) 1 h before the i.p. administration of NDPS (0.2 or 0.4 mmol/kg), NDHS (0.05 or 0.1 mmol/kg), NDHSA (0.05 or 0.1 mmol/kg) or vehicle. Indomethacin pretreatment potentiated the nephrotoxic potential of NDPS and its two metabolites, while dazmegrel pretreatment attenuated NDPS nephrotoxicity without marked effects on NDHS or NDHSA nephropathy. These results indicate that renal hemodynamic changes occur early in the development of NDPS nephrotoxicity and that autacoids are important modulators of NDPS- and NDPS metabolite-induced renal effects.

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl)succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPSA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h. However, NDPM caused a concentration-(25–100 μM) and time-dependent (0.25–4 h) loss in basal and nystatin-stimulated oxygen consumption and tubule viability. Investigations using isolated renal cortical mitochondria (RCM) showed that NDPM was a potent inhibitor of mitochondrial function. Isolated RCM respiring on pyruvate/malate and exposed to NDPM exhibited a concentration (25–100 μM) dependent decrease in state 3 and state 4 respiration. Inhibition of mitochondrial state 3 respiration by NDPM was mediated through site 1 of the respiratory chain. NDPM did not inhibit cytochrome c-cytochrome oxidase or the electron transport chain. These results indicated that NDPS, its known metabolites, and NDPMA were not directly toxic to rat RPT. However, the postulated metabolite NDPM, was a potent tubule cytotoxicant that inhibited mitochondrial function in isolated RCM and RPT and may produce cell death through this mechanism.

Nephrotoxicity of N-(3,5-dichlorophenyl)succinimide metabolites in vivo and in vitro

The experimental fungicide N-(3,5-dichlorophenyl)-succinimide (NDPS) has been shown to produce selective nephrotoxicity at least in part through the actions of one or more metabolites. The purpose of this study was to (1) determine the nephrotoxic potential of three known NDPS metabolites; N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), and N-(3,5-dichlorophenyl)malonamic acid (DMA) and (2) examine the role of renal biotransformation in NDPS-induced nephrotoxicity. In one set of experiments, male Fischer 344 rats were administered a single intraperitoneal (ip) injection of NDPS or a NDPS metabolite (0.2, 0.4, or 1.0 mmol/kg) or vehicle (sesame oil, 2.5 ml/kg) and renal function was monitored at 24 and 48 hr. Both NDHS and NDHSA administration (0.2 or 0.4 mmol/kg) resulted in nephrotoxicity similar to that produced by NDPS (0.4 or 1.0 mmol/kg). DMA administration resulted in only minor renal effects. Addition of NDPS to renal cortical slices prepared from naive Fischer 344 rats resulted in decreases in p-aminohippurate (PAH) and tetraethylammonium (TEA) accumulation at NDPS media concentrations of 10 −4 and 10 −5 m or greater, respectively. Pretreatment of rats with microsomal enzyme activity modifiers (phenobarbital, 3-methylcholanthrene, cobalt chloride, or piperonyl butoxide) had little effect on in vitro effects of NDPS on PAH or TEA accumulation. A pattern of PAH or TEA uptake similar to that observed for NDPS was observed in vitro with NDPS- d 4, a nonnephrotoxic analog of NDPS labeled on the succinimide ring with deuterium. Of the NDPS metabolites tested in vitro for nephrotoxicity, only NDHS produced decreases in PAH and TEA accumulation similar to those produced by NDPS. These results suggest that the NDPS metabolites NDHS and NDHSA are nephrotoxic compounds. However, the role of these metabolites in NDPS-induced nephrotoxicity remains to be determined. In addition, it appears that NDPS has direct effects on renal function, but these effects do not appear to be of major toxicological significance in vivo. Direct renal bioactivation of NDPS or its known metabolites to nephrotoxic species does not appear to occur in vitro.