Cytotoxicity of thiazolidinedione-, oxazolidinedione- and pyrrolidinedione-ring containing compounds in HepG2 cells.

Liver damage occurred in some patients who took troglitazone (TGZ) for type II diabetes. The 2,4-thiazolidinedione (TZD) ring in TGZ's structure has been implicated in its hepatotoxicity. To further examine the potential role of a TZD ring in toxicity we used HepG2 cells to evaluate two series of compounds containing different cyclic imides. N-phenyl analogues comprised 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT); 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO) and N-(3,5-dichlorophenyl)succinimide (NDPS). Benzylic compounds, which closely resemble TGZ, included 5-(3,5-dichlorophenylmethyl)-2,4-thiazolidinedione (DCPMT); 5-(4-methoxyphenylmethyl)-2,4-thiazolidinedione (MPMT); 5-(4-methoxyphenylmethylene)-2,4-thiazolidinedione (MPMT-I); 5-(4-methoxyphenylmethyl)-2,4-oxazolidinedione (MPMO); 3-(4-methoxyphenylmethyl)succinimide (MPMS) and 3-(4-methoxyphenylmethylene)succinimide (MPMS-I). Cytotoxicity was assessed using the MTS assay after incubating the compounds (0-250µM) with HepG2 cells for 24h. Only certain TZD derivatives (TGZ, DCPT, DCPMT and MPMT-I) markedly decreased cell viability, whereas MPMT had low toxicity. In contrast, analogues without a TZD ring (DCPO, NDPS, MPMO, MPMS and MPMS-I) were not cytotoxic. These findings suggest that a TZD ring may be an important determinant of toxicity, although different structural features, chemical stability, cellular uptake or metabolism, etc., may also be involved. A simple clustering approach, using chemical fingerprints, assigned each compound to one of three classes (each containing one active compound and close homologues), and provided a framework for rationalizing the activity in terms of structure.

Pyrrolidinediones reduce the toxicity of thiazolidinediones and modify their anti-diabetic and anti-cancer properties.

Thiazolidinediones have been established as a drug class of significant importance in the treatment of Type II diabetes mellitus and have more recently displayed emergent potential as anti-cancer agents. However, their toxicity has hampered clinical development and usage in both therapeutic areas. Studies to date have implicated that the thiazolidinedione ring is responsible for the generation of reactive metabolites after metabolism. As an attempt to improve their safety profiles, we considered the bioisosteric replacement of the thiazolidinedione ring with a chemically conserved pyrrolidinedione heterocyclic system. Using pyrrolidinedione analogs of the thiazolidinedione drugs troglitazone (TGZ), rosiglitazone (RGZ), and pioglitazone (PGZ), we evaluated their PPAR(γ) activities, anti-cancer properties as well as toxicological effects. Of significance, both pyrrolidinedione analogs demonstrated reduced toxicity. Pharmacologically, they also displayed diminished PPAR(γ) binding and ap2 gene expression in a mouse pre-adipocyte cell line 3T3-L1, but enhanced anti-cancer properties based on the suppression of liver cancer cell line (Huh-7) proliferation and the expression of tumor marker, afp. Overall, this study ascertains the general contribution of the thiazolidinedione ring to their cytotoxicity and proposes the applicability of the pyrrolidinedione ring as a selective and safer choice in anti-diabetic and cancer chemotherapeutics for future drug design.

[Study on the endocrine disrupting effect of dimethachlon].

OBJECTIVE: In order to study the endocrine disrupting effect of dimethachlon (NDPS) (estrogenic and antiandrogenic activity). METHODS: To investigate the estrogenic activity of NDPS by different experiments: (1) Carassius auratus received intraperitoneal injections with NDPS (0, 50, 100 and 200 mg/kg) at regular intervals (5 days) for the synthesis of VTG. The estrogenic effects of the treatment were investigated on VTG in plasma and liver measured by ELISA at 14th. (2) Added the positive control E2 (10(-7) mol/L), and NDPS (10(-10) mol/L, 10(-9) mol/L, 10(-8) mol/L, 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L and 10(-4) mol/L) to MCF-7 cells and determinated the proliferation of MCF-7 cells with MTT assay. (3) female mices 18-22 g in weight were castrated, then they were gavaged daily for 5 days with E2 (500 microg/kg) or NDPS (0, 125, 250 and 500 mg/kg) or peanut oil, then uterus was removed and weighed at 6th. Hershberger assay: male SD rats of 4 weeks age were castrated, after 2 weeks, the other groups were dosed daily for 7 days with testosterone propionate (1 mg/kg, sc) plus peanut oil (p.o) or plus NDPS (30 mg/kg, 60 mg/kg and 120 mg/kg, p.o) but one group of castrated rats were subcutaneously injected with peanut oil plus (sc) peanut oil (p.o) as control. Then, the adrenals, prostate, glans penis, seminal vesicle, levator ani and bulbocavernosus muscles were removed and weighed. RESULTS: Compared with control group. There have significantly increase in the plasma VTG concentration in 10 mg/kg of E2 treatment group as well as in 100 mg/kg and 200 mg/kg of NDPS 14 d after injection. In addition, a significantly increase in the liver VTG concentration was observed in the 200 mg/kg of NDPS treatment group. There was no significant alteration of Ca2+, total protein and liver protein content in plasma in all NDPS treatment groups, but E2 increased the plasma Ca2 level, that is higher than the control group. 10(-8) mol/L,10(-7) mol/L and 10(-6) mol/L NDPS could stimulate the proliferation of MCF-7 cell, and there were significant differences compared with control group. All NDPS treatment groups could increase the weight of uterus, but only 250 mg/kg group has signifgicant differences, compared with the control group. In Hershberger assay, relative weight of prostate, seminal vesicle, levator ani and bulbocavernous muscle in 60 mg/kg group and 120 mg/kg group were significantly decreased when compared with those in the control group. CONCLUSION: NDPS exerts endocrine disrupting effect including estrogenic activity and antiandrogenic activity.

The antiandrogenic activity of the fungicide N-(3, 5-dichlorophenyl) succinimide in in vivo and in vitro assays.

Antiandrogens can cause reproductive disorders in humans and wild animals. In the present study, we tested whether the fungicide N-(3, 5-dichlorophenyl) succinimide (NDPS) acts as an antiandrogen using in vitro and in vivo assays. A transient transfection system based on luciferase activity was utilized for in vitro analysis of the antiandrogeic activity of NDPS. Hershberger assay was used to analyze the antiandrogenic activity of NDPS in rats. The expressions of the androgen-responsive genes testosterone-repressed prostatic message-2 (TRPM-2) and prostate specific binding protein polypeptide C3 (PBP C3) in the rat ventral prostate were measured using real-time PCR. Our results indicated that NDPS can block 5-dehydrotestosterone (DHT)-induced androgen receptor (AR) activity in transiently transfected HepG2 cells (-5 log M). In the Hershberger assay, the weights of the seminal vesicles and levator ani/bulbocavernosus muscles were significantly decreased (P<0.05) in all NDPS groups, and the weights of the ventral prostate, dorsolateral prostate, and Cowper's glands were significantly decreased (P<0.05) in the 100 and 200 mg/kg NDPS groups. NDPS only decreased (P<0.05) the expression of PBP C3 and had no effect on the level of TRPM-2 (P>0.05). In conclusion, NDPS is a moderate antiandrogen that elicits antiandrogenic effects at least partly by antagonizing AR and increasing the expression of PBP C3.

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling.

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling. The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDA-SE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.

[The antiandrogenic effect of dimethachlon and its mechanism].

OBJECTIVE: To evaluate the antiandrogenic effect of heterocyclic fungicide dimethachlon and its mechanism. METHODS: A combination of in vivo and in vitro assays was selected. Hershberger assay was used to determine the antiandrogenic potential of dimethachlon in vivo. Six-week-old castrated male SD rats were administrated once daily for 7 days with testosterone propionate (TP, 100 micro g/d, sc) plus gavage doses of dimethachlon (50, 100 or 200 mg x kg(-1) x d(-1)), or procymidone (150 or 300 mg x kg(-1) x d(-1), positive control), or iprodione (100 mg x kg(-1) x d(-1), positive control), or flutamide (50 mg x kg(-1) x d(-1), positive control). Transcriptional activation assay in vitro was employed to determine the mechanism of antiandrogenic activity of dimethachlon. Human hepatoma liver cells HepG2 were transiently cotransfected with human androgen receptor (AR) expression plasmid and AR-dependent luciferase report plasmid. Transfected cells were exposed to various concentrations of dimethachlon or flutamide with or without dihydrotestosterone to induce the expression of luciferase gene. RESULTS: In Hershberger assay, dimethachlon, as well as other known antiandrogens, caused decrease in weight of androgen dependent organs or tissues. In 200 mg/kg group, the weight of seminal vesicle, ventral prostate, dorsolateral prostate, Cowper's gland, and levator ani plus bulbocavernosus muscles decreased by 57.8%, 44.8%, 43.9%, 30.1%, and 34.1% respectively, but did not decrease in the vehicle control group. The order of their antiandrogenic potencies was: flutamide > procymidone > dimethachlon > iprodione. In transcriptional activation assay, dimethachlon could inhibit dihydrotestosterone-dependent AR activity in transfected HepG2 cells in dose-effect relationship. The inhibiting potency of dimethachlon was about 1/100 of that of flutamide. CONCLUSION: Dimethachlon has antiandrogenic effect, and acts as an AR antagonist. Its antiandrogenic potency is lower than flutamide and procymidone, but higher than iprodione.

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.

A novel intravascular drug delivery method using endothelial biotinylation and avidin-biotin binding.

In this study, a novel intravascular drug delivery system was developed in which a drug injected from a catheter was fixed to the vasculature of the targeted tissue. Cellular proteins of viable endothelial cells were first biotinylated directly by biotinylation reagents, and then bound by an avidinated drug or, using avidin as a linker, a biotinylated drug. In the initial experiments, we studied in vitro the biotinylation of cultured bovine aortic endothelial cells (BAECs) by applying biotinylation reagents (NHS-LC-biotin or sulfo-NHS-LC-biotin) onto the washed intact BAEC monolayers and showed that the amount of biotin bound to the cells depended on the concentration of the biotinylation reagents applied. The cell-bound biotin decreased with time after the biotinylation. When fluorescein-labeled avidin (FITC-avidin) was applied to the biotinylated BAEC monolayers, the FITC-avidin readily bound to the cells. An LDH-release assay showed that sulfo-NHS-LC-biotin was only slightly cytotoxic to the BAECs and a colony formation assay showed only slight adverse effects of the reagent. In vivo studies were carried out on the renal arteries of normal rabbits. A solution of NHS-LC-biotin was injected through a catheter to one kidney to biotinylate its vasculature and the vehicle to the other as control, followed by a perfusion with saline. Finally, a solution of FITC-avidin was injected to both kidneys that were then reperfused with the blood flow following the withdrawal of the catheters. In the histological sections, more than 85% of glomeruli was stained with fluorescein in the biotinylated kidney, whereas no glomeruli were stained in the control. In the kidneys harvested 2 days after the same procedure, most glomeruli were still brightly stained. In the final experiment, biotinylated kidneys were injected with a solution of avidin, followed by a solution of fluorescein-biotin. Control kidneys had no prior biotinylation but received the same injections of avidin and fluorescein-biotin as above. More than 80% of glomeruli were stained in the biotinylated kidneys but none in the controls. This indicated that biotinylated drugs can be anchored to the biotinylated vasculature through avidin without being flushed away by blood flows. No apparent adverse effect was found in the functions of biotinylated kidneys. We propose that this drug delivery system is feasible for the treatment of some pathological conditions of blood vessels such as microvascular proliferation in malignant tumors and for continuous drug delivery in certain target organs.

Buthionine sulfoximine (BSO) and N-(3,5-dichlorophenyl)succinimide nephrotoxicity: temporal aspects of BSO administration and BSO effects on renal transport systems.

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure which is attenuated by pretreatment with the glutathione depletors, diethyl maleate or buthionine sulfoximine (BSO). In the present study, the temporal aspects of BSO attenuation of NDPS nephrotoxicity were investigated. In addition, the ability of BSO to alter the renal accumulation of selected organic ions was examined as a possible mechanism for BSO's ability to attenuate NDPS nephrotoxicity. In the first set of experiments, NDPS (0.2 or 0.4 mmol/kg) or vehicle (sesame oil, 2.5 ml/kg) was administered intraperitoneally (i.p.) to groups of male Fischer 344 rats (4-8 rats/group) 0.25 or 2 h prior to BSO (890 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. NDPS (0.4 mmol/kg) nephrotoxicity was markedly attenuated by administration of BSO at 0.25 h post-NDPS, but was not substantially altered by injection of BSO at 2 h post-NDPS. NDPS (0.2 mmol/kg)-induced renal effects were not potentiated by BSO injected at 0.25 h post-NDPS, and only 1 of 8 rats exhibited marked nephrotoxicity when BSO was administered at 2 h post-NDPS. In the second set of experiments, rats (4/group) were administered BSO (890 mg/kg, i.p.) or vehicle (0.9% saline, 10 ml/kg) and kidneys harvested at 2 or 5 h post-treatment. The ability of renal cortical slices to accumulate organic ions (p-aminohippurate [PAH], alpha-aminoisobutryic acid [AIB] or tetraethylammonium [TEA]) during a 90 min incubation was studied. Only TEA accumulation by renal cortical slices prepared from the 2 h post-treatment group was reduced. Studies were also conducted to examine the in vitro effects of BSO (10(-7)-10(-4) M) on the accumulation of PAH, AIB and TEA by renal cortical slices following 5, 15 or 90 min co-incubations of BSO and an organic ion BSO had no significant effects on the accumulation of any organic ion studied at any time point. These results indicate that BSO can still attenuate NDPS nephrotoxicity when administered at 0.25 h post-NDPS, but BSO loses effectiveness when given 2 h post-NDPS. These results also suggest that BSO is attenuating NDPS nephrotoxicity via glutathione depletion rather than altering renal accumulation of NDPS metabolites via renal PAH, TEA or AIB transporters.

Potential metabolism and cytotoxicity of N-(3,5-dichlorophenyl)succinimide and its hepatic metabolites in isolated rat renal cortical tubule cells.

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide and antimicrobial agent that produces nephrotoxicity in rats. The contribution of the kidney, if any, to the mechanism of toxicity of NDPS is not known. Therefore, the ability of isolated renal cortical tubule cells to metabolize NDPS and some of its known hepatic metabolites was studied. The cytotoxic potential of these compounds was also assessed. Renal cortical tubule cells were isolated by collagenase digestion and were incubated with the test compounds (2 mM) for 3 h. Metabolite formation was monitored by reversed phase HPLC and cell viability was assessed using trypan blue exclusion. The isolated kidney cells do not appear to metabolize NDPS or any of its known hepatic metabolites. In addition, none of these compounds were directly cytotoxic to the renal cells. However, the cells were susceptible to mercuric chloride (1 mM) and chloroform (125 or 200 mM). Intracellular glutathione levels were unaltered by the presence of NDPS in the incubations. These results suggest that NDPS and its metabolites are not directly toxic to the kidney and are not converted into the ultimate nephrotoxic species by the kidney. Extrarenal metabolism may, therefore, be critical to the expression of NDPS-induced nephrotoxicity.

The cytotoxicity of N-Pyridinyl and N-quinolinyl substituted derivatives of phthalimide and succinimide.

The N-pyridinyl and N-quinolinyl substituted derivatives of phthalimides and succinimides demonstrated cytotoxicity against the growth of a number of cultured cell lines. The substituted succinimides were more effective than the unsubstituted succinimide derivative in reducing cell growth. On the other hand, phthalimide demonstrated more potent cytotoxicity than its N-substituted derivatives. Three representative examples N-[2-pyridinyl-1-oxide) methyl] phthalimide 8, 1-[N-2-phthalimidoethyl]-3,4-dihydroiso-quinoline 12, and 1-[N-(2-(1,2,3,4-tetrahydro-2-quinolinyl)] ethylphthalimide 14 were shown to inhibit L1210 leukemia DNA synthesis whereas RNA synthesis was not inhibited at 25-100 uM. All three agents inhibited the activities of DNA polymerase alpha, PRPP-amido transferase, nucleoside kinases, and dihydrofolate reductase. The cellular pool levels of d[GTP], d[CTP], and d[TTP] were reduced after 60 minutes incubation at 100 uM. The DNA molecule itself was not a target of these agents.

Role of para-hydroxylation in phensuximide-induced urotoxicity in the Fischer 344 rat.

Phensuximide (PSX) is an antiepileptic agent which has been shown to induce hemorrhagic cystitis and mild nephrotoxicity following repeated administration in man or rats or when acutely administered to phenobarbital-pretreated rats. The purpose of this study was to explore the role of para-hydroxylation of the phenyl group of PSX in PSX-induced urotoxicity. Two PSX derivatives, 2-(4-fluorophenyl)-N-methylsuccinimide (FMPS) and N-methyl-2-(4-methylphenyl)succinimide (MMPS), were synthesized and evaluated for urotoxic potential. Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) a succinimide (0.4 or 1.0 mmol/kg) or vehicle and renal function monitored for 48 h. In a separate experiment, rats were pretreated with phenobarbital (75 mg/kg/day; 3 days, i.p.) prior to succinimide or succinimide vehicle. In non-phenobarbital pretreated rats, acute FMPS or MMPS treatment had little effect on renal function or morphology at the doses tested. Hematuria (+) was noted in the FMPS (1.0 mmol/kg) group on post-treatment day 2. However, in the phenobarbital-pretreated rats, FMPS (0.4 or 1.0 mmol/kg) induced marked hematuria (++) and increased proteinuria while having little or no effect on other renal functional parameters or renal morphology. At killing, bladders of treated rats were distended with bloody urine and exhibited hemorrhagic areas within the bladder wall. In phenobarbital-pretreated rats, MMPS administration had little effect on any renal functional parameter measured or urological morphology. These results suggest that para-hydroxylation does not contribute to the hemorrhagic cystitis induced by PSX.

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)succinimidyl)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)carbamoylmethyl)-1,4-th iazane-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) or 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 and NDPS metabolite as being the penultimate or ultimate nephrotoxic species.

Effect of buthionine sulfoximine on acute N-(3,5-dichlorophenyl)succinimide-induced nephrotoxicity in Fischer 344 rats.

The experimental agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) has been shown to be a nephrotoxicant in Fischer 344 rats. Results of a previous study conducted in our laboratory suggested that glutathione might be an important modulator of NDPS-induced nephrotoxicity. The purpose of this study was to examine the effect of DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione synthesis, on NDPS-induced renal effects. Male Fischer 344 rats received an intraperitoneal (i.p.) injection of BSO (890 mg/kg) in 0.9% saline (10 ml/kg) followed 2 h later by an i.p. injection of NDPS (0.4 or 1.0 mmol/kg) or sesame oil (2.5 ml/kg), and renal function monitored at 24 and 48 h. BSO pretreatment attenuated the diuresis, proteinuria, elevation in blood urea nitrogen (BUN) concentration and kidney weight, and decreases in organic ion accumulation by renal cortical slices induced by NDPS (0.4 or 1.0 mmol/kg) administration. Proximal tubular necrosis induced by NDPS administration also was attenuated by BSO pretreatment. These results indicate that BSO pretreatment attenuates NDPS-induced renal effects and that glutathione is important for modulating acute NDPS-induced nephropathy.

Role of glutathione in acute N-(3,5-dichlorophenyl) succinimide-induced nephrotoxicity in Sprague-Dawley and Fischer 344 rats.

N-(3,5-Dichlorophenyl)succinimide (NDPS), an experimental agricultural fungicide, has been shown to be a selective nephrotoxin in Sprague-Dawley and Fischer 344 rats. Previous studies have demonstrated that a toxic metabolite contributes to or is responsible for acute NDPS-induced nephrotoxicity. The purpose of this study was to investigate the role of glutathione in NDPS-induced renal effects. In 1 set of experiments, male Sprague-Dawley or Fischer 344 rats received a single intraperitoneal (i.p.) injection of NDPS (0.4 or 1.0 mmol/kg) or sesame oil (2.5 ml/kg). Rats were killed at 1, 3, 6 or 24 h, and reduced (GSH) and oxidized (GSSG) glutathione concentrations determined in liver and renal cortex. In both rat strains NDPS (0.4 or 1.0 mmol/kg) administration produced small decreases in GSH concentrations (1 and 3 h) but moderate increases in GSSG concentrations (1 and 3 h) in liver and kidney. At 24 h both GSH and GSSG concentrations were increased, particularly in kidney. In a second set of experiments, rats were pretreated with the glutathione depletor diethyl maleate (DEM) (0.4 ml/kg, i.p.) 1 h prior to NDPS (0.2, 0.4 or 1.0 mmol/kg, i.p.) or sesame oil (2.5 ml/kg, i.p.) administration, and renal function monitored at 24 and 48 h. DEM pretreatment attenuated the increase in urine volume (24 and 48 h), proteinuria, glucosuria, hematuria and elevated blood urea nitrogen (BUN) concentration produced by NDPS (0.4 or 1.0 mmol/kg) in both Sprague-Dawley and Fischer 344 rats. NDPS-induced increases in kidney weight also were generally prevented by DEM pretreatment. Proximal tubular necrosis produced by NDPS administration was reduced by DEM but not prevented. Pretreatment with the cysteine conjugate beta-lyase inhibitor amino-oxyacetic acid (0.5 mmol/kg, i.p.) 1 h prior to NDPS (0.4 or 1.0 mmol/kg) markedly attenuated all NDPS-induced effects on renal function and morphology. These results suggest that glutathione does not play a protective role against NDPS-induced renal effects and that a glutathione or cysteine conjugate of NDPS might contribute to NDPS-induced nephrotoxicity.

Combination effect of citrinin and other chemicals on rat kidney tumorigenesis.

Histological studies were made on the nephrotoxic effect of citrinin on the kidneys of rats, with or without previous treatment with the nephrotoxic chemicals, N-(3,5-dichlorophenyl)succinimide (NDPS) and N-nitrosodimethylamine (DMN). Oral administration of 0.02% or 0.05% citrinin alone caused signs of kidney injury but did not induce kidney tumors. On treatment with DMN alone, 8 of 14 rats (57.1%) developed kidney tumors; two (14.3%) were renal cell tumors, eight (57.1%) embryonal cell tumors, and one (7.1%) hemangioendothelioma. On the other hand, kidney tumors developed in 18 of 19 rats (94.7%) and 13 of 15 rats (86.7%) by the administration of 0.02% and 0.05% citrinin, respectively, after DMN. The tumors in these two groups were diagnosed histologically as renal cell tumors in 18 (94.7%) in group IV and 13 (86.7%) in group III, and as embryonal cell tumors in 14 (73.7%) in group IV and 9 (60.0%) in group III. Thus, in groups treated with citrinin after DMN the incidence of renal cell tumors was much greater and the incidence of embryonal cell tumors slightly greater than in the group treated with DMN alone. Kidney tumors developed in 4 of 18 rats (22.2%) treated with 0.02% citrinin after NDPS, but treatment with NDPS alone did not induce kidney tumors. Thus, treatment with citrinin changes the histological type and incidence of kidney tumors in rats induced by DMN. Moreover, this study confirms that citrinin in combination with NDPS can induce kidney tumor in rats, which was renal cell tumor (adenoma) histologically.