Sensitivity and biochemical characteristics of Sclerotinia sclerotiorum to propamidine.

Propamidine is an aromatic diamidine compound. In the current study, baseline sensitivity of Sclerotinia sclerotiorum to propamidine was determined using 78 strains collected from the oilseed rape fields without a previous history of propamidine usage. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.406 to 3.647µg/mL, with a mean of 1.616±0.217µg/mL. There was no correlation between sensitivity to propamidine and sensitivity to dimethachlon or carbendazim. After treated with propamidine, mycelia were thinner with irregular distortion and more branches; cell wall became thicker with uneven distribution of cytoplasm than untreated control. In addition, sclerotia production, cell membrane permeability and oxalic acid content significantly decreased. On detached oilseed rape leaves, propamidine exhibited better control efficacy than carbendazim at the same concentration whether the leaves were inoculated with carbendazim-sensitive or resistant strains. All the results showed that propamidine exhibited strong antifungal activity and potential application in controlling S. sclerotiorum. Importantly, these data will provide more information on understanding the mode of action of propamidine against S. sclerotiorum and should be valuable for development of new antifungal drugs.

Synthesis and anticonvulsant activity of new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones.

Twenty-two new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones were synthesized and tested for anticonvulsant activity. Initial anticonvulsant screening was performed using standard maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens in mice. Several compounds were tested additionally in the 6-Hz psychomotor seizure model. The neurotoxicity was determined applying the rotarod test. Excluding one compound, all other molecules were found to be effective in at least one seizure model. The most active were 1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (14), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3-methylpyrrolidine-2,5-dione (17), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3,3-dimethylpyrrolidine-2,5-dione (23) and 3,3-dimethyl-1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (26). These compounds showed high activity in the 6-Hz psychomotor seizure test as well as were active in the maximal electroshock and subcutaneous pentylenetetrazole (14 and 23) screens. Initial SAR studies for anticonvulsant activity have been discussed.

Synthesis and characterization of gelatin nanoparticles using CDI/NHS as a non-toxic cross-linking system.

Gelatin nanoparticles, cross-linked by a mixture of a water soluble carbodiimide (CDI) and N-hydroxysuccinimide (NHS) as a non-toxic cross-linking system, was prepared. The conventional two step desolvation method with acetone as the non-solvent was used. The mean size and size distribution as well as the morphology of the formed nanoparticles were evaluated and compared with those of nanoparticles cross-linked by glutaraldehyde (GA) as the most commonly used cross-linking agent. Furthermore, intrinsic viscosities of the nanoparticles cross-linked by CDI/NHS and GA were measured and compared under various conditions. The results showed the formation of smoother and more homogeneous nanoparticles with smaller size when CDI/NHS used as cross-linking agent under the same synthesis condition. Moreover, nanoparticles encapsulating paracetamol as a model drug were produced by the two different cross-linking agents and were characterized for drug entrapment and loading efficiencies and in vitro drug release. Both drug entrapment and loading efficiencies was higher in the CDI/NHS cross-linked nanoparticles; however, the release kinetics was comparable to that of nanoparticles cross-linked with GA. The differences in the characteristics of CDI/NHS and GA cross-linked nanoparticles were attributed to the different nature of network structures formed by the two cross-linking agents. On the whole, these results suggested that CDI/NHS cross-linked nanoparticles have high potential to be used for drug delivery application in preference to the nanoparticles synthesized by toxic cross-linking agents.

Immunogenicity Risk Assessment for PEGylated Therapeutics.

The objective of this manuscript is to provide the reader with two examples on how to present an immunogenicity risk assessment for a PEGylated therapeutic as part of Investigational New Drug (IND) application or during other stages of the drug development process. In order to provide context to the bioanalytical strategies used to support the PEGylated therapeutics presented here, a brief summary of information available for marketed PEGylated biologics is provided. Two case studies are presented, a PEGylated enzyme and a PEGylated growth factor. For the former, the risk assessment covers how to deal with a narrow therapeutic window and suggestions to utilize a PD marker as surrogate for neutralizing antibody assessments in Phase I. The latter has recommendations on additional analytes that should be monitored to mitigate risk of immunogenicity to endogenous counterparts.

Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.

The measurement of cell proliferation after mitogenic stimulation is an important parameter used in diagnosis of immunodeficiencies in clinical laboratory as well as in various fields of lymphocyte research. Recent methods try to overcome the radioactive assay using tritiated thymidine ((3)H) by flow-cytometric methods using different fluorochromes such as CFSE or even to substitute these direct methods by tracing the expression of cell-membrane activation markers associated with various steps of proliferation cycle. In our study we compared the (3)H assay with CFSE-staining method and expression of activation markers (CD69, HLA-DR, CD25, CD27, CD71, CD152, CD134 and CD195) on a sample of 128 consecutive patients and healthy controls evaluated in clinical laboratory. We also tested various concentrations of CFSE and its impact on proliferation activity and expression of activation markers. We found that CFSE in concentration from 37nM to 10microM decreases the proliferative capacity (expressed in cpm (3)H assay) due to the decreased viability of proliferating cells (measured as 7-AAD+) in concentration-dependent manner. Moreover, CFSE substantially modulates the expression of activation molecules (decreasing CD69, HLA-DR, CD25 the majority of examinated molecules). We found a good correlation between CFSE-staining method with (3)H assay, if CFSE low population is gated on CD3+ population (correlation coefficient 0.801), but only in samples with stimulation index (SI) higher then 25. In poorly proliferating samples (SI25) no correlation was found due to several false positive results in CFSE test. Statistically significant correlation between proliferation assessed as (3)H-thymidine incorporation and expression of activation markers was found in the case of CD25, CD27, CD38, CD152, CD71, still only in samples with higher proliferation activity (SI>25). No correlation was found with CD134, CD195, HLA-DR and CD69. We conclude that standard assay with (3)H-thymidine incorporation is unreplaceable assay in diagnosis of severe cellular immunodeficiencies as CFSE assay have high proportion of false positive results. Researchers tracing cell-membrane bound molecules on dividing cells stained by CFSE must take into account that CFSE may substantially modulate the expression of these markers and decrease the viability of stained cells.

[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.

Synthesis and anticonvulsant properties of new 1-(2-pyridinyl)- 3-substituted pyrrolidine-2,5-dione derivatives.

The synthesis and anticonvulsant properties of new 1-(2-pyridinyl)- succinimides [I-XXII] differently substituted at the position-3 of imide ring have been described. The profile of pharmacological activity of these compounds was examined by a maximal electroshock (MES) and pentylenetetrazole (scPTZ) tests, whereas their neurotoxicity was determined using a rotarod screen. The results obtained revealed that the anticonvulsant activity depended mainly on the kind of substituents at the position-3 of pyrrolidine-2,5-dione ring. The most active were 3,3-dialkyl-pyrrolidine-2,5-diones [IX-XVIII] as well as compounds with 3-methylcyclohexane moiety as a spiro nucleus at position-3 of the imide ring [I-IV]. The 3-cyclohexylsuccinimides [V-VIII] with cyclohexane ring as a flexible fragment were less active, whereas unsubstituated derivatives [XIX-XXII] were devoid of activity in both tests applied. In addition, the anti-seizure protection depended on the position of methyl group at the pyridine moiety. The most potent were compounds with the methyl substituent at the position-4 [II, VI, XVII] or -6 [XI, XIV]. It should be noted, that in the whole series the most active was 1-(4-methyl-2-pyridinyl)-3-cyclohexyl-pyrrolidne-2,5-dione [VI], which showed the anti-scPTZ protection at the dose of 30 mg/kg.

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 1.0mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3-4 x 10(6)cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1h 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.

Applicability of the dyes CFSE, CM-DiI and PKH26 for tracking of human preadipocytes to evaluate adipose tissue engineering.

Adipose tissue engineering with preadipocyte-loaded scaffolds requires adequate tracking of preadipocytes to allow evaluation and quantification of cell proliferation, expansion and differentiation in three-dimensional systems. To differentiate between graft and host cells, labeling of preadipocytes before implantation and tracking of these cells until harvest would be useful. Immunohistochemistry enables the differentiation between cells of different species but is time-consuming, expensive, elaborate, and not applicable for autologous transplantation. So far, there is no published method to use externally applied dyes for tracking of human preadipocytes in adipose tissue engineering. We tested the cell dyes PKH26, CM-DiI, and CFSE to analyze their applicability for labeling human preadipocytes. CM-DiI had toxicity levels of 45-70%, while 3-4% proliferating cells were stained on day 35. CFSE revealed clear cytoplasmic coloring in proliferating cells with 5-6% stained cells after 35 days and toxicity ranging from 55 to 90% dead cells. PKH26 demonstrates lowest levels of toxicity and best labeling results after 4 weeks in proliferating preadipocytes in monolayer. Although none of the dyes showed long-lasting labeling during proliferation, all three dyes demonstrated permanent staining in differentiated cells. The results reveal the problems of preadipocyte tracking with fluorescent dyes but justify the dye application for limited time periods.

Synthesis, Biological Evaluation and Pharmacokinetic Studies of Mefenamic Acid - N-Hydroxymethylsuccinimide Ester Prodrug as Safer NSAID.

BACKGROUND: Non steroidal anti-inflammatory drugs are the most widely prescribed drugs to manage pain and inflammatory conditions, but their long term use is associated with gastrointestinal toxicity. OBJECTIVES: The study aimed to synthesize an ester-based prodrug of a non steroidal anti-inflammatory agent, mefenamic acid in order to improve the therapeutic index vis a vis to overcome the side effects such as gastrointestinal irritation and bleeding associated with the use of mefenamic acid. METHODS: The ester prodrug (MA-NH) was prepared by condensing mefenamic acid with N-hydroxymethylsuccinimide in the presence of Phosphorus oxychloride. The pharmacokinetic profile, including stability and release of mefenamic acid and N-hydroxymethylsuccinimide from the ester prodrug (MA-NH) was studied by RP- HPLC in acidic medium (pH 1.2), basic medium (pH 7.4), 80 % v/v human plasma, 10 % w/v rat intestinal homogenate and 10 % w/v rat liver homogenate (pH 7.4). RESULTS: The chemical structure of the title compound was characterized by using modern spectroscopic techniques. The prodrug was found to be stable in acid medium, but it hydrolyzed and released sufficient quantities of the drug in alkaline medium. The prodrug produced lesser number of ulcers and showed improved analgesic and anti-inflammatory activity as compared to the parent drug. CONCLUSION: The results indicate that the synthesized prodrug (MA-NH) is better in terms of analgesic and antiinflammatory activities and with less GI toxicity than the parent drug.

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.

Oxidative damage in the guinea pig hippocampal slice.

Free radicals and active oxygen compounds are implicated in brain ischemia and head trauma. Previous studies have shown that free radicals, generated by radiation and through the Fenton reaction, produce both synaptic and postsynaptic damage in the hippocampal brain slice. To evaluate the contribution of oxidation to the observed damage, the actions of the oxidants, chloramine-T and N-chlorosuccinimide (NCS), were studied on electrophysiological responses in the hippocampal slice isolated from the brains of guinea pigs. Electrical stimulation of afferents to neurons of the CA1 region of hippocampus evoked a population postsynaptic potential (population PSP) in the dendritic layer and a population spike in the cell body layer. Chloramine-T (25-500 microM) and NCS (750-4000 microM) decreased the population spike in a dose-dependent manner (ED50 congruent to 125 microM and 1100 microM, respectively). Input/output curves revealed that both the population PSP were significantly reduced with both oxidants; but, the ability of the population PSP to produce a population spike was not impaired. These studies suggest that oxidation reactions can account for the synaptic component of the damage produced by free radicals but can not account for the postsynaptic effects.

Synthesis and anticonvulsant activity of imidooxy derivatives.

Previous results of anticonvulsant activity in several imidooxy carboxylates related to (aminooxy)acetic acid in young chicks, prompted an in-depth reinvestigation of these analogues in mice. A series of 22 succinimidooxy, phthalimidooxy, and naphthalimidooxy carboxylates were synthesized and evaluated for anticonvulsant activity by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). Methyl (succinimidooxy)acetate (2d), ethyl (succinimidooxy)acetate (2e), methyl (phthalimidooxy)acetate (3d), ethyl (phthalimidooxy)acetate (3e), and ethyl 2-(phthalimidooxy)propionate (3g), which were initially found to be active as anticonvulsants in young chicks were uniformly inactive in the Phase I seizure tests involving maximal electroshock (MES), pentylenetetrazol (scMet), or neurologic toxicity toxicity (Tox). Several newer analogues, ethyl (succinimidooxy)formate (2c) and methyl 3-(phthalimidooxy)-2-methylacrylate (4h) were found to be active in the scMet (3a) or both (4h) evaluations. Most interesting was the anticonvulsant results of N-(benzyloxy)-2-azaspiro[4,4] nonane-1,3-dione (5b), which displayed anti-MES activity and a protective index (TD50/ED50) of greater than 4.5.

Acute N-(3,5-dichlorophenyl)succinimide nephrotoxicity in female Fischer 344 rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an established nephrotoxicant in male Fischer 344 rats at i.p. doses of > or = mmol/kg. Since gender differences often exist in the susceptibility to toxicants, the nephrotoxic potential of NDPS was examined in female Fischer 344 rats. Rats (4-5/group) were administered NDPS (0.1, 0.2, 0.4, or 1.0 mmol/kg, i.p.) or vehicle (sesame oil, 2.5 ml/kg) and renal function monitored for 48 h. At a dose of 0.1 mmol/kg, NDPS had no effect on renal function. However, administration of NDPS at a dose of 0.2 or 0.4 mmol/kg resulted in marked nephrotoxicity characterized by diuresis, increased proteinuria, glucosuria, hematuria, elevated blood urea nitrogen (BUN) concentration and kidney weight, decreased organic ion accumulation and proximal tubular necrosis. NDPS treatment of 1.0 mmol/kg resulted in oliguric renal failure rather than polyuric renal failure in 3 of 4 rats. Proximal tubular damage was observed primarily in the S3 segment of the proximal tubule in NDPS-treated female rats, while in male rats the S1 and S2 segments are the initial renal targets. These results demonstrate that female Fischer 344 rats are more susceptible to NDPS nephrotoxicity than male Fischer 344 rats and that the site of the renal lesion is gender dependent.

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.

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 (approximately 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 (> or =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.

The effect of aromatic fluorine substitution on the nephrotoxicity and metabolism of N-(3,5-dichlorophenyl)succinimide in Fischer 344 rats.

N-(3,5-Difluorophenyl)succinimide (DFPS) is a non-toxic analogue of the nephrotoxic fungicide N-(3,5-dichlorophenyl)succinimide (NDPS). Although NDPS must be metabolized to produce renal damage, the metabolic fate of DFPS is unknown. These studies were therefore designed to examine the nephrotoxic potential of putative DFPS metabolites and to determine if DFPS is metabolized differently from NDPS. Male Fischer-344 rats were administered (1.0 mmol/kg. i.p. in corn oil) DFPS, N-(3,5-difluorophenyl)succinamic acid (DFPSA), N-(3,5-difluorophenyl)-2-hydroxysuccinimide (DFHS), N-(3,5-difluorophenyl)-2- or -3-hydroxysuccinamic acids (2- and 3-DFHSA, respectively), N-(3,5-difluoro-4-hydroxyphenyl)succinimide (DFHPS). N-(3,5-difluoro-4-hydroxyphenyl) succinamic acid (DFHPSA) or corn oil only (1.2 ml/kg). Although some of the compounds produced changes in renal function and histology, these alterations were not indicative of irreversible kidney damage. DFPSA, 2-DFHSA, 3-DFHSA and DFHPSA were detected in the urine of rats 3 h after administration of 0.2 mmol/kg [14C]DFPS. The same metabolites were produced by isolated rat hepatocytes, but not by renal proximal tubule cells. Formation of the oxidative metabolites in vitro was prevented by the cytochrome P450 inhibitor 1-aminobenzotriazole. It appears that DFPS undergoes hepatic biotransformation similar to NDPS and that some of its metabolites have reversible effects on renal proximal tubules.

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.

Interactions among nephrotoxicants: N-(3,5-dichlorophenyl)succinimide and cephaloridine.

Numerous studies have demonstrated the interactive potential between nephrotoxicants. The purpose of this study was to examine the interactive potential between two model nephrotoxicants, N-(3,5-dichlorophenyl)succinimide (NDPS) and cephaloridine (CPH). Male Fischer 344 rats (4 rats per group) were administered an intraperitoneal (i.p.) injection of CPH (500 mg/kg), NDPS (0.2 mmol/kg) or the appropriate vehicle 1 h prior to administration of an i.p. injection of NDPS (0.2, 0.4, or 1.0 mmol/kg), CPH (500, 750 or 1000 mg/kg) or the appropriate vehicle. Renal function was monitored at 24 and 48 h. Combination of non-nephrotoxic doses of CPH (500 mg/kg) and NDPS (0.2 mmol/kg) did not result in nephrotoxicity, regardless of which compound was administered first. NDPS (0.2 mmol/kg) weakly enhanced the nephrotoxicity observed following CPH (1000 mg/kg) injection but had little effect on CPH (750 mg/kg)-induced renal effects. However, CPH (500 mg/kg) markedly attenuated NDPS (0.4 or 1.0 mmol/kg)-induced nephrotoxicity. These results demonstrate that prior NDPS exposure has little effect on the outcome of CPH-induced renal effects, but prior CPH exposure can markedly alter the renal response to NDPS administration.

Effect of succinimide ring modification on N-(3,5-dichlorophenyl)succinimide-induced nephrotoxicity in Sprague-Dawley and Fischer 344 rats.

N-(3,5-Dichlorophenyl)succinimide (NDPS) has proven to be an effective experimental agricultural fungicide. However, NDPS produces marked nephrotoxicity in Sprague-Dawley and Fischer 344 rats. The purpose of this study was to determine the importance of an intact, unsubstituted succinimide ring for acute NDPS-induced nephrotoxicity. Structural modifications included ring opening, reduction of one or both carbonyl groups, breaking the ethylene carbon-carbon bond and mono- or dialkyl substitution on the succinimide ring. Sprague-Dawley or Fischer 344 rats were administered NDPS or an NDPS analog (0.1, 0.2, 0.4, 0.8 or 1.0 mmol/kg) or sesame oil (2.5 ml/kg, i.p.) and renal function was monitored at 24 h and 48 h. All structural modifications produced compounds with markedly reduced nephrotoxic potential in both Sprague-Dawley and Fischer 344 rats when compared to NDPS. However, N,N-diacetyl-3,5-dichloroaniline and N-(3,5-dichlorophenyl)pyrrolidine-2-one were more lethal than NDPS. The reduced renal effects of the NDPS analogs did not correlate with lipophilic character. These results indicate that an intact, unsubstituted succinimide ring is optimal for acute NDPS-induced nephrotoxicity.