[Effect of protein deficiency in rats on captan toxictiy. I. Acute toxicity of captan].
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Animals↗
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[Effect of protein deficiency on captan toxicity in rats. II. Activity of selected enzymes in rat serum and liver depending on the dietary level of protein and captan].
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Animals↗
Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.
RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.
Bacterial Proteins↗
Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity.
The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity.
Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Captan↗
Interaction of captan with mammalian microtubules.
Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.
Actin Cytoskeleton↗
Mammalian host- and fluid-mediated mutagenicity assays of captan and streptozotocin in Salmonella typhimurium.
The mutagenicity of captan and of streptozotocin was tested in vivo by reversion of hisG46 base-pair substitution histidine auxotrophs of Salmonella typhimurium in the peritoneal cavity or in blood, plasma or urine of rats or mice. Genetic response was determined by the frequency of revertants (quantitative test) or by the number of revertants per plate (semiquantitative test). In quantitative HMA captan gave negative results following 3 hourly 500 mg/kg s.c. doses or 1000 mg/kg oral dose in mice with the hisG46 mutant or 2000 mg/kg oral dose in rats with the hisG46, uvrB (TA1950) mutant. The positive control SZN induced many reversions at 0.5 mg/kg i.p. or 10 or 100 mg/kg oral doses. In semiquantitative in vivo blood or urine assays captan gave negative results after a 250 mg/kg oral dose with hisG46. SZN in the same experiment gave positive results in both semiquantitative and quantitative in vivo blood assays following 1000 mg/kg i.p. or 2000 mg/kg oral doses in the rat with TA1950. Rat blood mixed with captan for 45 min before adding TA1950 cells inactivated 1000 mug captan/ml but not 5000 mg/ml in the semiquantitative test. Corresponding figures in the quantitative test were 500 mu/ml and 1000 mug/ml. Rat plasma inactivated the mutagenicity of about 10 times less captan than rat blood. Human blood inactivated about as much captan as rat blood. The mutagenicity of captan was inactivated more efficiently than of SZN by blood. The results of the experiments suggested that captan's mutagenicity is probably inactivated by glutathione of the erythrocytes. Rat S-9 liver microsomal fraction also strongly decreased captan's mutagenicity in a semiquantitative test with the R factor, uvrB, hisG46 (TA100) mutant.
Administration, Oral↗
Carcinogenicity of captan.
Two studies on the carcinogenicity of the fungicide captan in animals were reviewed. The results and conclusions, which were based on my examination of the histological sections, showed that captan is highly carcinogenic in rats and mice. Neoplasms at all sites, as well as malignant neoplasms, were increased in both low and high dose captan-treated male and female rats. Benign and malignant neoplasms of the endocrine organs were increased in low and high dose male and female rats ingesting captan. Neoplasms of the adrenal and pituitary glands were increased. Increased incidences of benign and malignant neoplasms, and of malignant neoplasms only, were observed in the reproductive system of female rats ingesting captan. The incidence of these neoplasms was markedly increased in the mammary gland and ovary. Female rats given captan were more susceptible to the development of hepatic neoplasms than were male rats. Captan induced neoplasms of the duodenum in male and female mice. There also were toxic changes in rats. Captan-treated male rats were more susceptible to the induction of chronic renal disease than were female rats. Male rats also had a high incidence of particularly severe testicular atrophy as a result of the ingestion of captan. Such lesions interfere with the health of the rats and with the development of neoplasms.
Adrenal Gland Neoplasms↗
Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.
The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.
Captan↗
Toxicologic studies of N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (captan): its metabolism by rat liver drug-metabolizing enzyme system.
The degree of toxicity caused in rats by captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) administered intraperitoneally is greater than that induced by orally administered captan. With regard to its effect on the drug-metabolizing enzymes of rat liver, the activity of aniline hydroxylase and the level of cytochrome P-450 were found to decrease in the treated rats 24 h after a single oral dose (650 mg/kg). The loss was even greater in the animals receiving diethyl maleate 1 h prior to captan. Furthermore, usual increase in the activity of drug biotransformation enzymes seen after phenobarbital treatment appears to decrease in rats dosed with this funaicide. In vitro incubations of rat liver microsomes with captan resulted in a profound loss of cytochrome P-450 and the acitivty of benzphetamine N-demethylase as well as aniline hydroxylase. Although the inhibition of drug-metabolizing enzyme activity by captan was observed in microsomal incubations with or without NADPH, a detectable amount of carbonyl sulfide (COS) was found only in the incubations that contained captan plus NADPH. Carbonyl sulfide appears to arise from a captan-derived metabolite, thiophosgene (CSCl2), which decomposes to COS in aqueous solutions and in the presence of NADPH inhibits the activity of drug biotransformation enzymes.
Aniline Hydroxylase↗
Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase.
Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities. With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity. Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP. Conversely, inhibition of this polymerase activity was enhanced by template/primer. The calculated KdTTP of the uninhibited reaction was 5.6 microM. Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center. RNase H activity showed a sigmoidal relationship between activity and substrate concentration. Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan. Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action. Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.
Avian Leukosis Virus↗
Captan alters transcription in Escherichia coli permeabilized by toluene.
RNA synthesis was measured in toluenized E. coli by the incorporation of radiolabeled precursor into either acid precipitable or phenol extracted RNA. Exposure to captan (100 microM) caused a 2.6 fold increase in the apparent rate of RNA synthesis. When captan was tested for its effect on the initiation of RNA synthesis, using either rifampicin-treated cells or by measuring the incorporation of gamma [32P]ATP or gamma [32P]GTP, no change was observed in the number of RNA chains being initiated. Thus, captan does not exert its influence at the level of initiation of nascent chains. However, captan did have an effect on chain growth. From calculations of the incorporation of precursors molecules, RNAs isolated from treated cells were measured to be an average of 2.7 times longer than those from untreated cells. RNA chain lengths were also analyzed by polyacrylamide gel electrophoresis. By this latter technique it was also shown that cells exposed to captan synthesized RNAs that were longer than those of untreated cells. Alterations in the degradation of RNA molecules do not account for the captan mediated response in RNA synthesis.
Adenosine Triphosphate↗
Initiation of transcription in nuclei is inhibited by captan.
Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) was examined for its effects on bovine liver nuclear RNA synthesis. Transcription was measured by [3H] UTP incorporation into either acid insoluble product or isolated RNA. Captan (1 mM) was found to inhibit RNA synthesis 50% in intact nuclei and 70% in a hypotonic lysate of nuclei. Individual RNA polymerase activities were distinguished in both intact nuclei and hypotonic lysate by alpha-amanitin sensitivity. Captan inhibited RNA polymerase I and II activities to an equal extent in both intact and lysate systems. The sulfhydryl compound dithiothreitol (DTT) protected the RNA polymerase activities from inhibition by captan. Initiation of transcription in intact nuclei was measured by [gamma-32P] ATP incorporation into purified RNA and was found to be inhibited 75% by 1 mM captan. This report describes the inhibition of nuclear RNA polymerase activities by captan and suggests a possible mechanism for its toxic effect on eukaryotic polymerases.
Amanitins↗
Induction of gastric mucosal cell proliferation by the fungicide captan: role of tyrosine kinases.
Captan (1,2,3,6-tetrahydro-N-trichloromethylthiophthalmide), a widely used fungicide, has been shown to induce carcinoma in the gastrointestinal tract of rodents. However, little is known about the captan induction of early biochemical changes in the gastrointestinal tract. The present investigation examines the changes in gastric mucosal proliferative activity in 2-month-old Fischer 344 rats following a daily injection (s.c.) of captan (100 mg/kg body wt.) in DMSO while being infused (osmotic minipump) with the same compound (7.14 mg captan/kg body wt./h) for 2 weeks. The control rats received the vehicle the same way. The change in proliferative activity was related to tyrosine kinase (Tyr-k) activity and tyrosine-specific phosphorylation of protein(s) in gastric mucosal membranes since these intracellular events are thought to play an important role in proliferation, differentiation and transformation of cells. After 2 weeks of captan administration gastric mucosal DNA synthesis and thymidine kinase activity (indicators of proliferative activity) were increased by 330% (P less than 0.025) and 98% (P less than 0.025), respectively, when compared with the corresponding controls. Gastric mucosal DNA content was also increased by 90% (P less than 0.025) after administration of captan. These increases were associated with about 3-fold rise in Tyr-k activity and 2-fold increase in tyrosine phosphorylation of 6 mucosal membrane proteins with Mr of 105, 90, 60, 55, 48 and 37 kDa. We conclude that captan stimulates gastric mucosal cell proliferation, and activation of Tyr-k and tyrosine phosphorylation of certain membrane proteins may be important in the regulation of this process.
Animals↗
Protective effect of glutathione on the in vitro inhibition of hepatic cytochrome P-450 by captan.
The in vitro effect of various concentrations of captan on hepatic microsomal cytochrome P-450 from pehnobarbital-pretreated rats was studied. The I-50 value, namely the concentration of the inhibitor necessary to produce 50% loss of cytochrome P-450 was determined from theplotted inhibition curve. The presence of ethylenediaminetetraacetic acid (EDTA) in microsomal incubations prior to the addition of captan failed to prevent the loss of cytochrome P-450 by captan. In contrast, reduced glutathione (0.5 mM) added to microsomal incubations before captan (0.1 mM) afforded almost complete protection of cytochrome P-450 from captan inhibition. These data indicate that the inhibitory effect of captan on vitally important drug-metabolizing enzyme system, of which cytochrome P-450 is a major component, can be prevented by prior presence of reduced glutathione (GSH) but not of EDTA.
Animals↗
Determination of tetrahydrophtalimide and 2-thiothiazolidine-4-carboxylic acid, urinary metabolites of the fungicide captan, in rats and humans.
Capillary gas chromatographic (GC) methods using sulphur and mass selective detection for the qualitative and quantitative determination of tetrahydrophtalimide (THPI) and 2-thiothiazolidine-4-carboxylic acid (TTCA), urinary metabolites of the fungicide captan in rat and humans, were developed. Urinary detection limits were 2.7 micrograms/l for THPI and 110 micrograms/l for TTCA. Intraperitoneal and oral administration of captan to rats resulted in a 48-h cumulative urinary excretion of THPI of 1%-2% and 3%-9% of the dose, respectively. Cumulative urinary excretion of TTCA over 48 h ranged from 2% to 5% of the captan dose for the respective routes of administration. In urine of non-exposed human subjects, neither THPI nor TTCA could be detected. In urine of fruit-growers who were occupationally exposed to captan, both THPI and TTCA could be detected. Based on these results, THPI and TTCA are proposed as promising parameters for the biological monitoring of occupational exposure to captan.
Agriculture↗
Effects of the herbicide 2,4-DB and fungicide captan on reactions of mitochondria and chloroplasts.
The effects of the herbicide 4(2,4-dichlorophenoxy)butyric acid (2,4-DB) and fungicide N-(trichloromethyltio)-4-cyclohexene-1,2-dicarboximide (captan) on electron transport processes of mitochondria and chloroplasts have been investigated. Chloroplasts, isolated from spinach leaves (Spinacia oleracea L.), were treated with pesticide prior to the addition of electron acceptor and ADP. White potato (Solanum tuberosum L.) mitochondria were either incubated with pesticide before the addition of substrate, or they were treated with pesticide after the addition of substrate and ADP. Captan inhibited oxidation of malate by mitochondria and acted as an uncoupler. With succinate as sunstrate captan was found to stimulate state 4 respiration, as substrate captan was found to stimulate state 4 respiration, with the loss of coupled phosphorylation only at higher concentrations of fungicide. The herbicide 2,4-DB appeared to be 5 to 10 times less effective than captain. Both compounds inhibited phosphrylation-coupled succinate oxidation at higher concentrations and malate-coupled phosphorylation at lower concentrations. They acted as inhibitors of NADH-cytochrome c reductase. Both pesticides inhibited noncyclic electron transport in chloroplasts. The rate of ferricyanide reduction in the presence and absence of phosphorylating agents was reduced, and although the rate of ATP generation was reduced also, the P/2e ratio was not changed much under the influence of pesticides.
Butyrates↗
Personnel safety and foliage residue in an orchard spray program using azinphosmethyl and captan.
Residue levels of azinphosmethyl and captan were determined from blotter paper patches attached to the clothing of personnel participating in an orchard spray program. Average exposure of 1.74 mg/man/hr for azinphosmethyl and 1.94 mg/man/hr for captan were extrapolated from mean residue values obtained from analyzing the patches. Azinphosmethyl residue found on apple and peach foliage had been reached by 69% by the tenth day post-application, while captan residue had been reduced 50% for the same period. There was no evidence of a buildup of either azinphosmethyl or captan on treated foliage as the season progressed.
Agricultural Workers' Diseases↗