Differential immunomodulatory effects of six pesticides of different chemical classes on human monocyte-derived macrophage functions.

Exposure to pesticides through eyes, skin, ingestion and inhalation may affects human health by interfering with immune cells, such as macrophages. We evaluated, in vitro, the effect of six pesticides widely used in apple arboriculture on the functions of human monocyte-derived macrophages (hMDMs). hMDMs were cultured for 4 or 24 h with or without pesticides (0.01, 0.1, 1, 10 µmol.L-1). We showed that chlorpyrifos, thiacloprid, thiophanate, boscalid, and captan had little toxic effect at the tested concentrations, while dithianon had low-cytotoxicity at 10 µmol.L-1. While boscalid showed no effect on hMDMs function, thiophanate (0.01 µmol.L-1) stimulated with TPA and thiacloprid (1, 10 µmol.L-1) stimulated with zymosan activated ROS production. Chlorpyrifos, dithianon, and captan inhibited ROS production and TNF-α, IL-1ß pro-inflammatory cytokines. We established that dithianon (0.01-1 µmol.L-1) and captan (0.1, 1 µmol.L-1) induced mRNA expression of NQO1 and HMOX1 antioxidant enzymes. Dithianon also induced the mRNA expression of catalase, superoxide dismutase-2 at 10 µmol.L-1. Together, these results show that exposure to chlorpyrifos, dithianon, and captan induce immunomodulatory effects that may influence the disease fighting properties of monocytes/macrophages while pesticides such as thiacloprid, thiophanate and boscalid have little influence.

Induction of Systemic Resistance in Chickpea (Cicer arietinum L.) Against Fusarium oxysporum f. sp. ciceris by Antagonistic Rhizobacteria in Assistance with Native Mesorhizobium.

In the present study five potent rhizobacterial antagonists of Fusarium oxysporum f. sp. ciceris alone and in combination with Mesorhizobium (M) were evaluated for their potential to elicit the defence response reactions to reduce the total loss of plants and enhance the growth of two chickpea cultivars i.e. resistant GPF-2 and susceptible JG-41. Observations revealed that maximum phenolic, peroxidase (PO) and polyphenol oxidase (PPO) activity was induced after 30th day of germination. Maximum phenol concentration of 745.8 and 724.1 µg/gfw root tissues was recorded by Ps45 when co-inoculated with Mesorhizobium in both the varieties i.e. GPF-2 and JG-41 respectively. Isolates Ps45, Ps47 and Ps44 were found most promising to induce PO and PPO activity, in combination with Mesorhizobium and recorded superior over the fungicide with respect to negative control. Similar results were recorded for the phenylalanine ammonia lyase (PAL), maximally induced on 20th day after germination, where dual inoculation of Ps44+M and Ps45+M induced 57.0 and 54.2 nmol of cinnamic acid min-1 gfw-1 in GPF-2. However in case of JG-41, Ps45 and Ba1a exhibited highest PAL activity of 54.2 and 41.4 nmol of cinnamic acid min-1 gfw-1. Malonic aldehyde concentration in stem tissues at 30th day revealed that lipid peroxidation was effectively reduced in rhizobacterial treated plants compared to fungicide and negative control, signifying the role of antagonistic plant growth promoting rhizobacteria in reducing the stress and enhancing the plant's defence response to reduce the disease incidence and thus improving the plant growth and yield. Moreover the dual inoculations were observed superior over the fungicide treatment as well as single inoculations in terms of growth (root/shoot length and weight), signifying the synergistic effect of screened antagonists and native Mesorhizobium in suppressing the pathogen and thereby enhancing the plant growth.

Effects of Folpet, Captan, and Captafol on Human Aromatase in JEG-3 Cells.

BACKGROUND: Estradiol, produced by aromatase (CYP19A1), is very important for reproduction. Folpet, captan, and captafol belong to the phthalimide class of fungicides. They are used to protect the leaves of plants or fruits. They could be endocrine disruptors and may disrupt CYP19A1 activity. METHODS: In the present study, we investigated the effects of folpet, captan, and captafol on estradiol production and human CYP19A1 activity in JEG-3 cells. RESULTS: Folpet, captan, and captafol decreased estradiol production in JEG-3 cells in a concentration-dependent manner. Folpet, captan, and captafol inhibited human CYP19A1 with inhibitory concentration (IC50) values of 3.55, 10.68, and 1.14 µmol/L respectively. These chemicals competitively inhibited human CYP19A1. Molecular docking simulation analysis showed that they tended to bind to the steroid-binding pocket of the CYP19A1. However, the required concentrations may not be relevant to the negligible systemic exposures in humans to these chemicals. CONCLUSION: Folpet, captan, and captafol are potential inhibitors of human CYP19A1.

Screening and characterization of Xanthomonas oryzae pv. oryzae strains with resistance to pheazine-1-carboxylic acid.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) and can be effectively controlled by phenazine-1-carboxylic acid (PCA), an antibiotic secreted by Pseudomonas spp. PCA resistance in Xoo was investigated in this research. Only four PCA-resistant strains were obtained by extensive screening, and the resistance was genetically stable in only one of them (P4). P4 was also resistant to phenazine and 1-hydroxyphezine but not to captan, bismerthiazol, or streptomycin. The following were reduced in P4 relative to the parental wild type: growth, virulence, EPS production, extracellular cellulase production and activity, biofilm formation, and swimming ability. ROS accumulation was reduced, resistance to exogenous H2O2 was increased, and expression of catalase genes and catalase activities were increased in P4, suggesting that PCA resistance in P4 results from a reduction in ROS production and/or an increased ability to metabolize ROS following PCA treatment. Given the low risk of Xoo developing PCA resistance and the reduced virulence and fitness of the resistant strain, PCA can be used in alternation with other common bactericides to control BLB in rice fields.

Characterization of Trp(+) reversions in Escherichia coli strain WP2uvrA.

The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains. In contrast to Salmonella His(+) revertants, E.coli Trp(+) revertants have not been characterized at the molecular level. In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA). In spontaneous Trp(+) revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT). Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNA(Glu) (glnU), tRNA(Lys) (lysT) and tRNA(Tyr) (tyrT, tyrU), the Trp(+) reversion system with E.coli WP2uvrA detected five types of base substitutions, A.T-->T.A, A.T-->C.G, A.T-->G.C, G.C-->A.T and G.C-->T.A. About 30-50% of Trp(+) revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations. In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations. Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A.T base pairs (trpE65, lysT) and G.C base pairs (glnU, tyrT, tyrU).

Interaction of glutathione transferase P1-1 with captan and captafol.

Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.

Transformation of BALB/c 3T3 cells in vitro by the fungicides captan, captafol and folpet.

Cytotoxic and cell-transforming activities of the three fungicides, captan, captafol and folpet, have been studied in an experimental in vitro model by exposing BALB/c 3T3 cells to the chemicals with or without S-9 mix-induced bioactivation. Cytotoxicity of the three compounds was reduced in the presence of the metabolizing system. Each assayed pesticide displayed cell-transforming ability in the presence of the metabolizing system. The relative efficiency was: captafol > captan > folpet. Cell transformation was considered to be due to carcinogenesis-promoting activity. These data, obtained in a medium-term (6-8 weeks) experimental model, contribute to a better understanding of the action of the three pesticides in the multistep carcinogenesis process and provide more information concerning the oncogenic risk of these xenobiotic compounds for humans.

Estudo da influência de agrotóxicos sobre o desenvolvimento do Rhizobium leguminosarum biovar phaseoli / Study of the agrotoxics influence over the development of the Rhizobium leguminosarum biovar phaseoli

As bactérias fixadoras de nitrogênio atmosférico, via processo simbiótico, repassam N2 às leguminosas sob forma de amônia, proporcionando aumento na produçäo de alimentos protéicos, reciclagem biológica de nitrogênio do ar, evitando o alto custo da adubaçäo nitrogenada e o efeito potencialmente poluidor do nitrato lixiviado. Testou-se quatro estirpes de Rhizobium phaseoli frente a quatro fungicidas indicados para o tratamento de sementes de Phaseolus vulgaris L. (feijoeiro), no Estado do Rio Grande o sul (Brasil). Considerando-se as dosagens recomendadas dos fungicidas, houve crescimento bacteriano das quatro estirpes frente a Benomil e inibiçäo total frente a Captan; porém, para PCNB e Thiram, a resistência dependeu do tipo de estirpe. Observou-se que näo há influência de fungicidas sobre o desenvolvimento do Rhizobium phaseoli quando seleciona-se a estirpe adequada ao fungicida corretamente dosado.

1,8-Naphthalic anhydride antidote enhances the toxic effects of captan and thiram fungicides on Azospirillum brasilense cells.

The effects of ten fungicides, six herbicides and four insecticides on the nitrogen-fixing bacterium Azospirillum brasilense were examined. The fungicides captan and thiram were the most toxic among the compounds tested. Cell growth and nitrogenase activity of the bacterium were markedly inhibited by low concentrations of the two fungicides. Antidote 1,8-naphthalic anhydride increased by a factor of 2 the cellular level of glutathione. The addition of the antidote in the presence of captan or thiram caused a similar increase in the glutathione content, but at the same time enhanced the toxicity of the two fungicides.

Mutagenic evaluation of the pesticides captan, folpet, captafol, dichlofluanid and related compounds with the mutants TA102 and TA104 of Salmonella typhimurium.

A mutagenic evaluation of captan, folpet, captafol, dichlofluanid and related compounds was carried out using the Salmonella/mammalian microsome test using strains TA102 and TA104. These strains contain A-T base pairs at the site of the mutation in contrast to the other Salmonella tester strains that detect mutagens damaging G-C base pairs. In addition, the excision repair system of the TA102 strain is still intact. Captan and folpet were mutagenic in strain TA104, captafol was mutagenic in strain TA102, whereas the remaining test compounds (dichlofluanid, tetrahydrophthalimide and thiozolidine-4-carboxylic acid) were not mutagenic in either strain. In conclusion, we consider it of value to add these two strains to those already used in the Ames test in order to increase confidence in our ability to detect mutagens and to shed further light on their mechanism of action.

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.

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.

[Mutagenic activity of the fungicide captan]. / K voporsu o mutagennoi aktivnosti fungitsida kaptana.

A complex study of mutagenic activity of captan (a fungicide) in a series of standard test-systems has shown that the preparation induces gene mutations in certain Salmonella indicator strains (without metabolic activation), increases the frequency of mitotic crossing-over and gene conversion in yeasts (Saccharomyces), possesses a weak cytogenetic action on bone marrow cells in experimental animals, manifests no cytogenetic effect in the lymphocyte culture of human peripheral blood (including a system of microsomal activation). Genetic activity of captan cannot be a limiting criterion of its harmfulness.

[Cytogenetic action of the pesticides captan and benomyl in a lymphocyte culture of human peripheral blood in the absence and presence of a system of metabolic activation]. / Issledovanie tsitogeneticheskogo deistviia pestitsidov kaptana i benomila v kul'ture limfotsitov perifericheskoi krovi cheloveka v otsutstvie i pri nalichii sistemy metabolicheskoi aktivatsii.

Results of the investigation of mutagenic effects of fungicides captan and benomyl in the culture of human peripheral lymphocytes in vitro with and without metabolic activation showed that captan did not manifest cytogenetic activity in any experimental variants, while benomyl induced colchicine-like and weak clastogenic effect in the concentration 10 micrograms/ml in the presence of microsomal activating mixture only.

Cytostatic activity and metabolic effect of N-trichloromethylthio-4-cyclohexane-1,2-dicarboximide on Ehrlich ascites carcinoma cells.

Effect of N-trichloromethylthio-4-cyclohexane-1,2-dicarboximide (NTCD) on energy-yielding and energy-requiring processes in Ehrlich ascites carcinoma (EAC) cells have been investigated. At concentrations higher than 10 micrograms/ml NTCD causes a rapid and practically full inhibition of both aerobic glucose uptake and lactate formation. On the other hand, at concentrations lower than 10 micrograms/ml, these metabolic parameters are stimulated. The stimulation of glycolysis, according to our previous results, suggests the interference of NTCD with mitochondrial functions. This image is supported by the marked inhibitory effect on NTCD on respiration of isolated mitochondria. The inhibition of glycolysis with higher concentrations of NTCD is the consequence of inactivation of hexokinase (EC 2.7.1.1), eventually of 6-phosphofructokinase (FC 2.7.1.11). The described effects of NTCD are given into coherence with chemical modification of appropriate functional SH groups of EAC cells by the compound studied. Proportionally to the dose and time NTCD inhibits the synthesis of macromolecules in whole EAC cells as measured by the incorporation of labeled adenine and valine into the TCA-insoluble fractions. The inhibition of biosynthetic processes followed is the consequence of exclusion of key processes in the energy metabolism and leads to the loss of EAC cells transplantability.

The effect of the pesticides, Dexon, Captan and Roundup, on sister-chromatid exchanges in human lymphocytes in vitro.

3 pesticides at varying concentrations were tested for the induction of SCE in human lymphocytes in vitro. The fungicide, Dexon, sodium (4-(dimethylamino)phenyldiazene sulfate, was found to cause the greatest increase in SCE frequency and the response is dose related. The herbicide, Roundup, isopropylamine salt of N-(phosphonomethyl)glycine, had the lest effect on SCE requiring the use of much higher concentrations to produce an effect. Limited results were obtained with the fungicide Captan, cis-N-((trichloromethyl)thio)-4-cyclohexene-1, 2-dicarboximide because of toxic levels of either the fungicide or solvent used.

Sister-chromatid exchanges and chromosomal aberrations in cultured Chinese hamster cells treated with pesticides positive in microbial reversion assays.

The induction of sister-chromatid exchanges (SCEs), chromosomal aberrations and polyploids was investigated in cultured Chinese hamster cells treated with pesticides or a related compound positive in microbial reversion assays. The chemicals tested were captan, captafol, 1,2-dibromoethane (EDB), 1,2-dibromo-3-chloropropane (DBCP), 5-nitro-1-naphrhonitrile (NNN), p-dimethylaminobenzenediazo sodium sulfonate (DAPA), 2-hydrazinoethanol (HEH), vamidothion, dichlorovos (DDVP), N-nitroso-ethylenethiourea (N-nitroso-ETU), and 2,4-dinitrophenyl thiocyanate (NBT). A significant and dose-dependent increase in the frequency of SCEs and chromosomal aberrations was observed in the cell cultures treated with captan, captafol, EDB, DBCP, HEH, DDVP, vamidothion, DAPA or N-nitroso-ETU or NBT produced a significant increase in the frequency of polyploid cells, whereas the other agents did not. When compared with results from microbial reversion assays, a close correlation was observed between the ability to induce SCEs or chromosomal aberrations and the mutagenic potency in bacteria (r:0.71-0.84).

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.

Cytogenetic and dominant lethal studies on captan.

Possible mutagenic activity of captan was investigated by in vitro and in vivo cytogenetic studies and by the dominant lethal study in mice. In vitro cytogenetic study with cultured human diploid cells revealed a significant increase in the frequency of cells showing stickiness and a severe mitotic inhibition at concentrations of 3.0 and 4.0 microgram of captan per ml. although no chromosomal aberrations were observed. In in vivo cytogenetic study, no chromosomal aberrations were induced in the bone marrow cells of rats treated orally with captan at a single dose of 500, 1000 or 2000 mg/kg or at five consecutive doses of 200, 400 or 800 mg/kg/day. Dominant lethal study also failed to show any mutation induction after treatment of male mice with daily oral dose of 200 or 600 mg of captan per kg bw for five days.