Metabolism Of Chlorothalonil Research Articles

Effects of plant morphology, vitamin C, and other co-present pesticides on the deposition, dissipation, and metabolism of chlorothalonil in pakchoi.

Pesticide residues have been a focus of attention of food safety. Different varietal pakchoi plants grown in open fields were studied to understand effects of morphology, leaf wax content, and vitamin C on the deposition, dissipation, and metabolism of chlorothalonil. The loose pakchoi plants and flat leaves were conducive to pesticide deposition, but not plants with erect leaves. Chlorothalonil on nine varieties of pakchoi dissipated in the first-order kinetic with T1/2s of 1.4 ~ 2.0days. Vitamin C in pakchoi could promote the dissipation of chlorothalonil. Carbendazim could significantly promote the dissipation of chlorothalonil on pakchoi. Interestingly, four metabolites of chlorothalonil were identified in the pakchoi and the metabolic pathway was predicted by DFT calculations. The risk assessment showed that pakchoi were safe for consumption after 10days of application of the recommended dose. This work provides important information for the understanding of deposition, dissipation, and metabolism of chlorothalonil in pakchoi.

γ-Aminobutyric acid (GABA) improves pesticide detoxification in plants

It is important to ensure food safety to study the technology and mechanism of pesticide residues degradation in crops. Though γ-aminobutyric acid (GABA) has been widely reported to involve in plant stress resistance, whether exogenous application or endogenous regulation of GABA by gene-editing technology can promote the pesticide detoxification is not clear in plants. Using tomato and chlorothalonil (CHT) as research models, we discovered that exogenous application of GABA or endogenous elevation of GABA by knockout of pyruvate-dependent GABA transaminase promoted both CHT metabolism and plant stress tolerance to CHT. This is closely related to the active adaptation of GABA to CHT stress by regulating the plant GABA shunt pathway and polyamine pathway. The transcriptome data revealed 17 target genes that may be closely related to the involvement of GABA in CHT metabolism, including 4 peroxidases, 5 glycosyltransferases, 4 glutathione S-transferases, and 4 ABC transporters. In addition, the glutathione detoxification pathway and antioxidative enzyme also actively participated in the GABA-induced CHT detoxification process, which played an important role in relieving CHT stress. As a result, GABA significantly increased the photosynthetic capacity of tomato leaves under CHT stress. While studying photosynthesis, we unexpectedly found that GABA promotes stomatal closure in terms of decreased stomatal conductance and stomatal diameter. This result implies that GABA can reduce CHT absorption by regulating stomatal movement in leaves. Together, we provided a novel viewpoint that foliar application of GABA or metabolic engineering of GABA is an effective approach to reduce the risk of pesticide contamination in crop production.

Jasmonic acid promotes glutathione assisted degradation of chlorothalonil during tomato growth

Glutathione (GSH) biosynthesis and regeneration play a significant role in the metabolism of chlorothalonil (CHT) in tomatoes. However, the specific regulatory mechanism of GSH in the degradation of CHT remains uncertain. To address this, we investigate the critical regulatory pathways in the degradation of residual CHT in tomatoes. The results revealed that the detoxification of CHT residue in tomatoes was inhibited by buthionine sulfoximine and oxidized glutathione pretreatment, which increased by 26% and 46.12% compared with control, respectively. Gene silencing of γECS, GS, and GR also compromised the CHT detoxification potential of plants, which could be alleviated by GSH application and decreased the CHT accumulation by 33%, 25%, and 21%, respectively. Notably, it was found that the jasmonic acid (JA) pathway participated in the degradation of CHT regulated by GSH. CHT residues reduced by 28% after application of JA. JA played a role downstream of the glutathione pathway by promoting the degradation of CHT residue in tomatoes via nitric oxide signaling and improving the gene expression of antioxidant and detoxification-related enzymes. This study unveiled a crucial regulatory mechanism of GSH via the JA pathway in CHT degradation in tomatoes and offered new insights for understanding residual pesticide degradation.

Glutaredoxin S25 and its interacting TGACG motif-binding factor TGA2 mediate brassinosteroid-induced chlorothalonil metabolism in tomato plants

The presence of pesticide residues in fresh fruits and vegetables poses a serious threat to human health. Brassinosteroids (BRs) can reduce pesticide residues in plants, but the underlying mechanisms still remain unclear. Here, we identified a tomato glutaredoxin gene GRXS25 which was induced by 24-epibrassinolide (EBR) and chlorothalonil (CHT) in a way dependent on apoplastic reactive oxygen species (ROS). Silencing of GRXS25 in tomato abolished EBR-induced glutathione S-transferases (GSTs) gene expression and activity, leading to an increased CHT residue. Yeast two-hybrid and bimolecular fluorescence complementation assays showed protein-protein interaction between GRXS25 and a transcription factor TGA2. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that TGA2 factor bound to the TGACG-motif in the GST3 promoter. While silencing of TGA2 strongly compromised, overexpression of TGA2 enhanced expression of GST genes and CHT residue metabolism. Our results suggest that BR-induced apoplastic ROS trigger metabolism of pesticide residue in tomato plants through activating TGA2 factor via GRXS25-dependent posttranslational redox modification. Activation of plant detoxification through physiological approaches has potential implication in improving the food safety of agricultural products.

Interplay between mitogen-activated protein kinase and nitric oxide in brassinosteroid-induced pesticide metabolism in Solanum lycopersicum

Nitric oxide (NO) and mitogen-activated protein kinase (MPK) play important roles in brassinosteroid (BR)-induced stress tolerance, however, their functions in BR-induced pesticides metabolism remain unclear. Here, we showed that MPK activity and transcripts of SlMPK1 and SlMPK2 were induced by chlorothalonil (CHT), a widely used fungicide, in tomato leaves. However, cosilencing of SlMPK1/2 compromised the 24-epibrassinolide (EBR)-induced upregulation of detoxification genes and CHT metabolism in tomato leaves. In addition, cosilencing of SlMPK1/2 inhibited the accumulation of S-nitrosothiol (SNO), the reservoir of nitric oxide (NO) in plants, whereas tungstate, the inhibitor of nitrate reductase (NR), blocked EBR-induced SNO accumulation and MPK activity. Inhibiting the accumulation of NO by cPTIO, the specific scavenger and tungstate abolished the EBR-induced upregulation of detoxification genes, glutathione accumulation and CHT metabolism. The results showed that MPK and NR-dependent NO were involved in BR-induced CHT metabolism. Notably, there was a positive crosstalk between the MPK and NO production.

Glutathione biosynthesis and regeneration play an important role in the metabolism of chlorothalonil in tomato

Glutathione is one of the major endogenous antioxidants produced by cells. In plants, glutathione is crucial for both abiotic and biotic stress resistance, and also involved in the detoxification of xenobiotics in many organisms. However, as in vivo evidences of glutathione function are still lacking so far, its roles in plants are still poorly understood. In this study, we investigated the changes of thiols, glutathione homeostasis and transcripts of genes potentially involved in chlorothalonil (CHT) metabolism in tomato (Solanum lycopersicum L.). Two genes (GSH1, GSH2) encoding γ-glutamylcysteine synthetase and glutathione synthetase, respectively, and a gene for glutathione reductase (GR1) involved in glutathione regeneration were silenced by virus induced gene silencing (VIGS) approach. Silencing of GSH1, GSH2 and GR1 decreased glutathione contents and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), but increased CHT residues in plant tissues. The GSH1 and GR1 silenced plants showed the lowest GSH level and ratio of GSH/GSSG, respectively. Transcripts of P450, GST and ABC transporter genes as well as glutathione S-transferase (GST) activity were induced after CHT treatment. However, the increases of these transcripts were compromised in GSH1, GSH2 and GR1 silenced plants. This study indicates that glutathione not only serves as a substrate for CHT conjugation, but is also involved in regulation of transcripts of gene in pesticide metabolism via controlling redox homeostasis.

Effect of Potassium Dihydrogen Phosphate and Bovine Manure Compost on the Degradation of Chlorothalonil in Soil

The effects of potassium dihydrogen phosphate (KH2PO4) and bovine manure compost (BMC) on the degradation and metabolism of chlorothalonil were examined in a sandy loam soil under laboratory conditions. In non-sterilized, non-amended soil, chlorothalonil degradation half-life was 8.8 days. However, it was up to 19.0 days in sterilized non-amended soil, suggesting that the degradation rate was about 2-fold slower than that in non-sterilized non-amended soil. Biological mechanisms accounted for 54.7% of chlorothalonil degradation in non-sterilized soil, indicating that the indigenous microorganisms in soil play an important role in the degradation process. In non-sterilized soil, the more acutely toxic metabolite, 4-OH-chlorothalonil (HTI), of chlorothalonil started forming after the second day of incubation. The concentration of HTI reached its maximum level (2.9 μ g g− 1) at 10 days after treatment, and no further degradation of HTI was observed in the following span of 20 days (10–30 days of incubation). However, the degradation rate of chlorothalonil was decreased substantially by the addition of KH2PO4 with a half-life of 17.5 days. No formation of HTI was observed before 10 days of incubation and no significant difference of the metabolite concentration at the end of experiment was observed between KH2PO4-amended and non-amended treatments, which denoted that the addition of KH2PO4 did not reduce the formation of HTI in the longer incubation course. In BMC-amended soil, the degradation rate was about 1.8 times faster than in non-amended soil. At 30 days of incubation, both chlorothalonil and HTI were degraded to a lower level in BMC-amended soil than in non-amended soil. The application of farm litter is a common fertilizing practice in vegetable fields in China, and thus this practice could not only improve soil fertility but also promote the removal of chlorothalonil and its metabolite HTI to further increase safety in crop rotations.

Microbial degradation of chlorothalonil in agricultural soil: A laboratory investigation

The employment of pesticides for crop protection must be balanced by a total awareness of the detrimental effects they may have on the environment. Pesticide residues in soil, and metabolites resulting from microbial metabolism of such compounds, may contribute to pollution of streams, affect microbial diversity, and influence essential symbiotic associations between microorganisms and crops. Chlorothalonil, an extensively used fungicide, is classified as a probable human carcinogen by the US EPA. Microbial degradation of chlorothalonil residues in different types of agricultural soil was investigated in the laboratory. Bacterial plate count and GC analysis results indicated that indigenous soil bacteria were able to utilize chlorothalonil, although the rate of chlorothalonil metabolism appeared to be influenced by soil characteristics.

Urinary and Biliary Metabolic Patterns of Chlorothalonil in Germ-Free and Conventional Rats

The metabolic fate of chlorothalonil, a broad spectrum fungicide that is known to be metabolized via glutathione conjugation, was examined through the analysis of urine and bile metabolites. The role of digestive microflora in the metabolism of chlorothalonil was assessed by comparing the metabolic patterns in germ-free and conventional rats. Low urinary and biliary excretion of radioactivity was observed in both conventional and germ-free rats. However, the urinary excretion of radioactivity was higher in conventional than in germ-free rats. Radio-HPLC analysis of urine and bile showed a complex metabolic profile in both conventional and germ-free rats. Methylthio metabolites of chlorothalonil were determined in ethyl acetate extracts of urine and bile of conventional and germ-free rats. These metabolites were excreted in a higher amount in the urine of conventional rats than in the urine of germ-free rats. This study shows the complexity of chlorothalonil metabolism and the role of the digestive microflora in chlorothalonil metabolism.

Chlorothalonil Biotransformation by Gastrointestinal Microflora:In VitroComparative Approach in Rat, Dog, and Human

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum contact fungicide used in agriculture and horticulture. The role of digestive microflora in chlorothalonil metabolism was assessed byin vitroincubation of [14C]chlorothalonil with stomach, duodenum, and cecum contents from rat, stomach, duodenum, and colon contents from dog and with human feces and stomach contents using an incubation dose of 500 μg/g of digestive contents. Transformation of chlorothalonil mostly occurred in rat cecum contents, dog colon contents, and human feces, in which unchanged chlorothalonil accounted for 46.7, 29.7, and 22.6% of the radioactivity, respectively. In those incubations, the identified metabolites were: 2,5,6-trichloro-4-methylthioisophthalonitrile (0.8–3% of the radioactivity), 2,5,6-trichloro-4-thioisophthalonitrile (0–7.1%), 3-thia-1-cyano-2,5,6-trichloroisoindolinone (2.6–12.7%), 2,5,6-trichloro-4-hydroxy-isophthalonitrile (0–3.2%), and 2,5,6-trichloroisophthalonitrile (9.9–21.4%). The mercapto metabolites of chlorothalonil, methylated or not, were recovered from rat and dog duodenum contents (0.8 and 0.7%). Only traces of metabolic products were recovered from rat and human stomach contents. The monocysteine conjugate of chlorothalonil was detected from rat duodenum contents and accounted for 0.2% of the substrate. The formation of metabolites decreased when the digestive contents were heated before incubation to inactivate bacteria. Under these conditions, the monomethylthiotrichloroisophthalonitrile formation was suppressed, except in rat and dog duodenum contents (1.5 and 0.5%, respectively). The monothiotrichloroisophthalonitrile accounted for 0.1 to 0.4%. The monohydroxytrichloroisophthalonitrile accounted for 0.5 to 2%, the thia-indolinone metabolite for 3.9 to 8% and the dechlorinated chlorothalonil for 0.3 to 2.8%. These results suggest that intestinal microflora plays a significant role in chlorothalonil metabolism, which implies Cys β-lyases. The formation of methylthio metabolites is discussed, including thiol methylation in digestive contents.

Glutathione-mediated chlorothalonil detoxification in channel catfish gills

Maintenance of the glutathione (GSH) pathway is critical in guarding against the toxic effects of a variety of pollutants. However, despite their importance as an initial line of defense against waterborne toxicants, little is known regarding the role of fish gills in GSH-mediated xenobiotic metabolism. In these studies, the role of channel catfish gills in the detoxification of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil), an electrophilic fungicide, was examined. Chlorothalonil was metabolized in vitro by gill cytosolic and microsomal glutathione S-transferases (GSTs). Chlorothalonil metabolism by cytosolic fructions was reduced markedly when GSH was omitted from reaction mixtures. Microsomal metabolism of chlorothalonil did not occur in the absence of GSH and in the presence of an NADPH-regenerating system lacking GSH. Channel catfish exposed in vivo to chlorothalonil had increased gill GSH and cysteine concentrations after 72 h exposure, and increased gill gamma-glutamylcysteine synthetase (GCS) activity at 144 h of exposure. Our results show that the gills play an important role in the metabolism and detoxification of chlorothalonil in the channel catfish. Catfish can maintain elevated gill GSH concentrations in the presence of chlorothalonil by increasing gill cysteine concentrations.

Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish ( Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.

The toxicology and metabolism of chlorothalonil in fish. IV. Zinc coexposure and the significance of metallothionein in detoxication in Salmo gairdneri

Rainbow trout, Salmo gairdneri, were subjected to various regimes of exposure to zinc (0.36 mg/l) and 14C-chlorothalonil ( 14C-TCIN, 10 μg/l). Gel column chromatography of hepatic cytosol after exposure showed no binding of 14C-TCIN to metallothionein proteins with or without pre-exposure or with coexposure to zinc. 14C-TCIN did not induce the production of metallothionein with or without 96 h preexposure to 0.36 mg/l zinc, whereas induction did occur with zinc coexposure over 156 h. 14C-TCIN exposure did not affect cytosolic zinc levels, but exposure to zinc did reduce the level of cytosolic 14C-TCIN over the observed protein molecular weight range. It appears that metallothionein does not play a significant role in TCIN detoxication at this sublethal exposure level.

The toxicology and metabolism of chlorothalonil in fish. III. Metabolism, enzymatics and detoxication in Salmo spp. and Galaxias spp.

Hepatic glutathione S-transferase (GST) activity towards chlorothalonil, tetrachloroisophthalonitrile (TCIN), was established for the five fish species: Salmo gairdneri, S. trutta, Galaxias maculatus, G. truttaceus and G. auratus. Molecular weights and hepatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) of GST were determined in all species. Low level exposure to TCIN was found to induce higher GST activity in G. maculatus, G. truttaceus and S. gairdneri. Other properties of GST enzymes investigated in these fish included TCIN binding, reaction order and the effect of acephate on activity. The effect of TCIN exposure on liver thiol and glutathione (GSH) levels in S. gairdneri and G. truttaceus was investigated. Significant decreases occurred in liver thiol levels of fish when exposed to varying concentrations of TCIN for 96 h without feeding. Liver GSH levels increased in S. gairdneri exposed to 10 μg/l over 96 h with feeding. A marked increase in GST activity was observed during 96 h exposure to 10 μg/l. No significant changes in the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were observed during exposure to 10 μg/l TCIN in vivo. A transient decrease in activity was observed during exposure to 30 μg/l TCIN. The previously reported in vitro inhibition of GAPDH by TCIN was studied in the context of liver cytosolic levels of GAPDH and GSH. Hepatic GSH and GST activity levels toward 14C-TCIN and CDNB were compared in S. gairdneri, G. maculatus and G. auratus. The order of asymptotic LC 50 values for the three species agreed with that for total hepatic GST activity toward 14C-TCIN consistent with a detoxication role for GSH-TCIN conjugation. A proposal for a screening process for Australian freshwater fish based on detoxication enzyme activity is discussed.

The toxicology and metabolism of chlorothalonil in fish. II. Glutathione conjugates and protein binding

The existence of mono- and diglutathione conjugates of chlorothalonil (TCIN) in the bile of Salmo gairdneri exposed to 14C-TCIN is confirmed. A detailed analysis of the structural nature of the conjugates is described, using model thioether analogues. The degree of protein binding of TCIN in liver cytosol and bile is reported.

The toxicology and metabolism of chlorothalonil in fish. I. Lethal levels for Salmo gairdneri, Galaxias maculatus, G. truttaceus and G. auratus and the fate of 14C-TCIN in S. gairdneri

LC 50 values of chlorothalonil (TCIN) to Salmo gairdneri, Galaxias maculatus, G. truttaceus and G. auratus for 24, 48 and 96 h were determined using a flow-through apparatus. All values were lower than those previously reported for various fish species. The influence of decreased oxygen concentration and co-exposure of acephate on LC 50 values of TCIN to Salmo gairdneri was also investigated. Uptake of 14C-chlorothalonil in internal organs of Salmo gairdneri was measured during a 96 h exposure to 10 μg/l in water. The primary site of accumulation was the bile. Levels in all other organs were in the mg/kg range and increased with time of exposure. Protein binding was observed in the liver. Release of polar labelled metabolites was constant with time over 96 h depuration, with 0.5% being TCIN. All organ levels dropped during this time. The possible route of TCIN metabolism is discussed.