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Carbendazim (CBZ) and procymidone (PRO) are two widely used fungicides in agriculture. However, there are still gaps in knowledge regarding about the potential hazards of joint exposure to CBZ and PRO in animals. Here, 6-week-old ICR mice were exposed to CBZ, PRO and CBZ + PRO for 30 days, and metabolomics were performed to discover the mechanism by which the mixture enhanced the effects on lipid metabolism. Co-exposure to CBZ + PRO elevated the body weights, relative liver weights and relative epididymis fat weights, but not in the single exposure groups. Molecular docking analysis suggested that CBZ and PRO combined with peroxisome proliferator-activated receptor (PPARγ) at the same amino acid site as the agonist rosiglitazone. The RT-qPCR and WB results demonstrated that the levels of PPARγ were higher in the co-exposure group than in the single exposure groups. In addition, hundreds of differential metabolites were discovered by metabolomics and enriched in different pathways, such as pentose phosphate pathway and purine metabolism. A unique effect, a decrease in glucose-6-phosphate (G6P) that promoted more NADPH production, was observed in the CBZ + PRO group. These results demonstrated that exposure to CBZ + PRO caused more serious lipid metabolism disorder in the liver than exposure to a single fungicide, which could provide some new insight for the toxic effects after fungicides joint exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bromuconazole (BRO), as one of the typical triazole fungicides, has not been reported on its effects on aquatic organisms. In this study, zebrafish embryos were used as experimental objects to evaluate the toxicity of BRO. In the acute embryo toxicity test, it was observed that the heart rate and growing development were affected by BRO in a concentration-dependent manner, and the half-lethal concentration (LC50) of BRO at 96 h post-fertilization (hpf) was about 11.83 mg/L. Then, low concentrations of BRO (50 ng/L, 0.075 mg/L, 0.3 mg/L, 1.2 mg/L), which were set according to the LC50 and environmental related concentrations, were used to analyze the toxic effects on the different endpoints in larval zebrafish. Interestingly, the transcriptomic analysis found that most different expressed genes (DEGs) could be focused on the pathways of lipid metabolism, myocardial function, glycometabolism, indicating that heart function and lipid metabolism in larval zebrafish were disrupted by BRO. For supporting this idea, we re-exposed the transgenic zebrafish and WT zebrafish embryos, proved that BRO caused damage to heart development and lipid transport on morphological and genetic level, which was consistent with transcriptomic results. In addition, BRO exposure caused oxidative damage in the larvae. Taken together, BRO exposure could affect the myocardial contraction function and lipid transport in larval zebrafish, accompanied by disturbances in the level of oxidative stress, which was of great significance for improving the biotoxicological information of BRO., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The triazole fungicide prothioconazole (PTC) can cause adverse effects in animals, and its main metabolite prothioconazole-desthio (PTC-d) is even much more harmful. However, the toxic effects of PTC and PTC-d on the liver-gut axis of mice are still unknown. In the present experiment, we found that oral exposure to PTC and PTC-d increased total bile acids (TBAs) levels in the serum, liver, and feces. Correspondingly, the transcription of genes involved in bile acids (BAs) disposition was significantly influenced by PTC or PTC-d exposure. Furthermore, the BAs composition of serum BAs was analyzed by LC-MS, and the results indicated that PTC and PTC-d exposure changed the BAs composition, lowered the ratio of conjugated/unconjugated BAs, elevated the ratio of CA/b-MCA, and enhanced the hydrophobicity of BAs pool. 16s RNA gene sequencing of the DNA from colonic contents uncovered that PTC and PTC-d exposure altered the relative abundance and constitution of intestinal microbiota, increasing the relative level of Lactobacillus with bile salt hydrolase (BSH) activity. Furthermore, PTC and PTC-d exposure impaired the gut barrier function, causing an increase in mucus secretion. In particular, the effects of PTC-d on some endpoints in the BAs metabolism and gut barrier function had been proven to be more significant than the parent compound PTC. All these findings draw attention to the health risk of PTC and PTC-d exposure in regulating BAs metabolism, which might lead to some metabolic disorders and occur of related diseases in animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Difenoconazole is a commonly used triazole fungicide that consists of four stereoisomers [(2S,4S)-, (2S,4R)-, (2R,4R)-, and (2R,4S)-isomers] with different bioactivity. For example, the toxicity of the (2R,4S)-isomer to fish is approximately seven times higher than that of the (2S,4S)-isomer. However, the stereoselective toxic effects of difenoconazole stereoisomers on mammals have received little attention. In the present study, adult male mice were orally treated with a mixture of the four stereoisomers or each stereoisomer individually (0, 30, or 100 mg/kg/d) by gavage for 28 days. Pathological staining of the liver sections showed that the (2R,4R)-isomer caused lipid droplet accumulation. The mixture or each individual stereoisomers decreased the levels of amino acids and acyl-carnitine in serum. Moreover, the (2S,4R)-, (2R,4R)-, and (2R,4S)-isomers affected intestinal permeability, causing decreases in mucus secretion and tight junction protein expression in colon. Analysis of the gut microbiota composition showed that the stereoisomers caused decreases of OTU numbers and observed species at different levels. Interestingly, difenoconazole and its four stereoisomers reduced the relative abundance of Bacteroidetes at the phylum level and some short-chain fatty acid (SCFA)-producing bacteria. Taking the findings together, 2R-difenoconazole with strong bioactivity against pathogenic fungi also had significant effects in mammals, disrupting hepatic lipid metabolism, intestinal permeability, and gut microbiota. It is concluded that the health risks of the four difenoconazole stereoisomers to mammals should not be overlooked., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated mRNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the mRNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Epoxiconazole (EPX), as a broad-spectrum triazole fungicide, is widely used in agriculture to resist pests and diseases, while it may have potential toxicity to non-target organisms. In the present study, early developmental stage zebrafish were used as the subject organisms to assess the toxicity of EPX, and the possible mechanism of toxicity was also discussed by biochemical and transcriptomic analysis. Through embryo toxicity test, we had made it clear that the 96 h LC 50 of embryo was 7.204 mg/L, and acute exposure to EPX effected hatching rate, heartbeats, body length and even morphological defects. Then, by being exposed to EPX for 7 days at concentrations of 175 (1/40 LC 50 ), 350 (1/20 LC 50 ) and 700 (1/10 LC 50 ), biochemical parameters were affected, mainly manifested as increase of the triglyceride (TG) level and decrease of glucose content. Correspondingly, the transcription of genes related of glucose metabolism, lipid metabolism and cholesterol metabolism were also affected significantly in larval zebrafish. Moreover, some pathways, including lipid metabolism, glucose metabolism and amino acid metabolism were affected through transcriptome sequencing analysis in the larval zebrafish. Further data analysis based on the sequencing, EPX exposure also affected the expression of genes related to cell apoptosis. We further conformed that the bright fluorescence on the liver and bright spots near the liver by acridine orange staining. In addition, the mRNA levels of apoptosis related genes were also significantly affected in the EPX exposed larval zebrafish. Taken together, the work could provide an insight into toxic effects of EPX on the zebrafish larvae at embryo toxicity and transcriptional levels, providing some evidences for the toxic effects of triazole fungicides on non-target organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chlorothalonil (CTL), carbendazim (CBZ), and prochloraz (PCZ) are fungicides widely used in many countries. The use of these fungicides raises concerns because they are often applied together or used in the same agricultural area. However, the toxicity of these fungicides or mixtures, especially to aquatic organisms, has received limited attention. Here, embryonic and larval zebrafish were exposed to indicated concentrations of CTL, CBZ, and PCZ and their binary (CTL+CBZ, CTL+PCZ and CBZ+PCZ) and ternary (CTL+CBZ+PCZ) mixtures for 24 h. Based on metabolomics analysis, we observed that hundreds of metabolites were altered, and glycolysis metabolism and amino acid metabolism were the two most affected pathways. Interestingly, a total of 9 and 26 metabolites changed significantly in embryos and larvae treated with all fungicides, respectively. Among these altered metabolites, 2-aminoadipic acid (2-AAA) levels increased significantly in all groups, indicating that 2-AAA potentially represents a useful biomarker for evaluating the toxicity of fungicides. Furthermore, the joint effects of CTL+PCZ on embryos and larvae, especially on amino acid metabolism, were weaker than those in other groups, but combined treatment did not influence individual fungicidal activity. Data acquired from metabolomics provided important insight for understanding the mechanism by which fungicides or their mixtures affect zebrafish., (Copyright © 2020 Elsevier B.V. All rights reserved.)