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. 2023 Mar 30;11(4):328.
doi: 10.3390/toxics11040328.

Difenoconazole Exposure Induces Retinoic Acid Signaling Dysregulation and Testicular Injury in Mice Testes

Xiangqin Zheng  1 , Yuexin Wei  1 , Jiadong Chen  1 , Xia Wang  1 , Dinggang Li  1 , Chengjun Yu  1 , Yifan Hong  1 , Lianju Shen  1 , Chunlan Long  1 , Guanghui Wei  1 , Shengde Wu  1
Affiliations

Difenoconazole Exposure Induces Retinoic Acid Signaling Dysregulation and Testicular Injury in Mice Testes

Xiangqin Zheng et al. Toxics. .

Abstract

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes.

Keywords: GC-2 cell; apoptosis; difenoconazole; reproductive toxicity; retinoic acid signaling; testis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
DFZ exposure induces testicular injury. (A) Temporal graph of body weight, (B) body weight at the completion of treatment, (C) testicular organ coefficient, (D) sperm morphology (black arrows denote the malformed sperm), (E) sperm counts, (F) sperm deformity rate, and (G) serum testosterone concentration in different groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. indicates not statistically significant versus the control group.
Figure 2
Figure 2
DFZ exposure leads to pathological changes in testicular tissue. (A) H&E staining of testicular tissue (black markers indicate the disrupted germinal epithelium. Yellow arrows indicate the vacuolation of the germinal epithelium. Red arrows indicate the sloughing of germ cells. Asterisk indicates damaged seminiferous tubules), (B) diameter of seminiferous tubules, (C) number of spermatogenic epithelium layers, and (D) percentage of damaged seminiferous tubules in different groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. indicates not statistically significant versus the control group.
Figure 3
Figure 3
DFZ exposure induces increased apoptosis and abnormal meiosis in testicular tissues. (A) TUNEL staining of testicular tissue (the scale bar represents 100 µm) and (B) protein (Stra8 and Scp3) expression levels in different groups. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 versus the control group.
Figure 4
Figure 4
Effects of DFZ exposure on the testicular RA pathway. (A) RA, (B) RE, and (C) ROL concentrations in testicular tissues in different groups. (D) Relative mRNA expression level of key genes in RA pathway. (a–c) genes in RA synthesis, (d,e) RA receptors, and (f) genes in RA catabolism. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns. indicates not statistically significant versus the control group.
Figure 5
Figure 5
Toxic effects of DFZ on GC-2 cells. (A) Cell viability assay of GC-2 cell after exposure to different concentrations of DFZ for 24 h. (B) Altered cellular ultrastructure observed by transmission electron microscopy (the scale bar represents 2 µm). (C) RA, (D) ROL, and (E) RE concentrations in GC-2 cells in different groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the control group.
Figure 6
Figure 6
Bioinformatics analysis of the transcriptomic data of GC-2 cells in control and DFZ exposure groups. (A) Enriched terms related to RA pathway and apoptosis. (B) Heatmap, (C) Ontology clusters, and (D) network of DEGs enriched in RA pathway- and apoptosis-related terms.
Figure 7
Figure 7
Screening and identification of key genes related to RA pathway and apoptosis. (A) (a,b) Protein–protein interaction (PPI) and (c) cytoHubba MCC analysis of DEGs enriched in RA pathway- and apoptosis-related terms. (B) Heatmap and (C) qPCR validation of identified key genes. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the control group.
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References

    1. Carpenter C.P., Weil C.S., Smyth H.F.J. Toxicity of an imidazoline (or glyoxalidine) fungicide. A.M.A. Arch. Ind. Hyg. Occup. Med. 1951;4:494–503. - PubMed
    1. Zhang H., Qian M., Wang J., Yang G., Weng Y., Jin C., Li Y., Jin Y. Insights into the effects of difenoconazole on the livers in male mice at the biochemical and transcriptomic levels. J. Hazard. Mater. 2022;422:126933. doi: 10.1016/j.jhazmat.2021.126933. - DOI - PubMed
    1. Graham D.O., Wilson R.K., Ruma Y.N., Keniya M.V., Tyndall J.D.A., Monk B.C. Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate. J. Fungi. 2021;7:897. doi: 10.3390/jof7110897. - DOI - PMC - PubMed
    1. Dong F., Li J., Chankvetadze B., Cheng Y., Xu J., Liu X., Li Y., Chen X., Bertucci C., Tedesco D., et al. Chiral triazole fungicide difenoconazole: Absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil. Environ. Sci. Technol. 2013;47:3386–3394. doi: 10.1021/es304982m. - DOI - PubMed
    1. Pitombeira De Figueirêdo L., Athayde D.B., Daam M.A., van Gestel C.A.M., Guerra G.D.S., Duarte-Neto P.J., Espíndola E.L.G. Impact of temperature on the toxicity of Kraft 36 EC® (a.s. abamectin) and Score 250 EC® (a.s. difenoconazole) to soil organisms under realistic environmental exposure scenarios. Ecotox. Environ. Saf. 2020;194:110446. doi: 10.1016/j.ecoenv.2020.110446. - DOI - PubMed

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