Comparative Transcriptome Analysis to Investigate the Immunotoxicity Mechanism Triggered by Dimethomorph on Human Jurkat T Cell Lines

Abstract

Dimethomorph (DMM) is a broad-spectrum fungicide used globally in agricultural production, but little is known regarding the immunotoxicity of DMM in humans. In this study, the immunotoxicity of DMM on human Jurkat T cells was evaluated in vitro. The results indicated that the half-effective concentration (EC50) of DMM for Jurkat cells was 126.01 mg/L (0.32 mM). To further elucidate the underlying mechanism, transcriptomics based on RNA sequencing for exposure doses of EC25 (M21) and EC10 (L4) was performed. The results indicated that compared to untreated samples (Ctr), 121 genes (81 upregulated, 40 downregulated) and 30 genes (17 upregulated, 13 downregulated) were significantly differentially regulated in the L4 and M21 samples, respectively. A gene ontology analysis indicated that the significantly differentially expressed genes (DEGs) were mostly enriched in the negative regulation of cell activities, and a KEGG pathway analysis indicated that the DEGs were mainly enriched in the immune regulation and signal transduction pathways. A quantitative real-time PCR for the selected genes showed that compared to the high-dose exposure (M21), the effect of the low-dose DMM exposure (L4) on gene expression was more significant. The results indicated that DMM has potential immunotoxicity for humans, and this toxicity cannot be ignored even at low concentrations.

Use of Transcriptomics to Reveal the Joint Immunotoxicity Mechanism Initiated by Difenoconazole and Chlorothalonil in the Human Jurkat T-Cell Line

“…The cells were cultured in a cell culture incubator containing 5% CO 2 at 37 °C, maintained in the exponential growth phase by subculture 2–3 d intervals, and then used for subsequent experiments. The absence of mycoplasma was checked routinely using the Mycoplasma Stain Kit [ 21 ].…”

“…Briefly, 100 μL of activated Jurkat T cells was seeded into a 96-well plate (2 × 10 4 cells/well) and exposed for 36 h to a single pesticide at final concentrations of 0.5, 5, 25, 50, 100, 250, and 500 mg/L. The final dissolvent (acetone) concentration was adjusted to the same concentration and less than 0.1%, which exerted no effect on cell viability [ 21 ]. The cells exposed to 0.1% sterile ultrapure water and acetone were used as the blank and control groups, respectively.…”

“…The cells exposed to 0.1% sterile ultrapure water and acetone were used as the blank and control groups, respectively. After exposure, a 10% ( v / v ) CCK-8 solution was added to the well and re-incubated for 2 h [ 21 ]. Absorbance was measured at 450 nm in a ReadMax 500F enzyme-labeled instrument (Shanpu Biotechnology Co., Ltd., Shanghai, China).…”

“…The activated Jurkat T cells were inoculated in a 75 cm 2 cell culture flask with 60 mL medium at an initial concentration of 5 × 10 6 cells/bottle and exposed for 36 h to single or mixed pesticides at final concentrations of EC 10 and EC 25 , respectively. Upon removal of the culture medium after exposure, the cells were disrupted and homogenized in TRIzol ® reagent (Thermo Fisher, Waltham, MA, USA), and then RNA was isolated according to the operating manual [ 21 ]. Genomic DNA was eliminated using DNase I (TaKaRa, Dalian, China).…”

“…The transcription library was prepared according to the operating manual of the TruSeqTM RNA sample preparation kit. The libraries were size-selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplification using Phusion DNA polymerase (NEB, Waltham, MA, USA) for 15 PCR cycles [ 21 ]. After quantification using TBS-380 (Turner BioSystems, Inc. Sunnyvale, CA, USA), a paired-end RNA sequencing library was obtained by using a Nova Seq 6000 sequencer (2 × 150 bp read length).…”