2-Chloroethanol Induces Hepatic Toxicity by Disrupting Endoplasmic Reticulum Homeostasis Ameliorated by Dimethyl Sulfoxide

2-Chloroethanol Induces Hepatic Toxicity by Disrupting Endoplasmic Reticulum Homeostasis Ameliorated by Dimethyl Sulfoxide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 2-Chloroethanol Induces Hepatic Toxicity by Disrupting Endoplasmic Reticulum Homeostasis Ameliorated by Dimethyl Sulfoxide Tzung-Hsin Chou, Min-Hsiu Hu, Kuo-Tai Hua, Cheng-Chung Fang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5876858/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 2-Chloroethanol (2CE), a metabolite of ethylene oxide (EO), vinyl chloride (VC), and 1,2-dichloroethene (1,2-DCE), has an unclear toxic mechanism, complicating effective treatment of poisoning. This study examined the impact of acute 2CE exposure on endoplasmic reticulum (ER) homeostasis in liver cells. A single intraperitoneal injection of 130 mg/kg 2CE (approximately LD50) in mice caused severe liver damage and steatosis, along with increased ER stress and activation of the unfolded protein response (UPR) and autophagy. In H4IIEC3 rat hepatocytes, 2CE activated all three UPR pathways—IRE1, PERK, and ATF6—at both the gene and protein levels, and induced lysosomal accumulation, lipid droplet formation, and apoptosis. Among chemical chaperones tested, dimethyl sulfoxide (DMSO, 0.1–0.6%) showed the most potent therapeutic effects, reducing misfolded protein accumulation, alleviating ER stress, and suppressing apoptosis, even when autophagy was inhibited. These findings reveal that 2CE disrupts protein and lipid homeostasis in hepatocytes and highlight DMSO as a promising therapeutic agent for 2CE-induced toxicity. 2-chloroethanol Autophagy ER stress Unfolded protein response DMSO Chemical chaperone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Prior to regulations prohibiting its use, 2CE was directly used as a grape germination agent by farmers, leading to accidental poisoning (Deng et al. 2001 ). 2CE possesses a structure similar to ethanol, containing both alkyl chloride and alcohol functional groups. Its metabolism is believed to resemble that of ethanol, primarily involving oxidation to chloroacetaldehyde (CAA) by alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1), followed by further oxidation to chloroacetate (CA) by aldehyde dehydrogenase (ALDH) (Chen et al. 2010 ; Tang et al. 2019 ). 2CE is a contaminant of EO, which is used to sterilize medical equipment and fumigate grains. In the presence of environmental chloride ions, it produces 2CE as a contaminant (Cunliffe and Wesley 1967 ; Wesley et al. 1965 ). Furthermore, 2CE is a common metabolite of VC and 1,2-DCE, industrial chemicals known to cause liver diseases (Wang et al. 2021 ; Whysner et al. 1996 ). In recent years, VC or 1,2-DCE has been detected in groundwater near landfill sites, as well as in environmental and residential air, groundwater, and drinking water (Chen et al. 2015 ; Kielhorn et al. 2000 ). 2CE is implicated in the mechanisms underlying 1,2-DCE-induced toxicity, particularly in acute exposure scenarios (Sun et al. 2016 ), raising concerns about public exposure to these toxic substances. The ER is a critical organelle within liver cells, playing essential roles in protein and lipid synthesis, as well as detoxification (Alberts 2017 ). Maintaining ER integrity is crucial for liver health (Zhang et al. 2022 ). Exposure to hepatotoxic chemicals disrupts ER homeostasis, leading to increased ER stress and activation of the UPR (Deshmukh and Apte 2023 ). Several chemical toxins have been shown to trigger ER stress in metabolic organs, activating the three primary UPR pathways: PERK, IRE1, and ATF6 (Liang et al. 2023 ; Wang et al. 2022 ; Wang et al. 2023 ). Autophagy is subsequently activated, playing a key role in alleviating ER stress and influencing cell survival. Friedman et al. (Friedman et al. 1982 ) described a phenomenon resembling ER stress, involving the inhibition of hepatic protein synthesis and disaggregation of polysomes in response to 2CE. Recently, rapamycin, an autophagy activator, has been shown to alleviate 2CE-induced hepatic injury (Lang et al. 2019 ), suggesting a crucial role for both ER stress and autophagy in 2CE-induced liver injury. This study aimed to investigate the mechanisms of hepatic toxicity following acute exposure to 2CE and to explore potential therapeutic interventions. We found that acute exposure to 2CE induced severe hepatic steatosis in mice. The mechanism may be associated with the ER stress response induced by 2CE metabolism. To alleviate 2CE toxicity, we evaluated the effects of several chemical chaperones on 2CE-induced ER stress and identified DMSO as a uniquely effective therapeutic agent, potentially by maintaining ER homeostasis. These results provide a foundation for developing therapeutic strategies to counteract the adverse effects of 2CE and related compounds. Materials and methods Chemicals 2CE (#185744), DMSO (#D2650), chloroquine (#6628, CQ), 4-phenylbutyrate (#SML0309, 4-PBA), tauroursodeoxycholic acid (#580549, TUDCA), glycerol (#G6279) and trimethylamine N-oxide (#92277, TMAO) were purchased from Merck. BODIPY™ 493/503 (#D3922) and LysoTracker™ Red DND-99 (#L7528) were obtained from Thermo Fisher Scientific, Z-VAD-FMK (#FMK001) from R&D Systems, and Thioflavin T (#T17073, ThT) from TargetMol. Animal and Treatments Eight-week-old male Institute of Cancer Research (ICR) mice were acquired from BioLASCO Taiwan Co., Ltd. All animal experiments were conducted in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (Approval #20170452). After a one-week acclimatization period, the mice were randomly divided into two groups: the control group and the 2CE group. The 2CE group received a single IP injection of 130 mg/kg 2CE (LD50), while the control group received an equal volume of PBS. Twenty-four hours post-administration, five mice from each group were euthanized by isoflurane inhalation. Blood samples were collected via cardiac puncture. Serum was separated by centrifugation at 3000 x g for 15 minutes and subjected to biochemical analysis (aspartate aminotransferase, alanine aminotransferase, glucose, triglycerides, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol) using a Cobas c111 analyzer (Roche). For histological evaluations, liver samples were fixed overnight in 10% formalin and embedded in paraffin or frozen in optimal cutting temperature compound. For RNA-sequencing analysis, liver tissue from three additional animals per group was collected 7 hours post-treatment. Histopathological analysis Formalin-fixed liver samples were sectioned at a thickness of 4 µm and stained with hematoxylin and eosin (H&E). Oil Red O (ORO) staining was used to visualize lipid accumulation in frozen tissue sections. Briefly, sections were fixed with chilled 10% formalin for 10 minutes, rinsed with tap water, and then with 60% isopropanol. Subsequently, they were stained with 0.5% ORO (in 60% isopropanol) for 15 minutes, rinsed with 60% isopropanol, counterstained with hematoxylin for 3 minutes, and mounted. The sections were then examined under a light microscope (E600, Nikon). RNA-sequencing analysis Liver tissue was dissected, and total RNA was extracted using the TRIzol™ RNA isolation system (Thermo Fisher Scientific, 15596026). RNA-seq analysis was performed by BIOTOOLS Taiwan Co., Ltd. Briefly, RNA quality was assessed using the Qsep100 Analyzer from BiOptic Inc., Taiwan, to ensure sample integrity. Sequencing was performed on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads according to the manufacturer's instructions. Raw PE reads underwent quality control assessment using FastQC, followed by filtration using Trimmomatic (v0.38) to remove low-quality reads, trim adapter sequences, and eliminate poor-quality bases. The cleaned reads were aligned to the reference genome (Mus musculus, GRCm38) using HISAT2 (v2.1.0), and mapped reads were summarized to raw read counts using featureCounts (v2.0.0). To identify differentially expressed genes (DEGs) across sample groups, we utilized DESeq2 for relative log expression normalization. Genes with an absolute log fold change greater than 2 (|log2(FC)| > 1) and an adjusted P -value less than 0.05 were considered significantly differentially expressed. Data visualization was achieved through principal component analysis (PCA), volcano plots, and heatmaps. Pathway enrichment analysis was performed using g:Profiler (version: e111_eg58_p18_f463989d) with g:SCS multiple testing correction. A significance threshold of 0.05 was applied, and statistical significance was determined by considering all genes of the specified organism in the Ensembl database (Kolberg et al. 2023 ). Cell culture and 2CE treatment The rat liver cell line H4IIEC3 (CRL-1600) was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum, 20% horse serum, penicillin (50 units/ml, Gibco), streptomycin (50 µg/ml, Gibco), and 4 mM glutamine (Gibco) at 37°C in a humidified atmosphere containing 5% CO 2 . 2CE and DMSO were added to the cells at the indicated concentrations and incubation times following dilution in culture medium. CQ was dissolved in PBS and administered 1 hour prior to 2CE treatment. Western blotting Cells were washed with ice-cold PBS and lysed with radioimmunoprecipitation assay buffer (R0278, Sigma-Aldrich) supplemented with a protease inhibitor cocktail (ab201111, Abcam) and a phosphatase inhibitor cocktail (sc-45045, Santa Cruz). Lysates were rotated for 15 minutes at 4°C and then centrifuged at 16,000 x g for 30 minutes at 4°C. Protein concentration in the supernatants was determined using a bicinchoninic acid assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Protein samples (20 µg) were denatured in loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.005% bromophenol blue) at 95°C for 5 minutes and separated by electrophoresis on 10% or 12% SDS-polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at 25°C, followed by incubation with primary antibodies overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using a chemiluminescent HRP substrate kit (Millipore) and visualized with a BioSpectrum® imaging system. The following primary antibodies were used: anti-LC3B (#3868, Cell Signaling Technology), anti-phospho-eIF2α (Ser51, #3597, Cell Signaling Technology), anti-eIF2α (#2013, Cell Signaling Technology), anti-CHOP (#2895, Cell Signaling Technology), anti-ATF6 (#NBP1-40256, NOVUS), anti-PARP (#9542, Cell Signaling Technology), and anti-α-tubulin (T5168, Sigma-Aldrich). RT-qPCR and RT-PCR for XBP1 splicing Total RNA was extracted from cells using TRIzol™ reagent at the indicated time points. cDNA was synthesized from 1 µg of purified RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using a CFX Connect™ real-time PCR detection system (Bio-Rad). The specific primers used for each gene are listed in Supplementary Table S1 . The relative expression levels of target genes were determined using the comparative CT method with 18S rRNA as the endogenous control. RT-PCR for Xbp1 gene amplification was performed using a GeneAMP™ PCR System 9700 (Thermo Fisher Scientific). The following primers were used to amplify spliced and unspliced Xbp1 mRNA: 5’-CTCAGAGGCAGAGTCCAAGG-3’ and 5’-GGAAGATGTTCTGGGGAGGT-3’. α-Tubulin was used as an internal control. PCR products were separated by electrophoresis on 2.5% agarose gels and stained with DNA View (#TT-DNA01, TOOLS). DNA bands were visualized and quantified using the ProteinSimple AlphaImager HP system and ImageJ software (National Institutes of Health). BODIPY 493/503 and LysoTracker staining For neutral lipid droplet staining, cells were fixed with 4% paraformaldehyde (PFA) and subsequently stained with 2.5 µM BODIPY 493/503 (D3922, Thermo Fisher Scientific) for 1 hour at room temperature. After washing with PBS, cells were counterstained with Hoechst 33342. Stained cells were then mounted and imaged using an E600 fluorescence microscope (Nikon). For co-staining of lipid droplets and lysosomes, live cells were simultaneously incubated with 2.5 µM BODIPY™ 493/503 and 100 nM LysoTracker™ Red DND-99 (D3922, Thermo Fisher Scientific) for 30 minutes at 37°C. Following incubation, cells were washed and counterstained with Hoechst 33342. Images were acquired using a ZEISS LSM 880 Confocal Microscope equipped with a 63X oil-immersion objective lens. A total of four confocal Z-stacks were acquired with a step size of 0.4 µm. Maximum intensity projection images were generated and analyzed using ImageJ software (National Institutes of Health). Immunofluorescence staining Following drug treatment, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100. Cells were then blocked with 2.5% horse serum for 1 hour before incubation with primary antibodies against Lamp-1 (#sc-20011, Santa Cruz) and LC3B overnight at 4°C. The LC3 signal was amplified using the VectaFluor™ Excel Amplified Fluorescent Staining System (DK-1488, Vector), followed by incubation with appropriate secondary antibodies for 1 hour at room temperature. After washing, cells were counterstained with Hoechst 33342. Images were acquired using a LEICA SP8X Confocal Microscope equipped with a 100X oil-immersion objective lens. Cell viability Cells were seeded into six-well plates at a density of 7.5 x 10 5 cells per well and incubated overnight. 2CE and test drugs were added to the cells and incubated at 37°C for 24 hours. Subsequently, cells were trypsinized, stained with AccuStain solution (ADR-1000, NanoEnTek), and injected into AccuChips (AD4K-200, NanoEnTek). The number of viable cells was determined using an ADAM-MC automatic cell counter (Digital Bio). Cell viability of the untreated control group was considered as 100%. ThT staining Cells were grown on glass coverslips. Following treatment, the culture medium was supplemented with 10 µM ThT and incubated at 37°C for 40 minutes. Cells were then washed, fixed with 4% PFA for 20 minutes, and counterstained with Hoechst 33342. Coverslips were allowed to air-dry and mounted in aqueous mounting media. Images were acquired using a ZEISS Axio Observer Microscope equipped with a 40X objective lens. Statistical analysis Results are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using two approaches: Student's t-test (unpaired, two-tailed) for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for comparisons among multiple groups. Statistical significance was considered at a P -value of less than 0.05. Results 2CE induced hepatic steatosis in mice Twenty-four hours after a single IP injection of 2CE, mice exhibited severe hepatic steatosis (Fig. 1 A). Histological examination revealed marked cytoplasmic vacuolization and lipid droplet accumulation, as evidenced by H&E and ORO staining (Fig. 1 B-C). Serum biochemistry confirmed significant hepatic injury, characterized by elevated levels of liver enzymes, triglycerides, and cholesterol (Table S2 ). Notably, hypoglycemia was also observed, suggesting a potential disruption of glucose metabolism by 2CE. 2CE upregulated unfolding protein response (UPR) and autophagy genes in mice liver To investigate the role of ER stress in 2CE-induced liver injury, we performed RNA sequencing on mouse liver samples 7 hours post-2CE administration. PCA revealed a distinct separation between the 2CE and control groups in gene expression profiles (Fig. 2 A). Volcano plot analysis identified 1363 significantly upregulated genes and 1824 significantly downregulated genes (Fig. 2 B and Table S3 , P . adj < 0.05). Pathway enrichment analysis of biological process (BP) subcategories revealed significant enrichment in chaperone-mediated protein folding and protein refolding pathways (Fig. 2 C and Table S4 ). Notably, among the three UPR branches, the PERK pathway exhibited the most pronounced upregulation of key genes, including Atf4 , Ppp1r15a (Gadd34) , and Ddit3 (Chop) (Fig. 2 D). Given the established link between autophagy and ER stress (Gubas and Dikic 2022 ), we examined the expression of LC3/GABARAP family genes and observed consistent upregulation. Western blot analysis further confirmed the significant upregulation of LC3-II, phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and C/EBP homologous protein (CHOP) in liver protein extracts (Fig. 2 E-F), providing strong evidence for the activation of the UPR and autophagy pathways in response to 2CE exposure. 2CE induced UPR, autophagy and lipid accumulation in H4IIEC3 hepatoma cells To further investigate the cellular effects of 2CE, we utilized the rat liver cell line H4IIEC3 as a model system. After 24 hours of 2CE treatment, LC3-II protein levels were dose-dependently increased (Fig. 3 A). UPR markers, including p-eIF2α, CHOP, and cleaved ATF6, also exhibited an upward trend compared to the control. At the gene expression level, 2CE significantly induced the expression of ER stress-related genes (Map1lc3b (Lc3b), Hspa5, Eif2s1 (Eif2a), Atf4, Ppp1r15a (Gadd34), Ddit3 (Chop), Atf6, and Ern1 (Ire1)) in H4IIEC3 cells, consistent with the findings from animal experiments (Fig. 3 B). Additionally, 24 hours of 2CE treatment resulted in an increase in lipid droplet accumulation within the cytoplasm of H4IIEC3 cells, aligning with the acute hepatic steatosis observed in ICR mice exposed to 2CE (Fig. 3 C and 1 A). Chemical chaperone as an ER Stress modulator enhances cell viability following acute 2CE exposure Excessive ER stress can lead to diverse cellular damage and apoptosis. Several chemical chaperones have been identified as modulators of ER stress, effectively reversing stress-induced cellular damage. Among these, 4-PBA, TUDCA, glycerol, TMAO, and DMSO are the most extensively studied, with clinical applications in treating various diseases or serving as endogenous metabolites (Papp and Csermely 2006 ). Given that 2CE was shown to induce significant ER stress, we evaluated the protective effects of these chaperones against 2CE-induced cytotoxicity. While 4-PBA, TUDCA, glycerol, and TMAO exhibited no significant impact on cell viability (Figure S1 ), DMSO demonstrated a dose-dependent enhancement. Specifically, co-treatment with 0.1–0.6% DMSO and 16 mM 2CE resulted in an increase in cell viability from approximately 42–70% (Fig. 4 A). Similar improvements in cell viability were observed when cells were pre-treated with 0.3% DMSO one hour prior to or post-treated with DMSO two hours after 2CE exposure (Fig. 4 B). We further examined the effect of DMSO on modulating ER stress markers in cells exposed to 2CE. H4IIEC3 cells were treated with 16 mM 2CE, with or without 0.3% DMSO, for various time intervals. The results indicated that DMSO alleviated the phosphorylation of eIF2α and CHOP induction by 2CE (Fig. 4 C-D). Additionally, we investigated whether the key UPR regulator, Xbp1 , undergoes splicing into its active form ( Xbp1s ) following 2CE treatment. We observed that 2CE upregulated Xbp1s , an effect that was also mitigated by DMSO (Fig. 4 E-F). ER stress, often triggered by protein aggregation, disrupts cellular homeostasis (Ogen-Shtern et al., 2016). Using ThT staining, a gold-standard marker for protein aggregation (Arad et al., 2020), we evaluated the effect of DMSO on 2CE-induced protein aggregation. Significant protein aggregation around the nucleus was observed following 2CE treatment, which was alleviated by DMSO (Fig. 5 A-B). These findings suggest that DMSO reduces ER stress and the UPR by preventing protein aggregation. Furthermore, DMSO treatment reduced lipid droplet accumulation induced by 2CE exposure (Fig. 5 C-D). These results suggest that DMSO functions as an effective chemical chaperone, mitigating the cytotoxic effects of 2CE, likely through its modulation of ER stress. DMSO mitigated 2CE-induced dysregulated autophagic flux in H4IIEC3 cells The induction of autophagy by 2CE is characterized by a significant increase in LC3-II levels, prompting an investigation into the potential role of DMSO in modulating 2CE-induced autophagic flux. To evaluate the effects on lysosomal activity, we utilized LysoTracker™ to visualize lysosomes within H4IIEC3 cells. Exposure to 2CE led to markedly enlarged lysosomes, a hallmark of impaired autophagic flux, which was significantly alleviated by concurrent DMSO treatment (Fig. 6 A-B). To further evaluate the impact of DMSO on autophagy, we performed Western blot analysis for LC3-II and immunofluorescence staining to assess the co-localization of LC3B (autophagosome marker) and Lamp-1 (lysosomal marker). 2CE treatment significantly increased LC3B-Lamp-1 co-localization, reflecting enhanced autophagic flux. However, co-treatment with DMSO resulted in a reduction in LC3B-Lamp-1 co-localization (Fig. 6 C-D) and LC3-II conversion (Fig. 6 E-F), indicating that DMSO mitigated the dysregulated autophagic response induced by 2CE. These findings suggest that DMSO protects against 2CE-induced cytotoxicity by modulating upstream autophagic processes, thereby reducing LC3-II conversion and lysosomal enlargement. The therapeutic effects of DMSO may involve maintaining both ER and autophagy homeostasis Autophagy has been reported to improve ER stress and the UPR through ER-phagy (Gubas and Dikic 2022 ). To investigate the role of autophagic flux in DMSO-mediated cell survival, we inhibited autophagosome-lysosome fusion using CQ, which blocks autophagic flux. Despite CQ pre-treatment one hour prior to 2CE exposure, DMSO significantly improved cell viability and reversed eIF2α phosphorylation (Fig. 7 ). These results suggest that DMSO alleviates 2CE-induced ER stress independently of autophagic flux modulation. Interestingly, when autophagic flux was blocked by CQ, LC3 conversion and CHOP expression were inversely correlated with cell viability, suggesting that CHOP upregulation may result from CQ-induced toxicity. These findings suggest that DMSO enhances cellular survival by addressing ER stress upstream of the dysregulated autophagic flux induced by 2CE. Additionally, DMSO decreased p-eIF2α levels and the expression of apoptosis markers, including Caspase 3 and PARP (Fig. 7 B-C). To determine whether DMSO improves cell viability in 2CE-exposed cells by preventing ER stress-induced apoptosis, we used the pan-caspase inhibitor Z-VAD-FMK to block apoptosis. The effect of Z-VAD-FMK did not provide additional survival benefit beyond that of DMSO alone when used as the solvent (Figure S2 ), suggesting that DMSO improves cell viability by reversing ER stress-induced apoptosis in 2CE-exposed cells. Taken together, these results suggest that DMSO improves cell viability in 2CE-exposed cells by alleviating ER stress and modulating autophagic flux. Discussion Exposure to 2CE induces the generation of harmful metabolites, disrupting ER homeostasis and triggering a cascade of events: increased ER stress, UPR activation, and autophagy. These ultimately lead to cell death. Our findings emphasize the critical role of mitigating ER stress in countering these effects. Significantly, DMSO treatment alleviated ER stress and preserved cell viability, likely by acting as a chaperone-like molecule that assists protein folding and maintains ER function. This highlights the importance of targeting ER stress pathways to enhance cell survival under toxic conditions (Fig. 8 ). 2CE is a common metabolite of VC and 1,2-DCE, industrial chemicals known to cause liver diseases (Wang et al. 2021 ; Whysner et al. 1996 ). VC and 1,2-DCE have been detected in groundwater near landfill sites, as well as in environmental and residential air, groundwater, and drinking water, raising significant concerns about public exposure to these toxic substances. 2CE is believed to contribute to the toxic effects induced by 1,2-DCE (Sun et al. 2016 ). 2CE and its major metabolites, CAA and CA, have been shown to impair mitochondrial function and lead to cellular toxicity (Bhat et al. 1991 ; Sakai et al. 2005 ; Sood and O'Brien 1993 ). In addition to affecting cellular energy metabolism, 2CE has been shown to inhibit hepatic protein synthesis (Friedman et al. 1982 ) and the formation of halogenated fatty acids (Kaphalia and Ansari 1989 ). Furthermore, exposure to 2CE results in glutathione depletion, increased triglyceride levels, and the development of fatty liver in organisms (Andrews et al. 1983 ; Friedman et al. 1982 ). These perturbations can disrupt cellular protein and lipid homeostasis, hallmarks of ER stress (Mandl et al. 2013 ). Our results confirmed that 2CE induced ER stress, as evidenced by the upregulation of UPR markers and a significant accumulation of the autophagy marker LC3-II and lysosomes. This suggests that 2CE induces protein and lipid pathologies within the cell, prompting compensatory cellular responses, including the activation of the UPR and autophagy. 2CE is potently metabolized by ADH (Johnson 1967 ), and its toxicity is primarily driven by this enzymatic conversion. Alcohol serves as a competitive inhibitor of ADH, reducing 2CE's harmful effects (Johnson 1967 ). Current treatments for 2CE poisoning include 4-methylpyrazole (4MP), an ADH inhibitor, and N-acetylcysteine (NAC), an antioxidant, both of which have shown efficacy in laboratory studies (Chen et al. 2010 ). However, their optimal effectiveness relies on early administration, as delayed intervention, particularly after the conversion of 2CE to its toxic metabolite CAA, may exacerbate toxicity (Sood and O'Brien 1994 ). Despite the promise of these treatments, managing 2CE poisoning remains a clinical challenge, underscoring the need for timely intervention and further investigation into therapeutic strategies. Molecular chaperones play a crucial role in maintaining cellular protein homeostasis and ER stability, making them central to the study of protein-misfolding diseases (Chaudhuri and Paul 2006 ; Hendershot et al. 2024 ). Pharmacological chaperones are small, non-protein molecules specifically designed to bind to their target protein. In contrast, chemical chaperones are small molecules that non-specifically bind and stabilize proteins. Two major classes of chemical chaperones, osmolytes and hydrophobic compounds, function by sequestering water molecules or exploiting hydrophobic interactions to stabilize protein conformations (Arakawa et al. 2006 ; Sugiyama and Nishitoh 2024 ). Their ease of administration makes them a promising and widely applicable therapeutic strategy for treating a variety of protein-misfolding disorders. However, their mechanisms for modulating ER stress differ significantly. For instance, TUDCA mitigates ER stress by activating the PERK pathway, leading to the phosphorylation of eIF2α and subsequent upregulation of ATF4 expression. In contrast, 4-PBA induces eIF2α phosphorylation independently of PERK activation and results in minimal ATF4 expression. While neither significantly enhances ER chaperone activation, 4-PBA, unlike TUDCA, reduces rather than enhances cell viability under stress conditions like tunicamycin exposure and UV irradiation by promoting cell death through PARP cleavage. (Uppala et al. 2017 ). These observations demonstrate that different chemical chaperones modulate ER stress through distinct and context-dependent mechanisms. DMSO, in addition to being widely used as a solvent, also decreases protein aggregation. Numerous studies have demonstrated its capacity to stabilize mutant proteins, restore protein functionality, and reduce protein degradation caused by misfolding (Adsi et al. 2021 ; Bobak et al. 2016 ; Hwang et al. 2011 ; Kuribayashi et al. 2008 ; Lee et al. 2011 ; Simoes-Correia et al. 2008 ). Clinical evidence further supports DMSO's role in mitigating amyloid protein deposition (Amemori et al. 2006 ; Ravid et al. 1982 ; Regelson and Harkins 1997 ). For instance, in experiments with prion-infected mice, DMSO was found to prolong disease latency and reduce PrPSc accumulation (Shaked et al. 2003 ), highlighting its ability to alleviate protein misfolding disorders. Beyond its effects on protein misfolding, DMSO exhibits hepatoprotective properties against various toxic stimuli, including acetaminophen (Du et al. 2013 ; Yoon et al. 2006 ), chloroform (Lind and Gandolfi 1999 ), bromobenzene, carbon tetrachloride (Achudume 1991 ), and halothane (Lind and Gandolfi 1997 ). Notably, delayed administration of DMSO has also been shown to be beneficial, suggesting that it might provide a strategy for treating chemically induced liver injury (Lind et al. 2000 ; Lind and Gandolfi 1997 ). Furthermore, DMSO may also activate autophagy (Kang et al. 2017 ) and protect against fatty acid-induced hepatosteatosis by reducing protein aggregates and promoting autophagic responses (Song et al. 2012 ) and upregulating of heat shock proteins(Hallare et al. 2006 ; Yufu et al. 1990 ), thereby enhancing the cell's ability to manage the UPR. This broad spectrum of action suggests that DMSO does not inhibit specific chemicals or metabolic enzymes but rather alleviates common symptoms caused by toxic substances. Combined with its chaperone ability, DMSO has great potential to improve conditions in cases of toxic poisoning that lead to ER stress. Our study provides the first evidence that, among the chemical chaperones tested, DMSO uniquely modulates 2CE-induced ER stress and the autophagic response, leading to a significant decrease in hepatocyte death. Although the precise underlying mechanisms require further investigation, these findings offer a novel perspective for potential treatments of 2CE-induced toxicity and related chemical exposures. Additionally, this research provides valuable insights into therapeutic strategies for mitigating similar toxic effects caused by other substances. Conclusion Acute 2CE exposure caused hepatotoxicity, characterized by steatosis in ICR mice and cytotoxicity in H4IIEC3 cells. Mechanistically, 2CE activated the UPR and impaired autophagic flux, both contributing to cellular damage. Modulation of the UPR reduced 2CE-induced toxicity, while autophagy inhibition compromised these protective effects, highlighting the intricate interplay between these pathways. Among the chemical chaperones tested, DMSO uniquely enhanced cell survival by simultaneously alleviating ER stress and restoring autophagic homeostasis. These findings underscore the therapeutic potential of DMSO in mitigating 2CE-induced hepatotoxicity and provide new insights into its underlying mechanisms of action. Declarations Conflicts of interest The authors have no relevant financial or non-financial interests to disclose. Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Tzung-Hsin Chou, Min-Hsiu Hu and Kuo-Tai Hua. The first draft of the manuscript was written by Cheng-Chung Fang and Tzung-Hsin Chou. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This research was supported by grants from the National Taiwan University Hospital (grant numbers: 105 − 11, 105-S3178, 107-M4013, 108 − 15, 109 − 018, 110 − 20). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Tzung-Hsin Chou, Min-Hsiu Hu and Kuo-Tai Hua. The first draft of the manuscript was written by Cheng-Chung Fang and Tzung-Hsin Chou. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement We would like to express our sincere gratitude to the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital, for their invaluable technical support throughout this study. References Achudume AC. Effects of dimethyl sulfoxide (DMSO) on carbon (CCL4)-induced hepatotoxicity in mice. Clin Chim Acta. 1991;200:57-8. https://doi.org/10.1016/0009-8981(91)90335-a Adsi H, Levkovich SA, Haimov E, Kreiser T, Meli M, Engel H, Simhaev L, Karidi-Heller S, Colombo G, Gazit E, Laor Bar-Yosef D. Chemical Chaperones Modulate the Formation of Metabolite Assemblies. 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FEBS Letters. 1990;268:173-6. https://doi.org/https://doi.org/10.1016/0014-5793(90)81001-5 Zhang J, Guo J, Yang N, Huang Y, Hu T, Rao C. Endoplasmic reticulum stress-mediated cell death in liver injury. Cell Death Dis. 2022;13:1051. https://doi.org/10.1038/s41419-022-05444-x Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx FigureS2.docx TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.csv Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5876858","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406120761,"identity":"31d98d76-5d54-48b2-b155-39fa86c8d29c","order_by":0,"name":"Tzung-Hsin Chou","email":"","orcid":"","institution":"Department of Emergency Medicine, National Taiwan University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tzung-Hsin","middleName":"","lastName":"Chou","suffix":""},{"id":406120762,"identity":"b73de44f-a8b6-4e58-a87d-336b9dd65760","order_by":1,"name":"Min-Hsiu Hu","email":"","orcid":"","institution":"Department of Emergency Medicine, National Taiwan University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min-Hsiu","middleName":"","lastName":"Hu","suffix":""},{"id":406120767,"identity":"57a49c30-6246-4250-8ad7-1aa380a2c283","order_by":2,"name":"Kuo-Tai Hua","email":"","orcid":"","institution":"Graduate Institute of Toxicology, College of Medicine, National Taiwan University","correspondingAuthor":false,"prefix":"","firstName":"Kuo-Tai","middleName":"","lastName":"Hua","suffix":""},{"id":406120768,"identity":"ca476846-268e-4533-bc21-818e16ccb531","order_by":3,"name":"Cheng-Chung Fang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYNACNhsIzUOCljSIaiAhQayWwyRo4W/vPfbhR9n5PHuJBMYHb9sY6gwOENAiceZc8syec7eLeSQSmA3ntjFIENRiIJFjzMDbdjuxRyKBTZoXqMWMGC2Mf9vOgbSw/yZaCzNv2wGwLcxEaZE4c8aYWeZccmLPmYfNknPOSUjuJ6SFv73HmPFNmV1ie3vywQ9vymz4JRsIaEECjCC1xMbkKBgFo2AUjAK8AAAPUjj++G9s9QAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Emergency Medicine, National Taiwan University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Cheng-Chung","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2025-01-22 02:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5876858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5876858/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74909203,"identity":"c51d1869-976a-4877-9cc4-19542ae2b3a1","added_by":"auto","created_at":"2025-01-28 08:46:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211853,"visible":true,"origin":"","legend":"\u003cp\u003e2CE administration induces significant lipid accumulation in the liver of ICR mice. \u003cstrong\u003eA\u003c/strong\u003e Eight-week-old male ICR mice were IP injected with 130 mg/kg 2CE or PBS as a control. After 24 hours of exposure, the livers of 2CE-treated mice appeared noticeably paler compared to those of control mice. \u003cstrong\u003eB-C\u003c/strong\u003e Liver sections were stained with H\u0026amp;E or ORO. H\u0026amp;E staining revealed significant cytoplasmic vacuolization in the livers of 2CE-treated mice. ORO staining demonstrated pronounced lipid droplet accumulation (stained red) in the cytoplasm. Scale bar: 25 μm.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/a03ac926d4a79f7254f825e6.jpeg"},{"id":74909176,"identity":"9825a6c1-a3be-4558-a6ca-1c76eed3bf0a","added_by":"auto","created_at":"2025-01-28 08:46:42","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":291690,"visible":true,"origin":"","legend":"\u003cp\u003e2CE triggers ER stress rewiring in mice liver. \u003cstrong\u003eA\u003c/strong\u003e PCA of RNA-seq data revealed distinct clustering of liver samples from control and 2CE-treated mice. The analysis, based on three biological replicates per condition, clearly separated control (blue dots) and 2CE-treated (orange dots) samples. \u003cstrong\u003eB\u003c/strong\u003e Volcano plot of DEGs between control and 2CE-treated liver samples shows significant upregulation (red) and downregulation (blue) of genes in response to 2CE exposure. \u003cstrong\u003eC\u003c/strong\u003e Pathway enrichment analysis of upregulated DEGs using g:Profiler highlighted terms related to biological processes (BP), molecular function (MF), and cellular component (CC). \u003cstrong\u003eD\u003c/strong\u003e Heatmap showing the abundance changes of ER stress-related genes and LC3s observed in DEGs analysis between control and 2CE-treated liver samples. The color gradient indicates relative abundance, with green representing low and red representing high expression. *\u003cem\u003eP.\u003c/em\u003e\u003csub\u003e\u003cem\u003eadj \u003c/em\u003e\u003c/sub\u003e\u0026lt; 0.05, n = 3 biological replicates. \u003cstrong\u003eE-F\u003c/strong\u003e Western blot analysis was performed to detect changes in the expression levels of LC3-II, p-eIF2α, and CHOP in hepatic protein extracts from 2CE-treated mice. Densitometry analysis was used to quantify these changes. α-Tubulin served as the loading control. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, mean ± SEM, n = 3.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/7e0c4d8a16839767b0270f67.jpeg"},{"id":74909236,"identity":"281d84a4-306c-480a-b2c6-1c058442aea3","added_by":"auto","created_at":"2025-01-28 08:46:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":203692,"visible":true,"origin":"","legend":"\u003cp\u003e2CE induces autophagy, upregulates the UPR, and promotes lipid accumulation in rat liver cell line H4IIEC3. \u003cstrong\u003eA\u003c/strong\u003e H4IIEC3 cells were treated with 4, 8, 16, and 32 mM 2CE for 24 hours. Western blot analysis was performed to detect the autophagy marker LC3-II, UPR markers p-eIF2α, CHOP, and ATF6, and apoptosis markers PARP-C and Caspase 3-C (Cas 3-C). Protein expression levels were normalized to the control and are presented as fold change. \u003cstrong\u003eB\u003c/strong\u003e RT-qPCR was performed to determine the effect of 24 hours of 16 mM 2CE treatment on the mRNA levels of UPR genes, including Map1lc3b (Lc3b), Hspa5, Eif2ak3 (Perk), Eif2s1 (Eif2a), Atf4, Ppp1r15a (Gadd34), Ddit3 (Chop), Atf6, and Ern1 (Ire1), in H4IIEC3 cells. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, mean ± SEM, n = 3. \u003cstrong\u003eC\u003c/strong\u003e After fixation with 4% PFA, cells were stained with BODIPY 493/503 and Hoechst 33342 to visualize lipid droplets. Bright green spots represent lipid droplets, and blue indicates nuclei. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/723b00c9134d102e30210cd3.jpeg"},{"id":74909222,"identity":"5ccd8e45-afd1-440d-ba40-c5b044f5db1b","added_by":"auto","created_at":"2025-01-28 08:46:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220460,"visible":true,"origin":"","legend":"\u003cp\u003eDMSO increased cell viability following 2CE treatment and alleviated ER stress markers induced by 2CE. \u003cstrong\u003eA-B\u003c/strong\u003e H4IIEC3 cells were co-treated with 16 mM 2CE and 0.1-0.6% DMSO, or treated with 0.3% DMSO for varying durations. After 24 hours, cell viability was assessed using cell counting, with untreated cells serving as the 100% control. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3.\u003cstrong\u003e C\u003c/strong\u003eH4IIEC3 cells were treated with 16 mM 2CE with or without 0.3% DMSO for 1, 8, 16, and 24 hours. Western blot analysis was performed to detect expression changes in p-eIF2α and CHOP. \u003cstrong\u003eE\u003c/strong\u003e RT-PCR was conducted to examine changes in \u003cem\u003eXbp1s\u003c/em\u003e gene expression. \u003cstrong\u003eD, F\u003c/strong\u003eDensitometric analyses of Western blot and RT-PCR results were performed. Control values at each time point were normalized to 1, with α-tubulin used as the loading control. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/ca728e3a790c05443625531e.jpeg"},{"id":74910285,"identity":"8b9bd73c-2a4e-4f2e-b3fd-c76fb8ef971d","added_by":"auto","created_at":"2025-01-28 08:54:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":242284,"visible":true,"origin":"","legend":"\u003cp\u003eDMSO reduces the accumulation of unfolded proteins and lipids induced by 2CE. \u003cstrong\u003eA\u003c/strong\u003e H4IIEC3 cells were treated with 16 mM or 32 mM 2CE, with or without 0.3% DMSO for 24 hours, followed by ThT staining. Unfolded proteins were visualized as a green ring surrounding the nucleus (white arrow), with cell nuclei stained blue. Scale bar: 20 μm. \u003cstrong\u003eB\u003c/strong\u003e The experiment was independently conducted three times, with the number of fluorescence rings counted in each group based on 10-12 images. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 compared to the control group, and indicated groups (#\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Results are presented using a box and whisker plot, showing the range from minimum to maximum values. \u003cstrong\u003eC\u003c/strong\u003e H4IIEC3 cells were treated with 16 mM 2CE, with or without 0.3% DMSO. After 24 hours of treatment, lipid droplets were visualized using BODIPY 493/503 staining under confocal microscopy. Lipid droplets were stained green, and nuclei were counterstained with Hoechst (blue). Scale bar: 10 μm. \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of lipid droplet size was performed; the experiment was repeated three times, analyzing four images per group in each experiment. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/b540c6039287c9ce93876d19.jpeg"},{"id":74909205,"identity":"0670122f-63b8-45e2-ad95-7cc70d6bbb4c","added_by":"auto","created_at":"2025-01-28 08:46:44","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":288190,"visible":true,"origin":"","legend":"\u003cp\u003eDMSO mitigated 2CE-induced dysregulated autophagic flux in H4IIEC3 cells. \u003cstrong\u003eA\u003c/strong\u003e H4IIEC3 cells were treated with 16 mM 2CE, with or without 0.3% DMSO. After 24 hours of treatment, lysosomes were visualized using LysoTracker staining under confocal microscopy. Lysosomes were stained red and nuclei were counterstained with Hoechst (blue). Scale bar: 10 μm. \u003cstrong\u003eB\u003c/strong\u003e Quantitative analysis of lysosome size was performed; the experiment was repeated three times, analyzing four images per group in each experiment. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3. \u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence staining was performed to visualize the co-localization of the lysosomal marker Lamp-1 and the autophagic marker LC3B. Co-localization is indicated by yellow staining (highlighted by white arrows and enlarged in panel \u003cstrong\u003eD\u003c/strong\u003e). Scale bar: 2 μm. \u003cstrong\u003eE\u003c/strong\u003eH4IIEC3 cells were treated with 16 mM 2CE with or without 0.3% DMSO for 1, 8, 16, and 24 hours. Western blot analysis was performed to detect expression changes in the autophagy marker LC3B. \u003cstrong\u003eF\u003c/strong\u003e Densitometric analysis of Western blot results was performed, with control values at each time point set to 1. α-Tubulin was used as the loading control. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to control, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/96a560c9e0d4d2f2929e49ff.jpeg"},{"id":74910281,"identity":"5cda6b94-f449-4b9c-954a-266103a98044","added_by":"auto","created_at":"2025-01-28 08:54:44","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176912,"visible":true,"origin":"","legend":"\u003cp\u003eCQ does not eliminate the therapeutic effect of DMSO. \u003cstrong\u003eA\u003c/strong\u003e Following pre-treatment with the autophagy inhibitor CQ (10 μM) for 1 hour, cells treated with 16 mM 2CE and 0.3% DMSO were subjected to cell counting after 24 hours. Untreated cells served as the 100% baseline. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to the control group, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to 2CE; mean ± SEM, n = 3. \u003cstrong\u003eB-C\u003c/strong\u003e Western blot analysis and densitometry analysis of cellular proteins treated with CQ and 2CE with or without DMSO were performed to detect expression changes in LC3-II, p-eIF2α, CHOP, PARP-C, and Cas 3-C. The control group was set as 1, and α-tubulin was used as the loading control. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared to the control group and indicated groups (#\u003cem\u003eP\u003c/em\u003e or $\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Results are expressed as mean ± SEM, n = 3.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/8cabb3f6405bff1b6e0cfee1.jpeg"},{"id":74909211,"identity":"ed9d5aa4-ce82-4c41-a942-e007b7b4cf85","added_by":"auto","created_at":"2025-01-28 08:46:45","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":127477,"visible":true,"origin":"","legend":"\u003cp\u003eThis schematic illustrates the mechanism of 2CE-induced ER stress response and dysregulated autophagic flux, as well as the anticipated therapeutic effects of DMSO. 2CE triggers ER stress, activating the UPR, which subsequently leads to impaired autophagic flux. Excessive stress and autophagic dysfunction disrupt cellular homeostasis, ultimately resulting in apoptosis. During this process, lipid accumulation occurs, further exacerbating ER stress in a positive feedback loop. DMSO stands out among chemical chaperones, alleviating the aggregation of unfolded proteins and ER stress, while also mitigating 2CE-induced lipid accumulation, autophagic dysfunction, and apoptosis.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/a5f1b7df8bf19f975f91d4e2.jpeg"},{"id":78340650,"identity":"9d673ff3-5daa-4d54-9257-c73db3a10d79","added_by":"auto","created_at":"2025-03-12 08:38:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2641465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/c30c4f43-0aa2-4e9e-975b-ceed53e16dfb.pdf"},{"id":74909206,"identity":"9ec15af7-aa9a-4172-a48f-f78816d4f020","added_by":"auto","created_at":"2025-01-28 08:46:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":154986,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/da43ab6960f77f144f1493ea.docx"},{"id":74909178,"identity":"2feff606-5bc0-4090-a9d2-783365fe74c0","added_by":"auto","created_at":"2025-01-28 08:46:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":69518,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/92bdd912bf458a6c50a0d9bf.docx"},{"id":74909192,"identity":"62d94bc3-4ecb-40e9-a47f-16fb26bff84d","added_by":"auto","created_at":"2025-01-28 08:46:44","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11547,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/ae31493e417b5bc5a90f169f.xlsx"},{"id":74909224,"identity":"4524a5b6-6c59-41e8-935a-d6ed12df61a6","added_by":"auto","created_at":"2025-01-28 08:46:46","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11230,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/eb0674228e0da75e1322094e.xlsx"},{"id":74910286,"identity":"8e239af7-4f99-40d6-b942-62a535e997c4","added_by":"auto","created_at":"2025-01-28 08:54:45","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":837272,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/b51b71195a44486ee12d41aa.xlsx"},{"id":74909225,"identity":"01f58173-3730-4eed-a333-8839a52861c9","added_by":"auto","created_at":"2025-01-28 08:46:46","extension":"csv","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":32485,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-5876858/v1/778338bf87c6cbb1e5dd61cb.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"2-Chloroethanol Induces Hepatic Toxicity by Disrupting Endoplasmic Reticulum Homeostasis Ameliorated by Dimethyl Sulfoxide","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrior to regulations prohibiting its use, 2CE was directly used as a grape germination agent by farmers, leading to accidental poisoning (Deng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). 2CE possesses a structure similar to ethanol, containing both alkyl chloride and alcohol functional groups. Its metabolism is believed to resemble that of ethanol, primarily involving oxidation to chloroacetaldehyde (CAA) by alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1), followed by further oxidation to chloroacetate (CA) by aldehyde dehydrogenase (ALDH) (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2CE is a contaminant of EO, which is used to sterilize medical equipment and fumigate grains. In the presence of environmental chloride ions, it produces 2CE as a contaminant (Cunliffe and Wesley \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Wesley et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Furthermore, 2CE is a common metabolite of VC and 1,2-DCE, industrial chemicals known to cause liver diseases (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Whysner et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). In recent years, VC or 1,2-DCE has been detected in groundwater near landfill sites, as well as in environmental and residential air, groundwater, and drinking water (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kielhorn et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). 2CE is implicated in the mechanisms underlying 1,2-DCE-induced toxicity, particularly in acute exposure scenarios (Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), raising concerns about public exposure to these toxic substances.\u003c/p\u003e \u003cp\u003eThe ER is a critical organelle within liver cells, playing essential roles in protein and lipid synthesis, as well as detoxification (Alberts \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Maintaining ER integrity is crucial for liver health (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Exposure to hepatotoxic chemicals disrupts ER homeostasis, leading to increased ER stress and activation of the UPR (Deshmukh and Apte \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several chemical toxins have been shown to trigger ER stress in metabolic organs, activating the three primary UPR pathways: PERK, IRE1, and ATF6 (Liang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Autophagy is subsequently activated, playing a key role in alleviating ER stress and influencing cell survival. Friedman et al. (Friedman et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) described a phenomenon resembling ER stress, involving the inhibition of hepatic protein synthesis and disaggregation of polysomes in response to 2CE. Recently, rapamycin, an autophagy activator, has been shown to alleviate 2CE-induced hepatic injury (Lang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), suggesting a crucial role for both ER stress and autophagy in 2CE-induced liver injury.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the mechanisms of hepatic toxicity following acute exposure to 2CE and to explore potential therapeutic interventions. We found that acute exposure to 2CE induced severe hepatic steatosis in mice. The mechanism may be associated with the ER stress response induced by 2CE metabolism. To alleviate 2CE toxicity, we evaluated the effects of several chemical chaperones on 2CE-induced ER stress and identified DMSO as a uniquely effective therapeutic agent, potentially by maintaining ER homeostasis. These results provide a foundation for developing therapeutic strategies to counteract the adverse effects of 2CE and related compounds.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003e2CE (#185744), DMSO (#D2650), chloroquine (#6628, CQ), 4-phenylbutyrate (#SML0309, 4-PBA), tauroursodeoxycholic acid (#580549, TUDCA), glycerol (#G6279) and trimethylamine N-oxide (#92277, TMAO) were purchased from Merck. BODIPY\u0026trade; 493/503 (#D3922) and LysoTracker\u0026trade; Red DND-99 (#L7528) were obtained from Thermo Fisher Scientific, Z-VAD-FMK (#FMK001) from R\u0026amp;D Systems, and Thioflavin T (#T17073, ThT) from TargetMol.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal and Treatments\u003c/h3\u003e\n\u003cp\u003e Eight-week-old male Institute of Cancer Research (ICR) mice were acquired from BioLASCO Taiwan Co., Ltd. All animal experiments were conducted in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (Approval #20170452).\u003c/p\u003e \u003cp\u003eAfter a one-week acclimatization period, the mice were randomly divided into two groups: the control group and the 2CE group. The 2CE group received a single IP injection of 130 mg/kg 2CE (LD50), while the control group received an equal volume of PBS. Twenty-four hours post-administration, five mice from each group were euthanized by isoflurane inhalation. Blood samples were collected via cardiac puncture. Serum was separated by centrifugation at 3000 x g for 15 minutes and subjected to biochemical analysis (aspartate aminotransferase, alanine aminotransferase, glucose, triglycerides, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol) using a Cobas c111 analyzer (Roche). For histological evaluations, liver samples were fixed overnight in 10% formalin and embedded in paraffin or frozen in optimal cutting temperature compound. For RNA-sequencing analysis, liver tissue from three additional animals per group was collected 7 hours post-treatment.\u003c/p\u003e\n\u003ch3\u003eHistopathological analysis\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed liver samples were sectioned at a thickness of 4 \u0026micro;m and stained with hematoxylin and eosin (H\u0026amp;E). Oil Red O (ORO) staining was used to visualize lipid accumulation in frozen tissue sections. Briefly, sections were fixed with chilled 10% formalin for 10 minutes, rinsed with tap water, and then with 60% isopropanol. Subsequently, they were stained with 0.5% ORO (in 60% isopropanol) for 15 minutes, rinsed with 60% isopropanol, counterstained with hematoxylin for 3 minutes, and mounted. The sections were then examined under a light microscope (E600, Nikon).\u003c/p\u003e\n\u003ch3\u003eRNA-sequencing analysis\u003c/h3\u003e\n\u003cp\u003eLiver tissue was dissected, and total RNA was extracted using the TRIzol\u0026trade; RNA isolation system (Thermo Fisher Scientific, 15596026). RNA-seq analysis was performed by BIOTOOLS Taiwan Co., Ltd. Briefly, RNA quality was assessed using the Qsep100 Analyzer from BiOptic Inc., Taiwan, to ensure sample integrity. Sequencing was performed on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads according to the manufacturer's instructions. Raw PE reads underwent quality control assessment using FastQC, followed by filtration using Trimmomatic (v0.38) to remove low-quality reads, trim adapter sequences, and eliminate poor-quality bases. The cleaned reads were aligned to the reference genome (Mus musculus, GRCm38) using HISAT2 (v2.1.0), and mapped reads were summarized to raw read counts using featureCounts (v2.0.0). To identify differentially expressed genes (DEGs) across sample groups, we utilized DESeq2 for relative log expression normalization. Genes with an absolute log fold change greater than 2 (|log2(FC)| \u0026gt; 1) and an adjusted \u003cem\u003eP\u003c/em\u003e-value less than 0.05 were considered significantly differentially expressed. Data visualization was achieved through principal component analysis (PCA), volcano plots, and heatmaps. Pathway enrichment analysis was performed using g:Profiler (version: e111_eg58_p18_f463989d) with g:SCS multiple testing correction. A significance threshold of 0.05 was applied, and statistical significance was determined by considering all genes of the specified organism in the Ensembl database (Kolberg et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell culture and 2CE treatment\u003c/h3\u003e\n\u003cp\u003eThe rat liver cell line H4IIEC3 (CRL-1600) was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum, 20% horse serum, penicillin (50 units/ml, Gibco), streptomycin (50 \u0026micro;g/ml, Gibco), and 4 mM glutamine (Gibco) at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. 2CE and DMSO were added to the cells at the indicated concentrations and incubation times following dilution in culture medium. CQ was dissolved in PBS and administered 1 hour prior to 2CE treatment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were washed with ice-cold PBS and lysed with radioimmunoprecipitation assay buffer (R0278, Sigma-Aldrich) supplemented with a protease inhibitor cocktail (ab201111, Abcam) and a phosphatase inhibitor cocktail (sc-45045, Santa Cruz). Lysates were rotated for 15 minutes at 4\u0026deg;C and then centrifuged at 16,000 x g for 30 minutes at 4\u0026deg;C. Protein concentration in the supernatants was determined using a bicinchoninic acid assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Protein samples (20 \u0026micro;g) were denatured in loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.005% bromophenol blue) at 95\u0026deg;C for 5 minutes and separated by electrophoresis on 10% or 12% SDS-polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at 25\u0026deg;C, followed by incubation with primary antibodies overnight at 4\u0026deg;C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using a chemiluminescent HRP substrate kit (Millipore) and visualized with a BioSpectrum\u0026reg; imaging system. The following primary antibodies were used: anti-LC3B (#3868, Cell Signaling Technology), anti-phospho-eIF2α (Ser51, #3597, Cell Signaling Technology), anti-eIF2α (#2013, Cell Signaling Technology), anti-CHOP (#2895, Cell Signaling Technology), anti-ATF6 (#NBP1-40256, NOVUS), anti-PARP (#9542, Cell Signaling Technology), and anti-α-tubulin (T5168, Sigma-Aldrich).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRT-qPCR and RT-PCR for XBP1 splicing\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cells using TRIzol\u0026trade; reagent at the indicated time points. cDNA was synthesized from 1 \u0026micro;g of purified RNA using the iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using a CFX Connect\u0026trade; real-time PCR detection system (Bio-Rad). The specific primers used for each gene are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The relative expression levels of target genes were determined using the comparative CT method with 18S rRNA as the endogenous control.\u003c/p\u003e \u003cp\u003eRT-PCR for \u003cem\u003eXbp1\u003c/em\u003e gene amplification was performed using a GeneAMP\u0026trade; PCR System 9700 (Thermo Fisher Scientific). The following primers were used to amplify spliced and unspliced Xbp1 mRNA: 5\u0026rsquo;-CTCAGAGGCAGAGTCCAAGG-3\u0026rsquo; and 5\u0026rsquo;-GGAAGATGTTCTGGGGAGGT-3\u0026rsquo;. α-Tubulin was used as an internal control. PCR products were separated by electrophoresis on 2.5% agarose gels and stained with DNA View (#TT-DNA01, TOOLS). DNA bands were visualized and quantified using the ProteinSimple AlphaImager HP system and ImageJ software (National Institutes of Health).\u003c/p\u003e\n\u003ch3\u003eBODIPY 493/503 and LysoTracker staining\u003c/h3\u003e\n\u003cp\u003eFor neutral lipid droplet staining, cells were fixed with 4% paraformaldehyde (PFA) and subsequently stained with 2.5 \u0026micro;M BODIPY 493/503 (D3922, Thermo Fisher Scientific) for 1 hour at room temperature. After washing with PBS, cells were counterstained with Hoechst 33342. Stained cells were then mounted and imaged using an E600 fluorescence microscope (Nikon).\u003c/p\u003e \u003cp\u003eFor co-staining of lipid droplets and lysosomes, live cells were simultaneously incubated with 2.5 \u0026micro;M BODIPY\u0026trade; 493/503 and 100 nM LysoTracker\u0026trade; Red DND-99 (D3922, Thermo Fisher Scientific) for 30 minutes at 37\u0026deg;C. Following incubation, cells were washed and counterstained with Hoechst 33342. Images were acquired using a ZEISS LSM 880 Confocal Microscope equipped with a 63X oil-immersion objective lens. A total of four confocal Z-stacks were acquired with a step size of 0.4 \u0026micro;m. Maximum intensity projection images were generated and analyzed using ImageJ software (National Institutes of Health).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eFollowing drug treatment, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100. Cells were then blocked with 2.5% horse serum for 1 hour before incubation with primary antibodies against Lamp-1 (#sc-20011, Santa Cruz) and LC3B overnight at 4\u0026deg;C. The LC3 signal was amplified using the VectaFluor\u0026trade; Excel Amplified Fluorescent Staining System (DK-1488, Vector), followed by incubation with appropriate secondary antibodies for 1 hour at room temperature. After washing, cells were counterstained with Hoechst 33342. Images were acquired using a LEICA SP8X Confocal Microscope equipped with a 100X oil-immersion objective lens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eCells were seeded into six-well plates at a density of 7.5 x 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. 2CE and test drugs were added to the cells and incubated at 37\u0026deg;C for 24 hours. Subsequently, cells were trypsinized, stained with AccuStain solution (ADR-1000, NanoEnTek), and injected into AccuChips (AD4K-200, NanoEnTek). The number of viable cells was determined using an ADAM-MC automatic cell counter (Digital Bio). Cell viability of the untreated control group was considered as 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThT staining\u003c/h2\u003e \u003cp\u003eCells were grown on glass coverslips. Following treatment, the culture medium was supplemented with 10 \u0026micro;M ThT and incubated at 37\u0026deg;C for 40 minutes. Cells were then washed, fixed with 4% PFA for 20 minutes, and counterstained with Hoechst 33342. Coverslips were allowed to air-dry and mounted in aqueous mounting media. Images were acquired using a ZEISS Axio Observer Microscope equipped with a 40X objective lens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical comparisons were performed using two approaches: Student's t-test (unpaired, two-tailed) for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for comparisons among multiple groups. Statistical significance was considered at a \u003cem\u003eP\u003c/em\u003e-value of less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2CE induced hepatic steatosis in mice\u003c/h2\u003e \u003cp\u003eTwenty-four hours after a single IP injection of 2CE, mice exhibited severe hepatic steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Histological examination revealed marked cytoplasmic vacuolization and lipid droplet accumulation, as evidenced by H\u0026amp;E and ORO staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Serum biochemistry confirmed significant hepatic injury, characterized by elevated levels of liver enzymes, triglycerides, and cholesterol (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, hypoglycemia was also observed, suggesting a potential disruption of glucose metabolism by 2CE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2CE upregulated unfolding protein response (UPR) and autophagy genes in mice liver\u003c/h2\u003e \u003cp\u003eTo investigate the role of ER stress in 2CE-induced liver injury, we performed RNA sequencing on mouse liver samples 7 hours post-2CE administration. PCA revealed a distinct separation between the 2CE and control groups in gene expression profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Volcano plot analysis identified 1363 significantly upregulated genes and 1824 significantly downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, \u003cem\u003eP\u003c/em\u003e.\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05). Pathway enrichment analysis of biological process (BP) subcategories revealed significant enrichment in chaperone-mediated protein folding and protein refolding pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Notably, among the three UPR branches, the PERK pathway exhibited the most pronounced upregulation of key genes, including \u003cem\u003eAtf4\u003c/em\u003e, \u003cem\u003ePpp1r15a (Gadd34)\u003c/em\u003e, and \u003cem\u003eDdit3 (Chop)\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Given the established link between autophagy and ER stress (Gubas and Dikic \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we examined the expression of LC3/GABARAP family genes and observed consistent upregulation. Western blot analysis further confirmed the significant upregulation of LC3-II, phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and C/EBP homologous protein (CHOP) in liver protein extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F), providing strong evidence for the activation of the UPR and autophagy pathways in response to 2CE exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2CE induced UPR, autophagy and lipid accumulation in H4IIEC3 hepatoma cells\u003c/h2\u003e \u003cp\u003eTo further investigate the cellular effects of 2CE, we utilized the rat liver cell line H4IIEC3 as a model system. After 24 hours of 2CE treatment, LC3-II protein levels were dose-dependently increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). UPR markers, including p-eIF2α, CHOP, and cleaved ATF6, also exhibited an upward trend compared to the control. At the gene expression level, 2CE significantly induced the expression of ER stress-related genes (Map1lc3b (Lc3b), Hspa5, Eif2s1 (Eif2a), Atf4, Ppp1r15a (Gadd34), Ddit3 (Chop), Atf6, and Ern1 (Ire1)) in H4IIEC3 cells, consistent with the findings from animal experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, 24 hours of 2CE treatment resulted in an increase in lipid droplet accumulation within the cytoplasm of H4IIEC3 cells, aligning with the acute hepatic steatosis observed in ICR mice exposed to 2CE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChemical chaperone as an ER Stress modulator enhances cell viability following acute 2CE exposure\u003c/h2\u003e \u003cp\u003eExcessive ER stress can lead to diverse cellular damage and apoptosis. Several chemical chaperones have been identified as modulators of ER stress, effectively reversing stress-induced cellular damage. Among these, 4-PBA, TUDCA, glycerol, TMAO, and DMSO are the most extensively studied, with clinical applications in treating various diseases or serving as endogenous metabolites (Papp and Csermely \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Given that 2CE was shown to induce significant ER stress, we evaluated the protective effects of these chaperones against 2CE-induced cytotoxicity. While 4-PBA, TUDCA, glycerol, and TMAO exhibited no significant impact on cell viability (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), DMSO demonstrated a dose-dependent enhancement. Specifically, co-treatment with 0.1\u0026ndash;0.6% DMSO and 16 mM 2CE resulted in an increase in cell viability from approximately 42\u0026ndash;70% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similar improvements in cell viability were observed when cells were pre-treated with 0.3% DMSO one hour prior to or post-treated with DMSO two hours after 2CE exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e We further examined the effect of DMSO on modulating ER stress markers in cells exposed to 2CE. H4IIEC3 cells were treated with 16 mM 2CE, with or without 0.3% DMSO, for various time intervals. The results indicated that DMSO alleviated the phosphorylation of eIF2α and CHOP induction by 2CE (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Additionally, we investigated whether the key UPR regulator, \u003cem\u003eXbp1\u003c/em\u003e, undergoes splicing into its active form (\u003cem\u003eXbp1s\u003c/em\u003e) following 2CE treatment. We observed that 2CE upregulated \u003cem\u003eXbp1s\u003c/em\u003e, an effect that was also mitigated by DMSO (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eER stress, often triggered by protein aggregation, disrupts cellular homeostasis (Ogen-Shtern et al., 2016). Using ThT staining, a gold-standard marker for protein aggregation (Arad et al., 2020), we evaluated the effect of DMSO on 2CE-induced protein aggregation. Significant protein aggregation around the nucleus was observed following 2CE treatment, which was alleviated by DMSO (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). These findings suggest that DMSO reduces ER stress and the UPR by preventing protein aggregation. Furthermore, DMSO treatment reduced lipid droplet accumulation induced by 2CE exposure (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). These results suggest that DMSO functions as an effective chemical chaperone, mitigating the cytotoxic effects of 2CE, likely through its modulation of ER stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDMSO mitigated 2CE-induced dysregulated autophagic flux in H4IIEC3 cells\u003c/h2\u003e \u003cp\u003eThe induction of autophagy by 2CE is characterized by a significant increase in LC3-II levels, prompting an investigation into the potential role of DMSO in modulating 2CE-induced autophagic flux. To evaluate the effects on lysosomal activity, we utilized LysoTracker\u0026trade; to visualize lysosomes within H4IIEC3 cells. Exposure to 2CE led to markedly enlarged lysosomes, a hallmark of impaired autophagic flux, which was significantly alleviated by concurrent DMSO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e To further evaluate the impact of DMSO on autophagy, we performed Western blot analysis for LC3-II and immunofluorescence staining to assess the co-localization of LC3B (autophagosome marker) and Lamp-1 (lysosomal marker). 2CE treatment significantly increased LC3B-Lamp-1 co-localization, reflecting enhanced autophagic flux. However, co-treatment with DMSO resulted in a reduction in LC3B-Lamp-1 co-localization (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D) and LC3-II conversion (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F), indicating that DMSO mitigated the dysregulated autophagic response induced by 2CE. These findings suggest that DMSO protects against 2CE-induced cytotoxicity by modulating upstream autophagic processes, thereby reducing LC3-II conversion and lysosomal enlargement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eThe therapeutic effects of DMSO may involve maintaining both ER and autophagy homeostasis\u003c/h2\u003e \u003cp\u003eAutophagy has been reported to improve ER stress and the UPR through ER-phagy (Gubas and Dikic \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To investigate the role of autophagic flux in DMSO-mediated cell survival, we inhibited autophagosome-lysosome fusion using CQ, which blocks autophagic flux. Despite CQ pre-treatment one hour prior to 2CE exposure, DMSO significantly improved cell viability and reversed eIF2α phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggest that DMSO alleviates 2CE-induced ER stress independently of autophagic flux modulation. Interestingly, when autophagic flux was blocked by CQ, LC3 conversion and CHOP expression were inversely correlated with cell viability, suggesting that CHOP upregulation may result from CQ-induced toxicity. These findings suggest that DMSO enhances cellular survival by addressing ER stress upstream of the dysregulated autophagic flux induced by 2CE.\u003c/p\u003e \u003cp\u003eAdditionally, DMSO decreased p-eIF2α levels and the expression of apoptosis markers, including Caspase 3 and PARP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). To determine whether DMSO improves cell viability in 2CE-exposed cells by preventing ER stress-induced apoptosis, we used the pan-caspase inhibitor Z-VAD-FMK to block apoptosis. The effect of Z-VAD-FMK did not provide additional survival benefit beyond that of DMSO alone when used as the solvent (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting that DMSO improves cell viability by reversing ER stress-induced apoptosis in 2CE-exposed cells. Taken together, these results suggest that DMSO improves cell viability in 2CE-exposed cells by alleviating ER stress and modulating autophagic flux.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eExposure to 2CE induces the generation of harmful metabolites, disrupting ER homeostasis and triggering a cascade of events: increased ER stress, UPR activation, and autophagy. These ultimately lead to cell death. Our findings emphasize the critical role of mitigating ER stress in countering these effects. Significantly, DMSO treatment alleviated ER stress and preserved cell viability, likely by acting as a chaperone-like molecule that assists protein folding and maintains ER function. This highlights the importance of targeting ER stress pathways to enhance cell survival under toxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e2CE is a common metabolite of VC and 1,2-DCE, industrial chemicals known to cause liver diseases (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Whysner et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). VC and 1,2-DCE have been detected in groundwater near landfill sites, as well as in environmental and residential air, groundwater, and drinking water, raising significant concerns about public exposure to these toxic substances. 2CE is believed to contribute to the toxic effects induced by 1,2-DCE (Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2CE and its major metabolites, CAA and CA, have been shown to impair mitochondrial function and lead to cellular toxicity (Bhat et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sakai et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sood and O'Brien \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In addition to affecting cellular energy metabolism, 2CE has been shown to inhibit hepatic protein synthesis (Friedman et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) and the formation of halogenated fatty acids (Kaphalia and Ansari \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Furthermore, exposure to 2CE results in glutathione depletion, increased triglyceride levels, and the development of fatty liver in organisms (Andrews et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Friedman et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). These perturbations can disrupt cellular protein and lipid homeostasis, hallmarks of ER stress (Mandl et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our results confirmed that 2CE induced ER stress, as evidenced by the upregulation of UPR markers and a significant accumulation of the autophagy marker LC3-II and lysosomes. This suggests that 2CE induces protein and lipid pathologies within the cell, prompting compensatory cellular responses, including the activation of the UPR and autophagy.\u003c/p\u003e \u003cp\u003e2CE is potently metabolized by ADH (Johnson \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1967\u003c/span\u003e), and its toxicity is primarily driven by this enzymatic conversion. Alcohol serves as a competitive inhibitor of ADH, reducing 2CE's harmful effects (Johnson \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). Current treatments for 2CE poisoning include 4-methylpyrazole (4MP), an ADH inhibitor, and N-acetylcysteine (NAC), an antioxidant, both of which have shown efficacy in laboratory studies (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, their optimal effectiveness relies on early administration, as delayed intervention, particularly after the conversion of 2CE to its toxic metabolite CAA, may exacerbate toxicity (Sood and O'Brien \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Despite the promise of these treatments, managing 2CE poisoning remains a clinical challenge, underscoring the need for timely intervention and further investigation into therapeutic strategies.\u003c/p\u003e \u003cp\u003eMolecular chaperones play a crucial role in maintaining cellular protein homeostasis and ER stability, making them central to the study of protein-misfolding diseases (Chaudhuri and Paul \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hendershot et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Pharmacological chaperones are small, non-protein molecules specifically designed to bind to their target protein. In contrast, chemical chaperones are small molecules that non-specifically bind and stabilize proteins. Two major classes of chemical chaperones, osmolytes and hydrophobic compounds, function by sequestering water molecules or exploiting hydrophobic interactions to stabilize protein conformations (Arakawa et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugiyama and Nishitoh \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Their ease of administration makes them a promising and widely applicable therapeutic strategy for treating a variety of protein-misfolding disorders. However, their mechanisms for modulating ER stress differ significantly. For instance, TUDCA mitigates ER stress by activating the PERK pathway, leading to the phosphorylation of eIF2α and subsequent upregulation of ATF4 expression. In contrast, 4-PBA induces eIF2α phosphorylation independently of PERK activation and results in minimal ATF4 expression. While neither significantly enhances ER chaperone activation, 4-PBA, unlike TUDCA, reduces rather than enhances cell viability under stress conditions like tunicamycin exposure and UV irradiation by promoting cell death through PARP cleavage. (Uppala et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These observations demonstrate that different chemical chaperones modulate ER stress through distinct and context-dependent mechanisms.\u003c/p\u003e \u003cp\u003eDMSO, in addition to being widely used as a solvent, also decreases protein aggregation. Numerous studies have demonstrated its capacity to stabilize mutant proteins, restore protein functionality, and reduce protein degradation caused by misfolding (Adsi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bobak et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hwang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kuribayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Simoes-Correia et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Clinical evidence further supports DMSO's role in mitigating amyloid protein deposition (Amemori et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ravid et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Regelson and Harkins \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). For instance, in experiments with prion-infected mice, DMSO was found to prolong disease latency and reduce PrPSc accumulation (Shaked et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), highlighting its ability to alleviate protein misfolding disorders. Beyond its effects on protein misfolding, DMSO exhibits hepatoprotective properties against various toxic stimuli, including acetaminophen (Du et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yoon et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), chloroform (Lind and Gandolfi \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), bromobenzene, carbon tetrachloride (Achudume \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), and halothane (Lind and Gandolfi \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Notably, delayed administration of DMSO has also been shown to be beneficial, suggesting that it might provide a strategy for treating chemically induced liver injury (Lind et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Lind and Gandolfi \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Furthermore, DMSO may also activate autophagy (Kang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and protect against fatty acid-induced hepatosteatosis by reducing protein aggregates and promoting autophagic responses (Song et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and upregulating of heat shock proteins(Hallare et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yufu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), thereby enhancing the cell's ability to manage the UPR. This broad spectrum of action suggests that DMSO does not inhibit specific chemicals or metabolic enzymes but rather alleviates common symptoms caused by toxic substances. Combined with its chaperone ability, DMSO has great potential to improve conditions in cases of toxic poisoning that lead to ER stress.\u003c/p\u003e \u003cp\u003eOur study provides the first evidence that, among the chemical chaperones tested, DMSO uniquely modulates 2CE-induced ER stress and the autophagic response, leading to a significant decrease in hepatocyte death. Although the precise underlying mechanisms require further investigation, these findings offer a novel perspective for potential treatments of 2CE-induced toxicity and related chemical exposures. Additionally, this research provides valuable insights into therapeutic strategies for mitigating similar toxic effects caused by other substances.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAcute 2CE exposure caused hepatotoxicity, characterized by steatosis in ICR mice and cytotoxicity in H4IIEC3 cells. Mechanistically, 2CE activated the UPR and impaired autophagic flux, both contributing to cellular damage. Modulation of the UPR reduced 2CE-induced toxicity, while autophagy inhibition compromised these protective effects, highlighting the intricate interplay between these pathways. Among the chemical chaperones tested, DMSO uniquely enhanced cell survival by simultaneously alleviating ER stress and restoring autophagic homeostasis. These findings underscore the therapeutic potential of DMSO in mitigating 2CE-induced hepatotoxicity and provide new insights into its underlying mechanisms of action.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eContributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Tzung-Hsin Chou, Min-Hsiu Hu and Kuo-Tai Hua. The first draft of the manuscript was written by Cheng-Chung Fang and Tzung-Hsin Chou. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by grants from the National Taiwan University Hospital (grant numbers: 105\u0026thinsp;\u0026minus;\u0026thinsp;11, 105-S3178, 107-M4013, 108\u0026thinsp;\u0026minus;\u0026thinsp;15, 109\u0026thinsp;\u0026minus;\u0026thinsp;018, 110\u0026thinsp;\u0026minus;\u0026thinsp;20).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Tzung-Hsin Chou, Min-Hsiu Hu and Kuo-Tai Hua. The first draft of the manuscript was written by Cheng-Chung Fang and Tzung-Hsin Chou. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express our sincere gratitude to the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital, for their invaluable technical support throughout this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAchudume AC. Effects of dimethyl sulfoxide (DMSO) on carbon (CCL4)-induced hepatotoxicity in mice. Clin Chim Acta. 1991;200:57-8. https://doi.org/10.1016/0009-8981(91)90335-a\u003c/li\u003e\n\u003cli\u003eAdsi H, Levkovich SA, Haimov E, Kreiser T, Meli M, Engel H, Simhaev L, Karidi-Heller S, Colombo G, Gazit E, Laor Bar-Yosef D. Chemical Chaperones Modulate the Formation of Metabolite Assemblies. 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Cell Death Dis. 2022;13:1051. https://doi.org/10.1038/s41419-022-05444-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"2-chloroethanol, Autophagy, ER stress, Unfolded protein response, DMSO, Chemical chaperone","lastPublishedDoi":"10.21203/rs.3.rs-5876858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5876858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e2-Chloroethanol (2CE), a metabolite of ethylene oxide (EO), vinyl chloride (VC), and 1,2-dichloroethene (1,2-DCE), has an unclear toxic mechanism, complicating effective treatment of poisoning. This study examined the impact of acute 2CE exposure on endoplasmic reticulum (ER) homeostasis in liver cells. A single intraperitoneal injection of 130 mg/kg 2CE (approximately LD50) in mice caused severe liver damage and steatosis, along with increased ER stress and activation of the unfolded protein response (UPR) and autophagy. In H4IIEC3 rat hepatocytes, 2CE activated all three UPR pathways\u0026mdash;IRE1, PERK, and ATF6\u0026mdash;at both the gene and protein levels, and induced lysosomal accumulation, lipid droplet formation, and apoptosis. Among chemical chaperones tested, dimethyl sulfoxide (DMSO, 0.1\u0026ndash;0.6%) showed the most potent therapeutic effects, reducing misfolded protein accumulation, alleviating ER stress, and suppressing apoptosis, even when autophagy was inhibited. These findings reveal that 2CE disrupts protein and lipid homeostasis in hepatocytes and highlight DMSO as a promising therapeutic agent for 2CE-induced toxicity.\u003c/p\u003e","manuscriptTitle":"2-Chloroethanol Induces Hepatic Toxicity by Disrupting Endoplasmic Reticulum Homeostasis Ameliorated by Dimethyl Sulfoxide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 08:46:37","doi":"10.21203/rs.3.rs-5876858/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8924afde-3e2d-4dbd-bb79-a9cf609269aa","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-12T08:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-28 08:46:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5876858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5876858","identity":"rs-5876858","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}