Table 1.

In Vivo 1,4-Dioxane Exposure Studies Investigating Oxidative Stress and Genotoxicity in the Liver

Animal Model	DX Exposure Regime	Main Findings	Reference
Female Sprague-Dawley rats	100 mg/kg in drinking water for 6 weeks	Liver cytotoxicity  • Histopathology (portal edema, Kupffer cells activation, small focal hepatic necrosis associated with inflammatory cells infiltration)  • Increased serum ALP Liver genotoxicity  • No data reported Oxidative stress  • Increased marker of lipid peroxidation (MDA)  • Reduced catalase activity	Mnaa et al. (2016) [22]
Male gpt delta transgenic F344 rats	0.2, 2, 20, 200, 1000 or 5000 ppm in drinking water for 16 weeks	Liver cytotoxicity  • No treatment-related histopathology Liver genotoxicity  • Increased A:T- to -T:A transversion frequency by 1000 ppm  • Increased gpt mutation frequency, A:T- to -G:C transition and A:T- to -T:A transversion frequencies by 5000 ppm  • Increased mRNA level of DNA repair enzyme MGMT by 5000 ppm Liver Proliferation/preneoplastic lesion  • Increased mRNA level of PCNA by 5000 ppm  • Increased GST-P-positive foci by 5000 ppm Oxidative Stress  • No change in mRNA levels of 12 CYP enzymes  • No difference in 8-OHdG levels (measured by HPLC-ECD)	Gi et al. (2018) [23]
Male WT F344 rats	2, 20, 200, 2000, or 5000 ppm in drinking water for 16 weeks	Liver cytotoxicity  • No treatment-related histopathology Liver Proliferation/preneoplastic lesion  • Increased GST-P-positive foci by 2000 and 5000 ppm  • Increased BrdU labeling index by 5000 ppm	Gi et al. (2018) [23]
Male WT F344 rats	20, 200, or 5000 ppm in drinking water for 16 weeks	(Liver samples from Gi et al. 2018 were used for analyses)Liver genotoxicity  • Identified three DNA adducts (measured by HPLC-QTOF-MS) characteristic of DX treatment by 200 and 5000 ppm Oxidative Stress  • 8-oxo-dG is one of the three treatment-specific DNA adducts by 200 and 5000 ppm	Totsuka et al. (2020) [24]
Female BDF-1 mice	50, 500, and 5000 ppm in drinking water for 1 or 4 weeks	Liver cytotoxicity  • No gross histopathological change Liver genotoxicity  • Increased γH2AX-positive hepatocytes (DNA damage marker) by 5000 ppm at 1 and 4 weeks Oxidative Stress  • DEGs (measured by RNASeq analysis) enriched in GSH-mediated detoxification and NRF2-mediated oxidative stress response pathways by 5000 ppm at 4 weeks  • DAMs (measured by metabolomics analysis) enriched in metabolic pathways of nucleotide salvage synthesis and oxidative stress response by 5000 ppm at 4 weeks	Charkoftaki et al. (2021) [12]
Male B6 GclmKO and WT mice	1000 mg/kg/day by oral gavage for 1 week or 5000 ppm in drinking water for 12 weeks	Liver cytotoxicity (no difference between GclmKO and WT mice)  • Mild histopathology (single cell death and associated inflammation, ductal reactive change, and steatosis) at 12 weeks  • 2-fold increase in plasma ALT and AST activities at 12 weeks Liver genotoxicity (enhanced in GclmKO relative to WT mice)  • Increased 8-OHdG levels (by ELISA assay) at 12 weeks  • Elevated γH2AX/H2AX ratio (DNA damage repair marker) at 12 weeks Oxidative Stress (enhanced in GclmKO relative to WT mice)  • Transient induction of some NRF2-target antioxidant genes at 1 week  • Reduced GSH/GSSG ratio (index of oxidative stress) at 12 weeks  • Progressive induction of CYP2E1 and lipid peroxidation	Chen et al. (2022) [13]
Male B6 Cyp2e1KO mice	5000 ppm in drinking water for 1 week or 12 weeks	Liver cytotoxicity  • No treatment-related histopathology at 12 weeks  • No change in plasma ALT and AST activities at 12 weeks Liver genotoxicity  • Trend of increase in 8-OHdG levels (by ELISA assay) at 12 weeks  • Decreased γH2AX/H2AX ratio (DNA damage repair marker) at 12 weeks Oxidative Stress  • No induction of NRF2-targeted antioxidant genes  • No change in GSH/GSSG ratio (index of oxidative stress)  • No induction of lipid peroxidation	Wang et al. (Manuscript in preparation)

Abbreviations: 8-OHdG, 8-hydroxy-2′-deoxyguanosine; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate aminotransferase; BrdU, bromodeoxyuridine; CYP, cytochrome P450; CYP2E1, cytochrome P450 2E1; DAMs, differentially abundant metabolites; DEGs, differentially expressed genes; DX, 1,4-dioxane; ELISA, enzyme-linked immunosorbent assay; Gclm, glutamate-cysteine ligase modifier subunit; GSH, glutathione; GSSG, glutathione disulfide; GST-P, glutathione S-transferase placental-form; H2AX, H2A histone family member X; γH2AX, phosphorylation of the Ser-139 residue of H2AX; HPLC-ECD, high performance liquid chromatography with electrochemical detection; HPLC-QTOF-MS, high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer; MDA, malondialdehyde; MGMT, O6-methylguanine-DNA methyltransferase; NRF2, nuclear factor erythroid 2-related factor 2; PCNA, proliferating cell nuclear antigen; WT, wild type.