Molecular dosimetry of hemoglobin adducts in mice exposed to ethylene oxide

Ethylene oxide (EtO) is a widely used industrial compound with known carcinogenic potential in humans. Due to its high reactivity and short biological half-life, occupational exposure assessments (> 1 ppm) rely on the detection of stable biomarkers, such as N-(2-hydroxyethyl)-L-valine (HE-V), formed as hemoglobin adducts in blood. Existing analytical methods for HE-V detection often require large volumes of blood and purified hemoglobin due to limited sensitivity, restricting their application in characterizing the dose-response relationship between EtO exposure and HE-V accumulation-particularly at low environmental exposure levels relevant to assessing potential general population health risks. In this study, we aimed to characterize the molecular dosimetry of HE-V formation in B6C3F1 mice exposed by whole body inhalation to a broad range of concentrations of EtO: 0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm, 7 days/week for 4 weeks. To achieve this, we developed a sensitive LC-MS-based workflow for HE-V quantification, incorporating hemoglobin purification, HE-V release plus enrichment, and targeted mass spectrometric detection from as little as 10 μL of blood and 50 μg of extracted hemoglobin. A clear, dose-dependent increase in HE-V levels was observed following EtO exposure, with statistically significant elevations detected even at 0.05 ppm compared to endogenous background levels. At lower concentrations (0.5 to 1 ppm), HE-V levels increased linearly with dose, while higher concentrations (50 to 200 ppm) exhibited an upward-bending (increasing slope) dose response. No sex-specific differences were observed. Taken together, these findings indicate EtO exhibits linear systemic toxicokinetics at lower exposures that transition to nonlinear toxicokinetics in the range of higher exposures (likely due to saturation of glutathione-mediated detoxification), thus providing new quantitative insights to support improved risk assessments and toxicological evaluations of EtO exposure.