UC Irvine

Titanium dioxide (TiO2) nanoparticles are among the most widely manufactured nanomaterials, and inhaled particles can translocate from the lung into the systemic circulation, where they contact erythrocytes (red blood cells), the most abundant circulating cell. Despite this, erythrocytes remain an overlooked systemic target in the assessment of nanomaterial toxicity. Therefore, the main objective of this study was to characterize the concentration-dependent hematotoxicity of rutile TiO2 nanoparticles in human erythrocytes, and to determine whether cells that survive exposure carry sublethal membrane injury and functional impairment. We hypothesized that rutile TiO2 nanoparticles induce erythrocyte toxicity, characterized by premature cell death and hemoglobin impairment, through oxidative stress and ATP depletion, and increase erythrocyte susceptibility to secondary stressors. Erythrocytes isolated from 23 individual donors were exposed in vitro to rutile TiO2 nanoparticles (5–200 µg/mL; 15–20 nm grain size) for 24 hours. Hemolysis, eryptosis, intracellular oxidative stress and ATP were quantified by flow cytometry, spectrophotometry, and bioluminescence, and hemoglobin species and osmotic fragility were assessed in the surviving cells by multiwavelength spectrophotometry and graded hypotonic challenge. Concentration-response relationships were modeled by normalizing each sample’s responses to its own control and fitting a pooled constrained four-parameter logistic model. Exposure induced concentration-dependent hemolysis and eryptosis across all samples, accompanied by increased oxidative stress and intracellular ATP depletion. Eryptosis was the more sensitive endpoint, increasing at lower concentrations and more steeply than hemolysis. The cells that survived exposure were not unaffected: they showed more fragile membranes, indicating persistent sublethal injury despite remaining intact. Total hemoglobin and oxyhemoglobin declined across the exposed population, reducing its oxygen-carrying capacity, while the erythrocytes that remained carried a higher proportion of functional oxyhemoglobin and lower proportions of methemoglobin and carboxyhemoglobin, suggesting preferential loss of the cells with the most oxidized hemoglobin. Responses varied substantially between samples in both magnitude and slope, revealing considerable biological variability in cellular response. Applying standard uncertainty factors to the lower confidence bound of the eryptosis benchmark concentration resulted in an at-cell protective threshold of 0.16 µg/mL. These findings identify the erythrocyte as a direct and sensitive systemic target for TiO2 nanoparticles and demonstrate that assessment based on hemolysis alone can underestimate a nanomaterial’s hematotoxicity hazard. The results support integrating eryptosis and membrane fragility into future hazard assessment of blood-contacting nanomaterials and highlight the importance of accounting for inter-individual variability in health-protective threshold setting. This study uses rutile TiO2 as an example, but the approach and findings can be extended to other inhaled nanomaterials in the workplace.