Protein Carbonyls in Bronchoalveolar Lavage Fluid in Mice, Rats and Hamsters Following Inhalation of Pigmentary Titanium Dioxide Particles

Abstract

Elevation of protein carbonyls has been implicated in the clinical setting as a result of oxidant damage associated with a number of disease states in both humans and laboratory animals. Protein carbonyls, the product of oxidative modification of amino acid residues, may result from macrophage and neutrophil inflammatory responses to inhaled particles. We hypothesized that increased levels of protein carbonyl groups in the bronchoalveolar lavage fluid (BALF) may serve as a biomarker of oxidative stress in rodents exposed to extremely high airborne concentrations of poorly soluble particles (PSP) of low toxicity. The objective of the present study was to compare the BALF protein carbonyl levels in three rodent species following a subchronic PSP exposure known to result in pulmonary pathology in chronically exposed rats under similar conditions. Female Fischer 344 rats, B6C3F₁ mice, and Syrian golden hamsters were identically exposed by whole-body inhalation to concentrations of aerosolized pigmentary titanium dioxide (TiO₂)(MMAD and GSD, 1.42 and 1.3 μm, respectively) for 6 h/day and 5 days/wk for 13 wk. Groups of animals were exposed to 0, 10, 50, or 250 mg/m³ of pigmentary TiO₂. Levels of protein carbonyl groups in BALF were measured at the termination of the 13-wk exposure with an ELISA assay utilizing a 2,4-dinitrophenylhydrazine fluorescent probe. Protein carbonyl levels were elevated in rats at both the mid and high dose (50 and 250 mg/m³), while in mice and hamster the levels were elevated only at the high dose (250 mg/m³). The elevations in protein carbonyl levels paralleled changes in BALF-associated cytologic and biochemical inflammatory indices, including total protein levels and neutrophil counts. Inflammatory changes in all three species were limited to animals exposed to the highest concentrations of particles. Rats were the only species tested that had coincidental elevation of both protein carbonyls and a high inflammatory response measured in BALF following the 50-mg/m³ exposure. These results suggest that the measurement of protein carbonyl groups in BALF may be a useful biomarker of particle-induced oxidant change, although this endpoint should be used in conjunction with other oxidative endpoints as a total assessment of oxidant stress.