Gel electrophoresis and multiplexed fluorescent imaging analysis for the measurement of protein thiol oxidation induced in murine airway epithelial cells after treatment with the Clara cell toxicant, naphthalene (NA) or the non cytotoxic thiol depletor, diethylmaleate (DEM)

Oxidative stress leading to alterations in airway cell protein thiol (PSH) status has been implicated in NA cytotoxicity. This study identified specific thiol containing proteins altered by NA or DEM. Murine airway epithelial PSH and formation of oxidized thiols (PSSP, PSSG, PSOH, PSO 3 ) in response to NA (200 mg/kg, ip) or DEM (1 g/kg, ip) was measured using multiplexed fluorescent 2D gel electrophoresis. Proteins were labeled with SH reactive Cy3, disulfides and sulfenic acids were reduced and labeled with Cy5. Proteins were separated to measure significant changes in the SH/oxidized ratios of each spot using the ratio of maleimide labels. NA and DEM caused significant changes in PSH of 82 proteins total compared to control (p<0.05). DEM oxidized more proteins to a greater extent than those recovered from murine airway epithelial samples after NA. However, several proteins containing reactive thiols are oxidized to a greater extent by NA compared to DEM including those involved in transcription regulation (zinc finger proteins), protein synthesis/folding (ribosomal proteins, serine peptidase inhibitor), cell growth/cycle control, and apoptosis (Suv39h2 Histone‐lysine N‐methyltransferase, Bcl2‐associated transcription factor 1). We conclude that specific proteins/protein pathways are disrupted by oxidation/arylation in response to NA that are less affected by nontoxic doses of DEM. NIEHS 04311/04699