Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

Abstract

Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, b-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.Keywords: thiol oxidation; bronchial epithelial cells; naphthalene; diethyl maleate; proteomics Naphthalene (NA) is a well-studied environmental pollutant that, in mouse models, selectively results in necrotic lesions of nonciliated bronchiolar epithelial cells (Clara cells) regardless of route of exposure (oral, intraperitoneal, or inhalation) (1, 2). Similar toxicity is not observed in rat lung, which raises the issue of potential susceptibility of the respiratory tract of humans. Widespread exposure of humans to NA has been documented. NA is a byproduct of combustion of wood and gasoline (3) and is a significant component of jet fuel (1-3%). High concentrations of NA and structural congeners are found in mainstream and side-stream tobacco smoke (4). As demonstrated recently, food may be a source of human exposure as well (5). The Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey conducted a study in 2001 and 2002 of 2,748 United States citizens to determine their exposure to polycyclic aromatic hydrocarbons (PAHs). NA metabolites were detected in 100% of the population examined, and, of the 22 hydroxy-PAH metabolites measured, naphthols accounted for 75% of the total PAHs detected (6). Epidemiologic studies have failed to establish a link between NA exposure and lung...

Proteomics in mechanistic toxicology: History, concepts, achievements, caveats, and potential

“…As previously discussed, some proteins/subsets of proteins are found at a high frequency in this area of toxicoproteomics of industrial products, illustrating their central role in response to toxic injury. HSP proteins (mostly hsp70 and hsp27) are found in 14 of 30 papers , . Mitochondrial proteins, most often matricial enzymes, are found in 14 of 30 papers .…”

“…Mitochondrial proteins, most often matricial enzymes, are found in 14 of 30 papers . More surprisingly, the single protein enolase is found in 13 of 30 papers . Peroxiredoxins are found, frequently, in 10 of 30 papers and proteasome subunits in 10 of 30 papers [– ].…”

Thiol redox proteomics seen with fluorescent eyes: The detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

“…In this case, a combination of two fluorophores is needed: the first one acting as the blocking agent and whose signal will be associated with reduced thiols; and the second one added after reduction of the sample and whose signal will be associated with oxidised thiols [9,[35][36][37]. The ratio between the reduced/oxidised thiols is then used to express the degree of oxidation in each sample [36,37]. It is important to note that the first fluorophore must be used at a concentration suitable for blocking and not just for labelling, so that free reduced thiol groups are not left unblocked and capable of being labelled with the second fluorophore [7].…”

“…One study in murine airway epithelial cells addressed the toxicity of naphthalene, a common air pollutant with toxic effects [37]. Reduced thiols were labelled with one fluorophore and following reduction, the newly reduced thiols were labelled with another.…”

“…Reduced thiols were labelled with one fluorophore and following reduction, the newly reduced thiols were labelled with another. The ratio between the two signals was used to identify proteins that were oxidised by naphthalene, which include metabolic enzymes, structural proteins and proteins associated with the unfolded protein response [37].…”