Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label-free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 (http://proteomecentral.proteomexchange.org/dataset/PXD000846).

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is an antibacterial agent used in personal care products such as bar soaps. Small amounts of chemical are absorbed through the epidermis. Recent studies show that residues of reactive TCC metabolites are bound covalently to proteins in incubations with keratinocytes, raising concerns about the potential toxicity of this antimicrobial agent. To obtain additional information on metabolic activation of TCC, this study characterized the reactive metabolites trapped as glutathione conjugates. Incubations were carried out with (14)C-labeled TCC, recombinant CYP1A1 or CYP1B1, coexpressed with cytochrome P450 reductase, glutathione-S-transferases (GSH), and an NADPH-generating system. Incubations containing CYP1A1, but not 1B1, led to formation of a single TCC-GSH adduct with a conversion rate of 1% of parent compound in 2 hours. Using high-resolution mass spectrometry and diagnostic fragmentation, the adduct was tentatively identified as 3,4-dichloro-3'-glutathionyl-4'-hydroxycarbanilide. These findings support the hypothesis that TCC is activated by oxidative dehalogenation and oxidation to a quinone imine. Incubations of TCDD-induced keratinocytes with (14)C-TCC yielded a minor radioactive peak coeluting with TCC-GSH. Thus, we conclude that covalent protein modification by TCC in TCDD-induced human keratinocyte incubations is mainly caused by activation of TCC by CYP1A1 via a dehalogenated TCC derivative as reactive species.