Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

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

Efflux of Naphthalene Oxide and Reactive Naphthalene Metabolites from Isolated Hepatocytes1

by Renton et at. (1978) and Klaunig et at. (1981). Briefly, the mouse was treated i.v. 30 mm before the start of surgery with 100 U of heparin. The animal was anesthetized with ether, the abdomen and chest cavity opened and a cannula inserted Vol. 242 into the vena cava through the right atrium. To determine quantitatively the rate oftransfer of naphthalene 1,2-oxide across intact hepatocyte membranes, a method had to be developed that was capable of trapping naphthalene oxide on the exterior of the cell. The approach used in the current studies is summarized i

Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

<div><p>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.</p></div

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

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, β-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA

Overview of naphthalene metabolism and excretion.

<p>(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.</p

Extracted ion chromatograms of urinary naphthalene metabolites and deuterated internal standards.

<p>(A) QC Sample #2. Naphthalene mercapturate, N-acetyl glutathione conjugate, naphthol glucuronide, and naphthol sulfate at 40, 5, 70, 20 ng on column, respectively. (B) Pooled urine sample from day 6–7 of exposure treatments (n = 3 animals). Calculated amounts of each metabolite on column were: 124.8, 7.9, 45.3, 25.9 ng for mercapturic acid, N-acetyl GSH, naphthol glucuronide and naphthol sulfate, respectively.</p

Naphthalene metabolite-specific percentages of total quantified metabolite during repeated naphthalene exposure treatments (15 ppm x 4hrs daily) via inhalation.

<p>Naphthalene metabolite-specific percentages of total quantified metabolite during repeated naphthalene exposure treatments (15 ppm x 4hrs daily) via inhalation.</p

Parameters for LC/ESI-MS/MS analysis of urinary naphthalene metabolites and internal standards (IS).

<p>^a Molecular ion, [M-H]^-, acquired in negative ion ESI.</p><p>^bTo increase signal, product ion intensities were summed for quantification.</p><p>^cCollision energy used to produce product ions.</p><p>Parameters for LC/ESI-MS/MS analysis of urinary naphthalene metabolites and internal standards (IS).</p

Metabolic Activation of the Antibacterial Agent Triclocarban by Cytochrome P450 1A1 Yielding Glutathione Adducts

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