Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project focuses on understanding the mechanisms of action and cross species comparisons from rodents to non human primates to humans for the volatile aromatic hydrocarbon, naphthalene. There are several important sources of human naphthalene exposure which range from tobacco smoke, to fossil fuels, to degassing of coal tar waste sites to jet fuel. In fact it is the latter source that has raised significant concerns in the US DOD as jet fuels are 1-3% by weight naphthalene and estimates of the cost of removal would be $1.5-6 billion annually. Adducted proteins recovered from incubations of nasal epithelium and 14C-naphthalene were separated by 2D gel electrophoresis and imaged to register radioactive proteins. Proteins visualized by silver staining on complimentary nonradioactive gels were identified by peptide mass mapping. The levels of reactive metabolite binding in incubations of Rhesus ethmo- and maxilloturbinate are similar to those in incubations of target tissues including rat septal/olfactory regions and murine dissected airway incubations. Forty one adducted spots were identified from 2D gel separations of rat olfactory epithelial proteins; 23 of these were nonredundant. In monkeys, 19 spots were identified by mass spectrometry yielding 3 nonredundant identifications. Structural proteins (actin/tubulin) were prominent targets in both species. These studies identified potential target proteins which may serve as markers closely associated with toxicity. The large differences previously reported between the rates of naphthalene metabolism to water soluble metabolites in dissected airways from mice and monkeys are not reflected in similar differences in covalent adduct formation in the nose. This raises concerns that downstream metabolic/biochemical events are very similar between the rat, a known target for naphthalene toxicity and tumorigenicity, and the Rhesus macaque, a species similar to the human. PARTICIPANTS: Christina DeStefano Shields-technical Staff Dexter Morin-technical staff Alan Buckpitt-PI TARGET AUDIENCES: US EPA and corresponding agencies in the state of California US DOD Naphthalene coalition including asphalt workers, major oil companies, EPRI PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Impact: The EPA has determined that in the absence of additional and significant additional data, they will regulate naphthalene as a probable human carcinogen. There are still several issues that should be resolved prior to this assessment as the cost of reformulating fuels will be substantial.
Publications
- DeStefano Shields, C., Morin, D. and Buckpitt, A. (2010)Formation of covalently bound adducts from the cytotoxicant, naphthalene in nasal epithelium: Species comparisons. Env Health Persp. In press.
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Progress 05/01/07 to 12/31/08
Outputs
OUTPUTS: The studies described in this document focus on the metabolism and downstream reactions of naphthalene, a volatile aromatic hydrocarbon that is the most prevalent PAH in the atmosphere. Earlier work has demonstrated dose/concentration dependent acute cytotoxicity in rodents to non ciliated airway epithelial cells (mice) and to nasal epithelial cells (mice and rats) which is dependent upon metabolic activation by the cytochrome P450 monooxygenases. Long term exposure results in a slight increase in alveolar adenomas in female mice and in dose dependent adenomas and neuroblastomas in the nasal epithelium of rats. Epidemiologic studies have thus far failed to provide definitive evidence associating naphthalene exposure with untoward effects in the human, a situation likely associated with the relatively high incidence of lung disease in the US population. Thus, a number of key questions remain to be answered with respect to whether naphthalene has the potential to produce human respiratory disease. The studies outlined here were done to: 1) determine whether 5-phenyl-1-pentyne, a suicide substrate inhibitor for CYP2F is capable of significantly inhibiting naphthalene metabolism in nasal olfactory epithelial microsomal incubations and 2) compare the metabolism of naphthalene to reactive metabolites which become bound covalently to proteins in susceptible species (rat) and to non human primates (Rhesus macaques), a species with a respiratory system anatomically similar to the human. The first set of studies were done in support of other investigators modeling the uptake of naphthalene vapor in the nasal passages of rats. Administration of 5-phenyl-1-pentyne intraperitoneally to either male or female rats at a dose of 100 mg/kg produced a striking decrease (to 20-25% of control) in the rates of naphthalene metabolism in nasal olfactory microsomal incubations 2 hrs after dosing. These studies show that this dose and time of administration adequately block metabolism in the nasal olfactory epithelium allowing studies conducted by Professor Jon Morris (University of Connecticut) to measure uptake rates without confounding metabolism. Naphthalene is metabolized in nasal olfactory epithelium of both rats and monkeys to reactive metabolites that become bound covalently to protein. The total reactive metabolite binding is very similar in rat and monkey nasal epithelium and is only slightly lower than in mouse airways. A number of proteins were identified as adducted from incubation of rat nasal epithelilal cells. In addition to high abundance proteins (actin, tubulin), stress proteins (heat shock proteins), proteins associated with cellular energy production and signaling were adducted. Of the proteins identified in similar incubations with Rhesus nasal tissue only structural proteins have been identified thus far (actin/tubulin). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
From these studies we conclude that the 100 fold differences previously reported between the rates of target (dissected airways from mouse) and comparable areas of the rhesus macaque are not reflected in similar differences in the rates of covalent protein adduct formation in the nose. This work raises concern that downstream metabolic and biochemical events are very similar in the rat nasal epithelium, a known target for naphthalene toxicity and tumorigenicity, and the Rhesus, a species similar to the human.
Publications
- No publications reported this period
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