Superfund Research Program
Understanding the Mechanisms of Naphthalene-Induced Cytotoxicity
Background: Environmental health researchers are working to identify and quantify the relationships between exposure to toxic agents and resulting disease or dysfunction. This research is complicated by factors such as lack of reliable data on exposures; the reality of lifelong exposures to mixtures of contaminants; uncertainty with respect to the impact of chronic, low dose exposures; and difficulties in extrapolating from animal studies to human health impacts. One strategy to address these confounding issues is identification of molecular targets of environmental contaminants. Such information can clarify the mechanisms by which signal transduction pathways are perturbed and can provide meaningful biomarkers of exposure and effect.
Researchers at the University of California-Davis SBRP, led by Dr. Alan Buckpitt, are studying two model lung toxicants, naphthalene and nitronaphthalene. Both are inert compounds that require metabolic activation to cause cellular toxicity. Naphthalene and nitronaphthalene are combustion byproducts and are ubiquitous ambient air and groundwater pollutants, and naphthalene has been measured at high levels in ambient air samples during excavation, blending and treatment of materials at a Superfund site (manufactured gas facility). Naphthalene produces species- and tissue-selective injury, affecting nasal epithelium in both mice and rats, and affecting the pulmonary epithelium of mice but not rats. In comparison, nitronaphthalene is a more potent toxicant, and is not species- or tissue-selective, affecting nasal and pulmonary epithelium of both mice and rats.
Advances: Scientists in Dr. Buckpitt’s laboratory have conducted extensive studies to investigate the species- and tissue-selectivity of naphthalene cytotoxicity. Their approach relies on the development of sufficient information on the biochemical and metabolic events that lead to cytotoxicity. In the course of this work, Buckpitt’s team has developed new methods for preservation of the lung, allowing for transcriptome analysis of well-defined subcompartments. They have improved analysis of alterations in protein expression, and have developed and validated methods to selectively sample proteins from airway epithelium, facilitating observation of changes that occur in target epithelial cells challenged with toxicants.
Using these methods, Buckpitt’s laboratory has made several important discoveries. They demonstrated that inhalation of naphthalene vapors at levels at or above 2 ppm (that is, levels lower than the current OSHA 8-hour time-weighted standard of 10 ppm), leads to necrosis of mouse pulmonary bronchiolar epithelium cells.
The researchers focused on one of the cytochrome P450 proteins, CYP2F, which mediates the generation of cytotoxic metabolites of naphthalene and nitronaphthalene. They conducted comparisons of mouse and rat CYP2F, examining species-specific protein catalytic activity and ratios of metabolite stereoisomers. They found that the catalytic activity and stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. The researchers also conducted immunomapping studies to determine tissue-specific locations of protein activity and determined that, in rats, there is minimal CYP2F4 expression in the lungs. Together, these results indicate that the most important factor in determining species- and tissue-susceptibility to naphthalene injury is the level of CYP2F expression. Thus, the limited expression of CYP2F in the rat lung results in resistance to naphthalene-induced cytotoxicity.
Building on findings that naphthalene and nitronaphthalene toxicity is the result of their electrophilic metabolites covalently binding with proteins and interfering with cellular function, the UC-Davis researchers used proteomic approaches to identify airway epithelial proteins that are adducted by metabolites of nitronaphthalene in vivo. They identified a total 14 different protein spots representing 8 different gene products that were adducted by nitronaphthalene metabolites. Because protein adduct formation is not necessarily linked to toxicity, this study was designed to also provide information on the degree of adduction of specific proteins. The two most extensively adducted proteins (peroxiredoxin 6 and biliverdin reductase) are strongly associated with the first-line antioxidant defense of the cell.
Significance: The goals of this work are to develop biomarkers that are capable of indicating that an exposure to a lung toxicant has occurred and that the exposure is of sufficient duration and intensity to produce an adverse effect. To date, Dr. Buckpitt’s team has contributed important information for the understanding and assessment of the potential human health hazards of exposure to naphthalene and nitronaphthalene.
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To learn more about this research, please refer to the following sources:
- Baldwin RM, Shultz MA, Buckpitt AR. 2005. Bioactivation of the pulmonary toxicants naphthalene and 1-nitronaphthalene by rat CYP2F4. J Pharmacol Exp Ther 312(2):857-865. PMID:15509722
- Lee MG, Phimister AJ, Morin D, Buckpitt AR, Plopper CG. 2005. In situ naphthalene bioactivation and nasal airflow cause region-specific injury patterns in the nasal mucosa of rats exposed to naphthalene by inhalation. J Pharmacol Exp Ther 314(1):103-110. PMID:15833892
- Shultz MA, Zhang L, Gu Y, Baker GL, Fanucchi MV, Padua AM, Gurske WA, Morin D, Penn SG, Jovanovich SB, Plopper CG, Buckpitt AR. 2004. Gene expression analysis in response to lung toxicants: I Sequencing and microarray development. Am J Respir Cell Mol Biol 30(3):296-310. doi:10.1165/rcmb.2003-0214OC PMID:12947022
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