Grantee Research Project Results
Final Report: Increased Vulnerability of Neonates to Naphthalene and Its Derivatives
EPA Grant Number: R827442Title: Increased Vulnerability of Neonates to Naphthalene and Its Derivatives
Investigators: Fanucchi, Michelle V.
Institution: University of California - Davis
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1999 through September 30, 2002
Project Amount: $374,543
RFA: Children's Vulnerability to Toxic Substances in the Environment (1999) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
The overall objective of this research project was to identify the factors critical to establishing whether children need greater protection from environmental contaminants that produce lung toxicity in adults. Currently, in the United States, many children grow up in urban environments that have a high potential for exposure to a large number of toxic compounds derived from both industrial and military sources. Many of these compounds require metabolic activation to generate cytotoxic and carcinogenic derivatives. Current standards for acceptable levels of these contaminants in the environment are based on assessments of risk to the adult population. Little information is available regarding the relative susceptibility of children to these compounds. This research project focused on the Clara cell, one of the cell types in pulmonary airways. The Clara cell is a progenitor cell and is the pulmonary cell most likely to be affected by bioactivated toxicants. The Clara cell continues to develop and differentiate during the postnatal period. How do exposures to environmental contaminants during this period of development affect the Clara cell? Is it more susceptible to toxicant injury than a fully differentiated Clara cell of an adult, and if so, what are the critical factors that create the increased susceptibility? This research project was designed to answer these questions using two species (mice and rats) and four different environmental toxicants (naphthalene [NA], 1-nitronaphthalene [NN], 2-methylnaphthalene, and 2-isopropylnaphthalene). This research project addressed the following issues: (1) whether the increased susceptibility of Clara cells in neonates is chemical- and/or species-specific; (2) whether the increase in injury in the immature Clara cell is due to a difference in the balance of activation and deactivation enzymes in favor of the activation pathways; and (3) whether the increase in injury to immature Clara cells is due to a decreased ability to regulate glutathione (GSH) pools.
Summary/Accomplishments (Outputs/Outcomes):
Morphometric Analysis of Acute Toxicity
Adult and 7-day-old male Swiss Webster mice and Sprague Dawley rats were injected intraperitoneally with 0, 12.5, 25, 50, or 100 mg/kg NA or NN in corn oil and killed 24 hours posttreatment. Lungs were evaluated by high-resolution light microscopy. In adult mice, a few vacuolated and swollen cells were detected in bronchioles after 50 mg/kg NA, while in neonatal mice, vacuolated and swollen cells were detected after 25 mg/kg NA. Injury progressed in a dose-dependent fashion for both ages. In both adult and neonatal rats, there was no detectable injury following any dose of NA. Injury was detected in adult mice after 100 mg/kg NN and in neonatal mice after 12.5 mg/kg NN. No neonatal mice survived to the 24-hour time point at higher doses. A few vacuolated cells were detected in both bronchi and bronchioles after 25 mg/kg NN in adult rats. In neonatal rats, vacuolated and exfoliated cells were detected in bronchi after 12.5 mg/kg NN. This injury also progressed in a dose-dependent manner. We also evaluated the acute toxicity of 2-methylnaphthalene in 7-day-old and adult Sprague Dawley rats. Rats were injected intraperitoneally with 0, 20, 35, 70, or 140 mg/kg 2-methylnaphthalene. As with the other two compounds, neonates were more susceptible to pulmonary injury by 2-methylnaphthalene than were adults. In the 7-day-old rat terminal bronchioles, there was a dose-dependent decrease in the thickness of the epithelium and the mass of nonciliated cells as well as a dose-dependent increase in the mass of squamous and vacuolated cells. This injury also occurred in the adult rats; however, the dose-response curve was shifted to the right. To summarize, the order of sensitivity (greatest to least) for NA is: 7D mice >> adult mice >> 7D rats = adult rats; for NN: 7D mice >> 7D rats > adult rats >> adult mice; and for 2-methylnaphthalene: adult mouse > 7D rats > adult rats. We concluded that neonatal susceptibility to environmental pollutants is species- and chemical-specific, but cannot be reliably predicted based on adult susceptibility.
Metabolic Potential of Neonatal and Adult Lungs
1-Nitronaphthalene Metabolism. We evaluated the metabolism of NN in specific airway compartments (trachea, lobar bronchus, major daughter airways, minor daughter airways, terminal bronchioles, and parenchyma) of adult rats as well as mice. The isolated airways were incubated with 0.5 mM NN for 2 hours and metabolites were evaluated by high-performance liquid chromatography (HPLC). Our metabolism data correlated well with the histopathological data. We found slightly more total metabolism from the neonatal rat airways as compared to the adult rat airways. There was more covalent binding present in the airways from neonatal rats as compared to the adult rats. In contrast to the rat data, airways from neonatal and adult mice metabolize NN at the same rate, with the same amount of covalent binding.
P450 Expression. Based on Western blotting of microdissected airways, we found that 7-day-old rats had a similar baseline expression of cytochrome P450 isozymes 2B1, 2F2, and 2E1. We did find that 7-day-old rats had less pulmonary P450 isozyme 3A protein expression than adult rats.
Detoxification Enzyme Expression. We compared the distribution of immunoreactive protein and enzymatic activity of glutathione S-transferases (GST) and epoxide hydrolases in isolated distal airways during pre- and postnatal development in the lungs of mice. GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels on postnatal day 28 and slightly lower levels in adults. Immunoreactive protein of isozyme pi has peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity, however, increases with increasing age. Microsomal epoxide hydrolase protein expression increased in intensity with age, while activity was similar in airways from all ages. We concluded that in the mouse lung, cellular expression of GST varies by age and does not increase with increasing age, airway GST activity increases with increasing age and does not correlate with immunoreactive protein expression, and microsomal epoxide hydrolase activity does not increase with increasing age, although immunoreactive protein expression does.
GSH Expression and Ability To Resynthesize Following Toxic Insult
Naphthalene. To assess the distribution of pulmonary GSH expression in neonatal and adult animals, adult, 14- and 7-day-old male mice were injected intraperitoneally with 0, 25, or 100 mg/kg NA in corn oil to initiate Clara cell injury and killed at 1, 2, 3, or 6 hours posttreatment. Airway epithelium was evaluated for GSH content at specific airway generations (1, 9, 14-15, and 20-22) using quantitative epifluorescent imaging microscopy and HPLC. Adult and neonatal animals contained similar baseline levels of GSH in all airway generations studied except in generation 9, where 7-day-old mice had significantly more GSH. Treatment with NA resulted in an airway-specific decrease in the amount of GSH present in all ages. In adults and 14-day-old mice, the greatest decrease was observed in airway generations 20-22. In 7-day-old mice, the greatest decrease was seen in airway generation 1. We conclude that differentiating Clara cells of neonatal mice do not modulate cellular GSH pools in response to NA treatment in the same manner as differentiated Clara cells in adults and that this difference may play a role in the heightened sensitivity of differentiating Clara cells to NA injury.
1-Nitronaphthalene. Adult and 7-day-old male rats were treated with 0 or 100 mg/kg NN in corn oil to initiate nasal and pulmonary injury and killed at 1, 2, 3, 6, or 24 hours post-treatment. Nasal and airway epithelium was evaluated for GSH content at specific sites (nasal septum, maxilloturbinate, nasoturbinate, ethmoid turbinate, trachea, proximal airway generations 1-9, and distal airway generations 14-22) using HPLC coupled with electrochemical detection. Adult and neonatal rats contained similar baseline levels of GSH in all sites studied, except in proximal airways, where 7-day-old rats had significantly more GSH than adult rats. Treatment with NN resulted in a site-specific decrease in the amount of GSH present in both ages. In adult rats, the greatest decrease was observed in the maxilloturbinate, while in 7-day-old rats, the greatest decrease was seen in the proximal airways. We concluded that nasal and pulmonary cells of neonatal rats do not modulate cellular GSH pools in response to NN treatment in the same manner as nasal and pulmonary cells in adult rats and that this difference may play a role in the heightened neonatal sensitivity to NN injury.
Conclusions:
We have found that neonatal susceptibility to environmental pollutants is species- and chemical-specific, but cannot be reliably predicted based on adult susceptibility. Neonatal susceptibility also cannot be reliably predicated based on any one facet of xenobiotic metabolism. We believe that the evaluation of neonatal susceptibility to bioactivated toxicants must utilize an integrative approach.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 10 publications | 5 publications in selected types | All 5 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Evans MJ, Van Winkle LS, Fanucchi MV, Plopper CG. Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 2001;27(5):401-415. |
R827442 (2001) R827442 (Final) |
|
|
Evans MJ, Fanucchi MV, Van Winkle LS, Baker GL, Murphy AE, Nishio SJ, Sannes PL, Plopper CG. Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone. American Journal of Physiology-Lung Cellular and Molecular Physiology 2002;283(6):L1263-L1270. |
R827442 (Final) |
Exit Exit |
|
Fanucchi MV, Buckpitt AR, Murphy ME, Storms DH, Hammock BD, Plopper CG. Development of phase II xenobiotic metabolizing enzymes in differentiating murine Clara cells. Toxicology and Applied Pharmacology 2000;168(3):253-267. |
R827442 (2000) R827442 (Final) |
Exit |
|
Plopper CG, Fanucchi MV. Do urban environmental pollutants exacerbate childhood lung diseases? Environmental Health Perspectives 2000;108(6):A252-A253. |
R827442 (2000) R827442 (Final) |
Exit |
|
Plopper CG, Buckpitt A, Evans M, Van Winkle L, Fanucchi M, Smiley-Jewell S, Lakritz J, West J, Lawson G, Paige R, Miller L, Hyde D. Factors modulating the epithelial response to toxicants in tracheobronchial airways. Toxicology 2001;160(1-3):173-180. |
R827442 (2001) R827442 (Final) |
Exit Exit |
Supplemental Keywords:
chemicals, histology, pathology, biochemistry., RFA, Health, Scientific Discipline, Toxics, Toxicology, Environmental Chemistry, Genetics, Health Risk Assessment, HAPS, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, Pathology, glutathione pools, health effects, neonates, sensitive populations, cytochrome P450, infants, vulnerability, health risks, second hand smoke, Naphthalene, airway disease, gene-environment interaction, respiratory problems, exposure, air pollution, high resolution histopathology, children, childhood respiratory disease, children's vulnerablity, lung dysfunction, susceptibility, high performance liquid chromatography, cigarette smoke, bioactivated environmental toxicants, biomedical research, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.