Progress 08/01/98 to 07/31/04
Outputs
The cancer bioassay studies in mice showed a slight increase in bronchiolar alveolar adenomas and metaplasia of the nasal olfactory epithelium in response to naphthalene. As a result of these findings, an additional bioassay in rats was conducted and the data have been considered at the IARC meeting held in February 2002. Naphthalene was listd as a class 2B compound by the committee. This review underscored the need to conduct comparative metabolism studies in nasal epithelium. This review also supported the need to find out whether these compounds are toxic to primate lungs.
Impacts
In this project we explored the metabolism and cytotoxicity of naphthalene, 1-nitro-naphthalene and 4-ipomeanol in primate lungs in vivo and in vitro. We also cloned and expressed the major P450 isoforms from the monkey to determine their catalytic capability.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Studies in rodents have demonstrated the importance of cytochrome P450 monooxygenases in generating reactive metabolites which produce Clara cell injury. Pulmonary P450 activities in primates are much lower than those in rodents raising the issue of human relevance of rodent data. Few studies on P450 catalyzed activation of cytotoxicants in subcompartments of primate lung have been reported. Accordingly, monkey airway subcompartments including the trachea, proximal, medial, respiratory airways and parenchyma were incubated with naphthalene or 1-nitronaphthalene to define metabolism at both high (0.5 mM) and low (0.05 mM) substrate concentrations. There was a relatively even distribution of metabolizing activities for naphthalene across subcompartments but, at high concentrations of nitronaphthalene, more distal airways (medial through parenchyma) showed higher turnover. Dihydrodiol was the predominant water soluble metabolite of naphthalene generated at all airways
levels while covalently bound metabolites accounted for the greatest percentage of 1-nitronaphthalene metabolites. As anticipated, the amounts of metabolite covalently bound as a percentage of total metabolite formed increased dramatically with the 10 fold increase in substrate concentration. With both substrates, the rates of formation of water soluble metabolites were approximately 100 times less than those observed previously in rodents. We conclude that: 1there are significant quantitative differences between Rhesus and rodents in substrate turnover, 2 the distribution of metabolizing activities for naphthalene but not 1-nitronaphthalene are significantly different for rodents and primates, 3 a very high percentage of the metabolites generated, particularly for 1-nitronaphthalene,are bound covalently to cellular proteins.
Impacts
Our finding that dissected airways of Rhesus macaques metabolize naphthalene and 1-nitronaphthalene at rates which are 70-100 fold less than in rodents would, on first analysis, suggest that humans are not susceptible to these chemicals. However, the finding that a significant portion of the overall metabolism is directed at generating reactive, covalently bound metabolites is a concern
Publications
- Boland, B., Lin, C-Y, Morin, D., Plopper, C.G. and Buckpitt, A.: Site specific metabolism of naphthalene and 1-nitronaphthalene in dissected airways of Rhesus macaques. Submitted, expected 2004
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Progress 01/01/02 to 12/31/02
Outputs
The cancer bioassay studies in mice showed a slight increase in bronchiolar alveolar adenomas and metaplasia of the nasal olfactory epithelium in response to naphthalene. As a result of thee findings, an additional bioassay in rats has been completed and the report has been released recently. There was hyperplasia of the nasal olfactory epithelium and nasal tumors in both sexes which were dose dependent. the work conducted in the past year showing that CYP2F2 has substantially higher catalytic turnover than other nasal P450 monooxygenases tested, suggests that this may be an important P450 in the nose. This along with the studies showing that the atalytic activities of mouse and rat CYP2F proteins are highly similar and that the differences between mouse and rat susceptibility to naphthalene are likely related to differences in the amounts of protein present in epithelial cells of mouse vs rat suggest that understanding the quantities of these proteins in both lung
and nasal epithelium will be critical to determining whether naphthalene (and similar agents) is a health risk in humans.
Impacts
This project will help to determine whether naphthalene (and similar agents) is a health risk in humans.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The cancer bioassay studies in mice showed a slight increase in bronchiolar alveolar adenomas and metaplasia of the nasal olfactory epithelium in response to naphthalene. As a result of these findings, an additional bioassay in rats has been completed and the report has been released recently. There was hyperplasia of the nasal olfactory epithelium and nasal tumors in both sexes which were dose dependent. The work conducted in the past year showing that CYP2F2 has substantially higher catalytic turnover than other nasal P450 monooxygenases tested, suggests that this may be an important P450 in the nose. This along with the studies showing that the catalytic activities of mouse and rat CYP2F proteins are highly similar and that the differences between mouse and rat susceptibility to naphthalene are likely related to differences in the amounts of protein present in epithelial cells of mouse vs rat suggest that understanding the quantities of these proteins in both lung
and nasal epithelium will be critical to determining whether naphthalene (and similar agents) is a health risk in humans. It will be important to demonstrate that there are no catalytic differences between mouse and human/primate for these arguments to remain valid and this will be one of the issues we intent to focus on in the coming year.
Impacts
Nononcogenic pulmonary diseases are the third leading cause of death in the US and are a major factor in morbidity and disability. Although cigarette smoking is a major etiologic factor in these diseases, exposure to chemicals in the workplace and the environment may be important as well. This project will help to establish if rodent lung toxicants metabolically activated by P450 also produce focal injury in human lung.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Briefly, the research is designed to test three hypothesis: 1) the lung of the Rhesus macaque, which is anatomically and cellularly similar to human lung, is an appropriate surrogate for the human in evaluating chemical induced lung toxicity, 2) the cellular distribution of cytochrome P 450 monooxygenases in human and monkey is highly focal making the cells containing these proteins exquisitely sensitive to cytotoxicants requiring metabolic activation by this system, and 3) the catalytic activities of pulmonary cytochrome P450 monooxygenases in monkeys are similar to the human and dissimilar to rodents. We have optimized the expression of CYP2F4 (rat) in the baculovirus expression system and have established incubation conditions which yield maximal turnover of substrate. Maximal turnovers of 240/min have been obtained. Based on the data available so far, it appears that the differences between rats and mice in sensitivity toward naphthalene and derivatives is not
related to differences in the catalytic activity of the two proteins. Studies reported previously showed that naphthalene is metabolized with relatively high turnover (104/min) and low Km (3.7 microM). The focus of some of our work during the past year has been to expand some of these studies to include additional substrates to determine whether CYP2F2 was potentially important in the unique sensitivity of the mouse to a number of metabolically activated cytotoxicants including 2-methylnaphthalenes and 1-nitronaphthalene. In addition, we have examined the metabolism of larger polycyclic aromatic hydrocarbons by this protein including anthracene and benzo(a)pyrene. In all cases, epoxides were trapped with glutathione. Theoretically, 2-methylnaphthalene can be metabolized to 12 separate glutathione conjugates. Of those, 9 have been isolated from incubations of mouse liver microsomes in the presence of GSH and GST. All have been characterized as glutathione conjugates by mass
spectrometry. Of the 9 metabolites formed in liver microsomal incubations, 5 were generated by cytochrome P450 2F2. The rates of formation of these five metabolites were used to assess the catalytic activities of recombinant 2F2 with 2-methylnaphthalene. Kinetic studies indicated relatively high Kcat (67.6 min-1) and low Km (3.7 microM). Earlier work has shown that 1-nitronaphthalene is metabolized to both the 5,6- and the 7,8-epoxides. Glutathione conjugates derived from these epoxides have been fully characterized by both mass spectrometry and NMR spectroscopy. The ratio of epoxides generated at the 7,8-position to the 5,6- position by cytochrome P450 2F2 was 5:1. Cytochrome P450 2F2 also metabolizes 1-nitronaphthalene with relatively high turnover (17.1/min) and low Km (21.5 microM). Although anthracene and benzo(a)pyrene are both metabolized by cytochrome P450 2F2, the rates of metabolism of these two substrates are considerably lower than those of the naphthalene based substrates
(anthracene = 0.14 nmole/nmole/min. and benzo(a)pyrene = 0.04 nmole/nmole/min.) This work has provided detailed insights into the catalytic capabilities of cytochrome P450 2F2.
Impacts
The cancer bioassay studies in mice showed a slight increase in bronchiolar alveolar adenomas and metaplasia of the nasal olfactory epithelium in response to naphthalene. These findings supported the need to conduct the bioassay in another species- the rat. These studies have shown that there is a dose dependent increase in tumors of the lung and olfactory epithelium in rats been completed, the relevance for the human will still not be clear. In other work supported by the institute, we have found that mice exposed to naphthalene by inhalation are highly susceptible to the cytotoxicity of this compound. Exposures at concentrations well below the current occupational standard produce marked Clara cell necrosis. The goal of the work conducted with support from this program is to examine the cytochrome P450 monooxygenases in rodents, monkeys, and humans. The primary conclusion of the work conducted this past year indicates that the difference in susceptibility between
mice and rats is not related to striking differences in the catalytic capability of the P450 monooxygenases responsible for the metabolic activation of naphthalene. The work also shows that P450 2F2 may be of major importance in determining the unique sensitivity of the mouse to number of metabolically activated pulmonary cytotoxicants.
Publications
- Shultz, M., Choudary, P. and Buckpitt, A.: Role of murine CYP2F2 in the metabolic activation of naphthalene and in the metabolism of other xenobiotics. J. Pharmacol. Exp. Ther. 290: 281-288, 1999.
- Shultz, M.A., Chang, A., Morin, D. and Buckpitt, A.:Metabolic Capabilities of CYP2F2 with various pulmonary toxicants and its relative abundance in mouse lung sub-compartments. J. Pharmacol. Exp. Ther., in press (2001).
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Progress 01/01/99 to 12/31/99
Outputs
We have conducted 6 metabolism experiments with both naphthalene and 1-nitronaphthalene in airways obtained from Rhesus macaques. Work over the past year has focused primarily on characterizing the catalytic activities of P450 isoforms from the mouse, the rat and the monkey. P450 2F2 (mouse) has been expressed in Ti ni cells where 2F2 accounts for up to 20 percent of the total cellular proteins. Cytochrome P450 2F4 (rat) has been cloned, sequenced and expressed in Ti ni cells using a baculovirus expression system. While our original hypothesis was that the catalytic activity of P450 2F4 would be strikingly different from P450 2F2, it turns out that the catalytic activity of the recombinant rat enzyme is nearly identical to cytochrome P450 2F. We have begun studies to examine the distribution of P450 2F2 in mouse lung and to determine what percentage of the total P450 is represented by this protein
Impacts
Our findings this year indicate that the difference in susceptibility between mice and rats is not related to striking differences in the catalytic capability of the P450 monooxygenase responsible for the metabolic activation of naphthalene. The work also shows that P450 2F2 may be of major importance in determining the unique sensitivity of the mouse to number of metabolically activated pulmonary cytotoxicants.
Publications
- Schulz M, Choudary P and Buckpitt A. Role of murine CYP1F2 in the metabolic activation of naphthalene and in the metabolism of other xenobiotics. 1999. J Pharmacol Exp. Ther. In press.
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