Gender differences in naphthalene metabolism and naphthalene-induced acute lung injury

Van Winkle, LS, Gunderson, AD, Shimizu, JA, Baker, GL, Brown, CD.
Am J. Physiol Lung Cell Mol Physiol 282: L1122-L1134, 2002.

The purpose of this experiment was to determine whether or not the the process of naphthalene metabolism differs between male and female mice. The authors were specifically interested in determining if female mice are more susceptible to a single dose of naphthalene than male mice. Clara cells were the focus of the study because they contain Cytochrome P-450, already known to metabolize naphthalene in male mice. This experiment is unique for two reasons: 1) Female and not just male mice were used 2) The acute response of the lung to naphthalene was studied rather than the long-term response. The acute response is defined as changes occurring between 1 and 24 hours after exposure.

The authors note that the National Academy of Sciences has requested more research on gender-based differences as well as the use of more female subjects in research. This request is, in part, based on the observation of an increasing incidence of lung cancer in women compared to men. Lung cancer incidence in men appears to have become level in recent years. In addition, women tend to have more adenocarcinomas, which occur in the peripheral lung than do men. The current hypothesis for these findings is that females are inherently more susceptible than men to the components of tobacco smoke. Naphthalene is a PAH, a product of incomplete combustion and ubiquitous in the environment. One study has demonstrated increased incidence of naphthalene-induced broncho-alveolar lung adenomas in female but not male mice. The authors feel that examining the very early changes in the lungs after naphthalene exposure may explain the mechanism by which the later differences in naphthalene-induced adenomas occurred. In particular they are looking for gender related differences in susceptibility to the acute cytotoxicity of naphthalene.

Methods:
Animals and experimental design: Male and female Swiss-Webster mice were used. It should be noted that naphthalene was injected i.p. and not administered directly to the lung. The experiment was of a serial sacrifice design with sacrifices at 1,2,3,6, and 24 hr. post-treatment. The lungs were prepared either for standard 1 um light microscope histology studies or studies of cell permeability. Ethidium homodimer-1 labeled the nuclei of any cells with permeable membranes.

Techniques used: Quantitative histopathology was used to determine the number of normal and cytotoxic cells in the intrapulmonary lobar bronchus. The ratio of permeable to impermeable cells in certain airway levels was mapped with fluorescent microscopy and the morphology of the permeable cells studied. Measurement of cytochrome P-450 was used to assess naphthalene metabolism in microdissected airways. The metabolic products of naphthalene, naphthalene dihydrodiol and glutathione conjugate 2 were also measured with immunoblotting. Immunohistochemistry was used to assess the degree of differentiation marker proteins. Laser capture microdissection and RNA extraction, transcription and quantitation were also employed in this experiment.

Results:

The authors give detailed results with respect to location of morphological, histochemical and biochemical findings. Although not stated in the paper, this is important because naphthalene is known to be metabolized specifically by Clara cells. Clara cells in turn are found in specific, known locations in the mouse lung. Thus by focusing attention on these cells, findings are much more specific than they would be if all lung cells and larger or smaller airways were examined as well. The manuscript itself should be consulted for extensive photos of micrographs of the techniques used and the appearance of both normal and injured Clara cells.

In summary, the histological changes occurring between 1 and 24 hrs after naphthalene injection are described in both male and female mouse lung. It is noted that epithelial cells in the female are smaller than in the males. There are more permeable cells in the females and at airway bifurcations. Permeable cells in the males are found in the distal terminal bronchiole. There is no difference in CYP2F2 abundance in terminal bronchioles of male and female mice. Naphthalene metabolism is rated as greatest in female terminal bronchioles, next in male terminal bronchioles, next in female minor daughter bronchioles, and least in male minor daughter bronchioles.

Discussion:

The authors conclude that female mice are more susceptible than male mice to the same dose of naphthalene. The injury is earlier and the damaged cells are farther up the respiratory tree. The pattern of histological changes is different in male and female mice. Possible metabolic differences indicate that females produce more total products of naphthalene metabolism and more of the total product is naphthalene dihydrodiol. These metabolic differences could lead to the gender difference in sensitivity to naphthalene.

One obvious difference between male and female mice that may have a bearing on the results is the apparent difference in size of female mouse cells. The authors also speculate on many other possible reasons for increased susceptibility of female mice to naphthalene including different intracellular mechanisms of toxicity, differences in distribution of susceptible cells, and other biochemical differences in the metabolism of naphthalene. They note such theories as that of the "sentinel" cell at airway bifurcations. They also emphasize the need for studies of the effects of hormones on regulation of the CYP2F2 protein especially because young mice are known to be much more sensitive than adult mice to naphthalene.

In conclusion, the authors conclude that female mice are more susceptible to naphthalene toxicity than are males. These differences are related to differences in metabolism and to different patterns of airway damage between the genders as demonstrated in this manuscript. It is emphasized that gender differences should be considered when studying epithelial injury and repair in the lung.

By: Susan G. Shami, ScD, Senior Science Editor

Editorial comment: There is no doubt that it will be difficult to study the effects of inhaled particles and chemicals on the lung as well as the rest of the body in both male and female animals. However the results of this study as well as other studies reported on www.inhalation.net show clear gender differences in certain situations (See Gender and enhanced sensitivity to odors, Odor, Irritation and Perception of Health Risk. If the results of most of the studies in the fields of inhalation toxicology and respiratory biology are to be ultimately applied to findings in humans, it seems that gender differences must be addressed. The authors gave a good review of gender differences in lung disease in the manuscript, but even greater gender differences are known to exist in other human diseases. For example, autoimmune diseases are almost exclusively the misfortune of women as are some cancers.

By: Susan G. Shami, ScD

18 May 03