Humans are widely exposed to polycyclic aromatic hydrocarbons, commonly found in cigarette smoke and diesel exhaust. These can undergo site- and cell-specific metabolism to cytotoxic intermediates. Metabolism of naphthalene and Clara cell cytotoxicity have been extensively studied in male animals. To address whether male and female mice are equally susceptible to naphthalene, mice were injected with naphthalene, and lungs were examined 1, 2, 3, 6, and 24 h after treatment. By analysis of acute injury using differential permeability to fluorescent nuclear dyes and high-resolution histopathology, injury in female mice was found to be more extensive, occur earlier, and include permeable cells in proximal airways, including airway bifurcations. HPLC analysis of the products of cytochrome P-450 (CYP)-mediated metabolism in microdissected airways indicated that although both genders produced a predominance of products from CYP2F2, female mice produced more naphthalene dihydrodiol in distal airways, the primary sites of injury. We conclude that there are clear gender differences in susceptibility to naphthalene-induced injury and that differences in metabolism of naphthalene may play a role in elevated susceptibility in female mice.
- cytochrome P-450
- Clara cell
humans are exposed to a wide variety of environmental pollutants, many of which have a detrimental effect on the lung. Polycyclic aromatic hydrocarbons (PAHs), commonly found in cigarette smoke and diesel exhaust, can undergo site- and cell-specific metabolism to cytotoxic intermediates. Although metabolism of PAHs, such as naphthalene, and Clara cell cytotoxicity have been extensively studied in male animals, whether female animals are equally susceptible is not known. One clue that there may be differential responses in female animals exposed to PAHs can be found in the results of a toxicology and carcinogenesis study of naphthalene in mice (22): incidences of pulmonary alveolar/bronchiolar adenomas were increased in female, but not male, mice. It was concluded that there was some evidence of a carcinogenic effect of naphthalene in female, but not male, mice. Whether this long-term effect reflects differences in susceptibility to acute cytotoxicity between male and female animals is unknown.
Recent studies have shown that naphthalene is a prominent component of sidestream cigarette smoke. One study found naphthalene to be the most abundant chemical constituent in whole and filtered cigarette smoke (35). Lung cancer is now the principal cause of cancer death in men and women, and cigarette smoking is a major contributing factor. Current research suggests a pattern of sensitivity and long-term pathological outcomes to tobacco smoke in women that is different from that in men. Although lung cancer incidence is plateauing among men, it continues to rise among women (36). Cancers of the peripheral lung, adenocarcinomas, are more frequently observed in women than in men (16). Odds ratios for major lung cancer types are consistently higher for women than for men at every level of exposure (36). In addition, young girls have increased deficits in lung function compared with young boys after tobacco smoke exposure (15), and airway reactivity after smoke exposure is increased in women compared with men (23). Several studies have concluded that because differences in baseline exposure, smoking history, or body size do not account for the increased risk (particularly of cancer), these effects in women are probably due to increased susceptibility to substances in tobacco smoke (2, 5, 36). These studies and many others that demonstrate the disparate biology between men and women have prompted the National Academy of Sciences to generate a report calling for more research on gender-based differences and the inclusion of female subjects in research (21).
Although gender differences in pulmonary responses are clear after respiratory disease develops, the factors that result in increased susceptibility remain largely unknown. Among the factors that may influence susceptibility are the extent and pattern of acute injury. Our goal in this study was to determine whether there is a gender-based difference in susceptibility to the acute lung injury associated with naphthalene toxicity. Defining gender differences in susceptibility and repair is key to understanding the basis for gender differences in pulmonary disease. Our experiments were designed to address two primary questions: 1) Are female mice more susceptible than male mice to the same dose of naphthalene? 2) Does metabolism of naphthalene differ by gender?
Adult, 8- to 10-wk-old, male and female viral antibody-free Swiss Webster mice (CFW, Charles River Laboratories, Wilmington, MA) were housed in a high-efficiency particle air-filtered cage rack in American Association for Accreditation of Laboratory Animal Care-approved facilities. The animals were subjected to a 12:12-h light-dark cycle, and food and water were provided ad libitum for ≥5 days before use. Corn oil carrier or naphthalene (200 mg/kg) was injected intraperitoneally, and lungs were removed at 1, 2, 3, 6, or 24 h after treatment. For each time point, three mice were treated with naphthalene and one mouse was treated with corn oil. Lungs from corn oil-treated mice did not vary by time after treatment and, therefore, were analyzed together as one group, as described previously (32). The trachea was cannulated, and lungs were removed and fixed via tracheal cannula at 30 cm of pressure with 330 mosM Karnovsky's fixative (0.9% glutaraldehyde-0.7% paraformaldehyde in cacodylate buffer, pH 7.4) or 1% paraformaldehyde for 1 h. Lungs were stored in fixative until use. For cell permeability studies, the trachea was cannulated and the lungs were inflated to capacity in situ with 37°C ethidium homodimer-1 (5 μM; Molecular Probes, Eugene, OR) in Ham's F-12 nutrient mixture (Life Technologies, Grand Island, NY) for 20 min to label the nuclei of any membrane-permeable cells fluorescent red. Lungs were then lavaged three times with 37°C F-12 medium to remove any unincorporated ethidium and fixed at 30 cm of pressure with 330 mosM Karnovsky's fixative for 1 h (34). For permeability studies, one treated mouse was killed at each time point and a corn oil control mouse was killed at 1 h and another at 6 h. These studies were repeated three times. Lungs were stored in fixative in the dark until used.
High-Resolution Light Microscopy
The Karnovsky-fixed left lung lobe was sliced into three equal segments cut perpendicular to the long axis of the lobe, with each containing a portion of the first large intrapulmonary airway (32). These three pieces were postfixed in osmium tetroxide in Zetterquist's buffer, processed by large block methodology, and embedded in Araldite 502 epoxy resin. All specimens were sectioned at 1 μm and stained with methylene blue-azure II. High- and low-magnification images of fields (from 3 slides per animal and from 3 animals per group) containing terminal and distal bronchiolar epithelium and proximal bronchi were captured using an Olympus Provis computerized microscope in bright-field mode. Every airway on three high-resolution lung cross sections was examined. Representative airways were imaged and are shown for each animal.
The abundance of normal and cytotoxic epithelial cells in the intrapulmonary lobar bronchus of five male and five female mice was determined using methods discussed in detail by Hyde et al. (17). The entire circumference of the lobar bronchus was analyzed on an Olympus CAST grid system using a 36-point test grid and isotropic uniform random sampling. All measurements were made using high-resolution (×100) 1.0-μm sections and ≥15 fields per animal. The volume fraction (Vv) was calculated using the formula where Pp is the point fraction of Pn, the number of test points hitting the structure of interest (vacuolated cells), divided by Pt, the total number of points hitting the reference space (epithelium).
The right middle lobe from the ethidium-lavaged fixed lung was microdissected by cutting down the airway lumen to expose the long axis of the conducting airway tree, including the most distal six to eight conducting airway generations and the terminal bronchioles. The lung was counterstained with the nuclear binding fluorochrome Yo-Pro-1 (4 μM) for 20 min to label the remaining nuclei with green fluorescence. Samples were imaged using a scanning laser confocal microscope (Bio-Rad, Hercules, CA) with a ×10 long-working-distance water-immersion objective and a dual-filter set. With use of the dual-filter set (514 nm excitation-527 nm emission and 540 nm excitation-600 nm emission) two fluorescent wavelengths were captured. The laser scanned through the entire three-dimensional sample, producing a series of images (for both wavelengths) at sequential focal planes that were “stacked” one on top of the other to generate two separate pseudocolor (red and green) images of the sample (34). The two stacked images were then merged into one image to generate the image of red (permeable) cell nuclei in the context of the rest of the cell nuclei (green, impermeable). Each airway map was a composite of four to five fields collected in this manner.
High-Resolution Fluorescent Microscopy of Permeable Cells
Ethidium-stained tissue from key time points (sham-treated controls and 3 and 6 h after naphthalene treatment in female mice,n = 3 per group) was embedded in methacrylate resin after the laser scanning confocal images had been gathered to determine the morphology of the permeable cells. Blocks were sectioned at 1 μm and processed as described previously (34). Coverslips with a water-soluble, nonfluorescing mounting medium were applied. Using an Olympus BH-2 epifluorescent microscope and a wide-band ultraviolet (UV) fluorescence excitation-emission filter set, we imaged terminal bronchioles using a Cool-SNAP Pro color digital camera (Media Cybernetics, Del Mar, CA). Because of a phase shift in the wide-band UV excitation-emission filter set, the tissue autofluoresces blue and the ethidium-positive nuclei fluoresce pinkish-white.
Metabolism of Naphthalene
We measured cytochrome P-450 (CYP) activity toward naphthalene using microdissected airway regions. Briefly, the trachea was cannulated, and the lungs were removed en bloc. Lungs were filled with low-melting-temperature agarose (Seaplaque, Cambrex, East Rutherford, NJ) in 1% Waymouth's medium (GIBCO) and allowed to solidify on ice. Airways were then microdissected as described previously (26). Microdissected airway segments of minor daughter bronchioles and terminal bronchioles from five male and five female mice were incubated in sealed vials with a total volume of 0.5 ml at 37°C for 2 h. Incubations contained excess glutathione transferase (five 1-chloro-2,4-dinitrobenzene units) and glutathione (1 mM) to trap conjugates. The concentration of naphthalene was 0.5 mM. Vials containing cell culture medium, explants, and all other components, but lacking naphthalene, were used as controls. The reaction was stopped after 2 h with the addition of methanol. Samples were homogenized, and the supernatant was assayed by HPLC for formation of 1,2-dihydroxy-1,2-dihydronaphthalene (dihydrodiol) and glutathione conjugates from naphthalene oxide, as described previously (6) and modified by Shultz et al. (29).Conjugates 1 and 3 are from the 1S,2R-epoxide, and conjugate 2 is from the 1R,2S-epoxide (29). Only dihydrodiol and conjugate 2 were detected. Samples were normalized by protein content measured using a micro-Lowry assay (19).
Distal airways were isolated by microdissection of agarose-inflated lungs, as described previously (33). Dissected airways were homogenized in suspension buffer (0.1 M NaCl, 10 mM Tris, and 1 mM EDTA, pH 7.6) containing Complete mini-protease inhibitor cocktail tablets (Boehringer-Mannheim, Indianapolis, IN) and centrifuged at 9,000 g for 20 min. Supernatant was collected, and all samples were normalized by protein content that was measured using the method of Bradford (4). Soluble protein was diluted in 10% SDS sample buffer containing β-mercaptoethanol. Explant protein (10 μg) was loaded into each lane and separated by gel electrophoresis using a Bio-Rad Tris · HCl-buffered 15% polyacrylamide minigel with 6 μl of prestained broad-range SDS-PAGE standards. Proteins were electroblotted to polyvinyl difluoroacetate membranes (Bio-Rad) and immunostained using rabbit anti-CYP2F2 (20) and a horseradish peroxidase-linked goat anti-rabbit IgG secondary antibody (Amersham, Piscataway, NJ). Bands were detected with a chemiluminescence detection kit (NEN, Boston, MA) using methods suggested by the manufacturer. HPLC data from pooled minor daughter airways and pooled distal bronchioles were used to generate a value for each animal for the two naphthalene metabolism products detected in each airway level: naphthalene dihydrodiol and glutathioneconjugate 2. The value per animal was used to calculate the mean ± SD for each group (n= 5).
The presence of the differentiation marker proteins was detected using specific antibodies, rabbit anti-rat CC10, and rabbit anti-mouse CYP2F2 (20, 30), as described previously (32), on paraffin-embedded lung sections from three male and three female mice. The avidin-biotin peroxidase procedure was used to identify antibody binding sites. The procedure was the same as that outlined by the supplier (Vector Labs, Burlingame, CA). All primary antibodies were incubated overnight with the tissue at 4°C. Diaminobenzidine with nickel enhancement was used as the chromagen. To eliminate nonspecific binding of the primary antibody, sections were blocked with 1% BSA. Controls included substitution of PBS for the primary antibody. The optimal dilution at which there was positive staining with minimal background staining was determined separately for each antibody using a series of dilutions. Four lung cross sections were immunostained for each mouse, and sections from all the mice (male and female) were included in each immunochemical run. The immunohistochemistry was repeated twice for each antibody. In addition, to assess inter- and intra-animal variability, staining intensity and abundance were scored for up to 250 μm of every terminal bronchiole in each section from three male and three female mice at a series of dilutions of the CYP2F2 antibody. Scoring was as follows: 3 = most cells highly positive for CYP2F2; 2 = some cells highly positive or most cells positive but with decreased staining intensity; 1 = a few positive cells; 0 = no staining. The observer scoring the samples did not know which animals the slides represented. The average score for each animal was determined from all the terminal bronchioles evaluated (≥3), and this was then averaged per group (≥15 terminal bronchioles per group).
Naphthalene was purchased from Aldrich Chemical (Milwaukee, WI). Corn oil (Mazola) was manufactured by Best Foods/CPC International (Englewood Cliffs, NJ). Glutaraldehyde, paraformaldehyde, methylene blue, azure II, and Araldite 502 resin were obtained from Electron Microscopy Sciences (Fort Washington, PA).
Laser Capture Microdissection
Paraffin-embedded lung tissues from four male and four female mice were used for laser capture microdissection (LCM) of terminal bronchiolar epithelium. Six 7-μm-thick paraffin tissue sections from each mouse were placed on noncoated glass slides. Slides were deparaffinized in three changes of xylene, air-dried, and stored in a desiccator until LCM was performed. LCM was performed using a PixCell II system (Arcturus Engineering, Mountain View, CA) in fluorescent mode, using a wide band-pass UV excitation and emission filter set to visualize the tissue. The laser settings were as follows: 15-μm beam diameter, 2.0-ms pulse duration, and 70-mW power. All the terminal bronchioles (defined as up to 250 μm of the last conducting airway before the gas exchange region) present on the six sections for each mouse were captured. Samples were captured onto Capsure LCM caps (Arcturus Engineering), and nonspecific tissue adhering to the cap was removed with Capsure Pads (Arcturus Engineering) according to the manufacturer's instructions.
RNA Extraction, Transcription, and Quantitation
RNA was extracted using a PicoPure RNA Isolation Kit (Arcturus Engineering) according to the manufacturer's instructions. The isolated RNA was reverse transcribed in a 30-μl reaction using TaqMan RT reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Quantitative 5′-exonuclease-based real-time PCR was performed using an ABI Prism 5700 Sequence Detection System (PE Applied Biosystems). Primers and fluorescently labeled probes for CYP2F2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express software (PE Applied Biosystems). The following oligonucleotides were custom synthesized (Operon Technologies, Alameda, CA): 5′-AGCTTCGCTCCCAAGACTTG-3′ (forward primer), 5′-CCGTGAACACCGACCCATAC-3′ (reverse primer), and FAM-TGACCTCCCTCACCAAGCTTAGCAAGG-TAMRA (probe) for mouse CYP2F2 (GenBank accession no. BC011089) and 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward primer), 5′-CCTGCTTCACCACCTTCTTGA-3′ (reverse primer), and FAM-CCGCCTGGAGAAACCTGCCAAGTATG-TAMRA (probe) for mouse GAPDH (GenBank MUSGAPDH). Five microliters of the RT reaction were used for each PCR. Each reaction contained 1× TaqMan Universal PCR MasterMix (PE Applied Biosystems), 5′ primer (300 nM for CYP2F2 and GAPDH), 3′ primer (300 nM for CYP2F2 and 50 nM for GAPDH), and the probe (250 nM for CYP2F2 and GAPDH) in a 25-μl reaction. PCR was performed for 40 cycles with a combined annealing and extension temperature of 60°C (1 min) with a denaturing temperature of 95°C (15 s). Both genes were measured in duplicate PCR. A mouse lung airway cDNA was used to generate linear standard curves and determine the gene concentration in unknown samples. The reported CYP2F2 values are normalized to GAPDH.
Differences between male and female animals were determined using a two-tailed t-test with significance accepted atP < 0.05 (14).
Terminal bronchioles were defined as the most distal conducting airway generation contiguous with the alveolar duct. The epithelium in terminal bronchioles in female mice treated with vehicle (corn oil) was cuboidal (Fig. 1 A) and contained a majority of Clara cells. The thickness of the epithelium and the size of the epithelial cells were smaller in female than in male mice (cf. Figs. 1 and 2). At 1 h after naphthalene treatment, the epithelium lining terminal bronchioles was indistinguishable from that in sham-treated controls (Fig. 1 B). By 2 h after naphthalene treatment, terminal bronchiolar cells had a more rounded appearance, were swollen, and lacked pronounced Clara cell apical protrusions (Fig. 1 C). Very few cells contained even one cytoplasmic vacuole. By 3 h after naphthalene treatment, Clara cells contained apical membrane blebs that were light staining and lacked dark-staining organelles such as mitochondria or secretory granules (Fig. 1 D). Some cells contained vacuoles. Their cytoplasm remained darkly stained. At 6 h after naphthalene treatment, many of the Clara cells had vacuolated and exfoliated, leaving extremely attenuated squamated cells (Fig. 1,E and F). Any remaining Clara cells contained a light-staining cytoplasm and pyknotic nuclei. In some areas the basement membrane appeared denuded. By 24 h after naphthalene treatment, the terminal bronchiole, as well as adjacent proximal bronchioles (including bifurcations), was covered by squamated cells and contained an occasional vacuolated cell (Fig. 1, G andH). In the lobar bronchus at 24 h after naphthalene treatment, many vacuolated cells were present (Fig.3 D) and the epithelium was low cuboidal (Fig. 3 B). The thickness of the epithelium in the lobar bronchus, which was defined as the airway located at the proximal end of the airway tree where the airway enters the lobe, and the size of the epithelial cells were smaller in control female than in male mice (cf. Fig. 3, B and A). When the distribution of permeable cells was determined using laser scanning confocal microscopy, no permeable cells were detected in sham-treated control mice (Fig. 4 A). At 2 h after naphthalene treatment, several permeable cells were detected in the more proximal portions of the bronchioles, including airway bifurcations (Fig. 4 B). These permeable cells were detected singly or in a single line along the ridge of the bifurcation. At 3 h after treatment, permeable cells were more abundant throughout the terminal bronchiole, as well as at proximal airway bifurcations, in female than in male mice (cf. Figs. 4 C and5 B). Permeable cells in terminal bronchioles (Fig. 6,A and B) had characteristics of Clara cells, whereas those at proximal airway bifurcations did not (Fig. 6,C and D). By 6 h after naphthalene treatment, permeable cells increased in abundance and were distributed throughout the terminal bronchiole and more proximal airways, including airway bifurcations (Fig. 4 D). More permeable cells were detected in the terminal bronchioles at 6 h in female than in male mice (cf. Figs. 4 D and 5 C).
The epithelium lining terminal bronchioles in male control mice (sham treated with vehicle, corn oil) was cuboidal and composed of predominantly nonciliated bronchiolar (Clara) cells and some ciliated cells (Fig. 2 A). At 1 h after naphthalene injection, the epithelial cells varied little from controls but had a more rounded shape and lacked pronounced Clara cell apical protrusions (Fig.2 B). By 2 h after naphthalene injection, most Clara cells contained vacuolated cytoplasm. Ciliated cells did not contain vacuoles (Fig. 2 C). By 3 h after naphthalene injection (Fig. 2 D), many Clara cells had cytoplasmic vacuoles as well as light-staining apical blebs that lacked dark-staining organelles. The epithelium was of variable thickness, with cells ranging from low cuboidal (ciliated cells) to quite swollen (Clara cells). At 6 h after naphthalene injection (Fig. 2, E and F), the terminal bronchiolar epithelium contained many light-staining vacuolated Clara cells and a few regions where the Clara cells had exfoliated and the epithelium was squamated. By 24 h after naphthalene injury, the terminal bronchiolar epithelium was largely composed of low-cuboidal to squamated ciliated cells. Cell debris was present in the airway lumen, and vacuolated cells were rare (Fig. 2,G and H). In the lobar bronchus, at 24 h after naphthalene treatment, very few vacuolated cells were present in some animals (several had none; Fig.7 C) and the remainder of the epithelium resembled controls (Fig. 7 A). When the distribution of permeable cells was determined using laser scanning confocal microscopy, permeable cells in male mice first appeared in the most distal portions of the terminal bronchiole at 3 h after naphthalene treatment (Fig. 5 A). By 6 h after naphthalene treatment, permeable cells (red fluorescence) increased in abundance and were distributed primarily in the most distal portion of the terminal bronchiole (Fig. 5 B).
CYP2F2 Expression and Activity
Immunohistochemically, Clara cells in untreated male and female mice were characterized by their apical projections into the airway lumen and abundant expression of CYP2F2 and CC10 protein (Fig. 3,A and B). In female mice, expression of CC10 and CYP2F2 protein in terminal bronchiolar Clara cells was variable in abundance within the same animal (cf. Fig.8, C and D, withE and F) as well as between animals (Table1). To determine whether there was a difference in protein abundance, the intensity of CYP2F2 staining for male and female animals through a series of antibody dilutions was scored. Using this semiquantitative method, we did not detect a difference in CYP2F2 protein abundance in the terminal bronchioles of mice by gender. The abundance of CYP2F2 mRNA was measured using the recently described, sensitive method of Betsuyaku et al. (3) to measure gene expression in terminal bronchioles using LCM and quantitative RT-PCR. Using this method, we confirmed that there was no significant difference between mRNA levels for CYP2F2 in the terminal bronchioles of male and female mice (Fig.9), although there was a trend toward more mRNA in the female mice that was not significant because of the large variability. By Western blotting, we detected a single 50-kDa band of similar staining intensity in distal airway trees microdissected from male and female mice (Fig.10). This corresponds with the previously reported molecular mass of CYP2F2 in mouse airway microsomes (29). Similar levels of CYP2F2 were detected for all sham (corn oil)-treated male and female mice (Fig. 10 A). As expected in animals sensitive to naphthalene toxicity, 24 h after injection with naphthalene, the distal airways from all three female mice contained significantly less CYP2F2 than distal airways from male mice (Fig. 10 B). Total naphthalene metabolism as measured by HPLC detection of the products of metabolism was greater in the terminal bronchioles (Fig.11 B) than in the minor daughter bronchioles (Fig. 11 A) of male and female mice. Total naphthalene metabolism was ranked in the following order: female terminal bronchioles > male terminal bronchioles > female minor daughter bronchioles > male minor daughter bronchioles. Only two metabolic products were detected in airway incubations: naphthalene dihydrodiol and conjugate 2, reflecting the reported enantiomeric ratio of 66:1 for the 1R,2S-naphthalene oxide (conjugate 2) over the 1S,2R-naphthalene oxide (conjugates 1 and 3) for recombinant CYP2F2 (29). Similar amounts of conjugate 2 per milligram of protein were detected in male and female minor daughter airways and terminal bronchioles. However, more naphthalene dihydrodiol was produced from the female mouse airways (1.6-fold more in the terminal bronchioles of female than male mice). Naphthalene dihydrodiol production was ranked in the following order: female terminal bronchioles > male terminal bronchioles = female minor daughter bronchioles > male minor daughter bronchioles.
The goal of this study was to determine whether there is a gender-based difference in naphthalene susceptibility. Naphthalene is a common environmental contaminant (1), a member of a group of compounds that cause lung toxicity when metabolized by the CYP monooxygenase system (25), and abundant in tobacco smoke (35). The pattern of sensitivity and long-term pathological outcomes to tobacco smoke of women may be different from those of men (15, 23). Several studies have concluded that these effects in women may be due to increased susceptibility to substances in tobacco smoke (2, 5, 36). One aim of our study was to address the hypothesis that the lungs of female mice are more susceptible and respond differently to naphthalene, a prominent constituent of mainstream and sidestream tobacco smoke. We found that female mice were more susceptible than male mice to the same dose of naphthalene: injury occurred earlier and affected cells farther up the airway tree. In addition, the temporal pattern of acute cytotoxicity was different within the Clara cells themselves. In female mouse Clara cells exposed to naphthalene, the intracellular events occurred in the following order: Clara cell swelling, formation of apical cytoplasmic blebs, vacuolization, and exfoliation. In male mice, as reported previously, vacuolization (and smooth endoplasmic reticulum swelling) occurs before bleb formation (24, 34). We also found a possible metabolic basis for differential susceptibility to naphthalene. Female mice produce more products (total) of naphthalene metabolism, and more of the total product is composed of naphthalene dihydrodiol. These findings suggest that female mice are more susceptible to naphthalene and that the basis for this may be, in part, related to differences in naphthalene metabolism, distribution of susceptible cells, and/or a different intracellular mechanism of toxicity.
Female Swiss Webster mice have more injured cells in their conducting airways than male mice when given the same dose of naphthalene (200 mg/kg). This is especially apparent at 24 h after naphthalene injection, when female mice have injury up to, and including, cells in the lobar bronchus. Western blots from microdissected female mouse distal airways also show decreased CYP2F2 expression compared with male mice at the same time point after treatment. This is the expected result if more Clara cells are injured and exfoliated in the female mouse airways, resulting in a loss of CYP2F2-expressing cells. As has been reported previously in male mice, as the dose of naphthalene increases, injury includes more proximal airway levels (27). The pattern of injury (including cells in lobar bronchus) in females at 200 mg/kg naphthalene is similar to that reported for male mice treated with a 1.5-fold higher dose, 300 mg/kg (27). Previous studies with another strain of mouse, female FVB/n mice, treated with 200 mg/kg naphthalene reported injury in proximal airways such as trachea and bronchi (31). Although some of these differences in susceptibility may be attributable to strain variations, our present study suggests that gender is a contributing factor.
In addition to elevated susceptibility to naphthalene injury at 24 h after naphthalene injection, we found apparent differences in the distribution of injured cells during the initial stages of acute injury. In female mice, single permeable cells are detected at 2 and 3 h after naphthalene treatment in proximal airways, and lines of permeable cells are found at airway bifurcations. This is in contrast to the pattern seen in male mice, and reported previously (34), where permeable cells appear first at the distal-most portions of terminal bronchioles. Because the temporal pattern of acute injury varies between female and male mice, one possibility is that these injured cells also occur in male mice but were not detected at the time points examined (i.e., they occurred earlier). Further studies are needed to define whether the early presence of permeable cells in proximal airways is a gender-specific phenomenon. Differential susceptibility of this nature might be related, through the processes associated with airway remodeling, to the gender differences observed in pulmonary responses to tobacco smoke exposure (i.e., elevated lung cancer risk and airway hyperresponsiveness in women).
It is intriguing to speculate that there may be a population of cells, present at airway bifurcations, that function as sentinel cells for pulmonary injury. Airway bifurcations, as well as terminal bronchioles, represent potential foci for cytotoxicity from inhaled air pollutants (11). Cells at airway bifurcations could be regulated by the response of the sensitive, sentinel, cell. The bifurcation cells could be warned early in the injury process of the need to become resistant to pulmonary toxicants. Perhaps by upregulating certain tissue proteins (such as heat shock proteins) or by downregulating or decreasing expression of other proteins (such as CYP2F2), cells at bifurcations could modulate their responses. There is some support for survival of a resistant population in the pulmonary literature. Other researchers have reported that there is a population of Clara cells present at airway bifurcations in male (28) and female (31) mice that are “pollutant resistant.” Because the earliest time point examined was 6 h and the methods of injury detection did not look at the airway in three dimensions, they may have overlooked a small number of transiently present cytotoxic sentinel cells. Further studies are needed to address whether the permeable cells present at airway bifurcations are Clara cells or some other cell type.
The temporal pattern of acute injury was different within Clara cells of male and female mice. The sequence of intracellular events associated with toxicity was changed, in that Clara cells in female mice blebbed before pronounced cytoplasmic vacuolization took place. These differences could be due to a number of factors, including different intracellular targets of the active naphthalene metabolites and/or depleted glutathione in the female mice. Because female mouse cells appear to be smaller, it is possible that less glutathione was available. If less glutathione is available, this could lead to increases in susceptibility in the female cells. Both of these possibilities (differential metabolite binding and glutathione levels), as well as the possible influence of cell size on airway cell responses, remain to be investigated.
One major difference between male and female mice is in their metabolism of naphthalene. Total naphthalene metabolites and the amount of naphthalene dihydrodiol were larger in the female mice at both airway levels tested. Conjugate 2 and probably most of the dihydrodiol formed are due to metabolism of naphthalene to the 1R,2S-epoxide by the CYP enzyme CYP2F2.Conjugates 1 and 3, which were not detected in our assay, are generated at a much lower rate (1:66) by CYP2F2 and are the result of glutathione conjugation with the 1S,2R-oxide (29). Lack of detection of conjugates 1 and 3 is expected on the basis of the much greater formation of the 1R,2S-oxide by CYP2F2. The metabolism of naphthalene was stereospecific in both genders, producing similar amounts of conjugate 2 but significantly more naphthalene dihydrodiol in female mice. It is probable that the majority of the diol formed in the female mice is the product of epoxide hydrolase-dependent metabolism of the major epoxide formed by CYP2F2, the 1R,2S-epoxide. The 1R,2S-epoxide is the preferred substrate for epoxide hydrolase in mouse hepatocytes (8), and this may also be true in the lung. Increased diol formation in the female mice may also be related to a lower Michaelis-Menten constant (K m) of CYP2F2, increased levels of epoxide hydrolase, lower K m of epoxide hydrolase, or decreased glutathione levels in the female mice. We speculate that formation of excess naphthalene dihydrodiol in female mice may contribute to their elevated sensitivity to naphthalene injury. Chichester et al. (10) found that dihydrodiol alone was toxic to isolated Clara cells. The relation of this shift in naphthalene metabolism (to produce more dihydrodiol) in the female mice to the disparate sensitivity is unknown. One possibility is that naphthalene dihydrodiol can be further metabolized to the 1,2-naphthoquinone. Further studies are needed to define the relation of dihydrodiol formation to toxicity as well as the distribution and abundance of epoxide hydrolase, glutathione, and glutathione transferase in female mice.
Previous studies of gender-related differences in metabolism of compounds by the CYP monooxygenase system have focused primarily on other organ systems such as liver and kidney. When gender-specific lung metabolism is addressed, microsomes from whole lung homogenates are frequently used, thus blurring the effects of site-specific lung metabolism. Nevertheless, differences in metabolism of styrene to styrene oxide have been reported using microsomes derived from male and female mouse whole lung homogenates (9). In contrast to our present studies, the female mice formed less metabolite, styrene oxide, than the male mice. However, when the Clara cell-specific toxicant 1,1-dichloroethylene is metabolized to a toxic intermediate by CYP2E1 (18), this bioactivation occurs to a greater extent in microsomes from female than male mouse lungs (13). In correlation with the elevated metabolism in female mice, female mouse bronchiolar epithelium was much more susceptible to 1,1-dichloroethylene-mediated toxicity. Similar to our findings for CYP2F2, the expression of CYP2E1 in Western blots from male and female mice did not appear to differ greatly (13), yet bioactivation and toxicity were much greater in the female animals. This apparent discrepancy may be explained by two factors in both studies [as discussed previously by Forkert et al. (13)]: 1) limitations in the methods to detect a cytotoxic effect in individual Clara cells and 2) the possible contribution of differences in metabolite detoxification. It is important to keep in mind that immunodetectable protein levels do not always correlate directly with protein activity, particularly when the protein is also an enzyme. It is possible that theK m of CYP2F2 differs in male and female mice. It also cannot be ruled out at this time that the female mouse cells are more susceptible because they contain a different or a more accessible critical macromolecular target for reactive metabolite binding.
Male and female mice did not differ in the amount of total CYP2F2 detected by Western blot using homogenates of minor daughter airway trees containing terminal bronchioles and larger bronchioles. When site-specific expression of CYP2F2 in terminal bronchioles was examined using immunohistochemistry, female mice had a broader range of CYP2F2 abundance on a per cell and per animal basis, although the average abundance was not significantly different by gender (Table 1, Fig. 8). This was further confirmed at the mRNA level by LCM. The variability was not detected in the Western blots, because each lane is composed of more than one airway per animal, pooling high- and low-expressing terminal bronchioles with larger airways. The significance of this variability in CYP2F2 expression and its relation to the pattern of cytotoxicity are unknown. The foundations for the increased variability of CYP2F2 protein expression in terminal bronchioles in female mice may lie in regulation of this isozyme by steroid hormones. On the basis of previous studies in adult male mice, rats, and hamsters (7), we would expect metabolism of naphthalene and CYP2F2 protein expression/activity to correlate directly with toxicity (i.e., airways with high levels of CYP2F2-mediated naphthalene metabolism to the oxide had higher levels of toxicity). Thus we would expect the female mice to have higher levels of CYP2F2 protein expression in the target regions; this does not appear to be the case. It may be that only a few cells per airway express high levels of CYP2F2 in the female mice. Also, high levels of CYP2F2 are not always required for elevated susceptibility to naphthalene; young mice, which have low levels of CYP2F2 protein, are more susceptible than adult mice, which have high levels of protein (12). Further studies are needed to define whether CYP2F2 is hormonally regulated and how toxicity is produced in female mice.
Although gender differences in pulmonary responses are clear after respiratory disease develops, the factors that result in increased susceptibility remain largely unknown. Among the factors that may influence susceptibility are the extent and pattern of acute injury. Our goal in this study was to determine whether there is a gender-based difference in susceptibility to the acute lung injury associated with naphthalene toxicity. We conclude that there are clear gender differences in naphthalene responses in mice, with females having1) elevated sensitivity to naphthalene toxicity,2) a different pattern of airway epithelial injury that includes early presence of permeable cells in proximal airways and at airway bifurcations, and 3) differences in metabolism. Further studies are needed to determine the relationship of dihydrodiol formation and various activation and detoxification pathways to differential sensitivity on an airway level and cell-specific basis. These studies underscore the importance of evaluating the extent of injury by gender when studying airway epithelial injury and repair in the lung.
We thank Drs. A. Buckpitt, M. Evans, and C. Plopper for comments on the manuscript.
This study was supported by National Institute of Environmental Health Sciences Grants ES-04311 and ES-06700 and California Tobacco-Related Diseases Research Program Grant 6KT-0306. The University of California-Davis is a National Institute of Environmental Health Sciences Center for Environmental Health Sciences (Grant ES-05707), and support for core facilities used in this work is gratefully acknowledged.
Address for reprint requests and other correspondence: L. S. Van Winkle, Dept. of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616-8732 (E-mail:).
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