A Review of the Histopathology of Cigarette Smoke–Induced Lung Cancer in Rats and Mice

Abstract

In the past several years an increased number of lung tumors has been reported in laboratory studies of rats and mice after lifetime exposure to mainstream cigarette smoke. Proliferative epithelial lesions are present in the lungs of both species and are apparent antecedent lesions to benign and malignant tumors. Both species have alveolar epithelia hyperplasia, alveolar adenomas, and alveolar carcinomas. The incidence of all three are more in the rats. In addition, mice also have bronchiolar epithelial hyperplasia and bronchial papillomas not found in rats. Rats have a low incidence of squamous cyst that is not found in mice. Lung tumors in rats and mice are found at the end of the life span and rarely metastasize. The characteristics of the lung tumors, and the proliferative changes associated with the tumors, are important in helping understand the mechanisms of lung cancer induction. These studies in rats and mice allow new approaches to the study of cigarette smoke–induced changes in the lung.

BACKGROUND

Cancer of the lung is an important disease because of its high incidence in the human population and its poor response to treatment. The lung cancer incidence in women in the United States is increasing, even among nonsmokers. In spite of earlier diagnosis and new treatment regimes, response to treatment remains poor. Thus, lung cancer remains a high mortality disease. Lung cancer is also associated with several occupations and with environmental exposure to carcinogens (Ahsan and Thomas 2004; IARC 2004).

Studies in animals are important to identify causative agents, effective concentrations of carcinogens, and potential synergistic effects, and to determine the pathogenesis that aids in the development of new diagnosis and treatment procedures.

Recently reported studies at the Lovelace Respiratory Research Institute have demonstrated that prolonged exposure of both rats and mice results in an increased incidence of lung tumors as well as a chronic pulmonary disease and nasal tumors. The purpose of this paper is to compare the responses of rats and mice to prolonged inhalation of mainstream cigarette smoke.

METHODS

The methods have been fully described for rats (Mauderly et al. 2004) and mice (Hutt et al. 2005). Briefly, rats or mice were exposed (whole body) to either mainstream cigarette smoke diluted to the desired concentrations or to filtered air for 6 h per day, 5 days per week for 30 months. Smoke was generated from high-yield unfiltered research cigarettes obtained from the Tobacco Health Research Institute, Lexington, KY. Total particulate matter (TPM) was determined gravimetrically from filter samples taken three times daily in the exposure chambers and used to set exposure concentrations. In addition particle size distributions were measured periodically using a cascade impactor.

The experimental design for the study with rats is shown in Tab. 1. Male and female CDF (F344)/CrlBR rats were exposed to 250 or 100 TPM/m³ from high-yield unfiltered 1R3 research cigarettes. These exposure concentrations were estimated to equate to 3 to 4 packs/day and 1 to 2 packs/day, respectively, for a human smoker, based on particle weekly doses per unit of lung weight (Finch 1998).

The experimental design for the study with mice is shown in Tab. 2. Female B6C3F1 mice were exposed to 250 TPM/m³ from 2R1 research cigarettes. The experimental designs did not use the National Toxicology Program bioassay format, but the studies were part of a larger study of synergistic effects of cigarette smoke inhalation. The group sizes varied based on predicted synergistic effects.

In-life evaluations included body weights every 6 months and survival until 30 months. Postmortem evaluations included weights of the major organs and gross evaluation of all body cavities and major organs. The lungs were instilled with 4% paraformaldehyde. The fixed lung lobes (except the accessory lobe) were cut along the major axial airway and one histologic section of each was prepared. In addition, sections were taken from any gross lesions in the lung. Microscopic evaluations included examination of paraffin-embedded, 5 micron, hematoxylin and eosin–stained slides of the nasal cavity, pulmonary airways, and pulmonary parenchyma.

RESULTS

Study of F344 Male and Female Rats Exposed to Mainstream Smoke

A full presentation of results are shown in publications (Mauderly et al. 2004; March et al. 1999). Both the daily and study average TPM concentrations were maintained very close to target concentrations (244 ± 27 versus 250 mg/m³ and 98 ± 11 vesus 100 mg/m³, mean ± SD of daily values). The mass median aerodynamic diameters and geometric standard deviations of the particle sizes were 0.62 μm, 1.34 σ g for high concentration and 0.56 μm, 1.34 σg for high concentration. These results indicated that the exposure concentrations were as planned and that the particles were in the respirable range.

The median survival times for the exposed groups of the rats are shown in Tab. 3. The low-dose groups survived significantly longer than those in the high-smoke or sham-control groups. The survival of the females in the high-dose group was significantly shorter than the control female group.

The terminal body weights are shown in Tab. 4. The body weights were significantly lower for the males and females in both exposure groups. Limited, 1-week food consumption trials (one during exposure week 2 and one during exposure week 61) showed a dose-related reduction in food intake. The terminal lung weights are shown in Tab. 5. The lung weights of the high-exposed groups were significantly higher than the control lung weights, indicative of cellular reactions in the lung.

The non-neoplastic lesions in the lung were centered in the centriacinar region of the pulmonary parenchyma, around the terminal bronchiole. The lesions included small aggregates of golden pigment–filled alveolar macrophages, smoke granulomas consisting of large, focal aggregates of alveolar macrophages admixed with scattered inflammatory cells and associated epithelial hyperplasia and septal fibrosis, occasional chronic bronchiolitis, minimal emphysema (as diagnosed by enlargement of airspaces with fragmentation of a septa), and numerous epithelial proliferative lesions.

The proliferative lesions included foci of alveolar epithelial hyperplasia (Fig. 1A and B ) bronchiolar metaplasia of the alveolar epithelium in the centriacinus (Fig. 1C ) and rarely, squamous metaplasia of the alveolar epithelium (Fig. 1D ). Keratinizing squamous cysts were found in lungs of three rats in the high concentration smoke exposure.

The neoplastic pulmonary lesions were bronchioloalveolar adenoma, and bronchioloalveolar adenocarcinoma. The adenomas (Fig. 1E and F ) were characterized by a loss of normal alveolar architecture within a well-circumscribed focus of high epithelial density. The adenomas had an alveolar morphologic pattern. There was occasionally compression, but not invasion of adjacent tissues. The malignant neoplasms Fig. 1G and H ) obliterated the alveolar architecture by an area of high epithelial cell density with one of more features of malignancy, including atypia, anaplasia, and invasion of adjacent vessels. None metastasized outside the lung. The adenocarcinomas took several morphologic patterns: alveolar, papillary, tubular, and adenosquamous. Focal alveolar epithelial hyperplasia was common and appeared to be a precursor of the tumors.

The incidence of proliferative lesions, benign and malignant lung tumors was significantly increased in the exposed female rats, but not the male rats (Tab. 6). In the exposed females, the incidence of benign tumors was 8.6% and of malignant tumors was 4.9% for a total of 13.5% for all lung neoplasms. No lung neoplasms were found in the control rats.

Study of B6C3F1 Female Mice Exposed to Mainstream Smoke

A full presentation of the results has been shown in publications (Hutt et al. 2005; March et al. 1999). Both the daily and study average TPM concentrations were maintained very close to target concentration (254 ± 27 versus 250 mg/m³, mean ± SD of daily values). The mass median aerodynamic diameter and geometric standard deviation of the particle size was 0.59 μm, 1.32 σ g. These results indicated that the exposure concentrations were as planned and that the particles were in the respirable range.

The median survival time for the group of exposed mice was about 30 months, significantly longer than median survival time for the sham control group of about 26.5 months.

The terminal body weights (mean ± SE) were significantly lower for the exposed group (23 ± 1.9 g) than those of the sham control group (31.7 ± 0.4 g). In contrast, terminal lung weights (mean ± SE) from exposed mice (0.62 ± 0.002 g) were significantly greater than the mean terminal lung weights from the sham-control mice (0.40 ± 0.00l g).

The non-neoplastic lesions in the lungs of the mice were centered in the centriacinus as they were in the rat. The lesions included increased numbers of enlarged, often binucleate macrophages filled with smoke pigment, smoke granulomas, mild emphysema more severe than that of the rat, and numerous epithelial proliferative lesions.

The proliferative lesions included focal alveolar epithelial hyperplasia and bronchiolar epithelial hyperplasia. The alveolar hyperplasia (Fig. 2A ) was characterized by discrete foci of proliferating alveolar epithelial cells. The bronchiolar hyperplasia (Fig. 2B ) was characterized by proliferation of cuboidal or columnar cells forming small papillary protrusions into small conducting airways. Squamous metaplasia and keratinizing squamous cysts were not found.

The neoplastic lesions were pulmonary adenoma, bronchiolar papilloma, and pulmonary adenocarcinoma. The adenomas consisted of circumscribed areas of proliferating cells with distortion of the alveolar architecture and compression of adjacent parenchyma (Fig. 2C and D ). They had solid, papillary, or mixed morphologic growth patterns. The bronchiolar papillomas were characterized by fibrovascular cores lined by proliferation of cells, which projected into the lumen of the bronchioles (Fig. 2E and F ). They often appeared to arise from the epithelium of the bronchioles. These tumors were well differentiated and grew by expansion into the airways and not by invasion. The adenocarcinoma (Fig. 2G ) consisted of solid, papillary, and mixed morphologic types. They usually effaced the pulmonary architecture and had a distinct atypia and high mitotic rate. Invasion into the adjacent parenchyma, vessels, and the pleura typified many adenocarcinomas (Fig. 2H ). Local invasion of the surface of the heart, aorta, and other thoracic structures was not uncommon. Metastasis within the lung was not uncommon, occurring in 25% to 30% of mice in both sham and control groups. Extrapulmonary metastasis, however, was uncommon, 7.5% of the adenocarcinomas in the exposed mice and 11% of the adenocarcinomas in the control mice.

The incidence of proliferative lesions and benign and malignant lung tumors was significantly increased in exposed female mice (Tab. 7). In the exposed group (n = 326), the incidence of focal alveolar hyperplasia was 18%, of benign tumors (adenomas and papillomas) 22.5%, and of malignant tumors (adenocarcinomas) was 20%, for a total of 45% for all lung neoplasms. In the sham control group (n = 330) the incidence of focal alveolar hyperplasia was 1.8%, of benign tumors 6.7%, and of malignant tumors was 2.8%, for a total of 9.5% for all lung neoplasms.

DISCUSSION

These studies demonstrate that chronic whole-body inhalation exposure to mainsteam cigarette smoke can produce a statistically significant increase in benign and malignant lung tumors in female F344 rats and in female B6C3F1 mice. The main reason for the induction of lung tumors in these studies is most likely related to the greater time-integrated lung doses of particulate matter by using continuous 6 h per day, 5 days per week whole-body exposures for up to 30 months. Earlier studies using nose-only exposure systems could not achieve the high lung doses used in these two studies. For example, Chen et al. (1995) showed that the amount of radioactivity deposited in the lungs of rats exposed whole body for 40 min to radiolabled cigarette smoke was twice that deposited in the lungs of rats exposed in nose-only exposure tubes for the same length of time. In addition, the weekly exposure time (30 h) was much greater that in earlier studies using nose-only exposure. For example, 10 times the weekly exposure time used in the study by Dalby et al. (1980).

The 250 mg/m³ exposure concentration may have been near or at the maximum tolerated dose (MTD) for the female F344 rats. This exposure concentration was associated with a slight (~1 month), but significantly, reduced life span in the female rats. The MTD is generally defined as the highest dose (exposure) that does not cause life span shortening for causes other than neoplasia (Haseman and Lockhart 1994). For exposures to poorly soluble particles an additional criterion has been proposed for setting the exposure level at or below the minimum concentration causing retardation of particle clearance by overloading particle clearance mechanisms (Lewis et al. 1989). Identical exposures in a companion study were found to retard the clearance of inhaled poorly soluble plutonium-239 particles in females at both the high and low smoke exposure concentrations in females and at the high exposure concentration in males (Finch et al. 1998).

The female mice exposed to 250 mg/m³ had a median survival time significantly longer than the sham-control mice indicating that these animals were probably not at the MTD for them. The increased life span was most likely related to the reduce body weights in the smoke-exposed mice. The issue of particle overload and the reduction particle clearance do not seem to occur or be important in mice. However, the particle clearance in smoke-exposed mice has not been investigated.

The pulmonary tissue reaction of the rats, mice, and humans to the smoke inhalation is generally similar, but differences do occur. A summary of the proliferative and neoplastic lesions in the three species is show in Tab. 8. Comparisons are based on the two groups that had significant increases in pulmonary neoplasia, the female mice and the female rats and on literature reports for humans (Nikitin et al. 2004). Incidence of focal alveolar epithelial hyperplasia in the mice was double that of the rats both in controls and exposed. Keratin squamous cysts were not found in the mice, which is consistent with their absence in other carcinogenesis studies in mice. The incidence was 2.5% in the rats, which is consistent with the finding of squamous cysts in rats chronically exposed to high concentrations of relatively nontoxic diesel exhaust, carbon black, or titanium dioxide particles (Hahn 1996). Pulmonary adenomas of various morphologic patterns were also more numerous in the mice compared with the rats. No adenomas were found in the control rats, but were found in 8.6% of the exposed rats. Pulmonary adenomas were present in 6.7% of the control mice and 28% of the exposed mice. Bronchiolar papillomas were not present either the control or exposed groups of rats or in the control mice. In the exposed mice, however, the papilloma incidence was 4.5%. The tumors appear to arise from small papillary proliferations of the bronchiolar epithelium seen in many bronchioles. The adenocarcinomas were also more prominent in the control and exposed mice.

Hyperplasia and adenomas of the alveolar epithelium are common in mice and less frequent in rats and humans (Hahn 1998). The true incidence in humans is clouded by the difference in sampling at autopsy compared with rodents. Alveolar hyperplasia and adenoma are often found as incidental, and many times, multifocal lesions in patients who die from primary lung cancer. In lung cancer patients who smoked, anywhere from 10% of those with nonadenocarcinomas to 40% of those with adenocarcinomas have alveolar hyperplasia and adenomas. In contrast, 3% of lung cancer patients who never smoked had such lesions (Wistuba and Gazdar 2006). Tumors with squamous differentiation are common in human lung tumors, although decreasing in incidence with the decreasing in cigarette smoking and the changes in cigarettes in the past decades. Squamous cysts, seen in the rats exposed to cigarette smoke, have not been reported in humans (Boorman et al. 1996). These lesions are not analogous to the squamous cell carcinomas seen in humans exposed to cigarette smoke. Metastases of lung tumors to sites outside of the thoracic cavity are uncommon in mice and rare in rats. In contrast, human lung cancers are frequently aggressive and tend to metastasize widely throughout the body.

These results show that a significant increase in lung neoplasms can be induced in both rats and mice by prolonged exposure to high concentrations of mainstream cigarette smoke (Hecht 2005). The neoplastic lesions are accompanied by proliferative lesions of the pulmonary parenchyma that are undoubtedly precursors of the neoplasms. The results also lead the way to further studies of cigarette smoking in rats and mice for determining the mechanism of carcinogenesis, for development of chemopreventative agents, and for assessment of the effects of cigarette smoking when combined with other potential or known lung carcinogens.