Acute tobacco smoke-induced airways inflammation in spontaneously hypertensive rats.

Common laboratory rats and mice fail to develop persistent, progressive pulmonary inflammation found in chronic obstructive pulmonary disease as a result of tobacco smoke exposure. We hypothesized that spontaneously hypertensive rats would be more susceptible than normal Wistar Kyoto rats to acute tobacco smoke-induced pulmonary inflammation due to impaired apoptosis. Spontaneously hypertensive rats display systemic oxidative stress, inflammation, hypercoagulation, and immunosupression, similar to humans with chronic obstructive pulmonary disease. Male spontaneously hypertensive rats and Wistar Kyoto rats were exposed whole-body to tobacco smoke (total particulate concentration 75-85 mg/m(3)) or filtered air for 6 h/day for 2 or 15 days (3 days/wk). Tobacco smoke caused an increase in bronchoalveolar lavage fluid neutrophils at both time points in each strain. Significantly more neutrophils were noted in spontaneously hypertensive rats at 15 days compared to Wistar Kyoto rats. There was a trend of increase for macrophages in spontaneously hypertensive rats at both time points (significant at 2 days). TUNEL assay detected apoptotic cells in bronchoalveolar lavage fluid and lung tissue sections. The number of apoptotic neutrophils in airway walls and bronchoalveolar lavage fluid increased at 2 days in both strains, but at 15 days the effect was much lower in spontaneously hypertensive rats than in Wistar Kyoto rats. Tobacco smoke induces a greater inflammatory response associated with lower apoptotic neutrophils in the lungs of spontaneously hypertensive rats compared to Wistar Kyoto rats. The spontaneously hypertensive rat may be a more relevant animal model of acute tobacco smoke-induced airway inflammation than other laboratory rats.

A review of whole animal bioassays of the carcinogenic potential of naphthalene.

This report provides a summary of deliberations conducted under the charge for members of Module A participating in the Naphthalene State-of-the-Science Symposium (NS3), Monterey, CA, October 9-12, 2006. Whole animal bioassays have been performed by the National Toxicology Program in mice and rats to ascertain the carcinogenic potential of naphthalene by inhalation exposure. A statistically significant increased incidence of pulmonary alveolar/bronchiolar adenoma (a benign lesion), was observed among female mice; an observed increase among the males did not reach statistical significance. No nasal tumors were observed in either sex. A tumorigenic response was observed in both sexes of rats, in males an increased incidence of nasal respiratory epithelium adenoma (a benign rather than malignant lesion) and in females, olfactory epithelial neuroblastoma. Interpretations of these studies vary. On the one hand, evidence of extensive non-neoplastic response in both sexes of both species indicates cytotoxicity occurred at all doses, and strongly suggests that cytotoxicity played a significant role in the tumor responses observed in the target tissues. On the other hand, olfactory epithelial neuroblastoma has rarely been observed in NTP bioassays. This review seeks to develop a consensus understanding of the scientific evidence provided by these studies, taking into account that they have been used as the basis for quantitative human cancer risk assessment, and suggests scientific studies that, if performed, could resolve scientific uncertainties.

Lung tumors in 2 year old strain A/J mice exposed for 6 months to tobacco smoke.

Young adult strain A/J mice were exposed for 6 months in a whole-body inhalation chamber to a mixture of 89% sidestream and 11% mainstream cigarette smoke generated from Kentucky 1R4F research cigarettes. Chamber concentrations of smoke constituents were 158mg/m(3) of total suspended particulate matter (TSP). After an additional 4 months in air, some of the animals were killed. Lung tumor multiplicities in the smoke exposed animals were 1.8+/-0.2 versus 0.9+/-0.2 in controls. In animals kept beyond the age of 12 months, lung tumor multiplicities increased in both groups, but remained at all times twice the control values in the smoke exposed animals compared to controls (4.3+/-0.7 vs. 2.1+/-0.5 tumors per lung in 24 months old animals). Histopathology showed that, in 2 year old animals, still about 80% of tumors were of benign nature. No tumors were found in the nasal passages. It was concluded that tobacco smoke exposure not simply accelerates the development of lesions that eventually would have developed spontaneously, but induced de novo formation of lung tumors in A/J mice.

Carcinogenic activity of cigarette smoke gas phase and its modulation by beta-carotene and N-acetylcysteine.

Male strain A/J mice were exposed for six hours a day, five days a week for six months to either full tobacco smoke or to tobacco smoke drawn through a HEPA filter that removed more than 99% of particulate matter. After another four months in air, the animals were sacrificed and lung tumors were counted for calculation of multiplicities and incidences. Analysis of the chamber atmospheres showed that in the filtered smoke the concentrations of polycyclic aromatic hydrocarbons and tobacco smoke specific nitrosamines were reduced to from below 18% to even nondetectable levels of the original values measured in the unfiltered smoke. Aldehydes and other volatile organic compounds such as 1,3-butadiene, benzene, or acrolein were reduced to about 50 to 90% of the concentrations found in unfiltered smoke. Some potentially carcinogenic metals reached levels in filtered smoke ranging from 77% to less than 1% found in full smoke. The mice exposed to the filtered smoke atmosphere had practically identical lung tumor multiplicities and incidence as had the animals exposed to full smoke, significantly higher than in air exposed controls. Diets containing 0.5% beta-carotene or 0.4% N-acetylcysteine afforded some chemoprevention. It was tentatively concluded that 1,3-butadiene might be an important contributor to lung tumorigenesis in this mouse model of tobacco smoke carcinogenesis.

The complexities of an apparently simple lung tumor model: The A/J mouse.

A simple animal model of tobacco smoke carcinogenesis works as follows: Strain A/J mice are exposed for 5 months to tobacco smoke. They are then given a 4-month recovery period in air before being killed. Lung surface tumors are counted and lung tumor multiplicity (average number of tumors per lung, including non-tumor bearing animals) is calculated. Results obtained in four different laboratories during the past 8 years have consistently shown significant increases in lung tumor multiplicities in tobacco smoke exposed animals. While inhaling to tobacco smoke, strain A mice (but not some other strains) fail to gain weight and immediately after smoke exposure only have about 75% of control weight; however, when removed into air, they regain weight rapidly up to control levels. The counting of surface tumors only may occasionally underestimate total number of lung tumors and thus yield false negatives. At the end of the experiment, the mice are 1-year old and about 80% of the tumors are adenomas, the remainder adenomas with carcinomatous foci or adenocarcinomas. Tobacco smoke does not increase the percentage of adenocarcinomas. Studies with filtered tobacco smoke have suggested that benzo(a)pyrene or tobacco smoke-specific nitrosamines cannot account for lung carcinogenesis in mice; the most likely single agent to cause lung tumors is 1,3-butadiene. A major disadvantage of the assay is its low statistical power. While it is easy to detect a 70-100% decrease in lung tumor multiplicity caused by a chemopreventive agent using group sizes of 20-30 animals, the detection of smaller reductions (20-50%) would require group sizes in the hundreds. From all available evidence it must be concluded that the complex mixture of tobacco smoke, a known human carcinogen, is a rather weak rodent carcinogen.

Development of tobacco smoke-induced lung tumors in mice fed Bowman-Birk protease inhibitor concentrate (BBIC).

Male strain A/J mice were exposed, 6h a day, 5 days a week for 5 months to a mixture of 89% cigarette sidestream and 11% cigarette mainstream smoke and then allowed to recover for another 4 months in air. The animals were fed Bowman-Birk protease inhibitor concentrate (BBIC) at a concentration of 1% in AIN-93G diet either during smoke exposure, following smoke exposure or during the entire 9 months. At the end of the experiment, the incidence and multiplicity of lung tumors were determined. In a positive control experiment, strain A/J mice were injected with 3-methylcholanthrene (MCA) and fed a diet containing 1% BBIC; these animals were killed 5 months later. It was found that in the animals treated with MCA, BBIC decreased lung tumor multiplicities, whereas in the smoke exposed mice, BBIC did not modulate lung tumor development.

Short-term exposure to aged and diluted sidestream cigarette smoke enhances ozone-induced lung injury in B6C3F1 mice.

To determine the effects of aged and diluted sidestream cigarette smoke (ADSS) as a surrogate of environmental tobacco smoke (ETS) on ozone-induced lung injury, male B6C3F1 mice were exposed to (1) filtered air (FA), (2) ADSS, (3) ozone, or (4) ADSS followed by ozone (ADSS/ozone). Exposure to ADSS at 30 mg/m3 of total suspended particulates (TSP) for 6 h/day for 3 days, followed by exposure to ozone at 0.5 ppm for 24 h was associated with a significant increase in the number of cells recovered by bronchoalveolar lavage (BAL) compared with exposure to ADSS alone or ozone alone. The proportion of neutrophils and lymphocytes, as well as total protein level in BAL, was also significantly elevated following ADSS/ozone exposure, when compared with all other groups. Within the centriacinar regions of the lungs, the percentage of proliferating cells identified by bromodeoxyuridine (BrdU) labeling was unchanged from control, following exposure to ADSS alone, but was significantly elevated following exposure to ozone (280% of control) and further augmented in a statistically significant manner in mice exposed to ADSS/ozone (402% of control). Following exposure to ozone or ADSS/ozone, the ability of alveolar macrophages (AM) to release interleukin (IL)-6 under lipopolysaccharide (LPS) stimulation was significantly decreased, while exposure to ADSS or ADSS/ozone caused a significantly increased release of tumor necrosis factor alpha from AM under LPS stimulation. We conclude that ADSS exposure enhances the sensitivity of animals to ozone-induced lung injury.

A mouse lung tumor model of tobacco smoke carcinogenesis.

We examined the possibility of developing an animal model of tobacco smoke carcinogenesis. Male Balb/c and SWR mice were exposed for 5 months to tobacco smoke (6 h/day, 5 days/week; average concentration, 122 mg/m(3) of total suspended particulates [TSP]) followed by a recovery period of 4 months in air. In both strains there was an increase in lung tumor multiplicities and incidence, although statistical significance was only observed with lung tumor multiplicity in the SWR mice. An analysis of 11 previous and independently conducted assays with strain A/J mice that followed the same protocol was performed. In each experiment, lung tumor multiplicities were significantly higher in tobacco smoke-exposed mice compared with air-exposed controls, and a good correlation between exposure (average tobacco smoke concentrations multiplied by length of exposure in months) and lung tumor multiplicities was found. In 7 experiments involving tobacco smoke concentrations greater than 100 mg/m(3) of TSP, lung tumor incidences were 5 times higher than in control mice. Tobacco smoke-exposed mice had a smaller percentage of adenomas with carcinomatous foci or adenocarcinomas than air-exposed controls, and no differences between the two groups were found in an analysis of Ki-ras mutations. After 6 h of exposure to tobacco smoke, plasma cotinine levels in mice were comparable to those found in active human smokers. The lung tumor model might be suitable for future evaluation of chemopreventive agents or modified tobacco products.

The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants.

This study was designed to examine the role of the cytokine interleukin-6 (IL-6) in environmental air pollutant-induced pulmonary inflammation, injury, and repair. IL-6 knockout (KO) mice and wild-type (WT) mice were exposed to filtered air; aged and diluted cigarette smoke (ADSS), a surrogate for environmental tobacco smoke; ozone; or ADSS followed by ozone (ADSS/ozone). The proportion of monocytes and neutrophils recovered by bronchoalveolar lavage (BAL) as well as the level of total protein in BAL fluid were significantly increased in both IL-6 KO and WT mice following exposure to ozone or to ADSS/ozone. However, bromodeoxyuridine (BrdU) labeling within terminal bronchiolar epithelium and proximal alveolar regions in IL-6 KO mice exposed to ozone or to ADSS/ozone was significantly reduced compared with IL-6 sufficient mice (WT). WT mice treated with IL-6 antibodies also demonstrated a reduction in BrdU cell labeling similar to that observed in IL-6 KO mice following exposure to ozone or ADSS/ozone. Clara cell secretory protein (CCSP) abundance, a marker of Clara cell maturation and function, was markedly reduced in the terminal bronchiolar epithelium of WT mice following exposure to ADSS and/or ozone, whereas CCSP abundance was unchanged in IL-6 KO mice. We conclude that endogenous IL-6 in mice plays a critical role in the progress of lung inflammation/injury, but CCSP may also play a role to protect the lungs of mice exposed to toxic air pollutants. Data from this study further suggest that IL-6 antibody treatment modalities may be a means to attenuate pulmonary inflammation and injury.

Lung tumor development in mice exposed to tobacco smoke and fed beta-carotene diets.

In human clinical trials it was found that the putative chemopreventive agent beta-carotene not only failed to protect active smokers against the carcinogenic action of tobacco smoke, but actually increased their risk of developing lung cancer. In preclinical animal studies, beta-carotene had been effective against some chemically induced cancers, but not against tumors in the respiratory tract. We exposed male strain A/J mice to tobacco smoke at a concentration of 140 mg/m(3) of total suspended particulate matter, 6 h a day, 5 days a week, for either 4 or 5 months, followed by a recovery period in air for 4 or 5 months, or for 9 months without recovery period. beta-carotene was added in the form of gelatin beadlets to the AIN-93G diet either during or following tobacco smoke exposure at concentrations of 0.005, 0.05 and 0.5%. In the supplement-fed animals, plasma and lung levels of beta-carotene were higher than they were in animals fed control diets. Exposure to tobacco smoke increased rather than decreased plasma beta-carotene levels, but had no significant effect on lung levels. After 9 months, lung tumor multiplicities and incidence were determined. Tobacco smoke increased both lung tumor multiplicities and incidences, but beta-carotene failed to modulate tumor development under all exposure conditions. Animal studies in a model of tobacco smoke carcinogenesis would thus have predicted the absence of any beneficial effects of beta-carotene supplementation in current or former smokers, but would have failed to anticipate the increase in lung cancer risk.

Gestation stage-specific oxidative deoxyribonucleic acid damage from sidestream smoke in pregnant rats and their fetuses.

Transplacental exposure to environmental tobacco smoke (ETS) is a possible cancer risk factor in offspring. The authors exposed pregnant Sprague-Dawley rats to a relevant dose of ETS (1 mg/m3) from gestation day 4 to days 16 or 21. They then assayed tissues for levels of 8-oxo-2'-deoxyguanosine (8-oxo-dG), a marker of oxidative deoxyribonucleic acid damage. ETS exposure ending on gestation day 16 resulted in statistically significant increases in 8-oxo-dG in maternal liver and kidney and in fetal kidney. On gestation day 21, there were significant 8-oxo-dG increases in fetal liver and brain. These gestational stage- and tissue-specific increases of 1.2- to 1.4-fold are similar to the putative relative increases in risk of human cancers related to ETS.

Tobacco smoke-induced lung cancer in animals--a challenge to toxicology (?).

Tobacco smoke is a known human carcinogen that primarily produces malignant lesions in the respiratory tract, although it also affects multiple other sites. A reliable and practical animal model of tobacco smoke-induced lung cancer would be helpful for in studies of product modification and chemoprevention. Over the years, many attempts to reproduce lung cancer in experimental animals exposed to tobacco smoke have been made, most often with negative or only marginally positive results. In hamsters, malignant lesions have been produced in the larynx, but not in the deeper lung. Female rats and female B6C3F1 mice, when exposed over lifetime to tobacco smoke, develop tumors in the nasal passages and also in the lung. Contrary to what is seen in human lung cancers, most rodent tumors are located peripherally and only about half of them show frank malignant features. Distant metastases are extremely rare. Male and female strain A mice exposed to 5 months to tobacco smoke and then kept for another 4 months in air respond to tobacco smoke with increased lung tumor multiplicities. However, the increase over background levels is comparatively small, making it difficult to detect significant differences when the effects of chemopreventive agents are evaluated. On the other hand, biomarkers of exposure and of effect as well as evaluation of putative carcinogenic mechanisms in rats and mice exposed to tobacco smoke allow detection of early events and their modification by different smoke types or chemopreventive agents. The challenge will be to make such data broadly acceptable and accepted in lieu of having to do more and more long term studies involving larger and larger number of animals.

A/J mouse as a model for lung tumorigenesis caused by tobacco smoke: strengths and weaknesses.

Strain A/J mice have successfully been used to develop an animal model for tobacco smoke carcinogenesis. In 18 individual studies, reported by 4 different laboratories, a significant increase in lung tumor multiplicities following exposure from 50 to 170mg/m3 of total suspended tobacco smoke particulates was found in 15 studies (83 %) and a significant increase in lung tumor incidence in 10 studies (56%). However, tumor multiplicities are comparatively low (from an average of 1.1 to 2.8 tumors per lung). From a toxicological standpoint, this indicates that cigarette smoke is a weak animal carcinogen. Although the assay allowed one to detect substantial chemopreventive activity of a mixture of myo-inositol and dexamethasone, it was less successful in showing efficacy for several other agents.

The chemopreventive effects of orally administered dexamethasone in Strain A/J mice following cessation of smoke exposure.

Male Strain A/J mice were exposed for 6 mo, 6 h/d, 5d/wk to a mixture of cigarette sidestream and mainstream smoke with an average total suspended particulate concentration of 156 mg/m3. They then were removed into air and fed diet AIN93M containing 0.5 mg/kg of dexamethasone until killed 4 mo later for the evaluation of lung tumor multiplicities. In animals kept in air, an average of 1.3 tumors per lung was found, and in tobacco-smoke-exposed animals the average number of tumors per lung was 2.2 (p<.05). Addition of dexamethasone to the diet reduced lung tumor multiplicities in the tobacco smoke exposed animals to 1.4 (64% of control values), not quite statistically significant. In animals not exposed to tobacco smoke, however, dexamethasone significantly decreased lung tumor multiplicities to 46% of control values. In animals injected with the tobacco-smoke-specific carcinogen NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], dietary dexamethasone significantly reduced lung tumor multiplicities to 38% of controls. It is concluded that the dietary intake of dexamethasone against full tobacco smoke might show improved chemopreventive activity when combined with other agents.

MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and squamous metaplasia in the lungs of rats.

Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m(3) or 80 mg/m(3), 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers.

Lung tumors in A/J mice exposed to environmental tobacco smoke: estimated potency and implied human risk.

Directly inhaled tobacco smoke is a recognized human lung carcinogen, and epidemiological studies suggest relative risks of about 1.2-1.4 for nonsmoking spouses of smokers typically exposed to environmental tobacco smoke (ETS). While many individual ETS components have been shown experimentally to induce lung tumors, ETS itself was only recently shown to induce lung tumors in a series of studies in which strain A/J mice were exposed to well-defined ETS atmospheres. Data from these studies indicate that ETS exposure clearly can increase combined malignant and benign lung tumors in multiple experiments involving male and female A/J mice, and thus provide convincing evidence that ETS is a positive mouse carcinogen. Tumorigenic potencies estimated from these A/J mouse bioassay data predict a corresponding range of increased human risk (0.2-0.5%) that overlaps that implied by case-control studies showing increased lung cancer risks in lifelong nonsmokers married to smokers. In A/J mice exposed to a significantly tumorigenic ETS concentration, lung tumors were found to be significantly smaller than those in corresponding control mice, and mice so exposed for 9 months had significantly fewer tumors/animal than mice exposed for 5 months followed by 4 months in filtered ETS-free air. These findings support hypotheses that ETS does not promote growth of spontaneous neoplastic foci in A/J mice, and that ETS-induced lung-tumor risk in A/J mice occurs predominantly by genotoxic effects that can be suppressed by reduced cell proliferation associated with chronic, high-level ETS exposure. The results obtained add to evidence that A/J mouse lung tumors induced by ETS provide a relevant biological model of ETS-induced human lung tumors.