* Center for Health and the Environment, University of California, Davis, One Shields Avenue, Davis, California 95616; and
Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received January 18, 2002; accepted April 8, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: tobacco smoke; lung tumors; strain A/J mice; Balb/c mice; SWR mice; plasma cotinine; K-ras mutations; dose-response relationship; tobacco smoke inhalation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Attempts to duplicate the human experience in experimental animals have, for the most part, been unsuccessful. Mohr and Reznik (1978, p. 347), in their comprehensive review of the literature, concluded that, despite an enormous amount of work, "no researcher has succeeded as yet in producing a significant incidence of pulmonary tumors." In 1986 the International Agency for Research on Cancer (IARC) summarized the then-available evidence and found that of 4 rat studies judged to be adequate for critical analysis only one yielded unequivocal evidence for the carcinogenicity of tobacco smoke. Tumor incidence in the exposed group was below 10%. Hamsters developed laryngeal tumors but no tumors in the lower respiratory tract. Several mouse studies discussed in the IARC document failed to provide clear evidence for carcinogenicity by tobacco smoke, as did another large study conducted in a single laboratory (Henry and Kouri, 1986). Coggins, in a 1998 review of 14 chronic animal inhalation studies with mainstream cigarette smoke, indicated that "significant increases in the numbers of malignant tumors were not produced in the respiratory tract of rats or mice exposed chronically to inhalation of cigarette smoke" (Coggins, 1998
, p. 313).
It is acknowledged that there probably will always be a certain population of smokers who are unable or unwilling to quit. Efforts are underway to find new ways to achieve tobacco harm reduction. Along these lines, the tobacco industry has made major efforts in product improvement through the development of a less harmful cigarette (Wagner et al., 2000). For several reasons, it is desirable to have an animal model of tobacco smoke carcinogenesis. A committee of the Institute of Medicine recommended that animal models be used to test the potential adverse health effects of tobacco smoke or any proposed additives (Stratton et al., 2001
). Animal models of tobacco smoke carcinogenesis might also be useful in preclinical testing of chemopreventive agents. For example, animal studies with tobacco smoke showed that it is not always possible to predict effective chemoprevention if putative chemopreventive agents are only evaluated in lung tumor models induced by constituents of tobacco smoke (Witschi, 2000
). Animal studies showed that ß-carotene (Obermueller-Jevic et al., in press
) and N-acetylcysteine (Witschi et al., 1998
) are ineffective against tobacco smoke, an outcome that would have predicted the disappointing results of two major clinical trials (Omenn, 1998
; van Zandwijk et al., 2000
). Finally, it might be possible to develop risk assessments for environmental tobacco smoke exposure by complementing data from epidemiological studies with dose-response information derived from animal experiments. A recent attempt showed a surprisingly good correlation between tumorigenic potency of environmental tobacco smoke in animals and increased risk derived from human case-control studies (Bogen and Witschi, 2002
).
During the last few years, we have developed a murine model of tobacco smoke-induced lung carcinogenesis. The model uses strain A/J mice exposed to a mixture of cigarette sidestream and mainstream smoke. A key element in increasing the development of lung tumors after exposure to tobacco smoke was the design of a nonconventional protocol. Instead of being exposed for their entire lifespan to the smoke, the mice are exposed to tobacco smoke for 5 months only. After cessation of smoke exposure, the mice are allowed to recover in air for another 4 months before being evaluated for tumor development (Witschi et al., 1997a,1997b
). The results of these studies have been confirmed in a different laboratory (DAgostini et al., 2001a
). In the current study, we add evidence that a similar protocol might be successfully used in other mouse strains, and we present an overall analysis of 11 previous and independently conducted experiments using the strain A/J mouse model.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental Design for Carcinogenesis Studies
In most experiments, the original protocol for evaluation of tobacco smoke carcinogenesis was followed (Witschi et al., 1997a). Mice were exposed for 5 months (6 h/day, 5 days/week) to the tobacco smoke and then allowed to recover for another 4 months in air. In two additional experiments, the mice were kept for 9 months in tobacco smoke; concomitant controls were kept in air throughout. At the end of the experiment, they were killed by pentobarbital overdose. For analysis of tumor incidence and multiplicity, the lungs were manually expanded to inspiratory volume by intratracheal instillation of Tellyesniczkys fluid and fixed for at least 24 h in the same fixative before being transferred into 70% ethanol. After counting the tumors, a few lungs were selected and embedded in paraffin. Sections 5 µm thick were stained with hematoxylin-eosin (H&E) and examined under light microscope.
Exposure System
The tobacco smoke exposure system was identical to that used in our previous studies (Witschi et al., 1997a,1997b
,1998
,1999
,2000
). Briefly, mice were exposed to a mixture of 89% sidestream and 11% mainstream smoke generated from burning Kentucky 1R4F reference cigarettes (purchased from the Tobacco Research Institute, University of Kentucky, Lexington, KY). Chamber atmospheres were monitored for nicotine, CO, and total suspended particulates (TSP). Within the exposure chambers, all cages were periodically rotated so that each cage occupied at least once all possible locations within the exposure chambers. After an initial acclimatization period of 5 weeks involving increasing concentrations of tobacco smoke within the chamber, tobacco smoke concentrations used for the individual experiments ranged from 50 and 150 mg/m3 of TSP. The TSP concentrations for the individual experiments are listed in Tables 1 and 2
.
|
|
A 2:l aliquot of the lysate was used for the PCR. All of the reactions were carried out in a 100-µl reaction volume that consisted of reaction buffer (10 mmol Tris HCl, pH 8.0, 2.5 mmol MgCl2, 50 mmol KCl), 200 µM dNTPs, and two units of AmpliTaq Gold (Perkin Elmer Life Sciences, Boston, MA). Amplimers for exons 1 or 2 of the Ki-ras gene were added at a final concentration of 0.2 µM. The samples were overlaid with 100 µl of mineral oil and amplified by the standard procedure of Saiki et al.(1988). After denaturation for 2 min at 94°C, the samples were amplified by 40 cycles of denaturation for 1 min at 94°C, annealing for 2 min at 55°C, and extension for 2 min at 72°C, followed by a final extension step for 7 min at 72°C. Primers for exons 1 and 2 of Ki-ras were synthesized by Integrated DNA Technologies (Corallville, IA). Primer sequences for exon 1 were forward 5`-ATGACTGAGTATAAACTTGT, and reverse 5`-TCGTACTCATCCACAAAGTG, which produced a 98-bp fragment; primer sequences for exon 2 were forward 5`-TACAGGAAACAAGTAGTAATTGATGGAGAA, and reverse 5`-ATAATGGTGAATATCTTCAAATGATTTAGT, which produced a 171-bp fragment.
Each reaction included deparaffination procedure controls, which lacked tumor tissue but were mock extracted and taken through the entire protocol, and negative buffer controls for the PCR amplification reactions. All samples were amplified in a BioRad iQCycler thermocycler. The sizes of the PCR products were confirmed using 2% agarose gel.
Allele-specific oligonucleotide hybridization (ASO).
Thirty microliters of PCR products, diluted in 170 µl of sterile water, were heat denatured and blotted directly onto a Nytran membrane filter (Schleicher & Schuell, Keene, NH) using a Schleicher & Schuell minifold II slot blot apparatus. The amplified DNA products were fixed to the membrane by ultraviolet crosslinking, and the filters were prehybridized in 5 x SSC, pH 7.0, 50 mmol sodium phosphate, pH 7.0/5 x Denhardts solution, and 0.5% SDS/100 µg/ml of salmon sperm DNA at 37°C for 1.5 h. The filters were hybridized overnight in the same buffer containing 5 x 106 cpm/ml of a 20-bp oligonucleotide to mouse K-ras codons 12, 13, or 61 (Clontech, Palo Alto, CA). The oligomers were 5`-end-labeled with phosphorus 32 to a specific activity of more than 107 cpm/pmol using T4 polynucleotide kinase. After hybridization, the filters were washed under stringent conditions (3°C below the Tm) that allowed only fully matched probes to remain bound to DNA (Mattes and Miller, 2000; Miller et al., 1994
,2000
). The blots were visualized on a Molecular Dynamics PhosphorImager 445SI (Sunnyvale, CA). Results are based on two independent amplifications from the lysates to ensure that none of the observed mutations were the result of Taq-induced errors.
The Ki-ras gene was sequenced directly from the initial PCR products by the Wake Forest University School of Medicine DNA Sequencing and Gene Analysis Facility. Direct PCR sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Life Sciences) according to the manufacturers instructions. The DNA sequences was analyzed using DNASIS software (Hitachi Software Engineering America, Ltd., San Bruno, CA).
Plasma Cotinine Levels
A/J mice were exposed for 6 h to three different tobacco smoke concentrations (80, 120, and 160 mg/m3 of TSP). Immediately after tobacco exposure, half of the mice were deeply anesthetized with pentobarbital for blood collection. The other half were allowed to survive for another 18 h before being killed for blood collection. Plasma cotinine levels were measured using a commercially available enzyme-linked immunosorbent assay kit (Cotinine Serum Micro-Plate EIA, STC Technologies, Inc., Bethlehem, PA).
Statistical Analysis
For tumor analysis, the number of nodules visible on the lung surface were counted and the results expressed as tumor incidence (i.e., percentage of mice with one or several lung tumors) and tumor multiplicity (the average number of tumors per lung, including non-tumor-bearing animals). All numerical data were calculated as mean ± SD or SE. Comparisons of tumor multiplicity between tobacco smoke-exposed mice and air-exposed controls were made by by Welchs alternate test or, in the case of multiple comparisons, by analysis of variance followed by the Tukey-Kramer test. Tumor incidences were compared using Fishers exact test. A p value of 0.05 or less was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The results of this analysis are shown in Figure 2 and Table 2
. It can be seen that whenever we used the protocol in which mice were exposed to tobacco smoke first and then given a recovery period in air (split protocol), amounting to exposure-months ranging from 133 to 740, a good dose-response relationship was observed between exposure-months and lung tumor multiplicity (Fig. 2
). In every single one of the 11 experiments, the difference between the controls and the tobacco smoke-exposed mice was statistically significant (p < 0.05). Linear regression indicated a statistically significant increase in tumor multiplicity with dose (p < 0.01). However, in mice that were kept for the full 9 months in tobacco smoke (exposure-months of 1250 and 1350) and not allowed to recover in air, lung tumor multiplicities were not higher than in mice subjected to the split protocol, although their "dose" was almost twice as high. Lung tumor incidences for the experiments involving exposure to tobacco smoke followed by a recovery period in air are listed in Table 2
. In all experiments, lung tumor incidence was higher in the tobacco smoke-exposed mice (range, 58100%) than in the corresponding controls (range, 4280%). In the mice exposed to tobacco smoke concentrations below 100 mg/m3 of TSP, differences were not statistically significant, whereas in those exposed to higher concentrations (>100 mg/m3 of TSP), differences were statistically significant in 5 of 7 experiments.
|
|
|
Plasma Cotinine Concentrations
Plasma cotinine concentrations were measured in mice exposed for 6 h to tobacco smoke and then again in mice that had been removed from the smoke atmosphere for 18 h. The data are given in Table 5. Plasma cotinine levels were dose-dependent and fell once the mice were removed from the smoke atmosphere.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Murine lung tumors, on the basis of site of origin and histology, do not resemble human bronchial squamous cell carcinoma. Mouse lung tumors develop in the peripheral lung first as areas of hyperplasia and then grow into adenomas, eventually progressing to adenocarcinomas. At 1 year of age, approximately 80% of the tumors are adenomas and 20% are adenomas containing carcinomatous areas or are fully developed adenocarcinomas with solid, papillary, or mixed growth pattern (Witschi et al., 1997a). Invasion of adjacent tissues, blood vessels, or lymphatics by malignant cells is seen on occasion. If the mice were allowed to live out their lifespan, the tumors eventually would fill entire lobes and kill them. At this stage, all tumors are carcinomas (Foley et al., 1991
). The tumors thus resemble human lung adenocarcinoma, which has increased in incidence and by now is the most prevalent histological type of lung tumor observed clinically in the United States (Thun et al., 1997
). Existing mechanistic information on the multiple similarities between human and murine lung tumors in biochemistry, signal transduction pathways, and alterations in oncogenes reinforces the postulate that mouse lung tumors are a good model for adenocarcinoma of the human lung (Malkinson, 1998
). Lung tumors in strain A mice have become the most widely used animal model for the evaluation of chemopreventive agents (Stoner, 1998
).
A key element in the successful model development was a split protocol in which the mice were exposed to tobacco smoke first and then given a recovery period in air. This approach can be justified by the following observations. In mice exposed to tobacco smoke from 3.5 to 5 months and then given a recovery time in air, there is a very good dose response to the product of smoke concentration and length of exposure (see Figure 2 and Table 2
). Mice exposed to tobacco smoke for the full 9 months and not allowed to recover in air have a response that is similar to, but not higher than, that in mice subjected to the split protocol. This could indicate that inhalation of tobacco smoke not only initiates the tumorigenic process but also interferes with tumor progression. The data in Table 4
, presenting distribution of tumor types in several hundred specimens, show that tobacco smoke delays tumor progression: Significantly fewer carcinomas have been diagnosed in mice exposed to tobacco smoke compared with those kept in air. Interestingly, a similar effect on tumor progression could be achieved with chemopreventive agents (Conaway et al., 1998
). Several observations might explain the basically counterintuitive finding of tobacco smoke interfering with tumor development (DAgostini et al., 2001a
). One possibility is that smoke-induced stress imposed on the mice, coupled with caloric restriction, interferes with lung tumor development in strain A mice (Droms and Malkinson, 1991
; Pashko and Schwartz, 1996
). However, Figure 3
indicates that body weight, at least as determined at the end of an experiment, is not correlated with lung tumor multiplicity. Tobacco smoke is capable of inducing apoptosis in the epithelial cells of mice and rats exposed to tobacco smoke (DAgostini et al., 2001b
) as well as in human pulmonary cells (Vayssier et al., 1998
). Increased apoptosis could slow down tumor development by eliminating cells containing mutagenic and potentially procarcinogenic lesions, as might cytotoxicity caused by selected tobacco smoke constituents (e.g., acrolein). It remains speculative how this would apply to the natural history of lung cancer in smokers. It is interesting to note that, immediately after smoking cessation, former smokers are, for a few years, actually at higher risk for lung tumors than current or never smokers, although eventually the risk decreases (Wynder and Stellman, 1977
; Postmus, 1998
). In former smokers, the initial removal of the cytotoxic or apoptosis-enhancing elements present in tobacco smoke might allow tumors to proliferate more rapidly than they would in the presence of tobacco smoke. This should, of course, not be construed to imply that to quit smoking is not beneficial; it is. However, it might indicate that attention to thorough diagnostics and perhaps chemopreventive measures should particularly be directed toward former smokers.
Several studies have shown that mutations in K-ras are a frequent and early event in both murine and human lung adenocarcinomas (You et al., 1989). Studies using transgenic mice have clearly shown the importance of mutated K-ras genes for the initiation and maintenance of lung cancer (Fisher et al., 2001
). In addition, mutation in the K-ras gene shows a strong association with tobacco smoke in patients with adenocarcinoma of the lung (Ahrendt et al., 2001
). We have previously found that exposure to tobacco smoke at comparatively low concentrations (4 mg/m3 of TSP), which did not produce an increased tumor response, seemed to target mutations of the Ki-ras gene to exon 2 for mutations (Witschi et al., 1995
). We repeated this analysis in selected tumors in mice that had been exposed to a much higher, tumorigenic concentration of tobacco smoke (130 mg/m3 of TSP). We were surprised to find no difference in the mutation spectrum between air-exposed and smoke-exposed mice. Interestingly, we found no mutations in exon 1 in the air-exposed mice in this small study, thus accounting for the apparent lack of shift in the mutational spectrum observed previously (Witschi et al., 1995
). Several studies have shown that different carcinogens induce specific mutations at the Ki-ras gene locus (You et al., 1989
). Thus, although our result can be explained by the fact that the number of tumors examined in the current study was too small to detect a significant difference between the two treatment groups (9 tumors each in the control and smoke-exposed mice), the notion must also be entertained that Ki-ras mutations are not as dominant an event in tobacco smoke carcinogenesis in mouse lung tumors as is often assumed. In addition, because we selected the largest tumors for molecular analysis, we may have biased our study by specifically examining those tumors most likely to harbor the Arg61 mutant allele. Alternatively, it is also possible that tobacco smoke may promote the outgrowth of spontaneously mutated lesions in the A/J lung already predisposed to develop Ki-ras mutations. In the latter case, one would not expect the mutational spectrum between the air- and smoke-exposed mice to differ. Studies using the transplacental administration of the polycyclic aromatic hydrocarbon, 3-methylcholanthrene, to crosses between C57BL/6 and DBA mice (Leone-Kabler et al., 1997
; Miller et al., 2000
) or to Balb/c mice (Gressani et al., 1999
; Miller et al., 2000
) have documented differences in the Ki-ras mutational spectrum as a function of the histological stage of the tumors, organ site, and mouse strain. Further studies with larger numbers of tumors from different strains of mice are required to determine whether smoke exposure does have an effect on the mutational spectrum of the Ki-ras gene or merely promotes the proliferation of cells with preexisting mutations at this locus.
The plasma concentrations of the nicotine metabolite cotinine were evaluated after a single-day exposure to three different concentrations of tobacco smoke. The results were clearly dose-dependent. Plasma cotinine levels of approximately two to three times that in our mice (258273 ng/ml) were found in an experiment in which C57BLl/6j pun/pun mice were exposed for 4 h to similar concentrations of tobacco smoke (100 mg/m3 of total particulate matter; Jalili et al., 1998). The cotinine levels measured in our experiment correspond closely to cotinine levels found in active smokers and are much higher than those observed in passive smokers (Peacock et al., 1998
). This observation is not too surprising in view of the chamber concentrations of tobacco smoke used in the current experiments. The decline in plasma cotinine levels from the period immediately after smoking cessation to 18 h later was about the same as seen in smokers who quit smoking for a comparable length of time (Benowitz et al., 1983
).
The mouse lung tumor assay could help in the evaluation of the eventual full carcinogenic potential of new or modified tobacco products. To date, such information is usually developed by using mutagenesis assays and skin-painting studies (Wagner et al., 2000). Mouse skin tumors resemble lung tumors inasmuch as they develop in particularly sensitive mouse strains (e.g., SENCAR mice) and progress from benign papillomas to malignant neoplasms. Whereas in inhalation studies the full complex mixture of tobacco smoke can be evaluated, consisting of both gas phase and tar phase, skin-painting studies allow only the examination of smoke condensates. Information currently available suggests, however, that the gas phase is as carcinogenic as full tobacco smoke (Haussmann et al., 2001
; Leuchtenberger and Leuchtenberger, 1974
; Witschi et al., 1997b
). The positive tumor responses found in mice exposed to the gas phase alone also allows the conclusion that, even under conditions of whole body exposure, the carcinogenic response in the lung is due to inhaled carcinogens rather than particulate matter, which is deposited on the skin of mice and then ingested after grooming.
In conclusion, it is possible to produce lung tumors in different mouse strains by inhalation of tobacco smoke. A key element in the protocol is that mice are allowed to recover a few months in air after smoke exposure. Oxidative stress, possibly resulting in increased apoptosis or cytotoxicity, may interfere with tumor growth and progression as long as the mice are kept in the smoke atmosphere. Mice that are exposed almost twice as long to tobacco smoke and are not allowed to recover in air do not show an increased tumor response despite having been exposed to almost twice the tobacco smoke dose. Although it is not necessary to document, using animal studies, tobacco smoke as a lung carcinogenthis fact is widely recognized through the human experience alonethe mouse lung tumor system might be further explored for future studies on tobacco smoke toxicity and mechanisms of carcinogenic action as well as for preclinical evaluation of putative chemopreventive agents.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
This research was supported by grants from the National Institute of Environmental Health Sciences, National Institutes of Health and by the U.S. Environmental Protection Agency. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIEHS, NIH or the U.S. EPA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benowitz, N. L., Kuyt, F., Jacob, P. III, Jones, R. T., and Osman, A. L. (1983). Cotinine disposition and effects. Clin. Pharmacol. Ther. 34, 604611.[ISI][Medline]
Bogen, K. T., and Witschi, H. (2002). Lung tumors in A/J mice exposed to environmental tobacco smoke: Estimated potency and implied human risk. Carcinogenesis 23, 511519.
Coggins, C. R. E. (1998). A review of chronic inhalation studies with mainstream cigarette smoke in rats and mice. Toxicol. Pathol. 26, 307314.[ISI][Medline]
Conaway, C. C., Jiao, D., Kelloff, G. J., Steele, V. E., Rivenson, A., and Chung, F. L. (1998). Chemopreventive potential of fumaric acid, N-acetylcysteine, N-(4- hydroxyphenyl) retinamide and beta-carotene for tobacco-nitrosamine- induced lung tumors in A/J mice. Cancer Lett. 124, 8593.[ISI][Medline]
DAgostini, F., Balansky, R. M., Bennicelli, C., Lubet, R. A., Kelloff, G. J., and De Flora, S. (2001a). Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice. Int. J. Oncol. 18, 607615.[ISI][Medline]
DAgostini, F., Balansky, R. M., Izzotti, A., Lubet, R. A., Kelloff, G. J., and De Flora, S. (2001b). Modulation of apoptosis by cigarette smoke and cancer chemopreventive agents in the respiratory tract of rats. Carcinogenesis 22, 375380.
Droms, K. A., and Malkinson, A. M. (1991). Mechanisms of glucocorticoid involvement in mouse lung tumorigenesis. Exp. Lung Res. 17, 359370.[ISI][Medline]
Fisher, G. H., Wellen, S. L., Klimstra, D., Lenczowski, J. M., Tichelaar, J. W., Lizak, M. J., Whitsett, J. A., Koretsky, A., and Varmus, H. E. (2001). Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-ras transgene in the presence and absence of tumor suppressor genes. Genes Dev. 15, 32493262.
Foley, J. F., Anderson, M. W., Stoner, G. D., Gaul, B. W., Hardisty, J. F., and Maronpot, R. R. (1991). Proliferative lesions of the mouse lung: Progression studies in strain A mice. Exp. Lung Res. 17, 157168.[ISI][Medline]
Gressani, K. M., Leone-Kabler, S., OSullivan, M. G., Case, L. D., Malkinson, A. M., and Miller, M. S. (1999). Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and postnatally exposed to butylated hydroxytoluene. Carcinogenesis20, 21592165.
Haussmann, H. J., Rustemeier, K., and Elves, R. G. (2001). The use of risk analysis on selecting cigarette smoke compounds for reduction. Risk analysis in an interconnected world. Proceedings of the meeting of the Society for Risk Analysis, Seattle, December 15. Society for Risk Analysis, McLean, VA.
Henry, C. J., and Kouri, R. E. (1986). Chronic inhalation studies in mice: II. Effects of long-term exposure to 2R1 cigarette smoke on (C57BL/CumxC3H/AnfCum)F1 mice. J. Natl. Cancer Inst. 77, 203212.[ISI][Medline]
IARC (1986). Biological data relevant to the evaluation of carcinogenic risk to humans: 1. Carcinogenicity studies in animals. In IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Tobacco Smoking. Vol. 38, pp. 127139. International Agency for Research on Cancer, Lyon.
Jalili, T., Murthy, G. G., and Schiestl, R. H. (1998). Cigarette smoke induces DNA deletions in the mouse embryo. Cancer Res. 58, 26332638.[Abstract]
Leone-Kabler, S., Wessner, L. L., McEntee, M. F., DAgostino, R. B., Jr., and Miller, M. S. (1997). Ki-ras mutations are an early event and correlate with tumor stage in transplacentally-induced murine lung tumors. Carcinogenesis 18, 11631168.[Abstract]
Leuchtenberger, C., and Leuchtenberger, R. (1974). Differential response of Snells and C57 black mice to chronic inhalation of cigarette smoke. Pulmonary carcinogenesis and vascular alterations in lung and heart. Oncology29, 122138.[ISI][Medline]
Malkinson, A. M. (1998). Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp. Lung Res. 24, 541555.[ISI][Medline]
Mattes, W. B., and Miller, M. S. (2000). PCR: Methods and Limitations. In Genetic Polymorphisms and Susceptibility to Disease (M. S. Miller and M. T. Cronin, Eds.), pp. 1739. Taylor and Francis, London.
Miller, M. S., Baxter, J. L., Moore, J. W., Lewis, J. D., and Schuller, H. M. (1994). Molecular characterization of neuroendocrine lung tumors induced in hamsters by treatment with nitrosamines and hyperoxia. Int. J. Oncol. 4, 512.[ISI]
Miller, M. S., Gressani, K. M., Leone-Kabler, S., Townsend, A. J., Malkinson, A. M., and OSullivan, M. G. (2000). Differential sensitivity to lung tumorigenesis following transplacental exposure of mice to polycyclic hydrocarbons, heterocyclic amines, and lung tumor promoters. Exp. Lung Res. 26, 709730.[ISI][Medline]
Mohr, U., and Reznik, G. (1978). Tobacco carcinogenesis. In Pathogenesis and Therapy of Lung Cancer (C. C. Harris, Ed.), pp. 263368. Marcel Dekker, New York.
Obermueller-Jevic, U. C., Espiritu, I. E., Corbacho, A. M., Cross, C. E., and Witschi, H. P. (in press). Lung tumor development in mice exposed to tobacco smoke and fed ß-carotene diets. Toxicol. Sci.
Omenn, G. S. (1998). Chemoprevention of lung cancer: The rise and demise of ß-carotene. Annu. Rev. Public Health 19, 7399.[ISI][Medline]
Pashko, L. L., and Schwartz, A. G. (1996). Inhibition of 7,12-dimethylbenz[a]anthracene-induced lung tumorigenesis in A/J mice by food restriction is reversed by adrenalectomy. Carcinogenesis17, 209212.[Abstract]
Peacock, J. L., Cook, D. G., Carey, I. M., Jarvis, M. J., Bryant, A. E., Anderson, H. R., and Bland, J. M. (1998). Maternal cotinine level during pregnancy and birthweight for gestational age. Int. J. Epidemiol. 27, 647656.[Abstract]
Postmus, P. E. (1998). Epidemiology of lung cancer. In Fishmans Pulmonary Diseases and Disorders (A. P. Fishman, J. A. Elias, J. A. Fishman, M. A. Grippi, L. R. Kaiser, and R. M. Senior, Eds.), pp. 17071717. McGrawHill, New York.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491.[ISI][Medline]
Shimkin, M. B., and Stoner, G. D. (1975). Lung tumors in mice: Application to carcinogenesis bioassay. Adv. Cancer Res. 21, 158.[Medline]
Stoner, G. D. (1998). Introduction to mouse lung tumorigenesis. Exp. Lung Res. 24, 375383.[ISI][Medline]
Stratton, K., Shetty, P., Wallace, R., and Bondurant, S. (2001). Clearing the Smoke. Assessing the Science Base for Tobacco Harm Reduction. National Academy Press, Washington, DC.
Szabo, E. (2001). Lung epithelial proliferation: A biomarker for chemoprevention trials? J. Natl. Cancer Inst. 93, 10421043.
Thun, M. J., Lally, C. A., Flannery, J. T., Calle, E. E., Flanders, W. D., and Heath, C. W., Jr. (1997). Cigarette smoking and changes in the histopathology of lung cancer. J. Natl. Cancer Inst. 89, 15801586.
van Zandwijk, N., Dalesio, O., Pastorino, U., de Vries, N., and van Tinteren, H. (2000). EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. For the European Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups. J. Natl. Cancer Inst. 92, 977986.
Vayssier, M., Banzet, N., Francois, D., Bellmann, K., and Polla, B. S. (1998). Tobacco smoke induces both apoptosis and necrosis in mammalian cells: Differential effects of HSP70. Am. J. Physiol. 275, L771L779.
Wagner, B. M., Cline, M. J., Dungworth, D. L., Fischer, T. H., Gardner, D. E., Pryor, W. A., Rennard, S. I., and Slaga, T. J. (2000). A safer cigarette? A comparative study. A consensus report. Inhal. Toxicol. 12(Suppl. 5), 148.[Medline]
Wessner, L. L., Fan, M., Schaeffer, D. O., McEntee, M. F., and Miller, M. S. (1996). Mouse lung tumors exhibit specific Ki-ras mutations following transplacental exposure to 3-methylcholanthrene. Carcinogenesis17, 15191526.[Abstract]
Witschi, H. (2000). Successful and not so successful chemoprevention of tobacco smoke-induced lung tumors. Exp. Lung Res. 26, 743755.[ISI][Medline]
Witschi, H. (2001). A short history of lung cancer. Toxicol. Sci. 64, 46.
Witschi, H. P., and Espiritu, I. (in press). Development of tobacco smoke-induced lung tumors in mice fed Bowman-Birk protease inhibitor concentrate (BBIC). Cancer Lett.
Witschi, H. P., Espiritu, I., Maronpot, R. R., Pinkerton, K. E., and Jones, A. D. (1997a). The carcinogenic potential of the gas phase of environmental tobacco smoke. Carcinogenesis 18, 20352042.[Abstract]
Witschi, H. P., Espiritu, I., Peake, J. L., Wu, K., Maronpot, R. R., and Pinkerton, K. E. (1997b). The carcinogenicity of environmental tobacco smoke. Carcinogenesis 18, 575586.[Abstract]
Witschi, H., Espiritu, I., and Uyeminami, D. (1999). Chemoprevention of tobacco smoke-induced lung tumors in A/J strain mice with dietary myo-inositol and dexamethasone. Carcinogenesis 20, 13751378.
Witschi, H., Espiritu, I., Yu, M., and Willits, N. H. (1998). The effects of phenethyl isothiocyanate, N-acetylcysteine and green tea on tobacco smoke-induced lung tumors in strain A/J mice. Carcinogenesis 19, 17891794.[Abstract]
Witschi, H. P., Oreffo, V. I. C., and Pinkerton, K. E. (1995). Six-month exposure of strain A/J mice to cigarette sidestream smoke: Cell kinetics and lung tumor data. Fundam. Appl. Toxicol. 26, 3240.[ISI][Medline]
Witschi, H., Uyeminami, D., Moran, D., and Espiritu, I. (2000). Chemoprevention of tobacco-smoke lung carcinogenesis in mice after cessation of smoke exposure. Carcinogenesis 21, 977982.
Wright, D. K., and Manos, N. M. (1990). Sample preparation from paraffin-embedded tissues. In PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds.), pp. 153158. Academic Press, San Diego.
Wynder, E. L., and Stellman, S. D. (1977). Comparative epidemiology of tobacco-related cancers. Cancer Res. 37, 46084622.[Abstract]
You, M., Candrian, U., Maronpot, R. R., Stoner, G. D., and Anderson, M. W. (1989). Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse. Proc. Natl. Acad. Sci. U.S.A. 86, 30703074.[Abstract]