Effect of chemopreventive agents on separate stages of progression of benzo[
]pyrene induced lung tumors in A/J mice
R. D. Estensen1,4,
M. M. Jordan1,
T. S. Wiedmann2,
A. R. Galbraith1,
V. E. Steele3 and
L. W. Wattenberg1
1 Department of Laboratory Medicine and Pathology and 2 Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455, USA and 3 Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA
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Abstract
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The effects of aerosol budesonide and dietary myo-inositol on progression of benzo[
]pyrene (B[
]P) induced carcinogenesis were studied in A/J mouse lung. First, we determined when to intervene in the carcinogenesis process by exposing several animals to B[
]P at 100 and 150 mg/kg of body wt. Groups of these animals were necropsied from 1 to 36 weeks post-carcinogen. The presence of different categories of lung tumors was noted over the 36 week time period. Hyperplasia first appeared
6 weeks post-carcinogen followed by adenoma at 9 weeks, then by carcinoma at 26 weeks. From this temporal sequence we determined we could test for effects of preventive agents on progression to hyperplasia by intervening at 3 weeks, for effects on progression to adenoma by intervening at 6 weeks and for effects on progression to carcinoma by intervention at 12 weeks. Intervention at 3 weeks post-carcinogen with aerosolized budesonide delayed both hyperplasia and adenoma formation. Once hyperplasia appeared in budesonide treated animals, however, it increased at the same rate as in control animals, indicating a delay in progression. Progression from adenoma to carcinoma was reduced when budesonide was given 12 weeks post-carcinogen. Dietary myo-inositol failed to suppress progression from adenoma to carcinoma when started 12 weeks post-carcinogen. In summary, budesonide is a chemopreventive agent that has inhibitory effects on B[
]P induced carcinogenesis of the lung in A/J mice at all stages of progression from hyperplasia formation to cancer.
Abbreviations: B[
]P, benzo[
]pyrene; GSD, geometric standard deviation; MMAD, mass median aerodynamic diameter
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Introduction
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The model of carcinogenesis proposed by Berenblum (1) included initiation, considered a single event, followed by promotion that required more than one event. It is now generally believed that initiation occurs with a single exposure to carcinogen, which results in one or more genotoxic events. These events prepare the tissue for subsequent events, promotion, which lead tumors through several stages of escape from control of cell division to produce invasive tumors, which metastasize and kill the host. This post-initiation process was recognized as consisting of several stages by Foulds (24), who named this process progression. Progression is accompanied by changes in the biology, biochemistry and histopathology of tumors, which are associated with specific molecular changes as shown by Vogelstein and others (5,6). Finally, in animal and human systems, Lipkin (7) recognized that hyperplasia precedes the formation of histologically benign epithelial tumors (adenomas or papillomas), which will subsequently undergo dysplasia and finally progress to carcinoma.
Post-initiation stages of carcinogenesis, from hyperplasia to carcinoma, are targets for the chemical prevention of cancer. It is conceivable that some preventive agents could act over the whole range of tumorigenesis from hyperplasia to carcinoma. Some agents, however, may act at very narrow ranges, especially by preventing or delaying hyperplasia, progression of hyperplasia to adenoma or adenoma to carcinoma. Using the period before the appearance of each stage, we examined the effect of agents on the transition from earlier to later stages. Since most previous searches for chemopreventive agents in animals have been halted at early endpoints, such as adenoma or papilloma formation, we studied the effect of the synthetic glucocorticoid, budesonide, on the full range of the development of lung tumors (hyperplasia to carcinoma), produced by B[
]P in A/J mice. Additionally, we wanted to determine if myo-inositol, a compound found to inhibit pulmonary adenoma formation, would affect the later stage of progression from adenoma to carcinoma.
We report here that budesonide retards the appearance of hyperplasia. However, once hyperplasia appears in budesonide treated animals, its rate of increase is identical to controls. Thus, budesonide produces only a delay in progression when it is given before the stage of hyperplasia. When budesonide is given at 3 weeks, it inhibits the progression of hyperplasia to adenoma. When budesonide is given at 12 weeks, progression of adenoma (A) to adenoma with progression (A/P) and progression of adenoma with progression to carcinoma (C) are reduced. Our data on these two later progressions do not allow us to distinguish between suppression and delay. In the study of the effects of myo-inositol on late stage progression, it was found that addition of the compound to the diet at 12 weeks post-carcinogen did not alter progression from adenoma to carcinoma.
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Materials and methods
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Chemicals
The chemicals used were budesonide (>99% purity), myo-inositol (>99% purity) (Sigma Chemical Co., St Louis, MO) and B[
]P (>99% purity) (Aldrich Chemical Co., Milwaukee, WI).
General animal procedures
Female A/J mice obtained from Jackson Laboratories were used in all experiments. Animals were fed a semi-purified diet of 27% vitamin free casein, 59% starch, 10% corn oil, 4% salt mix (USP XIV) and a complete mixture of vitamins (Teklad, Madison, WI) as a diet. The mice were housed in a constant temperature facility with controlled lighting (light 12 h, dark 12 h). Animals were given three doses of B[
]P in 0.2 ml of cottonseed oil or cottonseed oil only by gavage over a 1 week period. The time interval between the first and second doses was 4 days and between the second and the third 3 days. Animals exposed to carcinogen received either 100, 125 or 150 mg/kg of body wt of B[
]P as indicated in the particular experiment. The first dose was given when the mice were between 8 and 14 weeks of age. Budesonide was administered by aerosol and myo-inositol was added to the diet.
Aerosol administration
Details of the aerosol apparatus (MiniHeart Nebulizer, Central Medical Services, Naperville, IL), Leibig condensers, tubing, water bath, nose cone and administration procedures have been described previously (8,9). Using this apparatus, mice were exposed 3 or 5 days a week to solutions containing either solvent alone (designated solvent control) or budesonide starting at various times post-carcinogen. The concentration of budesonide in the aerosol was determined by replacing the nose cone with a collection filter assembly using Whatman glass fiber filters as described previously (8,9). The aerosol concentration was determined spectrophotometrically after collection for 1 min.
Monitoring dose delivery
The dose delivered to the animal was estimated from the aerosol concentration (µg budesonide/l air) as follows:
where the respiratory minute volume was estimated with Guyton's formula (10), the exposure time was 1 min and the body weight was taken to be 0.023 kg. In this study, <10% of the dose calculated by this method is expected to be retained by the lung.
Aerosol particle sizing
A low flow rate cascade impactor (InTox, Albuquerque, NM) was used to determine the size distribution of the aerosol budesonide particles (8,9). The MMAD and geometric standard deviation (GSD) were obtained from the cumulative undersized mass collected given as a function of the logarithm of the cut-off diameter. For a budesonide solution of 1.4 mg/ml, the MMAD was 0.91 µm with a GSD of 2.1. At a solution concentration of 0.56 mg/ml, the MMAD was 0.78 µm with a GSD of 2.0.
Pathology
At the termination of experiments all animals were necropsied. The lungs were dissected free of other tissue and gross tumor counts on the surface of the lungs were performed either on unfixed lung or lung fixed in 10% formalin. All lobes of both lungs, after formalin fixation, were submitted for paraffin embedding and sectioned. A representative section or sections of all five lobes were evaluated according to the criteria of Foley et al. (11) as to the type of tumors, i.e. hyperplasia (increase in cell number with retention of alveolar pattern), adenoma (solid aggregation of benign neoplastic cells) or carcinoma (malignant cells in solid or gland like pattern). Additionally, in some experiments, the category, adenoma with progression, was included. An adenoma was considered adenoma with progression when a distinct clone of cells (10 or more) appeared with hyperchromatic nuclei and nuclear size greater than those found in cells of the adenoma. The total area of the lung tissue on each microscope slide was determined by photographing sections and calibration areas, then comparing relative areas, using Adobe Photoshop®. The numbers of specific tumor types per square centimeter were determined using the observed counts, evaluated as above, in each section.
Statistical analysis
Non-parametric tests were used to compare the different treatments, KruskalWallis analysis to test the null hypothesis of no difference among the groups. Since this null hypothesis was rejected, the Wilcoxon rank sum test for pairwise comparisons was used. No further adjustments for multiple comparisons were applied because the comparisons of interest had been determined a priori. Statistical analysis was carried out by means of a statistical software package (SAS), except the two tailed t-tests, which use the standard functions within Microsoft Excel®.
Choosing times of agent administration post-initiation
The time of appearance of various types of tumors was determined by exposing animals to either 100 or 150 mg/kg of B[
]P. Thereafter, animals were necropsied at various times post-carcinogen as Foley and co-workers had done previously using urethane as the carcinogen (11).
In Figure 1, the tumor responses of the A/J mouse to these two doses of B[
]P are plotted against time post-carcinogen. The total number of tumors at the 100 mg/kg dose of B[
]P appears to be slightly more than half the number seen at 150 mg/kg B[
]P. In addition, at 100 mg/kg B[
]P, the sequential appearance of hyperplasia, adenoma and carcinoma was more clearly separated than at the 150 mg/kg dose of B[
]P. At the 100 mg/kg dose of B[
]P, no hyperplasia appeared before 3 weeks, no adenoma appeared before 6 weeks, and no carcinoma before 12 weeks post B[
]P. In fact, most cancers did not appear until 26 weeks. This sequence of appearance of tumor types was similar to that reported by Foley et al. (11), therefore, we selected a dose of 100 mg/kg B[
]P for most subsequent experiments.

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Fig. 1. Time course of pulmonary tumor development after B[ ]P administration to female A/J mice as seen in hemotoxylineosin stained histology sections. Error bars indicate standard deviation and the number above is the n for each time period.
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Since our experiments depended on the progression of one tumor type to the next, it was important that our data confirmed the observations of Foley et al. (11). The hypothesis that progression from hyperplasia to adenoma and then to carcinoma occurred by progression of one tumor type to another (11) was supported in our observations that: (i) a decrease in hyperplasia was accompanied by an increase in adenomas and (ii) tumors were clearly transitional, i.e. a hyperplasia and adenoma or adenoma and adenoma with progression or adenoma with progression and carcinoma because both were present in the same tumor.
Based on the above analyses, we selected the times to add agents to the diet or to be given by aerosol. For example, we would begin administering an agent to test for prevention of hyperplasia when hyperplasia was absent in test animals (3 weeks). Likewise, we could test for prevention of adenoma by adding agent at 6 weeks or carcinoma at 12 weeks, i.e. at times before these lesions made an appearance.
We selected budesonide to test over the later phases of progression because in previous work it already had been shown to suppress tumor formation when A/J mice were killed at 16 weeks post B[
]P. Myo-inositol was also selected because of its ability to suppress tumor formation when present in the diet during the same period. To test for effects on late progression, the administration of both budesonide and myo-inositol were started 12 weeks post-carcinogen. Since myo-inositol and budesonide showed an additive inhibitory effect when given together from 1 to 16 weeks (9), we combined the two to test for an additive inhibition of progression from adenoma to carcinoma.
In the determination of budesonide effects on progression to hyperplasia and adenoma, animals were killed at 6, 12 and 16 weeks post-carcinogen. In the determination of effect on progression from adenoma to adenoma with progression and from adenoma with progression to carcinoma, mice were killed at 34 or 36 weeks post-carcinogen. In all specimens, gross tumors were counted and histologic preparations were made. Tumor number and type were enumerated per square centimeter of lung area as described above.
Determination of effect of budesonide on spontaneous formation of adenoma
Female A/J mice develop pulmonary adenomas in the absence of carcinogen administration. An experiment was performed to determine the effect of budesonide under these conditions. Animals that had not received carcinogen were randomized by weight at 19 weeks of age. Ten animals received no treatment and served as controls and 12 animals were given aerosol budesonide at a dose of 25 µg/kg, five times a week. They were weighed at weekly intervals. At 48 weeks all animals were killed. Tumors on the surface were counted on fresh tissue, using a dissecting microscope.
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Results
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Budesonide effect on hyperplasia and progression to adenoma
The results of the experiment to evaluate suppression of hyperplasia is seen in Figure 2. Animals were given aerosol budesonide three times per week beginning 3 weeks post-carcinogen and continuing to death. Both controls and treated animal were killed at 6, 12 and 16 weeks. Budesonide treated animals had significantly fewer tumors than solvent controls. However, once hyperplasia appeared in budesonide treated animals, the rate of formation of hyperplasia was identical to solvent controls (Figure 2a). Animals given budesonide beginning 3 weeks post-carcinogen also had significantly fewer adenomas than solvent controls (Figure 2b). The rate of adenoma formation remained low in budesonide treated animals during the 16-week period.
Budesonide effect on change to adenoma with progression and to carcinoma
As seen in Table I, aerosol budesonide prevented change of adenomas to adenoma with progression and to carcinomas when it was given three times a week at a dose of either 32 or 46 µg/kg body wt from 12 weeks post-carcinogen to 36 weeks post-carcinogen. There was no significant difference in hyperplasia or adenoma numbers at these two doses with a single exception. There were more hyperplasias in animals given the higher dose of budesonide than in solvent controls.
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Table I. Pulmonary tumors per square centimeter of lung tissue area 36 weeks after B[ ]P administration in female A/J mice given aerosol budesonide three times per week
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In a second experiment (Table II) animals were given budesonide at a lower dose, 25 µg/kg body wt, five times per week from 12 to 34 weeks post-carcinogen. In this experiment, a smaller response of late stage tumors was obtained, especially when adenomas with progression and carcinomas was considered as a single group. Inhibition by budesonide occurred and there was a dose response.
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Table II. Pulmonary tumors per square centimeter showing adenoma with progression or carcinoma 34 weeks after 100 mg/kg B[ ]P administered to female A/J mice
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Effect of budesonide on spontaneous tumors in A/J mice
Pulmonary tumors occur in A/J mice in the absence of administered carcinogen. As indicated in Table III, budesonide suppressed spontaneous tumor formation by a factor of two. Random histologic sections of lungs of these animals indicated that these tumors were adenomas. Note that the body weights of animals were the same in both groups.
Effect of myo-inositol on progression from adenoma to adenoma with progression and carcinoma
Table IV shows the effect of addition of 0.5% myo-inositol to the diet beginning at 12 weeks and continuing to 34 weeks following administration of B[
]P at a dose of 100 mg/kg. Myo-inositol by itself does not produce a significant effect on progression from adenoma to carcinoma. When the combination of myo-inositol and budesonide were administered, only animals given budesonide five times a week showed a decrease in progression from adenoma to adenoma with progression plus carcinoma.
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Table IV. Effect of dietary myo-inositol and aerosolized budesonide (25 µg/kg) on pulmonary tumors per square centimeter 34 weeks after B[ ]P administration to female A/J mice
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Discussion
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In experiments presented, it was found that aerosolized budesonide inhibits the full range of progression in B[
]P induced lung carcinogenesis in female A/J mice. This agent reduces the number of hyperplasias that appear in B[
]P treated mouse lung, reduces progression of hyperplasia to adenoma and reduces progression of adenoma to adenoma with progression and to carcinoma. Closer examination indicates that the effect on hyperplasia is only a delay, since once hyperplasia appears in budesonide treated animals, the rate of hyperplasia appearance is the same as that seen in control animals. In addition to its effects on B[
]P induced pulmonary carcinogenesis, budesonide also inhibited spontaneous lung carcinogenesis in the A/J mice.
Budesonide administration starting at 3 weeks post-carcinogen decreases the numbers of adenomas that occur after hyperplasia, either by delaying or suppressing the rate of progression of hyperplasia to adenoma. Pereira and colleagues (12) present data in A/J mice given vinyl carbamate, which suggest that budesonide's effect on transition of hyperplasia to adenoma is a delay. Our experiments terminated at 16 weeks were not carried on long enough to distinguish between delay and suppression of this progression. In the present work, progression of adenoma to adenoma with progression and to carcinoma was also decreased by budesonide treatment. Again rates of progression were not determined since only one endpoint was used. Our experiments also demonstrate that total dose of budesonide is important. Lower total dose as indicated by comparing Tables I, II and IV produces a smaller effect.
Myo-inositol alone or with aerosol budesonide failed to prevent carcinoma or adenoma with progression when added to the diet 12 weeks post-carcinogen. This failure occurred despite the fact that myo-inositol prevents the formation of adenoma by 3040% in animals when added to the diet 1 week post-carcinogen (12,13) when assayed at 16 weeks. Gunning and colleagues (14) also report the failure of dietary myo-inositol in preventing the progression from adenoma to carcinoma. From these results and our data, one may conclude that myo-inositol's effect both diminishes when present continuously in the diet and is ineffective on late progression.
The foregoing suggests that previous searches for preventive agent using the early endpoint of tumors seen at 16 weeks may miss effects on later stages of cancer progression. Furthermore, it suggests that differing mechanisms of inhibition may result in an inhibition of a narrower portion of the spectrum of progression. In the case of myo-inositol, for example, the initial effect on progression is lost over a period of 24 weeks (14) and is ineffective in our study when added late in progression.
In a thorough theoretical discussion of chemoprevention, Lippman and Hong (15) have indicated that either delay or suppression or combinations of the two are possible mechanisms for chemopreventive effects. In fact, budesonide's effect, seen in Figure 2, delay of hyperplasia is identical to data they present. These authors further maintain that delay alone will make an agent useful in the treatment of high-risk patients.
Others have studied the effects of various agents on tumor progression in the A/J mouse model. Yang et al. (16) and Wang et al. (17) have demonstrated that green tea given in the drinking water delays or prevents progression in lung tumors of A/J mice as well as tumors of the skin of carcinogen-treated, UV-promoted SKF mice. Gunning et al. (14) have demonstrated that addition of budesonide at times from 4 to 20 weeks post-carcinogen reduces either multiplicity or progression of mouse lung tumors in A/J mice given the carcinogen vinyl carbamate. In studies of glucocorticoids in other epithelial tumor models Slaga et al. (18) demonstrated inhibition of papilloma formation in mouse skin with topical glucocorticoids and Mira-y-Lopez et al. (19) showed that systemic hydrocortisone inhibited mammary tumor growth in mice and halted or reversed growth in established tumors.
In summary, we have demonstrated that chemopreventive agents may selectively effect one or more specific stages of tumor progression. Future investigations of agents in screening assays should include the full range of progression.
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Notes
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4 To whom correspondence should be addressed Email: esten001{at}umn.edu 
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Acknowledgments
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This work was supported by the National Cancer Institute Contract No. N01-CN-85069.
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Received June 5, 2003;
revised October 2, 2003;
accepted October 12, 2003.