REPORTS

Retinoid Signaling and Activator Protein-1 Expression in Ferrets Given ß-Carotene Supplements and Exposed to Tobacco Smoke

Xiang-Dong Wang, Chun Liu, Roderick T. Bronson, Donald E. Smith, Norman I. Krinsky, Robert M. Russell

Affiliations of authors: X.-D. Wang, N. I. Krinsky, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University and Department of Biochemistry, Tufts University School of Medicine, Boston, MA; C. Liu, R. T. Bronson, D. E. Smith, R. M. Russell, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University.

Correspondence to: Xiang-Dong Wang, M.D., Ph.D., Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111 (e-mail: Wang_CN{at}HNRC.TUFTS.EDU).


    ABSTRACT
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Epidemiologic studies have demonstrated that individuals who eat more fruits and vegetables and/or have high levels of serum ß-carotene have a lower risk of cancer, especially lung cancer. However, recent human intervention studies using ß-carotene supplements have shown an increase in the risk of lung cancer among smokers and asbestos workers. In this study, we used an animal model system to evaluate the hazard associated with a combination of high-dose ß-carotene supplementation and tobacco smoking. METHODS: Ferrets were given a ß-carotene supplement, exposed to cigarette smoke, or both for 6 months. Cell proliferation and squamous metaplasia in lung tissue were assessed by examination of proliferating-cell nuclear antigen expression and histopathologic examination, respectively. ß-Carotene and retinoid concentrations in lung tissue and plasma samples were analyzed by high-performance liquid chromatography. Expression of genes for retinoic acid receptors (RARs) and activator protein-1 (encoded by the c-Jun and c-Fos genes) in lung tissue specimens was examined by western blotting. RESULTS: A strong proliferative response in lung tissue and squamous metaplasia was observed in all ß-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared with control groups, all three treatment groups had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited 18%-73% reductions in RARß gene expression; however, RAR{alpha} and RAR{gamma} gene expression was not reduced. Ferrets given a ß-carotene supplement and exposed to tobacco smoke had threefold to fourfold elevated expression of the c-Jun and c-Fos genes. CONCLUSIONS: Diminished retinoid signaling, resulting from the suppression of RARß gene expression and overexpression of activator protein-1, could be a mechanism to enhance lung tumorigenesis after high-dose ß-carotene supplementation and exposure to tobacco smoke.



    INTRODUCTION
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 
Does ß-carotene supplementation increase rather than decrease human lung cancer rates? Since Peto et al. (1) first raised the hypothesis that dietary ß-carotene reduces human cancer rates, a large body of observational epidemiologic studies has demonstrated that individuals who eat more fruits and vegetables rich in carotenoids and/or who have higher levels of serum ß-carotene have a lower risk of cancer, particularly lung cancer [reviewed in (2)]. However, there is also some contradictory evidence from recent human intervention studies using ß-carotene supplements (20-30 mg/day). An increase in the risk of lung cancer among smokers who took ß-carotene supplements was reported in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (3,4) and among smokers and asbestos workers in the Beta-Carotene and Retinol Efficacy Trial (5,6) but not among male physicians in the United States in the Physicians' Health Study (11% of whom were current smokers) (7). The failure of these intervention studies to demonstrate a protective effect for supplemental ß-carotene could be due to many factors, but it is more difficult to explain the apparent enhancement of lung carcinogenesis by ß-carotene supplementation in smokers; i.e., is it due to ß-carotene itself or to metabolites of ß-carotene, or is there a different metabolism of ß-carotene in the lungs of smokers versus nonsmokers?

Whether there is a true hazard associated with high doses of ß-carotene should be evaluated by controlled studies in animal model systems. One problem is the lack of an appropriate animal model to mimic human ß-carotene absorption and metabolism because most laboratory animals break down ß-carotene in their intestine and thus absorb almost none intact. The ferret, however, mimics the absorption and tissue metabolism of ß-carotene in humans (8) and has been used for studies of tobacco smoking and inhalation toxicology (9,10). Therefore, the ferret is an appropriate model system to study the effect of an interaction between ß-carotene and smoking and to investigate possible harmful effects and the mechanism(s) involved, which cannot be performed in humans.

In this study, ferrets were given ß-carotene supplements and exposed to cigarette smoke for 6 months. We investigated 1) whether there is a relationship between high-dose ß-carotene supplementation and lung epithelial lesions in normal ferrets and ferrets exposed to cigarette smoke in vivo, 2) whether smoking modifies ß-carotene and retinoid metabolism and the formation of oxidative products in vivo and in vitro, and 3) if a harmful effect of high doses of ß-carotene is observed, what possible biologic bases or mechanism(s) may be responsible for the adverse effect.


    M ATERIALS AND METHODS
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 
Animals and Study Groups

Male adult ferrets (1.0-1.2 kg) from Marshall Farms (North Rose, NY) were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited animal facility at the Human Nutrition Research Center on Aging at Tufts University. The animals were maintained individually in suspended stainless-steel cages measuring 24 x 24 x 18 inches. Before the experiment began, all animals were quarantined for a minimum of 2 weeks to ascertain health status. All experimental procedures, including exposure to smoke, were approved by the Animal Care and Use Committee at the Human Nutrition Research Center on Aging at Tufts University, and the procedures were conducted under the supervision of the Animal Care and Use Committee. Twenty-four male ferrets were randomly assigned to one of four groups (each containing six animals) that were treated as follows: 1) chronic exposure to cigarette smoke for 6 months; 2) ß-carotene supplementation for 6 months; 3) both treatments for 6 months; and 4) neither treatment for 6 months. During the 6-month experimental period, ferret body weights were recorded weekly. After the 6-month experimental period, all ferrets were killed by puncturing the abdominal aorta while the animals were under deep isoflurane anesthesia.

Exposure to Cigarette Smoke

Ferrets were exposed to cigarette smoke twice in the morning and twice in the afternoon (10 cigarettes over a 30-minute period, each time) in a chamber connected to a smoking device, adapted from a previous study (11). Cigarette smoke was drawn out of the cigarettes (10 cigarettes; Standard Research Cigarettes, Tobacco and Health Research Institute, University of Kentucky, Lexington) with a vacuum pump and then exhausted into the chamber. The volume of the chamber was approximately 100 x 80 x 70 cm. Six ferrets were exposed to smoking in the chamber during each trial. During the first 2 weeks of the study, the number of cigarettes was gradually increased to 10 cigarettes over a 30-minute period twice in the morning and twice in the afternoon and then remained the same for the rest of the 6-month experimental period. The ferrets not exposed to smoke were housed in a separate room and underwent the same procedures as the animals exposed to smoke, except they received no exposure to smoke. To assess smoke-exposure efficiency in the ferret, we measured levels of urinary nicotine metabolites by use of a diethylthiobarbituric acid method as described previously (12). During the 6 months of study, the nicotine metabolite concentration in ferret urine was monitored weekly.

ß-Carotene Supplementation

All-trans-ß-carotene (type IV; Sigma Chemical Co., St. Louis, MO) above that contained in the basal diet (Purina Ferret Chow; Ralston Purina, St. Louis, MO) was dissolved into 1 mL of corn oil and fed orally (not gavaged) to the ferrets every morning for 6 months. The ferrets in the control group were fed the basal diet plus 1 mL of corn oil only. The concentrations of ß-carotene and vitamin A in the ferret basal diet were controlled by using one lot of ferret diet. The calculation of ß-carotene dose between humans and ferrets was based on total absorption of intact ß-carotene (8): 1) In the group fed the basal diet containing low ß-carotene, the dry ferret food contained 0.8 µg of ß-carotene/g of food, as determined by high-performance liquid chromatography (HPLC) analysis in our laboratory. Because the daily food intake of ferrets (for a 1.0 to 1.2-kg ferret) was about 200 g per day, the average intake of ß-carotene from the basal diet during the experimental period was 0.16 mg/kg per day (0.8 µg/g x 200 g per day). Since the total absorption of ß-carotene by ferrets is about five times less than that by humans, the ß-carotene intake in the ferret was equivalent to an intake of 2.3 mg of ß-carotene per day in a 70-kg human. 2) In the ß-carotene-supplemented group (2.4 mg of ß-carotene/kg per day including the ß-carotene in the basal diet), to mimic human trials, we fed 2.4 mg of ß-carotene/kg per day (15 times higher than the 0.16 mg of ß-carotene/kg per day for the group fed a low ß-carotene diet) to ferrets. In the ferret, this higher dose of ß-carotene was equivalent to an intake of 30 mg of ß-carotene per day in a 70-kg human.

Plasma and Lung Sample Extraction and HPLC Analysis

ß-Carotene, retinol, retinyl esters, and retinoic acid in blood and lung tissue homogenates were assayed by HPLC as described previously (13,14) with modification. Briefly, 100 µL of an ethanolic solution of 0.5 N KOH and 0.5 mL of H2O were added to 2.0 mL of ferret plasma, followed by the addition of the internal standards echinenone and retinyl acetate in 50 µL of ethanol. The mixture was extracted by adding 2 mL of hexane and then centrifuging for 5 minutes at 800g at 4 °C. The hexane layer was removed, and the residue was acidified by adding 50 µL of 6 N HCl. A second extraction was performed with 2 mL of hexane. The two extractions were pooled, dried under nitrogen, and resuspended in 50 µL of ethanol for injection into the HPLC system. The lung tissues were homogenized in a mixture of ice-cold HEPES buffer and methanol, 2 : 1 (vol/vol). After homogenization, samples of lung were extracted twice without saponification by use of 6.0 mL of CHCl3/CH3OH, 2 : 1 (vol/vol). The internal standards (echinenone and retinyl acetate) were added to the samples before the extraction. The two extracts were collected and evaporated under nitrogen. A 50-µL aliquot of the extract reconstituted with ethanol was injected into the HPLC system. A gradient reverse-phase HPLC system was used for the analysis of retinol, retinyl palmitate, and plasma retinoic acid. The gradient procedure at a flow rate of 1 mL/minute was as follows: 100% solvent A (acetonitrile/tetrahydrofuran/water, 50 : 20 : 30 [vol/vol], with 0.35% acetic acid and 1% ammonium acetate in water), 3 minutes; this was followed by a 6-minute linear gradient to 40% solvent A and 60% solvent B (acetonitrile/tetrahydrofuran/water, 50 : 44 : 6 [vol/vol], with 0.35% acetic acid and 1% ammonium acetate in water), a 12-minute hold at 40% solvent A/60% solvent B, and then a 7-minute gradient back to 100% solvent A. In this HPLC system, retinoic acid, retinol, retinyl palmitate, and ß-carotene were eluted at 6.8 minutes, 8.6 minutes, 18.6 minutes, and 20.1 minutes, respectively. A Waters 490E multiwavelength spectrophotometer detector was set at 340 nm for the retinoids and 380 nm, 400 nm, and 450 nm for the carotenoids. An additional Waters 994 programmable photodiode array detector was used to measure absorption spectra. Individual carotenoids and retinoids were identified by coelution with standards and were quantified relative to the internal standard (echinenone for the carotenoids and retinyl acetate for retinoids) by determining peak areas calibrated against known amounts of standards. In all experiments, all procedures were carried out under red light to prevent photodamage to the compounds.

In Vitro ß-Carotene Incubation Procedure

Incubation was as described previously (15). The apocarotenoid and retinoid products were analyzed by HPLC.

Histopathology and Immunohistochemistry

The right upper lobe of each lung was inflated and fixed by intratracheal instillation of 10% formalin. The samples were embedded in paraffin wax. Sections (5 µm) were made with an AO microtome and stained with hematoxylin-eosin for histopathologic examination. For immunohistochemistry analysis, 5-µm-thick paraffin sections were deparaffinized, rehydrated, and incubated with 0.3% H2O2 in absolute methanol for 30 minutes at 37 °C to inhibit endogenous peroxidase. The sections were incubated for 60 minutes at 37 °C with an anti-Pan cytokeratin monoclonal antibody (mixture; Sigma Chemical Co.) that reacts with simple cornified squamous epithelia. The sections were then rinsed with phosphate-buffered saline (PBS) and incubated with a peroxidase-labeled goat anti-mouse immunoglobulin antibody (Bio-Rad Laboratories, Richmond, CA) at a dilution of 1 : 1000 in PBS, in which sections were incubated for 30 minutes at 37 °C. Thereafter, the tissue sections were stained with HistoMark BLACK substrate solutions and counterstained with contrast green solution (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). After being air dried and mounted in xylene-based mounting medium, the sections were examined independently by two investigators by light microscopy. In our animal study, we focused on the examination of lung squamous metaplasia, which provides strong evidence for a precancerous lesion and potential tumorigenesis. Since the areas of squamous metaplasia lesion are spotty and localized, we treated a sample as positive when any keratinized squamous metaplasia lesion (histologic examination plus confirmations by immunohistochemistry with anti-keratin antibody) was observed in the right upper lobe of lung. Otherwise the sample was considered negative.

Nuclear Protein Preparations and Western Blot Analysis

Nuclear protein extracts from the lungs of the ferrets in each of the four groups were prepared as described previously (16) with modification. Briefly, the lung tissues were homogenized gently in a PolytronTM homogenizer (Brinkmann Instruments, Inc., Westbury, NY) with ice-cold buffer A (10 mM Tris-HCl [pH 7.5], 10% glycerol, 10 mM KCl, 10 mM monothioglycerol, 1 mM phenylmethylsulfonyl fluoride, leupeptin [0.5 µg/mL], and aprotinin [0.5 µg/mL]), and nuclei were collected by centrifugation (15 minutes at 1200g). Nuclear pellets were solubilized in buffer B (10 mM Tris-HCl [pH 7.5], 10% glycerol, 600 mM KCl, 1 mM dithiothreitol, 10 mM monothioglycerol, 1 mM phenylmethylsulfonyl fluoride, leupeptin [0.5 µg/mL], and aprotinin [0.5 µg/mL]) for 60 minutes. The extracts were centrifuged for 30 minutes at 100 000g, and the resulting supernatants are referred to as the nuclear extract. All the above procedures were carried out at 4 °C. Protein quantitation was performed by use of the bicinchoninic acid protein assay kit (Pierce Co., Rockford, IL). Western blot analyses were carried out by using monoclonal or polyclonal antibodies against proliferating-cell nuclear antigen (PCNA), retinoic acid receptor (RAR) {alpha}, RARß, RAR{gamma}, c-Jun, and c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA) and quantified by densitometry, as described previously (17).

Validation of Ferret Model and Statistical Analysis

Before we conducted the experiment reported herein, we carried out an in vivo pilot study in 12 ferrets (three ferrets per group) for either 45 days (one ferret in each group) or 3 months (two ferrets in each group). We validated that the ferret is an excellent model for studying the role of ß-carotene in lung carcinogenesis for the following reasons: 1) ß-Carotene levels in plasma of the ferrets after ß-carotene supplementation increased 17- to 22-fold, which is similar to the increase observed in some of the human trials (up to 18-fold increase) after supplementation with comparable doses of ß-carotene (3). 2) Plasma and tissue levels of retinol, retinoic acid, and ß-carotene in the control ferret were within the range found in the normal human, except that the ferret has high levels of circulating retinyl esters, which is a general phenomenon in carnivores. 3) The lung architecture and formation of oxidative metabolites of ß-carotene in ferrets are similar to those in humans, as reported earlier (10,13). 4) The concentration of urinary cotinine equivalents in the smoke-exposed ferrets (about 14.3 µg/mL of urine) was similar to that found in humans smoking 1.5 packs of cigarettes per day (18). In these 12 ferrets, we observed a decrease in retinoic acid concentration (50%) and RARß expression (87% decrease) in the lungs of all ß-carotene-supplemented ferrets with or without smoke exposure, compared with the control group. We also observed squamous metaplasia lesions in the lungs of all ß-carotene-supplemented ferrets with (three of three ferrets) or without (three of three ferrets) smoke exposure, compared with the control group (none of three ferrets) and the group exposed to smoke alone (none of three ferrets). From these preliminary data, the final sample size of six ferrets chosen in this report was one that would allow a 99% chance of detecting statistically significant differences among the groups at a .05 level of significance if there was a 50% reduction in both retinoic acid level and RARß expression in the lungs in the ß-carotene-supplemented ferret group that was exposed to smoke compared with the control group.

Results were expressed as means ± standard deviations, and statistically significant differences were compared with an analysis of variance among the four groups and Student's t test between two groups at P<.05. All P values are two-sided.


    RESULTS
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 
There were no differences in body weight among the four groups of ferrets at the start of the study or during or after 6 months of treatment (data not shown). The assessment of smoke-exposure efficiency showed that there were no differences in the concentration of urinary cotinine equivalents in ferrets that were exposed to cigarette smoke or exposed to smoke plus a ß-carotene supplement (14.3 ± 0.6 versus 14.2 ± 0.5 µg/mL of urine).

Table 1Go shows that there were large increases in the concentration of ß-carotene in both plasma and lung tissue in the ferrets that had been supplemented with ß-carotene for 6 months. Plasma ß-carotene increased 22-fold after ß-carotene supplementation. However, plasma ß-carotene levels were statistically significantly lower (63%) in the ß-carotene-supplemented animals that were exposed to cigarette smoke than in the ß-carotene-supplemented animals that were not exposed to cigarette smoke. Similarly, ß-carotene levels in lung tissue were statistically significantly lower in ferrets exposed to smoke than in ferrets not exposed to smoke in the ß-carotene-supplemented and nonsupplemented animals (Table 1)Go. The concentration of plasma retinoids (retinoic acid, retinol, and retinyl palmitate) did not differ among the four groups; however, the concentrations of retinoic acid in the lung tissue were statistically significantly lower in the all three treatment group as compared with the control group (Table 1).Go


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Table 1. Concentrations of ß-carotene and retinoids in four groups of ferrets after 6 months of treatment*

 
To assess whether the dramatic decrease in ß-carotene was due to enhanced breakdown or metabolism of ß-carotene (especially by excentric cleavage) by smoke exposure, we conducted in vitro incubations of all-trans-ß-carotene with the postnuclear fractions from lung tissue from ferrets exposed or not exposed to smoke (Fig. 1).Go The results showed that the formation of ß-apo-14'-, ß-apo-12'-, ß-apo-10'-, and ß-apo-8'-carotenals was threefold higher (P<.05) in lung extracts from the ferrets exposed to smoke (93 ± 7, 354 ± 55, 196 ± 9, and 50 ± 4 pmol/mg of protein [mean ± standard deviation], respectively) (Fig. 1, C)Go than in the control ferrets (30 ± 5, 102 ± 4, 69 ± 12, and 22 ± 4 pmol/mg of protein) (Fig. 1, B).Go



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Fig. 1. High-performance liquid chromatography (HPLC) analysis of carotenoids in ferret lung postnuclear fractions (LPNFs) after incubation with 10 µM ß-carotene. Samples are control without LPNF (A), normal LPNF (B), and LPNF from ferrets exposed to smoke (C). The incubation of LPNF and HPLC analysis were as described previously (15). Peak 1 is ß-apo-14'-carotenal; peak 2 is ß-apo-12'-carotenal; peak 3 is ß-apo-10'-carotenal, peak 4 is ß-apo-8'-carotenal, peak 5 is retinyl acetate (internal standard), and peak 6 is ß-carotene. The numbers in small type in panels A, B, and C show elution times for each molecular species more precisely. Au = absorbance units.

 
We examined lung sections from the four groups of ferrets (Fig. 2).Go The results show that smoke exposure caused a mild aggregation and proliferation of macrophages in the lung tissue of ferrets (Fig. 2, H).Go However, localized proliferation of alveolar cells (type II pneumocytes) and alveolar macrophages and keratinized squamous epithelium were observed in all six ferrets in the group given a high-dose ß-carotene supplement (Fig. 2, D and E).Go Furthermore, severe focal proliferation of alveolar cells, squamous metaplasia, and destruction of alveolar walls were observed in all six smoke-exposed ferrets that were given a high-dose ß-carotene supplement (Fig. 2, J and K).Go Keratinized squamous metaplasia was confirmed by immunohistochemistry with anti-keratin antibody in the lung sections of all ferrets given either high-dose ß-carotene alone (Fig. 2, F)Go or high-dose ß-carotene with additional smoke exposure (Fig. 2, L).Go



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Fig. 2. Pathologic changes in ferret lung tissue resulting from feeding of ß-carotene (D, E, F), smoke exposure (G, H, I), or both (J, K, L) for 6 months compared with the untreated control group (A, B, C). Hematoxylin-eosin-stained sections at original magnification of x5 are shown in panels A, D, G, and J. Hematoxylin-eosin-stained sections at original magnification of x50 are shown in panels B, E, H, and K. In panels C, F, I, and L, immunohistochemical staining for keratin (at original magnification of x50) is shown. The ferrets were treated as positive if any keratinized squamous metaplasia lesion (histologic examination plus confirmations by immunohistochemistry with anti-keratin antibody) was observed in the right upper lobe of lung. The squamous metaplasia lesions were observed in both the high-dose ß-carotene-supplemented group (D, E, F) (six positive samples of six total samples) and the smoke-exposed group with high-dose ß-carotene supplementation (J, K, L) (six positive samples of six total samples) compared with the smoke-exposed group (G, H, I) (no positive sample of six total samples) or the untreated control group (A, B, C) (no positive sample of six total samples).

 
To assess a potential increase in cell proliferation in whole lung, we analyzed PCNA expression from the four groups of ferrets. PCNA expression increased 3.7-fold or 1.8-fold (Fig. 3)Go in the lungs of ß-carotene-supplemented ferrets with or without smoke exposure, respectively.



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Fig. 3. Effect of ß-carotene feeding, smoke exposure, or the combination on proliferating-cell nuclear antigen (PCNA) expression, retinoic acid receptors (RARs) (RAR{alpha} and RARß) gene expression, and activator protein-1 (encoded by c-Jun and c-Fos) expression in the lung of ferrets. Representative western blot analyses are shown that used anti-PCNA, anti-RAR{alpha}, anti-RARß, anti-c-Jun, and anti-c-Fos antibodies. Lane 1, control; lane 2, smoke-exposed; lane 3, high-dose ß-carotene feeding; and lane 4, smoke-exposed with additional high-dose ß-carotene feeding. The size of the detected PCNA was 36 kilodaltons (kDa). The size of the detected RAR{alpha} and RARß was 53 kDa. The sizes of the detected c-Jun and c-Fos were 39 kDa and 62 kDa, respectively. The intensity of the protein signal was determined by densitometry analysis (six samples in each group). The relative protein values in the three treatment groups were calculated as percent ± standard deviation (SD) compared with the untreated group as 100%.

 
We examined both RAR gene expression and activator protein-1 (AP-1 [c-Jun/c-Fos]) gene expression in the lungs of the four groups of ferrets. The results show that RARß gene expression (Fig. 3),Go but not RAR{alpha} or RAR{gamma} (data not shown), was down-regulated in the three treatment groups (smoke-exposed, 18%; high-dose ß-carotene, 62%; or both, 73%) compared with the control group. We also found that c-Jun and c-Fos (Fig. 3)Go expression was up-regulated threefold to fourfold in the ferrets given a ß-carotene supplement and exposed to smoke compared with the control animals.


    DISCUSSION
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 
In this study, cigarette smoke exposure decreased the elevated levels of ß-carotene in both plasma and lung in ferrets given the ß-carotene supplement and decreased the levels of ß-carotene in the lung in the unsupplemented group (Table 1).Go ß-Carotene can be cleaved in mammalian tissues mainly at the central double bond (C-15,15') (19,20) and also at excentric double bonds (e.g., C-13',14', C-11',12', C-9',10', and C-7',8') (13,21) to form retinoids and ß-apo-carotenoids, which have structures that are similar to retinoids. Our results (Fig. 1)Go of in vitro incubations of all-trans-ß-carotene with the postnuclear fractions of lung tissue from ferrets exposed or not exposed to smoke showed that one reason for the dramatic decrease in ß-carotene was the enhanced formation of ß-carotene oxidative metabolites. These data indicate that the free-radical-rich atmosphere in the lung of cigarette smokers can lead to conditions that modify ß-carotene metabolism to form an abundance of metabolites that are structurally similar to retinoids. Therefore, it is possible that cigarette smoke, ß-carotene supplementation, or both may interfere with the metabolism of tissue ß-carotene and retinoic acid and, therefore, retinoid signaling. This notion was supported by our observation that the concentration of retinoic acid in the lung tissue was decreased significantly in all three treatment groups compared with the control group (Table 1)Go. Such a process could occur after the induction of cytochrome P450 enzymes, as was demonstrated (22,23) after administration of carotenoids. It has been reported that ß-apo-8'-carotenal, an excentric cleavage product of ß-carotene, is a strong inducer of cytochrome P450 enzymes (23). Such an induction of cytochrome P450 enzymes might occur with high-dose ß-carotene supplementation because the formation of ß-apo-8'-carotenal was 2.5-fold higher in lung extracts from ferrets exposed to smoke than in control ferrets (Fig. 1).Go

Vitamin A deficiency is known to result in replacement of the mucociliary epithelium with keratinized squamous epithelium in the tracheobronchial mucosa (24). We therefore reasoned that the decreased lung concentration of retinoic acid, which is a ligand for two classes of nuclear receptors—the RARs (RAR{alpha}, RARß, and RAR{gamma}) and the retinoid X receptors (RXR{alpha}, RXRß, and RXR{gamma}) (25)—may cause diminished retinoid signaling, enhanced lung cell proliferation, and potentially tumor formation. Our results show that localized keratinized squamous metaplasia (a precancerous lesion) was observed in all ferrets in the high-dose ß-carotene-supplemented group with or without exposure to smoke (Fig. 2)Go and support our hypothesis. These lesions correlated with our observation that PCNA expression increased 1.8-fold in the lungs of ß-carotene-supplemented ferrets and 3.7-fold in the lungs of ferrets supplemented with ß-carotene and exposed to cigarette smoke (Fig. 3).Go Furthermore, we demonstrated that the expression of RARß but not RAR{alpha} or RAR{gamma} (data not shown) was down-regulated in the three treatment groups compared with the control group (Fig. 3).Go These data indicate that, in contrast to the up-regulation of RARß gene expression by retinoic acid, the down-regulation of RARß expression in ferret lung after high-dose ß-carotene feeding, smoke exposure, or both could be a mechanism for enhancement of lung tumorigenesis after high-dose ß-carotene supplementation and cigarette smoke exposure. Several lines of evidence have demonstrated that RARß plays an important role in normal lung development (26-28). Primary lung tumors and lung cancer cell lines lack RARß expression, and such loss of expression may be an early event in lung carcinogenesis (26,29-31). Most RARß expression abnormalities involve reduced or absent expression of the RARß2 isoform, the most abundant isoform in normal human lung tissue (26,27). Furthermore, restoration of RARß2 in a RARß-negative lung cancer cell line has been reported to inhibit tumorigenicity in nude mice (32). Because a role for RARß as a tumor suppressor gene has been proposed (33), loss of a tumor suppressor function, by mutation or transcriptional repression, could lead to enhanced cell proliferation and potentially to tumor formation. However, in our study, smoke exposure alone caused only a mild aggregation and proliferation of macrophages in the lung tissue of ferrets, while the retinoic acid level of the lung was decreased. This result raises the possibility that some of the excentric cleavage products of ß-carotene could act as weak ligands (and/or agonists) and interfere with retinoic acid binding activity, a subject that requires further investigation.

Lung carcinogenesis is also associated with an alteration in retinoid signaling involving the AP-1 complex (34), which mediates the signal from growth factors, inflammatory peptides, oncogenes, and tumor promoters, usually resulting in cell proliferation. AP-1 transcriptional activity can be inhibited by retinoid treatment (35). This inhibitory effect on AP-1 activity by retinoids contributes to the suppression of human bronchial epithelial squamous metaplasia (34). We found that expression of c-Jun and c-Fos was up-regulated threefold to fourfold in the ß-carotene-supplemented ferrets that were exposed to smoke compared with the control animals (Fig. 3).Go This overexpression of AP-1 was positively associated with squamous metaplasia (Fig. 2)Go and with increased expression of PCNA (Fig. 3)Go and was inversely associated with the expression of RARß (Fig. 3)Go in the lungs of the ß-carotene-supplemented ferrets that were exposed to smoke, although the associations were not strong in ferrets supplemented with ß-carotene alone. This observation implies a causal relationship between diminished retinoid signaling and an overexpression of AP-1 by high-dose ß-carotene supplementation, resulting in enhancement of lung squamous metaplasia and potential tumorigenesis.

Our finding that high-dose ß-carotene supplementation alone could result in precancerous lesions was surprising, since one human intervention trial (7), which did not include many smokers, did not reveal any adverse effects of ß-carotene supplementation in terms of excess cancer or death. Since there was no assessment for precancerous lesions in the lung tissue in this human intervention trial, the possibility that preneoplastic lesions occurred cannot be excluded and should be addressed in further follow-up studies of this population.

In summary, one of the possible mechanisms responsible for the harmful effect of ß-carotene supplementation in smokers demonstrated in this study is that the free-radical-rich atmosphere in the lungs of cigarette smokers enhances ß-carotene oxidation and the formation of excentric cleavage oxidative metabolites. These metabolites might cause diminished retinoid signaling by down-regulating RARß expression and the retinoic acid level in lung tissue and by up-regulating AP-1 and, therefore, accelerating lung tumorigenesis. Our findings shed light on the potential harmful effects of high-dose ß-carotene supplementation, particularly as it relates to cigarette smoke, and offer a mechanistic understanding of what was observed in the human ß-carotene experiments. Conversely, if low levels of ß-carotene excentric cleavage products are produced in the cells, as would be the case when one consumes ß-carotene from a carotenoid-enriched diet, this form of carotenoid intake could be beneficial and antiproliferative because low levels of excentric cleavage products of ß-carotene can, in and of themselves, give rise to some retinoic acid (14,36,37).


    NOTES
 
Support in part by Public Health Service grant R01CA-49195 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by U.S. Department of Agriculture contract 53-3K06-01.

We thank Drs. Jeffery Settleman, Shumin Zhang, and Elizabeth J. Johnson for helpful advice.


    REFERENCES
 Top
 Abstract
 Introduction
 M aterials and Methods
 Results
 Discussion
 References
 

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Manuscript received April 17, 1998; revised July 28, 1998; accepted November 2, 1998.


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