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).
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ABSTRACT |
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INTRODUCTION |
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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.
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M ATERIALS AND METHODS |
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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) , RARß, RAR
, 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.
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RESULTS |
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Table 1 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)
.
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).
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DISCUSSION |
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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 receptorsthe RARs (RAR, RARß, and
RAR
) and the retinoid X receptors (RXR
, RXRß, and
RXR
) (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)
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).
Furthermore, we demonstrated that the expression of RARß
but not RAR
or RAR
(data not shown) was down-regulated in the
three treatment groups compared with the control group (Fig. 3).
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). This overexpression of AP-1 was
positively associated with squamous metaplasia (Fig. 2)
and with
increased expression of PCNA (Fig. 3)
and was inversely associated with
the expression of RARß (Fig. 3)
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).
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NOTES |
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We thank Drs. Jeffery Settleman, Shumin Zhang, and Elizabeth J. Johnson for helpful advice.
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Manuscript received April 17, 1998; revised July 28, 1998; accepted November 2, 1998.
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