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Carcinogenesis Advance Access originally published online on May 16, 2006
Carcinogenesis 2006 27(12):2383-2391; doi:10.1093/carcin/bgl074
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dual role of ß-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2

P. Palozza*, S. Serini, S. Trombino3, L. Lauriola1, F.O. Ranelletti2 and G. Calviello

Institute of General Pathology, Catholic University School of Medicine Rome, Italy
1 Institute of Pathology, Catholic University School of Medicine Rome, Italy
2 Institute of Histology, Catholic University School of Medicine Rome, Italy
3 Department of Pharmaceutical Sciences, University of Calabria Cosenza

*To whom correspondence should be addressed Email: p.palozza{at}rm.unicatt.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results from some intervention trials indicated that supplemental ß-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100–150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of ß-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying ß-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6–25 µg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), ß-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50–100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of {alpha}-tocopherol (25 µM) prevented the prooxidant effects of the carotenoid. ß-Carotene auto-oxidation, measured as formation of 5,6-epoxy-ß,ß-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

Abbreviations: MDA, malondialdehyde; ROS, reactive oxygen species; TBA, 2-thiobarbituric acid


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Although epidemiological studies evidenced that ß-carotene may be protective against several types of cancers (1), results from two randomized trials, the {alpha}-Tocopherol, ß-Carotene cancer prevention (ATBC) trial in Finland (2) and the beta-carotene and retinol efficacy trial (CARET) in the United States (3) showed that supplemental ß-carotene increased the risk of lung cancer in chronic smokers. Moreover, experiments on cigarette smoke-exposed ferrets fed ß-carotene supplements have demonstrated increases in molecular markers of cell proliferation as well as histopathological changes in lung tissues (4). In addition, in Balb/c 3T3 cells, ß-carotene has been reported to act as an enhancer of cell transforming activity of cigarette smoke condensate (5). Finally, in immortalized fibroblasts exposed to cigarette smoke condensate, the carotenoid has been reported to modify p53-related pathways of cell proliferation and apoptosis with consequent increase in cell growth (6). All these findings raised much debate on the underlying mechanism(s) by which the carotenoid may promote carcinogenesis. Several hypotheses have been proposed to explain the negative results of ß-carotene in association with cigarette smoke. The carotenoid may (i) activate and/or induce phase I carcinogen-bioactivating enzymes, including activators of cigarette smoke carcinogens, such as polycyclic aromatic hydrocarbons (7,8); (ii) decrease the absorption of other carotenoids with better antioxidant profile (9); and (iii) induce at high doses the formation of metabolites, which may cause diminished retinoic acid signaling by down-regulating RARß expression and up-regulating AP-1 with consequent possible acceleration of lung tumorigenesis (4). A further attractive hypothesis that may be put forward to explain the promoting effects of the carotenoid on cell growth is that at high serum levels of ß-carotene, as those reported in both CARET and ATBC trials, prooxidant characteristics of the carotenoid may become evident, yielding reactive oxygen species (ROS) (10,11). In accordance with this hypothesis, ß-carotene antioxidant chemistry is known to display a striking dependence on oxygen tension (12). The carotenoid acted as an effective antioxidant at low pO2, while it could be readily autoxidized and could exhibit prooxidant properties at high pO2 in several biological models, including homogeneous solutions (13), isolated membranes (14) and intact cells (15,16).

Moreover, tar is known to contain high concentrations of stable radicals (17–19) and it is continually being deposited in the lung of smokers and associates with DNA damage (20). The high concentration of ROS in smoke is probably one of the major factors contributing to a high incidence of lung cancer in smokers. In accordance with this, smokers possess higher levels of oxyradicals, including reactive nitrogen species and other oxidants, and lower levels of antioxidants in their lungs than do non-smokers (18,20–22). Moreover, a prooxidant character of ß-carotene could be further enhanced by a high carotenoid concentration. Mayne (1) observed that the high dose given as a supplement in the ATBC and CARET trials resulted in carotenoid blood levels much higher (3.0 and 2.1 mg/l in ATBC and CARET, respectively) than those reported for the US population (0.05–0.5 mg/l). Consistent with these findings, lung tissue from chronic smokers may represent a biological environment which may tip the ß-carotene antioxidant–prooxidant balance toward a prooxidant state: it exhibits a relatively high oxygen tension (100–150 mmHg) and accumulates high levels of cigarette smoke condensate (tar) (20). Finally, the ability of ß-carotene to induce over-generation of ROS via cytochrome P450 activity appeared to be relatively high in lung tissues (8). In the lung, the relatively high pO2 combined with ROS from tobacco smoke condensate may be conducive for ß-carotene oxidative breakdown products, acting as propagators of free-radical formation.

In this study, we investigated the interactions existing between cigarette smoke and ß-carotene, under different oxygen tensions, in isolated lung membranes. For this purpose, rat lung microsomes, enriched with different concentrations of ß-carotene, were exposed, under varying oxygen tensions, to aqueous cigarette tar.

Aqueous extracts of cigarette tar have been used to model the types of species that lung molecules are exposed to in smokers' lungs (20). With respect to gas-phase smoke, in which radicals are transient and rapidly vaporized and exhaled, the effects of aqueous cigarette tar that lead to the formation of different stable free radicals, including a quinone/hydroquinone complex capable of reducing molecular oxygen to produce superoxide, hydrogen peroxide and hydroxyl radicals, may last for several hours or even days. Moreover, in the lung it is likely that gas-phase components rapidly vaporize. On the contrary, the lung fluid continuously washes over the micro-deposits and extracts water-soluble components gradually over a long period. Dimethyl sulfoxide was used to extract the aqueous cigarette tar rather than PBS, because, although the redox-sensitive tar components are soluble in water, extraction is more extensive in this solvent.

In this model, we measured the production of malondialdehyde (MDA), a potential mutagenic compound, and conjugated diene formation, as indicators of oxidative stress as well as we investigated the rates of ß-carotene depletion and oxidation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
ß-Carotene and {alpha}-tocopherol were obtained from Fluka Chemica-Biochemica (Buchs, Switzerland). Ethylenediaminetetracetic acid (EDTA), butylated hydroxytoluene (BHT), 1,1,3,3-tetramethoxypropane, ammonium acetate and 2-thiobarbituric acid (TBA) were obtained from Sigma (Sigma Chemical Co, St Louis, MO). Trichloroacetic acid (TCA) and HCl were obtained from Fisher Scientific (Fairlawn, NJ). Tetrahydrofuran (THF) (99.9%) and tetrabutylammonium dihydrogen phosphate were purchased from Aldrich Chemical Co (St Louis, MO). Hexane, methanol, acetonitrile, isobutyl alcohol, 2-propanol, chloroform and ethanol were HPLC grade and obtained from Fluka Chemika-Biochemika (Buchs, Switzerland). The purity of ß-carotene was verified to be 97% by HPLC. Both the solutions of ß-carotene and {alpha}-tocopherol (Fluka Chemika-bioChemika) were prepared immediately before each experiment.

tar preparation
The particulate phase of cigarette smoke condensate (tar) was provided by BAT Italia (British American Tobacco, Rome, Italy). It was obtained by mechanically smoking cigarettes, using a smoking machine (Cerulean, ASM 516 model). Cigarettes were smoked using the puff profile (one 35 ml puff/min) to a butt length of 2.3 cm, as indicated by BAT protocol. The experimental conditions during smoking were 22°C temperature and 60% humidity. The tar from 20 cigarettes was trapped on filters and then extracted with deionized water. The aqueous solutions were filtered through a Whatman (0.2 µm) filter and dried in vacuum at room temperature. The dried cigarette total particulate matter was re-dissolved in dimethyl sulfoxide (DMSO) and stored at –20°C.

HPLC separation of aqueous tar components was performed using a Hypersil ODS column (200 x 4.6 mm) connected to a Perkin Helmer HPLC equipped with a diode array detector. The eluent consisted of water and acetonitrile with an initial composition of 3% acetonitrile over 12 min at a constant flow rate of 1.0 ml/min. The column was washed with 100% acetonitrile for 20 min after each injection (15 µl). HPLC separation of aqueous tar components allowed positive identification of 1,2-dihydroxybenzene (catechol) (0.95 ± 0.1 mM) and 1,4-dihydroxybenzene (hydroquinone) (1.55 ± 0.2 mM) by spiking the extracts with authentic compounds and by comparing the UV spectra at 280 nm recorded with a diode array detector in chromatograms obtained from aqueous tar extracts and authentic compounds.

Microsomal preparation
Lung microsomes were prepared from male Wistar rats. The animals were fed a non-purified commercial diet (Altromin-Rieper diet, Rieper Company, Bz, Italy). The composition of the diet was (% w/w) as follows: crude protein, 23; fat, 5.5; fiber, 5; minerals, 8; carbohydrates, 58.5; and water, 12. The vitamin mixture added to the diet was 2.5 g/kg (vitamin E, 0.1 g/kg) and the mineral mixture was 0.52 g/kg. Rats weighing 180–220 g were killed by cervical dislocation. Lungs were collected on ice immediately after killing and were minced with scissors to small pieces and washed extensively with 0.15 M NaCl. The minced lungs were homogenized with three volumes of ice-cold buffer, 0.25 M sucrose, 5 mM HEPES and 0.5 mM EDTA, pH 7.5, using the Potter–Elvehjem homogenizer for 20 s and transferred to a centrifuge tube. The homogenate was spun at 8000x g for 10 min to remove nuclei and cell debris, the pellet was discarded and the supernatant was centrifuged at 18 000x g to remove mitochondrial pellet. Microsomes were sedimented from supernatant at 105 000x g for 60 min. After washing, they were dispersed in 0.1 M potassium phosphate buffer, pH 7.5, to provide a protein concentration of ~4 mg/ml and stored at –80°C to minimize possible auto-oxidation of lipid components. Microsomal proteins were determined according to the Bradford method.

Incorporation of ß-carotene into microsomal membranes
ß-Carotene was added to the microsomes as indicated previously (14). Briefly, aliquots of ß-carotene in nitrogen-saturated CHCl3/CH3OH (2:1, v/v) were evaporated to dryness in a Potter–Elvehjem homogenizer under a stream of nitrogen. Microsomes in phosphate buffer were added and gently homogenized at 0°C until all the ß-carotene was homogenously dispersed in the microsomes. When microsomes enriched with ß-carotene were ultracentrifuged, the ß-carotene spun down with the microsomes, demonstrating that it was incorporated into or associated with microsomal membranes. The concentration of ß-carotene was verified by HPLC as indicated below, after the extraction of microsomes with one volume of methanol and three volumes of hexane/diethyl ether (1:1). Nearly 100% of the added ß-carotene was found associated with the membranes after microsomal re-sedimentation.

Incubation conditions
Lipid peroxidation was induced by the addition of tar, at the concentrations indicated, to microsomal membranes (2.0 mg/ml) suspended in an incubation medium containing 0.1 M phosphate buffer, pH 7.5. tar was delivered to microsomes as DMSO solutions. Control microsomes received an amount of DMSO equal to that present in tar-treated ones. The amount of DMSO given to the microsomes was not >0.1% (v/v) and did not alter microsomal viability (data not shown).

The reaction mixtures were shaken in the dark at 37°C for 1–3 h, as indicated. Incubations were performed under air (pO2 = 150 mmHg) or a constant humidified flow of either 100% O2 or a N2/O2 mixture, where pO2 = 15 mmHg and, when indicated, pO2 = 50, 75, 100 and 450 mmHg. The pO2 of the incubations was measured with YSI model 5300 biological oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH) equipped with Clark type electrodes prepared with high sensitivity membranes.

When indicated, {alpha}-tocopherol, at the required concentrations, was added to the microsomes and homogenized as a THF solution, containing 0.025% BHT to prevent the formation of peroxides (6). Control microsomes received an amount of THF equal to that present in {alpha}-tocopherol-treated ones. The amount of THF added to the microsomes was not >0.1% (v/v) and did not affect lipid peroxidation. After microsomal centrifugation, the amount of {alpha}-tocopherol associated with microsomal membranes was determined by HPLC, as indicated below.

MDA formation
MDA was extracted and analyzed as indicated (23). Briefly, aliquots of 1 ml of microsomal suspension (2.0 mg proteins) were mixed with 3 ml of 0.5% TCA and 0.5 ml TBA solution (two parts 0.4% TBA in 0.2 M HCl and one part distilled water) and 0.07 ml of 0.2% BHT in 95% ethanol. Samples were then incubated in a 90°C bath for 45 min. After incubation, the TBA–MDA complex was extracted with 3 ml of isobutyl alcohol. The isobutanol extract was mixed with methanol (2:1) prior to injection in an HPLC system. The column was packed with Supelcosil LC-18 material, 3 µm particle size, in a 15 cm x 4.6 mm cartridge format (Supelco, Bellefonte, PA). A 2 cm cartridge precolumn containing 5 µm LC-18 Supelcosil packing was used. The mobile phase was a 1:1 (v/v) mixture of methanol and double-distilled water, with the addition of tetrabutylammonium dihydrogen phosphate (0.05%, w/v), as an ion-pairing reagent. The TBA–MDA adduct was detected by a fluorimeter set to an excitation wavelength of 515 nm and an emission wavelength of 550 nm. At a flow rate of 1 ml/min, the retention time of the TBA–MDA adduct was 5 min. MDA concentration was calculated from a calibration curve generated from a peak height of the MDA standard, prepared by the hydrolysis of 1,1,3,3'-tetramethoxypropane.

Neither ß-carotene nor {alpha}-tocopherol interfered with the MDA assay, because no MDA formation was detected in the medium containing the antioxidants alone in the absence of the microsomes.

Conjugated diene formation
Aliquots of 1 ml of microsomal suspension (2.0 mg proteins) were extracted in chloroform/methanol (2:1, v/v) and conjugated dienes were measured by monitoring the increase of the absorbance at 233 nm. The reference cuvettes lacked the microsomes but contained the prooxidant (tar) and when indicated, ß-carotene and/or {alpha}-tocopherol. In addition, another set of cuvettes contained microsomes to monitor their spontaneous autoxidation. All of the cuvettes were incubated at 37°C, and the 233 nm readings due to oxidized products of ß-carotene and that due to autoxidation were subtracted from the sample cuvettes to obtain the 233 nm reading attributable to conjugated dienes. The difference spectrum obtained was used for the determination of conjugated dienes, using {Sigma} = 25 200 M/cm (24).

ß-Carotene assay
The extraction of ß-carotene from microsomes was performed as indicated by Palozza et al. (16). Sample was dissolved in methanol, and a 20 µl aliquot was analyzed by reverse phase HPLC with spectrophotometric detection on a Perkin–Elmer LC-295 detector at 450 nm (ß-carotene content) and at 350 nm (ß-carotene 5,6-epoxide) (25). The column was packed with Alltech C18 Adsorbosphere HS material, 3 µm particle size, in a 15 x 0.46 cm cartridge format (Alltech Associates, Deerfield, IL). A 1 cm cartridge precolumn, containing 5 µm C18 Adsorbosphere packing was used. Analyses were done by gradient elution, the initial mobile phase was 85% acetonitrile/15% methanol, with the addition of 30% 2-propanol at 8 min. Ammonium acetate, HPLC grade, 0.01%, was added to the initial mobile phase.

{alpha}-Tocopherol assay
The extraction of tocopherol from microsomes was performed as indicated earlier (15). The sample was dissolved in methanol and a 20 µl aliquot was analyzed by reverse phase HPLC with fluorescence detection on a Perkin Elmer 650-LC fluorescence detector with excitation at 295 nm and emission at 340 nm. {alpha}-Tocopherol was eluted with 100% methanol on an Alltech C18 3 µm (Alltech Associates, Deerfield, IL) column.

Statistical analysis
Results were expressed as means ± SEM. Multifactorial two-way ANOVA was adopted to assess any difference in Figures 1, 3 and 4–6, and Table 1. When the F-tests were significant (P < 0.05), post hoc comparisons of means were made using Tukey's Honestly Significant Differences test. Data in Figure 2 were analyzed using one-way ANOVA. When significant values were found (P < 0.05), post hoc comparison of means were made using the Fisher's test. Differences were analyzed using Minitab Software (Minitab, State College, PA).


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  		Table I Effect of varying ß-carotene concentrations on malondialdehyde production in rat lung microsomal membranes exposed to different oxygen partial pressures in the absence and in the presence of tar

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of varying pO2 on tar-induced lipid peroxidation
After 3 h of incubation at 37°C, MDA production was increased by pO2 in lung microsomes and this effect became more remarkable in the presence of tar (Figure 1A). In particular, at the highest concentration of tar tested (25 µg/ml), the rate of peroxidation increased by 20% at 15 mmHg, by 35% at 150 mmHg and almost doubled at 760 mmHg. The time course of tar-induced MDA formation was linear, at least for 3 h, under all three pO2 conditions (Figure 1B).


Figure 1
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  		Fig. 1 Effect of cigarette tar on malondialdehyde (MDA) production in rat lung microsomal membranes exposed to 15, 150 and 760 mmHg. (A) The levels of MDA in microsomes treated with various concentrations of tar for 3 h. (B) The levels of MDA in microsomes treated with tar (25 µg/ml) for different periods of time. The values were the means ± SEM, n = 9. The treatment/concentration (A) and treatment/time (B) interactions were significant (P < 0.05). Values not sharing a letter were significantly different (A: P < 0.005; B: P < 0.003) (Tukey's test).

 
Effect of ß-carotene on tar-induced lipid peroxidation under different pO2
We then examined the effect of ß-carotene on tar-initiated lipid peroxidation in lung microsomes under different pO2. The carotenoid was used at the concentration of 10 nmol/mg protein, which represents the amount normally found in lung tissue after supplementation (4). Microsomes with and without ß-carotene were incubated in the absence and in the presence of tar (25 µg/ml) for 3 h at 37°C in the dark (Figure 2) under 15, 150 or 760 mmHg pO2. In the absence of tar, ß-carotene significantly (P < 0.05) inhibited MDA formation under the low pO2. However, it reduced and completely lost such an ability under 150 and 760 mmHg, respectively. In the presence of tar, the carotenoid still exhibited significant (P < 0.05) antioxidant effects under 15 mmHg, but progressively increased MDA formation by increasing pO2. Tar-induced MDA production was significantly (P < 0.05) enhanced by the carotenoid by ~10% under air and by ~44% under 760 mmHg.


Figure 2
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  		Fig. 2 Effects of cigarette tar and ß-carotene, alone and in combination, on malondialdehyde (MDA) production in rat lung microsomes exposed to 15, 150 and 760 mmHg. Microsomes were treated with tar at the concentration of 25 µg/ml and ß-carotene at the concentration of 10 nmol/mg protein for 3 h. The values were the means ± SEM, n = 11. Values not sharing a letter were significantly different (P < 0.05, Fisher's test).

 
Using MDA formation at 3 h as an index of lipid peroxidation, we also compared the effect of varying ß-carotene concentration (1–10 nmol/mg protein) on spontaneous and tar-induced lipid peroxidation in lung microsomes at the different pO2 (Table 1). The carotenoid alone acted as an antioxidant under 15 mmHg, protecting membranes from spontaneous MDA formation in a dose-dependent manner. Such a protection was progressively lost by increasing pO2. The carotenoid in combination with tar still maintained its antioxidant properties under 15 mmHg. However it induced dose-dependent prooxidant effects by increasing pO2. Under 150 mmHg, a significant increase in tar-induced MDA formation was observed at a carotenoid concentration of 7.5 nmol/mg prot, while under 760 mmHg, such an effect was found even starting from a carotenoid concentration of 1 nmol/mg prot.

The effects of pO2 ranging from 15 to 760 mmHg on MDA production of lung microsomes enriched with ß-carotene (10 nmol/mg protein) in the absence (A) and in the presence (B) of tar (25 µg/ml) for 3 h are shown in Figure 3. The two panels inside Figure 3A and B are representative of pO2s ranging only from 15 to 150 mmHg. In the absence of tar, antioxidant effects by the carotenoid were observed under pO2 lower than 150 mmHg. At higher pO2s, ß-carotene completely lost its ability in protecting lung microsomes from spontaneous lipid peroxidation. In the presence of tar, the carotenoid clearly acted as an antioxidant under pO2 ranging from 15 to 50 mmHg. It completely lost its antioxidant ability under pO2 ranging from 75 to 100 mmHg and became a strong prooxidant by further increases of the pO2. Interestingly, oxygen pressure increased in a linear manner the effects of tar and ß-carotene on MDA production.


Figure 3
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  		Fig. 3 Effects of varying pO2 on malondialdehyde (MDA) production in rat lung microsomal membranes treated with ß-carotene alone (A) and in combination with cigarette tar (B). The panels inside the figures represent MDA production under pO2 only ranging from 15 to 150 mmHg. Microsomes were treated with ß-carotene at the concentration of 10 nmol/mg protein and tar at the concentration of 25 µg/ml and for 3 h. The values were the means ± SEM, n = 5. The interaction treatment/pO2 concentration was significant (P < 0.05). Values not sharing a letter were significantly different (A: P < 0.005; B: P < 0.002) (Tukey's test).

 
We also evaluated the effects of ß-carotene, alone and in combination with tar, under different pO2s, on another marker of lipid oxidation. In particular, we also measured the production of conjugated dienes, early products in the lipid peroxidation process in the absence and in the presence of tar (25 µg/ml) at 3 h of incubation in lung microsomes (Figure 4). Under 15 mmHg, ß-carotene (10 nmol/mg protein) inhibited tar-induced conjugated diene formation. Such an effect was lost at 150 mmHg and again the carotenoid exhibited clearly prooxidant effects under 760 mmHg, confirming the results obtained on MDA. On the other hand, a progressive loss of the carotenoid ability in inhibiting spontaneous conjugated diene formation was observed by increasing pO2.


Figure 4
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  		Fig. 4 Effects of cigarette tar and ß-carotene, alone and in combination, on conjugated diene production in rat lung microsomes exposed to 15 mmHg, 150 mmHg and 760 mmHg. Microsomes were treated with ß-carotene at the concentration of 10 nmol/mg protein and tar at the concentration of 25 µg/ml for 3 h. The values were the means ± SEM, n = 5. The interaction treatment/pO2 concentration was significant (P < 0.05). Values not sharing a letter were significantly different (A: P < 0.005) (Tukey's test).

 
Effect of {alpha}-tocopherol on the prooxidant activity of ß-carotene in combination with tar
The effects of {alpha}-tocopherol (25 µM) on spontaneous and tar-induced MDA production in the absence and in the presence of ß-carotene (10 nmol/mg protein) under 150 mmHg and under 760 mmHg pO2 in lung microsomes are shown in Figure 5. Microsomes were incubated for 3 h. No significant changes in spontaneous MDA formation by {alpha}-tocopherol were observed in lung microsomes in the absence of tar and ß-carotene. In contrast, {alpha}-tocopherol induced a significant inhibition of MDA production in microsomes exposed to tar, alone and in combination with the carotenoid, under both the two pO2. However, it appears that {alpha}-tocopherol is more effective at 150 mmHg compared with 760 mmHg as a radical scavenger. The inhibition of MDA formation by {alpha}-tocopherol was dose-dependent, starting from 10 µM, and reached the maximum at the concentration of 25 µM (data not shown). A further increase of {alpha}-tocopherol concentration up to 30 µM did not modify the antioxidant potency of the tocopherol in our model (data not shown).


Figure 5
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  		Fig. 5 Effects of {alpha}-tocopherol on malondialdehyde (MDA) production in rat lung microsomal membranes treated with tar alone and in combination with ß-carotene under 150 and 760 mmHg. Microsomes were treated with tar at the concentration of 25 µg/ml and ß-carotene at the concentration of 10 nmol/mg protein for 3 h. The values were the means ± SEM, n = 6. The interaction treatment/concentration was significant (P < 0.05). Values not sharing a letter were significantly different (A: P < 0.001; B: P < 0.002) (Tukey's test).

 
Effect of varying pO2 on ß-carotene oxidation in the absence and in the presence of tar
To determine the effect of varying pO2 on ß-carotene oxidation, lung microsomes pre-enriched with ß-carotene (10 nmol/mg protein), were incubated in the absence (panels A and B) and in the presence (panels C and D) of tar (25 µg/ml) for different periods of time under 15, 150 and 760 mmHg pO2 and the carotenoid consumption (panels A and C) as well as 5,6-epoxy-ß,ß-carotene formation (panels B and D) was measured (Figure 6). The rate of ß-carotene consumption was time-dependent and increased by enhancing pO2. Such an effect was much more remarkable in microsomes exposed to tar. On the other hand, the rate of 5,6-epoxy-ß,ß-carotene formation was strongly enhanced by pO2. In the absence of tar, this effect was extremely remarkable from 15 to 150 mmHg pO2 and less evident from 150 to 760 mmHg pO2. In the presence of tar, a slighter increase in 5,6-epoxy-ß,ß-carotene formation was observed from 15 to 150 mmHg pO2 with respect to tar-untreated microsomes and under 760 mmHg pO2, the formation of this product was even lower than that observed under 150 mmHg pO2. These data seem to suggest that both radical trapping and auto-oxidation reactions consume the carotenoid.


Figure 6
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  		Fig. 6 ß-Carotene loss (A, C) and 5,6-epoxy-ß,ß-carotene formation in rat lung microsomal membranes exposed to ß-carotene alone (A, B) and in combination with tar (C, D) under 150 and 760 mmHg for different periods of time. Microsomes were treated with ß-carotene at the concentration of 10 nmol/mg protein and tar at the concentration of 25 µg/ml and for 3 h. The values were the means ± SEM, n = 7. The interaction treatment/time was significant (P < 0.05). Values not sharing a letter were significantly different (A: P < 0.005; B: P < 0.003; C: P < 0.005; D: P < 0.002) (Tukey's test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The mechanism by which ß-carotene may result in an increased lung cancer incidence and mortality among smokers has to be defined. In this study, we show that oxygen tension may deeply influence the ability of ß-carotene to modulate cigarette smoke condensate (tar)-induced lipid peroxidation. In particular, we demonstrated that the carotenoid, at concentrations close to those found in vivo after supplementation, may promote the formation of highly toxic lipid peroxidation products in the presence of cigarette tar at the oxygen pressure present in lung.

ROS have been reported to play an important role in several models of lung injury (26). When the production of ROS exceeds the ability of the antioxidant system to eliminate them, oxidative stress can occur leading to lung injury. Peroxidative destruction of cell membranes may lead to loss of their functional integrity and to the formation of early products of lipid peroxidation, such as conjugated dienes and terminal products of lipid peroxidation, such as MDA, some of which have been implicated in carcinogenesis. In particular, it has been reported that MDA possesses both mutagenic and pro-carcinogenic properties because its ability to interact with functional groups of a variety of cellular compounds, including the amino groups of proteins and nucleic acid bases, the N bases of phospholipids and the SH groups of sulphydryl compounds (27).

A 3 h incubation of isolated lung microsomal membranes was able to induce a spontaneous MDA production. Such a production was much lower than that observed in other tissues, such as liver, as we reported previously. This can be presumably due to the low ratio of peroxidable PUFAs to vitamin E in lung microsomes. In accordance with this, Kornbrust et al. (28) showed that highly oxygenated tissues, such as lung and heart microsomes, peroxidized at a much lower extent than liver, kidney, testes and brain microsomes. Moreover, Leedle and Aust (29) demonstrated that rat lung microsomes were much more resistant to lipid peroxidation than those from liver in both enzymatic and non-enzymatic systems. The spontaneous MDA production was strongly increased by increasing pO2. This finding is in agreement with previous observations from other authors. Freeman and Crapo (30) provide evidence for an association between hyperoxia and the increased production of partially reduced species of oxygen by the lung and lung-isolated membranes. Moreover, the formation of thiobarbituric acid-reactive substances (TBARs) in the homogenates of lung was strongly stimulated at 100% compared with 20% oxygen (31). Finally, aqueous cigarette tar extract caused marked decrease in erythrocyte deformability with concomitant increase of membrane lipid peroxidation (32).

In our study, it is clearly shown that tar acted as a cellular stressor, increasing lipid peroxidation, measured as both conjugated diene formation and MDA production, in lung microsomes. The prooxidant action of tar was dose- and time-dependent and strongly increased by increasing pO2. This is presumably due to the fact that cigarette tar consists of a mixture of different stable free radicals, including a quinone/hydroquinone complex, which is capable of reducing molecular oxygen to produce superoxide, hydrogen peroxide and hydroxyl radicals (18). An increased lipid peroxidation as a consequence of a cigarette tar exposure has been reported previously in vitro and in vivo studies. The levels of conjugated dienes were significantly higher in the lung tissues of rat exposed to cigarette tar with respect to those of untreated rats (33). Cigarette smoke exposure also induced peroxidation of microsomal lipids, as evidenced by the formation of conjugated dienes, MDA and fluorescent pigments (34). Moreover, coal tar extracts were reported to lead to the in vivo free-radical generation and lipid peroxidation in the striatum, cerebellum and liver of mice (35).

In lung microsomes, pO2 influenced the ability of ß-carotene to modulate both spontaneous and tar-induced lipid peroxidation as well as it affected ß-carotene consumption and the interaction of the carotenoid with {alpha}-tocopherol. In the absence of tar, ß-carotene inhibited MDA formation under low pO2, but this ability was progressively lost by increasing pO2. This finding is in agreement with other previous observations. A high pO2 reduced the antioxidant efficiency of ß-carotene as well as a low pO2 increased it in several models, including homogeneous solutions (12,13), liposomes (36), isolated membranes (14) and intact cells (15,16). The loss of ß-carotene antioxidant efficiency in these conditions under high pO2 could be explained by the effect of an increased rate of lipid peroxidation, which was 3-fold higher at 760 mmHg than at 15 mmHg. However, in tar-exposed lung microsomes, clearly prooxidant effects by the carotenoid were observed by increasing pO2, suggesting that ß-carotene can play an active role in propagating lipid peroxidation processes. A significant increase in MDA production by a combination of ß-carotene and cigarette tar was observed in lung microsomes at pO2 higher than 100 mmHg. This finding seems to be particularly interesting in view of the fact that the pO2 of non-pulmonary tissues ranged from 5 to 71 mmHg, but it became ~105–150 mmHg in lung tissue. Therefore, it is reasonable to think that a combination of tar and ß-carotene may bring about an increased production of lipid peroxidation products in lung tissue from chronic smokers, exacerbating the oxidative pulmonary damage induced by tar alone.

Our data seem to show that the prooxidant effects of the carotenoid in lung microsomes were also dependent on ß-carotene concentration. In our experimental conditions, microsomes were enriched in vitro with carotenoid concentrations which can be reached in vivo by supplementation (4). In ferrets fed a low (37) and high (4) dose ß-carotene diet, lung levels were 2.83 and 22.3 nmol/g tissue, respectively. When the concentration of carotenoids was determined in lung tissue from autopsies of human subjects in age from 4 months to 86 years, total carotenoid content ranged from 0.1 to 8.4 nmol/g tissue and ß-carotene was one of the most predominant carotenoids found (38). Clearly, it has been reported that ferrets as well as humans possess a much more efficient mechanism to accumulate ß-carotene in their lung tissue than have other species, including rats and mice. Although some reports show that lung appears to be a target organ for ß-carotene even in rats, reaching levels from 0.5 to 5.763 nmol/g wet tissue (39), the different carotenoid metabolism between the different species might account for the different carcinogenic effect of an association between ß-carotene and cigarette smoke observed in some studies. Lung tumour development in mice exposed to tobacco smoke (40) or to NNK (41) was not enhanced by ß-carotene supplementation. It should be considered, however, that the highest lung levels of the carotenoid reported in mice (40) were 0.26 nmol/g lung tissue, which were nevertheless several times lower than the lung levels in ferrets (4). Concomitantly, the lack of prooxidant effects of ß-carotene in human bronchial cells exposed to gas-phase cigarette smoke may be due to the low concentration of the carotenoid found in these cells (42). In fact, carotenoid addition resulted in cellular ß-carotene levels of ~400 pmol/mg prot, a concentration much less than that used in this study (42). Interestingly, ferrets have been reported to show a squamous metaplasia after high-dose ß-carotene supplementation and exposure to tobacco smoke (4). Concomitantly, the serum concentrations of ß-carotene in the two trials where an increase of lung cancer incidence was demonstrated, were markedly higher (3.0 and 2.1 mg/l in ATBC and CARET, respectively, than those reported for the US population (0.05–0.5 mg/l) and for the Physicians' Health Study (1.2 mg/l), where no increase in lung cancer risk was seen from ß-carotene supplementation (1).

Our finding that {alpha}-tocopherol may prevent the prooxidant effects of an association of ß-carotene and cigarette tar strongly supports the possibility of interactions between {alpha}-tocopherol and ß-carotene in tar-induced lipid peroxidation. Several findings evidence that ß-carotene treatment may influence intracellular tocopherol status. In isolated membranes enriched with the carotenoid, the concomitant presence of {alpha}-tocopherol facilitated the antioxidant properties of the carotenoid (43) and/or reverted its prooxidant effects on lipid peroxidation observed under high pO2 (14). In both these studies, an increased loss of {alpha}-tocopherol in the presence of the carotenoid was observed, strongly supporting the hypothesis that {alpha}-tocopherol might be consumed to retard the formation of carotenoid-radical cations and/or the further degradation of the carotenoid to oxidation products. From these observations, it is possible that the lack of preventive effects of ß-carotene and {alpha}-tocopherol in combination on lung cancer incidence in the ATBC trial (2) is due to the fact that {alpha}-tocopherol was administered to chronic smokers at a concentration which is not sufficient to counteract the prooxidant effects of the carotenoid. Moreover, it should be also mentioned that antioxidants, other than {alpha}-tocopherol, may play a role in the protection of the prooxidant effects of ß-carotene in lung tissue and that chronic exposure to cigarette smoke has been reported to deeply modify intracellular redox status, by modulating the expression and the activity of antioxidant enzymes and the content of other endogenous antioxidants. In particular, it has been observed that cigarette smoke condensate induced a strong decrease in reduced glutathione content in lung tissue accompanied by enhanced levels of oxidized glutathione (44). Such an effect, together with the observation that a high pO2 is able to increase total glutathione content in human bronchial epithelial cells (45), strongly supports the hypothesis that a complex network of intracellular antioxidants may cooperate in preventing the oxidative damage induced by smoke in lung tissue.

The loss of the antioxidant effects of ß-carotene in lung membranes under relatively high pO2 could be due to an increased formation of ß-COO., as suggested by Burton and Ingold (12). These authors hypothesized that the prooxidant effect of ß-carotene could be due to a high formation of chain-carrying ß-carotene peroxyl radicals (ß-COO.) over the carbon-centered radicals (ß-C.), able to trap free radicals. Although potential ß-COO. forms have not yet been isolated, several adducts and products arisen from the reaction of ß-carotene with free-radical species have been described elsewhere (25,36,46,47). In particular, several studies show that tobacco smoke oxidized carotenoid molecules, forming different oxidation products (4,42,48,49). It has been hypothesized that the decreased antioxidant efficiency of ß-carotene at high pO2 could be due to an increased ß-carotene auto-oxidation, which consumed the molecule without scavenging free radicals (42). Reactions of auto-oxidation occurred in our model and they were deeply accelerated by increasing pO2, as observed by the increased formation of 5,6-ß,ß-carotene epoxide in lung microsomes enriched with the carotenoid alone. However, this hypothesis does not explain the prooxidant effect of ß-carotene in association with cigarette tar. Moreover, ß-carotene auto-oxidation was even decreased in ß-carotene enriched microsomes exposed to tar. Thus, it is possible that, in the presence of relatively high concentrations of oxygen, tar-induced oxidation products of ß-carotene could be responsible for an increased lipid peroxidation in lung microsomes.

Several findings seem to support the hypothesis that smoke may increase the prooxidant character of high concentrations of ß-carotene. The carotenoid increased DNA oxidative damage and modified p53-related pathways of cell proliferation and apoptosis in cells exposed to tobacco smoke condensate (6). Moreover, the carotenoid, in its oxidized form, acted as an inducer of oxidative DNA damage and as a pro-carcinogenic agent (50). Carotenoid oxidation products could arise in the free-radical-rich environment in the lungs of cigarette smokers and could be responsible for the apparent pro-carcinogenic effects of high-dose ß-carotene supplements.

Further studies using biopsies of lung tissue isolated from chronic smokers supplemented with high-dose ß-carotene could be necessary to verify the importance of these findings in physiological conditions. Although ß-carotene could be incorporated into biological membranes differently in vitro than in vivo, these data point out that the oxygen pressure present in lung may promote a prooxidant character of ß-carotene in the presence of cigarette tar.


   Acknowledgments
 
This work was supported by MURST-ex 60%.

Conflict if Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 15, 2005; revised April 26, 2006; accepted May 5, 2006.


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