In vitro investigations into the interaction of ß-carotene with DNA: evidence for the role of carbon-centered free radicals

Jos C. S. Kleinjans1, Marcel H. M. van Herwijnen, Jan M. S. van Maanen{dagger}, Lou M. Maas, Theo M. C. M. de Kok, Harald J. J. Moonen and Jacob J. Briedé

Department of Health Risk Analysis and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands

1 To whom correspondence should be addressed Email: j.kleinjans{at}grat.unimaas.nl


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Supplementation by ß-carotene has unexpectedly appeared to increase lung cancer risk among smokers. In order to explain this it has been suggested that at high serum levels of ß-carotene, prooxidant characteristics of ß-carotene may become manifest, yielding reactive oxygen species (ROS) and inducing oxidative DNA damage. It has further been hypothesized that cigarette smoke carcinogens such as benzo[a]pyrene (B[a]P) and/or B[a]P metabolites, may directly react with ß-carotene. Furthermore, ß-carotene oxidation products may have a role in the bioactivation of B[a]P analogous to the peroxide shunt pathway of cytochrome P450 supported by cumene hydroperoxide. The aim of this study was to assess the effects of ß-carotene on the formation of B[a]P–DNA adducts and oxidative DNA damage in vitro in isolated DNA, applying as metabolizing systems rat liver and lung metabolizing fractions and lung metabolizing fractions from smoking and non-smoking humans. We established that ß-carotene in the presence of various metabolizing systems was unable to induce oxidative DNA damage (8-oxo-dG), although ß-carotene is capable of generating ROS spontaneously in the absence of metabolizing fractions. We also could not find an effect of ß-carotene on DNA adduct formation induced by B[a]P upon metabolic activation. We could, however, provide evidence of the occurrence of a carbon-centered ß-carotene radical which was found to be able to interact with B[a]P and to intercalate in DNA.

Abbreviations: B[a]P, benzo[a]pyrene; dG, 2'-deoxyguanosine; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DMSO, dimethylsulfoxide; DRZ, diagonal radioactive zone; ESR, electron spin resonance; HPLC-ECD, high performance liquid chromatography with electrochemical detection; 8-oxo-dG, 7-hydroxy-8-oxo-2'-deoxyguanosine; PAHs, polycyclic aromatic hydrocarbons; PEI, polyethyleneimine; POBN, {alpha}-(4-pyridyl-1-oxide)-N-t-butylnitrone; ROS, reactive oxygen species; SOD, superoxide dismutase; THF, tetrahydrofuran


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several intervention studies assessing the anticarcinogenic properties of ß-carotene in humans unexpectedly found a higher risk for lung cancer in ß-carotene supplemented smokers than in non-supplemented controls (1,2). Also, experiments on cigarette smoke-exposed ferrets fed ß-carotene at comparable doses as applied in these human intervention studies have demonstrated increases in molecular markers of cellular proliferation as well as histopathological changes in lung tissue (3). This has raised much debate on the underlying mechanisms by which ß-carotene, despite its well-established antioxidant and anticarcinogenic capacities in experimental animals (4), may promote, rather than inhibit, chemical carcinogenesis. Various hypotheses for such molecular interactions have been suggested.

  1. ß-carotene may activate and/or induce phase I carcinogen-bioactivating enzymes, including activators of cigarette smoke carcinogens such as polycyclic aromatic hydrocarbons (PAHs), and this may furthermore be associated with the generation of oxidative stress (5).
  2. At high serum levels of ß-carotene, as obtained during these clinical trials applying ß-carotene as a single agent, prooxidant characteristics of ß-carotene may become manifest, yielding reactive oxygen species (ROS) (2,3,6).
  3. Linking the previous hypotheses, it has been suggested that cigarette smoke carcinogens such as benzo[a]pyrene (B[a]P) may react directly with ß-carotene. In particular, ß-carotene oxidation products may have a role in the bioactivation of B[a]P analogous to the peroxide shunt pathway of cytochrome P450 supported by cumene hydroperoxide (7,8).

It is furthermore suggested that these interactions lead to reactive intermediates of B[a]P metabolism and/or ROS, causing chemical damage to lung epithelial DNA, e.g. B[a]P–DNA adducts and oxidative lesions, predominantly 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG), which are considered to be promutagenic (9,10) and thereby contribute to the initiation and promotion of carcinogenesis. This may explain the co-carcinogenic effects of ß-carotene as observed in the clinical trials on smokers. Further, these interactions may differ between tissues: although ß-carotene-induced cytochrome P450 activity appeared to be relatively high in lung tissue of Sprague–Dawley rats and was associated with the overgeneration of superoxide (5), in vivo the liver appears to be the most susceptible organ, possibly due to the fact that it accumulates the highest ß-carotene levels (11). Further, there may exist interspecies differences since cytochrome P450-inducing effects of ß-carotene could not be observed in liver of mice (12) or Wistar rats (13), leaving unanswered the question of to what extent these ß-carotene-mediated interactions with B[a]P metabolism and generation of ROS occur in human target tissues.

The aim of this study was to assess the effect of ß-carotene, at concentrations achieved during the clinical trials, on the formation of B[a]P–DNA adducts and 8-oxo-dG in vitro in isolated DNA, applying as metabolizing systems phenobarbital/5,6-benzoflavone-induced liver and lung S9 mix from male Lewis rats and lung S9 mix from smoking and non-smoking humans. Furthermore, we addressed the question of whether ß-carotene oxidation products, rather than non-oxidized ß-carotene, acts as the inducing agent (7). B[a]P–DNA adducts were analyzed by means of the 32P-postlabeling method (14) and the formation of 8-oxo-dG by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD) (15). At these concentrations of both non-oxidized ß-carotene and ß-carotene oxidation products we did not observe any effect on the formation of B[a]P–DNA adducts or 8-oxo-dG. In order to obtain more information on the generation of ROS during incubations with and without B[a]P, we applied ESR spectroscopy in the absence and presence or rat liver S9 mix. In the absence of this metabolizing system ß-carotene induced oxygen radical formation. However, under neither circumstance in the presence of the metabolic fraction,were there indications of a positive effect of ß-carotene on ROS generation or B[a]P–DNA adduct formation, but we could provide evidence of the occurrence of a carbon-centered ß-carotene radical which probably interacts with DNA through intercalation. The relevance with respect to the postulated co-carcinogenic effect of ß-carotene remains to be proven.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Glass capillaries (100 µl) for ESR spectroscopy were purchased from Brand AG (Wertheim, Germany). B[a]P, salmon sperm DNA and the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and {alpha}-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) were from Sigma (St Louis, MO). Solutions of DMPO in nitrogen-flushed Milli-Q purified water were further purified by charcoal treatment. Stock concentrations of DMPO were determined spectrophotometrically by using the extinction coefficient {varepsilon} = 7700 M–1 cm–1 at 234 nm. ß-carotene was purchased from CalBiochem (Merck Eurolab, Oslo, Norway). Spleen phosphodiesterase II was obtained from Worthington Biochemical Co. (Lakewood, NJ). T4 polynucleotide kinase was from Amersham (Braunschweig, Germany). Polyethyleneimine (PEI)–cellulose plates were from Macherey-Nagel (Düren, Germany) and [{gamma}-32P]ATP with a specific activity of ~7000 Ci/mmol was obtained from ICN (Meckenheim, Germany). Superoxide dismutase (SOD) and catalase was from Boehringer-Mannheim (Mannheim, Germany).

ß-carotene was dissolved in tetrahydrofuran (THF) at a stock concentration of 1.0 mg/ml, which was confirmed by UV-Vis spectrometry. Contact with oxygen was prevented under any circumstance and solutions were consequently deoxidized by gassing with N2. For the preparation of ß-carotene oxidation products, this was subsequently diluted in Tris buffer and strongly stirred at room temperature for 24 h. Complete oxidation of ß-carotene was confirmed by UV-Vis spectrometry.

Single-stranded salmon sperm DNA was obtained by denaturation of double-stranded DNA at 95°C for 10 min, immediately followed by chilling on ice. Spectrophotometrical determination showed that 96% of the DNA was converted into its single-stranded conformation.

Incubations
S9 microsomal mixtures were prepared by differential centrifugation, using standard procedures, from pooled lung and liver of three male Lewis rats after 5 days' induction with phenobarbital/5,6-benzoflavone, as well as from human non-tumorous lung epithelial biopsies, taken during surgery of lung cancer patients in the Academic Hospital Maastricht.

The incubation concentration range of ß-carotene was derived from data on background plasma ß-carotene concentrations in humans and on plasma levels of ß-carotene as obtained during human intervention studies (1,2), as well as on incubation concentrations used in a previous in vitro study (16), and consisted of 0 (THF solvent control), 170, 3000 and 15 000 µg/l. The same incubation range was applied for oxidized ß-carotene.

The optimal incubation concentration of B[a]P to generate B[a]P–DNA adducts after metabolic activation was selected by evaluating a concentration–effect range of 0 (DMSO solvent control), 1, 10, 100 and 1000 µM, and appeared to be 10 µM B[a]P.

Test concentrations of ß-carotene and/or B[a]P were incubated in duplicate in Tris buffer (40 mM, pH 8.0) supplemented with 2 mM MgCl2 for 1 h at 37°C with 400 µg/ml salmon sperm DNA with and without 2 mg/ml rat liver, rat lung or human lung S9 fractions in the presence of a NADPH generating system containing 0.5 mM NADP (Roche), 5 mM glucose 6-phosphate (Boehringer-Mannheim) and 1.0 U/ml glucose 6-phosphate dehydrogenase (Roche, Mannheim, Germany). As positive controls for the induction of oxidative DNA damage a hydroxyl radical generating system (293 mM H2O2 and 1, 2 or 5 µM FeSO4) and a superoxide generating system (40 µM xanthine and 40 mU xanthine oxidase) were used.

At the end of the incubation period, DNA was isolated by means of repetitive extraction with radical-free phenol, phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) and precipitated with 2 vol of 100% cold ethanol and 1/30 vol of 3 M sodium acetate (pH 5.3). Precipitated DNA was rinsed with 70% ethanol, dissolved in either 2 or 5 mM Tris (pH 7.4) and stored at –20°C until determination of 8-oxo-dG or 32P-postlabeling analysis.

32P-postlabeling assay
The 32P-postlabeling assay was performed as described earlier (14). Briefly, 10 µg DNA was digested to deoxyribonucleoside 3'-monophosphates using calf spleen phosphodiesterase (2.8 µg) and micrococcal endonuclease (0.4 U). Half of the digest was treated with nuclease P1 (6.25 µg) and subsequently labeled with 50 µCi/sample [{gamma}-32P]ATP in the presence of T4 polynucleotide kinase (5 U). Radiolabeled adducted nucleotide biphosphates were separated by two-dimensional chromatography on PEI–cellulose sheets using the following solvent systems: D1, 1 M NaH2PO4, pH 6.5; D2, 8.5 M urea, 5.3 M lithium formate, pH 3.5; D3, 1.2 M lithium chloride, 0.5 M Tris, 8.5 urea, pH 8.0; D4, 1.7 M NaH2PO4, pH 6.0. To ensure the efficiency of nuclease P1 treatment and excess ATP, an aliquot of the digest was one-dimensionally chromatographed on PEI–cellulose sheet using a solvent system of 0.12 M NaH2PO4, pH 6.8. For quantification purposes, two [3H]B[a]P-7,8-dihydrodio-9,10-epoxide-modified DNA standards (1 adduct per 107 and 108 unmodified nucleotides) were run in parallel in all experiments. Quantification was performed using a phosphoimager (Molecular DynamicsTM, Sunnyvale, CA). Quantitatively, half of the detection limit for the diagonal radioactive zone (DRZ) (0.25 adducts per 108 nucleotides) was taken as the level of adducts for samples which showed neither a DRZ nor an adduct spot in their adduct maps. Nucleotide quantification was done by labeling the remaining half of the digested DNA with [{gamma}-32P]ATP in the presence of T4 polynucleotide kinase, followed by one-dimensional chromatography on PEI–cellulose sheet using a solvent system of 0.12 M NaH2PO4, pH 6.8. Results are expressed as numbers of adducts per 107 nucleotides.

HPLC-ECD for determining 8-oxo-dG
HPLC-ECD of 8-oxo-dG was based on a method described earlier (15). Briefly, after extraction, DNA was digested to deoxyribonucleosides by treatment with nuclease P1 (0.02 U/µl) and alkaline phosphatase (0.014 U/µl). The DNA extraction procedure has been optimized in order to minimize artificial induction of 8-oxo-dG. This was achieved by using radical-free phenol, minimizing exposure to oxygen and by evaluating the effect of 1 mM deferoxamine mesylate and 20 mM TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), all factors which prevent the induction of positive artefacts in the analysis of 8-oxo-dG (European Standards Committee on Oxidative DNA Damage). The digest was then analysed by HPLC-ECD, using a SupelcosilTM LC-18S column (250 x 4.6 mm) (Supelco Park, Bellefonte, PA) and a DECADE lectrochemical detector (Antec, Leiden, The Netherlands). The mobile phase consisted of 10% aqueous methanol containing 94 mM KH2PO4, 13 mMK2HPO4, 26 mM NaCl and 0.5 mM EDTA. Elution was performed at a flow rate of 1.0 ml/min with a lower detection limit of 40 fmol absolute for 8-oxo-dG or 1.5 residues/106 2'-deoxyguanosine (dG). 8-Oxo-dG was detected at a potential of 850 mV and dG was simultaneously monitored at 260 nm. Results are expressed as percentages of the ratios of 8-oxo-dG to dG, relative to control ratios.

Electron spin resonance (ESR) spectroscopy
ESR spectra were recorded at room temperature in glass capillaries on a Bruker EMX 1273 spectrometer equipped with an ER 4119HS high sensitivity cavity and 12 kW power supply operating at X band frequencies. The modulation frequency of the spectrometer was 100 kHz. Instrumental conditions for the recorded spectra were as follows: magnetic field, 3490 G; scan range, 60 G; modulation amplitude, 1 G; receiver gain, 1 x 105; microwave frequency, 9.85 GHz; power, 50 mW; time constant, 40.96 ms; scan time, 20.97 s; number of scans, 50. Quantitation of the spectra (in arbitrary units) was performed by peak height measurements using the WIN-EPR spectrum manipulation program. Radical formation was followed by performing incubations of 15 µg/ml ß-carotene and 10 µM B[a]P in the presence of 100 mM spin trap (POBN or DMPO) and in the absence and presence of salmon sperm DNA, as described above. The effects of SOD (500 U) and catalase (1000 U) on radical formation were also investigated. Samples for ESR detection were taken from the incubates at the time intervals indicated. In control experiments, ß-carotene and/or B[a]P was replaced by equal amounts of the vehicles, THF and DMSO, respectively.

Statistics
Statistical significance of differences between the intensities of spin adduct signals were calculated by one-factor ANOVA. Statistical evaluation of differences in 8-oxo-dG levels and B[a]P–DNA levels in salmon sperm DNA between respective incubations was performed by means of the Mann–Whitney U-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ESR analysis of ROS formation
When ß-carotene or ß-carotene pre-oxidized at room temperature for 24 h was incubated with the spin trap DMPO for 1 h at 37°C, a small but non-significant increase in DMPO-OH production beyond solvent control levels was observed in the presence of ß-carotene, with no increase in the presence of ß-carotene oxidation products (see Table I). This indicates a slight potential of ß-carotene for generating ROS spontaneously. When a co-incubation of ß-carotene or ß-carotene oxidation products with B[a]P was performed, no influence of the presence of B[a]P on DMPO-OH production was observed (see Table I). B[a]P itself also did not increase DMPO-OH above background control levels.


View this table:
[in this window]
[in a new window]
 
Table I. Formation of DMPO-OH during incubation of ß-carotene (15 µg/ml), ß-carotene oxidation products and B[a]P (10 µM) for 1 h at 37°Ca

 
Effect of ß-carotene on 8-oxo-dG levels in salmon sperm DNA
Figure 1 shows levels of salmon sperm 8-oxo-dG upon incubation with ß-carotene and oxidized ß-carotene in comparison to respective controls in the presence of metabolizing systems derived from rat liver and rat lung, as well as from smoker or non-smoker lung tissue. No statistically significant changes in the relative 8-oxo-dG levels are observed. Additionally, the average absolute 8-oxo-dG/dG ratio in control experiments was 72 ± 36 x 10–6 8-oxo-dG/dG. As positive controls, the hydroxyl radical generating system with 1, 2 and 5 mM FeSO4 dose-dependently induced 8-oxo-dG/dG levels of 5.4, 18.5 and 32.9 x 10–4 8-oxo-dG/dG, respectively, while the superoxide radical generating system induced a ratio of 3.9 x 10–4, thereby demonstrating the sensitivity of the assay. The average absolute 8-oxo-dG/dG ratio in incubations with ß-carotene, ß-carotene oxidation products, B[a]P or the combinations of ß-carotene with B[a]P or ß-carotene oxidation products with B[a]P were 73 ± 34 x 10–6 8-oxo-dG/dG and were not increased if compared with the absolute level in control experiments. Also, co-incubation of ß-carotene or ß-carotene oxidation products with B[a]P in the presence of rat liver activating system had no effect on 8-oxo-dG levels in salmon sperm DNA (data not shown).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 1. ß-carotene microsomal metabolism-induced oxidative DNA damage. 8-Oxo-dG levels in salmon sperm DNA were measured after incubation with the indicated concentrations of ß-carotene or ß-carotene oxidation products, in the presence of metabolizing S9 fractions prepared as described in Materials and methods. S9 fractions were obtained from (A) rat liver, (B) rat lung, (C) human lung biopsy material of three non-smoking lung cancer patients and (D) human lung biopsy material of a smoking lung cancer patient.

 
Effect of ß-carotene on the formation of B[a]P–DNA adducts in salmon sperm DNA
Figure 2 shows data on the formation of B[a]P–DNA adducts in the presence of the respective metabolizing systems during co-incubation of 10 µM B[a]P with increasing concentrations of ß-carotene or ß-carotene oxidation products. No significant changes in B[a]P–DNA levels in the presence of ß-carotene or ß-carotene oxidation products were observed: the apparent increase in adduct level at 0.17 µg/ml ß-carotene as well as the decrease at 15 µg/ml ß-carotene in the presence of the rat liver metabolizing system do not gain statistical significance. Furthermore, in in vitro experiments in which we incubated human embryonic lung cells with 10 nM B[a]P, ß-carotene in increasing concentrations of 0.17, 2 and 3 µg/ml had no significant impact on B[a]P–DNA adduct formation either (data not shown).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. B[a]P–DNA adducts levels in salmon sperm DNA upon co-incubation of B[a]P and ß-carotene. B[a]P–DNA adduct levels in salmon sperm DNA were measured after incubation with the indicated concentrations of B[a]P in combination with ß-carotene or ß-carotene oxidation products, in the presence of metabolizing S9 fractions prepared as described in Materials and methods. S9 fractions were obtained from (A) rat liver, (B) rat lung, (C) human lung biopsy material of 3 non-smoking lung cancer patients and (D) human lung biopsy material of a smoking lung cancer patient.

 
Generation of a carbon-centered ß-carotene-derived radical
When incubations of ß-carotene and B[a]P were performed with a NADPH-supported rat liver S9 fraction for 1 h at 37°C in the presence of the spin trap DMPO, at 0 time of incubation a small ESR signal was observed which disappeared with increasing time and which could not be characterized, but showed features of a carbon-centered radical adduct. Since POBN is a much more effective spin trap for carbon radicals in S9 fractions than DMPO (17), we continued experiments with the spin trap POBN. At 0 time of incubation of ß-carotene with S9 fractions, immediately after mixing the components of the incubation mixture, a high level of a POBN spin adduct was observed, which increased above background control levels in the presence of the solvent THF (see Table II). The ESR spectrum showed the characteristics of a POBN carbon-centered radical adduct: AN = 15.6 G, AH = 2.7 G (see Figure 3). These results indicate that immediately after incubation with S9 fraction ß-carotene is oxidized and produces carbon-centered radicals which are immediately trapped by POBN. The level of the POBN-spin trapped radicals of ß-carotene decreased at 30 and 60 min of incubation to background levels. This decrease in POBN-spin trapped radical levels is most probably a consequence of the previously reported reduction in POBN-trapped methyl radicals by rat liver microsomes (18). Addition of SOD, or SOD combined with catalase, to the incubation mixture of S9 and ß-carotene did not decrease the POBN-trapped radicals (data not shown), indicating that the trapped radicals are not superoxide radicals, which can also be produced during oxidation of ß-carotene (see Table I), but indeed a carbon-centered radical of ß-carotene itself. When ß-carotene oxidation products were incubated with S9 mix and a NADPH generating system, no increase in POBN-trapped radical formation relative to the control value was observed.


View this table:
[in this window]
[in a new window]
 
Table II. Formation of POBN-spin trapped carbon-centered radicals during incubation of ß-carotene (15 µg/ml), ß-carotene oxidation products and B[a]P (10 µM) with NADPH-supported rat liver S9 fraction for 1 h at 37°Ca

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Electron spin resonance spectrum of the POBN-spin trapped ß-carotene carbon-centered radical. ß-carotene (15 µg/ml) was added to a Tris-buffered rat liver S9 fraction (2 mg/ml) in the presence of a NADPH generating system and the spin trap POBN (100 mM). Immediately after addition, the ESR signal was measured using the spectrometer settings described in Materials and methods. The hyperfine splitting constants were AN = 15.6 G and AH = 2.7 G.

 
Interaction of the carbon-centered ß-carotene-derived radical with B[a]P
When ß-carotene was co-incubated with B[a]P in the presence of NADPH-supported rat liver S9 fraction, a statistically significant decrease in formation of the POBN-trapped radicals in comparison with incubation of ß-carotene alone was observed (see Table II). Incubation of B[a]P alone with S9 did not result in increased POBN-trapped radicals above the control value. These results indicate that the radical fragments produced by oxidation of ß-carotene by S9 mix react with B[a]P or with metabolites of B[a]P formed in the presence of S9. At 30 and 60 min incubation of ß-carotene with S9, the level of the POBN-trapped radicals decreased to 0 in the presence of B[a]P. Furthermore, no effect of co-incubation of B[a]P with ß-carotene oxidation products was observed. This again indicates that during oxidation of ß-carotene by NADPH-supported S9 fraction, radical fragments are produced from ß-carotene which are spin trapped by POBN and which can react with B[a]P or metabolites of B[a]P.

Interaction of the carbon-centered ß-carotene-derived radical with DNA
Using the procedure described for the skatolyl radical of indole-3 acetic acid (19), the interaction of the carbon-centered radical of ß-carotene with DNA was studied (see Figure 3). Incubation of ß-carotene with double-stranded salmon sperm DNA in the presence of NADPH-supported rat liver S9 fraction led to inhibition of POBN-trapped carbon-centered radical formation at 0 time of incubation; incubation with 200, 600 and 1000 µg/ml DNA resulted in 30, 60 and 56% inhibition of the levels of ß-carotene carbon-centered radicals, respectively, while incubation with single stranded salmon sperm DNA did not result in quenching of the POBN-trapped carbon-centered radical signal at all (Figure 4). Also, incubation with the mononucleotides dG, dA, dC and dT, did not result in a decrease in POBN-trapped radicals, suggesting that the decreasing effect of salmon sperm DNA might be due to intercalation.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. Quenching of the POBN-spin trapped ß-carotene carbon-centered radical by increasing concentrations of double-stranded and single-stranded salmon sperm DNA. ß-Carotene (15 µg/ml) was added to a Tris-buffered rat liver S9 fraction (2 mg/ml) in the presence of a NADPH generating system, the spin trap POBN (100 mM) and various concentarions of double-stranded or single-stranded salmon sperm DNA. Immediately after addition, ESR signals were measured using the spectrometer settings described in Materials and methods. Peak heights in ESR spectra were normalized for control levels and expressed as percentages.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our ESR data show that ß-carotene has a slight capacity for producing ROS spontaneously, but it also appears that in the presence of microsomes at incubation concentrations comparable with serum ß-carotene levels obtained during human intervention trials no induction of oxidative DNA damage occurs in isolated DNA in vitro. Previous studies on the relationship between ß-carotene and oxidative DNA damage show conflicting results.

In Chinese hamster ovary cells in vitro, ß-carotene has been proven to possess antioxidative capacity against endogenous and H2O2-induced ROS (20). However, it was also shown in human HT29 cells that at higher incubation concentrations, e.g. >2 µg/ml, ß-carotene loses its antioxidative properties and may generate oxidative DNA damage instead (21). Actually, induction of 8-oxo-dG in calf thymus DNA upon incubation with 200 µM vitamin A (retinol) has been reported, but for this, the presence of Cu2+ in the incubation mixture appeared to have a pivotal role, 8-oxo-dG levels being considerably increased in particular after an incubation period of 6 h (6). However, without showing their data, the same authors reported that ß-carotene has a less efficient effect in comparison with retinol. Further, carotenoids also appear to be capable of inducing oxidative stress in cultured rat Sertoli cells (22) and human immortalized keratinocytes upon challenge with visible light (23). Lastly, while ß-carotene oxidation products have been reported to spontaneously induce oxidative DNA damage, but very slowly, in human foreskin fibroblasts at high concentrations (24), we could not demonstrate increases in salmon sperm 8-oxo-dG levels by ß-carotene oxidation products in the presence of rat or human metabolizing systems.

In experimental animals ß-carotene has been shown to have minor inhibitory effects on chemically induced formation of 8-oxo-dG in lung (25) and none in liver (26); no data on induction of oxidative DNA damage by ß-carotene in experimental animals have yet been presented.

Conflicting data on the relationship between ß-carotene and oxidative DNA damage have also been reported in humans. In cross-sectional studies, negative (27) and positive (27) associations, as well as no relationship (29,30) have been observed. In human intervention studies in which ß-carotene was administered either as a single compound or in mixtures, most studies showed no or inhibitory effects on oxidative DNA damage, both in smokers (31,32) and in non-smokers (27,33), while an increase in 8-oxo-dG levels in lymphocytes from smokers upon administration of antioxidant nutrient supplements has been demonstrated (34). However, it has to be taken into consideration that lymphocytic 8-oxo-dG levels in smokers tend to be decreased in comparison with non-smokers (35), making peripheral lymphocytes a poor target to study modulation of oxidative DNA damage in smokers.

In conclusion, it can be assumed that ß-carotene has weak prooxidant properties but it remains highly questionable that ß-carotene is capable of inducing promutagenic oxidative lesions in the target tissue in vivo.

We also failed to demonstrate that either ß-carotene or ß-carotene oxidation products promote the formation of promutagenic DNA adducts by the cigarette smoke carcinogen B[a]P, implying that our data do not support a role of ß-carotene oxidation products in the suggested peroxide shunt pathway of cytochrome P450 leading to one electron activation of B[a]P (7). In experiments in vitro utilizing human embryonic lung cells co-incubated with B[a]P and ß-carotene no significant impact on B[a]P–DNA adduct formation was found either.

With respect to effects of ß-carotene on B[a]P–DNA adduct formation, only inhibition has been reported in various in vitro models (7,16), while in experimental animals high dietary intake of ß-carotene appeared to have no effect on B[a]P–DNA adduct formation in the target tissues (36). In cross-sectional studies on human smokers, overall no association between blood ß-carotene levels and B[a]P–DNA adduct levels in peripheral lymphocytes has been demonstrated (37,38), but in GSTM1-deficient smokers an inverse relationship has been shown (39). Furthermore, in a recently published 6 month intervention study in heavy smokers applying a multivitamin formulation including ß-carotene, a negative, but non-significant decrease in PAH–DNA adduct levels in blood mononuclear or oral cells could be established (40). The results of experiments with combinations of ß-carotene and B[a]P as presented in this study, a decrease in the level of carbon-centered radicals formed from ß-carotene, lend support to the hypothesis that the decreasing effect of ß-carotene on binding of B[a]P to DNA is caused by scavenging of B[a]P metabolites by ß-carotene (7). However, it is suggested here that radical fragments of ß-carotene react with B[a]P and/or B[a]P metabolites, rather than the intact olefinic chain of ß-carotene, as proposed by others (7). The lack of effect of scavenging of B[a]P and/or B[a]P metabolites by ß-carotene oxidation products additionally supports this hypothesis.

Obviously, no differences in the influence of ß-carotene on DNA oxidation or B[a]P–DNA adduct formation upon activation by rat liver versus rat lung versus human lung S9 fractions could be established. Rat liver S9 fractions appeared to possess the strongest activity with regard to metabolic activiation of B[a]P, probably due to strong CYP1A2 activity (41), followed by rat lung S9 fractions; human lung S9 fractions appear to have a very low capacity to oxidize B[a]P, which is in accord with earlier reports showing that, for instance, cytochrome P450 levels in human lung S9 fractions are 0–20% of levels in rat lung S9 fractions (42).

It has been suggested that metabolic activation of B[a]P, specifically through one-electron oxidation, induces oxidative stress (8). However, our experiments do not support this hypothesis: either with or without rat liver S9 fractions in the simultaneous presence of B[a]P, no increase in ROS generation could be demonstrated by means of ESR (Tables I and II). Also, no induction of 8-oxo-dG in salmon sperm DNA by B[a]P in the presence of rat liver or lung activating systems or human lung S9 fractions (Figure 1) could be observed. This is confirmed by findings in human bronchial epithelial cells in vitro (43), as well as in intact humans exposed to B[a]P either through smoking (40) or occupational conditions (44), in whom no increased levels of 8-oxo-dG could be shown.

We have observed a carbon-centered radical generated by ß-carotene upon activation by rat liver S9 fraction, which is quenched by double-stranded DNA but not by single-stranded DNA and nor by individual nucleotides; this suggests that this carbon-centered ß-carotene radical probably interacts with DNA through intercalation. In general, agents which intercalate between DNA base pairs appear to be able to induce frameshift mutations, but their capacity to do so is weak if they do not simultaneously possess the capacity of forming DNA adducts through covalent binding (4547). We have found no evidence of covalent binding to DNA by this carbon-centered ß-carotene-derived radical yet, but we suggest that ß-carotene–DNA intercalation may contribute to the mutagenic effects of DNA adducts formed by carcinogens such as B[a]P, thereby explaining the observed co-carcinogenic effects in ß-carotene-supplemented smokers. It has also to be considered that our other data demonstrate that this carbon-centered ß-carotene radical may inactivate B[a]P metabolites, thereby potentially reducing B[a]P-related cancer risk. Consequently, in vivo there may exist a delicate balance between the cancer-promoting and anticarcinogenic properties of ß-carotene, which have to be further explored. Therefore, future research will be aimed at further chemico-biological characterization of this particular ß-carotene–DNA adduct.


    Notes
 
{dagger} Jan van Maanen died unexpectedly on November 5, 2002. This article is to honor his memory. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. {alpha}-Tocopherol, ß-Carotene Cancer Prevention Study Group (1994) The effect of vitamin E and beta-carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med., 33, 1029–1035.[CrossRef]
  2. Omenn,G.S., Goodman,E., Thongquist,M.D. et al. (1996) Effects of a combination of ß-carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med., 334, 1150–1155.[Abstract/Free Full Text]
  3. Liu,C., Wang,X., Bronson,R.T., Smith,D.E., Krinsky,N.I. and Russell,R.M. (2000) Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets. Carcinogenesis, 21, 2245–2253.[Abstract/Free Full Text]
  4. Vainio,H. and Rautalahti,M. (1998) An international evaluation of the cancer preventive potential of carotenoids. Cancer Epidemiol. Biomarkers Prev., 7, 725–728.[Abstract]
  5. Paolini,M., Cantelli-Forti,G., Perocco,P., Pedulli,G.F., Abdel-Rahman,S.Z. and Legator,M.S. (1999) Co-carcinogenic effect of ß-carotene. Nature, 398, 760–761.[CrossRef][ISI][Medline]
  6. Murata,M. and Kawanishi,S. (2000) Oxidative DNA damage by vitamin A and its derivative via superoxide generation. J. Biol. Chem., 275, 2003–2008.[Abstract/Free Full Text]
  7. Salgo,M.G., Cueto,R., Winston,G.W. and Pryor,W.A. (1999) Free beta-carotene and its oxidation product have different effects on microsome mediated binding of benzo[a]pyrene to DNA. Radiat. Biol. Med., 26, 162–173.
  8. Cavalieri,E.L., Rogan,E.G., Devanesan,P.D., Cremonesi,P., Cerny,R.L., Gross,M.L. and Bodell,W.J. (1990) Binding of benzo[a]pyrene to DNA by cytochrome P450-mediated catalyzed one-electron oxidation in rat liver and nuclei. Biochemistry, 29, 4820–4827.[ISI][Medline]
  9. Denissenko,M.F., Pao,A., Tang,M. and Pfeiffer,G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science, 274, 430–432.[Abstract/Free Full Text]
  10. Kuchino,Y., Mori,F., Kasai,H., Inoue,H., Iwai,S., Miura,K., Ohtsuka,E. and Nishimura,S. (1987) Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature, 32, 77–79.
  11. Paolini,M., Antelli,A., Pozzetti,L., Spetlova,D., Perocco,P., Valgimigli,L., Pedulli,G.G. and Cantelli-Forti,G. (2001) Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats. Carcinogenesis, 22, 1483–1495.[Abstract/Free Full Text]
  12. Astorg,P., Gradelet,S., Leclerc,J. and Siess,M.H. (1997) Effects of provitamin A or non-provitamin A carotenoids on liver xenobiotic-metabolizing enzymes in mice. Nutr. Cancer, 27, 245–249.[ISI][Medline]
  13. Gradelet,S., Leclerc,J., Siess,M.N. and Astorg,P. (1996) Beta-Apo-8'-carotenal, but not beta-carotene, is a strong inducer of liver cytochromes P4501A1 and 1A2 in rat. Xenobiotica, 26, 909–919.[ISI][Medline]
  14. Besarati Nia,A., Maas,L.M., Van Breda,S.G.J., Curfs,D.M.J, Kleinjans,J.C.S., Wouters,E.F.M. and van Schooten,F.J. (2000) Applicability of induced sputum for molecular dosimetry of exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic DNA adducts in smokers and nonsmokers. Cancer Epidemiol. Biomarkers Prev., 9, 367–372.[Abstract/Free Full Text]
  15. De Kok,T.C.M.C., Ten Vaarwerk,F.J., Zwingman,I., Van Maanen,J.M.S. and Kleinjans,J.C.S. (1994) Peroxidation of linoleic, arachidonic and oleic acid in relation to the induction of oxidative DNA damage and cytogenetic effects. Carcinogenesis, 15, 1399–1404.[Abstract]
  16. Lahiri,M. and Bhide,S.V. (1993) Effect of four plant phenols, beta-carotene and alpha-tocopherol on 3(H)benzopyrene-DNA interaction in vitro in the presence of rat and mouse liver postmitochondrial fraction. Cancer Lett., 73, 35–39.[CrossRef][ISI][Medline]
  17. Augusto,O., Beilan,H.S. and Ortiz de Montellano,P.R. (1982) The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation. J. Biol. Chem., 257, 11288–11295.[Abstract/Free Full Text]
  18. Novakov,C.P. and Stoyanovsky,D.A. (2002) Comparative metabolism of N-tert-butyl-N-[1-(1-oxypyidin-4-yl)-ethyl]- and N-tert-butyl-N-(1-phenyl-ethyl)-nitroxide by the cytochrome P450 monooxygenase system. Chem. Res. Toxicol., 15, 749–753.[CrossRef][ISI][Medline]
  19. Folkes,L.K., Dennis,M.F., Stratford,M.R.L., Candeias,L.P. and Wardman,P. (1999) Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro. Biochem. Pharmacol., 57, 375–382.[CrossRef][ISI][Medline]
  20. Cozzi,R., Ricordy,R., Aglitti,T., Gatta,V., Perticone,P. and De Salvia,R. (1997) Ascorbic acid and beta-carotene as modulators of oxidative damage. Carcinogenesis, 18, 223–228.[Abstract]
  21. Lowe,G.M., Booth,L.A., Young,A.J. and Bilton,R.F. (1999) Lycopene and beta-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses. Free Radic. Res., 30, 141–151.[ISI][Medline]
  22. Dal-Pizzol,F., Klamt,F., Benfato,M.S., Bernard,E.A. and Moreira,J.C.F. (2001) Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells. Free Radic. Res., 34, 395–404.[ISI][Medline]
  23. Pflaum,M., Kielbassa,Ch., Garmyn,M. and Epe,B. (1998) Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects. Mutat. Res., 408, 137–146.[ISI][Medline]
  24. Yeh,S.L. and Hu,M.L. (2001) Induction of oxidative DNA damage in human foreskin fibroblast Hs68 cells by oxidized beta-carotene and lycopene. Free Radic. Res., 35, 203–213.[ISI][Medline]
  25. Nagashima,M., Kasai,H., Yokota,J., Nagamachi,Y., Ichinose,T. and Sagai,M. (1995) Formation of an oxidative DNA damage, 8-hydroxydeoxyguanosine, in mouse lung DNA after intratracheal instillation of diesel exhaust particles and effects of high dietary fat and beta-carotene on this process. Carcinogenesis, 16, 1441–1445.[Abstract]
  26. Sai-Kato,K., Umemura,T., Takagi,A., Hasegawa,R., Tanimura,A. and Kurokawa,Y. (1995) Pentachlorophenol-induced oxidative DNA damage in mouse liver and protective effect of antioxidants. Food Chem. Toxicol., 33, 877–882.[CrossRef][ISI][Medline]
  27. Collins,A.R., Olmedilla,B., Southon,S., Granado,F. and Duthie,S.J. (1998) Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis, 19, 2159–2162.[Abstract]
  28. Bianchini,F., Elmståhl,S., Martinez-Garciá,C., Van Kappel,A.L., Douki,T., Cadet,J., Ohshima,H., Riboli,E. and Kaaks,R. (2000) Oxidative DNA damage in human lymphocytes: correlations with plasma levels of alpha-tocopherol and carotenoids. Carcinogenesis, 21, 321–324.[Abstract/Free Full Text]
  29. Loft,S., Vistisen,K., Ewertz,M., Tjønnelund,A., Overvad,K. and Poulssen,H.E. (1992) Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis, 13, 2241–2247.[Abstract]
  30. Poulssen,H.E., Loft,S., Priemé,H., Vistisen,K., Lykkesfeldt,J., Nyyssonen,K. and Salonen,J.T. (1998) Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion. Free Radic. Res., 29, 565–571.[ISI][Medline]
  31. Van Poppel,G., Poulsen,H.E., Loft,S. and Verhagen,H.J. (1995) No influence of beta-carotene on oxidative DNA damage in male smokers. J. Natl Cancer Inst., 87, 310–311.[ISI][Medline]
  32. Duthie,S.J., Ma,A., Ross,M.A. and Collins,A.R. (1996) Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res., 56, 1291–1295.[Abstract]
  33. Pool-Zobel,B.L., Bub,A., Müller,H., Wollowski,I. and Rechkemmer,G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis, 18, 1847–1850.[Abstract]
  34. Welch,R.W., Turley,E., Sweetman,S.F., Kennedy,G., Collins,A.R., Dunne,A., Livingstone,M.B.E., McKenna,P.G., McKelvey-Martin,V.J. and Strain,J.J. (1999) Dietary antioxidant supplementation and DNA damage in smokers and nonsmokers. Nutr. Cancer, 34, 167–172.[CrossRef][ISI][Medline]
  35. Besarati Nia,A., Van Schooten,F.J., Schilderman,P.A.E.L. et al. (2001) A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms. Carcinogenesis, 22, 395–402.[Abstract/Free Full Text]
  36. Wolterbeek,A.P.M., Roggeband,R., Baan,R.B., Feron,V.J. and Rutten,A.A.J.J.L. (1995) Relation between benzo[a]pyrene–DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high beta-carotene diet. Carcinogenesis, 16, 1617–1622.[Abstract]
  37. Grinberg-Funes,R.A., Singh,V., Perera,F.P., Bell,D.A., Young,T.L., Dickey,Ch., Wang,L.W. and Santella,R.M. (1994) Polycyclic aromatic hydrocarbon–DNA adducts in smokers and their relationship to micronutrient levels and the glutathione-S-transferase M1 genotype. Carcinogenesis, 15, 2449–2454.[Abstract]
  38. Wang,Y., Ichiba,M., Oishi,H., Iyadomi,M., Shono,N. and Tomokuni,K. (1997) Relationship between plasma concentrations of beta-carotene and alpha-tocopherol and life-style factors and levels of DNA adducts in lymphocytes. Nutr. Cancer, 27, 69–73.[ISI][Medline]
  39. Mooney,L.A., Bell,D.A., Santella,R. et al. (1997) Contribution of genetic and nutritional factors to DNA damage in heavy smokers. Carcinogenesis, 18, 503–509.[Abstract]
  40. Jacobson,J.S., Begg,M.D., Wang,L.W., Wang,Q., Agarwal,M., Norkus,E., Singh,V.N., Young,T., Yang,D. and Santella,R.M. (2000) Effects of a 6-month vitamin intervention on DNA damage in heavy smokers. Cancer Epidemiol. Biomarkers Prev., 9, 1303–1311.[Abstract/Free Full Text]
  41. Shimada,T., Inoue,I., Suzuki,Y., Kawai,T., Azuma,E., Nakajima,T., Shindo,M., Kurose,K., Sugii,A. and Yamagishi,Y. (2002) Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6 J mice. Carcinogenesis, 23, 1199–1207.[Abstract/Free Full Text]
  42. Powis,G. and Jansson,I. (1982) Hepatic cyt 450 monooxygenase system. In Schenkman,J.B. and Kupfer,D. (eds), International Encyclopedia of Pharmacology and Therapeutics. Pergamon Press, Oxford, p. 699.
  43. Arora,A., Willhite,C.A. and Liebler,D.C. (2001) Interactions of beta-carotene and cigarette smoke in human bronchial epithelial cells. Carcinogenesis, 22, 1173–1178.[Abstract/Free Full Text]
  44. Marczynski,B., Rihs,H., Rossbach,B., Hölzer,J., Angerer,J., Scherenberg,M., Hoffmann,G., Brüning,T. and Wilhelm,M. (2002) Analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in white blood cells of occupationally exposed workers: comparison with ambient monitoring, urinary metabolites and enzyme polymorphisms. Carcinogenesis, 23, 273–281.[Abstract/Free Full Text]
  45. McCoy,E.C., Rosenkranz,E.J., Petrullo,L.A. and Rosenkranz,H.S. (1981) Frameshift mutations: relative roles of simple intercalation and of adduct formation. Mutat. Res., 90, 21–30.[CrossRef][ISI][Medline]
  46. DeMarini,D.M., Cros,S., Paoletti,C., Lecointe,P. and Hsie,A.W. (1983) Mutagenicity and cytotoxicity of five antitumor ellipticines in mammalian cells and their structure–activity relationships in Salmonella. Cancer Res., 43, 3544–3552.[Abstract]
  47. Snyder,R.D. and Diehl,M.S. (2000) The bleomycin amplification assay in V79 cells predicts frameshift mutagenicity of intercalative agents. Mutagenesis, 15, 203–205.[Abstract/Free Full Text]
Received September 8, 2003; revised February 12, 2004; accepted February 14, 2004.