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Carcinogenesis Advance Access originally published online on December 4, 2008
Carcinogenesis 2009 30(2):308-314; doi:10.1093/carcin/bgn270
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The carotenoid β-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells

Yolanda Lorenzo1,2, Amaia Azqueta1, Luisa Luna3, Félix Bonilla2, Gemma Domínguez2 and Andrew R. Collins1,*

1 Department of Nutrition, Faculty of Medicine, University of Oslo, PO Box 1046 Blindern, 0316 Oslo, Norway
2 Department of Medical Oncology, Hospital Universitario Puerta de Hierro, 28035 Madrid, Spain
3 Department of Molecular Biology, Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Rikshospitalet Medical Centre, 0027 Oslo, Norway

* To whom correspondence should be addressed. Tel: +47 22851360; Fax: +47 22851341; Email: a.r.collins{at}medisin.uio.no


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The role of dietary antioxidants in human health remains controversial. Fruits and vegetables in the diet are associated with lower rates of chronic disease, and this is often attributed to their content of antioxidants, and a resulting protection against oxidative stress. However, large-scale human trials with antioxidant supplements have shown, if anything, an increase in mortality. We have investigated the biological properties of β-cryptoxanthin, a common carotenoid, in cell culture model systems, using the comet assay to measure DNA damage. At low concentrations, close to those found in plasma, β-cryptoxanthin does not itself cause damage, but protects transformed human cells (HeLa and Caco-2) from damage induced by H2O2 or by visible light in the presence of a photosensitizer. In addition, it has a striking effect on DNA repair, measured in different ways. Incubation of H2O2-treated cells with β-cryptoxanthin led to a doubling of the rate of rejoining of strand breaks and had a similar effect on the rate of removal of oxidized purines by base excision repair. The latter effect was confirmed with an in vitro assay: cells were incubated with or without β-cryptoxanthin before preparing an extract, which was then incubated with substrate DNA containing 8-oxo-7,8-dihydroguanine; incision was more rapid with the extract prepared from carotenoid-preincubated cells. No significant increases were seen in protein content of human 8-oxoguanine DNA glycosylase 1 or apurinic endonuclease 1. The apparent cancer-preventive effects of dietary carotenoids may depend on the enhancement of DNA repair as well as antioxidant protection against damage.

Abbreviations: APE1, apurinic endonuclease 1; BER, base excision repair; DMSO, dimethyl sulphoxide; FPG, formamidopyrimidine DNA glycosylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; OGG1, 8-oxoguanine DNA glycosylase 1; 8-oxoGua, 8-oxo-7,8-dihydroguanine; PBS, phosphate-buffered saline; SB, strand break


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Diet certainly plays an important role in preventing cancer, but the mechanism is still not clear. Epidemiological evidence links consumption of fruits and/or vegetables with decreased risk of cancer (1).

Reactive oxygen species produced in cells, largely as by-products of metabolic processes, constantly threaten the integrity and correct functioning of cellular DNA (2). Several oxidant species have the capacity to produce promutagenic lesions in DNA (3), which may play a significant role in the development of cancer. Fruits and vegetables contain relatively high amounts of components with inherent antioxidant properties, such as vitamin C, carotenoids and flavonoids, which has led to the suggestion that dietary antioxidants may augment cellular antioxidant defences and help protect cellular components from oxidative damage. Antioxidant activity is therefore considered to play an important role in the protective effects of fruits and vegetables against cancer (4,5).

β-Cryptoxanthin, one of the six major carotenoids routinely measured in human serum (β-carotene, lycopene, lutein, β-cryptoxanthin, zeaxanthin and {alpha}-carotene), is obtained primarily from citrus fruits (6), but is also found in corn, peas and some yellow coloured animal products such as egg yolk and butter. Like other carotenoids, β-cryptoxanthin is an antioxidant and may help prevent free radical damage to biomolecules including lipids, proteins and nucleic acids. Retinoids, the cleavage products of carotenoids, as well as being antioxidants, have in some cases (including β-cryptoxanthin) vitamin A activity and may play an important role in the prevention and treatment of certain cancers (7,8).

There are several prospective studies investigating the possible effect of β-cryptoxanthin (and other carotenoids) on risk of various cancers. Toniolo et al. (9) found that levels of β-cryptoxanthin and lutein in serum were linked with reduced risk of breast cancer in a dose-dependent fashion. A similar negative association of breast cancer risk with plasma carotenoid levels was reported by Tamimi et al. (10). β-Carotene, lycopene, β-cryptoxanthin, zeaxanthin and lutein are reduced in colorectal adenomas, suggesting that mucosal carotenoids could serve as biomarkers for predisposition to colorectal cancer (11). Women with high circulating concentrations of β-cryptoxanthin and tocopherol may be at a reduced risk of cervical atypical squamous cells of undetermined significance (12). However, Dorgan et al. (13) found a positive association between several plasma carotenoids, including β-cryptoxanthin, and risk of squamous cell carcinoma in a 5 year follow-up trial of patients who had already had a non-melanoma skin cancer.

Supplementation trials with carotenoids, aimed at reducing risk of cancer, have had disappointing results. A recent meta-analysis (14) showed, if anything, an increase in cancer risk following supplementation with β-carotene, vitamin A and vitamin E, singly or in combination. Thus, carotenoids in general must be regarded with circumspection, and further investigations of their modes of action are warranted.

Here, we use the comet assay (alkaline single-cell gel electrophoresis) to investigate the ability of β-cryptoxanthin to protect against DNA oxidation damage in cultured Caco-2 and HeLa cells and also the possible enhancement of DNA repair (a second line of defence). The comet assay is a simple, fast and reliable method for measuring DNA strand breaks (SBs). A simple modification, incorporating digestion of DNA with lesion-specific enzymes, endonuclease III and formamidopyrimidine DNA glycosylase (FPG), allows measurement of oxidized pyrimidines and oxidized purines, respectively (15). DNA repair can be studied with the comet assay in different ways. If cells are subjected to damage (strand breakage or base oxidation) and then incubated to allow them to repair, the lesions remaining can be measured at intervals. Alternatively, an in vitro approach can be used, in which a cell extract is incubated with lysed agarose-embedded substrate cells containing specific damage. The ability of the extract to introduce DNA breaks in the substrate cells reflects the activity of the enzymes responsible for the initial steps of repair, i.e. removing the lesion. The assay was tested on Ogg1– and wild-type mouse fibroblast cell lines (16): there was essentially no incision activity in the extract from the mutant cells, confirming that it measures principally 8-oxoguanine DNA glycosylase 1 (OGG1) enzyme activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
HeLa cells (derived from human cervical cancer) and Caco-2 cells (derived from human colon carcinoma) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were maintained as monolayer cultures at 37°C in a humidified atmosphere with 5% CO2 and routinely passaged by trypsinization when nearly confluent.

Comet assay
Cells were trypsinized and resuspended in phosphate-buffered saline (PBS) at 106 cells/ml. Thirty microlitre of cell suspension was mixed with 140 µl of 1% low-melting point agarose, and two 70 µl aliquots were dropped onto a microscope slide precoated with 1% normal melting point agarose. Glass cover slips were placed on the drops of agarose, which were left to set at 4°C. The cover slips were removed and the agarose-embedded cells lysed for 1 h in 2.5 M NaCl, 0.1 M Na2EDTA, 0.1 M Tris and 1% Triton X-100, pH 10 at 4°C. This removes membranes and most cellular components, including histones, leaving the DNA attached to the nuclear matrix as a nucleoid. After lysis, slides for measurement of oxidized bases were washed three times in enzyme buffer [0.1 M KCl, 0.5 mM Na2EDTA, 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) and 0.2 mg/ml bovine serum albumin, pH 8.0] and then incubated for 30 min at 37°C with 45 µl of FPG or 45 µl of enzyme buffer [FPG was prepared as a crude extract from Escherichia coli containing an over-producing plasmid, obtained from Serge Boiteux (CEA, Fontenay aux Roses, France). It was not purified, as this would reduce its stability. Optimum incubation conditions were established using nucleoids with an excess of 8-oxo-7,8-dihydroguanine (8-oxoGua), prepared as described below for the in vitro repair assay.] In the in vitro assay for DNA repair, a similar (but shorter) incubation was carried out with cell extract (see below). For measurement of SBs, agarose-embedded cells were simply lysed. All slides were then placed in a horizontal electrophoresis tank and the DNA was allowed to unwind for 40 min in cold alkaline electrophoresis solution (0.3 M NaOH and 1 mM Na2EDTA, pH >13). Electrophoresis was carried out in this solution for 30 min at 25 V and ~300 mA, in a cold room. The slides were washed in cold 0.4 M Tris buffer (pH 7.5) for 10 min to neutralize excess alkali and for 10 min in water and left to dry overnight.

Gels were stained with 25 µl of 4',6-diamidino-2-phenylindole (1 µg/ml), covered with a cover slip and coded. Using a fluorescence microscope, 100 comets (50 on each gel) were visually classified into five categories, 0–4, representing increasing relative tail intensities. Summing the scores (0–4) of 100 comets therefore gives an overall score of between 0 and 400 arbitrary units (15).

Measuring the effect of β-cryptoxanthin on endogenous and induced DNA damage
Stock solutions of β-cryptoxanthin (Extrasynthese, Lyon, France) for all experiments were prepared in dimethyl sulphoxide (DMSO) in the dark. The final concentration of DMSO in the culture medium was <1%. HeLa and Caco-2 cells were incubated with 0, 1, 4, 10 or 25 µM β-cryptoxanthin for 1 h at 37°C in the dark. The comet assay was performed to assess the effect of β-cryptoxanthin on endogenous DNA SBs and—with the inclusion of FPG digestion—the effect on oxidized purines. To examine the ability of β-cryptoxanthin to protect against induced oxidative damage, preincubated cells were washed with PBS and treated on ice with H2O2 (25 µM for HeLa cells and 15 µM for Caco-2 cells) for 5 min to induce SBs or with 1 µM Ro (photosensitizer Ro19-8022, from F.Hoffmann-La Roche, Basel, Switzerland) plus visible light from a 500 W tungsten–halogen source (2.5 min for HeLa cells and 1.5 min for Caco-2 cells) at 33 cm to induce 8-oxoGua. The comet assay was performed without or with FPG to measure SBs or 8-oxoGua, respectively. Three independent experiments were performed.

Cellular repair assays
Rejoining of SBs was followed after treating cells with H2O2, and repair of oxidized bases after treating them with Ro plus light. In each case, three independent experiments were performed.

HeLa and Caco-2 cells were treated with H2O2 (30 µM for HeLa cells and 20 µM for Caco-2 cells) for 5 min on ice to induce SBs. H2O2 was washed off with PBS and cells were incubated with 1 or 4 µM β-cryptoxanthin for 0, 5, 15, 30 and 45 min for HeLa cells and 0, 15, 30, 45 min and 1 h for Caco-2 cells at 37°C in the dark.

To follow repair of oxidized bases, cells were treated with 1 µM Ro plus light (2.5 min for HeLa cells and 1.5 min for Caco-2 cells) to induce oxidized bases. Ro was washed off with PBS and cells were incubated with 1, 4, 10 or 25 µM β-cryptoxanthin for 0, 0.5, 1, 2 and 4 h for HeLa cells and 0, 1, 2, 4 and 8 h for Caco-2 cells at 37°C in the dark.

Measuring in vitro repair
This in vitro assay (16) provides a measure of the incision activity of a cell extract provided with a DNA substrate from cells containing specific damage—in this case, 8-oxoGua. These cells are embedded in agarose, lysed and incubated with the extract. Incision at damage sites is detected as SBs using the comet assay.

Preparation of substrate cells.
Near-confluent HeLa cells were treated with 1 µM Ro and irradiated on ice with visible light (4 min at 33 cm) to induce 8-oxoGua, as the substrate for base excision repair (BER). Cells were then suspended in freezing medium (Dulbecco’s modified Eagle’s medium with 20% serum and 10% DMSO) at 106 cells/ml, frozen slowly to –80°C in 0.7 ml aliquots and kept at this temperature until needed. Near-confluent HeLa cells without treatment were also frozen in the same way.

Preparation of cell pellets and extracts.
HeLa and Caco-2 cells were incubated with 0, 1 or 4 µM β-cryptoxanthin for 2 h at 37°C in the dark. They were washed with PBS, trypsinized and resuspended in cold PBS at 106/ml. Cells were dispensed in equal aliquots of 1 ml, centrifuged at 14 000g, 4°C for 5 min, and the supernatant was completely removed leaving an almost dry pellet. Pellets were frozen by dropping into liquid nitrogen and stored at –80°C. On the day of the experiment, a pellet was thawed and resuspended in 33 µl of extraction buffer (45 mM HEPES, 0.4 M KCl, 1 mM ethylenediaminetetraacetic acid, 0.1 mM dithiothreitol and 10% glycerol, pH 7.8) to which Triton X-100 was added to 0.25% just before use. Vortex mixing for 5 s at top speed was followed by incubation for 5 min on ice and centrifugation at 14 000g, 4°C for 5 min. Twenty-eight microlitre of supernatant was removed and combined with 110 µl of cold reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM ethylenediaminetetraacetic acid and 0.2 mg/ml bovine serum albumin, pH 8).

The reaction.
Pairs of gels containing ~2 x 104 substrate HeLa cells, with or without treatment with Ro plus light, in 70 µl of 1% low-melting point agarose in PBS were set on agarose-precoated glass slides, and cells were lysed for 1 h as in the standard comet assay (see above). Slides were washed for 3x 5 min with reaction buffer. Thirty microlitre of extract was placed on each gel, covered with a cover slip and incubated for 10 min in a humid chamber at 37°C. To check whether β-cryptoxanthin present in the extract directly induces DNA breaks, substrate nucleoids from damaged or undamaged HeLa cells were treated with β-cryptoxanthin. In this case, β-cryptoxanthin in 10 µl DMSO at concentrations of 0.1, 0.4, 1 and 4 µM was mixed with extraction buffer plus 0.25% Triton X-100 and then diluted with reaction buffer following the same procedure as the one used to make extracts from cell pellets. The incubation was performed exactly as with the extracts.

As a negative control, 30 µl of a mixture (1:4) of extraction buffer plus 0.25% Triton X-100 and reaction buffer was used, and as a positive control FPG. After incubation, alkaline unwinding and electrophoresis proceeded as in the standard comet assay. Three independent experiments were performed using extracts from different cell cultures.

Western blotting
Whole-cell extract.
Aliquots of 107 HeLa and Caco-2 cells, untreated or after incubation for 2 h with 4 µM β-cryptoxanthin, were centrifuged and snap frozen as described above. Pellets were resuspended in 1 ml of cold radioimmunoprecipitation assay buffer [0.15 M NaCl, 25 mM Tris, 1% NP40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 2 mM dithiothreitol, 1x proteinase inhibitor cocktail (Sigma-Aldrich, St Louis, MO), pH 7.5] and left on ice for 10 min. The suspension was sonicated and centrifuged at 16 000g for 20 min at 4°C to remove cell debris.

Nuclear extract.
Frozen pellets were resuspended in an equal volume of cold nuclear extraction buffer 1 (10 mM KCl, 10 mM HEPES, 1.5 mM MgCl2, 1 mM dithiothreitol and 1x proteinase inhibitor cocktail, pH 8.0) and left on ice for 15 min. The suspension was then forced four times through a 23-gauge needle, centrifuged at 16 000g for 20 s and the pellet resuspended in two-third of the original cell pellet volume of nuclear extraction buffer 2 (0.42 M NaCl, 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 25% glycerol and 1x proteinase inhibitor cocktail, pH 8.0). The suspension was stirred at 4°C for 30 min and centrifuged at 16 000g for 20 min at 4°C.

Western blots.
Protein content in the supernatants was measured with the Bio-Rad Protein Microassay. Aliquots of 60 µg were taken for hOGG1 detection, and aliquots of 10 µg for apurinic endonuclease 1 (APE1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). They were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using an XCell II blot module (Invitrogen, Carlsbad, CA). Primary antibodies used were polyclonal anti-hOGG1 (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-APE1 (Abcam, Cambridge, UK) and monoclonal anti-GAPDH (Ambion, Applied Biosystems, Foster City, CA). The secondary apurinic-conjugated antibodies were goat anti-mouse (Santa Cruz Biotechnology) and goat anti-rabbbit (Sigma-Aldrich). Immunoblots were detected by enhanced chemifluorescence (GE Healthcare, Piscataway, NJ) and phosphorimaging (Typhoon 9410 variable mood imager system, GE Healthcare). Typhoon scans and images were processed and quantified with ImageQuant TL.

Statistical analysis
Statistical analysis was performed using the software SPSS 11.0. Data are presented as means ± SDs from three independent experiments. Total comet scores of the different groups were compared using the non-parametric Mann–Whitney U-test. P ≤ 0.05 was set as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
DNA damage
DNA damage, in the form of SBs and oxidized bases, was measured just after incubation of HeLa and Caco-2 cells with different concentrations of β-cryptoxanthin for 1 h (Figure 1). β-Cryptoxanthin did not cause SBs or base oxidation in HeLa cells (Figure 1A) or Caco-2 cells (Figure 1B). Incubation with β-cryptoxanthin for several hours did not induce any damage (data not shown).


Figure 1
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  		Fig. 1. DNA damage (SBs and FPG-sensitive sites) in HeLa (A) and Caco-2 cells (B) treated for 1 h with different concentrations of β-cryptoxanthin. Standard deviation values based on the results of three independent experiments are shown.

 
Protection against DNA damage
We tested HeLa and Caco-2 cells for their resistance to DNA oxidation by treating them for 5 min with H2O2 on ice to induce DNA SBs. There were significantly lower levels of DNA breaks in cells preincubated with β-cryptoxanthin compared with control cells (Figure 2A), indicating an enhanced antioxidant defence.


Figure 2
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  		Fig. 2. Effect of 1 h incubation with β-cryptoxanthin on sensitivity of HeLa and Caco-2 cells to oxidative attack by H202 (25 µM for HeLa and 15 µM for Caco-2) (A) or by Ro (1 µM Ro plus 2.5 min of light for HeLa and 1.5 min of light for Caco-2) (B). Standard deviation values based on the results of three independent experiments are shown. *Indicates significant reduction in H2O2- or Ro-induced breaks (P ≤ 0.05). C-, untreated cells.

 
Ro plus light induces 8-oxoGua and gives a high level of SBs with FPG. When β-cryptoxanthin-pretreated cells were treated with Ro plus light and directly analyzed in the comet assay, a clear effect on DNA migration was seen; β-cryptoxanthin caused a concentration-related decrease in Ro-induced DNA damage (Figure 2B).

Cellular repair
The ability of HeLa and Caco-2 cells to rejoin SBs induced by H2O2 in the absence and presence of β-cryptoxanthin was followed by measuring the residual damage after different times of incubation (Figure 3). In cells without β-cryptoxanthin, the t1/2 for rejoining of breaks was ~10 min for HeLa and 18 min for Caco-2; in β-cryptoxanthin-treated cells, t1/2 decreased to 4 min for HeLa (both 1 and 4 µM) and to 10 and 4 min for Caco-2 (1 and 4 µM, respectively). In control cells, the level of SBs was constant during the entire repair incubation time, indicating that preparation and subsequent processing of the cells did not cause significant DNA damage.


Figure 3
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  		Fig. 3. Cellular repair of H2O2-induced damage. HeLa (A) and Caco-2 cells (B) were treated with H2O2 for 5 min on ice. The cells were incubated at 37°C with or without β-cryptoxanthin to allow repair to occur, and the remaining damage was measured at intervals. Standard deviation values based on the results of three independent experiments are shown. In HeLa cells, there was a significant decrease in residual breaks at both β-cryptoxanthin concentrations at times 5, 15 and 30 min. In Caco-2 cells, this applied over the whole incubation period. C-, untreated cells.

 
We treated cells with Ro plus light to induce 8-oxoGua, and followed BER by measuring residual FPG-sensitive sites over a period of incubation with and without β-cryptoxanthin (Figure 4). Cells treated with β-cryptoxanthin repaired the damage more quickly than untreated cells; in HeLa cells, the t1/2 of ~135 min without β-cryptoxanthin decreased to 80 and 65 min with 1 and 4 µM β-cryptoxanthin, respectively. In Caco-2 cells, the t1/2 decreased from ~260 to 155 and 125 min with 1 and 4 µM β-cryptoxanthin, respectively.


Figure 4
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  		Fig. 4. Cellular repair of 8-oxoGua. HeLa (A) and Caco-2 cells (B) were treated with Ro plus light to induce 8-oxoGua in DNA. The cells were incubated at 37°C with or without β-cryptoxanthin to allow repair to occur, and the remaining 8-oxoGua was estimated as FPG-sensitive sites. Standard deviation values based on the results of three independent experiments are shown. In HeLa cells, the decrease was significant only at 2 h (for both 1 and 4 µM β-cryptoxanthin). In Caco-2 cells, significant differences were seen at all incubation times for 4 µM β-cryptoxanthin and at 2 and 8 h for 1 µM β-cryptoxanthin. C-, untreated cells.

 
In vitro repair
The capacity of HeLa and Caco-2 cell extracts for BER of oxidized bases was assessed with and without β-cryptoxanthin pretreatment using an in vitro assay. HeLa and Caco-2 cells were incubated for 2 h with 0, 1 or 4 µM β-cryptoxanthin before preparation of a whole-cell extract. Extracts were then incubated with substrate nucleoids containing 8-oxoGua or without lesions, and incision was detected using the comet assay. Mixed extraction plus reaction buffer and FPG were used as negative and positive controls. Figure 5 shows a significant increase in incision by the extracts of cells treated with β-cryptoxanthin in nucleoids containing 8-oxoGua (Figure 5A), even after the low concentration of 1 µM, but no increase in breaks in nucleoids without lesions (Figure 5B).


Figure 5
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  		Fig. 5. In vitro DNA repair: incision by HeLa or Caco-2 cell extracts incubated for 10 min with gel-embedded nucleoid DNA containing 8-oxoGua (A) or nucleoids without lesions (B). Cells were incubated with β-cryptoxanthin (0, 1 and 4 µM) for 2 h before preparing extracts. Standard deviation values based on the results of three independent experiments are shown. *P ≤ 0.05 (comparison of extracts with and without β-cryptoxanthin preincubation).

 
β-Cryptoxanthin per se did not induce any breaks in nucleoids containing 8-oxoGua or nucleoids without lesions (data not shown).

Western blots
The glycosylase primarily associated with repair of oxidized purines in eukaryotes is OGG1. Removal of the base is followed by cutting at the apurinic site by APE1 (17). The increased BER of 8-oxoGua might be due to an increase in cellular concentration of these proteins. We therefore carried out western blots on whole-cell extract and nuclear extract from HeLa and Caco-2 cells with and without pretreatment with β-cryptoxanthin. Results are shown in Figure 6. APE1 was readily detected in both whole-cell and nuclear extracts. hOGG1 was much less intense and was especially difficult to detect in whole-cell extract (therefore only results with nuclear extract are shown). After normalizing band intensities against the reference anti-GAPDH, only minor differences were seen (the most pronounced being an ~50% decrease in anti-APE1 intensity in whole-cell extract from Caco-2 cells with β-cryptoxanthin). We conclude that the increase in OGG activity of the cell extract is not related to a detectable increase in enzyme concentration.


Figure 6
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  		Fig. 6. Western blots. Top: nuclear extracts; middle: whole-cell extracts; lanes 1–4, anti-APE1; lanes 5–8, anti-GAPDH (reference). Bottom: nuclear extracts; lanes 1–4, anti-hOGG1; lanes 5–8, anti-GAPDH. Lanes 1 and 5, HeLa cells untreated; lanes 2 and 6, HeLa cells pretreated with β-cryptoxanthin. Lanes 3 and 7, Caco-2 cells untreated; lanes 4 and 8, Caco-2 cells pretreated with β-cryptoxanthin. After normalizing band intensities with reference to GAPDH, ratios of intensities with/without β-cryptoxanthin were; nuclear extract: APE1, HeLa, 0.8; APE1, Caco2, 0.8; hOGG1, HeLa, 1.2; hOGG1, Caco2, 1.0. Whole-cell extract: APE1, HeLa, 1.3; APE1, Caco2, 0.5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
In addition to clinical studies, where carotenoids have been investigated in terms of cancer risk, numerous investigations have focused on DNA damage as an end point, rather than disease incidence. Both observational studies and intervention trials have been carried out.

Plasma carotenoid concentrations were measured in a group of 40 healthy Spanish subjects (18). Total carotenoids, lutein and β-carotene (though not β-cryptoxanthin) showed significant negative correlations with endonuclease III-sensitive sites (oxidized pyrimidines) in lymphocyte DNA. Similarly, Haegele et al. (19) reported inverse associations of lutein and β-cryptoxanthin with 8-oxodGuo in DNA measured by high-performance liquid chromatography. Also using high-performance liquid chromatography, Mecocci et al. (20) found that 8-oxodGuo in lymphocyte DNA was high and most plasma carotenoids were low in patients with Alzheimer disease compared with matched healthy controls—and that among the patients there was an inverse correlation between 8-oxodGuo and plasma concentrations of several carotenoids. Urinary 8-oxodGuo—a biomarker for oxidative stress (though the origin of the 8-oxodGuo has not been proven)—was measured by Thomson et al. (21) in a group of breast cancer survivors; there were negative correlations with several plasma carotenoids, including β-cryptoxanthin. It should be remembered that, in association studies of this sort, plasma carotenoids may simply be a marker of consumption of fruits and vegetables, and some other unidentified components may be responsible for the effect on DNA damage.

In an interesting contradictory study, Bianchini et al. (22) investigated groups of women in Spain and Sweden and found significant positive correlations between 8-oxodGuo in lymphocyte DNA and carotenoid concentrations in plasma.

The results of intervention trials, with carotenoid supplements or carotenoid-rich foods and with DNA oxidation as an end point, have been mixed. Pool-Zobel et al. (23) reported a decrease in pyrimidine oxidation during supplementation with carrot juice. Twelve weeks of supplementation with carotenes, lutein or lycopene increased plasma concentrations of carotenoids but failed to decrease the level of oxidized bases in lymphocyte DNA (18). Thompson et al. (24) gave a high fruit and vegetable diet for 14 days and found raised plasma carotenoid levels and decreased DNA oxidation in lymphocytes. Van den Berg et al. (25) supplemented volunteers with a vegetable burger and fruit drink for 3 weeks; plasma levels of several carotenoids rose, but there was no change in DNA oxidation. Porrini et al. (26) demonstrated an increased resistance of lymphocyte DNA to H2O2-induced breakage after supplementation with tomato puree (rich in lycopene).

In our experiments applying β-cryptoxanthin to cells in culture, there was a clear, dose-dependent antioxidant-protective effect, decreasing the breaks induced by H2O2 even at a concentration as low as 1 µM (in the case of Caco-2 cells). Perhaps more impressively, Ro-induced base oxidation was also markedly decreased, in both cell types at 1 µM. The mean plasma concentrations of β-cryptoxanthin vary from country to country and between men and women (27), the highest levels in that study being found in women in Spain (0.42 µM). Thus, our effective concentration is close to the physiological range. Bearing in mind that β-cryptoxanthin is one of several carotenoids that probably act as antioxidants in concert, our results are likely to have biological relevance.

Effects of carotenoids on DNA repair have received less attention than antioxidant actions. We reported an apparent enhancement of SB rejoining in lymphocytes isolated from some but not all subjects 24 h after a single large dose of β-carotene, lutein or lycopene (28,29); but we concluded that this effect might simply be an antioxidant-protective effect against additional damage induced by atmospheric oxygen since the freshly isolated lymphocytes were subject to a sudden increase in ambient oxygen concentration.

This consideration does not apply to the cultured cells used in the current investigation. β-Cryptoxanthin at 1 µM approximately doubled the rate at which HeLa and Caco-2 cells rejoin H2O2-induced SBs and had a proportionately similar effect on the intrinsically much slower repair of oxidized bases. That this truly reflects an increase in repair activity is confirmed by the experiments in which cell extracts were incubated in vitro with substrate DNA containing 8-oxoGua; β-cryptoxanthin preincubation of the cells significantly stimulated incision activity. Control experiments exclude the possibility that β-cryptoxanthin itself causes breaks or that it induces non-specific nuclease activity in the extracts. The increase in activity was not explained by any detectable increase in concentration of hOGG1 or of APE1, the two enzymes involved in removing 8-oxoGua and incising the DNA. This agrees with our previous finding that an increase in OGG activity resulting from kiwifruit consumption was not accompanied by any change in expression of hOGG1 or APE1 (30) and with the report of Paz-Elizur et al. (31) that there is a poor correlation between OGG1 activity and hOGG1 messenger RNA levels. It is possible that β-cryptoxanthin is involved in the post-translational regulation of OGG1 or APE1. OGG1 is regulated by phosphorylation (32) and also by acetylation (33), and APE1, as well as being inactivated by phosphorylation (34), is subject to redox regulation by reduction/oxidation at a cysteine adjacent to the active site (35).

Astley et al. (36) treated Molt-17 human lymphocytes (a permanent line) with β-carotene, β-cryptoxanthin or lutein and found no evidence of antioxidant protection against DNA damage, although apparently carotenoids were required for any SB rejoining to occur over a 2 h incubation after H2O2 treatment. There was no sign of an increase in repair activity in an in vitro assay (with oxidation damage in the substrate). Subsequently, lymphocytes from volunteers following various dietary regimes were tested; after a 3 week intervention with cooked carrots (200 g daily), in vitro repair was enhanced, and after mixed carotene capsules, SB rejoining was more rapid (37).

In summary, in our cell culture model system, we report effects of β-cryptoxanthin on levels of DNA oxidation that are consistent with a significant role as an antioxidant in vivo. In addition, β-cryptoxanthin has a striking stimulatory effect on two kinds of DNA repair—SB rejoining and excision repair of oxidized bases. The repair incision activity of a whole-cell extract is also stimulated by this phytochemical. Thus, the cancer-protective role of carotenoids, as seen in observational epidemiological studies, is probably in part at least the result of a stimulation of DNA repair.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Formación de Personal Investigador scholarship of the Ministerio de Educación, Política Social y Deporte of Spain to Y.L.


   Acknowledgments
 
Ro 19-8022 was kindly provided by F.Hoffmann-La Roche.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received June 23, 2008; revised November 20, 2008; accepted November 20, 2008.


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A. Azqueta, Y. Lorenzo, and A. R. Collins
In vitro comet assay for DNA repair: a warning concerning application to cultured cells
Mutagenesis, July 1, 2009; 24(4): 379 - 381.
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