Centre de recherche, Institut Universitaire de Gériatrie de Sherbrooke (Pavillon d'Youville) 1036, rue Belvédère Sud, Sherbrooke, Québec J1H 4C4, Canada
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Abstract |
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Abbreviations: EC, electrochemical detection; FBS, fetal bovine serum; GCMS, gas chromatographymass spectrometry; ODS, octadecylsilyl; 5-OH-dCyd, 5-hydroxy-2'-deoxycytidine; 8-oxo-dGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; PBS, phosphate-buffered saline.
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Introduction |
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The formation of endogenous oxidative DNA damage in cells involves a Fenton reaction that requires two basic components: H2O2 and metal ions (e.g. Fe2+ or Cu+). The resulting damaging species of this reaction appear to be hydroxyl radicals, although metal ion peroxide complexes cannot be ruled out (6). On the basis of model studies with ionizing radiation, the reaction of hydroxyl radicals with nucleic acids leads to a multitude of base and sugar modifications (7,8). In a biological setting, DNA damaging Fenton reactions must occur in close proximity to DNA because hydroxyl radicals react rapidly with nearly all biological compounds and thus do not diffuse very far from their site of generation. Therefore, the formation of oxidative DNA damage in cells is expected to depend on the rate of production of free radicals and oxidants, the probability that these species diffuse to DNA and the movement of reactive metal ions from cellular sources to DNA. Additionally, free radicals and oxidants induce the release of metal ions, which in turn generate more reactive species. For instance, during oxidative stress, reactive iron is liberated from heme-containing compounds, iron storage proteins and proteins containing iron-sulfur clusters, and this can contribute to oxidative damage (9,10).
The majority of biological studies dealing with oxidative DNA damage have focused on 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo), a major oxidation product of dGuo in DNA (11). This modification probably contributes to spontaneous mutagenesis because enzymes with similar substrate specificity appear to function in the removal of 8-oxo-dGuo in bacteria, yeast and mammalian cells, and disruption of this repair system leads to a specific mutator phenotype (12,13). In comparison, the biological consequences of 5-hydroxy-2'-deoxycytidine (5-OH-dCyd), which arises from the oxidation of dCyd in DNA, are not clearly understood. The yield of 5-OH-dCyd is at least 5-fold lower than that of 8-oxo-dGuo when DNA is exposed to various oxidants (14). DNA containing 5-OH-dCyd is a substrate for endonuclease III and VIII and probably for homologous enzymes in yeast and mammalian cells (15,16). Previous studies suggested that 5-OH-dCyd is potentially mutagenic because it miscodes during DNA synthesis with synthetic oligonucleotides using polymerase I Klenow fragment (17) and causes CT transitions (2.5%) when incorporated into bacteria (18). In contrast, a recent study (19) suggested that the frequency of mutations for 5-OH-dCyd is relatively low in bacteria (0.05%). It should also be noted that mammalian cells can tolerate a level of nuclear 5-OH-dCyd that is 20-fold above baseline without any adverse effects with respect to cell growth, which suggests that this lesion is not very genotoxic (20). Thus, the potential mutagenesis of 5-OH-dCyd lesions remains to be established. Nevertheless, 5-OH-dCyd may be considered as a marker of oxidative DNA damage related to mutagenesis because its formation and DNA repair are similar in many ways to the formation and DNA repair of 5-OH-dUrd and dUrd 5,6-glycols. The latter lesions are highly mutagenic since, in contrast to 5-OH-dCyd, they have lost the exocyclic amino group that is involved in base pairing with G in duplex DNA.
Epidemiological studies consistently point to an inverse association for the consumption of fruits and vegetables and cancer incidence, an effect that can be attributed to a variety of anticarcinogenic ingredients including dietary fiber, folic acid, vitamin A and various antioxidants (vitamin C, vitamin E, carotenoids, selenium) (21). The majority but not all of epidemiological studies support a beneficial effect of antioxidants micronutrients (22,23). In the present study, we report on the analysis of intracellular antioxidants, including glutathione and ascorbate, and oxidative DNA damage, including 8-oxo-dGuo and 5-OH-dCyd, as measured from freshly separated lymphocytes obtained from 105 healthy subjects. The results reveal an inverse correlation between intracellular antioxidants and oxidative DNA damage in human lymphocytes.
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Materials and methods |
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The criteria for acceptance of subjects into our study were that they did not smoke, nor take medication or antioxidant supplements, and generally, the subjects were required to be in good health as ascertained during an interview. The average age of the group was 43 years old with a range of ages from 20 to 86 years. The group consisted of 68 women and 37 men. Blood donations were collected by venipuncture in the morning from fasting individuals using five 20 ml vacutainer tubes that contained citrate buffer as an anticoagulant (total 100 ml). The lymphocytes were immediately isolated from the blood by techniques used in general-practice today by immunologists (24). First, blood was divided into three fractions (~30 ml each) into 50 ml Falcon tubes, and the tubes were centrifuged at 300 g for 15 min to remove plasma. The plasma was defribinated by the addition of CaCl2 (33 mM, final concentration), coated onto 250 ml Falcon tissue culture flasks, and incubated for at least 30 min before used for the removal of monocytes (below). To the blood sample, 1015 ml of PBS (completing the volume to 30 ml) and 15 ml of a solution of 2% dextran were added, and the sample was mixed thoroughly. The erythrocytes were allowed to sediment out for 30 min at 37°C. The resulting cell suspension was layered onto Ficoll-Hypaque (Pharmacia Biotech Uppsala, Sweden), with a density of 1.077, and centrifuged at 750 g for 20 min without braking. The mononuclear layer was collected and the cells were washed twice with RPMI (Roswell Park Memorial Institute) media (Gibco BRL, Grand Island, NY) at 300 g for 15 min. The cells (lymphocytes and monocytes) were then incubated at 37°C in RPMI media with 10% fetal bovine serum (FBS) (ICN, Costa Mesa, CA) for 50 min using plasma coated flasks (above) and the lymphocytes were removed from the monocytes that remained attached to the surface of the flask. The resulting lymphocytes were <90% viable as determined by trypan blue exclusion. In addition, it should be noted that there was no indication of hemolysis throughout purification and that there was no visible trace of red blood cells in the final preparation of lymphocytes.
Ten million lymphocytes were washed with PBS and nuclei buffer solution (0.25 M sucrose, 10 mM EDTA and 1 mM CaCl2) and then lysed with 0.2% Triton X-100 in the latter buffer (10 min at 4°C). The nuclei were spun down at 2000 g and stored at 80°C. To extract DNA, the nuclei were first treated with 0.8 mg/ml proteinase K (Boehringer #745 723, Indianapolis, IN) for 1 h at 37°C in lysis buffer composed of urea (8 M), NaCl (0.4 M), TrisHCl (0.2 M; pH 7.9), N-lauroylsarcosine (1%) and CaNa2EDTA (20 mM) (Applied Biosystems #743 876). The DNA sample was purified by liquidliquid extraction with a mixture of ultrapure phenol, chloroform and water (80:10:10) (Applied Biosystems #745 123, Foster City, CA). After the addition of 0.3 M (pH 4.5) acetate buffer (Aldrich #38,012-1, Milwaukee, WI) and an equal volume of cold HPLC grade isopropanol (15°C), the mixture was held at 15°C for 1 h and then centrifuged at 10 000 g for 30 min at 4°C. DNA was recovered and subsequently dried and stored at 80°C until analysis.
Intracellular antioxidants glutathione and ascorbate were measured according to the method of Rose and Bode (25). Ten million cells were washed three times with ice-cold chelex treated PBS, suspended in 200 mM phosphate buffer pH 2 containing 0.1 mM EDTA, and subjected to three freezethaw cycles (4 to 80°C). Glutathione and ascorbate were separated by HPLC using an octadecylsilyl (ODS) Inertsil 5 µm 150x4.6 mm internal diameter column (CSC, Montreal) with 200 mM phosphate, pH 3.0, as the mobile phase. The HPLC system consisted of a M6000 pump (Waters, Milford, MA) and a L-ECD-6A amperometric electrochemical detector (Shimadzu, Kyoto, Japan) set at 1.1 V versus AgCl2 reference electrode. The concentration of protein in cell lysates was determined by the Bradford assay (Bio-Rad), which was automated using an AS 3000 autosampler (Hitachi, Tokyo, Japan) and a U 3000 spectrophotometer (Hitachi). The standards were stored at 80°C and the same ones were used for the entire study.
Oxidative DNA damage was measured by HPLC with electrochemical and UV detection using a similar procedure as described (14). Samples were processed in sets of 12. Between 40 and 60 µg samples of DNA were dissolved in 60 µl of acetate buffer (10 mM, pH 4.5; Aldrich #38,012-1) and digested down to deoxyribonucleosides, first by treatment with Nuclease P1 (10 U, Pharmacia Biotech #27-0852-01) for 20 min at 50°C, and then by treatment with alkaline phosphatase (10 U, Boehringer #405 612) in ammonium acetate buffer (100 mM pH 7.0, Aldrich #37,233-1) for 40 min at 37°C. The amount of Nuclease P1 was in excess of that required to completely release 8-oxo-dGuo and 5-OH-dCyd from freshly extracted DNA. After digestion, the mixture was adjusted to pH 5.5 by the addition of 5 µl of 0.2 M phosphoric acid. The samples were transferred into glass 150 µl inserts and immediately frozen at 80°C. They were analyzed on the same day of digestion in two groups of six samples. Before analysis, the samples were maintained at 4°C inside an autosampler. Both oxidative lesions, 5-Oh-dCyd and 8-oxo-dGuo were quantitated by HPLC on the same chromatography run using a step gradient such that 5-OH-dCyd was eluted in the first 20 ml with 0.1% methanol in phosphate buffer (25 mM, pH 5.5) whereas 8-oxo-dGuo was subsequently eluted in 57 ml with 6.0% methanol in the same buffer. The separation was achieved using an ODS-AQ 5 µm 250x4.6 mm internal diameter column (YMC, Wilmington, NC). The HPLC system consisted of the following modules: 600S controller, 616 pump, 717 plus autoinjector, 510 UV detector (Waters), and Coulochem II detector equipped with a 5011 analytical cell (ESA Associates, Chelmsford, MA), controlled and operated by millennium 2010 chromatography manager (Waters). In order to optimize the detection of both 8-oxo-dGuo and 5-OH-dCyd, the electrochemical cell was set at 0.05 (cell #1) and 0.35 V (cell #2) versus Pd reference electrode. Oxidative DNA damage was determined from the amount of 5-OH-dCyd or 8-oxo-dGuo divided by the amount of dCyd, measured by electrochemical and UV detection, respectively. Every fourth analysis, synthetic standards were injected to correct for any changes in the response of the electrochemical detector.
For statistical analysis, data were fit to a line using least square regression analysis, with the aid of Microsoft Excel software. The statistical significance was determined using a two-tailed test of the null hypothesis that the true correlation is zero. A probability of <0.05 was considered significant. Grouped data are expressed as the mean ± SD.
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Results |
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The average level of 8-oxo-dGuo was 4.5 ± 1.8 fmol per 106 dGuo and that of 5-OH-dCyd was 2.9 ± 1.4 fmol per 106 dGuo for lymphocytes obtained from 105 healthy subjects (Table I). The observed level of 8-oxo-dGuo is comparable with that reported by others using similar methods of DNA digestion and HPLC analysis with EC. In particular, Collins et al. (28) observed a level of 4.3 ± 3.0 (n = 36) 8-oxo-dGuo/106 dGuo in lymphocyte DNA. Similarly, Nakajima et al. (29) reported 2.7 ± 1.2 (n = 92) 8-oxo-dGuo/106 dGuo whereas Asami et al. (30) reported 3.1 ± 1.6 (n = 10) 8-oxo-dGuo/106 dGuo when taking white blood cells as the source of DNA. The lower value of 8-oxo-dGuo reported by Asami et al. compared with our value, may be caused by the extraction procedure. The extraction of DNA using high concentrations of NaI has been shown to reduce the level of 8-oxo-dGuo in the DNA of rat liver by ~50% compared with the common phenol-based method (31). Similarly, we observed lower levels of 8-oxo-dGuo as well as 5-OH-dCyd for the analysis of DNA damage in lymphocytes when comparing our method using phenol with the NaI method using the DNA Extractor WB kit (Wako Pure Chemical Industries, Japan). However, there was an indication from preliminary experiments that NaI or its byproducts destroyed 8-oxo-dGuo and 5-OH-dCyd in DNA during extraction. In particular, NaI extraction of previously extracted calf thymus DNA that contained a pre-existing quantity of damage led to a significant decrease in the level of 8-oxo-dGuo (20%) and 5-OH-dCyd (90%).
The levels of 8-oxo-dGuo and 5-OH-dCyd were significantly correlated in lymphocyte DNA (Figure 2; r = 0.52, P < 0.001). From this relationship, the ratio of 8-oxo-dGuo to 5-OH-dCyd was 1.4 (1/0.72). In contrast, the ratio of these two lesions is much higher when DNA undergoes oxidation in solution. For example, when DNA is exposed to ionizing radiation, the ratio of 8-oxo-dGuo to 5-OH-dCyd is 14, and when DNA is treated with Fenton reagents, Fe2+ or Cu+, and/or H2O2, and/or ascorbate, the corresponding ratio lies between 5 and 8. Also, 8-oxo-dGuo is produced in much greater yields than 5-OH-dCyd during photosensitization since guanine is the most easily oxidizable base in DNA (32). Thus, it is doubtful that either 8-oxo-dGuo or 5-OH-dCyd arise from artifactual oxidation during sample preparation because this would give a high ratio of 8-oxo-dGuo to 5-OH-dCyd, similar to that observed when DNA undergoes oxidation induced by radiation or Fenton reagents.
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Discussion |
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It is unlikely that HPLCEC grossly overestimates the level of 8-oxo-dGuo. Firstly, auto-oxidation is minimal in our study because the removal of oxygen or the addition of metal chelators had no significant effect on the level of 8-oxo-dGuo in cellular DNA. Secondly, it is doubtful that 8-oxo-dGuo and 5-OH-dCyd arise from artifactual oxidation during sample preparation because the ratio with 8-oxo-dGuo:5-OH-dCyd is 1.4 in cellular DNA whereas the ratio is at least 5-fold greater when DNA undergoes oxidation in solution induced by ionizing radiation or Fenton reagents. The nearly equal levels of 8-oxo-dGuo and 5-OH-dCyd in cellular DNA implies that the former may be more efficiently removed by DNA repair. Finally, the measurement of 8-oxo-dGuo in cellular DNA by either the comet assay or the alkali-elution method, in combination with purified DNA repair enzymes, leads to levels of damage that are ~10-fold lower than by HPLCEC (28,33). The reason for this discrepancy is not clear. Although formamidopyrimidine DNA N-glycosylase (fapy) efficiently removes 8-oxo-dGuo from oxidized DNA, it is not clear whether this occurs to the same extent for DNA that contains endogenous damage. A reason why oxidative damage persists in cellular DNA may be because it is partly resistant to removal by DNA repair depending on the specific context or sequence of the damage. For example, it is known that fapy efficiently excises 8-oxo-dGuo from 8-oxo-dGuo-C base pairs in duplex DNA but not from 8-oxo-dGuo-A base pairs, which may persist to some extent in cellular DNA (37). Thus, the comet assay or the alkali elution method with DNA repair enzymes may underestimate 8-oxo-dGuo in cellular DNA.
Whether the separation of lymphocytes from blood has an effect on the level of oxidative DNA damage is difficult to determine. During separation, lymphocytes are exposed to various potentially damaging agents or conditions, including changes in volume and pressure caused by centrifugation, changes in temperature (3722°C) and changes in oxygen tension, i.e. the concentration of oxygen may be expected to increase 5-fold in going from in vivo to in vitro. Nevertheless, it should be noted that the separation of lymphocytes from blood is not sufficient by itself to induce either proliferation or apoptosis in cell culture (in contrast, granulocytes or monocytes are much more sensitive toward activation). The work of Collins et al. (28) suggests that the transition of going from oxygen tension in vivo to that in vitro has only a minor effect on the level of oxidative DNA damage in lymphocytes. They compared the level of oxidative DNA damage for lymphocytes isolated from blood taken before and 2 h after consumption of 1 g of vitamin C on the hypothesis that an increased level of antioxidants would protect lymphocytes against oxidative stress in vitro. However, the data showed no significant difference in the initial level of either single strand breaks or oxidized pyrimidines estimated by the comet assay, although there were noticeable changes in the initial level of H2O2-induced damage and the subsequent removal of this damage. Another important point is whether the incubation of lymphocytes in culture has an effect on oxidative DNA damage. The final step in the separation of lymphocytes from blood involves the removal of monocytes, which requires the incubation of cells in culture for 50 min. However, it is doubtful that such a short period of incubation has any effect on oxidative DNA damage since there is no observable effect on damage even for lymphocytes in culture for as long as 24 h on the basis of single strand breaks or oxidized pyrimidines measured by the comet assay, as well as fapy-sensitive sites measured by the alkali elution method (28,33). In contrast, primary lymphocytes in cell culture for 1824 h are more vulnerable to H2O2-induced DNA damage (38). The effects at long incubation times though can probably be attributed to a dramatic drop in the activity of several antioxidant enzymes, glutathione reductase, glutathione peroxidase and catalase. Interestingly, the addition of antioxidants, particularly flavanoids, to primary lymphocytes in cell culture significantly inhibits the formation of oxidative DNA damage as measured by the comet assay (38,39). This indicates that antioxidants are able to protect lymphocytes against H2O2-induced oxidative DNA damage.
The levels of intracellular glutathione and ascorbate as well as oxidative DNA damage were different in lymphocytes, granulocytes and monocytes (Table I). In particular, the level of 8-oxo-dGuo in granulocytes and monocytes was significantly higher than that in lymphocytes. This may be caused in part by the activation and increased oxidant production of granulocytes and monocytes during their separation from blood. However, the ratio of 8-oxo-dGuo:5-OH-dCyd was different in all categories of white blood cells, which may possibly reflect differences in DNA repair between these cell types. Intriguingly, there was no apparent correlation between the levels of glutathione and ascorbate with oxidative DNA damage in these cells. For example, the level of glutathione was 70% higher in monocytes than in lymphocytes whereas the former showed slightly higher levels of 8-oxo-dGuo. Also, it was surprising that Jurkat T cells in culture have about the same level of nuclear 8-oxo-dGuo as primary lymphocytes whereas the former have no detectable ascorbate. These results underline the importance of separating white blood cells into their different major groups in order to evaluate antioxidants and oxidative DNA damage.
Although the interaction of antioxidants and oxidative DNA damage is complex, it is reasonable to assume that glutathione and ascorbate play a role in neutralizing free radicals and oxidants that cause DNA damage, because they are present inside cells at relatively high concentrations and they react efficiently with reactive oxygen species. The ability of these antioxidants to protect against oxidative DNA damage has been shown in various model systems (11). For example, the administration of ascorbate has been shown to reduce oxidative damage in kidney DNA induced by potassium bromate (40). Ascorbate also reduces damage in liver DNA after treatment with redox-cycling estradiol derivatives (41). Contrastingly, Cadenas et al. observed no change in the level of 8-oxo-dGuo in the DNA of rat liver despite a 60-fold diet-induced variation in the level of ascorbate in the same organ (42). The notion that glutathione protects against oxidative DNA damage in rodents is shown by the effect of buthionine sulfoximine, which reduces intracellular glutathione and significantly increases 8-oxo-dGuo in various organs (43). Also, an exceptional study by Garcia de la Asuncion et al. (44) showed that the ratio of oxidized to reduced glutathione in mitochondria was strongly correlated with the level of 8-oxo-dGuo in mitochondrial DNA and that the administration of vitamin C and vitamin E reversed the levels of oxidative damage for both glutathione and DNA in aged rats to the levels observed in young rats.
In humans, there have also been a number of interesting studies directly linking antioxidants to oxidative DNA damage. For example, dietary supplementation with a cocktail of vitamin C (0.1 g/day), vitamin E (0.28 g/day) and beta-carotene (0.025 g/day) significantly reduced oxidative DNA damage in lymphocytes in both non-smokers and smokers as measured by the modified comet assay for oxidized pyrimidines (45). The subjects with a supplement compared with those without were also more resistant to ex vivo H2O2-induced oxidative DNA damage. Similarly, an increase in the consumption of vegetable products that are high in carotenoids, such as tomato and carrot juice, was shown to reduce the baseline level of oxidized pyrimidines by as much as 3-fold (46). There have also been negative or uncertain results. For example, the level of 8-oxo-dGuo decreased whereas the level of 8-oxo-7,8-dihydroadenine increased in lymphocyte DNA upon supplementation of donors with vitamin C. Paradoxically, these results suggest that vitamin C exerts anti- and pro-oxidant effects in vivo (35). In addition, a supplement of vitamin C together with iron was reported to increase the level of certain oxidative base lesions while decreasing the level of other lesions (47). However, the conclusions of the latter two studies should be reconsidered in view of the fact that GCMS analysis is prone to extensive auto-oxidation (e.g. the levels of 8-oxo-dGuo in the latter studies are 60-fold higher than those in the present study). Finally, the effect of dietary antioxidants has been evaluated by urinary 8-oxo-dGuo as a marker of oxidative DNA damage. The results of an epidemiological study indicated that vitamin C based on dietary evaluation was negatively correlated with the urinary excretion of 8-oxo-dGuo (48). In contrast, other studies have not reported a significant difference in urinary 8-oxo-dGuo upon dietary supplementation with 250 mg of vitamin C in smokers despite the fact that smokers have lower plasma vitamin C and higher urinary 8-oxo-dGuo (49,50).
In the present study, we have examined the endogenous levels of antioxidants and oxidative DNA damage in human lymphocytes from healthy volunteers. Also, we have focused on the measurement of intracellular rather than plasma levels of antioxidants because the former is more relevant to oxidative DNA damage. The results demonstrate that both glutathione and ascorbate are negatively correlated to endogenous oxidative DNA damage in human lymphocytes, which provides compelling evidence that these antioxidants protect against oxidative DNA damage. Moreover, the dependence of oxidative DNA damage on intracellular glutathione and ascorbate, and the large variation of antioxidants, suggest that this damage can be modulated in the human population. Assuming that oxidative DNA damage contributes to cancer, it should be possible to prevent cancer by maintaining sufficiently high levels of intracellular glutathione and ascorbate.
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Acknowledgments |
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Notes |
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References |
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