Received 5 August 1996. Oxidative DNA damage in human cells: the influence of antioxidants and DNA repair. A. R. Collins, S. J. Duthie, L. Fillion, C. M. Gedik, ...
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absorption of photons at UVB and UVC wavelengths, guanine is very resistant to modification but adenine can undergo photoaddition reactions with neighbouring thymine or adenine bases. T h e s e reactions occur with very low quantum yields in the region of 20 PmoVeinstein. Consequently, the resulting TA*, AA* and A=A photoadducts should constitute no more than about 2% of the total photolesions produced in native DNA by direct excitation; they are therefore likely to be of marginal biological importance.
1 Taylor, J.-S. (1994)ACC.Chem. Res. 27,76-82 2 Cadet, J. and Vigny, P. (1990)in Bioorganic Photochemistry (Morrison, H., ed.), vol. 1, pp. 1-272,John Wiley & Sons, New York 3 Sage, E. (1993)Photochem. Photobiol. 57,163-174 4 Davies, R. J. H. (1995)Biochem. SOC.Trans. 23, 407-418 5 Kochevar, I. E. and Dunn, D. A. (1990)in Bioorganic Photochemistry (Morrison, H., ed.), vol. 1, pp. 273-315,John Wiley & Sons, New York 6 Paillous, N. and Vicendo, P. (1993)J. Photochem. Photobiol. B: Biol. 20,203-209 7 Devasagayam, T. P. A., Steenken, S., Obendorf, M. S. W., Schulz, W. A. and Sies, H. (1991) Biochemistry 30,6283-6289 8 Kasai, H., Yamaizumi, Z., Berger, M. and Cadet, J. (1992)J. Am. Chem. SOC.114,9692-9694 9 Chung, M.-H., Kiyosawa, H., Ohtsuka, E., Nishimura, S. and Kasai, H. (1992)Biochem. Biophys. Res. Commun. 188, 1-7 10 Friedmann, T. and Brown, D. M. (1978)Nucleic Acids Res. 5,615-622
1 1 Kamiya, H. and Kasai, H. (1995)Jpn. J. Toxicol. Environ. Hlth. 41,307-319 12 Harm, W. (1980)Biological Effects of Ultraviolet Radiation, Cambridge University Press, Cambridge 13 Cadet, J., Anselmino, C., Douki, T. and Voituriez, L. (1992)J. Photochem. Photobiol. B: Biol. 15, 277-298 14 Gorner, H. (1994)J. Photochem. Photobiol. B: Biol. 26, 117-139 15 Bose, S.N., Davies, R. J. H., Sethi, S. K. and McCloskey, J. A. (1983)Science 220,723-725 16 Bose, S. N.,Kumar, S., Davies, R. J. H., Sethi, S. K. and McCloskey, J. A. (1984)Nucleic Acids Res. 12,7929-7947 17 Koning, T. M. G., Davies, R. J. H. and Kaptein, R. (1990)Nucleic Acids Res. 18,277-284 18 Zhao, X.,Nadji, S., Kao, L.-F. and Taylor, J.-S. (1996)Nucleic Acids Res. 24,1554-1560 19 Bose, S.N. and Davies, R. J. H. (1984)Nucleic Acids Res. 12,7903-7914 20 Gut, I. G., Farmer, R., Huang, R. C. and Kochevar, I. E. (1993)Photochem. Photobiol. 58,313-317 21 Zhao, X.and Taylor, J.-S. (1996)Nucleic Acids Res. 24, 1561-1565 22 Kumar, S.,Sharma, N. D., Davies, R. J. H., Phillipson, D. W. and McCloskey, J. A. (1987) Nucleic Acids Res. 15,1199-1216 23 Kumar, S.,Joshi, P. C., Sharma, N. D., Bose, S. N., Davies, R. J. H., Takeda, N. and McCloskey, J. A. (1991)Nucleic Acids Res. 19,2841-2847 24 Sharma, N. D. and Davies, R. J. H. (1989)J. Photochem. Photobiol. B: Biol. 3, 247-258 25 Clingen, P. H. and Davies, R. J. H. (1996)J. Photochem. Photobiol. B: Biol., in the press Received 5 August 1996
Oxidative DNA damage in human cells: the influence of antioxidants and DNA repair A. R. Collins, S. J. Duthie, L. Fillion, C. M. Gedik, N. Vaughan and S. G. Wood Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 I 9SB, Scotland, U.K.
Introduction O n e of the most important insults to which cellular DNA is subjected is oxidative damage, resulting from attack by reactive oxygen species, i.e. free radicals, of exogenous or endogenous origin. Oxidative damage is implicated in the Abbreviations used: FPG, formamidopyrimidine glycosylase; GC-MS, gas chromatography with mass spectrophotometric detection; 8-OH-dG, 7,8-dihydro-8-oxodeoxyguanosine; SCGE, single-cell gel electrophoresis.
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earliest stages of carcinogenesis [ 11. Epidemiological evidence linking the high incidence of certain cancers with a low intake of fruit and vegetables [Z-61 can be explained, at least in part, by the presence in these foods of various antioxidant micronutrients (vitamin C, carotenoids, vitamin E, flavonoids and other polyphenolics), which are believed to decrease the amount of free radicals - particularly hydroxyl radicals - reaching the DNA. Attempts to test this hypothesis of antioxidant protection by means of intervention trials have had mixed
DNA Damage and Mutagenesis
results. Although a supplement of vitamin E, vitamin C and selenium appeared to protect against oesophageal cancer in a trial in Linxian, China [7], /I-carotene was significantly linked with an increased incidence of lung cancer in the much-publicized 'Finnish study' [8]. Using a more focused molecular epidemiological approach, in which oxidative DNA damage was measured directly in human subjects (as a putative index of cancer risk), we found that a supplement of vitamin C, p-carotene and vitamin E taken together for 20 weeks significantly decreased base oxidation in lymphocyte DNA [9] as well as rendering the lymphocytes more resistant to H202-induced damage to DNA in vitro . What we measure in lymphocytes, or any other tissue or cell type, is the steady-state level of DNA damage, a dynamic equilibrium reflecting both input of damage and its removal by cellular repair processes. (A non-equilibrium situation, with input greater than repair of damage, might occur temporarily as a result of infection or some other sudden increase in oxidative stress, and a slow accumulation of damage might occur with age.) Measuring the steadystate level of damage does not tell us about the overall rate at which damage is inflicted on the DNA. The only way to estimate this, at least in vivo, is probably to look at the released products of repair of the damage, since this will normally reflect the amount of damage incurred. Urine contains the oxidized products of nucleic acid breakdown, and 7,8-dihydro-8-oxodeoxyguanosine (8-OH-dG) in particular has been regarded as a quantitative indicator of DNA damage related to oxidative stress [ 10,111, although the precise origin of the urinary oxidized nucleosides has been questioned [12]. The steady-state level of damage may be the most relevant parameter from the point of view of the aetiology of cancer. As the DNA is replicated, unrepaired damage is fixed as permanent changes or mutations. In addition, repair may be inaccurate and itself introduce errors. The total damage inflicted is also, however, of crucial importance if, for example, we are interested in the effect of antioxidant protection against damage. Ideally, we need to know the relative contributions to a given steady-state level of damage from the modulation of damage by antioxidants, and from removal of damage by repair. The possibility that the capacity for repair of oxidative damage might itself vary, either at an
intrinsic level between individuals or as a result of environmental or dietary factors, has yet to be seriously addressed. Here we will draw attention to some of the problems involved in measuring DNA damage and estimating repair capacity in human subjects.
Materials and methods Cell isolation and culture
Lymphocytes or leucocytes were isolated from peripheral blood, collected from volunteers by venepuncture, using density-gradient-centrifugation techniques as described ([9,13]; C. M. Gedik, unpublished work). In some experiments, lymphocytes were incubated in Dutch modified RPMI 1640 medium (ICN Flow, Thame, Oxfordshire, U.K.) with 10% foetal calf serum. GM1899A human hymphoblastoid cells were grown in suspension in the same medium. HeLa (human transformed epithelial) cells were cultured as monolayers in Glasgow-modified Eagle's minimal essential medium (ICN Flow) with 5% foetal calf serum and 5% newborn calf serum. Penicillin and streptomycin were present in all media, and cells were incubated (unless otherwise stated) at 37 "C and 5% C 0 2 in air. Measurement of 8-OH-dG
DNA was isolated from leucocytes (which had been stored in 0.4 M NaCVlO mM Tris/2 mM EDTA, pH 8.0, containing 1 mM 1,lO-phenanthroline at -80 "C under nitrogen) using a proteinase K/high-salt precipitation method [ 141. The DNA was hydrolysed to nucleosides [14] and the hydrolysate was analysed by HPLC on a Ct8 reverse-phase Apex ODS 3 pm column (15 cm x 0.46 cm; Jones, Hengoed, Mid-Glamorgan, Wales, U.K.) with a 2 cm x 0.4 cm guard column. The mobile phase was 50 mM potassium phosphate, pH 5.5, with 8% methanol at a flow rate of 0.75 mumin. 8-OH-dG was detected with a Coulochem I1 electrochemical detector (ESA, Chelmsford, MA, U.S.A.). Results are expressed as a ratio of 8-OH-dG to unoxidized dG measured with a Holochrome UV detector (Gilson Medical Electronics, Middleton, WI, U.S.A.) at 254 nm. Single-cell gel electrophoresis (SCGE)
The procedure for SCGE, usually known as the comet assay, has been described in detail, including the incubation of nucleoids with lesion-
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specific endonucleases [ 141. Analysis by visual assessment of the extent of movement of DNA into the comet tail (reflecting DNA break frequency) for 100 comets gives a score of between 0 and 400 (minimal to maximal damage, representing a few hundred to a few thousand breaks per cell). Alternatively, the percentage DNA in the tails of 25 comets per sample is assessed using the computerized image analysis system Komet 3.0 (Kinetic Imaging Ltd, Liverpool, U.K.).
Glutathione and antioxidant enzymes
Glutathione was assayed on acid-precipitated cellular material by a spectrophotometric method [ 151. Catalase, glutathione reductase and Sedependent glutathione peroxidase activities were measured in cell homogenate as described [ 16-18].
Results and discussion Measuring oxidative damage to bases in DNA
The three most common approaches to the measurement of oxidized bases in DNA isolated from cells are: gas chromatography with mass spectrophotometric detection (GC-MS); HPLC with electrochemical detection; and the use of lesion-specific repair endonucleases to introduce DNA breaks at the sites of damaged bases, the breaks then being measured by a variety of methods, such as alkaline elution [19], nick translation [20] and SCGE (see below). As discussed by us and others previously [12,14,21], there are serious discrepancies in the results obtained by the different approaches, particularly when measuring 8-OH-dG. GC-MS has been reported to be prone to oxidation artifacts occurring during derivatization of the hydrolysed DNA, and if these are eliminated, GC-MS and
HPLC give similar results [21]. We have recently re-examined the HPLC approach, using methods for isolation and hydrolysis of DNA that decrease the chance of oxidation occurring in vitro. T h e isolation does not employ phenol-chloroform extraction, since these reagents have been implicated in spurious oxidation [22]. T h e high sensitivity of the latest model of electrochemical detector is essential for detection of the small peak of 8-OH-dG in leucocyte DNA from normal subjects; also, it is important to distinguish this peak from an unknown compound that, we find, runs very close to the position of 8-OH-dG. T h e analysis of 8-OH-dG in the white blood cell DNA from a group of 36 volunteers gives a mean +S.D. of 0.43 f0.30 per lo5 dG, at the low end of the range of published data (Table 1) [23-261, and corresponding to about 13000 per cell. A very recent report, using an anaerobic isolation method, gives a value of about 0.2 per lo5 dG 1271. T h e other method we have used is SCGE, or the comet assay, which detects DNA strand breaks by virtue of their ability to relax supercoiled loops in nucleoids (lysed and saltextracted nuclei) embedded in agarose. Relaxed loops are drawn out by electrophoresis to form a tail, and the amount of DNA in the tail reflects the number of breaks present. Treatment of the nucleoids in the gel with endonuclease I11 (specific for oxidized pyrimidines) or with formamidopyrimidine glycosylase (FPG) , which recognizes 8-OH-dG, allows detection of the enzyme-sensitive sites converted into DNA breaks [14]. Samples of lymphocytes from the same 36 volunteers, analysed by SCGE, gave mean fS.D. values of percentage tail DNA of 12.6f6.7 for baseline DNA breakage, 21.3 f7.1 with endonuclease I11 digestion, and 19.1 f6.8 with FPG. Using a calibration of the comet assay based on X-irradiation [14], we estimate the net
Table I
8-OH-dG concentrations in human leucocyte DNA 8-OH-dG per lo5dG
Number of determinations
S.E.
S.D.
Reference
0.33
10
-
0.08
8 I .07
2
-
-
79
-
0.23
48
0.26
-
23 24 25 26
36
-
0.30
Present study
3.9 0.43
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D N A Damage and Mutagenesis
numbers of endonuclease 111- and FPG-sensitive sites as 1120 and 870 per cell respectively. This is an order of magnitude lower than the yield of 8-OH-dG; the origin of this discrepancy is not understood. If the steady-state level of oxidative base damage is low, this is consistent with the existence of an effective base excision repair process in cells, and with the fact that several of the damaged bases are potentially mutagenic. It is clearly to the cell's advantage to eliminate them as quickly and as completely as possible. It is worth pointing out that, in cultured cells such as HeLa, the level of oxidized purines and pyrimidines is barely detectable, either by HPLC or by SCGE ([28,29]; C. M. Gedik and S. G. Wood, unpublished work).
the relatively defective repair in the latter has not been further explored. In the case of lymphocytes, there is apparently only a slow removal of H20z-induced strand breaks over 24 h (Figure 2). The modified comet assay, with endonuclease 111, indicates no removal of oxidized bases in this period. The observation of a slight but consistent increase in damage (both strand breaks and oxidized pyrimidines) in cells that were not treated with HzOz (Figure 2A) raises the possibility that lymphocytes, once in culture in vitm, suffer from oxidative stress resulting from the increased oxygen tension in the medium compared with blood. The hyperoxic environment has been recognized as likely to induce toxicity [32,33], but until now Figure I
Measuring repair of oxidative DNA damage
Although urinary 8-OH-dG may reflect the overall DNA repair activity of the organism, the question of possible variation in or modulation of cellular repair capacity is best investigated at the cellular level, challenging cells in vitro with DNA-damaging agent and monitoring removal of the damage during a period of incubation. H z 0 2 is commonly used to introduce oxidative damage. Figure 1 shows that the immediate response to H202,in terms of damage inflicted, seems to vary according to cell type. (The comet assay was used in its basic form to measure strand breaks.) When treated for 30 min at 4 "C, lymphocytes are more resistant than either the GMl899A lymphoblastoid cell line or HeLa transformed epithelial cells. (We have shown elsewhere [30] that the resistance of lymphocytes is particularly marked in a subpopulation that requires very high H202doses to produce any damage, the rest of the population behaving more like HeLa or GM1899A cells.) At 37"C, however, the yield of damage in HeLa cells is much reduced, whereas lymphocytes and GM1899A cells show, if anything, slightly more damage. It seems that, in HeLa cells at the higher temperature, antioxidant enzymes decrease the level of reactive oxygen, and/or cellular repair acts rapidly on the damage that is inflicted. By giving just 5 min treatment with H202at 4 "C and then monitoring the level of damage on subsequent incubation at 37"C, we have shown [31] that the difference in behaviour between HeLa and GM1899A cells is the result of a very rapid repair of strand breaks that occurs during the incubation period in HeLa but not in lymphoblastoid cells. The reason for
DNA damage (strand breaks) incurred by different cell types treated with H202:dependence on incubation temperature GM I899A lymphoblastoid cells (A), HeLa cells (6) and peripheral lymphocytes (C) were treated with H202for 30 mtn, at either 37 "C or 4 "C, before analysis of D N A damage using the comet assay 100 comets were assessed visually, the arbitrary units relate t o the mean relative amount of D N A in the comet tails, which reflects D N A break frequency Bars indicate S E M
A
-1
R
r;
2wr I 150
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effects at the level of DNA stability have not been examined. We have monitored the repair of oxidative damage in H202-treated hymphocytes on incubation in an atmosphere of 5% oxygen, and found a dramatic increase in the rate of removal of strand breaks; in addition, untreated cells then show a decrease in damage to a very low level (L. Fillion and A. R. Collins, unpublished work). Figure 2 In vitro repair of oxidative DNA damage in human lymphocytes: effect of in vivo vitamin C supplementation Lymphocytes were isolated from blood taken just before (0,W , 0, 0 ,-) and 2 h after (A,A,0, +, ----) consumption of a I g tablet of vitamin C. 0 , 0, A,0, lymphocytes were treated with 0.1 mM H 2 0 2for 5 min on ice, and then incubated in medium at 37 "C for up to 24 h. W , 0 , A,+, incubation without H202treatment. At intervals, samples were taken for analysis of strand breaks ( 0 ,W , A,A) and oxidized pyrimidines (0, 0 , 0, +) with the comet assay. Results were converted into breaks per I O9 Da (see [ I41 for calibration). Subjects were divided into two groups: the majority. who showed a modest increase in plasma vitamin C concentration (A), and two, whose vitamin C level almost doubled (6). Mean values are shown, with bars indicating S.E
1.5
I
-
1
0
I
I
I
I
1
5
10
15
20
25
I
I
I
I
I
5
10
15
20
25
Incubation time (h)
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If high oxygen tension is causing damage to DNA in vitro, then antioxidants carried over from the in vivo environment might protect against this damage. We tested the significance of antioxidant protection by carrying out a small-scale supplementation trial with vitamin C. Blood was taken before and 2 h after a single 1 g dose of vitamin C. Plasma ascorbate was measured, and lymphocytes were isolated for an assessment of repair of strand breaks and oxidized pyrimidines (shown in Figure 2). The relative increase in plasma ascorbate varies considerably. Two cases out of eight show an increase of over 90%, almost double the mean increase of the other subjects. The two samples giving the high plasma vitamin C response also show a significant enhancement of the rate of removal of strand breaks (P
