Mutagenesis, Vol. 18, No. 2, 139-143,
March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
A study on the effects of seasonal solar radiation on exposed populations
1 DNA Repair Laboratory, Institute of Biology, National Center for Scientific Research ‘Demokritos’, 153 10 Aghia Paraskevi, Athens, Greece, 2 Department of Cell Biology, School of Biology, University of Athens, Greece and 3 Department of Immunology and Histocompatibility, ‘Aghia Sofia’ Hospital, Athens, Greece
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Abstract |
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In the present study the effects of seasonal solar radiation (summer and winter) on exposed populations of two different age groups (20–25 and 40–55 years old) were investigated. In addition, the effects of external factors, such as hydrogen peroxide (H2O2) and
-irradiation, as well as the repair efficiency of human lymphocytes from these populations, was also evaluated. Our results show that the amount of DNA damage appears to be influenced by the exposure to solar radiation, with the summer exposure being the most damaging. Age was also found to be a significant factor, with the older population being more susceptible to solar radiation than the younger one. Season does not appear to affect the sensitivity to external DNA-damaging agents, while age does. Age was also found to have an effect on the DNA repair capacity of the examined populations. |
Introduction |
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Sunlight is the energy source of most life forms. This life-giving force has, however, the potential for destroying life itself.
There is overwhelming evidence that sunlight is a human carcinogen (IARC, 1992
; Longstreth et al., 1998) and in terms of absolute numbers is one of the most significant carcinogens to which populations are exposed.
Skin cancer is the most common neoplasm in Caucasians in the USA, with a lifetime risk nearly equal to that of all other cancers combined (Scotto et al., 1982). Sun exposure is the major environmental agent implicated in induction of non-melanoma skin cancer. While sun exposure begins early in life, the average patient with non-melanoma skin cancer is 
60 years old (Jonason et al., 1966; Scotto et al., 1982).
The UV component of solar radiation is divided for convenience into three regions, UVA (315–400 nm), UVB (280–318 nm) and UVC (<280 nm) (IARC, 1992). The shorter the wavelength, the more energy it transmits and the more damage it causes. However, shorter wavelengths are more readily blocked, either by the atmosphere or outer layers of skin (Green et al., 1999). Solar radiation gives rise to cellular DNA damage by direct and indirect mechanisms (Moan and Peak, 1989; Tyrrell and Keyse, 1990). The direct excitation of DNA generates predominantly cyclobutane pyrimidine dimers (CPD), the pyrimidine pyrimidone (6–4) photoproduct and the Dewar isomer, which are without doubt of principal importance for the cytotoxic, mutagenic and carcinogenic effects of shortwave UV-radiation (UVC and UVB) (Doninger et al., 1981; Anathaswamy and Pierceall, 1990). The 6–4 photoproduct is induced at shorter wavelengths, but is isomerized to the Dewar isomer by longer wavelength UVB and even UVA. UVC forms the same direct photoproducts as UVB but in different proportions, with negligible levels of the Dewar isomer (Clingen et al., 1995). On the other hand, indirect mechanisms are responsible for DNA damage and genotoxic effects at long wavelengths (UVA and visible light), at which DNA absorbs only weakly or not at all (Wells and Han, 1984; Jones et al., 1987). In this range of the spectrum various oxidative DNA modifications such as 8-hydroxyguanine (8-oxoG), strand breaks, site of base loss and DNA–protein crosslinks are generated (Cadet et al., 1992; Hattori-Nakakuki et al., 1994).
Research groups investigating the effects of environmental factors (including sunlight) in relation to DNA damage have used a variety of techniques. Since DNA damage induced by these agents is often tissue- and cell type-specific, an optimal assay would be capable of detecting DNA damage in individual cells obtained under a variety of experimental conditions (Tice, 1995). Several cytogenetic techniques, such as sister chromatid exchange (SCE), chromosomal aberration and micronuclei assay, have been used to evaluate the type and degree of such mutagenic behavior at a chromosomal level (Barale et al., 1993). The single cell gel electrophoresis (SCGE), or Comet, assay has also been used in similar studies (Betti et al., 1994; Andreoli et al., 1997; Calderon-Garciduenas et al., 1997; Sram et al., 1998; Wojewodzka et al., 1998).
In the present study we investigated the levels of DNA damage on peripheral blood lymphocytes of two populations aged 20–26 and 40–55 years exposed to solar radiation in two different seasons (summer and winter). The effects of additional external factors (hydrogen peroxide and -irradiation) were also examined.
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Materials and methods |
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Chemicals and media
Plastics were from Corning (New York, NY). RPMI 1640 medium with phenol red, fetal calf serum, phytohemagglutinin (PHA) and trypan blue were obtained from Biochrom KG (Berlin, Germany). Lymphoprep was supplied by Nycomed (Oslo, Norway). L-Glutamine, penicillin and streptomycin were from ICN Flow (Irvine, UK). Low and normal melting point agarose were supplied by Gibco BRL (UK). Phosphate-buffered saline (PBS) tablets, dextrose, hydrogen peroxide (H2O2) and 4',6-diamidine-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma Chemical Co. (St Louis, MO).
Studied population
The sample consisted of a total of 40 non-smoking men of two different age groups. Half of them were 20–26 years old and the other half 40–55 years old.
The participants examined were selected with the use of a detailed questionnaire including questions on health, lifestyle, diet, age, hours of exposure to the sun (on average they were engaged in outdoor activities for 6 h each day during the summer), etc. This questionnaire is similar to that published by the International Commission for Protection against Environmental Mutagens and Carcinogens (Carrana and Natarajan, 1988). Only individuals following a similar lifestyle, on a well-balanced diet and with no other medical or genetic problems were included in the study.
Two blood samples were collected, the first during the third week of March (after the person was exposed to the minimum total solar radiation during the winter) and the second during the third week of September (after the same person was exposed to the maximum total radiation during the summer). These dates were chosen based on data from NASA on evaluation of seasonal erythema dose of UV solar radiation (Herman, 2001). Prior to the study, all the individuals gave informed consent and blood samples were collected and further manipulated in accordance with ethical standards.
Lymphocyte isolation, cryopreservation and thawing
The blood samples (5 ml), which were obtained from each volunteer at the beginning of a working day by venipuncture using heparinized vacutainers, were diluted 1:1 with RPMI 1640 (pH 7.3) and kept on ice for 15 min. Lymphocytes were separated by centrifugation using 5 ml of Lymphoprep, at 200 g for 30 min. Buffy coats were removed and washed twice with RPMI 1640. Lymphocytes suspended in RPMI were counted in a hemocytometer and then cryopreserved. For the cryopreservation, the cell suspension was centrifuged at 200 g for 5 min and the cell pellet was resuspended at 10x106 cell/ml in freezing medium consisting of 10% DMSO, 40% RPMI and 50% fetal calf serum. The cell suspension was transferred to plastic freezing vials in aliquots of 2x106 cells. Vials were placed in a Cryo 1°C freezing container, then directly in a –70°C freezer so as to achieve a –1°C/min cooling rate and stored afterwards at –70°C.
Vials were retrieved as needed and submerged in a 37°C water bath until the last trace of ice had melted. The thawed cells were quickly transferred to conical centrifuge tubes containing 15 ml of pre-chilled thawing medium consisting of 50% fetal calf serum, 40% RPMI and 10% dextrose (one tube/vial). Cells were centrifuged at 200 g for 10 min at 4°C and the cell pellet was resuspended in ice-cold PBS (pH 7.3) in order to be used for the Comet assay. Cell viability, using Trypan blue, was found to be >95% at each time point of the study (Visvardis et al., 1997).
Hydrogen peroxide (H2O2) and -irradiation treatments and repair
Thawed lymphocytes suspended in ice-cold PBS were exposed to H2O2 for 5 min on ice in microcentrifuge tubes (105 cells/tube) or to -irradiation from a 60Co source delivering a dose of 3.99 Gy/min. To examine DNA repair after DNA damage caused by exposure of the lymphocytes to H2O2 or -irradiation, cells were resuspended in 1 ml of RPMI 1640 supplemented with 20% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin, distributed in a 24-well plate (Corning) and incubated at 37°C in a humidified atmosphere of 5% CO2 for 2 h with the addition of PHA at a final concentration of 2.4 µg/ml in each well at the start of incubation (Piperakis et al., 1999).
Single-cell gel electrophoresis (SCGE)
The SCGE assay was performed under alkaline conditions using an adaptation of the method described previously by Collins et al. (1995). Cells with or without H2O2 and -irradiation treatment were suspended in 1% low melting point agarose in PBS (pH 7.4) at 37°C and 100 µl were pipetted onto a microscope slide pre-coated with 100 µl of 1% agarose. The agarose was allowed to set on ice for 10 min and the slide was immersed in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, NaOH to pH 10.0 and 1% Triton-X-100) for 1 h at 4°C, to remove cellular proteins. Slides were initially placed in an electrophoresis tank containing 0.3 M NaOH and 1 mM Na2EDTA (pH > 13) for 40 min. Subsequently, the tank was set at 25 V (1 V/cm, 300 mA) for 30 min at an ambient temperature of 4°C. The slides were then washed three times for 5 min each with 0.4 M Tris–HCl (pH 7.5) at 4°C before staining with 5 µg/ml DAPI (Piperakis et al., 1999).
Evaluation of DNA damage
DAPI-stained nucleoids were examined at 400x magnification with a Wang epi-fluorescence microscope (Wang BioMedical, The Netherlands), equipped with an excitation filter of 350 nm and a barrier of 420 nm. One hundred comets on each slide were scored visually as belonging to one of five predefined classes according to tail intensity and were given a value of 0, 1, 2, 3 or 4 (from undamaged, 0, to maximally damaged, 4). Thus, the total score for 100 comets could range from 0 (all undamaged) to 400 (all damaged) in arbitrary units (Piperakis et al., 2000). The percentage of DNA in the tail was also estimated using an image analysis system (Kinetic Analysis, UK) connected to a computer with a suitable program.
Statistical analysis
For each donor, 300 comets (100 comets/slide, triplicate slides per treatment) were used to evaluate DNA damage and repair. Mean scores, in arbitrary units (± SD) were calculated from the respective values of the three slides, as well as from the image analysis estimates. A multivariate analysis of variance (GLM repeated measures) as well as a non-parametric test (Kruskal–Wallis) were used to evaluate differences in the distribution of DNA damage and repair. Both tests gave similar results. For all statistical analyses, a level of at least 0.05 was used to determine significance (Piperakis et al., 2000).
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Results |
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After 5 min treatment of lymphocytes with doses of 0, 50, 100 and 150 µM H2O2 the mean extent of basal and H2O2-induced DNA damage was measured. These values are presented in Figure 1
. There is a noticeable and significant difference in the extent of DNA damage in both age groups (20–26 and 40–55 years old) after exposure to solar irradiation (P < 0.001). The basal DNA damage is more extensive in the older group in relation to the younger one, indicating that age influences the amount of DNA damage accumulated (P < 0.005). This result is in agreement with Piperakis et al. (1998).
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In terms of H2O2-induced DNA damage, the extent of damage was proportional to the dose and independent of seasonal variation. It is noticeable that the difference between the old and young groups was retained.
After 2 h incubation of the lymphocytes from all groups a DNA repair capacity was revealed, with the younger group being more efficient than the older one. However, the difference found during the treatment with H2O2 was retained among the groups.
Treatment of the old and young age groups with -radiation (Figure 2) revealed a statistically significant difference (P < 0.001), indicating that age influenced the amount of DNA damage accumulated. The amount of DNA damage increased proportionally according to the dose of -radiation (P < 0.001) but was independent of season. In general the results obtained after -radiation were analogous to those obtained with H2O2 treatment.
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Damage caused by
-radiation treatment was repaired after 2 h incubation. However, the difference found among the groups during the treatment was retained.
The percentage of DNA in the comet tail was also estimated using an image analysis system (Kinetic Analysis, UK). Tables I and II give the mean values for the March populations while Tables III and IV give the mean values for the September populations. The results correspond with those found by measuring arbitrary units.
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Figure 3
shows the dispersion of the basal DNA damage according to age. In order to measure the dispersion we first calculated the variance for the younger [(
2) variance = 0.0123] and older (variance = 4.644) age groups. The values found differ significantly. The estimated coefficients of variation (CV) [CV = SD ()/averagex100%] are CV1 = 2.56% and CV2 = 21.53% for the younger and the older age groups, respectively. These values also differ significantly. Treatment with H2O2 and -irradiation retained the significant dispersion difference (data not shown) between younger and older age groups.
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Discussion |
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The thinning of the stratospheric ozone shield and the resulting increase in UVB radiation reaching the Earth are expected to have direct health effects on humans. Exposure to the UV radiation of sunlight plays a major role in the appearance of skin cancer and in premature aging of the skin. The incidence of all kinds of skin cancer, which constitute the commonest form of cancer among white populations, increases with greater exposure to UVB (Longstreth, 1990
).
Evidence for the involvement of DNA photoproducts in human skin carcinogenesis originally came from the work of Cleaver (1968), who showed that patients with xeroderma pigmentosum have a propensity to develop light-induced cancers early in life. It was pointed out that cells of patients with Cockayne syndrome were also hypermutable by UV (Arlett, 1980; Bridges, 1990). Bech-Thomsen et al.(1993) found that a 1 week holiday in the Canary Islands in May increased epidermal thickness, spontaneous DNA synthesis and DNA strand breaks in lymphocytes after the holiday. Arlett et al. (1993) showed that T lymphocytes are exquisitely hypersensitive to the DNA-damaging and lethal effects of UVB radiation.
In our present work the effects of solar radiation on lymphocytes of the examined populations was found to be influenced by seasonal variations.
Longitudinal studies by Betti et al. (1995) and Frenzilli et al. (1997) found that more DNA damage was detected in samples obtained during the summer months than in samples obtained during other times of the year. Moller et al. (1998, 2002) have found that DNA strand breaks and alkali-labile sites in mononuclear blood cells are influenced by the time of sampling, suggesting that exposure to solar radiation in the sunnier months of the year contributes to changes in DNA damage and DNA repair in these cells.
The occurrence of endogenous DNA oxidation via reactive oxygen species, released during normal metabolism, has been suggested as a possible cause of cancer (Ames, 1989). H2O2 can penetrate the cell membrane easily and initiate the generation of highly reactive species through the transition metal-catalyzed Haber–Weiss reaction (Meneghini, 1988). In order for the H2O2 to produce DNA damage, a sufficient concentration must be available to overwhelm the antioxidant capacity of the cell. This consists of antioxidant enzymes (catalase), scavenger molecules and the ability of the cells to remove altered molecules by turnover (Hersey et al., 1983).
H2O2-induced DNA damage in our case was analogous to the dose used for all the treated populations. Season does not affect the sensitivity to H2O2.
-Irradiation is able to break the DNA directly by deposition of energy in the deoxyribosephosphate backbone. Single-strand breaks and double-strand breaks are produced. However, most of the energy is deposited in water, leading to hydroxyl radicals, which can subsequently react with bases and sugars in DNA producing base modifications, sites of base loss (AP sites) and strand breaks (Visvardis et al., 1997). Comet analyses of -irradiated lymphocytes from the examined populations showed response according to the dose used, with the season having no effect whatsoever.
The levels of DNA damage and repair capacity have been widely considered to be factors closely related to aging (Arlett et al., 1993). According to some predictions of the DNA-related theories of aging, cells from older individuals should be expected to have increased levels of basal DNA damage and a possibly reduced DNA repair efficiency (Taylor et al., 1988). These results agree with Piperakis et al. (1999) and with our present findings. It was also interesting to find that as a consequence of aging the dispersion of basal DNA damage is far greater in the older population compared with the younger one. An American study comprising 41 individuals in the age range 24–93 years old ( Singh et al., 1991) detected a 12% increase in the basal level of DNA damage. A similar effect of age was also observed in hepatocytes from rats, where the subset of highly damaged cells was greater in older individuals, although the mean basal value did not increase with age (Higami et al., 1994). However, most of the studies carried out, albeit with a small age range, failed to detect such an effect (Hartmann et al., 1998; Moller et al., 1998; Pitarque et al., 1999). An interesting observation by Singh et al. (1990) suggested that cells from older individuals have less resistance to DNA damage by ex vivo X-ray exposure.
In conclusion, our findings show that seasonal solar radiation and aging have a significant effect on human populations. The effect of external factors (H2O2 and -irradiation) is, however, independent of season but not of age. Finally, repair was found to be reduced in older populations.
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Acknowledgments |
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We would like to thank N.Psiouris, A.Iroglidis and E.Petrakou for their great help during the accomplishment of this study. This work was partially supported by EU grant no. ERBICISCT960300 and by the Vardinogianio Foundation.
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Notes |
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4 To whom correspondence should be addressed. Tel: +30 211 6503626; Fax: +30 211 8075978; Email: piper{at}mail.demokritos.gr
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Received on April 2, 2002; revised on September 19, 2002; accepted on October 31, 2002.
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