Demonstration of UV-dimers in human skin DNA in situ 3 weeks after exposure

Kari Hemminki1,3, Guogang Xu1, Laura Kause2, Leena M. Koulu2, Chunyan Zhao1 and Christer T. Jansen2

1 Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden and
2 Department of Dermatology, University of Turku, 20520 Turku, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data on DNA repair rates of specific types of DNA lesions are very limited in humans in situ. Rate of repair of UV-induced DNA damage was followed in the skin of 17 volunteers up to 3 weeks of UV exposure, using a 32P-postlabelling technique for the determination of specific photoproducts. The subjects of skin phototypes I and IV were exposed to 40 mJ/cm2 of solar simulating radiation on buttock skin, and biopsies were taken at 0 h, 48 h and 3 weeks of exposure for the analysis of two cyclobutane pyrimidine dimers, TT=C and TT=T, and two 6-4 photoproducts, TT-C and TT-T, as trinucleotides. Repair rates were heterogeneous for different photoproducts. T=T dimers were repaired slower than C=T dimers, and 2.3–9.0% of the initial T=T damage remained unrepaired after 3 weeks, and was detectable in 16/17 subjects. The identity of the identified photoproducts was confirmed by a photochemical reversion assay. Damage level correlated with skin types, type I being more sensitive than type IV in an age-matched comparison. This is the first time the persistence of defined human DNA damage is demonstrated up to 3 weeks. Long-lasting DNA damage increases the likelihood of mutations.

Abbreviations: CPD, cyclobutane pyrimidine dimers; TT=C and TT=T, cyclobutane dimers; SSR, solar simulating radiation; MED, minimal erythemal dose; NER, nucleotide excision repair.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Long-lasting DNA damage is likely to be most harmful for the induction of mutations. However, practically all data on DNA repair rates in humans are derived from cultured cells, because methods have not been available for quantitative assay of DNA repair of specific types of DNA damage in humans (1–4). Among the in vivo assays, either UV-endonuclease cleavage or immunological techniques have been used but the results have been contradictory; the reported half-lives of photoproducts have ranges from 1 h to 30 h (5–8). However, none of the studies extended measurements beyond 7 days, and generally, data on the persistence of DNA damage in humans in situ are almost completely lacking (1,2,4). A novel DNA repair test was recently introduced, which was based on the quantification of specific UV radiation (UVR)-induced photoproducts in human skin biopsies by the 32P-postlabelling technique (9). For the determination of a repair rate, a single dose of UVR was administered on the back of volunteers and the removal of damage was followed with biopsies taken at various periods of time after UV exposure. UV damage was quantified as specific radioactive fractions in high performance liquid chromatography (HPLC). Standards were available for the identification of two cyclobutane pyrimidine dimers (CPDs) and two 6-4 photoproducts. The results obtained showed that repair kinetics of CPDs and 6-4 photoproducts encompassed fast and slow components, probably relating to the genomic heterogeneity of nucleotide excision repair (NER) (1,10). CPDs were removed considerably slower than 6-4 photoproducts, with 50% removal times of ~15 and 5 h (8,10), respectively; dimers at TT sites were repaired slower than those at TC sites (10). A 20-fold interindividual variation in repair rates was observed but age, and history of basal cell carcinoma and melanoma did not appear to affect repair rates when measured up to 48 h (11–13).

Solar UVR is a well-established etiologic agent for skin cancer in humans, operating through DNA damage, damage repair and photoimmunosuppression (14). Epidemiological studies have shown a higher incidence of skin cancer in fair-skinned populations (i.e. skin types I and II) compared with the pigmented counterparts (e.g. skin type IV) (15,16). This difference is likely to be related to the level of DNA damage induced by UVR in fair and dark skin, in line with our previous results (7,17). However, the differences between skin types were not large, and because the studies were designed for other purposes, the effect of skin type was not properly addressed.

The present study has two aims. Because our previous data show that substantial amounts of CPDs are detectable in human skin in situ after 48 h (12,13) and that repair kinetics apparently consist of multiple components, we wanted to test whether CPDs can be found as late as 3 weeks after exposure. In in vitro experiment CPDs have been found even 20 days after UVC exposure (18). The second aim was to quantify the level of DNA damage in persons of different skin types. The volunteers were selected to the kinetic study to include skin types I and IV, which was verified by phototesting. In this paper we demonstrate that UV irradiation of human skin leads to long-lasting DNA damage, which may increase the probability of harmful mutagenic and carcinogenic effects.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
SSR source
A broadband Philips (HP 3136) solarium was used as the SSR source. The spectral distribution of the SSR resembled the spectrum of noon summer sunlight in Helsinki (19).

Study population
Ten subjects were selected from 16 healthy university students (ages 21–29 years) after phototesting for skin type. Each of them gave informed consent. The minimal erythemal dose (MED) of SSR was defined as a just visibly perceptible erythema at 24 h. The assessment was done on unexposed buttock skin of 1 x 1 cm2, using a square root 2 incremental dose series (7, 10, 14, 20, 28, 40 and 56 mJ/cm2). For skin type I, MED was 10 mJ/cm2 or less; for skin type IV, MED was 40 mJ/cm2 or more. Each skin type group included five persons. The mean ages were 24.6 and 22.4 years for skin types I and IV, respectively. However, in order to increase the number of subjects for the repair kinetics part of the study, seven further volunteers were recruited, all skin type I but older than the above groups, i.e. 30 to 69 years. Based on our previous study, age has not influenced the rate of repair in skin in situ (11).

Sampling of skin biopsies
All participants were irradiated with 40 mJ/cm2 of SSR on buttock skin site. One skin biopsy was taken immediately after irradiation (within 5 min) from each subject and put into dry ice at once, then kept in a –20°C freezer until DNA extraction. At 48 h and 3 weeks, another two skin biopsies were taken at the exposed sites. A control sample (without irradiation) was also collected from all the participants.

DNA extraction and photoproduct measurement
DNA extraction from epidermis was performed as described before (20). DNA isolation included steps by ethanol precipitation, which would have removed any highly fragmented DNA. The levels of SSR-induced DNA damage (CPDs) were quantified using the 32P-postlabelling method, which was described elsewhere (12). CPDs (T=C and T=T) and 6-4 photoproducts (T-C and T-T) were assayed as trinucleotide with an unmodified nucleotide (thymine) in the 5'-side, i.e. TT=C and TT=T. For each 32P-postlabelling assay 3 µg DNA was digested and labeled. Each sample was measured twice. After labeling, 10 Ml water was added to each sample and then completely injected into an HPLC system for analysis. The products were detected with an online Beckman radioisotope detector. For calculation of CPD levels, the HPLC peaks of interest were integrated with Beckman System Gold software and the external synthetic standards of TT=C, TT=T, TT-C and TT-T were used for quantification.

Statistical methods
Two-sided Wilcoxon rank sum for independent groups was used for statistical analysis (20).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among the volunteers, the mean MED for skin type IV was four times higher than that for skin type I, i.e. 43 mJ/cm2 versus 10 mJ/cm2, respectively. After a single SSR dose (40 mJ/cm2), close to two times higher levels of both TT=C and TT=T immediately after SSR were found in the skin type I group compared with the skin type IV group (P = 0.005, Wilcoxon rank sum test, Table IGo). For both skin type groups, TT=T was more abundant than TT=C (P = 0.005 for skin type I and P = 0.05 for skin type IV). The levels of 6-4 photoproducts were <1/10 of those of CPDs (Table IGo). Even 6-4 photoproducts were at higher levels in DNA of skin type I compared with skin type IV but due to large variation, the differences were not significant. No measurable UV-damage was observed on control samples from any of the volunteers, taken on unirradiated sites, adjacent to the exposed sites (Table IGo).


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Table I. Comparison of photoproduct levels (mean ± SD, per 106 nucleotides) at different time points after SSR between skin types I and IV
 
In the skin type I group, 2.4% of TT=T remained after 3 weeks of repair, while in the skin type IV group 8.9% remained (P = 0.05, two sided Wilcoxon test, Table IGo). All TT=C was repaired by 3 weeks. Most 6-4 photoproducts were repaired by 48 h. To boost the number of subjects for the kinetic part of the study, we included seven additional subjects of skin type I into Table IGo. However, these persons were older that those in the skin type comparison, and should not be included in that part of the study. Even in this group TT=T remained after 3 weeks, and the level was 9% from the initial level (P = 0.05 from the other skin type I group).

Before irradiation, no CPDs were present in the control skin biopsy as shown in the HPLC radiogram (Figure 1Go, control). The retention times were about 15.5 min for TT=C and 22.0 min for TT=T (as indicated by the arrows). At 0 h, large peaks were detected for both TT=C and TT=T (Figure 1Go, 0 h). After 48 h, the bulk of TT=C and TT=T was repaired, shown by diminishing peaks. A clear TT=T peak remained at 3 weeks, whereas TT=C had disappeared (Figure 1Go, arrow in the last panel). We present here a chromatogram from one subject as evidence for the presence of TT=T after 3 weeks of UVR. Yet, 16/17 participants showed detectable levels of TT=T at 3 weeks. The mean repair kinetics among the subjects of different skin types are shown in Figure 2Go. The repair rates of TT=C were faster than those of TT=T; repair of TT=T appeared to be slower for individuals with skin type IV compared with type I. The difference between repair rates for TT=T and TT=C, measured at 3 weeks was highly significant (P < 0.01, Table IGo).



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Fig. 1. Profiles of cyclobutane pyrimidine dimers (CPDs) in DNA from human skin biopsies in high performance liquid chromatography (HPLC). Arrows show the two kinds of CPDs (TT=C, 15.5 min and TT=T, 22 min) determined in the present study. Control, profile in unirriadated skin biopsy; 0 h, skin biopsy collected immediately after 40 mJ/cm2 of SSR; 48 h and 3 weeks are the skin biopsies collected after 48 h and 3 weeks post-SSR. All four HPLC chromatograms were from one subject (skin type I).

 


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Fig. 2. Repair rates of cyclobutane dimers (CPDs) TT=C and TT=T in skin type I and skin type IV. The levels of CPD at 0 h were normalized to 100%. The percentage of unrepaired CPD at 48 h and 3 weeks was plotted for TT=C (top panel) and TT=T (bottom panel). The bars are standard deviation of five subjects at each time point (48 h and 3 weeks).

 
In order to confirm that the fraction identified as TT=T at 3 weeks was indeed the authentic product; we carried out a reversion test using a high dose (10 kJ/m2) of UVC. This converts the dimer back to an unmodified TT. The reversion experiment with a synthetic standard TT=T is shown in Figure 3A and BGo in which TTT is recovered as a late eluting fraction. A sample from human DNA, 3 weeks after UV-exposure, is shown in Figure 3CGo. After reversion, the isolated human fraction is recovered as TTT (Figure 3DGo), demonstrating that a true photoproduct was identified.



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Fig. 3. Identification of TT=T cyclobutane dimer from human skin. A [32P]HPLC chromatogram of standard TT=T prepared as in (5) (A). The result of a reversion test on the isolated fraction of TT=T fraction from (A), irradiated with a dose of 10 kJ/m2 of UVC; TTT indicates the position of the reverted unmodified TTT trimer (B). An HPLC chromatogram of DNA from a skin biopsy taken at 3 weeks after SSR; TT=T is indicated with an arrow (C). The result of UVC irradiation of the isolated human TT=T fraction from (C), irradiated with a dose of 10 kJ/m2; TTT indicates the position of the reverted TTT (D).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 32P-postlabelling method used in the present study measures dimers as trinucleotide, e.g. as TT=C, TT=T, TT-C and TT-T, because cross-linked nucleotides are poor substrates for polynucleotide kinase that carries out the labeling step (9). Unmodified 5'-nucleotides label well and we use the above synthetic trinucleotides as external standards in the assay. The 5'-unmodified nucleotide can be any one of the four nucleotides (A, C, G, T) but we have selected the same nucleotide (T) for both T=C and T=T to minimize the flanking nucleotide effects (22). We have used the method for 6-4 photoproducts as well but their initial levels are less and repair rates are faster compared with CPDs, making a simultaneous assay of dimers and 6-4 photoproducts unfeasible in a long-term study (10). An important question is the reliability of the method, particularly because the reported interindividual variability has been large (11–13). We have several pieces of evidence on the high methodic reproducibility and accuracy. In repeated analyses of the same samples the variation is <30% (23). Two samples taken from the same individuals at the same time or at different times agree with a correlation coefficient of 88% and ~70%, respectively (24). Urinary excretion of thymidine dimers, measured with the postlabelling method, correlated closely with the dose of monitored sun exposure (25). When we have compared the postlabelling method with gas chromatography-mass spectrometry analysis in a coded analysis of in vivo ethylene oxide adducts, the correlation was 95% and the absolute levels agreed (26). To our knowledge, none of the available antibodies against UV-damage have been validated with an independent quantitative technique. Validation results with some other DNA adduct antibodies have been discouraging (27).

The results were unambiguous on the two questions we set out to answer, questions that have not been addressed in vivo in humans before with damage-specific quantitative methods. Firstly, skin of 16/17 subjects contained T=T dimers even 3 weeks after UVR, which is in agreement with our recent work on melanoma patients and controls (28). In work based on antibodies, antibody-positive cells reached background level in 10 days (29). In the present study, the authentic nature of the products was confirmed by a reversion assay, in which human TT=T was recovered as TTT after a high dose of UVC. The level TT=T in human skin was 8.9% of the initial level for subjects with skin type IV and 2.4% for age-matched group of skin type I, the difference being of borderline statistical significance. The older group of skin type I individuals had 9.0% of TT=T remaining after 3 weeks. It is unlikely that there is a true difference in the rate of DNA repair between subjects of different skin types, or the two groups of skin type I, because for the T=C no difference was found between skin types I and IV, nor have we found such a difference earlier when repair has been followed up to 48 h (11). It may be pointed out that the amount of T=T dimers remaining after 3 weeks is a minimal estimate because we cannot control for the dilution of dimers because of cell replication and shedding of dead cells from epidermis, i.e. the rates of repair are overestimated. The repair of 6-4 photoproducts is considerably faster than that of CPDs, and only traces of 6-4 photoproducts remained even at 48 h (10). The differential repair rates for dimers and 6-4 photoproducts, are strong evidence that repair in living cells has been measured. If most of the photodamage originated from dead cells, the rate of removal of any damage would be expected to be uniform. Moreover, DNA purification involves ethanol precipitation, which would remove oligonucleotides.

The results show that the NER system in humans consists of many functional components which was already observed when kinetics were followed up to 48 h (10). The first 50% of dimers are removed within 15–50 h but the remaining component is slow and is likely to extend far beyond 3 weeks for T=T dimers. Based on work with cultured cells, the fast early components could be, at least in part, due to transcription coupled repair and the slow late components to global genome repair (1). For the fixation of mutations, long-lasting damage may be more deleterious than transient damage. Because transcription coupled repair is targeted towards the transcribed strand, the slowly repaired untranscribed stand may be particularly vulnerable to mutagenesis. Consistently, nearly all UV-`signature' mutations in the p53 gene from skin cancer in xeroderma pigmentosum patients are located on the non-transcribed stand (30). The faster repair of TT=C as compared with TT=T in situ in human skin has been a consistent finding also in our earlier studies (10–13) and is yet another aspect of heterogeneity in the NER system (1). Apparently the NER complex is very sensitive to subtle conformational differences causing DNA distortions (21). Another explanation may be that many highly repetitive satellite DNA sequences are AT-rich with more TT than TC nucleotide pairs (31); such satellites are often non-coding and their repair may be slow (1).

The second question related to the level of DNA damage depending on skin type, which was here determined, both anamnestically and by phototesting. Skin type is determined by erythemal response and it is unclear whether skin reddening and DNA damage correlate (21,32,33). The earlier studies suggest more damage in fair skinned individuals but the results have remained inconclusive due to uncertainties in skin type assignment or population size (12,13,34). However, constitutional pigmentation is a clear protective factor against UV-damage in skin (17). The present results are clear on this point: individuals with skin type I are close to twice as sensitive to UV-damage compared with those of skin type IV. These data agree with epidemiological findings on skin types and risks of skin cancer and melanoma (15,16).

The present study demonstrates the applicability of the recently developed assay for UV-damage in studies on important variables in susceptibility to photocarcinogenesis. The relatively high levels of damage inflicted in DNA of individuals with skin type I are likely to be one mechanistic explanation for sensitivity to skin cancer. The slow rate of repair of T=T dimers may be related to the probability of mutations in skin cells, assuming that the number of mutational events relates to the product of time and number of lesions. After these first results a number of new questions arise concerning the interindividual variation in this slow repair and its possible links to skin cancer and melanoma susceptibility.


    Notes
 
3 To whom correspondence should be addressed at: Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden
Email: kari.hemminki{at}cnt.ki.se Back


    Acknowledgments
 
This study was supported by the Swedish Radiation Protection Institute and the Swedish Cancer Society.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 24, 2001; revised December 7, 2001; accepted December 10, 2001.





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