In Situ Repair of Cyclobutane Pyrimidine Dimers and 6–4 Photoproducts in Human Skin Exposed to Solar Simulating Radiation
1999; Elsevier BV; Volume: 112; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1999.00523.x
ISSN1523-1747
AutoresVladimir J.N. Bykov, Kari Hemminki, John M. Sheehan, Antony R. Young,
Tópico(s)Science, Research, and Medicine
ResumoDNA repair is crucial to the integrity of the human genome. The ultraviolet radiation portion of solar radiation is responsible for the rising incidence of skin cancer, one of the most common types of cancer in humans. We applied a recently developed 32P-post-labeling technique to measure the in situ DNA repair efficiency of solar-simulated radiation induced cyclobutane pyrimidine dimers and 6–4 photoproducts in the skin of nine healthy volunteers with skin type II. Our results show about 6-fold interindividual variations in the level of DNA damage after exposure to an equal biologic dose – 2 minimal erythema doses. The kinetics of DNA repair indicated a base sequence dependence of the repair process. The DNA repair efficiency showed a 20-fold difference in volunteers. An age-related decrease of DNA repair capacity was observed; however, the data are limited due to a small number of subjects and a narrow age range. The variable response in DNA damage levels and individual differences in DNA repair efficiency suggest a susceptible subgroup of people probably with a higher skin cancer risk. DNA repair is crucial to the integrity of the human genome. The ultraviolet radiation portion of solar radiation is responsible for the rising incidence of skin cancer, one of the most common types of cancer in humans. We applied a recently developed 32P-post-labeling technique to measure the in situ DNA repair efficiency of solar-simulated radiation induced cyclobutane pyrimidine dimers and 6–4 photoproducts in the skin of nine healthy volunteers with skin type II. Our results show about 6-fold interindividual variations in the level of DNA damage after exposure to an equal biologic dose – 2 minimal erythema doses. The kinetics of DNA repair indicated a base sequence dependence of the repair process. The DNA repair efficiency showed a 20-fold difference in volunteers. An age-related decrease of DNA repair capacity was observed; however, the data are limited due to a small number of subjects and a narrow age range. The variable response in DNA damage levels and individual differences in DNA repair efficiency suggest a susceptible subgroup of people probably with a higher skin cancer risk. cyclobutane pyrimidine dimers solar simulating radiation cyclobutane pyrimidine dimers 6–4-[pyrimidine-2′-one] pyrimidine photoproducts Dewar isomer Epidemiologic and experimental studies show that solar ultraviolet radiation (UVR) is a major cause of human skin cancer (Vitasa et al., 1990Vitasa B.C. Taylor H.R. Strickland P.T. et al.Association of nonmelanoma skin cancer and actinic keratosis with cumulative solar ultraviolet exposure in Maryland watermen.Cancer. 1990; 65: 2811-2817Crossref PubMed Scopus (227) Google Scholar;IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 1992IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Solar and Ultraviolet Radiation. IARC, Lyon1992: 73-138Google Scholar;Kraemer et al., 1994Kraemer K.H. Lee M.-M. Andrews A.D. Lambert W.C. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer.Arch Dermatol. 1994; 130: 1018-1021Crossref PubMed Scopus (447) Google Scholar). UVR-specific dipyrimidine lesions such as cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts are likely to be important in the pathogenesis of nonmelanoma skin cancer as mutations, e.g., in the p53 gene, at dipyrimidine sites occur with high frequency in nonmelanoma skin tumors (Dumaz et al., 1993Dumaz N. Drougard C. Sarasin A. Daya-Grosjean L. Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients.Proc Natl Acad Sci USA. 1993; 90: 10529-10533Crossref PubMed Scopus (233) Google Scholar;Ziegler et al., 1993Ziegler A. Leffell D.J. Kunala S. et al.Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers.Proc Natl Acad Sci USA. 1993; 90: 4216-4220Crossref PubMed Scopus (642) Google Scholar). In addition, the inability to repair efficiently pyrimidine dimers and 6–4 photoproducts in xeroderma pigmentosum results in an incidence of skin cancer that is at least 1000 times greater than normal (Mitchell and Nairn, 1989Mitchell D.L. Nairn R.S. The biology of the (6–4) photoproduct.Photochem Photobiol. 1989; 49: 805-819Crossref PubMed Scopus (482) Google Scholar;Ananthaswamy and Pierceall, 1990Ananthaswamy H.N. Pierceall W.E. Molecular mechanisms of ultraviolet radiation carcinogenesis.Photochem Photobiol. 1990; 52: 1119-1136Crossref PubMed Scopus (322) Google Scholar;Kraemer, 1997Kraemer K.H. Sunlight and skin cancer: another link revealed.Proc Natl Acad Sci USA. 1997; 94: 11-14Crossref PubMed Scopus (329) Google Scholar). Some evidence from skin cancer in normal patients suggests that decreased DNA repair capacity may relate to susceptibility to skin cancer (Alcalay et al., 1990Alcalay J. Freeman S.E. Goldberg L.H. Wolf J.E. Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma.J Invest Dermatol. 1990; 95: 506-509Abstract Full Text PDF PubMed Google Scholar;Wei et al., 1993Wei Q. Matanoski G.M. Farmer E.R. Hedayati M.A. Grossman L. DNA repair and aging in basal cell carcinoma: a molecular epidemiology study.Proc Natl Acad Sci USA. 1993; 90: 1614-1618Crossref PubMed Scopus (367) Google Scholar,Wei et al., 1995Wei Q. Matanoski G.M. Farmer E.R. Hedayati M.A. Grossman L. DNA repair capacity for ultraviolet light-induced damage is reduced in peripheral lymphocytes from patients with basal cell carcinoma.J Invest Dermatol. 1995; 104: 933-936Abstract Full Text PDF PubMed Scopus (67) Google Scholar). CPD have also been associated with UVR-induced immunoregulatory molecules such as tumor necrosis factor-α (Kibitel et al., 1998Kibitel J. Hejmadi V. Alas L. O’Connor A. Sutherland B.M. Yarosh D. UV-DNA damage in mouse and human cells induced the expression of tumor necrosis factor α.Photochem Photobiol. 1998; 67: 541-546Crossref PubMed Scopus (67) Google Scholar) and immunosuppression in mouse (Kripke et al., 1992Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.Proc Natl Acad Sci USA. 1992; 89: 7516-7520Crossref PubMed Scopus (441) Google Scholar). Such immunosuppression is known to play a major part in mouse photocarcinogenesis (Kripke, 1994Kripke M.L. Ultraviolet radiation and immunology: something new under the sun.Cancer Res. 1994; 54: 6102-6105PubMed Google Scholar) and is suspected of having a major role in human skin cancer (Streilein et al., 1994Streilein J.W. Taylor H.J.R. Vincek V. et al.Relationship between ultraviolet radiation-induced immunosuppression and carcinogenesis.J Invest Dermatol. 1994; 103: 107S-111SAbstract Full Text PDF PubMed Scopus (88) Google Scholar). Research into the repair of DNA lesions in human epidermis in situ has been impeded by the lack of sensitive and lesion-specific quantitative assays. Methods for the detection of UVR-induced DNA damage and/or repair include the measurement of DNA strand breaks resulting from endonuclease sensitive sites or DNA repair synthesis (Lambert et al., 1979Lambert B. Ringborg U. Skoog L. Age-related decrease of ultraviolet light-induced DNA repair synthesis in human peripheral leukocytes.Cancer Res. 1979; 39: 2792-2795PubMed Google Scholar;Freeman, 1988Freeman S.E. Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ.J Invest Dermatol. 1988; 90: 814-817Abstract Full Text PDF PubMed Google Scholar), the use of antibodies raised against UVR lesions (Strickland, 1985Strickland P.T. Immunoassay of DNA modified by ultraviolet radiation: a review.Environ Mutagenesis. 1985; 7: 599-607Crossref PubMed Scopus (9) Google Scholar;Freeman et al., 1987Freeman S.E. Gange R.W. Sutherland J.C. Sutherland B.M. Pyrimidine dimer formation in human skin.Photochem Photobiol. 1987; 46: 207-212Crossref PubMed Scopus (36) Google Scholar;Bruze et al., 1989Bruze M. Emmet E.A. Creasey J. Strickland P.T. Cyclobutane-dithymidine induction by solar-simulated UV radiation in human skin: II. Individual responses.J Invest Dermatol. 1989; 93: 341-344Crossref PubMed Google Scholar;Matsunaga et al., 1990Matsunaga T. Mori T. Nikaido O. Base sequence specificity of a monoclonal antibody binding to (6–4) photoproducts.Mutation Res. 1990; 235: 187-194Crossref PubMed Scopus (50) Google Scholar;Strickland et al., 1992Strickland P.T. Nikaido O. Matsunaga T. Boyle J.M. Further characterization of monoclonal antibody indicates specificity for (6–4) dipyrimidine photoproducts.Photochem Photobiol. 1992; 55: 723-727Crossref PubMed Scopus (10) Google Scholar;Clingen et al., 1995Clingen P.H. Arlett C.F. Roza L. Mori T. Nikaido O. Green M.H.L. Induction of cyclobutane pyrimidine dimers, pyrimidine (6–4) pyrimidone photoproducts and Dewar valence isomers by natural sunlight in normal human mononuclear cells.Cancer Res. 1995; 55: 2245-2248PubMed Google Scholar;Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten C.S. The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 1307-1313Crossref PubMed Scopus (129) Google Scholar) and the host-cell reactivation assay in isolated lymphocytes (Wei et al., 1993Wei Q. Matanoski G.M. Farmer E.R. Hedayati M.A. Grossman L. DNA repair and aging in basal cell carcinoma: a molecular epidemiology study.Proc Natl Acad Sci USA. 1993; 90: 1614-1618Crossref PubMed Scopus (367) Google Scholar;Grossman and Wei, 1995Grossman L. Wei Q. DNA repair capacity (DRC) as a biomarker of human variational responses to the environment.in: Voss J.-M.H. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Springer, New York1995: 329-347Google Scholar;Moriwaki et al., 1996Moriwaki S.I. Ray S. Tarone R.E. Kraemer K.H. Grossman L. The effect of donor age on the processing of UV-damaged DNA by cultured human cells: reduced DNA repair capacity and increased DNA mutability.Mutation Res. 1996; 364: 117-123Crossref PubMed Scopus (146) Google Scholar). Each of these techniques has drawbacks, e.g., the endonuclease sensitive sites can be applied only to the limited number of lesions and the antibody techniques are semiquantitative. In this study we have used a novel 32P-post labeling technique to follow the loss of dipyrimidine DNA lesions in human skin tissue (Bykov and Hemminki, 1995Bykov V.J. Hemminki K. UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection.Carcinogenesis. 1995; 16: 3015-3019Crossref PubMed Scopus (23) Google Scholar,Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA,B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar). The advantages of the 32P-post labeling method are high sensitivity, specificity, requirement of a low amount of DNA, and the ability to study different photoproducts in one sample. The combination of the 32P-post labeling with high performance liquid chromatography (HPLC) analysis provides a powerful tool in terms of sensitivity, resolution, reproducibility and quantitation for adduct determination (Bykov et al., 1998aBykov V.J. Lindgren C. Tobin D. Hemminki K. Sensitive 32P-HPLC technique shows base sequence dependent differences in photolesion repair in human keratinocytes.Chem–Biol Interactions. 1998; 110: 71-84Crossref PubMed Scopus (7) Google Scholar). Two sequence specific CPD and two sequence-specific 6–4 photoproducts were identified and measured in human skin biopsies after exposure to physiologically relevant doses of solar simulating radiation (SSR). We selected volunteers of skin type II (burn easily and tan poorly) as they are more prone to skin cancer than people who are more sun-tolerant and tan well such as skin types III and IV. The UVR source was SSR from an Oriel (Leatherhead, U.K.) Solar Simulator, the optical design of which gives a field of even irradiance (290–400 nm) of ≈10 mW per cm2 at the skin surface of which about 10% is UVB (290–320 nm). SSR doses were routinely monitored with a broad band thermopile radiometer (Medical Physics, Dryburn Hospital, Durham, U.K.) and calculated by making comparisons with spectroradiometric determinations (Bentham Instruments, Reading, U.K.) as described byYoung et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten C.S. The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 1307-1313Crossref PubMed Scopus (129) Google Scholar. The emission spectrum is shown in Figure 1 ofYoung et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten C.S. The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 1307-1313Crossref PubMed Scopus (129) Google Scholar. Convolution of the SSR spectrum with the CIE action spectrum for erythema (McKinley and Diffey, 1987McKinley A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.Cie J. 1987; 6: 17-22Google Scholar) shows that 92% of the erythema effective energy is UVB induced. The study was approved by the Ethics Committee of St Thomas’ Hospital, London, U.K. and all volunteers gave informed consent. Volunteers, details of which are in Table 1, were selected according to the following criteria. Inclusion: ages 20–34 with skin types II. Exclusion: pregnancy, a history of nude tanning whether by sunlight or sunbeds, any medication within 7 d prior to start of the study with the exception of oral contraception (volunteers D and I), any investigational drug within 28 d of the start study. The 24 h just perceptible minimal erythemal dose (MED) was assessed on previously unexposed buttock skin sites (1 cm × 1 cm) using a geometric series of six exposure doses with increments of √2. In all cases erythema was also assessed by a reflectance device (Dia-Stron, Andover, U.K.).Table 1Induction rate of TT=T cyclobutane dimers in human skin immediately after irradiationVolunteerSexAgeSkin typeMED, J per cm2TT=T per nucleotides per J per cm2AF25II48.1 × 10–7BM25II2.85.5 × 10–7CM33II2.86.1 × 10–7DF21II41.4 × 10–7EF28II2.82.5 × 10–7FF20II42.0 × 10–7GM24II45.4 × 10–7HF21II41.6 × 10–7IF34II2.82.0 × 10–7 Open table in a new tab Volunteers were exposed to 2 MED SSR on five previously unexposed buttock sites (1 cm × 1 cm) at different times so that 4 mm punch biopsies could be taken at the same time, under local anesthesia, at 0 h (within 15 min), 2 h, 6 h, 24 h, and 48 h post-irradiation. In addition, a control biopsy was taken from a non-SSR exposed site. Immediately after sampling, biopsies were frozen in liquid nitrogen and stored at –80°C. The epidermis was separated from dermis and DNA extraction was performed as described (Bykov et al., 1998aBykov V.J. Lindgren C. Tobin D. Hemminki K. Sensitive 32P-HPLC technique shows base sequence dependent differences in photolesion repair in human keratinocytes.Chem–Biol Interactions. 1998; 110: 71-84Crossref PubMed Scopus (7) Google Scholar). For each 32P-post labeling assay 2.5 μg DNA was digested as previously described in full byBykov and Hemminki, 1995Bykov V.J. Hemminki K. UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection.Carcinogenesis. 1995; 16: 3015-3019Crossref PubMed Scopus (23) Google Scholar. The samples were phosphorylated in a total volume of 2 μl containing 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 1.3 pmol [γ32P]ATP (3000 Ci per mmol) and 6 U T4 polynucleotide kinase. Incubation was performed at 37°C for 1 h. The HPLC assay was performed on a Beckman instrument operated with System Gold software and coupled to a radioisotope detector module 171. A Luna C18(2) column, manufactured by Phenomenex (2 × 250 mm, 5 μm particle size), connected to a Kromasil C18 precolumn, manufactured by Phenomenex (2 × 50 mm, 5 μm particle size) with a precolumn filter were used. A switching valve was introduced between the analytical column and precolumn (Mauthe et al., 1996Mauthe R.J. Marsch G.A. Turteltaub K.W. Improved high-performance liquid chromatography analysis of 32P-postlabeled 2-amino-1-methyl-6-phenylimidazo [4, 5–b] pyridine–DNA adducts using in–line precolumn purification.J Chromatogr B. 1996; 679: 91-101Crossref PubMed Scopus (7) Google Scholar). The Cool Pocket, manufactured by Keystone Scientific was used to maintain temperature of the analytical column at +40°C. The labeled products were diluted with water up to 10 μl and injected on to the HPLC. The initial separation of photoproducts from an excess of unreacted isotope was performed on the precolumn during 2.5 min. At this time point, employing the switching valve, the analytical column was connected to the precolumn. Separation of photoproducts was performed employing gradient elution with a buffer (0.5 M ammonium formate, 20 mM ortho-phosphoric acid, pH 4.6) that was mixed with methanol. The initial elution was isocratic with 100% buffer at 4 min a linear methanol gradient (0%–1.4%) was pumped for 14 min, followed by an increase to 8.5% methanol over 15 min. The column was washed for 10 min with methanol, followed by gradient to 100% water for 7 min and finally switched back to the initial buffer. The flow rate was 0.2 ml per min. The products were detected by a Beckman 32P radioisotope detector. For calculations of product yields the HPLC peaks areas were integrated with Beckman System Gold software. Radioactivity from the unirradiated control biopsy was subtracted as background. The results were corrected for recovery with external standards (Bykov et al., 1995Bykov V.J. Kumar R. Försti A. Hemminki K. Analysis of UV-induced photoproducts by 32P-postlabelling.Carcinogenesis. 1995; 16: 113-118Crossref PubMed Scopus (36) Google Scholar). According to our previous study the recovery of whole procedure is more than 70% for cyclobutane dimers (Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA,B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar). Each sample was analyzed two times. The obtained values represented mean ± 40% (mean ± SEM) in repeated analyses of the same samples. For characterization of photoproducts in human biopsies, photochemical behavior of the detected compounds was tested. HPLC fractions of assumed identity were collected. Half of the sample was reanalyzed by HPLC while the other half was subjected to physical tests by using UV treatment. The HPLC fractions coeluting with CPD were irradiated by 10 kJ per m2 UVC radiation. The HPLC fractions coeluted with 6–4 photoproducts were irradiated by 30 kJ per m2 UVB radiation. The group mean values were compared by using paired t-tests. Linear regression analysis was employed to study correlation between induction of different photoproducts and to study correlation between age and DNA repair efficiencies. The nonlinear regression method based on the Levenberg–Marquardt algorithm employing the Nelder model Equation 1 was used to study DNA repair efficiencies of CPD. Y=x+ab0+b1(x+a)+b2(x+a)2(eqn 1) where a, b0, b1, b2 are parameters;x-independent variable, Y-dependent variable. Fifty per cent repair time was determined from the regression plots as the time when photoproduct levels decreased from 100% to 50%. All statistical operations were performed by Microcal TM Origin software. The DNA digestion procedure released CPD and 6–4 photoproducts as trinucleotides with a nonmodified nucleotide at the 5′-end of the photoproduct, which guaranteed efficient labeling by polynucleotide kinase (Bykov and Hemminki, 1995Bykov V.J. Hemminki K. UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection.Carcinogenesis. 1995; 16: 3015-3019Crossref PubMed Scopus (23) Google Scholar,Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA,B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar). In HPLC analysis of human samples synthetic standards were used to identify the photoproducts as shown in Figure 1. The migration of two CPD TT=T and TT=C and two 6–4 photoproducts TT–T and TT–C is shown in Figure 1 from a biopsy obtained after an SSR dose of 2 MED. The HPLC fractions coeluting with the available standards were tested for photochemical reactivity as previously reported (Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA,B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar). UVC irradiation is able to break the intradimer bond and release a nonmodified product. This reaction is specific for CPD allowing identification of these compounds (Setlow, 1966Setlow R.B. Cyclobutane-type pyrimidine dimers in polynucleotides.Science. 1966; 153: 379-386Crossref PubMed Scopus (217) Google Scholar). Figure 2 shows that UVC irradiation of the HPLC fraction comigrating with TT=T Figure 2a released a nonmodified trinucleotide TTT Figure 2b. Similar results were obtained for the HPLC fraction comigrating with TT=C. TT=C, however, will photochemically revert most likely to TTU due to deamination process, and the employed HPLC method was not designed to separate this from TTC. 6–4 photoproducts undergo isomerization to Dewar photoproducts with high doses of UVB irradiation (Taylor and Cohrs, 1987Taylor J.-S. Cohrs M.P. DNA, light and Dewar pyrimidinones: The structure and biological significance of TpT3.J Am Chem Soc. 1987; 109: 2834-2835Crossref Scopus (148) Google Scholar;Mitchell and Nairn, 1989Mitchell D.L. Nairn R.S. The biology of the (6–4) photoproduct.Photochem Photobiol. 1989; 49: 805-819Crossref PubMed Scopus (482) Google Scholar). UVB irradiation converted the HPLC fraction comigrating with TT–T Figure 3a into its corresponding Dewar isomer TT*T Figure 3b, c. The incomplete conversion to a Dewar isomer suggests that the peak corresponding to TT–T may additionally contain some other photoproducts. Similar conversion procedure was used to identify TT–C.Figure 3Identification of TT–T 6–4 photoproduct in human skin.32P-HPLC chromatogram of purified TT–T fraction from SSR-irradiated skin biopsy (a). The result of UVB irradiation of TT–T fraction at a dose of 30 kJ per m2, the release of Dewar isomer indicated by TT*T (b). Chromatogram of the TT*T Dewar isomer (c).View Large Image Figure ViewerDownload (PPT) Induction of the four different photoproducts correlated highly with each other (r = 0.92–0.99). The volunteers received a dose of 2 MED, resulting in average adduct levels: TT=C, 3.9 ± 0.9 photoproducts per 106 nucleotides; TT=T, 2.6 ± 0.6 photoproducts per 106 nucleotides; TT–T, 0.5 ± 0.1 photoproducts per 106 nucleotides; TT–C, 0.7 ± 0.2 photoproducts per 106 nucleotides (mean ± SEM, n = 9). The abundance of photoproducts in the volunteers followed the order: TT=C > TT=T > TT–C > TT–T. The difference in the induction rates between TT=C and TT=T was small but statistically significant according to a paired t-test (p = 0.002); the difference between TT–C and TT–T was not significant (p > 0.1). Levels of TT=T CPD in biopsies from nine individuals per unit physical dose are shown in Table 1. Notably, there was an almost 6-fold difference between the highest and lowest adduct level between the individuals. The volunteers received two physical doses, 5.6 J per cm2 and 8.0 J per cm2 (1:1.4). The respective means of TT=C photoproduct levels at these doses were 3.1 ± 0.8 and 4.4 ± 1.5 per 106 (1:1.4) nucleotides, show evidence for a direct dose relationship. DNA repair kinetics of the two CPD are shown in Figure 4 as average values for nine individuals. Similarly, DNA repair kinetics of two 6–4 photoproducts are shown in Figure 5. For CPD there appeared to be an initial slow phase, followed by a rapid repair phase. For 6–4 photoproducts no lag phase was noted and repair was fast. These data were used to calculate time required for 50% repair of damage. TT=C was 50% repaired in 14.9 ± 3.5 h; TT=T–, 17.2 ± 5.1 h; TT–T–, 7 ± 2.3 h; TT–C–, 5.2 ± 1.9 h (mean ± SEM, n = 9) Figure 6. The difference in 50% DNA repair efficiency between CPD, TT=C, and TT=T, was not statistically significant according to a paired t-test (p > 0.1). The difference in 50% DNA repair efficiency between 6 and 4 photoproducts, TT–T and TT–C, was statistically significant according to a paired t-test at the p = 0.1 level (p = 0.069) with TT–C being faster.Figure 56–4 photoproducts repair in human skin is rapid with ≥50% complete within 6 h. There is an indication that TT–C repairs faster than TT–T; however, difference has reached statistical significance only at p < 0.1 level. Mean ± SEM (n = 9).View Large Image Figure ViewerDownload (PPT)Figure 6Average data on DNA repair efficiency in humans. Volunteers were exposed to 2 MED SSR. The values are the means of 50% removal of photoproducts, mean ± SEM (n = 9). According to paired t-test, the difference in DNA repair efficiency between CPD was not statistically significant (p > 0.1) and between 6 and 4 photoproducts it reached significance at p = 0.1 level (p = 0.069).View Large Image Figure ViewerDownload (PPT) The individual DNA repair efficiencies are shown in Figure 7. The time for 50% removal of photoproducts ranged substantially among individuals: TT=C, 2.8–39.5 h; TT=T, 2.1–47.1 h; TT–T, 1.7–18.3 h; TT–C, 1.5–16.9 h. After 48 h, 24% ± 5% of TT=C and 41% ± 8.8% of TT=T (mean ± SEM, n = 9) remained in skin, and there was a direct relationship between individuals with slow repair and the levels of photoproducts remaining at 48 h. The percentage of CPD persisting in skin after 48 h was spanning a range: 3.3%–41.8% for TT=C and 6.9%–47.5% for TT=T. The difference in the repair efficiency between TT=C and TT=T at 48 h was statistically significant according to a paired t-test at the p = 0.01 level, with TT=C being repaired faster. The efficiency of repair of CPD correlated negatively with ages of the six female volunteers. The correlation coefficients were –0.73 and –0.64 for TT=C and TT=T CPD. The regression analysis was not performed for male volunteers because only three men were included. In this group of volunteers men repaired DNA faster than women. The average time for 50% removal of photoproducts in the female group was 18.8 ± 4.3 h for TT=C, 21.9 ± 6.6 h for TT=T, 8.9 ± 3.2 h for TT–T, and 7.0 ± 2.6 h for TT–C (mean ± SEM, n = 6). The average time for 50% removal of photoproducts in the male group was 7.1 ± 3.4 h for TT=C, 7.7 ± 4.9 h for TT=T, 3.2 ± 1.5 h for TT–T, and 1.7 ± 0.2 h for TT–C (mean ± SEM, n = 3). The advantages of our 32P-post labeling HPLC technique over previous methods (Freeman, 1988Freeman S.E. Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ.J Invest Dermatol. 1988; 90: 814-817Abstract Full Text PDF PubMed Google Scholar;Bruze et al., 1989Bruze M. Emmet E.A. Creasey J. Strickland P.T. Cyclobutane-dithymidine induction by solar-simulated UV radiation in human skin: II. Individual responses.J Invest Dermatol. 1989; 93: 341-344Crossref PubMed Google Scholar;Alcalay et al., 1990Alcalay J. Freeman S.E. Goldberg L.H. Wolf J.E. Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma.J Invest Dermatol. 1990; 95: 506-509Abstract Full Text PDF PubMed Google Scholar;Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten C.S. The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 1307-1313Crossref PubMed Scopus (129) Google Scholar) include high sensitivity, requirement of a small amount of DNA, and ability to quantitate a variety of dipyrimidine photoproducts in one sample. Furthermore, there is the ability to identify the adducts separated by HPLC, as shown in the present work on the human skin for CPD and 6–4 photoproducts. The main disadvantage of this technique is that it requires the disruption of the epidermis so it is not possible to locate DNA photodamage within the epidermis. Recent studies, however, have shown that UVB readily induces CPD in the basal layer (Young et al., 1998aYoung A.R. Chadwick C.A. Harrison G.I. Nikaido O. Ramsden J. Potten C.S. The similarity of action spectra for thymine dimers in human epidermis and erythema suggest that DNA is the chromophore for erythema.J Invest Dermatol. 1998; 111: 982-988Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) including melanocytes (Young et al., 1998bYoung A.R. Potten C.S. Nikaido O. Parsons P.G. Boenders J. Ramsden J.M. Chadwick C.A. Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers.J Invest Dermatol. 1998; 111: 936-940Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The expression of DNA photodamage per nucleotide per J per cm2 may not reflect total DNA damage or DNA damage per cell because of possible variations due to physical factors such as epidermal thickness. Recent studies, however, have shown that the formation of thymine dimers is epidermal layer independent at wavelengths ≥ 300 nm and th
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