Induction and Excretion of Ultraviolet-Induced 8-Oxo-2′-deoxyguanosine and Thymine Dimers In Vivo: Implications for PUVA
2001; Elsevier BV; Volume: 116; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2001.01251.x
ISSN1523-1747
AutoresMarcus S. Cooke, Mark D. Evans, Kayuri Patel, Angela Barnard, Joseph Lunec, Robert M. Burd, P.E. Hutchinson,
Tópico(s)Skin Protection and Aging
ResumoMolecular epidemiology has linked ultraviolet-induced DNA damage with mutagenesis and skin carcinogenesis. Ultraviolet radiation may damage DNA in one of two ways: either directly, leading to lesions such as cyclobutane thymine dimers (T T), or indirectly, via photosensitizers that generate free radical species that may ultimately produce such oxidative lesions as 8-oxo-2′-deoxyguanosine. We report the results of a pilot, case control study in which seven, healthy, human volunteers (skin type II; aged 23–56 y; three male, four female) received a suberythemal dose of whole body irradiation from ultraviolet-A-emitting fluorescent tubes used in psoralen plus ultraviolet A therapy. First void, mid-stream urine samples were collected pre-exposure and daily postexposure, for up to 13 d. Analysis of urinary 8-oxo-2′-deoxyguanosine and cyclobutane thymine dimers was by competitive enzyme-linked immunosorbent assay (interassay coefficient of variation ≤ 10%) and compared with a matched, control group of unirradiated individuals. A maximal increase in levels of urinary 8-oxo-2′-deoxyguanosine was seen 4 d post-ultraviolet exposure. A subsequent reduction was noted, before finally returning to baseline. Similarly, cyclobutane thymine dimer levels peaked 3 d postexposure, before returning to baseline. In contrast to the 8-oxo-2′-deoxyguanosine analysis, however, a second peak was noted at days 9–11, before again returning to baseline. This is the first report examining urinary 8-oxo-2′-deoxyguanosine and cyclobutane thymine dimers following ultraviolet exposure of healthy human subjects. This work illustrates the induction and time course for excretion of ultraviolet-induced lesions, perhaps alluding to repair and ultimately offering the potential to define psoralen plus ultraviolet A dosage regimes in terms of minimizing DNA damage and hence cancer risk. Molecular epidemiology has linked ultraviolet-induced DNA damage with mutagenesis and skin carcinogenesis. Ultraviolet radiation may damage DNA in one of two ways: either directly, leading to lesions such as cyclobutane thymine dimers (T T), or indirectly, via photosensitizers that generate free radical species that may ultimately produce such oxidative lesions as 8-oxo-2′-deoxyguanosine. We report the results of a pilot, case control study in which seven, healthy, human volunteers (skin type II; aged 23–56 y; three male, four female) received a suberythemal dose of whole body irradiation from ultraviolet-A-emitting fluorescent tubes used in psoralen plus ultraviolet A therapy. First void, mid-stream urine samples were collected pre-exposure and daily postexposure, for up to 13 d. Analysis of urinary 8-oxo-2′-deoxyguanosine and cyclobutane thymine dimers was by competitive enzyme-linked immunosorbent assay (interassay coefficient of variation ≤ 10%) and compared with a matched, control group of unirradiated individuals. A maximal increase in levels of urinary 8-oxo-2′-deoxyguanosine was seen 4 d post-ultraviolet exposure. A subsequent reduction was noted, before finally returning to baseline. Similarly, cyclobutane thymine dimer levels peaked 3 d postexposure, before returning to baseline. In contrast to the 8-oxo-2′-deoxyguanosine analysis, however, a second peak was noted at days 9–11, before again returning to baseline. This is the first report examining urinary 8-oxo-2′-deoxyguanosine and cyclobutane thymine dimers following ultraviolet exposure of healthy human subjects. This work illustrates the induction and time course for excretion of ultraviolet-induced lesions, perhaps alluding to repair and ultimately offering the potential to define psoralen plus ultraviolet A dosage regimes in terms of minimizing DNA damage and hence cancer risk. Commission Internationale de l'Éclairage 8-oxo-2′-deoxyguanosine nucleotide excision repair cyclobutane thymine dimers transcription-coupled repair It is primarily the ultraviolet (UV) region of the solar spectrum that is implicated in the induction of skin cancer (Farr et al., 1986Farr P.M. Friedmann P.S. Solar radiation-induced disorders.in: Thody A.J. Friedmann P.S. Scientific Basis of Dermatology. Churchill Livingstone, London1986: 262-289Google Scholar). DNA is a primary target for UV radiation and may be damaged either by direct absorption of UV, generating such lesions as cyclobutane thymine dimers (T T) (Cadet et al., 1990Cadet J. Vigny P. Photochemistry of nucleic acids.in: Morrison H. Bioorganic Photochemistry. Wiley, New York1990: 1-272Google Scholar), or indirectly, via reactive intermediates, to produce such oxidative lesions as 8-oxo-2′-deoxyguanosine (8-oxodG) (Rosen et al., 1996Rosen J.E. Prahalad A.K. Williams G.M. 8-oxodeoxyguanosine formation in the DNA of cultured cells after exposure to H2O2 alone or with UVB or UVA radiation.Photochem Photobiol. 1996; 64: 117-122Crossref PubMed Scopus (68) Google Scholar;Zhang et al., 1997Zhang X. Rosenstein B.S. Wang Y. et al.Induction of 8-oxo-7,8-dihydro-2′-deoxyguaosine by ultraviolet radiation in calf thymus DNA and HeLa cells.Photochem Photobiol. 1997; 65: 119-124Crossref PubMed Scopus (106) Google Scholar), although many other lesions can also occur (Dizdaroglu, 1991Dizdaroglu M. Chemical determination of free radical-induced damage to DNA.Free Rad Biol Med. 1991; 10: 225-242Crossref PubMed Scopus (459) Google Scholar). Both T T and 8-oxodG are mutagenic (Kuchino et al., 1987Kuchino Y. Mori F. Kasai H. et al.Misreading of DNA templates containing, 8-hydroxydeoxyguanosine at the modified base and at adjacent residues.Nature. 1987; 349: 77-79Crossref Scopus (713) Google Scholar;Tornaletti and Pfeifer, 1994Tornaletti S. Pfeifer G.P. Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer.Science. 1994; 263: 1436-1440Crossref PubMed Scopus (278) Google Scholar) and hence potentially carcinogenic (Alcalay et al., 1990Alcalay J. Freeman S.E. Goldberg L.H. et al.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;Guyton and Kensler, 1993Guyton K.Z. Kensler T.W. Oxidative mechanisms in carcinogenesis.Br Med Bull. 1993; 49: 523-544Crossref PubMed Scopus (427) Google Scholar). The degree of involvement of either oxidative or nonoxidative damage in UV-induced skin cancer, however, is not clear. A role for such damage is supported by molecular epidemiology with skin cancers from sun-exposed sites frequently showing C→T and CC→TT transitions (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1636) Google Scholar), potentially derived from either mechanism (Wang et al., 1988Wang D. Kreutzer D.A. Essigmann J.M. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions.Mutat Res. 1988; 400: 99-115Crossref Scopus (417) Google Scholar;Reid and Loeb, 1993Reid T.M. Loeb L.A. Tandem double CC → TT mutations are produced by reactive oxygen species.Proc Natl Acad Sci USA. 1993; 90: 3904-3907Crossref PubMed Scopus (134) Google Scholar;Nataraj et al., 1995Nataraj A.J. Trent J.C. Anathaswamy H.N. p53 mutations and photocarcinogenesis.Photochem Photobiol. 1995; 62: 218-230Crossref PubMed Scopus (138) Google Scholar). The treatment of psoriasis with psoralens in conjunction with UVA (PUVA) is associated with a long-term, increased risk of cutaneous malignancy, such as squamous cell carcinoma (SCC), in part related to cumulative UVA exposure (Henseler et al., 1987Henseler T. Christophers E. Hönigsmann H. et al.Skin tumours in the European PUVA study.J Am Acad Dermatol. 1987; 16: 108-116Abstract Full Text PDF PubMed Scopus (118) Google Scholar;Stern and Lange, 1988Stern R.S. Lange R. Members of the Photochemotherapy Follow-up Study: Non-melanoma skin cancer occurring in patients treated with PUVA five to ten years after first treatment.J Invest Dermatol. 1988; 91: 120-124Abstract Full Text PDF PubMed Google Scholar;Chuang et al., 1992Chuang T.-Y. Heinrich L.A. Schultz M.D. et al.PUVA and skin cancer.J Am Acad Dermatol. 1992; 26: 173-177Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Whereas in vitro studies have shown that mutations induced by PUVA are of the form T→A, located at 5′TpA sites (Sage and Bredberg, 1991Sage E. Bredberg A. Damage distribution and mutation spectrum: the case of 8-methoxypsoralen and UVA in mammalian cells.Mutat Res. 1991; 132: 1257-1259Google Scholar), several studies examining mutations in SCC from PUVA patients report a significant level of the characteristic C→T and CC→TT transitions (Nataraj et al., 1997Nataraj A.J. Wolf P. Cerroni L. et al.p53 mutation in squamous cell carcinomas from psoriasis patients treated with psoralen + UVA (PUVA).J Invest Dermatol. 1997; 109: 238-243Crossref PubMed Scopus (62) Google Scholar;Wang et al., 1997Wang X.M. McNiff J.M. Klump V. et al.An unexpected spectrum of p53 mutations from squamous cell carcinomas in psoriasis patients treated with PUVA.Photochem Photobiol. 1997; 66: 294-299Crossref PubMed Scopus (34) Google Scholar). This would appear to further implicate an exclusive UV mechanism, rather than involvement of psoralens, in SCC formation, although the data are still contentious (Peritz and Gasparro, 1999Peritz A.E. Gasparro F.P. Psoriasis. PUVA and skin cancer – molecular epidemiology: the curious question of T→A transversions.J Invest Dermatol. 1999; 4: 11-16Abstract Full Text PDF Scopus (19) Google Scholar). Dosimetry of PUVA is currently based on considerations of skin type, the induction of erythema, and total UV dosage. The most important variable, however, is likely to be DNA damage and it is not known what impact current practice has on this. We have developed noninvasive methods of estimating T T and 8-oxodG (Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), which potentially may facilitate biomonitoring of treatments such as PUVA, identify ''at risk'' individuals, and examine the mechanisms behind UV-induced mutagenesis/carcinogenesis. Although shown to correlate well with high performance liquid chromatography and electrochemical detection (HPLC-EC) for 8-oxodG quantitation (Evans et al., 2000Evans M.D. Cooke M.S. Akil M. et al.Aberrant processing of oxidative DNA damage in systemic lupus erythematosus.Biochem Biophys Res Comms. 2000; 273: 894-898https://doi.org/10.1006/bbrc.2000.3078Crossref PubMed Scopus (55) Google Scholar; Ochi et al. submitted 1Ochi H, Yoshikawa T, Cutler R, Takeuchi M, Ramarathnam N: Development of a monoclonal antibody ELISA for the quantification of 8-hydroxy-2′-deoxyguanosine, submitted.1Ochi H, Yoshikawa T, Cutler R, Takeuchi M, Ramarathnam N: Development of a monoclonal antibody ELISA for the quantification of 8-hydroxy-2′-deoxyguanosine, submitted.), immunochemical approaches to the detection of urinary DNA lesions offer significant benefits over HPLC-EC and gas chromatography-mass spectrometry (GC-MS), which require extensive prepurification prior to analysis and are not readily amenable to routine clinical analysis. Furthermore, current HPLC-EC (Degan et al., 1991Degan P. Shigenaga M.K. Park E.-M. et al.Immunoaffinity isolation of urinary, 8-hydroxy-2′-deoxyguanosine and, 8-hydroxyguanine and quantitation of, 8-hydroxy-2′-deoxyguanosine in, DNA, by polyclonal antibodies.Carcinogenesis. 1991; 12: 865-871Crossref PubMed Scopus (129) Google Scholar;Loft et al., 1993Loft S. Fischer-Nielsen A. Jeding I.B. et al.8-Hydroxydeoxyguanosine as a urinary biomarker of oxidatve DNA damage.J Tox Envir Health. 1993; 40: 391-404Crossref PubMed Scopus (202) Google Scholar) and GC-MS (Ravanat et al., 1999Ravanat J.-L. Guicherd P. Tuce Z. et al.Simultaneous determination of five oxidative DNA lesions in human urine.Chem Res Toxicol. 1999; 12: 802-808Crossref PubMed Scopus (99) Google Scholar) methods for the detection of 8-oxodG are limited to recognizing the lesion solely as the deoxynucleoside, not in a single-stranded oligomeric form reported to derive from nucleotide excision repair (NER) of DNA and accounting for the higher 8-oxodG levels seen with enzyme-linked immunosorbent assay (ELISA) (Cooke et al., 2000aCooke M.S. Evans M.D. Herbert K.E. et al.Urinary 8-oxo-2′-deoxyguanosine – source, significance and supplements.Free Rad Res. 2000; 32: 381-397Crossref PubMed Scopus (181) Google Scholar). Indeed, ELISA-based measurement of urinary 8-oxodG is a well-established procedure with basal levels in healthy, human subjects showing consensus between studies (Cooke et al., 2000aCooke M.S. Evans M.D. Herbert K.E. et al.Urinary 8-oxo-2′-deoxyguanosine – source, significance and supplements.Free Rad Res. 2000; 32: 381-397Crossref PubMed Scopus (181) Google Scholar). Such chromatographic techniques and GC-MS in particular, however, offer absolute identification of lesions, in addition to quantitation. At present, there are no reported HPLC-EC or GC-MS methods for the detection of urinary T T. Previously, we have examined levels of T T and 8-oxodG in the urine of psoriatic patients (Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and found T T levels, but not 8-oxodG, to be significantly elevated. The principal aim of this study was to establish lesion induction and the time course for removal and appearance in the urine following a single exposure to a therapeutic source of UV radiation. From a previous study (Tagesson et al., 1995Tagesson C. Kalleberg M. Klintenberg C. et al.Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients.Eur J Cancer. 1995; 31A: 934-940Abstract Full Text PDF PubMed Scopus (158) Google Scholar), we expected to observe an increase in urinary 8-oxodG, and potentially T T, within the first week following irradiation. We also aimed to validate the assay for urinary T T reported previously (Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and to conduct a preliminary examination of the potential sources of elevated urinary T T seen previously in psoriatic patients. Ethical approval was granted by the Leicestershire Area Health Authority Ethics Committee. Fourteen healthy, human volunteers (skin type II,Fitzpatrick, 1988Fitzpatrick T.B. The validity and practicality of sun reaction skin types I through VI.Arch Dermatol. 1988; 124: 869-871Crossref PubMed Scopus (2657) Google Scholar) were recruited following written, informed consent. The age of subjects, consisting of seven males and seven females, ranged from 23 to 56 y. The subjects were stratified block randomized and allocated into those to be exposed to UV and a matched, sham-irradiated, control group. They had not received significant sunlight exposure prior to the study and were asked to avoid exposure throughout the study. Indeed, the study was performed when ambient sunlight and seasonal exposure was minimal. The appropriate subjects were given whole body exposure to a dose of 15 J per cm2 CIE (Commission Internationale de l'Éclairage,McKinlay and Diffey, 1987McKinlay A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.CIE J. 1987; 6: 17-22Google Scholar) UV from UVA-emitting fluorescent tubes (Philips 100 W Cleo Professional) routinely used in PUVA therapy. This dose was suberythemal for all subjects. Spectroradiometric analysis of the tubes (Hospital Lamp Supplies, Leicester, U.K.) gave a range from 320 to 405 nm (λmax 350 nm) (Figure 1). A spike was noted at 313 nm, however, although this represented only 0.5% of the total lamp output. Daily, first void, mid-stream urines were collected from all subjects prior to and for 13 d following irradiation. Upon collection, samples were stored, without any additives, at -80°C prior to analysis. Following thawing, the samples were split into two batches, one of which was centrifuged (300 × g for 10 min). The samples were then analyzed by competitive ELISA for 8-oxodG (centrifuged samples), according to the manufacturer's protocol (JaICA, Fukuroi City, Japan) (Cooke et al., 1998Cooke M.S. Evans M.D. Podmore I.D. et al.Novel repair action of vitamin C upon in vivo oxidative DNA damage.FEBS Letts. 1998; 439: 363-367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), or T T (noncentrifuged samples;Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Briefly, the analysis of T T was performed using single-stranded UVC DNA as the solid phase antigen, bound to a 96-well Nunc Immuno Maxisorp, ELISA plate (Life Technologies, Paisley, Scotland) by incubation. Following blocking of free sites, the urine samples were applied to the plate, along with the antiserum specific for thymine dimers (Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), diluted 1:5000. Primary antiserum binding was quantified using a peroxidase-labeled goat antirabbit secondary antibody, in conjunction with orthophenylenediamine (0.5 mg per ml in 0.05 M phosphate citrate, pH 5.0, and containing 0.03% wt/vol sodium perborate) substrate solution. The reaction was stopped with 2 M H2SO4 and the resulting absorbance was read at 492 nm using a plate reader. The final data included correction for background values. In order to correct for variations in urine concentration, urinary creatinine was assessed by a procedure based upon the Jaffe reaction (Department of Chemical Pathology, Leicester Royal Infirmary NHS Trust, Leicester, U.K.;Henry, 1974Henry J.B. Clinical chemistry principles and techniques. 1974: 550Google Scholar) and results were expressed per unit creatinine. Summary statistics were based upon t test analysis of the area under the curves. Secondary analysis comprised general linear model analysis of variance with subsequent comparison between means using Fisher's least significant difference test and t test analysis between groups. Analyses were performed using Minitab version 12 and GraphPad Prism, version 2.01 (GraphPad Software, San Diego, CA). Validation of the competitive ELISA assay for urinary T T was performed. Analysis was repeated on a separate day but with the same samples, and the corrected urinary T T values were compared (r2 = 0.97, p < 0.0001). The intra-assay coefficient of variation was 5.63%, whereas inter-assay variability was shown to be 10.30%. Inter- and intra-assay variability of the 8-oxodG assay is <10%, as determined by the manufacturer (JaICA). The effect of centrifugation of urine prior to T T analysis was examined. It was shown that levels of T T in centrifuged samples showed a significant (p < 0.0001) correlation (r2 = 0.76), illustrating that although centrifugation of samples reduced total levels of lesion, it did so proportionally. The manufacturer of the 8-oxodG kit recommends that urines are centrifuged to remove particulates prior to analysis; such particulates did not interfere with the T T assay, enabling analysis of noncentrifuged urines and further limiting sample manipulation. In the absence of a calibration curve for the T T assay allowing absolute quantitation, results from both assays were expressed in terms of percentage change, corrected for creatinine, enabling direct comparison between lesions. No change in urinary creatinine levels was seen during the period of study, or between the treatment groups. Differences in urinary 8-oxodG and T T excretion between the UV-exposed and unexposed individuals were investigated by use of a summary statistic. Comparison using the unpaired t test of the areas under the curves for T T and 8-oxodG excretion against time (Figure 2, Figure 3) illustrated significant differences between mean levels of 8-oxodG and T T during the first 7 d of the study, in the exposed versus unexposed groups (p = 0.003 and p < 0.0001, CI = 0.0773%-0.1227% and 0.2942%-0.3172%, respectively). Analysis of area under the curve for days 8–14 revealed no significant difference between urinary 8-oxodG levels in exposed versus unexposed subjects (p = 0.2), although a significant difference was noted for the T T levels (p < 0.0001, CI = 0.2748%-0.3118%).Figure 3Competitive ELISA analysis of urinary T T. Healthy, human volunteers (n = 7) were exposed to a single suberythemal dose of UV. Daily first void urine samples were collected prior to exposure (day 0) and subsequently over a period of 13 d. Urine samples were also collected from an age- and sex-matched, unexposed control group (n = 7) over the same period of study. Values are corrected to represent percentage change from pre-exposure levels (day 0). Bars represent mean ± standard deviations. Asterisks indicate a significant (*p = 0.05) difference from pre-exposure values (day 0), determined by Fisher's least significant difference test, and a significant (**p < 0.001; ***p < 0.0001) difference between UV-exposed and unexposed volunteers, determined by t test. Both are the results of secondary statistical analysis.View Large Image Figure ViewerDownload (PPT) Upon inspection, an upward trend in levels of urinary 8-oxodG was noted, as measured by ELISA, reaching a maximum 4 d post-UVA exposure, compared to pre-exposure (day 0; Figure 2). Secondary statistical analysis of urinary 8-oxodG levels at day 4 were significantly (p < 0.0001) increased compared to levels in the unexposed group. Levels subsequently fell below baseline at 9 d postexposure, which was significant in terms of baseline (p = 0.05), but not in the unexposed group. Finally the levels returned to baseline. In contrast there was no significant variation in urinary 8-oxodG levels amongst the control (unirradiated) subjects, over the 14 d studied. Similarly, T T levels, as measured by ELISA, peaked 3 d following irradiation, deemed significant by secondary statistical analysis (p = 0.05, compared to pre-exposure; p < 0.0001 compared to unexposed group), before returning to baseline (Figure 3). In contrast with the 8-oxodG analysis, however, a second significant (p = 0.05) increase was noted at days 9–11, again highly significant compared to the unexposed, control group (p = 0.0002). Following this increase, levels again returned to baseline. Control, unirradiated subjects showed no significant variation in their urinary T T levels over the 14 d period. Urinary creatinine levels, used to correct for urine concentration, did not vary, in either group of subjects, during the period of study. This is the first report examining urinary lesion levels derived from both direct and indirect UV-induced mechanisms (T T and 8-oxodG, respectively) following UV exposure of healthy human subjects. In a previous report, the assay for urinary T T was shown to identify differences in levels of excretion between patients with psoriasis, atopic dermatitis, and healthy control individuals (Ahmad et al., 1999Ahmad J. Cooke M.S. Hussieni A. et al.Urinary thymine dimers and, 8-oxo-2′-deoxyguanosine in psoriasis.FEBS Letts. 1999; 460: 549-553Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We have further validated this assay in terms of inter- and intra-plate variability and the effects of sample preprocessing are reported. The validation results support the potential use of this assay within a clinical context and the ELISA format makes it amenable to the simultaneous analysis of a large number of samples. Currently, there is no form of biomonitoring for PUVA therapy, other than perhaps PUVA lentigines (Lever and Farr, 1994Lever L.R. Farr P.M. Skin cancers or premalignant lesions occur in half of high-dose PUVA patients.Br J Dermatol. 1994; 131: 215-219Crossref PubMed Scopus (67) Google Scholar). Given the increased risk of SCC development as a result of cumulative PUVA treatment, a better indicator of risk is clearly required. The factors that influence PUVA-related SCC and basal cell carcinoma development appear multifarious (recently reviewed byMurphy, 1999Murphy G.M. Skin cancer in patients with psoriasis – many intertwined risk factors.Br J Dermatol. 1999; 141: 1001-1003https://doi.org/10.1046/j.1365-2133.1999.03531.xCrossref PubMed Scopus (4) Google Scholar) and include the total UVA dosage, the age at which PUVA treatment began, and previous use of arsenicals (Bruynzeel et al., 1991Bruynzeel I. Bergman W. Hartevelt H.M. et al.'High single dose' European PUVA regimen also causes an excess of non-melanoma skin cancer.Br J Dermatol. 1991; 124: 49-55Crossref PubMed Scopus (89) Google Scholar). The ability of an individual to repair treatment-induced DNA lesions is likely to be a particularly important factor. Indeed, recent studies have supported this hypothesis, demonstrating lower repair capacity in psoriatic patients with skin cancer (Møller et al., 1998Møller P. Knudsen L.E. Frentz G. et al.Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis.Mutat Res. 1998; 407: 25-34Crossref PubMed Scopus (68) Google Scholar;Dybdahl et al., 1999Dybdahl M. Frentz G. Vogel U. et al.Low DNA repair is a risk factor in skin carcinogenesis: a study of basal cell carcinoma in psoriasis patients.Mutat Res. 1999; 433: 15-22Crossref PubMed Scopus (44) Google Scholar). Both studies required the isolation of peripheral blood mononuclear cells, an invasive procedure, and their subsequent in vitro analysis involving the comet assay, unscheduled DNA synthesis, or host cell reactivation assays, all of which are time-consuming and labor-intensive. In contrast, immunoassays represent a sensitive and specific approach to the study of DNA damage, with antibodies described that recognize 8-MOP- and 6,4,4′-trimethylangelicin-derived adducts (Gasparro and Santella, 1988Gasparro F.P. Santella R.M. Immunoassay of DNA damage.Photochem Photobiol. 1988; 48: 321-328Crossref PubMed Scopus (5) Google Scholar), 8-oxodG, and T T in various biologic systems (Cooke et al., 2000bCooke M.S. Mistry N.M. Ladapo A. et al.Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes.Free Rad Res. 2000; 33: 369-381Crossref PubMed Scopus (32) Google Scholar). There is precedent for examining urinary 8-oxodG, as a marker of oxidative stress, in various disease states, using HPLC-EC, GC-MS, ELISA (reviewed inCooke et al., 2000aCooke M.S. Evans M.D. Herbert K.E. et al.Urinary 8-oxo-2′-deoxyguanosine – source, significance and supplements.Free Rad Res. 2000; 32: 381-397Crossref PubMed Scopus (181) Google Scholar), and liquid chromatography with tandem mass spectrometry (Renner et al., 2000Renner T. Fechner T. Scherer G. Fast quantitation of the urinary marker of oxidatve stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection.J Chromatog B. 2000; 738: 311-317Crossref PubMed Scopus (75) Google Scholar), with the proven potential for such measurements to suggest the presence of repair defects (Evans et al., 2000Evans M.D. Cooke M.S. Akil M. et al.Aberrant processing of oxidative DNA damage in systemic lupus erythematosus.Biochem Biophys Res Comms. 2000; 273: 894-898https://doi.org/10.1006/bbrc.2000.3078Crossref PubMed Scopus (55) Google Scholar). A time course for postinsult lesion induction and appearance in the urine of healthy individuals has not been established previously. In this report we describe a significant increase in urinary 8-oxodG of healthy volunteers, 4 d after exposure to a suberythemal dose of UV. This timescale is consistent with another report in which cancer patients were exposed to therapeutic ionizing radiation and 8-oxodG excretion was monitored (Tagesson et al., 1995Tagesson C. Kalleberg M. Klintenberg C. et al.Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients.Eur J Cancer. 1995; 31A: 934-940Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Urinary 8-oxodG may be derived either from sanitization of the nucleotide pool or from the action of a specific endonuclease or NER (Cooke et al., 2000aCooke M.S. Evans M.D. Herbert K.E. et al.Urinary 8-oxo-2′-deoxyguanosine – source, significance and supplements.Free Rad Res. 2000; 32: 381-397Crossref PubMed Scopus (181) Google Scholar). It has also been argued, however, that 8-oxodG may derive from oxidation of DNA released from dead cells (Lindahl, 1993Lindahl T. Instability and decay of the primary structure of DNA.Nature. 1993; 362: 709-715Crossref PubMed Scopus (4004) Google Scholar), and yetShigenaga et al., 1989Shigenaga M.K. Gimeno C.J. Ames B.N. Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.Proc Natl Acad Sci USA. 1989; 86: 9697-9701Crossref PubMed Scopus (675) Google Scholar have shown that dG is not artefactually oxidized in the systemic circulation. Furthermore, in this study, a significant decrease in urinary 8-oxodG, compared with baseline, was noted at day 10. This might be explained by a possible over-compensation by insult-induced DNA repair systems, a phenomenon previously noted following vitamin C supplementation (Cooke et al., 1998Cooke M.S. Evans M.D. Podmore I.D. et al.Novel repair action of vitamin C upon in vivo oxidative DNA damage.FEBS Letts. 1998; 439: 363-367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), but cannot easily be explained in the context of oxidation of dG in DNA released from dead cells. Similarly, the immunoassay for urinary T T demonstrated a peak of excretion 3 d following exposure; however, a second larger peak was also noted at days 9–11. These data suggest that two mechanisms are operating, which are distinct for each lesion in terms of induction times. Repair of T T has been reported to occur via NER (Galloway et al., 1994Galloway A.M. Liuzzi M. Paterson M.C. Metabolic processing of cyclobutyl pyrimidine dimers and (6–4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.J Biol Chem. 1994; 269: 974-980Abstract Full Text PDF PubMed Google Scholar), with their accelerated removal by transcription-coupled repair (TCR,Van Hoffen et al., 1995Van Hoffen A. Venema J. Meschini R. et al.Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6–4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts.EMBO J. 1995; 14: 360-367Crossref PubMed Scopus (201) Google Scholar). Such repair processes generate a lesion-containing oligomer some 24–29 bases long (Huang et al., 1992Huang J.-C. Svoboda D.L. Reardon J.T. et al.Human nucleotide excision nuclease removes thymine dimers from DNA incising the 22nd phosphodiester bond 5′ and the 6th phosphodiester bond 3′ to the photodimer.Proc Natl Acad Sci USA. 1992; 89: 3664-3668Crossref PubMed Scopus (361) Google Scholar), which rapidly becomes subject to exonucleolytic attack.Galloway et al., 1994Galloway A.M. Liuzzi M. Paterson M.C. Metabolic processing of cyclobutyl pyrimidine dimers and (6–4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.J Biol Chem. 1994; 269: 974-980Abstract Full Text PDF PubMed Google Scholar speculate that degradation of the oligomer would continue until the dimer is encountered, resulting in a 6- or 7-mer.Le Page et al., 2000Le Page F. Kwoh E.E. Avrutskaya A. et al.Transcription-coupled repair of, 8-oxoguanine. Requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.Cell. 2000; 101: 159-171Abstract Full Text PDF PubMed Scopus (281) Google Scholar recently established that TCR initiates the rapid removal of lesions, such as 8-oxodG and T T, that obstruct transcription and that TCR is a major repair pathway for 8-oxodG. If the increase in urinary lesions reported in this study is due to an induction of repair, NER or TCR are likely to be the mechanisms responsible, given the coincident peak of excretion around days 3 and 4. This phenomenon, in addition to the biphasic response seen for T T, requires further investigation. The analysis of urinary T T following UV exposure has not been previously reported. Data examining the in vivo removal of T T from the DNA of human skin, however, have suggested an initial rapid removal over the first few hours that subsequently slowed, but removed more than 90% of lesions over 24 h (Sutherland et al., 1980Sutherland B.M. Harber L.C. Kochevar I.E. Pyrimidine dimer formation and repair in human skin.Cancer Res. 1980; 40: 3181-3185PubMed Google Scholar;D'Ambrosio et al., 1981D'Ambrosio S.M. Whetstone J.W. Slazinski L. et al.Photorepair of pyrimidine dimers in human skin in vivo.Photochem Photobiol. 1981; 34: 461-464Crossref PubMed Scopus (61) Google Scholar;Reusch et al., 1988Reusch M.K. Meager K. Leadon S.A. et al.Comparative removal of pyrimidine dimer from human epidermal keratinocytes in vivo and in vitro.J Invest Dermatol. 1988; 91: 349-352Abstract Full Text PDF PubMed Google Scholar). In contrast, the study ofYoung et al., 1996Young A.R. Chadwick C.A. Harrison G.I. et al.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 (127) Google Scholar reported a much slower rate for T T removal, extending over a 7 d period. These results are not necessarily comparable to ours. These studies were measuring lesion loss from DNA whereas our results are also influenced by postexcision processing, which must occur for the lesion to appear in the urine. Nevertheless, our findings do not appear to be inconsistent with the in vivo skin studies. Finally, we report the induction of T T by a UV source whose spectral characteristics fall within the CIE description of UVA (320–400 nm), excepting a spike at 313 nm that represented only 0.5% of the total energy emitted. There are two possible explanations for this: T T production by UVA or from the small UVB component of the spectral output. The spike at 313 nm represents the basis of an important caveat relating to potential UVB contamination of UVA lamps used in PUVA, likely to be effective at inducing T T.Tyrrell, 1973Tyrrell R.M. Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation.Photochem Photobiol. 1973; 17: 69-73Crossref PubMed Scopus (146) Google Scholar reported the induction of T T in cells exposed to 365 nm UV, withFreeman et al., 1985Freeman S.E. Gange R.W. Matzinger E.A. et al.Production of pyrimidine dimers in skin of humans exposed to UVA.Photochem Photobiol. 1985; 42: 90SGoogle Scholar extending this to include 385 nm and 405 nm, although a subsequent publication refuted these findings, based upon experimental conditions used (Hacham et al., 1990Hacham H. Freeman S.E. Gange R.W. et al.Does exposure of human skin in situ to 385 or 405nm UV induce pyrimidine dimers in DNA?.Photochem Photobiol. 1990; 52: 893-896Crossref PubMed Scopus (15) Google Scholar). Given that DNA weakly absorbs UVA (Sutherland and Griffin, 1981Sutherland J.C. Griffin K.P. Absorption spectrum of DNA for wavelengths greater than 300 nm.Radiat Res. 1981; 86: 399-409Crossref PubMed Scopus (155) Google Scholar), an indirect, photosensitizer-based mechanism of T T induction seems possible (Peak et al., 1984Peak M.J. Peak J.G. Moehring P. et al.Ultraviolet action spectra for DNA dimer induction, lethality and mutagenesis in Escherichia coli with emphasis on the UVB region.Photochem Photobiol. 1984; 40: 613-620Crossref PubMed Scopus (110) Google Scholar). Work byMoysan et al., 1991Moysan A. Viari A. Vigny P. et al.Formation of cyclobutane thymine dimers photosensitised by pyridopsoralens quantitative and qualitative distribution within DNA.Biochemistry. 1991; 30: 7080-7088Crossref PubMed Scopus (41) Google Scholar demonstrated the induction of T T following UVA irradiation of pyridopsoralens, although found that none were produced with UVA alone; nor have they been found, in vitro at least, with other psoralens (Costalat et al., 1990Costalat R. Blais J. Ballini J.P. et al.Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: a triplet-triplet energy transfer mechanism.Photochem Photobiol. 1990; 51: 255-262Crossref PubMed Scopus (28) Google Scholar). Although this remains a possible route for T T production during PUVA therapy in vivo, it suggests that UVA in conjunction with endogenous sensitizers is a possible source for the T T noted in our study. Irrespective of the mechanism of production, it is notable that the UV component of a common PUVA system can independently produce T T. Work is ongoing to elucidate the significance of this. This pilot study has illustrated the removal of T T and 8-oxodG and kinetics of their appearance in urine, post-UV exposure, suggestive of differential repair. We have also revealed the induction of T T and 8-oxodG by a therapeutic UV(A) source used in PUVA therapy. In a therapeutic context, it would appear that PUVA does indeed induce T T and oxidative damage, at least via a UV effect, independent of any additional psoralen plus UV mechanism. These are potential causes of the mutations seen in PUVA-derived SCC (Wang et al., 1997Wang X.M. McNiff J.M. Klump V. et al.An unexpected spectrum of p53 mutations from squamous cell carcinomas in psoriasis patients treated with PUVA.Photochem Photobiol. 1997; 66: 294-299Crossref PubMed Scopus (34) Google Scholar), but whether this is via contamination of the UVA source with shorter wavelengths or via a UVA-dependent photosensitizer mechanism requires investigation. The authors gratefully acknowledge financial support from the Leicestershire Dermatology Research Fund and the Scottish Office. The authors thank Paul Whitaker, Leicester Royal Infirmary, NHS Trust, for analysis of urinary creatinine. The authors would like to thank Nick Taub, Department of Epidemiology and Public Health, University of Leicester, for his advice regarding statistical analysis of the data.
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