Quantification of Urinary Thymidine Dimers in Volunteers After Ultraviolet Radiation Using a New UPLC-MS/MS-based Method.

BACKGROUND/AIM: Solar ultraviolet radiation (UVR) is a carcinogen and irradiation of the skin results in DNA damage. Cyclobutane pyrimidine dimers (CPDs), including thymidine dimers, are among the most frequent forms of DNA damage. When CPDs are formed, the nucleotide excision repair system is activated and CPDs are excreted in the urine. Here, we developed a mass spectrometry-based method to quantify thymidine dimers in the urine and tested the method on a small group of volunteers after whole-body UVR exposure. PATIENTS AND METHODS: Years of research resulted in a method based on the "dilute-and-shoot" principle and ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. The whole body of each of eight healthy volunteers was exposed to 1.5-2.0 standard erythema doses (SEDs) of UVR for 3 consecutive days. Morning urine was collected on Day 1 (before irradiation) and on the following 7-9 days. Prior to analysis, sample preparation consisted of a simple dilution. A tandem quadrupole mass spectrometer coupled to UPLC was used for quantitative analysis in the multiple reaction monitoring mode. RESULTS: After 3 consecutive days of 1.5-2 SEDs, the highest level of thymidine dimer excretion occurred on Day 6 (0.7 ng/ml urine). Compared with baseline, significantly more thymidine dimers were excreted every day until Day 8 (p<0.016). Our method quantifies thymidine dimers that are excreted as dimers (i.e., not degraded further) after nucleotide excision repair. CONCLUSION: This is the first published mass spectrometry-based method for quantifying thymidine dimers in the urine after whole-body UVR exposure.

Concurrent beneficial (vitamin D production) and hazardous (cutaneous DNA damage) impact of repeated low-level summer sunlight exposures.

BACKGROUND: The concurrent impact of repeated low-level summer sunlight exposures on vitamin D production and cutaneous DNA damage, potentially leading to mutagenesis and skin cancer, is unknown. OBJECTIVES: This is an experimental study (i) to determine the dual impact of repeated low-level sunlight exposures on vitamin D status and DNA damage/repair (via both skin and urinary biomarkers) in light-skinned adults; and (ii) to compare outcomes following the same exposures in brown-skinned adults. METHODS: Ten white (phototype II) and six South Asian volunteers (phototype V), aged 23-59 years, received 6 weeks' simulated summer sunlight exposures (95% ultraviolet A/5% ultraviolet B, 1·3 standard erythemal doses three times weekly) wearing summer clothing exposing ~35% body surface area. Assessments made were circulating 25-hydroxyvitamin D [25(OH)D], immunohistochemistry for cyclobutane pyrimidine dimer (CPD)-positive nuclei and urinary biomarkers of direct and oxidative (8-oxo-deoxyguanosine) DNA damage. RESULTS: Serum 25(OH)D rose from mean 36·5 ± 13·0 to 54·3 ± 10·5 nmol L-1 (14·6 ± 5·2 to 21·7 ± 4·2 ng mL-1 ) in phototype II vs. 17·2 ± 6·3 to 25·5 ± 9·5 nmol L-1 (6·9 ± 2·5 to 10·2 ± 3·8 ng mL-1 ) in phototype V (P < 0·05). Phototype II skin showed CPD-positive nuclei immediately postcourse, mean 44% (range 27-84) cleared after 24 h, contrasting with minimal DNA damage and full clearance in phototype V (P < 0·001). The findings did not differ from those following single ultraviolet radiation (UVR) exposure. Urinary CPDs remained below the detection threshold in both groups; 8-oxo-deoxyguanosine was higher in phototype II than V (P = 0·002), but was unaffected by UVR. CONCLUSIONS: Low-dose summer sunlight exposures confer vitamin D sufficiency in light-skinned people concurrently with low-level, nonaccumulating DNA damage. The same exposures produce minimal DNA damage but less vitamin D in brown-skinned people. This informs tailoring of sun-exposure policies.

Urinary levels of thymine dimer as a biomarker of exposure to ultraviolet radiation in humans during outdoor activities in the summer.

The incidence of skin cancer is rising rapidly in many countries, presumably due to increased leisure time exposure to solar ultraviolet radiation (UVR). UVR causes DNA lesions, such as the thymine dimer (T=T), which have been causatively linked to the development of skin cancer. T=T is clearly detectable in urine and may, thereby, be a potentially valuable biomarker of UVR exposure. The objective of this study was to evaluate the relationship between UVR exposure and urinary levels of T=T in a field study involving outdoor workers. Daily ambient and personal exposure of 52 beach lifeguards and agricultural workers to UVR were determined (employing 656 personal polysulphone dosimeters). In 22 of these subjects, daily urinary T=T levels (120 samples) were measured, the area of skin exposed calculated and associations assessed utilizing mixed statistical models. The average daily UVR dose was approximately 600 J/m(2) (7.7 standard erythemal doses), i.e. about 20% of ambient UVR. T=T levels were correlated to UVR dose, increasing by about 6 fmol/µmol creatinine for each 100 J/m(2) increase in dose (average of the three preceding days). This is the first demonstration of a relationship between occupational UVR exposure and urinary levels of a biomarker of DNA damage. On a population level, urinary levels of T=T can be used as a biomarker for UVR exposure in the field.

Quantification of ultraviolet radiation-induced DNA damage in the urine of Swedish adults and children following exposure to sunlight.

CONTEXT: DNA damage following exposure to ultraviolet radiation (UVR) is important in skin cancer development. The predominant photoproduct, cyclobutane thymine dimer (T=T), is repaired and excreted in the urine, where it provides a biomarker of exposure. OBJECTIVE: To quantify urinary T=T levels after recreational sunlight exposure in adults and children. METHODS: Average UVR doses were measured with personal dosimeters. Urinary T=T was analysed with (32)P-postlabelling. RESULTS: Background levels of T=T increased significantly following exposure to sunlight. Amounts of T=T in urine of children and adults were not significantly different after adjusting for area of skin exposed and physiological differences. UVR dose and amounts of T=T correlated for both adults and children. CONCLUSION: Recreational exposure to sunlight in Sweden induces levels of DNA damage, clearly detectable in urine.

Immunochemical detection of UV-induced DNA damage and repair.

Because of a substantial rise in the incidence of skin cancer in the United Kingdom and elsewhere a greater awareness of the role of sun-induced cutaneous genetic damage has developed. This, in turn, has increased interest in the cellular mechanisms responsible for tumorigenesis, and the need to develop experimental methodologies to investigate these mechanisms. DNA represents a most important cellular target for ultraviolet radiation (UVR), leading to the formation of various DNA damage products. A number of these products, such as the cyclobutane pyrimidine dimer, have been implicated in the pathogenesis of various UVR-related conditions. In this chapter we detail a number of methods for assessing UVR-induced DNA damage using two antisera which recognize cyclobutane thymine dimers (T-T). Immuno-approaches have a number of benefits over chromatographic techniques, and have been applied herein to quantitatively and qualitatively assess the presence of T-T in cultured keratinocytes, human skin, and urine, providing information about lesion induction and repair.

Urinary thymidine dimer as a marker of total body burden of UV-inflicted DNA damage in humans.

High levels of DNA damage are induced in human skin following exposure to UV radiation. Cyclobutane thymidine dimer (T = T) is the most common of these lesions, which are enzymatically removed as oligonucleotides from DNA and further degraded before excretion in urine. Analysis of such repair products in the urine could serve as a biomarker of total body burden of UV exposure. The aim of this study was to examine the kinetics of T = T excretion following a single tanning session in a commercial solarium and to validate the method by delivering different doses. Ten individuals used the solarium for a total of 35 sessions of body tanning. Urine was collected before UV exposure and daily thereafter (up to 5 or 11 days) and T = T was analyzed using a very sensitive and quantitative (32)P-postlabeling technique combined with high-performance liquid chromatography. Following exposure, T = T levels increased dramatically and reached a peak 3 days later; afterwards, the T = T levels gradually decreased. The total amount of T = T excreted differed about 5-fold among subjects given an equal dose. A 50% excretion time was calculated using the excretion data for the first 5 days and it was found to be between 55 and 76 hours for different individuals. There was a good correlation between the amount of T = T excreted during days 1 to 5 and the delivered UV dose. Reducing exposure time to 50% lowered the amount of T = T to 47%; if half of the lamps were covered, T = T decreased to 44%. Our data show that urinary T = T could be a suitable noninvasive biomarker for UV exposure; a finding which could also be applicable to studies in children.

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo.

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.

Cyclobutane thymidine dimers are present in human urine following sun exposure: quantitation using 32P-postlabeling and high-performance liquid chromatography.

Cyclobutane thymidine dimer (T=T) is the major DNA photoproduct formed in human skin after solar radiation. We have developed a 32P-postlabeling method suitable for quantitating T=T in human urine with a detection limit of about 0.5 fmol per 10 microl urine. The method was used in the present study to measure the daily T=T urinary level of two volunteers over a 15 d period, including frequent sun exposures ranging from 0 to 5 h daily. T=T was not detected before or immediately (4 h) after the initial sun exposure but was first observed in urine samples collected 18 h after the initial exposure. Thereafter, urinary T=T levels gradually increased up to a peak reached about 3 d after the maximum sun exposure. The levels decreased during the following days but were still detectable 8 d after the last sun exposure. About 70-75% decrease in excreted T=T was observed after 8 d. The T=T levels measured in urine were lower but in the same order of magnitude as the levels expected after a theoretical calculation based on previous published results and reasonable assumptions. This study shows the occurrence of cyclobutane thymidine dimers in human urine after skin exposure to solar radiation.

Urinary thymine dimers and 8-oxo-2'-deoxyguanosine in psoriasis.

Psoralen in conjunction with UVA (PUVA) is perhaps the most effective treatment for psoriasis. It is, however, a risk factor for skin cancer in these patients and there is a need to develop non-invasive assays reflective of treatment-induced DNA damage. We report here the assessment of two important lesions, thymine dimer (T<>T) and 8-oxo-2'-deoxyguanosine (8-OHdG), in the urine of psoriasis patients. It was found that, once corrected for urine concentration, the psoriatic group had significantly higher (P<0. 0001) urinary levels of thymine dimers compared to the control group. No significant differences in urinary 8-OHdG levels were noted between the psoriatic, atopic dermatitis and control groups. Therefore biomonitoring of therapy from the very start with this simple and non-invasive assay could perhaps be an effective measure of the risk involved with the treatment allowing optimization for minimal-risk therapy.