2.1 Study design and subjects

The participants in this study were healthy volunteers living in the Copenhagen area of Denmark who were going on vacation in February 2023 to destinations with a generally higher UV index (5–11 +) than Denmark (UV index = 1). Destinations included Thailand, the Caribbean, the USA (Florida), and the Canary Islands. Inclusion criteria were as follows: age ≥ 18 years, vacation ≥ 1 week, and written informed consent.

Exclusion criteria were as follows: UVR exposure from a vacation or from using sun beds within 4 weeks prior to the study start, pregnancy, breastfeeding, immunosuppression, skin diseases, taking medication that interferes with DNA repair, or increased photosensitivity.

One urine sample was collected pre-holiday, while three subsequent samples were collected post-holiday. Before their vacation, participants were provided with a measuring cup to quantify the volume of their morning urine, and four 50 mL tubes were provided for storage of urine samples. The participants also received a personal UVR dosimeter to be worn on the wrist. In addition, the participants received data sheets to document their demographic data, urine sample volumes, and the time of urination immediately prior to providing the collected sample (which reflected the time over which the urine sample provided had been accumulating in the body). Furthermore, the participants were asked to keep a diary with information about their clothing and factor of sunscreen used over three 4-h time intervals (07:00–11:00 h, 11:00–14:00 h, and 14:00–18:00 h) during all days of their vacation. The documents given to the participants are shown in the supporting information section.

2.2 Chemicals

Thymidylyl-3′-5′-thymidine ammonium (Carbosynth Limited, Berkshire, UK), isotope-labeled (13C2) cis-syn-thymidine dimers (Toronto Research Chemicals, Toronto, ON, Canada), creatinine (Merck KGaA, Darmstadt, Germany), and isotope-labeled (D3) creatinine (Merck KGaA) were used.

2.3 Sample collection and preparation

Urine samples were collected on the morning of the last day before the vacation and on the first three mornings after returning from vacation. The urine samples were stored at − 18 °C in freezers at the homes of the volunteers before being transferred to the hospital. After transfer to the hospital, the samples were thawed, distributed into Eppendorf tubes, and stored at − 20 °C until analysis.

Because unlabeled standard of cis-syn-thymidine dimers was unavailable, 30 mg Thymidylyl-3′-5′-thymidine ammonium was dissolved in 15 mL water with 18 μL acetophenone. A total of 15 mL of this solution was irradiated with ultraviolet B in a UV 200 L cabin (Waldmann, Villingen-Schwenningen, Germany) for 3 h resulting in a mixture of thymidine photoproducts including cis-syn, trans-syn thymidine dimers, and the 6–4 photoproducts from TpT, similar to previously described methods [11, 14]. In the following cis-syn-thymidine dimers are abbreviated T<>T.

2.4 Ultra-performance liquid chromatography tandem mass spectrometry2.4.1 T<>T analysis

The ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method used here was adapted from a previously published method by Lerche et al. [11]. The UPLC-MS/MS analysis was performed on an Acquity UPLC I-Class system (Waters, Milford, MA, USA) coupled to a Xevo TQ-XS tandem quadrupole mass spectrometer (Waters, Manchester, UK) using an electrospray ionization interface as the ion source.

For the T<>T analysis, 180 µL of urine sample was mixed with 10 µL of 400 ng/mL isotopically labeled internal standard and 10 µL of water, resulting in a concentration of 20 ng/mL internal standard. Calibration plots were created using standard concentrations of 32 ng/mL, 16 ng/mL, 8 ng/mL, 4 ng/mL, 2 ng/mL, 1 ng/mL, 0.5 ng/mL, 0.25 ng/mL, and 0.125 ng/mL, by adding known concentrations of standards to blank urine. After mixing the samples, these were diluted with 1400 µL 1% formic acid and transferred to LC–MS vials for analysis with three technical replicates performed for each sample. A linear calibration plot was obtained for T<>T in the range 0.25–32 ng/mL, with R2 > 0.985.

The parameters for the mass spectrometer were set to the following values: source temperature, 150 °C; desolvation temperature, 650 °C; desolvation gas flow rate, 1000 L/h; cone gas flow rate, 150 L/h; capillary voltage, 0.5 kV; and detector gain, 1.

The LC separation was performed on an Atlantis Premier BEH C18 AX VanGuard FIT column (1.7 μm, 2.1 × 100 mm; Waters, Milford, MA, USA) at a temperature of 55 °C. The mobile phases consisted of (A) 0.2% formic acid in 10 mM ammonium formate and (B) methanol. Separation was performed with gradient elution from 0 to 50% B over 3 min, followed by a 2-min washing step with 98% B and re-equilibration to initial conditions for 1.5 min. For the analysis, the mass spectrometer was operated using multiple reaction monitoring in negative ion mode using the m/z 545 → 79 transition of the T<>T and the m/z 547 → 79 transition of the 13C-labeled internal standard; the cone voltage was set to 84 V and the collision energy to 48 eV.

2.4.2 Creatinine analysis

For the creatinine analysis, a urine sample was diluted 2500-fold in milliQ water. A total of 500 µL of the diluted sample was mixed with 500 µL 100 ng/mL deuterated internal standard to generate a 50 ng/mL internal standard solution. Calibration plots were created by diluting with water to generate standard solution concentrations of 800 ng/mL, 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, and 12.5 ng/mL. The diluted samples were transferred to LC–MS vials for analysis, with three technical replicates performed for each sample.

The parameters for the mass spectrometer were set to the following values: source temperature, 150 °C; desolvation temperature, 500 °C; desolvation gas flow rate, 1000 L/h; cone gas flow rate, 150 L/h; capillary voltage, 0.5 kV; and detector gain, 1.

The LC separation was performed at a temperature of 55 °C. The mobile phases consisted of (A) 0.2% formic acid in 10 mM ammonium formate and (B) methanol. Separation was performed by isocratic elution with 100% A over 2 min, followed by a 1-min washing step with 100% B and re-equilibration to initial conditions for 1 min. For the analysis, the mass spectrometer was operated using multiple reaction monitoring in positive ion mode using the m/z 114 → 44 transition of creatinine and the m/z 117 → 47 transition of the deuterated internal standard; the cone voltage was set to 40 V and the collision energy to 14 eV.

2.5 Personal UV radiation exposure

Personal UVR exposure during vacations was measured using wrist-borne dosimeters [15]. The dosimeters measured UVR exposure as standard erythema doses (SEDs) every 5 s and recorded a mean reading every 5 min. The UVR dosimeters (SunSaver 3, Bispebjerg Hospital, Copenhagen, Denmark) were updated models of a previous dosimeter [16] and they were calibrated [17] before the study. The action spectra for producing CPDs and erythema in human skin are very similar [18]; therefore, we assume a linear relationship exists between erythema-weighted UVR reaching the skin and CPD formation in the skin. To adjust for the different clothing worn by participants during the study period, exposed body surface areas (m2) were calculated using the diary information clothing codes provided by the participants and the corresponding exposed skin fraction, as previously described by Datta et al. [19].

2.6 Conversion and correction of data2.6.1 UVR data

The measured personal UV dose in SEDs was converted to erythema-weighted Joules delivered to the skin by adjusting for exposed body surface area in m2.

To account for differences in the clothing worn by participants, these conversions were calculated separately for each of the three time slots in each day using the clothing codes recorded by participants and the following equation:

$$}\left( } \right) = }\left( }} \right)\frac}}}}^ }}}\left( }^ } \right)\,}$$

where SED represents the standard erythema dose, 1 SED = 100 J/m2 erythema weighted; BSA represents the body surface area. UV dose (J) represents erythema-weighted joule corrected for exposed skin area.

Further conversions were also applied to UV doses to account for self-reported sunscreen use. For this purpose, two different approaches were used:

1) Correction of UV dose using the labeled sun protection factor (SPF).

$$}\left( } \right) = \frac}\left( }} \right)\frac}}}}^ }}}}}}}\left( }^ } \right)\,}$$

2) Effective SPF was calculated from the labeled SPF based on previous reports of sunscreen application thickness for Scandinavians going on a vacation in the sun [20], using the formula for effective SPF described by Petersen et al. (Effective SPF = labeled SPF0,79/2) [8]:

$$}\left( } \right) = \frac}\left( }} \right)\frac}}}}^ }}}}}}}\left( }^ } \right) \,}$$

The UV doses were corrected for sunscreen use only when the participants had applied sunscreen to their bodies, not just to their faces.

2.6.2 T<>T data

To correctly correlate the measured T<>T concentrations with personal UVR exposure, the measured levels were corrected either by using (1) creatinine measurements or (2) reported urine volumes. For this purpose, three methods were used and compared:

1) Correction based on the urinary flow rate of the volunteers (calculated from urine volume and the time over which the urine sample provided had been accumulating in the body):

$$} < > }\left( }}}}}}} \right) = } < > }\left( }}}}}}} \right)}\left( }}}}}} \right)24\left( }}}}}} \right)$$

2) Correction for expected daily creatinine excretion based on metadata (sex, age, and weight):

$$} < > }\left( }}}}}}} \right) = \frac} < > }\left( }}}}}}} \right)}}}\left( }}}}}}} \right)/}\left( }}}}}}} \right)^ }}$$

†The expected quantity of creatinine was calculated using excretion data sourced from Kampmann et al. [21] based on weight, sex, and age.

3) Correction based on creatinine excretion as a ratio:

$$\frac} < > }}}} }}} = \frac} < > }\left( }}}}}}} \right)}}}/}}}}}}\left( }}}}}}} \right)}}}/}}}}}$$

2.7 Statistical analysis

The statistical data analysis was performed using IBM SPSS statistical software (ver. 29; IBM, Armonk, NY, USA). Age, BSA, UV dose (SED), and UV dose (J) were tested for normal distribution using Kolmogorov–Smirnov Test and p values > 0.2; therefore, descriptive data are presented as means ± standard deviation. The optimal relationship (i.e., greatest r2 value) between the measured T<>T in the urine and personal UVR exposure follows a power model (double logarithmic) after testing linear, logarithmic, inverse, power, and exponential models. The UV doses were tested as accumulated UV doses for the last 3–10 vacations days, both with and without correction for sunscreen use. The urine post-vacation T<>T sample days were tested individually and as totals from days 1 and 2, and from days 1, 2, and 3. The effects of age, skin phototype, and sex were investigated using multiple regression analyses. All tests were two sided and a p value < 0.05 was considered statistically significant.

2.8 Ethics

This study was conducted in accordance with the Declaration of Helsinki. This study was approved by the Danish Research Ethics Committee (H-20076172), the Danish Knowledge Center for Data Reviews (P-2021-591), and it was registered on ClinicalTrials.gov (NCT05277961). Written informed consent was obtained from all participants.