Quantification of DNA Repair Products in the Urine After Ultraviolet Radiation of the Skin
Phase: NA
Conditions: Ultraviolet Rays; Injury, DNA Adduct Formation, DNA Damage, Radiation Induced, DNA Damage
Sponsor: Bispebjerg Hospital

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Study Protocol 
 
Project title: 
 
Determination of DNA repair products in urine after UV 
irradiation of the skin 
 
 
Sponsor and principal investigator 
Catharina M. Lerche, MSc Pharm, PhD 
Bispebjerg Hospital, University of Copenhagen 
Department of Dermatology, D92 
Nielsine Nielsensvej 17, Entrance 9 
DK-2400 Copenhagen NV 
Direct: + 45 21470444 
Cell: + 45 28207100 
Mail: catharina.margrethe.lerche@regionh.dk 

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Participants: 
Peter A. Philipsen, MSc (EE), PhD   
Bispebjerg Hospital, University of Copenhagen 
Department of Dermatology, D92 
Nielsine Nielsensvej 17, Entrance 9 
DK-2400 Copenhagen NV 
Mail: Peter.Alshede.Philipsen@regionh.dk 
 
Professor Hans Christian Wulf, Dr Med et Dr Pharm 
Bispebjerg Hospital, University of Copenhagen 
Department of Dermatology, D42 
Nielsine Nielsensvej 9, Entrance 4 
DK-2400 Copenhagen NV 
Mail: h.wulf@regionh.dk  

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Anthropologist Nynne Frederiksen 
Bispebjerg Hospital, University of Copenhagen 
Department of Dermatology, D92 
Nielsine Nielsensvej 17, Entrance 9 
DK-2400 Copenhagen NV 
Mail: nyfred@cancer.dk 
 
 

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1. Titel 
Determination of DNA repair products in urine after UV irradiation of the skin. 
2. Purpose 
Problem definition 
DNA damage is formed in the skin by sun exposure 1. The most common DNA damage is called thymine 
dimers (Figure 1). Many of these injuries are repaired by "Nucleotide excision repair" (NER) and 
excreted through the urine 2. The importance of this repair mechanism is seen in patients with defects 
in their NER, who have a very high risk of developing skin cancer. We want to be able to quantify the 
amount of thymine dimers in the urine after ultraviolet (UV) irradiation of the skin. With the 
establishment of such a test system, it will eventually be possible to test potential photoprotective 
substances or potential photocarcinogenic substances based on damage measured in the urine after a 
known UV dose. We would like to investigate when the highest secretion of this type of DNA damage 
occurs using 6 different irradiation regimens. It has previously been possible to quantify thymine 
dimers in the urine using an isotope method, but that method is no longer available and it is desirable 
to avoid isotopes 3. We have developed a new method based on mass spectrometry (UHPLC-MS). 
 

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Figure 1. DNA damage of the thymine dimer type occurs by UV irradiation of the skin. The damage will 
often be repaired and excreted in the urine.  
Hypothesis  
Our hypothesis is that maximum excretion of thymine dimers in the urine occurs 1-4 days after UV 
irradiation of the skin.  
Endpoints and rationales  
The primary endpoint is quantification of thymine dimers in the urine before irradiation as well as 
every day for up to 7 days after UVR irradiation with six different irradiation regimens in healthy 
subjects. The 2-3 days where the concentration of excreted thymine dimers is greatest will be the days 
to collect urine in future studies. One of the six irradiation regimens will consist of healthy volunteers 
going sun holiday outside of Denmark. The volunteers will receive a UV-dosimeter which measures the 
UV dose at their location. The urine collection will follow the same procedure as the other groups, 
upon returning to Denmark. 

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The secondary endpoints are changes in redness and pigment, measured non-invasively using surface 
reflectance measurement. 
The 6 irradiation regimens will be compared so that the greatest amount of thymine dimers can be 
determined with the least change in pigment and redness. 
Background 
Ultraviolet (UV) radiation causes DNA damage in the skin that can eventually lead to skin cancer 1. 
Thymin dimer is a DNA damage that is only formed when UV rays hit DNA in the skin cells 1. 
Nucleotide excision repair (NER) repairs and removes thymine dimers in the individual cell, which are 
then excreted in the urine 2. Thymine dimers in the urine are a biomarker for the total DNA repair3. In 
the past, a method for quantifying thymine dimers in urine using isotope labeling3 has existed. The 
method was very sensitive but had a long analysis time and in addition it is an advantage from a work 
environment to avoid working with radioactive isotopes3.  
We have succeeded in developing a new method of analysis using Ultra High-pressure Liquid 
Chromatography Mass Spectrometry (UHPLC-MS). This method has high sensitivity and high 
selectivity while having a fast analysis time. In addition, this equipment is capable of analyzing 
multiple DNA modifications. Furthermore, there is high sensitivity and high selectivity. This study is a 
proof-of-concept study in which we would like to investigate when the most optimal time for urine 
collection is after six different irradiation regimens. The perspective is to be able to use the method to 
find new potential photo-protective as well as photocarcinogenic substances. 
3. Method 
Study design for healthy subjects 
The subjects will be recruited recruited by a notice on Bispebjerg's hospital website and by 
anthropologist Nynne Johanne Sahl Frederiksen. 
For five of the six irradiation regimens the subjects will be divided into 2 groups of 8-10 people (group 
1-5). Group 1 is irradiated 3 times with 1 standard erythema dose (SED). 1 SED corresponds to approx. 
10 min sun around kl. 13 on a good Danish summer day. Group 2-5 will be irradiated once with up to 3 
SED, which corresponds to approx. 30 min around kl. 13 on a good Danish summer day. Group 6 
consists of subjects going on sun holiday. 
Subjects in group 1-5 are irradiated in a full body UV cabin (Waldmann, Willing-Schwenningen, 
Germany) with 26 F85 / 100W UV6 tubes (290-350 nm, broad spectrum), TL-01 (290-380 nm, Philips, 
Eindhoven, The Netherlands) or TL-12 (280-380, Philips, Eindhoven, The Netherlands). Dosages arre 

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measured out by multiplying the CIE erythema spectrum with the UV-spectra to achieve the same 
biological effect in the skin. The irradiation time is set on a timer clock, so that the UV cabin switches 
off after the desired irradiation time. The irradiation time is 13 seconds for 1 SED and 39 seconds for 3 
SED. The UV cabin is measured using calibrated equipment, which is also used to measure our UV 
cabins in the clinic, which are used for patients. The subjects stand up in the cabin and have a screen 
on so that their eyes and face are not exposed to radiation. When lighting, subjects should only wear 
underwear, which for men are underpants / boxer shorts, while for women it is bras and panties.   
The following groups will be irradiated: 
Group 1: 3x1SED in underwear corresponding to approx. 80% irradiated body surface area 
Group 2: 1x3SED in underwear corresponding to approx. 80% irradiated body surface area 
Group 3: 3x3SED in underwear corresponding to approx. 80% irradiated body surface area 
Group 4: 3x3SED wearing shorts and bra/naked torso corresponding to approx. 60% irradiated body 
surface area 
Group 5: 3x3SED wearing shorts and T-shirt corresponding to approx. 40% irradiated body surface 
area 
Group 6: up to 30 subjects who are already planning a sun vacation. The individuals should behave in 
the sun as they normally do and not do anything differently. The subjects will be provided with a 
dosimeter, which measures the UV dose at their location. The test subjects will also receive a form 
where they have to note their clothing/sunscreen consumption. The subjects will be asked 4 questions 
before they go on vacation (appendix 1) and must undergo a qualitative interview (appendix 2) when 
they return from vacation and receive their urine test results. 
The experiment is performed between October and March in Denmark, to avoid that the subjects do 
not simultaneously receive UV radiation from the sun and thus can form DNA damage. Subjects must 
collect morning urine in dispensed containers and must store it in their own freezer until the final 
visit. 
Morning urine samples (2x 50 ml) will be collected before irradiation, labeled as day 1 and up to day 
10 for groups 1, 3, 4, and 5, and day 8 for group 2. Group 6 will collect a urine sample in Denmark 
before the vacation starts and then collect urine as the other 5 groups do when they are back in 
Denmark. A total of 1000 ml of urine (2 x 50 ml x 10) will be collected for groups 1, 3, 4, 5, and 6, and 
800 ml of urine (2 x 50 ml x 8) will be collected for group 2. Before and after each illumination are 

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measured pigment and redness on the subjects as well as on final visits. Pigment and redness 
measurement are performed on the back, chest and buttocks. 
4. Statistical considerations 
Previous studies have observed an increase of 1.7 nmol with a standard deviation (SD) of 1.3 nmol 4, 
with a power of 0.80, as well as significance level of 0.05 in a paired test, then 7 persons must complete 
the study when we set power to 0.80 and have a significance level of 0.05 and uses the paired test 
(Power and Sample Size Calculation, PS, Vanderbilt University; version 3.1.2). As the study lasts up to 
10 days, dropouts can be expected and therefore 8-10 subjects are included in each of the groups 1-5, 
as well as up to 30 subjects in group 6. There is an expectation of a larger variability in group 6 due to 
the varying doses the subjects will receive during their vacation. 
5. Subjects  
A total up to 80 subjects are recruited to participate in the study. Men and women aged 18 or over who 
meet the criteria for inclusion and exclusion will be included. The subjects are recruited through 
Bispebjerg Hospital's intranet and by anthropologist Nynne Frederiksen. Subjects will be invited to an 
initial screening visit where they will receive oral information about the study by trial supervisor 
Catharina Lerche, Peter Philipsen or Nynne Frederiksen. The meeting takes place in a separate room to 
ensure a safe and quiet environment. Subjects will be informed that they can bring an assistant if they 
wish. Written informed consent will be ensured, and takes place at least 24 hours after the screening 
visit. 
Inclusion criteria 
Over the age of 18 
Written informed consent from the subject. 
Exclusion criteria 
Immunosuppressed individuals 
Sun holiday / ski holiday / solarium 4 weeks before trial start  
Skin disease  
Medicines that cause photosensitivity or affect DNA repair 
Pregnancy 
 
6. Risks, side effects and disadvantages in the short and long term  

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There is a slight risk of short-term redness after irradiation, as well as after staying in the sun. The UV 
doses (3 SED) have been chosen so that almost all Danes will avoid redness. The total UV dose 
corresponds to a ½ hour stay in the sun in the middle of the day during the summer in Denmark. All 
subjects will be informed both orally and in writing about the risks and possible side effects.  
Extraction of new biological material or collection of biological material from already existing 
biobank  
No biobank. The urine samples are analyzed immediately after collection. Excess biological material is 
destroyed immediately after analysis.                
7. Quality control  
The Helsinki II Declaration will be respected as well as the standards of good clinical research. 
Respect for privacy as well as for physical and mental integrity in patients is maintained. The 
study will be registered at the Knowledge Center for Data Reporting, the Capital Region, in the 
electronic system Pactius.  
 
8. Data protection and storage  
 
The general data protection regulation will be respected. Research using data from the capital 
region is considered public research. Use and distribution of data collected in this study will be 
discussed with patients during the consent process. The project is reported to the Knowledge 
Center for Data Reporting, the Capital Region of Denmark in the electronic system Pactius. A 
list of the subjects' names, study ID and date of birth is prepared. All collected data will be 
anonymised and protected by Danish legislation regarding the handling of personal data and 
the Health Act. Data are registered and stored for 5 years after completion of the study at the 
Department of Dermatology, Bispebjerg Hospital. 
 
9.  Economy 
It is Pharmacist Catharina Lerche, Professor Hans Christian Wulf and Engineer Peter Philipsen who 
have taken the initiative for the experiment and they are all paid by the department of 
dermatology, Bispebjerg hospital. There is no contribution from companies to this experiment. An 
external grant of 500,000 DKK has been received from the Danish Research Center For Skin Cancer 
(https://vfhk.org/en), which is intended to cover the salary for Nynne Frederiksen as well as 
chemicals for urine analysis. Catharina Lerche has a grant from the Lundbeck Foundation, which 
covers a taxable compensation of 750-1,500 DKK per participant as inconvenience compensation. 

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750 DKK will be paid if the exposure is completed and 4 urine samples are provided for groups 1-
6. 1,000 DKK will be paid if the exposure is completed and all urine samples are provided for 
groups 1-5. For group 6, 1,500 DKK will be paid if all urine samples are provided, and both the 
vacation and interview are completed.   
10. Information for subjects  
The principle investigator, Catharina M. Lerche, is responsible for providing clear oral and 
written information about goals, designs and risks of the study, as stated in "Information and 
consent to participate in health science research projects" by the Ministry of Health and the 
Elderly. In undisturbed surroundings, the participant will be made aware of their right to have 
one / or more attendants present, that participation is voluntary and that withdrawal is 
possible at any time during the study. Subjects are given sufficient reflection time (at least 24 
hours). Subjects will be asked to sign a consent form.  
 
11. Publication of results  
Positive, negative and conclusive results will be published. The aim is to publish and present 
the results in a peer-reviewed international dermatology journal and / or at dermatological 
conferences. The intellectual property rights to the results belong to Bispebjerg Hospital. 
Publications will comply with the Vancouver Guidelines.  
 
12. Ethics  
Personal information and urine samples are processed in accordance with the Personal Data 
Act and the Health Act. The project is also implemented with minimal health and safety risks 
for the participants. It is expected that the potential risks of adverse effects in this study are 
very small. The potential for evidence-based future gains and the future perspectives that this 
study may provide should be weighed against trial participation. Danish laws on patients' 
rights and compensation are followed. 
 
References 
(1)  Brash, D. E.; Rudolph, J. A.; Simon, J. A.; Lin, A.; McKenna, G. J.; Baden, H. P.; Halperin, A. J.; Pontén, 
J. A Role for Sunlight in Skin Cancer: UV-Induced P53 Mutations in Squamous Cell Carcinoma. 
Proc. Natl. Acad. Sci. U. S. A. 1991, 88 (22), 10124–10128. 
(2)  Martens, M. C.; Emmert, S.; Boeckmann, L. Sunlight, Vitamin D, and Xeroderma Pigmentosum. In 
Sunlight, Vitamin D and Skin Cancer; Reichrath, J., Ed.; Advances in Experimental Medicine and 
Biology; Springer International Publishing: Cham, 2020; pp 319–331. 
https://doi.org/10.1007/978-3-030-46227-7_16. 
(3)  Le Curieux, F.; Hemminki, K. Cyclobutane Thymidine Dimers Are Present in Human Urine 
Following Sun Exposure: Quantitation Using 32P-Postlabeling and High-Performance Liquid 

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Chromatography. J. Invest. Dermatol. 2001, 117 (2), 263–268. https://doi.org/10.1046/j.1523-
1747.2001.01416.x. 
(4)  Petersen, B.; Wulf, H. C.; Triguero-Mas, M.; Philipsen, P. A.; Thieden, E.; Olsen, P.; Heydenreich, J.; 
Dadvand, P.; Basagaña, X.; Liljendahl, T. S.; Harrison, G. I.; Segerbäck, D.; Schmalwieser, A. W.; 
Young, A. R.; Nieuwenhuijsen, M. J. Sun and Ski Holidays Improve Vitamin D Status, but Are 
Associated with High Levels of DNA Damage. J. Invest. Dermatol. 2014, 134 (11), 2806–2813. 
https://doi.org/10.1038/jid.2014.223. 
 
 
 

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Appendix 1: 
Baseline Questions Before Vacation: 
1. Have you ever had or do you currently have cancer? 1.1 If yes: Have you ever had or do you 
currently have skin or melanoma cancer? 
2. Do you know people close to you (family, friends, close colleagues, etc.) who have or have had 
cancer? If yes: 2.1 Who (specify relationship):_________ (e.g., my sister or my friend - Multiple 
relationships can be mentioned) 2.2 Have any of the above had skin or melanoma cancer? 
3. On a scale from 0-10, how significant do you consider your risk of developing skin or melanoma 
cancer? (0: No risk, 10: High risk) 
4. How often do you think about your risk of developing skin or melanoma cancer? (Options: Often, 
occasionally, rarely, never) 
 
 

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Appendix 2: 
Qualitative Interview: 
The interviews will revolve around the main questions: 
 
What happens to the target audience's risk perception when they are informed about UV-induced 
DNA damage after sun exposure? 
 
Was it successful in making the abstract risk tangible through this bio-feedback method? 
Theme 1: Everyday Life and Relationships: To gain a comprehensive understanding of the informant's 
world, questions will be asked about their daily life, work, hobbies, family life, etc. 
Theme 2: Health History and Habits: To understand what might have influenced the informant's 
understanding of risk regarding illness, questions will be asked about the informant's health and medical 
history, current health habits, with a specific focus on sun and tanning bed habits, and behavior during sun 
vacations. Additionally, questions will be asked about secondary exposure to cancer diseases (especially 
skin or melanoma cancer) among close relations. 
Theme 3: Reactions to Urine Test Results: To investigate reactions to the results of the informant's urine 
test, questions will be asked about the informant's understanding of the test results, potential intended 
behavioral changes, and risk perception regarding skin or melanoma cancer.