Mutagenesis, Vol. 17, No. 3, 189-191,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Measurement of cyclobutane thymidine dimers in melanocytic nevi and surrounding epidermis in human skin in situ
Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden and 1 Department of Dermatology, Päijät-Häme Central Hospital, FIN-15850 Lahti, Finland
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Abstract |
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The numbers of melanocytic nevi, localized benign proliferations of melanocytes, have been shown to be associated with an increased risk for development of melanoma. In the present study we have developed an alternative post-labelling method for determination of levels of cyclobutane thymidine dimers (T=T) as dinucleotides at sensitivities sufficient for analysis of human skin samples. Using the developed method, the induction of T=T was determined in melanocytic nevi in situ and surrounding skin, obtained from seven subjects, after exposure to solar simulating radiation. The T=T level in nevi was found to be 1- to 4.5-fold lower than that in surrounding skin and the difference was statistically significant (Student's t-test, P < 0.05).
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Introduction |
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Solar radiation plays an important role in life on Earth. However, besides its beneficial effects, it also has several deleterious effects on humans, such as ageing of the skin, local and systemic immunosuppression and induction of skin cancer. For human health it is very important to maintain the balance between the beneficial and harmful effects of solar radiation (Diffey, 1998
). Skin cancer is increasing, especially in developed countries, due to several factors, like increasing sunbathing and sun tourism (de Gruijl, 1999). Ultraviolet radiation (UVR), the most biologically active component of sunlight, is an important cause of skin cancers and the DNA damage induced by it may play a direct role in the initiation of skin cancers (Kraemer, 1997). The main products induced by UV are dipyrimidine lesions: cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine–pyrimidone photoproducts. These lesions may influence cellular functions such as replication, transcription and DNA repair (Tornaletti and Pfeifer, 1995). Photodamage is mainly repaired by the nucleotide excision repair system (Sarasin, 1999). Unrepaired or misrepaired UV-induced DNA damage can deliver misinformation during replicative bypass, leading to fixation of mutations that in turn relate to cancer.
There is growing evidence that the numbers of melanocytic nevi, the localized benign proliferations of melanocytes, are strongly associated with an increased risk for development of melanoma (Garbe et al., 1994; Grulich et al., 1996; Tucker et al., 1997; Briollais et al., 2000; Naldi et al., 2000). However, information about the induction of photoproducts in human nevi in situ is not available. In our previous studies, the formation and repair of cyclobutane thymidine dimers (T=T) were investigated in human skin DNA (Bykov et al., 1999; Xu et al., 2000a, 2000b). However, T=T was assayed as trinucleotides with an unmodified nucleotide (thymine) on the 5'-side, i.e. TT=T, which represents only a fraction of the total level of T=T. The aim of the present study was to develop an alternative method to analyse UV photoproducts by measuring T=T as dinucleotides at sensitivities sufficient for analysis of human skin samples. Using the developed method, the induction of T=T was determined in melanocytic nevi in situ and surrounding skin after exposure to solar simulating radiation (SSR).
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Materials and methods |
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Study population
The study was approved by the Medical Ethics Committee of Päijät-Häme Central Hospital, Lahti, Finland. All participants gave their informed consent. Altogether seven healthy volunteers (three female and four male) were included in the study. Their mean age was 45 years (range 20–64 years).
Solar simulating radiation (SSR) and UV exposures
A broadband Philips (HP 411/A) solarium was used as the SSR source to induce photoproduct formation in the moles and their surroundings. The spectral distribution of the lamp, which closely mimics the spectral distribution of noon summer sunlight in Helsinki (Snellman et al., 1995), was measured (280–400 nm) prior to the study at 30 cm using a spectroradiometer, 97.8% of the irradiance being UVA and 2.2% UVB. The unweighted UVB irradiance was 0.38 mW/cm2 and UVA irradiance 17 mW/cm2. The respective CIE (Commission Internationale de l'Éclairage) weighted figures were 0.03 mW/cm2 for UVB and 0.01 mW/cm2 for UVA. The lamp emitted no UVR at wavelengths below 290 nm. All the moles and their surrounding tissue were exposed to a dose of 40 mJ/cm2 CIE of SSR.
Sampling of skin biopsies
Before sampling, the diameters of the target moles were measured and the moles were assessed for colour, border and symmetry. Only symmetrical, benign-looking moles with no recent change in outlook were accepted for the study. The clinical type of the mole (junctional, compound or intradermal) and their colour were defined prior to UV irradiation, thus three moles were regarded as junctional, three were compound and one was intradermal. In each subject, one mole with its surrounding skin was irradiated using a dose of 40 mJ/cm2 CIE. Immediately after the UV exposure a 4 mm punch biopsy was taken using lidocaine + epinephrine local anesthesia. All the skin biopsies were stored at–20°C until DNA extraction.
DNA hydrolysis
After separation of the epidermis from the dermis with a blunt scalpel, DNA extraction from the epidermis was performed as described previously (Bykov and Hemminki, 1995). DNA was hydrolysed as described (Bykov and Hemminki, 1995) in order to obtain trinucleotides containing a dimeric lesion. Thus incubation with snake venom phosphodiesterase (3.7 mU/µg DNA in 8 mM Tris–HCl, pH 7.5, 3.2 mM MgCl2, final concentration) was carried out for 4 h at 37°C. Then prostatic acid phosphatase (25 mU/µg DNA) was added (20 h incubation at 37°C). As an additional step, not present in the original protocol, a third incubation was done with spleen phosphodiesterase (4 mU/µg DNA in 16 mM ammonium acetate, pH 5, final concentration) for 2 h at 37°C, to remove the base in front of the dimer. After digestion of the DNA all enzyme reactions were stopped in a boiling water bath for 10 min. Ethanol precipitation of the enzymes was achieved by adding 100 µl of freezer-cold absolute ethanol to the samples. The samples were kept for 40 min at –20°C and then spun at 14 000 r.p.m. for 15 min at 4°C. The supernatant was collected and dried in a vacuum centrifuge.
As shown previously (Le Curieux and Hemminki, 2001), the cyclobutane thymidine dimer cannot be directly labelled by T4 polynucleotide kinase. Therefore, reversion to the parent dinucleotide, TpT, which is easily labelled, was carried out by irradiation with UVC at 254 nm. A Stratalinker UV Crosslinker 2400 was used for the irradiation with almost monochromatic 254 nm light. Reversion took place by irradiating for 10 min at 40 mJ/m2/s. The reversion rate ranged from 30 to 50%. The results were normalized based on recovery of the standard.
The DNA samples were radioactively labelled on the 5'-side with a 32P phosphate group, using the protocol described previously (Bykov and Hemminki, 1995).
Chromatographic system
The system used for HPLC consisted of an ISCO model 2360 gradient programmer, an ISCO model 2350 pump and an ISCO CV4 capillary absorbance UV detector. This system was connected to a Packard flow scintillation analyser 500 TR series. The 32P-labelled DNA samples were analysed using a C18 Luna column (5 µm, 2x250 mm) from Phenomenex (Genetec, Kungsbacka, Sweden). This column was connected to a C18 Kromasil pre-column (5 µm, 2x50 mm; Phenomenex) and a pre-column filter. The pre-column and analytical column were separated by a switch system. During the first 7 min the eluate from the pre-column, predominantly consisting of unreacted 32P isotope, was discarded to prevent contamination of the analytical column, thus lowering the baseline and improving sensitivity. The column was eluted isocratically for 5 min with 95% buffer (200 mM ammonium formate, 20 mM o-phosphoric acid, pH 4.6) and 5% methanol, followed by a gradient to 18% methanol over 60 min and then to 100% methanol over 10 min.
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Results |
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Since T=T cannot be labelled directly, a reversion step in which T=T was reverted to its parent compound TpT by irradiation was performed before labelling. Consequently, quantification of T=T in human samples was based on determination of TpT.
In Figure 1 a spiking experiment is shown for the TpT standard and a fraction collected from a human skin sample. Co-elution of the standard with the second peak in the collected fraction shows that the peak present in the human skin sample is indeed TpT. Equal amounts of standard and collected fraction were used for spiking. Two additional experiments were carried out to verify the authenticity. Firstly, co-elution of the human product with the TpT standard was confirmed in two independent HPLC systems. Secondly, the human product was converted back to T=T by irradiation with UVB: the HPLC profile was identical to the standard TpT.
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Figure 2
shows an example of a chromatogram of nevus and surrounding skin samples of the same subject. The upper chromatogram shows the surrounding skin, while the lower shows the nevus sample. An arrow indicates the TpT peak, formed from a cyclobutane thymidine dimer. The TpT peak in the surrounding skin sample is 
4.5 times higher than that in the nevus.
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The T=T levels in nevi and surrounding skin of all subjects are summarized in Table I
. The mean T=T level in nevi was 4.7 ± 3.2, and 9.3 ± 3.0/106 nt in surrounding skin. The difference in T=T level was statistically significant (Student's t-test, P < 0.05) and was found to be 1- to 4.5-fold lower in nevi than in surrounding skin.
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To test the reproducibility of the method, two experiments were carried out on surrounding skin samples containing sufficient amounts of DNA for parallel measurements. One was carried out using four human skin samples (mean T=T level 6.4 ± 0.6/106 nt). The other experiment was carried out with three samples (mean T=T level 8.5 ± 1.9/106 nt). This resulted in mean variation/mean values of 10 and 22%, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The aim of this project was to develop an alternative method to measure UV-induced cyclobutane pyrimidine dimers at sensitivities sufficient for analysis of human skin samples. Previously, a method in which trinucleotides are measured (Bykov and Hemminki, 1995
) was used. Even though successfully used in many human studies (Bykov et al., 1999; Xu et al., 2000a, 2000b), the method has the disadvantage that any photoproduct is present in four fractions, because the third nucleotide can be any one of the four DNA nucleotides. As investigated before in human urine samples (Le Curieux and Hemminki, 2001), it is possible to analyse T=T dimers and (6–4) photoproducts by reverting them to the parent compound (TpT) and then post-labelling the TpT radioactively. The advantage of analysis of photodamage in skin samples over analysing it in urine samples is that there is no delay due to repair mechanisms and transport of the photoproducts to the urinary system. We were only able to detect TpT in these human samples. T=C and C=T dimers are probably not stable enough and thus are degraded during the procedure.
The advantage of this newly developed method is that it is possible to measure the total amount of T=T. In the trinucleotide method, one measures only 25% of the total amount of T=T, assuming that the frequencies of AT=T, CT=T, GT=T and TT=T are similar. The gain in sensitivity will be particularly useful in DNA repair studies in which the disappearance of photoproducts is being followed.
The results of the analysis of nevi and surrounding skin samples show that the level of T=T is significantly lower in nevi than in surrounding skin, with a difference of 1- to 4.5-fold. This may be due to the melanin content of nevi. This is in agreement with an in vitro study (Kobayashi et al., 1993) that showed that melanin reduces the formation of photoproducts in UV-irradiated melanoma cells in a melanin concentration-dependent manner. In human skin in situ it has been shown that constitutional pigmentation efficiently guards DNA against the formation of photoproducts (Bykov et al., 2000). The large interindividual variation in the levels of T=T in both nevi and surrounding skin may relate to individual susceptibility to UV. In the present study, the ratio between T=T level in surrounding skin and in nevi is comparable to our previous study in which similar samples were analysed using the trinucleotide method (Zhao et al., 2002). These results confirm the usefulness of the new method.
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Acknowledgments |
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The work was supported by the Swedish Cancer Society and the Swedish Radiation Protection Institute.
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Notes |
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2 To whom correspondence should be addressed. Tel: +46 8 6089243; Fax: +46 8 6081501; Email: kair hemminki{at}cnt.ki.se
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References |
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Received on September 17, 2001; accepted on December 12, 2001.
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