Formation of the main UV-induced thymine dimeric lesions within isolated and cellular DNA as measured by high performance liquid chromatography-tandem mass spectrometry

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.     

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.     

紫外线B区对小牛胸腺DNA损伤的拉曼光谱研究

用拉曼光谱检测了远紫外区UVB(280m～320nm)对小牛胸腺DNA的损伤,并对这个区段紫外线对DNA的不同照射时间时的损伤特征进行了比较分析。实验中所用的紫外线强度与太阳光强度相当。结果表明,辐照3h以内时,UVB对DNA的构型有较明显的损伤,这可能是受到嘧啶碱基损伤的影响。相对而言,UVB对脱氧核糖和碱基的损伤要严厉得多。就碱基对的受损伤程度来说,嘧啶碱受损最严重,部分证明了环丁烷嘧啶二聚体和6-4光产物的形成。经过3h UVB照射,AT碱基对和和胞嘧啶环的堆叠程度有所瓦解,一些碱基对受到修饰。而一些拉曼特征峰强度的反复波动,则说明了长时间的紫外照射可以导致部分DNA光复活的产生。进一步分析发现,UVB以一种较快的方式对DNA产生损伤。     

Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320-340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290-320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC-MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (lambda > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidine dimers (CPDs) are formed in a 1:1:3:10 ratio. In addition, we demonstrate that, in contrast to UVB and sunlight, UVA generates CPDs with a large predominance of TT CPDs, which strongly suggests that they are formed via a photosensitized triplet energy transfer. Moreover, UVA induces neither (6-4) photoproducts nor their Dewar isomers via direct absorption. We also show that UVA photons contained in sunlight, rather than UVB, are implicated in the photoisomerization of (6-4) photoproducts, a quickly repaired damage, into poorly repaired and highly mutagenic Dewar photoproducts. Altogether, our data shed new light on the deleterious effect of UVA.     

The greater lethality of UVB radiation to cultured human cells is associated with the specific activation of a DNA damage-independent signaling pathway

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.     

单细胞凝胶电泳联合原子力显微镜观测UVB诱导的细胞DNA损伤模式变化

目的：检测UVB诱导的真核细胞DNA损伤。方法：采用单细胞凝胶电泳与原子力显微镜。结果：不同照射剂量的UVB引起的真核细胞DNA损伤模式不同。在0～20J／m2照射剂量范围内DNA无损伤;在20--360J／m2照射剂量范围内DNA损伤程度加快;当照射剂量超过360J／m2时DNA损伤速度减慢,实验组之间无显著性差异,出现“平台”。原子力显微镜的观察结果表明随着UVB照射剂量的增加,DNA结构的变化经历了断裂、交联与断裂并存的损伤增强趋势。当照射能量达到280J／m2时细胞DNA大都形成断片,并相互交联在一起。这一结果表明彗星电泳检测到的UVB照射剂量达到一定剂量后,DNA损伤出现”平台”的原因可能是此时DNA发生了链内或链间交联。结论：不同照射剂量的UVB造成的细胞DNA损伤模式不同;原子力显微镜是一种比较直观的观测DNA损伤的方法。借助原子力显微镜我们可以深入了解单细胞凝胶电泳检测的原理,为DNA损伤检测提供更优良的检测手段。     

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.     

Molecular response of nasal mucosa to therapeutic exposure to broad-band ultraviolet radiation

Ultraviolet radiation (UVR) phototherapy is a promising new treatment for inflammatory airway diseases. However, the potential carcinogenic risks associated with this treatment are not well understood. UV-specific DNA photoproducts were used as biomarkers to address this issue. Radioimmunoassay was used to quantify cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts in DNA purified from two milieus: nasal mucosa samples from subjects exposed to intranasal phototherapy and human airway (EpiAirway™) and human skin (EpiDerm™) tissue models. Immunohistochemistry was used to detect CPD formation and persistence in human nasal biopsies and human tissue models. In subjects exposed to broadband ultraviolet radiation, DNA damage frequencies were determined prior to as well as immediately after treatment and at increasing times post-treatment. We observed significant levels of DNA damage immediately after treatment and efficient removal of the damage within a few days. No residual damage was observed in human subjects exposed to multiple UVB treatments several weeks after the last treatment. To better understand the molecular response of the nasal epithelium to DNA damage, parallel experiments were conducted in EpiAirway and EpiDerm model systems. Repair rates in these two tissues were very similar and comparable to that observed in human skin. The data suggest that the UV-induced DNA damage response of respiratory epithelia is very similar to that of the human epidermis and that nasal mucosa is able to efficiently repair UVB induced DNA damage.     

Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.     

Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis

In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.     

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.     

Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three-photon near-infrared absorption

The high-resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near-diffraction-limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell–cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near-infrared radiation and suggest how it may be used for new biological investigations.     

Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA.

Hot piperidine is often used to cleave abasic and UV-irradiated DNA at the sites of damage. It can inflict non-specific damage on DNA, probably because it is a strong base and creates significant concentrations of hydroxyl ions which can attack purines and pyrimidines. We show that several other amines can cleave abasic DNA at or near neutral pH without non-specific damage. One diamine, N,N'-dimethylethylenediamine, efficiently cleaves abasic DNA at pH 7.4 by either beta- or beta,delta-elimination, depending on temperature. Using end-labelled oligonucleotides we show that cleavage depends mainly on elimination reactions, but that 4',5'-cyclization is also significant. This reagent also cleaves at photoproducts induced by UVC and UVB, producing the same overall pattern as piperidine, but with no non-specific damage. It should prove valuable in locating low levels of photoproducts in DNA, such as those induced by natural sunlight.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.     

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.     

Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are the two main classes of mutagenic DNA damages induced by UVB radiation. Numerous studies have been devoted so far to their formation and repair in human cells and skin. However, the biochemical methods used often lack the specificity that would allow the individual study of each of the four CPDs and 6-4PPs produced at TT, TC, CT and CC dinucleotides. In the present work, we applied an HPLC-mass spectrometry assay to study the formation and repair of CPDs and 6-4PPs photoproducts in primary cultures of human keratinocytes and fibroblasts as well as in whole human skin. We first observed that the yield of dimeric lesions was slightly higher in fibroblasts than in keratinocytes. In contrast, the rate of global repair was higher in the last cell type. Moreover, removal of DNA photoproducts in skin biopsies was found to be slower than in both cultured skin cells. In agreement with previous works, the repair of 6-4PPs was found to be more efficient than that of CPDs in the three types of samples, with no observed difference between the removal of the TT and TC derivatives. In contrast, a significant influence of the nature of the two modified pyrimidines was observed on the repair rate of CPDs. The decreasing order of removal efficiency was the following: C<>T>C<>C>T<>C>T<>T. These data, together with the known intrinsic mutational properties of the lesions, would support the reported UV mutation spectra. A noticeable exception concerns CC dinucleotides that are mutational hotspots with an UV-specific CC to TT tandem mutation, although related bipyrimidine photoproducts are produced in low yields and efficiently repaired.     

Short interspersed DNA element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin.

We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000-80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1-3 kJ m-2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m-2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1-6 kJ m-2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.