CORRELATION OF UVC AND UVB CYTOTOXICITY WITH THE INDUCTION OF SPECIFIC PHOTOPRODUCTS IN T-LYMPHOCYTES AND FIBROBLASTS FROM NORMAL HUMAN DONORS

Abstract— By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.     

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions(†)

The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.     

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra‐S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6‐4 pyrimidine–pyrimidone photoproducts was highest in DNA from UVC‐irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA–UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8‐oxo‐7,8‐dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.     

N4‐Methylation of Cytosine Drastically Favors the Formation of (6‐4) Photoproducts in a TCG Context

Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5‐methylcytosine (5mC), some bacterial species contain in their genome N⁴‐methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV‐induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6‐4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non-radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB-induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

Effects of cytosine methylation on pyrimidine dimer formation in DNA

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)pyrimidone photoproducts ([6-4]PD) was quantified in the duplex homopolymers polydeoxyadenosine:polydeoxythymidine, polydeoxyguanosine:polydeoxycytidine and polydeoxyguanosine:polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6-4)PD after irradiation with UVC light and of cytosine CPD and (6-4)PD after irradiation with UVB light. The data suggest that CPD and (6-4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.     

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation,

This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2‐deoxyribose moieties of DNA in isolated cells and skin. The UVA‐induced generation of 8‐oxo‐7,8‐dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal‐catalyzed Haber–Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single‐strand breaks at the exclusion, however, of direct double‐strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA‐induced DNA damage could include single and more complex lesions arising from one‐electron oxidation of the guanine base together with aldehyde adducts to amino‐substituted nucleobases.     

Ultraviolet B but not A radiation activates suppressor B cells in draining lymph nodes

Immunosuppressive doses of solar-simulated UV radiation activate lymph node B cells that can suppress primary immunity by inhibiting the function of dendritic cells. The aim of this study was to determine the waveband responsible for activation of these suppressor B cells. We exposed C57BL/6 mice to various doses of either UVA or UVB radiation and analyzed the number and activation state of lymph node antigen-presenting cells (APC). Immunosuppressive doses of UVB but not UVA activated B cells as assessed by major histocompatibility complex II (MHC II) expression and doubled their numbers in draining lymph nodes. Higher doses of UVA that were not immunosuppressive actually suppressed B cell activation. Our results show that UVA and UVB suppress systemic immunity via different mechanisms. Lymph node B cells are activated in response to immunosuppressive doses of UVB but not UVA. Thus, the activation state of lymph node APC appears to be important for UV immunomodulation.     

QUANTUM YIELDS AND SECONDARY PHOTOREACTIONS OF THE PHOTOPRODUCTS OF dTpdT, dTpdC AND dTpdU

Abstract— The photoproducts of the dinucleoside monophosphates, dTpdT, dTpdC and dTpdU, have been purified by high performance liquid chromatography and characterized by UV absorption spectroscopy, fast atom bombardment mass spectrometry and by secondary thermal and photoreactions. Four types of photoproducts were analyzed: (1) cyclobutane dimers including cis-syn isomers and two diastereomers of the trans-syn isomers; (2) 6-4 photoadducts and the corresponding Dewar valence isomers; (3) photohydrates comprising two diastereomers and (4) a new photoproduct resembling nucleobase amine adducts, which occurs only for dTpdC. The quantum yields of formation of these photoproducts and for some secondary photoreactions were measured by kinetic analysis of the photoproduct yield as a function of photon fluence. These results indicate that cis-syn cyclobutane dimers are the photoproducts formed with highest efficiency with dT[p]dC dimers being formed with 50–75% the efficiency of dT[p]dT dimers. The 6-4 photoadducts are formed with 5–10% the efficiency of cis-syn cyclobutane dimers and the 6-4 photoadduct of dTpdC is formed two to three times more efficiently than that of dTpdT. Photohydrates are also formed efficiently due to an equilibrium between stacked and unstacked complexes of the dinucleoside monophosphates. It is shown that three of these photoproducts, namely the cyclobutane dimers of dTpdC, the 6-4 photoadducts and the possible nucleobase amine adduct, undergo photolysis in the UV-B region resulting in either photoreversion or secondary photoreaction.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

An immunoassay based on CE–LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL⁻¹) of DNA under a low UVB fluence of 65 J m⁻² for CPDs or 195 J m⁻² for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.     

Direct and indirect effects of UV radiation on DNA and its components.

In this survey, emphasis was placed on the main photoreactions of nucleic acid components, involving both direct and indirect effects. The main UVB- and UVA-induced DNA photoproducts, together with the mechanisms of their formation, are described. Information on the photoproduct distribution within cellular DNA is also provided, taking into account the limitations of the different analytical methods applied to monitor the formation of the DNA damage. Thus, the formation of the main DNA dimeric pyrimidine lesions produced by direct absorption of UVB photons was assessed using a powerful HPLC-tandem mass spectrometry assay. In addition, it was found that UVA photooxidation damage mostly involves the guanine residues of cellular DNA as the result of singlet oxygen generation by still unknown endogenous photosensitizers.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Electrospray-mass spectrometry characterization and measurement of far-UV-induced thymine photoproducts

Far-UV-induced formation of dimeric pyrimidine photoproducts within DNA is a major cause of the carcinogenic effects of solar light. The chemical structure of this class of lesion has been mostly determined by studies on model compounds. The present work is aimed at providing mass spectrometry data on the thymine-thymine photoproducts, including the diastereoisomers of the cyclobutane dimer, the (6-4) adduct, the related Dewar valence isomer and the spore photoproduct. Fragmentation mass spectra of the modified bases, nucleosides, dinucleoside monophosphates and dinucleotides were recorded following electrospray ionization with either triple-quadrupolar or ion-trap detection. The results showed differences in fragmentation pattern between the different types of photoproducts. In addition, a drastic effect of the diastereoisometry was observed for the cyclobutane dimers. A sensitive detection technique has been developed for the analysis of dinucleoside monophosphate photoproducts by high-performance liquid chromatography associated with mass spectrometry in the negative mode with multiple reaction-monitoring detection.     

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm^?1 M^?1 at 300 nm) compared with the UVA region (2016 cm^?1 M^?1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.     

6‐Formylindolo[3,2‐b]carbazole (FICZ) is a Very Minor Photoproduct of Tryptophan at Biologically Relevant Doses of UVB and Simulated Sunlight

Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring‐opened and oxidized photoproducts. One of them, 6‐formylindolo[3,2‐b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biologic processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC‐tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02 and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered the only endogenous photoproduct responsible for the induction of AhR‐dependent responses in UV‐irradiated cells.     

Far-UV photochemistry and photosensitization of 2'-deoxycytidylyl-(3'-5')-thymidine: isolation and characterization of the main photoproducts.

Far-UV irradiation of 2'-deoxycytidylyl-(3'-5')-thymidine (dCpT) gave rise to the pyrimidine (6-4) pyrimidone adduct and its Dewar valence isomer as the main photoproducts. The absolute configuration of the former adduct was determined and its photoisomerization studied. A comparison of the alkali lability of both compounds showed that hydrolysis of the phosphodiester bond occurs for the Dewar valence isomer but not for its (6-4) precursor. In addition, the trans-syn and cis-syn cyclobutane dimers of dCpT were obtained by acetophenone photosensitization and characterized. Finally, the deamination rate constants for this series of compounds were shown to be dramatically influenced by the nature and the configuration of the photoproducts.