The relative cytotoxicity of (6-4) photoproducts and cyclobutane dimers in mammalian cells

Abstract— The significance of the pyrimidine(6-4)pyrimidone photoproduct in mammalian cell killing is considered. Photochemical data indicate that the(6–4) photoproduct is induced at a substantial frequency compared to the cyclobutane dimer and that the action spectra for the induction of both lesions are equivalent. The repair of(6–4) photoproducts in various normal and UV-hypcrsensitive mammalian cell lines, including several recently derived somatic cell hybrids and transformants, is presented. The sensitivity of these cells to ultraviolet irradiation correlates better with the capacity to repair(6–4) photoproducts than cyclobutane dimers. These data are used to support that idea that the(6–4) photoproduct is one of the major cytotoxic lesions induced in DNA by ultraviolet light.     

Resistance of the genome of Escherichia coli and Listeria monocytogenes to irradiation evaluated by the induction of cyclobutane pyrimidine dimers and 6-4 photoproducts using gamma and UV-C radiations

The effect of gamma and UV-C irradiation on the production of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) in DNA was investigated to compare the natural resistance of the genome of a Gram-positive bacterium and a Gram-negative bacterium against irradiation. Solution of pure DNA and bacterial strains Listeria monocytogenes and Escherichia coli were irradiated using gamma and UV-C rays. Extracted DNA from bacteria and pure DNA samples were then analysed by ELISA using anti-CPDs and anti-6-4 PPs monoclonal antibodies. The results show that gamma rays, as well as UV-C rays, induce the formation of CPDs and 6-4 PPs in DNA. During UV-C irradiation, the three samples showed a difference in their sensitivity against formation of CPDs (P≤0.05). Pure DNA was the most sensitive while the genome of L. monocytogenes was the most resistant. Also during UV-C irradiation, the genome of L. monocytogenes was the only one to show a significant resistance against formation of 6-4 PPs (P≤0.05). During gamma irradiation, for both types of lesion, pure DNA and the genome of E. coli did not show significant difference in their sensitivity (P>0.05) while the genome of L. monocytogenes showed a resistance against formation of CPDs and 6-4 PPs.     

Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.     

Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial, Monodelphis domestica

Radioimmunoassays were used to investigate the repair of cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone photoproducts ((6-4] photoproducts) in the epidermis of the South American opossum, Monodelphis domestica. In the absence of photoreactivating light, both types of photodamage were excised with similar kinetics, 50% of the damage remaining 8 h after UV irradiation in vivo. Exposure of UV-irradiated skin to photoreactivating light resulted in removal of most of the cyclobutane dimers and an enhanced rate of (6-4) photoproduct repair. Photoenhanced excision repair of non-dimer damage increases the range of biologically effective lesions removed by in vivo photoreactivation.     

Melanin content of cultured human melanocytes and UV-induced cytotoxicity

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.     

Formation of cyclobutane pyrimidine dimers and 8-oxo-7,8-dihydro-2'-deoxyguanosine in mouse and organ-cultured human skin by irradiation with broadband or with narrowband UVB

Narrowband UVB (NB-UVB) is a newly developed UVB source that, in addition to the previously used broadband UVB (BB-UVB), has been effectively used in phototherapy of various skin diseases. Besides its therapeutic effectiveness, NB-UVB also has some adverse effects that should be evaluated. As with all phototherapies, the photocarcinogenic potential of NB-UVB is the major concern. To assess the carcinogenic potential we measured the DNA damage induced by the two UVB sources because exposure of cells to UVB directly or indirectly induces DNA damage such as cyclobutane pyrimidine dimers (CPD) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. These types of DNA damage cause mutations of oncogenes and tumor suppressor genes, which can lead to photocarcinogenesis. In the present study we measured the yield of CPD and the oxidative DNA damage marker, 8-oxodGuo, in organ-cultured human skin and in mouse skin after exposure to NB-UVB or BB-UVB at therapeutically equivalent doses. We show that a 10-fold higher dose of NB-UVB yields a similar amount of CPD compared with BB-UVB in two types of samples examined. In contrast to CPD, the formation of 8-oxodGuo after irradiation with NB-UVB at a 10-fold higher dose is 1.5-3 times higher than that caused by BB-UVB. These results suggest that although NB-UVB at equivalent erythema-edema doses is not more potent in inducing CPD formation than is BB-UVB, NB-UVB may generate a higher yield of oxidized DNA damage.     

Naringenin protects HaCaT human keratinocytes against UVB-induced apoptosis and enhances the removal of cyclobutane pyrimidine dimers from the genome

Many naturally occurring agents are believed to protect against UV-induced skin damage. In this study, we have investigated the effects of naringenin (NG), a naturally occurring citrus flavonone, on the removal of UVB-induced cyclobutane pyrimidine dimers (CPD) from the genome and apoptosis in immortalized p53-mutant human keratinocyte HaCaT cells. The colony-forming assay shows that treatment with NG significantly increases long-term cell survival after UVB irradiation. NG treatment also protects the cells from UVB-induced apoptosis, as indicated by the absence of the 180 base pair DNA ladders and the appearance of sub-G1 peak using agarose gel electrophoresis and flow cytometric analysis, respectively. The UVB-induced poly (ADP-ribose) polymerase-1 (PARP-1) cleavage, caspase activation and Bax/Bcl2 ratio were modulated following NG treatment, indicating an antiapoptotic effect of NG in UVB-damaged cells that occurs at least in part via caspase cascade pathway. Moreover, treatment of UVB-irradiated HaCaT cells with NG enhances the removal of CPD from the genome, as observed by both direct quantitation of CPD in genomic DNA and immuno-localization of the damage within the nuclei. The study provides a molecular basis for the action of NG as a promising natural flavonoid in preventing skin aging and carcinogenesis.     

Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose-dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 +/- 8, 36 +/- 10, 57 +/- 12 and 80 +/- 10, and total percent dermal cells positive for CPD ranged from 1 +/- 1, 7 +/- 3, 16 +/- 3 and 20 +/- 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.     

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.     

Electron-transfer induced repair of 6-4 photoproducts in DNA: a computational study

The mechanism employed by DNA photolyase to repair 6-4 photoproducts in UV-damaged DNA is explored by means of quantum chemical calculations. Considering the repair of both oxetane and azetidine lesions, it is demonstrated that reduction as well as oxidation enables a reversion reaction by creating anionic or cationic radicals that readily fragment into monomeric pyrimidines. However, on the basis of calculated reaction energies indicating that electron transfer from the enzyme to the lesion is a much more favorable process than electron transfer in the opposite direction, it is suggested that the photoenzymic repair can only occur by way of an anionic mechanism. Furthermore, it is shown that reduction of the oxetane facilitates a mechanism involving cleavage of the C-O bond followed by cleavage of the C-C bond, whereas reductive fragmentation of the azetidine may proceed with either of the intermonomeric C-N and C-C bonds cleaved as the first step. From calculations on neutral azetidine radicals, a significant increase in the free-energy barrier for the initial fragmentation step upon protonation of the carbonylic oxygens is predicted. This effect can be attributed to protonation serving to stabilize reactant complexes more than transition structures.     

Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate

The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.     

The dose dependence of cyclobutane dimer induction and repair in UVB-irradiated human keratinocytes

UVB and UVA components of the solar spectrum or from artificial UV-sources might be important etiological factors for the induction and development of skin cancer. In particular, deficiencies in the capacity to repair UV-induced DNA-lesions have been linked to this phenomenon. However, until now only limited data are available on the biological and physical parameters governing repair capacity. We have, therefore, developed a flowcytometric assay using fluorescence-labeled monoclonal antibodies to study the dose-dependence of induction and repair of UVB-induced cyclobutane pyrimidine dimers in a spontaneously immortalized keratinocytic cell line (HaCaT). Our results show that the kinetics of recognition and incision of UVB-induced DNA lesions slows down by a factor of about 3 in a dose range of 100-800 J m-2. Furthermore, a thorough analysis of repair kinetics indicates that this reduction in repair capacity might not be dependent on saturation of enzymatic repair capacity (Michaelis-Menten) but may be caused by a UV-induced impairment of enzymes involved in DNA repair. Because this effect is evident in vitro at doses comparable to the minimal erythemal dose in vivo, our results might have significant impact on risk assessment for UV-induced carcinogenesis.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

Photodynamic activities of silicon phthalocyanines against achromic M6 melanoma cells and healthy human melanocytes and keratinocytes

Dichlorosilicon phthalocyanine (Cl2SiPc) and bis(tri-n-hexylsiloxy) silicon phthalocyanine (HexSiPc) have been evaluated in vitro as potential photosensitizers for photodynamic therapy (PDT) against the human amelanotic melanoma cell line M6. Each photosensitizer is dissolved in a solvent-PBS mixture, or entrapped in egg-yolk lecithin liposomes or in Cremophor EL micelles. The cells are incubated for 1 h with the sensitizer and then irradiated for 20 min, 1 h or 2 h (lambda > 480 nm, 10 mW cm-2). The photocytotoxic effect is dependent on the photosensitizer concentration and the light dose. Higher phototoxicity is observed after an irradiation of 2 h: treatment with a solution of photosensitizer (2 x 10(-9) M) leads to 10% (HexSiPc in egg-yolk lecithin liposomes) or 20% (Cl2SiPc in DMF-PBS solution) cell viability. After 1 h incubation and 20 min of light exposure, the photodynamic effect is connected with the type of delivery system used. For HexSiPc, lower cell viability is found when this photosensitizer is entrapped in egg-yolk lecithin instead of solvent-PBS or for Cremophor EL micelles with Cl2SiPc. Liposome-delivered HexSiPc leads to lipid damage in M6 cells, illustrated by an increase of thiobarbituric acid-reacting substances (TBARs), but the change is not significant with Cremophor EL. The same is observed for the antioxidative defences after photodynamic stress. The cells irradiated with HexSiPc entrapped in liposomes display an increase of superoxide dismutase (SOD) activity and a decrease of glutathione (GSH) level, glutathione peroxidase (GSHPx) and catalase (Cat) activities.     

Electron tunneling pathways and role of adenine in repair of cyclobutane pyrimidine dimer by DNA photolyase

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a superexchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds, while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways, and the critical role of adenine ensure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron-transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding that the forward and futile back-electron transfer is in the normal region and that the final electron return of the catalytic cycle is in the inverted region.     

4-甲胺基-6-三氟甲基-2-甲砜基嘧啶的合成及其晶体结构

以三氟乙酰乙酸乙酯和硫脲为原料,经环化、氯化和亲核取代反应制得中间体4-甲胺基-6-三氟甲基-2-甲硫基嘧啶(3); 以间氯过氧苯甲酸为氧化剂,3经氧化反应合成了新化合物--4-甲胺基-6-三氟甲基-2-甲砜基嘧啶(4),其结构经¹H NMR,MS,元素分析和X-射线单晶衍射表征。 4（CCDC：973037）属单斜晶系,空间群:P₂(1)/c,晶胞参数a=5.068 7(4) , b=15.296 8(11) , c=13.833 9(10) , β=13.833 9(10)°, V=1 065.81(14) ³, Z=4, Dc＝1.591 g·cm-³, μ=0.336 mm^-1, F(000)=520, R₁=0.051 1, wR₂=0.135 3。     

Ab initio study of the (5R)- and (5S)-TT pyrimidine h5(6-4) pyrimidone photoproducts. Implications on the design of new biologically relevant analogues

A computational study of a series of N(1)- and/or C(6)-alkyl-5,6-dihydrothymine diastereomers at theory levels up to MP4(SDTQ)/6-31G//HF/6-31G and MP2/6-311G//HF/6-31G has demonstrated the respective importance of the substituents at positions 1, 5, and 6 on the energetically favored conformation of each isomer. Results obtained both in the gas and condensed phase indicate that unsubstitution of the N(1)-position favors a half-chair conformation with the C(5) -and C(6)-substituents in the equatorial position. On the other hand, in the case of the (6S)-1,6-dimethyl-5,6-dihydrothymine, the C(6)-substituent adopts the axial position to minimize its van der Waals interactions with the N(1)-substituent. Furthermore, if the configuration at the C(5)-dihydrothymine position has no resultant influence on the total molecular free energy, when a pyrimidone substituent is introduced at the dihydrothymine C(6)-position, additional repulsive forces between the C(5)- and C(6)-substituents make the diaxially substituted half-chair conformation the most energetically favorable one. These results indicate that the observed C(6)-axially substituted conformation of the thymine-thymine pyrimidine h(5)(6-4) pyrimidone photoproducts is not necessarily induced by the macrocyclic structure. They also nicely explain the formation mechanism of these photoproduct derivatives, and allow the prediction of the conformation of new analogues.