DNA targeted UVA photosensitization: characterization of an extremely photopotent iodinated minor groove binding DNA ligand

Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.     

γH2AX,an Accurate Marker That Analyzes UV Genotoxic Effects on Human Keratinocytes and on Human Skin

The phosphorylated form of histone H2AX, γH2AX, is a component of the DNA repair system. Most studies have focused on the role of γH2AX during cell transformation and human cancer, but little is known about its role in keratinocytes and the skin during UV irradiation. We analyzed the response to UV irradiation focusing on the phosphorylation of histone H2AX both in vitro, in keratinocyte cultures and in artificial epidermis, and then in vivo, in human skin. Acute UVB irradiation of human keratinocytes increased the phosphorylation of H2AX in a dose-dependent manner; two types of γH2AX response were observed either in vitro or in vivo. After a low nonapoptotic UVB irradiation, cells contained phosphorylated H2AX and arrested their cell cycle to repair the DNA damages. For a stronger and proapoptotic UVB irradiation, keratinocytes dramatically increased the phosphorylation of H2AX and committed apoptosis. Our results indicate that γH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.     

反相高效液相色谱法分离2-(氟苯基)-5-甲基苯并恶唑和2-(氯苯基)-5-甲基苯并恶唑的位置异构体

基于商品化的普通色谱柱建立了2-(氟苯基)-5-甲基苯并恶唑和2-(氯苯基)-5-甲基苯并恶唑邻、间、对位置异构体的分离检测方法。色谱柱为Inertsil ODS-SP C₁₈(250 mm×4.6 mm, 5 μm),以乙腈(A)和水(B)为流动相,在60%A~80%A间线性梯度洗脱15 min,流速为1.5 mL/min,柱温40℃,检测波长为310 nm。在质量浓度为2~200 mg/L范围内,2-(氟苯基)-5-甲基苯并恶唑邻、间、对位的异构体、2-(氯苯基)-5-甲基苯并恶唑邻、间、对位的异构体具有良好的线性关系,6种化合物的检出限(S/N=3)依次为0.0307、0.0293、0.0315、0.0226、0.0237、0.0226 mg/L。该法既为5-甲基苯并恶唑与氟苯或氯苯碳氢活化偶联反应制备的异构体混合物提供了一个快速检测的方法,又为2-芳基苯并恶唑类异构体的分离检测提供了参考。     

Phosphorylation of Histone H2AX Generated by Linear Alkylbenzene Sulfonates and its Suppression by UVB Exposure

We previously demonstrated that the nonionic surfactants, nonylphenol polyethoxylates (NPEOs) induced the phosphorylation of histone H2AX (γ‐H2AX), accompanied by DNA double‐strand breaks (DSBs), and that exposure to ultraviolet (UV) degraded NPEOs, which sometimes enhanced their DNA‐damaging ability. In this study, we showed that linear alkylbenzene sulfonates (LAS), general anion surfactants, also generated DSBs with γ‐H2AX, and this ability was attenuated by UVB exposure. In the human breast adenocarcinoma cell line, MCF‐7, γ‐H2AX was generated in a dose‐dependent manner immediately after cells were treated with LAS, and this was attributed to the formation of DSBs and was independent of cell cycle phases. The ability to generate γ‐H2AX was markedly reduced in LAS exposed to UVB. HPLC analysis revealed that LAS were a mixture of various alkyl chain lengths, the peaks of which were detected at individual retention times. UVB evenly decreased all peaks of LAS, without migration of peaks to other retention times, which indicated that UVB may degrade the benzene ring of LAS, but did not shorten the alkyl chains. UVB is an important environmental factor in the degradation of LAS exhibiting the ability to induce DSBs, the most serious type of DNA damage.     

Electron-induced isomerisation of dichlorobenzene on Cu(111) and Ag(111)

The constitutional isomerisation of single dichlorobenzene molecules adsorbed on the surfaces of Ag(111) and Cu(111) between their meta- and para-isomers is induced and investigated by means of a low temperature scanning tunneling microscope. On both substrates similar isomerisation thresholds are found indicating that the excitation mechanism of this reaction does not depend significantly on the underlying substrate. The isomerisation threshold voltage of (170 +/- 7) meV most likely corresponds to excitation of a C-C stretch mode whose gas-phase energies we calculated ab initio to lie at 174 and 172 meV for meta- and para-isomers respectively. Though the reaction is found to be localized on the submolecular scale, it depends heavily on the second substituent both in terms of excitation energy and reaction outcome.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.     

Nonylphenol Polyethoxylates Degraded by Three Different Wavelengths of UV and Their Genotoxic Change—Detected by Generation of γ‐H2AX

UV rays in sunlight are an important factor in the degradation of chemicals. In this study, we investigated the degradation of nonionic surfactants, nonylphenol polyethoxylates (NPEOs) with 10 or 70 ethylene oxide (EO) units using UVA, B and C, and their genotoxic change based on phosphorylation of histone H2AX (γ‐H2AX), a marker of DNA damage. NPEOs were degraded dependent on the energy of UV, that is, UVC having the highest energy was most effective, whereas UVA having the lowest energy caused little change. The EO side chain of NPEO(70) was broken near the benzene ring by UV, producing NPEOs with a shortened EO chain (around 10 units). The generation of γ‐H2AX reflected the pattern of degradation; shortening of the EO chain changed NPEO(70) into an inducer for γ‐H2AX, and degradation of NPEO(10) attenuated the genotoxicity. The γ‐H2AX generated by NPEO(10) and UV‐degraded NPEO(70) was independent of the cell cycle. The formation of DNA double strand breaks detected by gel electrophoresis was consistent with the results for γ‐H2AX. These results suggested that UV rays can make NPEOs harmless or genotoxic according to the degradation of the EO side chain, the effects being dependent on wavelength.     

DNA双链断裂的组成与自由基清除效能的关系

ＤＮＡ双链断裂（ＤＳＢ）是重要的辐射生物学效应之一 ,它是导致细胞死亡和变异的主要因素［１,２］．大量研究表明 ,ＤＳＢ由αＤＳＢ和 βＤＳＢ两部分组成 ,分别为电离辐射能量沉积的单击作用 (包括ＬＭＤＳ机制和单自由基传递机制 )和双击作用所致［３ -５］．但对不同的生物体系 ,由于靶体积大小等不一 ,α、β有不同的值．从根本上讲 ,α、β与电离辐射的径迹如粒子的种类、ＬＥＴ等有关 ,α在中等ＬＥＴ时最大 ,而 β则在低ＬＥＴ辐射时占优势［６］．研究α、β 与生物体系之间的关系 ,将有助于加深人们对电离辐射物理化学效应的…     

Only complete rejoining of DNA strand breaks after UVC allows K562 cell proliferation and DMSO induction of erythropoiesis

DNA strand breaks are early intermediates of the repair of UVC-induced DNA damage, however, since they severely impair cellular activities, their presence should be limited in time. In this study, the effects of incomplete repair of UVC-induced DNA strand breaks are investigated on K562 cell growth and the induction of erythroid differentiation by addition of DMSO to the cell culture medium. The kinetics were followed after UV irradiation by single cell gel electrophoresis, and in total cell population by alkaline or neutral agarose gel electrophoresis. Shortly after exposure, an extensive fragmentation occurred in DNA; DNA double strand breaks were negatively correlated with recovery time for DNA integrity. DNA damage induced by UVC 9J/m2 rapidly triggered necrosis in a large fraction of irradiated K562 cells, and only 40% of treated cells resumed growth at a very low rate within 24h of culture. The addition of DMSO to the culture medium of cells 15min after UVC, when DNA strand break repair was not yet complete, produced apoptosis in >70% of surviving cells, as determined by TUNEL assay. Conversely, if DMSO was added when the resealing of DNA strand breaks was complete, surviving K562 cells retained full growth capacity, and their progeny underwent erythroid differentiation with normal levels of erythroid proteins, delta-aminolevulinic acid dehydrase and hemoglobin. This study shows that the extent of DNA strand break repair influences cell proliferation and the DMSO induced erythroid program, and the same UVC dose can have opposite effects depending on cellular status.     

Gel electrophoresis method for quantitation of gamma ray induced single- and double-strand breaks in DNA irradiated in vitro

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single- and double-strand breaks formed by gamma-rays for DNA irradiated in vitro. For single-strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double-strand breaks doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single-strand breaks is linear while the dose response function for double-strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.     

Substituent positional variations and the lithographic properties of poly(methylstyrene-CO-chloromethylstyrene) resist systems

The lithographic performances of structurally different copolymers of methylstyrene (ortho-, meta- and para-isomers) and chloromethylstyrene (meta- and para-isomers) have been assessed. Linear correlations of the data, based on Charlesby's theory of radiation-induced crosslinking of polymers, demonstrate that sensitivity and contrast are functionally related for this system. Variation of the structure of the chloromethylstyrene component of the copolymers had little effect on the lithographic parameters, but the effect of structural variation of the methylstyrene component was pronounced, the order of increasing sensitivity being ortho < meta < para.     

γH2Ax: biomarker of damage or functional participant in DNA repair "all that glitters is not gold!"

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important for the assembly of repair complexes at DNA double strand breaks (DSBs). The formation and functional role of γH2Ax after other kinds of DNA damage, especially UV light, where DSBs are rare, is less clear. Following UV light in the UVB and UVC ranges, complex distributions of γH2Ax can be identified, quite unlike the discrete enumerable foci seen after ionizing radiation. Several distinct distributions of γH2Ax occur: a low level nuclear-wide distribution of γH2Ax occurs during nucleotide excision repair; irregular focal distributions occur at arrested replication forks; high intensity nuclear-wide γH2Ax occurs in association with S-phase apoptosis. The intensity and distributions of γH2Ax vary according to the activity of excision repair, bypass polymerase and apoptotic caspases. The frequency of DSBs at arrested replication forks is low but highly variable in different cell types, and probably caused by enzymatic action. Despite the prominence of S139 phosphorylation following UV damage, mutation of this site has no influence on the UV damage response indicating that γH2Ax is a biomarker but not a participant in the UV-DNA damage response.     

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.     

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.     

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY-INDUCED DNA DOUBLE-STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K-12

Abstract— We have recently reported that DNA double-strand breaks arise enzymatically during the course of excision repair in uvr ⁺ strains of Escherichia coli K-12. Survival curves for ultraviolet (UV) irradiated E. coli K-12 pol⁺ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double-strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double-strand breaks in both strains. These data support the hypothesis that enzymatically-induced DNA double-strand breaks may be the lesion ultimately responsible for UV-induced cell killing in the pol⁺ strain of E. coli K-12. and perhaps also in the polA1 strain.     

Determination of the isomeric distribution and associated impurities in commercial diethylbenzenes by capillary gas chromatography

A capillary gas chromatographic method for the quantitative analysis of diethylbenzene fractions is described. Estimation of ortho-, meta- and para-isomers and other C₉ and C₁₀ aromatic impurities is covered. The conditions developed involve the use of a capillary column of Carbowax-1540 (300 feet × 0.01 inch) under isothermal operation. The retention index data for a number of aromatics are presented at four temperatures (90, 100, 110 and 120°C). The method offers a good choice for any level of concentration both for isomers and impurities commonly encountered within a reasonable analysis time.     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.