UVB照射后小鼠脾淋巴细胞DNA损伤程度的检测

用强度为30 μW/cm2、60 μW/cm2、90 μW/cm2、120 μW/cm2、150 μW/cm2的中波红斑效应紫外线(UVB)分别照射小鼠脾淋巴细胞5 min、15 min、30 min,采用单细胞凝胶电泳法(SCGE)检测UVB照射对细胞DNA的损伤,结果照射5 min和15 min时,DNA损伤程度与照射强度呈正相关;而照射30 min时,30 μW/cm2组DNA损伤程度最高,60 μW/cm2、90 μW/cm2、120 μW/cm2组损伤程度有所降低,而150 μW/cm2组又出现了高DNA损伤.认为通过SCGE来检测UVB照射对小鼠脾淋巴细胞的损伤程度,可广泛应用于环境毒理学中对DNA具有损伤作用的分析,为环境生物学研究辐射对生物体的影响提供借鉴.     

核苷酸切除修复对UVB光损伤的识别机制

中波紫外线（UVB）会对皮肤造成各种损伤,这些都根源于UVB对皮肤细胞DNA的光损伤。光损伤产物主要有环丁烷嘧啶二聚体（CPD）和64光产物（6-4PP）两类,还包括少量的氧化损伤。CPD和6-4PP的修复是由核苷酸切除修复（NER）执行的。NER可分为全基因组核苷酸切除修复（GGR）和转录耦联核苷酸切除修复（TCR）两个亚途径。识别因子XPC通过一种不直接识别损伤本身的机制在GGR识别过程中发挥作用;在TCR识别过程中强调了关键因子CSB单体及二聚体两种形式的转换。在染色质水平上,DDB介导的泛素化作用是NER识别过程中重要的调控要素。另外,完成使命的识别因子的最终走向也是NER途径中的一个重要环节。通过分析上述生化过程,较清楚地总结了GGR及TCR对UVB导致的光损伤的识别机制。     

原子力显微镜观测紫外线诱导的K562肿瘤细胞DNA损伤

利用彗星电泳检测出UVB、UVC短时间照射会使肿瘤细胞的DNA发生断裂,而长时间照射之后彗星电泳无法检测到碎片,推测可能是由于DNA分子交联的原因[1],国内外尚无定论.为了更直观的研究这种现象,提取了UVB,UVA照射后K562细胞的DNA,并调节到合适的浓度在原子力显微镜下观测.实验结果表明UVB对K562肿瘤细胞DNA损伤的影响呈现时间/剂量效应,较短时间照射主要产生DNA的链断裂,较长时间辐射则主要产生DNA链的交联.UVC对K562肿瘤细胞DNA的损伤大于UVB.UVC短时照射即可引起DNA的断裂和交联,较长时间辐射主要产生交联和一些断裂;长时间照射不但产生大量交联,同时有大量断裂产生,并发生凝缩和缠绕等结构破坏.     

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。     

紫外线诱导的DNA损伤与皮肤癌的发生(1)

日光中紫外线(ultraviolet radiation,UV)诱导的DNA损伤是导致皮肤癌发生的一个重要因素。在皮肤癌细胞中发现,调控细胞增殖、分化和凋亡的特异性基因出现与紫外线损伤相关的变异。根据日光中紫外线波长的不同,可将其分为3种：UVA,UVB和UVC。UVB是导致DNA损伤的主要类型。目前已证实一些原癌基因和抑癌基因由于紫外线引起的DNA损伤而发生变异。UV诱导产生皮肤癌是一个复杂的过程,涉及到许多细胞和分子水平上的变化,同时还与DNA的修复作用相关。     

用EndoV和碱电泳法检测哺乳动物细胞嘧啶二聚体的切除修复

紫外线(UV)辐射是自然环境中重要的DNA致伤因子。UV辐射在DNA造成的最主要一类损伤产物是环丁烷嘧啶二聚体(pyrim-idine dimer,PD),它是由DNA中一条多核着酸链上两相邻嘧啶碱基各自的C5和C6共价连接形成的环丁烷结构。哺乳动物细胞主要通过切除修复途径移除PD,恢复DNA的正常结构。一种从噬菌体T4感染的E.coli中提取的T4核酸内切酶V(EndoV)能特异识别PD,并在该损伤位点切断磷酸二醋键,造成单链断裂。本文即以EndoV为探针,以其敏感位点(endonuclease-sensitive-site,ESS)     

损伤DNA 的“SOS”修复

活体细胞内的DNA会受到许多外界因素,诸如射线、带电离子、化学药物等的损伤。在活细胞中,有一系列原有的或由损伤因素诱导产生的酶,能对DNA的各种损伤进行不同形式的修复。易错修复(error-prone DNA repair),即修复时容易发生错误。它是M.Radman首先发现的。他用紫外线照射λ噬菌体和ΦX174噬菌体,然后用这种噬菌体去感染经紫外线轻度照射过的大肠杆菌,结果是被照射过的λ噬菌     

DNA光修复酶修复紫外损伤的DNA

DNA光修复酶在蓝光驱动下,利用黄素腺嘌呤二核苷酸（FAD）分子的黄素酶作为催化辅助因子,来修复紫外线诱导的环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮的DNA损伤产物。通过无根发育树,综述了DNA光修复酶/隐花色素家族的分类;详细地阐述两种DNA光修复酶的结构、光损伤后产生的嘧啶二聚体的结构及光修复过程;最后回顾了DNA光修复酶的研究现状并展望该领域的发展前景。     

UVB影响中华大蟾蜍早期胚胎发育的初步研究

为了研究UVB对两栖类动物早期胚胎发育的影响,以中华大蟾蜍为研究对象,用UVB分别以不同强度和不同时间照射其二细胞期胚胎,观察记录胚胎的发育过程、统计死亡率并在形态学上对UVB照射诱导的畸胚进行分析。结果表明,30μW／cm^2强度的UVB分别照射二细胞期胚胎20s、60s和80μW／cm。强度分别照射20s、60s、120s后,对中华大蟾蜍早期胚胎发育有促进作用,使发育过程中的死亡率降低,畸胚出现的类型较少且畸变程度低;80μW／cm。强度的UVB照射二细胞期胚胎600s,100μW／cm^2强度的UVB照射20s、60s、600s,120μw／cm。强度的UVB照射20s,导致胚胎发育过程中死亡率明显升高,畸胚数量多且畸变程度高。结论：UVB照射剂量和时间不同,对中华大蟾蜍早期胚胎发育的影响有明显差异。     

UV—A区段紫外线照射对DNA影响的拉曼光谱分析

本文检测了鲱鱼精DNA水溶液经不同时间UV－A紫外辐射后的拉曼光谱,研究结果表明,该区段紫外辐射比用UV－A和UV－B共同照射对DNA的影响要小,主链构象基本稳定。但经较长时间辐射仍会对鲱鱼精DNA造成损伤,受影响的部位主要是脱氧核糖和胸腺嘧啶碱基部分,UV－A对脱氧核糖的影响与UV－A加UV－B共同照射的结果作比较后,可以说明UV－A对脱氧核糖的损伤有累积的效应,而对于胸腺嘧啶的影响,从其各个指标的分析来看,有损伤但程度较小。本实验说明UV－A辐射条件下没有嘧啶二聚体的形成,也不存在6,4光产物形成的证明,但对于Dewar异构体的形成,有部分证明,与Taylor(1994)报道的结果相一致,UV－A没有造成DNA单链断裂现象。     

Raman spectroscopic analysis of the effect of ultraviolet irradiation on calf thymus DNA

Raman spectroscopy was used for the first time to detect the effect of independent UVA (ultraviolet-A: 320-400nm) and UVB (ultraviolet-B: 280-320 nm) irradiation on the calf thymus DNA in aqueous solution. After both UVA and UVB irradiation for 1h or 3h, the damage to the conformation of DNA was moderate, but the reduction of the B-form DNA component was obvious. Both UVA and UVB caused significant damage to the deoxyribose moiety and bases, among which the pyrimidine base pairs were more seriously affected. There appeared to be preferential damaging sites on DNA molecules caused by UVA and UVB irradiation. UVA irradiation caused more damage to the deoxyribose than UVB irradiation, while UVB irradiation caused more significant damage to the pyrimidine moiety than UVA irradiation. After UVB irradiation for 3h, unstacking of the AT base pairs and the cytosine ring took place, severe damage to the thymine moiety occurred, and some base pairs were modified. Moreover, with either UVA or UVB irradiation for 3h,the photoreactivation of DNA occurred. The damage to the DNA caused by UVB was immediate, while the damage caused by UVA was proportional to the irradiation duration. The experimental results partly indicate the formation of some cyclobutane pyrimidine dimers and (6-4) photoproducts.     

Comparison of DNA damage responses following equimutagenic doses of UVA and UVB: a less effective cell cycle arrest with UVA may render UVA-induced pyrimidine dimers more mutagenic than UVB-induced ones

Mechanisms of UVA-mutagenesis remain a matter of debate. Earlier described higher rates of mutation formation per pyrimidine dimer with UVA than with UVB and other evidence suggested that a non-pyrimidine dimer-type of DNA damage contributes more to UVA- than to UVB-mutagenesis. However, more recently published data on the spectra of UVA-induced mutations in primary human skin cells and in mice suggest that pyrimidine dimers are the most common type of DNA damage-inducing mutations not only with UVB, but also with UVA. As this rebuts a prominent role of non-dimer type of DNA damage in UVA-mutagenesis, we hypothesized that the higher mutation rate at UVA-induced pyrimidine dimers, as compared to UVB-induced ones, is caused by differences in the way UVA- and UVB-exposed cells process DNA damage. Therefore, we here compared cell cycle regulation, DNA repair, and apoptosis in primary human fibroblasts following UVB- and UVA-irradiation, using the same physiologic and roughly equimutagenic doses (100-300 J m(-2) UVB, 100-300 kJ m(-2) UVA) we have used previously for mutagenesis experiments with the same type of cells. ELISAs for the detection of pyrimidine dimers confirmed that much fewer dimers were formed with these doses of UVA, as compared to UVB. We found that cell cycle arrests (intra-S, G1/S, G2/M), mediated at least in part by activation of p53 and p95, are much more prominent and long-lasting with UVB than with UVA. In contrast, no prominent differences were found between UVA and UVB for other anti-mutagenic cellular responses (DNA repair, apoptosis). Our data suggest that less effective anti-mutagenic cellular responses, in particular different and shorter-lived cell cycle arrests, render pyrimidine dimers induced by UVA more mutagenic than pyrimidine dimers induced by UVB.     

Lethal and sub-lethal effects of UVB on juvenile Biomphalaria glabrata (Mollusca: Pulmonata)

Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects.     

Sensitivity of rice to ultraviolet-B radiation

BACKGROUND: Depletion of the stratospheric ozone layer leads to an increase in ultraviolet-B (UVB: 280-320 nm) radiation reaching the earth's surface, and the enhanced solar UVB radiation predicted by atmospheric models will result in reduction of growth and yield of crops in the future. Over the last two decades, extensive studies of the physiological, biochemical and morphological effects of UVB in plants, as well as the mechanisms of UVB resistance, have been carried out. SCOPE: In this review, we describe recent research into the mechanisms of UVB resistance in higher plants, with an emphasis on rice (Oryza sativa), one of the world's most important staple food crops. Recent studies have brought to light the following remarkable findings. UV-absorbing compounds accumulating in the epidermal cell layers have traditionally been considered to function as UV filters, and to play an important role in countering the damaging effects of UVB radiation. Although these compounds are effective in reducing cyclobutane pyrimidine dimer (CPD) induction in plants exposed to a challenge exposure to UVB, certain levels of CPD are maintained constitutively in light conditions containing UVB, regardless of the quantity or presence of visible light. These findings imply that the systems for repairing DNA damage and scavenging reactive oxygen species (ROS) are essential for plants to grow in light conditions containing UVB. CONCLUSION: CPD photolyase activity is a crucial factor determining the differences in UVB sensitivity between rice cultivars. The substitution of one or two bases in the CPD photolyase gene can alter the activity of the enzyme, and the associated resistance of the plant to UVB radiation. These findings open up the possibility, in the near future, of increasing the resistance of rice to UVB radiation, by selective breeding or bioengineering of the genes encoding CPD photolyase.     

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.     

Ultraviolet light‐induced impairment of goldfish embryo development and evidence for photorepair mechanisms

Goldfish Carassius auratus embryos were subjected to artificial ultraviolet‐B (UVB) radiation (280–320 nm) at various times during development to evaluate the effects on production of anatomically normal larvae. The UVB radiation used in these experiments included a higher proportion of shorter wavelengths compared to the natural spectrum. The development of embryos exposed to UVB for 2 or 4 h at 26 h post‐fertilization was severely impaired whereas similar exposures at 50 or 74 h post‐fertilization had no effect. A 2 h exposure to UVB commencing at 2 h post‐fertilization did not adversely affect embryonic development whereas a 4 h exposure to a lower dose did. At 50 h post‐fertilization, when embryos were normally resistant to UVB, denial of access to visible light and UVA before, during and after exposure to UVB caused impairment of development. Analysis of DNA fragment length after incubation with an endonuclease suggested that UVB damage at 50 h was caused by formation of pyrimidine dimers. This study demonstrated that the sensitivity of goldfish embryos to UVB varied during development and that resistance to UVB in later developmental stages included a photorepair mechanism.     

高等植物UV-B效应研究进展

本文概述了植物ＵＶ－Ｂ效应近年来的研究进展,ＵＶ－Ｂ对植物生理过程的影响表现为抑制细胞伸长,降低光合作用,引起植物细胞内活性氧代谢的紊乱,膜脂过氧化作用增强。植物种间、种内都存在ＵＶ－Ｂ敏感性差异。ＵＶ－Ｂ对植物ＤＮＡ的损伤主要是形成嘧啶二聚体。ＵＶ－Ｂ可诱导紫外吸收化合物的合成,积累,并对植物基因表达有重要调节作用。     

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.     

The presence of two modes of binding to calf thymus DNA by metal-free bleomycin: a low frequency Raman study

Double-stranded DNA is targeted by bleomycin in cancer cells and ambiguity exists as to its mode of DNA binding. A conventional Raman study was performed on drug/DNA complexes in which the low frequency spectral region (560-930 cm(-1)) was examined at two temperatures (19 and 30 degrees C). At 30 degrees C, a global Raman hypochromism was observed consistent with partial intercalation of the bithiazole moiety. At 19 degrees C, Raman hypochromism (increased base pair stacking) was detected for bands associated with GC base pairs while Raman hyperchromism (base pair destacking) was evident for bands associated with AT base pairs. These results suggest that intercalation of the bithiazole moiety occurs with greater disruption of the more efficiently stacked AT base pairs at the lower temperature. Evidence for minor groove binding was indicated by an increase in the population of bands corresponding to C3' endo sugar conformations resulting from drug induced local desolvation of the DNA polymer.     

Ultraviolet radiation-mediated damage to cellular DNA

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1O2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.