Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel eletrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of ⁶⁰Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Dominant lethal effect of 60Co gamma radiation in Biomphalaria glabrata (SAY, 1818)

The dominant lethal effects of gamma radiation of 60Co in the snail Biomphalaria glabrata were studied. Three groups of 13 wild-type snails were irradiated with single doses of 2.5; 10 and 20 Gy. Crossings were carried out at intervals of 7, 17, 23, 30 and 36 days after irradiation. The dominant lethal effect was observed only at the first crossing occurring 7 days after irradiation with 2.5 Gy. With 10 and 20 Gy, the induction of lethal mutations was detected at 7, 17 and 23 days after irradiation; a dose-response effect was observed. The effect was stronger 7 days after irradiation, decreasing in the succeeding crossings up to 30 days. Cell-killing effects on germ cells were detected in the crossings at 23 days and 30 days after irradiation with 20 Gy. After 36 days, frequencies of malformations resumed background levels; crossing rates partially recovered. These results show that gamma radiation affected all the stages of spermatogenesis. Germ cells at later phases were more sensitive to the mutagenic effect of radiation and the cell killing effects were observed on the youngest cells. This response was similar to the highly homogeneous pattern observed in widely different species and allowed us to estimate some parameters of spermatogenesis in B. glabrata.     

Multifunctional Centromere Binding Factor 1 Is Essential for Chromosome Segregation in the Human Pathogenic Yeast Candida glabrata

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.     

Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure

Somatic intrachromosomal recombination can result in inversions and deletions in DNA, which are important mutations in cancer. The pKZ1 chromosomal inversion assay is a sensitive assay for studying the effects of DNA damaging agents using chromosomal inversion as a mutation end-point. We have previously demonstrated that the chromosomal inversion response in pKZ1 spleen after single low doses of X-radiation exposure does not follow the linear no-threshold dose–response model. Here, we optimised a chromosomal inversion screening method to study the effect of low dose X-radiation exposure in pKZ1 prostatic tissue. In the present study, a significant induction in inversions was observed after ultra-low doses of 0.005–0.01 mGy or after a high dose of 1000 mGy, whereas a reduction in inversions to below the sham-treated frequency was observed between 1 and 10 mGy exposure. This is the first report of a reduction to below endogenous frequency for any mutation end-point in prostate. In addition, the doses of radiation studied were at least three orders of magnitude lower than have been reported in other mutation assays in prostate in vivo or in vitro. In sham-treated pKZ1 controls and in pKZ1 mice treated with low doses of 1–10 mGy the number of inversions/gland cross-section rarely exceeded three. Up to 4 and 7 inversions were observed in individual prostatic gland cross-sections after doses ≤0.02 mGy and after 1000 mGy, respectively. The number of inversions identified in individual cross-sections of prostatic glands of untreated mice and all treated mice other than the 1000 mGy treatment group followed a Poisson distribution. The dose–response curves and fold changes observed after all radiation doses studied were similar in spleen and prostate. These results suggest that the pKZ1 assay is measuring a fundamental response to DNA damage after low dose X-radiation exposure which is independent of tissue type.     

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.     

Evaluation of DNA damage in a population of bats (Chiroptera) residing in an abandoned monazite mine

Ionising radiation has the ability to induce DNA damage. While the effects of high doses of radiation of short duration have been well documented, the biological effects of long-term exposure to low doses are poorly understood. This study evaluated the clastogenic effects of low dose ionising radiation on a population of bats (Chiroptera) residing in an abandoned monazite mine. Bats were sampled from two chambers in the mine, where external radiation levels measured around 20 microSv/h (low dose) and 100 microSv/h (higher dose), respectively. A control group of bats was sampled from a cave with no detectable radiation above normal background levels. The micronucleus assay was used to evaluate residual radiation damage in binucleated lymphocytes and showed that the micronucleus frequency per 500 binucleated lymphocytes was increased in the lower radiation-exposed group (17.7) and the higher radiation-exposed group (27.1) compared to the control group (5.3). This study also showed that bats exposed to radiation presented with an increased number of micronuclei per one thousand reticulocytes (2.88 and 10.75 in the lower and high radiation-exposed groups respectively) when compared to the control group (1.7). The single-cell gel electrophoresis (comet) assay was used as a means of evaluating clastogenecity of exposure to radiation at the level of individual cells. Bats exposed to radiation demonstrated increased DNA damage as shown by the length of the comet tails and showed an increase in cumulative damage. The results of the micronucleus and the comet assays indicated not only a statistically significant difference between test and control groups (P<0.001), but also a dose-dependent increase in DNA damage (P<0.001). These assays may thus be useful in evaluating the potential clastogenecity of exposure to continuous low doses of ionising radiation.     

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.     

Protection from solar simulated radiation-induced DNA damage in cultured human fibroblasts by three commercially available sunscreens

Exposure to solar radiation can produce both acute and chronic changes in the skin, including sunburn, edema, immunosuppression, premature skin aging, and skin cancer. At the cellular level, solar radiation can produce adverse structural and functional changes in membrane proteins and lipids and in chromosomal and mitochondrial DNA. The increasing awareness of these adverse effects has led the public to demand better photoprotection. In this study, the alkaline comet assay was used to evaluate the photoprotective effects of three commercially available sunscreens at sun protection factors (SPF) 15 and 30. Human fibroblasts were used as target cells to conveniently study the effects of solar simulated radiation on DNA damage in the presence and absence of sunscreens. When human fibroblasts were exposed to various doses of solar simulated radiation, DNA damage, as measured in sunscreen-protected cells by the comet assay, was not significantly different from that detected in unexposed cells. At 1.0 and 1.5 minimal erythemal doses (MED), all sunscreens, at both SPF 15 and 30, provided nearly 100% photoprotection to the fibroblasts. Further studies are required to elucidate the role of UVA in the production and repair of DNA damage in cells exposed to sunlight.     

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.     

Schistosoma mansoni: use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata.

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely Bg/II, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.     

Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.     

Effects of combined radiofrequency radiation exposure on the cell cycle and its regulatory proteins

The aim of this study was to investigate whether single or combined radio frequency (RF) radiation exposure has effects on the cell cycle and its regulatory proteins. Exposure of MCF7 cells to either single (837 MHz) or combined (837 and 1950 MHz) RF radiation was conducted at specific absorption rate values of 4 W/kg for 1 h. During the exposure period, the chamber was made isothermal by circulating water through the cavity. After RF radiation exposure, DNA synthesis rate and cell cycle distribution were assessed. The levels of cell cycle regulatory proteins, p53, p21, cyclins, and cyclin‐dependent kinases were also examined. The positive control group was exposed to 0.5 and 4 Gy doses of ionizing radiation (IR) and showed changes in DNA synthesis and cell cycle distribution. The levels of p53, p21, cyclin A, cyclin B1, and cyclin D1 were also affected by IR exposure. In contrast to the IR‐exposed group, neither the single RF radiation‐ nor the combined RF radiation‐exposed group elicited alterations in DNA synthesis, cell cycle distribution, and levels of cell cycle regulatory proteins. These results indicate that neither single nor combined RF radiation affect cell cycle progression. Bioelectromagnetics 32:169–178, 2011. © 2010 Wiley‐Liss, Inc.     

Radiosensitization and production of DNA double-strand breaks in U87MG brain tumor cells induced by tetraiodothyroacetic acid (tetrac)

We describe the steady-state levels and molecular and cellular repair of DNA double-strand breaks (DSBs) in tetraiodothyroacetic acid (tetrac)-treated human U87MG glioblastoma cells after x-irradiation in vitro. This study was conducted to provide a basis for our previous observation of radiosensitization and inhibition of cellular recovery after irradiation of tetrac-exposed GL261 murine brain tumor cells. We used the neutral comet assay to assess DSBs, and found that the steady-state DSB levels as indicated by the mean tail moment after a 1 h application of 2 nM tetrac at 37oC was increased from a value of 6.1 in control cells to 12.4 in tetrac treated cells at 0 radiation dose. However, at all radiation doses, the induction curves of DSBs were parallel, suggesting that no interaction of tetrac with the initial physical-chemical actions of ionizing radiation occurred. Flow cytometric measurements indicated that this increase was not due to alterations in the relative percentages of U87MG cells throughout the cell cycle. In split-dose DNA repair studies we found that tetrac decreased the repair rate of U87 cells by a factor of 72.5%. This suggests that the radiosensitization from graded single doses of x-rays occurs as a consequence of tetrac inhibition of the post-irradiation repair process. These results link the previously noted changes in cellular endpoints to a molecular endpoint. That is, tetrac produces increased numbers of DSBs in the unirradiated steady-state coupled with a decreased repair rate of DSBs in fractionated radiation experiments.     

Protection by WR-3689 against gamma-ray-induced intestinal damage: comparative effect on clonogenic cell survival, mouse survival, and DNA damage

The aminophosphorothioate WR-3689 was characterized for its ability to protect mouse jejunal cells in vivo from single doses of X or gamma radiation. First, the effect of the drug on the survival of jejunal stem cells was examined using a clonogenic end point, the crypt microcolony assay. When WR-3689 was administered 30 min prior to whole-body irradiation, the number of surviving crypt cells was markedly increased at all doses of the drug, although protection began to level out at doses larger than 600 mg/kg. Protection was maximal when the drug was given 30 min before whole-body irradiation and declined rapidly with both shorter and longer intervals. Protection factors (PFs) were obtained by measuring survival curves for clonogenic crypt cells as a function of radiation dose; WR-3689 given 30 min before whole-body irradiation protected jejunum in the microcolony assay with a PF of 1.26 +/- 0.02, 1.50 +/- 0.10, and 1.65 +/- 0.10 at doses of 200, 400, and 800 mg/kg, respectively. Next, the effect of WR-3689 on the survival of jejunal stem cells was determined by assaying the survival of mice given X-ray doses to the whole abdomen in the range leading to death from the gastrointestinal syndrome. The PFs based on the LD50 values for 11-day survival were 1.31 +/- 0.05 (200 mg/kg) and 1.48 +/- 0.05 (400 mg/kg). Crypt-cell survival and animal survival were thus modified to a similar extent by this agent. Finally, the effect of WR-3689 on the induction of DNA single-strand breaks (SSBs) in jejunal cells was measured using an adaptation of the alkaline elution methodology. In mice treated with WR-3689 (400 or 800 mg/kg) 30 min prior to whole-body irradiation with 10 Gy there was no significant reduction in the number of DNA SSBs induced either in samples of the jejunum or in the cycling crypt cells, providing further evidence that there is no simple relationship between the modification of DNA SSBs and the survival of jejunal stem cells.     

Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8-100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated.     

Pdr1蛋白N端携带Flag标签的光滑假丝酵母的构建

近年来,光滑假丝酵母已成为第二位引起侵袭性真菌感染的病原体。光滑假丝酵母对唑类药物(临床一线抗真菌药物)的敏感性低且易发生耐药,一直是研究的热点。介导光滑假丝酵母对唑类药物耐药的关键基因是转录因子pdr1,其功能性突变会使Pdr1蛋白功能过度活跃,导致下游唑类药物外排泵基因高表达,从而对唑类药物耐药。本研究利用同源重组技术,构建在基因pdr1的5′端定点插入3×Flag标签的重组菌株2a2和2b2,为后续利用免疫染色质共沉淀技术寻找Pdr1直接调控基因奠定基础。结果表明,3×Flag标签添加到Pdr1蛋白N端可成功表达Flag-Pdr1蛋白;与野生型菌株相比,表达Flag-Pdr1的菌株对氟康唑的耐药性增强。此外,与野生型菌株相比,表达Flag-Pdr1的菌株中cdr1和pup1基因表达水平显著上升,提示在Pdr1蛋白N端加Flag标签能使其功能活跃,表明N端对Pdr1蛋白功能具有重要意义。