Biomarkers for translational oncology - Peggy Olive's contribution.

PURPOSE: Peggy Olive of the BC cancer research center (BCCRC), Vancouver, Canada, dedicated her career to improving the efficiency of radiation in the treatment of cancer. Keenly interested in the study of hypoxic cell radiosensitizers, she recognized the importance of DNA repair in improving the efficacy of radiotherapy. At the BCCRC she developed two methods for clinical practice that detect and quantitate DNA damage in mammalian cells. The alkaline comet assay and phosphorylated gamma histone H2AX (γH2AX) protein foci staining were two sensitive and attractive techniques that she attempted to apply in clinical practice. CONCLUSION: Peggy Olive was able to establish the comet and the γH2AX assays as prospective predictive biomarkers in the application of personalized radiation treatment and improved cancer treatment outcomes. Nevertheless, several studies with a large number of samples are required before application of these biomarkers in routine radiotherapy could become a reality. The advent of 'omis' and microchip technologies envisage successful outcomes of future research in this direction.

Peggy Louise Olive: A journey through the 'comet' and beyond.

Peggy Olive from the British Columbia Cancer Research Center in Canada is credited with the development of a method to measure DNA damage in individual cells based on the technique of microelectrophoresis that she named the 'comet assay'; a well-accepted method to measure DNA damage, hypoxia and apoptosis. A multifaceted person and an ardent campaigner of environmental issues, Peggy has contributed significantly to several areas of radiobiology related to the treatment of cancer, her expertise being tumor hypoxia and gamma H2AX foci as a biomarker in radiotherapy.

Gene expression in Catla catla (Hamilton) subjected to acute and protracted doses of gamma radiation.

Studies on transcriptional modulation after gamma radiation exposure in fish are limited. Cell cycle perturbations and expression of apoptotic genes were investigated in the fish, Catla catla after acute and protracted exposures to gamma radiation over a 90day period. Significant changes in gene expression were observed between day 1 and 90 post-exposure. Gamma radiation induced a significant down-regulation of target genes gadd45α, cdk1 and bcl-2 from day 1 to day 3 after protracted exposure, whereas it persists till day 6 upon acute exposure. From day 12 onwards, Gadd45α, cdk1 and bcl-2 genes were up-regulated following protracted exposure, indicating DNA repair, cell-cycle arrest and apoptosis. There exists a linear correlation between these genes (gadd45α - r=0.85, p=0.0073; cdk1 - r=0.86, p=0.0053; bcl-2 - r=0.89, p=0.0026) at protracted exposures. This is the first report on the dual role of bcl-2 gene in fish exposed to acute and protracted radiation and correlation among the aforementioned genes that work in concert to promote 'repair' and 'death' circuitries in fish blood cells.

Nucleoplasmic bridges and tailed nuclei are signatures of radiation exposure in Oreochromis mossambicus using erythrocyte micronucleus cytome assay (EMNCA).

Gamma radiation-induced genetic perturbations in aquatic vertebrates is largely unknown at low-dose rate, especially in the wake of a nuclear disaster and/or other environmental outbreaks. Freshwater fish, Oreochromis mossambicus subjected to low-dose rate (2 mGy/min) at 2.5-, 5-, and 10-Gy doses, were analyzed for "exposure signatures" in blood samples drawn on days 3, 6, 12, 18, and 30, respectively. Significant dose-dependent increments in micronuclei frequency and other anomalies such as nucleoplasmic bridges and tailed nuclei were observed and exhibit a strong positive correlation, suggesting that they could be used as prospective signatures of radiation exposure. Similarly increased incidence of apoptosis and DNA repair machinery circuits at high and low doses were noted. This work highlighted "cytogenetic signatures" in fish and the sensitivity of these endpoints toward low-dose rate of radiation exposure.

Relative biological effectiveness of radon: An in vitro study using chromosome aberrations as a biomarker.

PURPOSE: The need to determine a reliable relative biological effectiveness (RBE) value for alpha exposures has become important as reports remain controversial. Although a radiation-weighting factor of 20 has been designated for alpha particles, uncertainty exists on realistic value of the RBE of alpha radiation. The aim of this study was to estimate RBE values for radon using chromosome aberrations as the endpoint in respect to various dose rates of gamma radiation. MATERIALS AND METHODS: Human blood samples were exposed ex vivo to different doses of radon ranging from 0.0011-0.008 Gy. Blood samples were also exposed to gamma radiation with dose rates of 1, 0.1, 0.01 and 0.001 Gy/min. Chromosome aberrations in giemsa-stained first division metaphase preparations were scored. RESULTS: Dose response curves for dicentric chromosomal aberration yields were generated for both radon and gamma rays. Radon dose rates were compared with gamma dose rates to deduce RBE values. The values obtained were 16, 25, 29 and 38 for reference gamma dose-rates of 1, 0.1, 0.01 and 0.001 Gy/min, respectively. CONCLUSIONS: The study indicates that an RBE value of radon can range between 16 and 38, if one were to consider chromosome aberrations as an effective biomarker of risk due to ionizing radiation.

Cytogenotoxicity assessment of monocrotophos and butachlor at single and combined chronic exposures in the fish Catla catla (Hamilton).

Cytogenotoxic effects in the form of micronuclei and deformed nucleus, nuclear buds, binucleated cells, vacuolated nucleus, vacuolated cytoplasm, echinocytes, and enucleus induced by two compounds belonging to two different chemical classes of agrochemicals (monocrotophos and butachlor) at sublethal concentrations (0.625, 1.3, and 2.3 ppm and 0.016, 0.032, and 0.064 ppm) in single and combined chronic exposures were studied under laboratory conditions for a period of 35 days in the economically important Indian fish Catla catla. Statistically significant duration-dependent increases in the frequencies of micronucleus (MN) and other cytological anomalies were observed. Compared to single exposures, a twofold increase in micronuclei frequency was noted at combined exposures indicating the synergistic phenomenon. Binucleated and enucleated cells appeared only in fishes exposed to sublethal concentrations of butachlor. The present study is the first of its kind in exploring a significant positive correlation between micronuclei and other nuclear anomalies suggesting them as new possible biomarkers of genotoxicity after agrochemical exposures. The study highlights the sensitivity of the assay in exploring various predictive biomarkers of genotoxic and cytotoxic events and also elicits the synergistic effects of agrochemicals in apparently healthy fishes. C. catla can be considered as a suitable aquatic biomonitoring sentinel species of contaminated water bodies.

Gamma radiation induced cell cycle perturbations and DNA damage in Catla Catla as measured by flow cytometry.

Gamma radiation induced cell cycle perturbations and DNA damage in Catla catla were analyzed in erythrocytes at different time points using flow cytometry (FCM). Protracted exposure to radiation induced damage between days 12 and 45. Disturbances in cell cycle machinery, i.e., proportional increase and decrease in Gap0 or quiescent/Gap1 (G0/G1), Synthesis (S) and Gap2/Mitotic (G2/M) phases were observed at both acute and protracted treatments. Both acute and protracted exposures induced apoptosis with a notable significance between days 3 and 6 at protracted and on day 45 at acute doses. Fish exposed protractedly avail some DNA repair mechanisms than acutely exposed. This is the first study to analyze radiation induced DNA damage under laboratory conditions and suggests that flow cytometry can also be an alternate tool to screen genotoxicity induced by ionizing radiation in fish.

Synergistic effect of radon in blood cells of smokers - an in vitro study.

Epidemiological studies indicate that the risk of lung cancer among smokers increases with exposure to residential radon. The present study aimed to investigate the synergetic effect between smoking and radon. Blood samples from smokers and non-smokers were exposed to different concentrations of radon ranging from 0 to 189MBq/m(3) corresponding to doses ranging from 0.2 to 15.2mGy. Chromosome aberrations in first division metaphase preparations were scored. The frequency of dicentrics in radon-exposed smoker cells was found to be higher than non-smokers by factor of 3.8. The present study is the first of its kind to investigate the interaction of radon and smoking sans confounding factors, as smoker cells were exposed in vitro to radon.

Gamma radiation induced micronuclei and erythrocyte cellular abnormalities in the fish Catla catla.

Ionizing radiation induced DNA damage in fishes is a scarcely studied topic and very few studies are available in fishes exposed to ionizing radiation using the erythrocyte micronucleus assay under laboratory conditions. Since radionuclides released accidentally or during a nuclear disaster can contaminate inland water bodies, biomonitoring methods are required for assessing the impacts of high and low levels of radiation that may ultimately result in ionizing radiation exposure to both humans and non-human biota. Fresh water fish, Catla catla were subjected to protracted (0.002 Gy/min) and acute (3.2 Gy/min) gamma radiation to a total dose of 5 Gy. Peripheral blood samples were collected at different intervals (days 3, 6, 12, 18, 30, 45, 90, 135, 202) and analyzed by the erythrocyte micronucleus assay. Nuclear anomalies observed were micronuclei (MN), deformed nuclei (DN), nuclear bud (NBu), nuclear bridge (NBr), vacuolated nucleus (VN), binucleated cell (BNC), apoptotic cells (AC) while cytoplasmic abnormalities detected were vacuolated cytoplasm (VC), anisochromasia (AN), echinocytes (EC) and enucleus (EN). Both exposures caused a statistically significant increase in nuclear and cytoplasmic abnormalities that correlated with micronucleus and other nuclear anomalies. However, the extent of damage is higher after an acute exposure lasting for a longer period leading to apoptosis. Nuclear and cytoplasmic abnormalities are the resultants of gamma radiation induced genotoxicity and cytotoxicity.

Cytogenetic damage in human blood lymphocytes exposed in vitro to radon.

The effect of radon in inducing DNA damage was investigated in vitro by two well-established cytogenetic assays. Blood samples were irradiated with radon using a novel irradiation assembly. Doses varied between 0 and 127 mGy for chromosome aberration (CA) assay and 0 and 120 mGy for cytokinesis blocked micronucleus (CBMN) assay. Dose-rates varied between 0.000054 and 0.708 mGy/min. After the irradiation period of 3h, excess radon gas was released and cultures were initiated using standard procedures. Chromosome aberrations such as dicentrics, excess acentric fragments, acentric rings, centric rings, chromatid breaks were observed. Micronuclei, nucleoplasmic bridges and nuclear buds were scored by the CBMN assay. A significant increase in the frequency of dicentrics, excess acentric fragments and centric rings was observed with increasing radon dose, whereas total acentric rings plus double minute and chromatid breaks/cell were not significantly elevated. In CBMN assay, the frequency of micronuclei was found to be significantly raised whereas that of nucleoplasmic bridges and nuclear buds were not. Nucleoplasmic bridges and nuclear buds tended to increase with dose but did not achieve statistical significance. There was a strong positive correlation between nucleoplasmic bridges and dicentrics (P<0.028) or rings (P<0.0001) and between micronuclei and acentric fragments (P<0.0005). The study shows that radon is capable of inducing significant chromosome damage at very low doses and dose-rates.

A simple method to irradiate blood cells in vitro with radon gas.

The design and dosimetry of a novel in vitro radon irradiation facility to investigate cytogenetic damage induced by radon and progeny is described. The system offers a 4pi geometry for uniform irradiation, proving a versatile and convenient facility for irradiation of whole blood cells in suspension or media. Doses can be controlled as exact volumes of the gas can be dispensed and measured by the Lucas cell. Irradiation of blood samples could be carried out in a unique and safe manner and the present study is an attempt to demonstrate the usefulness of a facility to expose blood lymphocytes in vitro to radon and its decay products for chromosome aberration analysis. The preliminary results obtained using this facility are presented. Results show an increase in dicentric frequency with increasing concentration of radon (r = 0.97, P < 0.0001). Multiple aberrations in a single cell, characteristic of high linear energy transfer radiation, confirm the effect of alpha exposure.

Dose-rate effect on the induction of HPRT mutants in human G0 lymphocytes exposed in vitro to gamma radiation.

The influence of dose rate on expression time, cell survival and mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was evaluated in human G(0) peripheral blood lymphocytes exposed in vitro to gamma rays at low (0.0014 Gy/min) and high (0.85 Gy/min) dose rates. A cloning assay performed on different days of postirradiation incubation indicated an 8-day maximum expression period for the induction of HPRT mutants at both high and low dose rates. Cell survival increased markedly with decreasing dose rate, yielding D(0) values of 3.04 Gy and 1.3 Gy at low and high dose rates, respectively. The D(0) of 3.04 Gy obtained at low dose rate could be attributed to the repair of sublethal DNA damage taking place during prolonged exposure to low-LET radiation. Regression analysis of the mutant frequency yielded slopes of 12.35 x 10(-6) and 3.66 x 10(-6) mutants per gray at high and low dose rate, respectively. A dose and dose-rate effectiveness factor of 3.4 indicated a marked dose-rate effect on the induced HPRT mutant frequency. The results indicate that information obtained from in vitro measurements of dose-rate effects in human G(0) lymphocytes may be a useful parameter for risk estimation in radiation protection.

Studies on the HPRT mutant frequency in T lymphocytes from healthy Indian male population as a function of age and smoking.

Mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the peripheral blood lymphocytes obtained from 44 healthy individuals (23 non-smokers and 21 smokers) of an Indian male population was studied using T-lymphocyte cloning assay. It was found that lnMF increased with age at a rate of 2.5% per year (P <0.001). Blood samples from smokers showed a significant (P <0.037) increase in HPRT mutant frequency (MF) (10.43 +/- 4.74 x 10(-6)) as compared to that obtained from non-smokers (7.69 +/- 3.69 x 10(-6)). This study also showed a significant (P <0.027) inverse correlation between lnMF and non-selected cloning efficiency (CE). However, with respect to age no variation was observed in cloning efficiency. The results obtained in this study showed a good comparison with those reported in different populations of the world.

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay.

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.