Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.).

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Evaluation of gamma radiation-induced DNA damage in Aedes aegypti using the comet assay.

The study was undertaken to evaluate gamma radiation-induced DNA damage in Aedes aegypti. The comet assay was employed to demonstrate the extent of DNA damage produced in adult male A. aegypti exposed to seven different doses of gamma radiation, ranging from 1 Gy to 50 Gy. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in all samples; the extent of damage being proportional to the dose of gamma radiation the organism received, except in those treated with 1 Gy. The highest amount of DNA damage was noticed at 1 h postirradiation, which decreased gradually with time, that is, at 3, 6 and 12 h postirradiation. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the postirradiation time increased. The comet assay serves as a sensitive and rapid technique to detect gamma radiation-induced DNA damage in A. aegypti. This could be used as a potential biomarker for environmental risk assessment.

Effects of gamma radiation on the early developmental stages of Zebrafish (Danio rerio).

The zebrafish is gaining importance as a popular vertebrate model organism and is widely employed in ecotoxicological studies, especially for the biomonitoring of pollution in water bodies. There is limited data on the genetic mechanisms governing the adverse health effects in regards to an early developmental exposure to gamma radiation. In the present study zebrafish (Danio rerio) embryos were exposed to 1, 2.5, 5, 7.5 and 10Gy of gamma radiation at 3h post fertilization (hpf). Different developmental toxicity endpoints were investigated. Further, expression of genes associated with the development and DNA damage i.e. (sox2 sox19a and p53) were evaluated using Quantitative PCR (qPCR). The significant changes in the expression of sox2 sox19a and p53 genes were observed. This data was supported the developmental defects observed in the zebrafish embryo exposed to gamma radiation such as i.e. increased DNA damage, decreased hatching rate, increase in median hatching time, decreased body length, increased mortality rate, increased morphological deformities. Further, study shows that the potential ecotoxicological threat of gamma radiation on the early developmental stages of zebrafish. Further, it revealed that the above parameters can be used as predictive biomarkers of gamma radiation exposure.

Effect of gamma radiation on life history traits of Aedes aegypti (L.).

Aedes aegypti is an important vector for Dengue and Dengue hemorrhagic fever. Considering its medical importance and its relevance as a model system, this study was undertaken to evaluate the impact of different doses of gamma radiation for three generations of A. aegypti. Two to three days old virgin males of A. aegypti were irradiated with 15 doses of gamma radiation, ranging from 1 to 50 Gy and were immediately mass mated with the same aged virgin females. Observations were made for changes on their life history traits, particularly fecundity, hatchability, adult emergence, sex ratio and longevity, for three generations. Adult males exposed 30, 35, 40, 45 and 50 Gy doses showed a significant decrease in fecundity in F0 generations. While hatchability was observed to have decreased with increasing radiation doses from 3 Gy onwards in the F1 generation, samples irradiated with 30, 35, 40, 45 and 50 Gy maintained significant decline in hatchability in their succeeding generations, F2 and F3 also. Similarly, a decline was observed in adult emergence from 3 Gy onwards in all three generations. A male favoring sex ratio distortion was observed at the doses of 35, 40, 45 and 50 Gy in all three generations. Following exposure to 4 Gy, parental males and the resultant progeny showed increased longevity by 10.56 and 8.66 days respectively. Similarly, the F1 generations of samples irradiated with 30, 35 and 40 Gy exhibited an increase in longevity by 7.16, 7.44 and 6.64 days respectively. Dose response curve for fertility among the three generations was drawn and presented. The effect of radiological exposure on the life history traits of A. aegypti varies with dose for the three generations studied. These results have potential implications in mutational studies and risk assessment and also contribute to a better understanding towards employment of the sterile insect technique in A. aegypti, plausibly paving the way to an effective mosquito genetic control program.

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation.

Effects of acute gamma radiation on the reproductive ability of the earthworm Eisenia fetida.

Earthworms are the most suitable biological indicators of radioactive pollution because they are the parts of nutritional webs, and are present in relatively high numbers. Four months old Eisenia fetida were exposed to different doses of gamma radiation, namely 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 & 60 Gy to study the effects of radiation on different reproductive parameters. The number of cocoons laid and the hatchlings emerged were recorded for all the selected doses. There was no reduction in cocoon production, however; decreasing size and weight of the cocoons was observed from the samples exposed to 20 Gy and above doses. Significant reductions in the hatchlings were recorded in earthworms exposed to 10 Gy and above doses. The dose response curves for a percentage reduction in hatchlings were constructed. Exposure to radiation dose of 1 and 2 Gy did not show any reduction, however, there was ≈10%, ≈50% and ≈90% decrease in the hatchlings in samples exposed to 3, 15 and 45, 50, 55 and 60 Gy doses respectively. Delayed hatchability was also reported at al exposure level. Histology of irradiated earthworms revealed that the structural damage in the seminal vesicles was prominent at the exposed dose of 3 Gy onwards with complete degeneration on exposure to 60 Gy of gamma radiation.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples.

Age-dependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice.

Age-dependent changes in chromosomal damage in bone marrow - a self-proliferating tissue - in the form of spontaneously occurring micronucleated erythrocytes, and DNA damage in peripheral blood were examined in male and female Swiss mice. In the erythrocyte population in the bone marrow, polychromatic (immature) erythrocytes showed a significant increase in the frequency of micronuclei as a function of age of the mice (1-20 months). The increase in micronucleus frequency was less in normochromatic (mature) erythrocytes. The female mice showed a higher frequency of micronuclei than the male mice in all the age groups examined. However, the female to male ratio of micronucleus frequencies in total erythrocytes as well as in polychromatic erythrocytes decreased with age. DNA damage, measured as tail moment in the single-cell gel electrophoresis in peripheral blood of different age groups of mice (1, 6, 12 and 18 months) showed a gradual increase with age. Female mice showed more DNA damage than 1-month and 18-month-old male mice. In conclusion, these results show that there is an accumulation of genetic damage in bone marrow and DNA damage in peripheral blood of mice during ageing, and that females show more alterations than males.

Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay.

Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study reveals that gamma radiation induces single strand breaks in DNA as measured by alkaline comet assay in bivalves and comet assay serves as a sensitive and rapid method to detect genotoxicity of gamma radiation. This study further indicates that both M. casta and P. malabarica exhibit almost identical sensitivity to gamma radiation as measured by DNA damage.

Protective effects of selenocystine against γ-radiation-induced genotoxicity in Swiss albino mice.

Selenocystine (CysSeSeCys), a diselenide aminoacid exhibiting glutathione peroxidase-like activity and selective antitumor effects, was examined for in vivo antigenotoxic and antioxidant activity in Swiss albino mice after exposure to a sublethal dose (5 Gy) of γ-radiation. For this, CysSeSeCys was administered intraperitoneally (i.p.) to mice at a dosage of 0.5 mg/kg body weight for 5 consecutive days prior to whole-body γ-irradiation. When examined in the hepatic tissue, CysSeSeCys administration reduced the DNA damage at 30 min after radiation exposure by increasing the rate of DNA repair. Since antigenotoxic agents could alter the expression of genes involved in cell cycle arrest and DNA repair, the transcriptional changes in p53, p21 and GADD45α were monitored in the hepatic tissue by real-time PCR. The results show that CysSeSeCys alone causes moderate induction of these three genes. However, CysSeSeCys pretreatment resulted in a suppression of radiation-induced enhancement of p21 and GADD45α expression, but did not affect p53 expression. Further analysis of radiation-induced oxidative stress markers in the same tissue indicated that CysSeSeCys significantly inhibits lipid peroxidation and prevents the depletion of antioxidant enzymes and glutathione (GSH) levels. Additionally, it also prevents radiation-induced DNA damage in other radiation sensitive cellular systems like peripheral leukocytes and bone marrow, which was evident by a decrease in comet parameters and micronucleated polychromatic erythrocytes (mn-PCEs) frequency, respectively. Based on these observations, it is concluded that CysSeSeCys exhibits antigenotoxic effects, reduces radiation-induced oxidative stress, and is a promising candidate for future exploration as a radioprotector.

In vivo radioprotection studies of 3,3'-diselenodipropionic acid, a selenocystine derivative.

3,3'-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to gamma-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of gamma-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45alpha and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body gamma-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay.

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2Gy of gamma rays delivered at a dose rate of 0.316Gy/min using a (60)Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney. Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.

Protection against radiation oxidative damage in mice by Triphala.

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.

Computerized image analysis software for the comet assay.

Single-cell gel electrophoresis (SCGE) or the comet assay is a powerful tool for the detection of DNA single- and double-strand breaks and base damage and for investigating the kinetics of DNA strand break rejoining in human and animal model systems. It is a versatile technique that can be applied in various areas of biomedical research. This chapter highlights the importance of computerized analysis and data processing for the comet assay and describes the criteria used for manual evaluation of comets and their limitations compared with the computer-based analysis. It describes in detail SCGE-Pro, a semiautomatic software developed in our laboratory for comet evaluation and data processing. For comparison, some of the commercially available software for analysis of data from the comet assay is also described.

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay.

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668Gy/min between 0 and 4 degrees C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r(TM)=0.999 and r(TL)=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.

Radioadaptive response in human lymphocytes in vitro.

Exposure to low doses of radiation and/or chemicals can prime an organism to withstand the stress of a subsequent exposure to higher doses of the same agent. In the case of radiation, this phenomenon has been called radioadaptive response. Cytogenetic studies have been undertaken in human lymphocytes to investigate adaptive response (AR) to ionizing radiation, in particular to seek the role of variables such as priming dose, cell cycle stage, and age and gender of the donor. We demonstrated that pre-exposure of lymphocytes in whole blood cultures to very low doses in the range of about 1 cGy (priming or adaptive dose [AD]) reduced the frequency of micronuclei in binucleated cells induced by 100 cGy--that is, produced an AR in these cells in vitro. However, pre-exposure of cells to 10.0 cGy did not reduce the chromosomal damage (micronuclei) induced by the challenging dose (CD) of 100 cGy under the same protocol, thus exhibiting an inverse dose-response relationship. There was marked variability in the AR among the individuals investigated in the study. The extent of AR also depended on the stage of cell cycle exposed to the CD of radiation. Maximum AR was observed when CD of 100 cGy was given 4 hours after AD, 30 hours following the mitogenic stimulation of lymphocytes. The least AR was observed when CD was given 48 hours after stimulation. Interestingly, AR was also found to be dependent on the age of the donor, a decrease in AR being observed with an increasing age. No significant difference in AR was observed between male and female donors. To understand the molecular events underlying AR, protein synthesis patterns were studied in human lymphocytes subjected to mitogen, heat, or radiation stress. Heat shock (45 degrees C, for 15 min) induced 7 proteins with molecular weights ranging from 40 to 80 kDa, while treatment with phytohemagglutinin (40 microg/mL) showed induction of 2 proteins of molecular weights 38 and 48 kDa, respectively. However, exposure of human lymphocyte cultures to gamma radiation did not significantly induce synthesis of any protein, up to 800 cGy dose. Lack of induction of proteins by gamma radiation in human lymphocytes contrasts with the previous reports showing X-ray radiation-enhanced gene expression in melanoma cells and/or human tumor fibroblasts.

Inhibition of radiation-induced DNA damage in plasmid pBR322 by chlorophyllin and possible mechanism(s) of action.

Naturally occurring compounds capable of protecting DNA against ionizing radiation and chemical mutagens have considerable potential for prevention of mutation-based health impairment including cancer and other degenerative diseases. Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been examined for its ability to protect DNA against radiation induced strand breaks using an in vitro plasmid DNA system. Gamma-radiation, up to a dose of 6 Gy (dose rate 1.25 Gy/min), induced a dose-dependent increase in single-strand breaks (ssbs) in plasmid pBR322 DNA. CHL per se did not induce, but inhibited radiation-induced ssbs in a concentration-dependent manner; 500 microM giving about 90% protection. The protection afforded by CHL was comparatively less than that of trolox, a water-soluble analogue of alpha-tocopherol. To elucidate the underlying mechanism(s), reaction of CHL with the radiation-derived hydroxyl radical (.OH) and deoxyribose peroxyl radical (ROO.) was studied by pulse radiolysis. CHL exhibited a rate constant of 6.1+/-0.4x109 M-1 s-1 with.OH and 5.0+/-1.3x107 M-1 s-1 with ROO. To our knowledge, this is the first report providing direct evidence of free radical-scavenging properties of CHL. The results showed that CHL, effectively protects plasmid DNA against ionizing radiation, in an in vitro system independent of DNA repair or other cellular defense mechanisms. The ability of CHL to scavenge. OH and ROO., may contribute to its protective effects against radiation induced DNA damage in the pBR322 system.

Radio-adaptive response in human lymphocytes in vitro.

An attempt has been made to investigate the adaptive response to ionizing radiation in the human lymphocytes in vitro using cytochalasin-B blocked micronucleated binucleate cells (mn-BNCs) as a cytogenetic end point. Whole blood samples drawn from healthy donors, of either sex were irradiated in vitro at a dose of 1 cGy (adaptive or conditioning dose) Cobalt-60 gamma radiation (dose rate 1.12 cGy/min) at about 26 hr after mitogenic stimulation. After 31 hr of their initiation, groups of cultures were subsequently exposed to a challenging dose of 100 cGy gamma radiation (dose rate 82 cGy/min.). Eight males in the age group ranging from 25 to 55 years and eight females (age group 25 to 29 years), have been analysed during this study. Analysis of data revealed 40.6% reduction in the frequency of mn-BNCs among the males with a range from 25.7% to 54.7%. In case of females, also the per cent reduction varied from 26.3% to 49.0%, with a mean value of 33.7%. Pooling the data from males and females gave an overall reduction of 37.1% in the frequency of radiation induced mn-BNCs due to pre-exposure to 1 cGy radiation.

Studies on the migration of micronucleated erythrocytes from bone marrow to the peripheral blood in irradiated Swiss mice.

Micronucleated polychromatic (mn-PCE) and normochromatic erythrocytes (nm-NCE) were enumerated in the bone marrow and peripheral blood of Swiss male mice at different time intervals following whole-body (1.0 Gy) gamma-irradiation. Polychromatic cells migrated to the peripheral blood soon after their formation in the bone marrow and mn-PCE achieved a frequency close to that of the bone marrow with a delay of about 12 h. The optimal time for peripheral sampling was found to be about 36 h after radiation exposure. The frequency of mn-NCE in bone marrow and peripheral blood showed only a moderate and gradual increase till 60 h, and was much lower in the latter. In another experiment, mice irradiated with 0.42 Gy gamma-rays (0.21 Gy/h) once a day for 5, 10 or 15 days (5 days per week) showed a cumulative dose-dependent increase in the levels of mn-NCE in the peripheral blood, sampled at 7 or 21 days after the last exposure. These observations demonstrate persistence and accumulation of mn-PCE in the peripheral blood of mice during repeated exposure to ionizing radiation, and the sampling could be delayed up to several days after the last exposure. Thus, peripheral mn-PCE, scored between 24-48 h following irradiation, can be conveniently used to measure acute chromosomal damage induced by ionizing radiation in the bone marrow erythroblasts of mice, while peripheral mn-NCE are suited to monitor accumulated damage during chronic/repeated exposure.