mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (<Superscript>56</Superscript>Fe ions)

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. ⁵⁶Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy) or ¹³⁷Cs γ rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of ⁵⁶Fe ions or 3 Gy of ¹³⁷Cs γ rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, ⁵⁶Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than ¹³⁷Cs γ rays in inducing chromosomal damage.     

Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation

We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.     

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.     

Cytogenetic effects of extremely low doses of plutonium-238 alpha-particle irradiation in CHO K-1 cells

CHO K-1 cells were irradiated during the G1 phase with 0.5-6 rad of alpha particles. There was no appreciable cell killing in this low dose range. Significantly increased frequencies of sister-chromatid exchanges were induced by doses as low as 0.5 rad of alpha-particle irradiation, whereas increased numbers of chromosomal aberrations were observed following exposure to 2 rad. These results suggest that very low doses of alpha radiation may lead to radiation-induced genetic alterations.     

Characteristic cytogenetic effects of small doses of ionizing radiation

A study was made of the frequency of chromosome aberrations in human lymphocyte culture after gamma-irradiation (60Co) with doses ranging from 0.05 to 1.0 Gy at dose--rates of 0,005, 0.05 and 0.5 Gy/min. The frequency of structural changes in chromosomes at low doses was higher than it was expected in the case of extrapolating the effect produced by high to low doses of radiation; within the dose range from 0.1 to 0.5 Gy a plateau was registered for aberrations of the exchange type (dicentrics and rings). The abnormal character of the dose dependence of the yield of chromosome aberrations persisted with all three dose - rates under study.     

Complementation of chromosomal aberrations in AT/NBS hybrids: inadequacy of RDS as an endpoint in complementation studies with immortal NBS cells

Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare autosomal recessive hereditary disorders characterized by radiosensitivity, chromosomal instability, immunodeficiency and proneness to cancer. Although the clinical features of both syndromes are quite distinct, the cellular characteristics are very similar. Cells from both NBS and AT patients are hypersensitive to ionizing radiation (IR), show elevated levels of chromosomal aberrations and display radioresistant DNA synthesis (RDS). The proteins defective in NBS and AT, NBS1 and ATM, respectively, are involved in the same pathway, but their exact relationship is not yet fully understood. Stumm et al. (Am. J. Hum. Genet. 60 (1997) 1246) have reported that hybrids of AT and NBS lymphoblasts were not complemented for chromosomal aberrations. In contrast, we found that X-ray-induced cell killing as well as chromosomal aberrations were complemented in proliferating NBS-1LBI/AT5BIVA hybrids, comparable to that in NBS-1LBI cells after transfer of a single human chromosome 8 providing the NBS1 gene. RDS observed in AT5BIVA cells was reduced in these hybrids to the level of that seen in immortal NBS-1LBI cells. However, the level of DNA synthesis, following ionizing radiation, in SV40 transformed wild-type cell lines was the same as in NBS-1LBI cells. Only primary wild-type cells showed stronger inhibition of DNA synthesis. In summary, these results clearly indicate that RDS cannot be used as an endpoint in functional complementation studies with immortal NBS-1LBI cells, whereas the cytogenetic assay is suitable for complementation studies with immortal AT and NBS cells.     

High frequency of spontaneous translocations revealed by FISH in cells from patients with the cancer-prone syndromes ataxia telangiectasia and Nijmegen breakage syndrome.

The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 +/- 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 +/- 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 +/- 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 +/- 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 +/- 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.     

Radiosensitivity of ataxia telangiectasia and Nijmegen breakage syndrome homozygotes and heterozygotes as determined by three-color FISH chromosome painting

A three-color chromosome painting technique was used to examine the spontaneous and radiation-induced chromosomal damage in peripheral lymphocytes and lymphoblastoid cells from 11 patients with ataxia telangiectasia (AT) and from 14 individuals heterozygous for an AT allele. In addition, cells from two homozygous and six obligate heterozygous carriers of mutations in the Nijmegen breakage syndrome gene (NBS) were investigated. The data were compared to those for chromosome damage in 10 unaffected control individuals and 48 cancer patients who had not yet received therapeutic treatment. Based on the well-documented radiation sensitivity of AT and NBS patients, it was of particular interest to determine whether the FISH painting technique used in these studies allowed the reliable detection of an increased sensitivity to in vitro irradiation of cells from heterozygous carriers. Peripheral blood lymphocytes and lymphoblastoid cells from both the homozygous AT and NBS patients showed the highest cytogenetic response, whereas the cells from control individuals had a low number of chromosomal aberrations. The response of cells from heterozygous carriers was intermediate and could be clearly differentiated from those of the other groups in double-coded studies. AT and NBS heterozygosity could be distinguished from other genotypes by the total number of breakpoints per cell and also by the number of the long-lived stable aberrations in both AT and NBS. Only AT heterozygosity could be distinguished by the fraction of unstable chromosome changes. The slightly but not significantly increased radiosensitivity that was found in cancer patients was apparently due to a higher trend toward rearrangements compared to the controls. Thus the three-color painting technique presented here proved to be well suited as a supplement to conventional cytogenetic techniques for the detection of heterozygous carriers of these diseases, and may be superior method.     

Influence of dose rate on the induction of simple and complex chromosome exchanges by gamma rays

Single-color painting of whole chromosomes, or protocols in which only a few chromosomes are distinctively painted, will always fail to detect a proportion of complex exchanges because they frequently produce pseudosimple painting patterns that are indistinguishable from those produced by bona fide simple exchanges. When 24-color multi-fluor FISH (mFISH) was employed for the purpose of distinguishing (truly) simple from pseudosimple exchanges, it was confirmed that the acute low-LET radiation dose-response relationship for simple exchanges lacked significant upward curvature. This result has been interpreted to indicate that the formation of simple exchanges requires only one chromosome locus be damaged (e.g. broken) by radiation to initiate an exchange-not two, as classical cytogenetic theory maintains. Because a one-lesion mechanism implies single-track action, it follows that the production of simple exchanges should not be influenced by changes in dose rate. To examine this prediction, we irradiated noncycling primary human fibroblasts with graded doses of (137)Cs gamma rays at an acute dose rate of 1.10 Gy/min and compared, using mFISH, the yield of simple exchanges to that observed after exposure to the same radiation delivered at a chronic dose rate of 0.08 cGy/min. The shape of the dose response was found to be quasi-linear for both dose rates, but, counter to providing support for a one-lesion mechanism, the yield of simple aberrations was greatly reduced by protracted exposure. Although chronic doses were delivered at rates low enough to produce damage exclusively by single-track action, this did not altogether eliminate the formation of complex aberrations, an analysis of which leads to the conclusion that a single track of low-LET radiation is capable of inducing complex exchanges requiring up to four proximate breaks for their formation. For acute exposures, the ratio of simple reciprocal translocations to simple dicentrics was near unity.     

Evasion of early cellular response mechanisms following low level radiation-induced DNA damage

DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the "inverse dose rate effect," an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATM-associated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors.     

Cytogenetic Effects of γ-Radiation in Onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Seedlings

The effect of γ-radiation on the cytogenetic parameters of root meristem cells of onion seedlings was studied in laboratory experiments (Allium-test). An increase in the overall frequency of chromosomal aberrations and micronucleus frequencies in seedling cells at low γ-radiation doses (≤0.1 Gy) was detected for the first time. At a maximum absorbed dose of 13 Gy, chromosomal aberrations were detected in the majority of cells in the anaphase and telophase stages of the cell cycle, and the number of cells with multiple aberrations increased. The main contribution to the overall frequency of chromosomal aberrations, in addition to multiple aberrations, is made by the bridge-type aberrations, fragments, and lagging chromosomes. The data obtained allow using the cytogenetic indices of Allium cepa seedlings to assess the biological effects of lowdose γ-radiation.     

Evidence that unrejoined DNA double-strand breaks are not predominantly responsible for chromosomal radiosensitivity of AT fibroblasts

To examine more fully the nature of chromosomal radiosensitivity in ataxia telangiectasia (AT) cells, we employed 24-color combinatorial painting to visualize 137Cs gamma-ray-induced chromosome-type aberrations in cells of two AT and one normal primary human fibroblast strains irradiated in log-phase growth. As a measure of misrejoined radiation-induced DSBs, we quantified exchange breakpoints associated with both simple and complex exchanges. As a measure of unrejoined DSBs, we quantified breakpoints from terminal deletions as well as deletions associated with incomplete exchange. For each of these end points, the frequency of damage per unit dose was markedly higher in AT cells compared to normal cells, although the proportion of total breaks that remained unrejoined was rather similar. The majority of breakpoints in both cell types were involved in exchanges. AT cells had a much higher frequency of complex exchanges compared to normal cells given the same dose, but for doses that resulted in approximately the same level of total breakpoints, the relative contribution from complex damage was also similar. We conclude that although terminal deletions and incomplete exchanges contribute to AT cell radiosensitivity, their relative abundance does not-in apparent contrast to the situation in lymphoblastoid cells-overwhelmingly account for the increased damage we observed in cycling AT fibroblasts. Thus, from a cytogenetic perspective, a higher level of unrepaired DSBs does not provide a universal explanation for the radiation-sensitive AT phenotype.     

The ATM kinase signaling induced by the low-energy β-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induced by the energetic β-particles emitted by (32)P

Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter (32)P-orthophosphate for 30min is ～1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter (33)P-orthophosphate is ～0.18Gy IR. We show that low-energy β-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ～0.36Gy β-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Genome Instability in Ataxia Telangiectasia (A-T) Families: Camptothecin-Induced Damage to Replicating DNA Discriminates between Obligate A-T Heterozygotes,A-T Homozygotes and Controls

Previously we used the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells primarily by inducing double strand breaks (DSBs) in replication forks, to show that ataxia telangiectasia (A-T) fibroblasts are defective in the repair of this particular subclass of DSBs. CPT treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges, viewed as prematurely condensed G2 chromosomes (G2 PCC), compared with normal cells where aberrations are mostly chromatid breaks. Here we show that A-T lymphoblastoid cells established from individuals with different mutations in the ATM gene also exhibit increased levels of chromosomal exchanges in response to CPT, indicating that the replication-associated DSBs are misrepaired in all these cells. From family studies we show that the presence of a single mutated allele in obligate A-T heterozygotes leads to intermediate levels of chromosomal exchanges in CPT-treated lymphoblastoid cells, thus providing a functional and sensitive assay to identify these individuals.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

Chromosomal analysis of Chinese hamster V79 cells exposed to multiple gamma-ray fractions: induction of adaptive response to mitomycin C.

Chinese hamster V79 cells were irradiated daily with 0.3 Gy of gamma-rays 5 times per week for 12 weeks (total 18 Gy). These cells were challenged with an additional dose of 15. Gy gamma-rays or treated with 5 micrograms/ml of mitomycin C (MMC) for 2 h. In spite of the high total accumulated dose of gamma-rays, the number of chromosomal aberrations and sister-chromatid exchanges (SCEs) did not significantly increase in the preirradiated cells, as compared to control cells. If preirradiated cells were challenged with an additional 1.5 Gy of gamma-rays, an insignificant decrease in the yield of chromatid aberrations was observed. In contrast, preirradiated cells became significantly more resistant to the induction of chromosomal damage when challenged with mitomycin C. Our results suggest that multiple fractions of gamma-rays can induce the adaptive response to mitomycin C in preirradiated cells.     

Induction of chromosome aberrations and micronuclei in human peripheral blood lymphocytes at low dose of radiation

The chromosome damage induced by the doses of y-irradiation 6)Co in peripheral blood lymphocytes was studied using different cytogenetic assays. Isolated lymphocytes were exposed to 0.01-1.0 Gy, stimulated by PHA, and analysed for chromosome aberrations at 48 h postirradiation by metaphase method, at 49 h--by the anaphase method, at 58 h by micronucleus assay with cytochalasin B and, additionally, micronuclei were counted at 48 h on the slides prepared for the metaphase analysis without cytochalasin B. Despite of the quantitative differences in the amount of chromosome damage revealed by different methods all of them demonstrated complex nonlinear dose dependence of the frequency of aberrant cells and aberrations. At the dose range from 0.01 Gy to 0.05-0.07 Gy the cells had the highest radiosensitivity mainly due to chromatid-type aberration induction. With dose increasing the frequency of the aberrant cells and aberrations decreased significantly (in some cases to the control level). At the doses up to 0.5-0.7 Gy the dose-effect curves have become linear with the decreased slope compare to initial one (by factor of 5 to 10 for different criteria) reflecting the higher radioresistance of cells. These data confirm the idea that the direct linear extrapolation of high dose effect to low dose range--the procedure routinelly used to estimate genetic risk of low dose irradiation--cannot be effective and may lead to underestimation of chromosome damage produced by low radiation doses. Preferences and disadvantages of used cytogenetic assays and possible mechanisms of low ionising radiation doses action were discussed.     

Vicia faba chromosome damage induced by low doses of gamma radiation

The rate of radiation damage to chromosomes by low doses of gamma rays (0.01-0.30 Gy) was studied in the root tips ofVicia faba. As criteria of the effect of ionizing radiation, the frequency of sister chromatid exchanges (SCEs), incidence of chromosomal aberrations and the number of micronuclei were evaluated and compared in irradiated cells. The results obtained confirmed that the analysis of SCEs did not represent an efficient indicator of radiation damage to chromosomes. On the contrary, the formation of chromosomal aberrations and micronuclei was effectively stimulated by low radiation doses, there being linear dose-effect relationships in the low doses region used.     

The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.