The mechanisms of adaptive response. Estimation of capacity of human blood lymphocytes to radiation adaptive response using different criteria

Blood lymphocytes of 15 healthy donors have been investigated for the ability to decrease their radiosensitivity after treatment with low dose irradiation named radioinduced adaptive response (AR). The unstable chromosome aberrations were used to evaluate the radiosensitivity change after irradiation of cells with low adaptive dose (5 cGy) and subsequent high challenge dose (1.0 Gy) in comparison with the effect of challenge irradiation only. Three indexes were used: the frequency of cells with aberrations in all analyzed cells (A), the number of chromosome aberrations per cell (B) and the number of chromosome aberrations per one aberrant cell (C). It has been discovered that all donors examined can be divided into four groups: 1--individuals which cells did not show AR by all indexes used; 2--individuals which cells showed AR by indexes A and B, but not C; 3--AR was demonstrated by indexes B and C; 4--AR was confirmed by all three indexes. Generally accepted repair model for AR formation explains only the case of donor groups 3 and 4, but can not explain the mechanism leading to the case of group 2. For understanding this mechanism, the distribution of metaphases by the number of chromosome aberrations per cell was analyzes for each donor. It was shown that the part of cells without aberrations in group 2 donors increased significantly after treatment with the adaptive and challenge irradiation in comparison with that after irradiation with challenge dose only. The conclusion is that in this case AR is formed as a result of change in the frequency 0 cell class--population shift. The analogous shift was observed in the distributions of metaphases for all donors of the group 4, but was absent in the group 3 donors. The data obtained suggest that AR of blood lymphocytes might be a result of several processes: activation of submutational genome damage repair; population shifts manifested by the change in the part of undamaged cells; and, possibly, activation of apoptotic cell death. The complex nature of AR affects each of radiosensitivity evaluation criteria to a different extent.     

Cytogenetic effect of mitomycin C and cytosine arabinoside on mice of different genotypes]

Cytogenetic effect of mitomycin C (MC) and cytosine arabinoside (CA) on bone marrow cells of male mice of the strains 101/HY, C57BL/6Y C,3H/SnY and of the (C3HX101) F1 hybrids was studied. The frequencies of cells with chromosome aberrations after the treatment with MC at a 5 mg/kg dose were 54,4%; 41,8%; 40,4% and 26,8% in 101H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. The frequencies of cells with chromosome aberrations after the treatment with CA at a 500 mg/kg dose were 25,2%; 17,8%; 10,8% and the 101/H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. Both mutagens induced the greatest number of chromosome aberrations in the 101/H strain and the smallest number in the F1 hybrid (C3HX101). A positive correlation was established between the levels of induced and spontaneous chromosome lesions.     

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.     

Effect of lipid hydroperoxides on the yield of chromosome aberrations in irradiated cells

The combined effect of linoleic acid hydroperoxide and gamma-radiation on Ehrlich ascites tumor cells was not additive with regard to the formation of chromosome aberrations. When cells were preincubated in the presence of a subliminal hydroperoxide dose of 2.10(-5) M the number of aberrant cells increased after irradiation.     

Cellular reactions induced by chronic exposure to 14C beta-rays

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Cytogenetic effects of UV laser radiation with wavelengths of 248, 223 and 193 nm on mammalian cells

Ten hours after irradiation of mouse cornea with doses of 0.09 to 1.5 J/cm2 the incidence of cells with chromosome aberrations increased linearly with dose and amounted to 11.7% at 248 nm, 5.5% at 223 nm and 2.6% at 193 nm per 1 J/cm2. No induced chromosome aberrations occurred 72 hr following irradiation. Within the dose range from 3.0 to 18 J/cm2 the cytogenetic effect of radiation was less manifest than that with the doses mentioned above, the frequency of chromosome aberrations being independent of either wave length or radiation dose and amounted to 2.5 to 3.0%.     

Induction of chromosome aberrations in human lymphocytes by cyclophosphamide action in vivo and in vitro

The dose dependences of chromosome aberrations rate were investigated in lymphocytes of oncological patients after cyclophosphamide (CP) administration. It was shown that the rate of chromosome aberrations and the number of disruptions per cell in vivo and in vitro increases exponentially with the dose. At the same time, the parameters of regression equations coincide. This evidences that CP has the similar effect in vivo and in vitro.     

Chromosome aberrations in human lymphocytes at a various duration of cultivation after irradiation

Human peripheral blood lymphocytes were exposed to 60Co gamma-rays (a dose of 3 Gy) and cultivated during seven days in the presence of PHA and BrdU. It was shown that the metaphases of the first and second mitosises occurred during cultivation of the irradiated and unirradiated lymphocytes, being evidence about of irregularity of the coming into division of various fractions of lymphocytes. The time of cultivation did not influence a rate of aberrations in metaphases of the first and second mitosises of the irradiated lymphocytes. During the first and the subsequent mitosises the number of exchange chromosome aberrations decreased and reached a control level in metaphases of the fourth and fifth mitosises. The number of paired fragments at second and third mitosises increased a little and started to decrease only in metaphases of the fourth and fifth mitosises. The decrease in chromosome aberrations with prolongation of the cultivation of lymphocytes after irradiating is a consequence of elimination of cells with chromosome damages during sequential mitotic divisions.     

Aberrations induced by fission neutrons in human peripheral lymphocytes

Analysis of dose-response relationship was carried out for chromosome aberrations produced in human peripheral lymphocytes by fission neutrons at doses of 25, 50, 100 or 200 rad.Statistical treatment showed experimental data to be fitted by a regression curve described by the mathematical model Y = a+bD. A linear relation to dose characterized both one-break and two-break aberration yields. Numerical values of coefficients are reported for yields of dicentrics, chromosome fragments, minutes, aberrant cells, total number of aberrations, and total breakage.Based on chromosome fragments and aberrant cells, relative biological efficiency (RBE) value derived for fission neutrons relative to 180 kV X-rays for chromosome fragments was 2.53, and for aberrant cells it was 2.80.     

Mechanism of adaptive response: Estimation of human lymphocyte adaptive response to radiation using various criteria

Blood lymphocytes of 15 healthy donors have been investigated for their ability to reduce radiosensitivity after low-dose irradiation-radio-induced adaptive response (AR). The frequency of unstable chromosome aberrations was used to evaluate cell radiosensitivity after the irradiation of cells in low adaptive (5 cGy) and high challenge (1 Gy) doses in comparison with the effect of challenge irradiation only. Three indexes have been used, i.e., (A) the frequency of cells with aberrations per total analyzed cell, (B) the number of chromosome aberrations per one cell, (C) and the number of chromosome aberrations per one aberrant cell. It was found that the donors can be divided in the four following groups: 1. AR was not estimated any of the indexes used; 2. AR was estimated with indexes A and B, but not C; 3. AR was shown by indexes B and C; 4. AR was evident with all three indexes. The generally accepted AR repair model only explains the appearance of group-3 and-4 donors, but not group-2. For the purpose of understanding the AR mechanisms and the difference in AR estimations with various criteria, the metaphase distribution by the number of chromosome aberrations has been analyzed for each donor. It was shown that, in group-2 donors, the number of cells without aberrations after adaptive and challenge irradiations was significantly higher than after irradiation with a challenge dose only. Thus, in this group, AR is formed as a result of the changed frequency of cells in the 0 class (population shift). A similar shift is observed in the metaphase distribution in the donors of group 4, but not in group 3. The data obtained show that AR is probably a result of several processes, including the activation of the reparation of premutational genome damages, population shifts evident in the frequency of undamaged cells, and, possibly, the activation of apoptotic cell death. The complex character of AR is reflected to different degrees in each criterion of radiosensitivity.     

Comparison of frequencies of radiation-induced stable chromosomal aberrations in somatic and germ tissues of the mouse

Radiation-induced stable chromosome aberrations have been studied in the testes and bone-marrow of the mouse (Mus musculus). 60 days after whole-body irradiation with a dose of 400 rad X-rays, the frequency of visible chromosome aberrations in bone-marrow cells was 19.8%. The frequency of chromosome aberrations in spermatogonia of the same mice, scored as multivalents in spermatocytes, was considerably lower — only 4.7%. The possible mechanisms underlying this marked difference in translocation yield are discussed.     

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n male symbol =23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers' testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose-effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.     

Possible mutagenic action of a tularemia vaccine

No increase in the number of chromosome aberrations in the bone marrow cells of the Wistar rats was observed on the 1st, 2nd, 7th and 15th days after epidermal and intradermal immunization by tularemic live vaccine. Subcutaneous injection of great quantities of tularemic microbic cells which were not used in practice increased the number of cells with chromosome aberrations only on the second day.     

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.     

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n♂=23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers’ testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose–effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.     

Persistence of chromosome rearrangements in peripheral lymphocytes from patients treated with melphalan for ovarian carcinoma

Summary Chromosome aberrations were studied in peripheral lymphocytes from 50 patients treated with melphalan against ovarian carcinoma. The chromosome analyses were carried out 4–132 months (mean 57 months) after the end of melphalan therapy. Most of the patients were studied several times during four years. The mean frequency of cells with chromosome and chromatid aberrations was 5.4% in the patients and 2.3% in an untreated control group. The highest aberration frequency (average 18%) was found in a patient who later developed gastric carcinoma. The dominating types of berrations in the patients were chromosome exchanges occurring as single marker chromosomes or as multiple chromosome rearrangements. These types of aberrations were found in only 0.3% of the control cells as compared to 3.8% of the patient cells. Patients with a high total dose of melphalan (above 420 mg) and a long duration of the therapy (average 22.5 months) had a higher frequency of cells with aberrations (6.3%) than patients with a lower total dose (below 420 mg) and a shorter therapy (12 months) (4.2%). No additive effect of radiation therapy was observed on the aberration frequency.This work was supported by grants from the Swedish Cancer Society (1179), and the Swedish Medical Research Council (3681)     

Chromosomal aberrations in cultured human lymphocytes treated with aldicarb, a carbamate pesticide

Aldicarb was tested for its ability to induce chromosomal aberrations in human peripheral lymphocytes in vitro, in the presence of an exogenous metabolic activation system. The pesticide induced an increase in the number of chromatid and chromosome breaks. The increase was higher in the presence of S9 mix. A positive linear association between frequencies of abnormal cells and dose of aldicarb was observed.     

Radiobiological effects of a low-energy ion beam on wheat

The radiobiological effects of a low-energy nitrogen ion (N⁺) beam on wheat were studied, particularly with regard to the induction of chromosome aberrations. The results demonstrated that the three test varieties showed different sensitivities to ion implantation, and a higher dose of ion implantation had a marked effect on the germination and survival rate of the seeds exposed. The germination rate and survival rate curve basically followed a similar trend in the same variety. Cytological analysis indicated that ion beams were effective in producing chromosome aberrations. The frequencies of mitotic or meiotic cells with chromosome aberrations increased linearly with increasing doses. The aberration types included, for example, acentric fragments, chromosome deletions, lagging chromosomes, chromosome bridges and micronuclei. In the root tip cells, aberrations chiefly consisted of acentric fragments and deletions. Chromosome bridges and lagging chromosomes were the main aberration phenomena observed in the pollen mother cells. The highest frequencies of root tip cells and pollen mother cells with chromosome aberrations were 15.2% and 39.8%, respectively. Changes in morphology and mutant were also observed in the plants derived from exposed seeds. Received: 10 April 2000 / Accepted: 10 October 2000