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.     

Methods of scoring induced chromosome structural changes in barley

In barley, a material widely used in mutation and chromosomal aberration studies, the method most frequently used for scoring induced chromosomal changes is still anaphase analysis.In this paper, data obtained after treatment of barley with gamma-rays and ethyleneimine (EI) and comparative scoring of aberrations in metaphase and anaphase are reported and discussed.It is evident that the metaphase aberrations induced by gamma-rays and ethyleneimine, due probably to their specific location, showed a differential manifestation during anaphase. Thus, after treatment with ethyleneimine a great portion of the induced aberrations, being located preferentially at the centromere regions, gave no scorable bridges, and an apparent excess of fragments was observed at anaphase. After gamma-irradiation the differences between metaphase and anaphase scoring were mainly due to a large portion of fragments escaping detection.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Evaluation of the mutagenic potential of the antichagasic drug Rochagan in healthy and chagasic rodents

Benznidazole (bz) is the active component of the antichagasic drug Rochagan. Tests were carried out to detect the induction of chromosomal aberrations and micronuclei in rodent bone marrow cells and peripheral blood cells, respectively. Rats were exposed to acute treatment with Rochagan by gavage at total doses of 150, 300, 1500, 2000 and 3000 mg bz/kg body weight and killed at different times. In the chronic treatments, healthy and chagasic Balb/c mice were treated with Rochagan by gavage at a dose of 100 mg bz/kg/day for 10 and 25 days. No significant increase in frequency of chromosomal aberrations in bone marrow cells or of micronuclei in peripheral blood cells was detected in the animals acutely or chronically exposed to Rochagan in vivo.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17beta-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24h with Vitamin E (Vit E), and (4) pre-treatment for 4h with 17beta-estradiol (17beta-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2-20 mg/kg bw). These doses corresponded to 0.4-4% of the LD50 in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 x 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 x 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a 'hit and go' manner. Furthermore, pre-treatment of animals with 17beta-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.     

Micronuclei in human lymphocytes: the Co-60 gamma-ray dose-response

Dose-response for micronuclei in cytokinesis-blocked lymphocytes after in vitro irradiation of whole blood from 3 donors with Co-60 gamma-rays in the range 0–5.0 Gy was established. The numerical relationship between radiation induced chromosomal aberrations, and micronuclei is also examined. An increased frequency of micronuclei following low doses of gamma-irradiation is reported from a study of 41 radiation workers.     

Chromosome aberrations in spermatogonia and sperm abnormalities in Curacron-treated mice

Curacron is an organophosphorus pesticide widely used in cotton fields. In order to assay its mutagenic potential in mammalian germ cells chromosomal aberrations in spermatogonial cells and sperm abnormalities were examined in mice after Curacron treatment. For studying chromosomal aberrations mice were treated both acutely (single treatment) and subacutely (for 5 consecutive days) with 3 dose levels of Curacron, 12, 36 and 72 mg/kg. Curacron was found to produce a significant increase in structural chromosomal aberrations after acute and subacute treatments. This increase was dose-dependent. A dose-dependent inhibition in mitotic activity in spermatogonia was also found. For studying sperm abnormalities mice were treated for 5 consecutive days with 20, 40 and 60 mg/kg. Morphological sperm abnormalities increased significantly after treatment with Curacron. The increase was dose-dependent. An inhibition of 40.2% in sperm count and of 74.5% in sperm motility occurred after treatment with 60 mg/kg Curacron. These results show that Curacron has a damaging effect on spermatogonial cells as well as on sperm morphology.     

A comparative study on the genotoxic effect of pyrimethamine in bone marrow and spermatogonial mice cells

Pyrimethamine is an antimalarial agent widely used in clinical therapy. We aimed to compare its mutagenic potential in mammalian spermatogonial and bone marrow cells. For studying chromosomal aberrations mice were treated acutely (single treatment) with 4 dose levels of pyrimethamine (5, 10, 20 and 40 mg/kg). Pyrimethamine was found to produce a significant increase in structural chromosomal aberrations after acute treatment in bone marrow cells of mice (p < 0.001). It also induced chromosome abnormalities in spermatogonial cells (p < 0.05) at the highest dose.     

A behavioural study of the effect of pentadecapeptide BPC 157 in Parkinson's disease models in mice and gastric lesions induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine.

The effect of a stomach pentadecapeptide, BPC 157, on Parkinson's disease in mice was investigated, along with its salutary activity on stomach lesions induced by parkinsongenic agents. Parkinsongenic agents, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30.0 mg x kg(-1)b.w. i.p. once daily for 6d, and after 4d once 50.0 mg x kg(-1)b.w. i.p.) or reserpine (5.0 mg x kg(-1)b.w. i.p.) were applied i.p. BPC 157 (1.50 microg or 15.0 ng x kg(-1)b.w. i.p.) was applied 15 min before or alternatively 15 min after each MPTP administration. In reserpine studies, BPC 157 (10.0 microg or 10.0 ng x kg(-1)b.w. i.p.) was given either 15 min before reserpine or in the already established complete catalepsy 24 h thereafter. BPC 157 strongly improved the MPTP-impaired somatosensory orientation and reduced the MPTP-induced hyperactivity, and most importantly, MPTP-motor abnormalities (tremor, akinesia, catalepsy -otherwise very prominent in saline control), leading to almost complete abolition of otherwise regularly lethal course of MPTP treatment in controls. Likewise, in reserpine experiments, BPC 157 strongly prevented the development of otherwise very prominent catalepsy and when applied 24 h thereafter reversed the established catalepsy. In addition, a reduction of reserpine-hypothermy (BPC 157 pre-treatment) and reversal of further prominent temperature fall (BPC 157 post-treatment) have been consistently observed. Taking together these data, as the two most suitable animal models were consistently used and since the high effectiveness was demonstrated in pre- and post-treatment, microg and ng regimens, BPC 157 as an organoprotector should be further therapeutically investigated. Additionally, given in either regimen, pentadecapeptide BPC 157 strongly attenuated the stomach lesions in mice that otherwise consistently appeared in mice treated with the parkinsogenic neurotoxin MPTP.     

The effect of isoniazid (INH) on Chinese hamster and mouse spermatogonia

Acute and chronic treatment of Chinese hamsters and mice with 5, 25 and 125 mg/kg isoniazid (INH) given by oral intubation did not enhance the chromosomal aberration frequencies in spermatogonia. The structural and numerical aberration rates remained in the range of spontaneous events even after the chronic treatment over 12 weeks with 3 x 125 mg/kg INH per week. This dose is much higher than normally used for prevention and therapy in man (3--16 mg/kg).     

Caffeine effects on the chromosomal aberrations induced in vivo by sarcolysine and methotrexate

Wistar adult rats bearing Guérin T8 ascite tumours were intravenously inoculated with 3 mg/kg b.w. sarcolysine and respectively 5 mg/kg b.w. methotrexate on the 7th day after ascite cells grafting. Four hours after cytostatic administration, 5 mg/kg b.w. caffeine was intravenously given. Cytogenetic observations concerning the frequency and the type of induced chromosomal aberrations were performed 24 hrs after cytostatic administration, both in the animals treated with only sarcolysine, methotrexate and caffeine and in those double-treated with cytostatics and caffeine. Chromosome examinations were also performed in untreated controls. Both in the sarcolysine- and methotrexate- treated tumors, the induced chromosome lesions were enhanced by caffeine administration, but this effect was very obvious in the methotrexate experiments and rather weak in the sarcolysine treatments (see table). This different effect of caffeine might be due to the different mechanisms by which sarcolysine and methotrexate are interfering in DNA replicating processes.     

Phosphatidylserine externalization during bone marrow cell death after treatment of mice with WR-2721 and gamma-rays

The cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment were assessed in the bone marrow of adult male Swiss mice exposed to a single 6 Gy dose of 60 Co gamma-rays, and treated intraperitoneally with the aminothiol WR-2721 (Amifostine, S-2-/3-aminopropylamino/ethyl phosphorothioic acid), at a dose of 400 mg/kg body weight, 30 min prior to gamma-irradiation. The bone marrow cells were stained with a combination of fluoresceinated annexin V (annexin V--FITC) and propidium iodide (PI) at 3 h, 7 h, and 24 h after treatment of mice with WR-2721 and 60Co gamma-irradiation. The number of early apoptotic cells (annexin V--FITC positive/PI negative), and late apoptotic and necrotic cells (annexin V--FITC positive/PI positive), was increased at 3 h after exposure of mice to 60Co gamma-rays and thereafter declined with the frequency of apoptotic and necrotic cells remaining lower in WR-2721 pre-treated mice. Using the annexin V--FITC flow cytometric assay, the radioprotective effect of WR-2721 against induction of apoptosis and necrosis in normal cells of the haematopoietic system was shown.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

In vivo postirradiation protection by a vitamin E analog, alpha-TMG

The water-soluble vitamin E derivative alpha-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with alpha-TMG against a range of whole-body lethal (8.5-12 Gy) and sublethal (1-5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.     

Anticlastogenic activity of beta-carotene against cyclophosphamide in mice in vivo

beta-Carotene (BC), a natural food colourant and an antioxidant, acts as an antimutagen/anticarcinogen in several test systems. The anticlastogenic activity of BC against cyclophosphamide (CP) was studied in bone marrow cells of mice in vivo. Seven days' oral priming with BC (2.7 and 27 mg/kg b.w.) followed by an acute treatment with cyclophosphamide (25 mg/kg b.w.; i.p.) inhibited clastogenicity. The values of chromosomal aberrations and micronucleated polychromatic erythrocytes were consistently lower than the sum of the expected values of BC and CP given individually. This antagonistic response indicates anticlastogenic activity of BC against CP.     

Mutagenic effect of cyclophosphan on bone marrow cells of irradiated rats]

The rate of chromosome aberrations in bone marrow cells of male rats was investigated in 24 hours after the cyclophosphan intraperitoneal injection (25 mg/kg). Cyclophosphan was given to rats exposed earlier (15 days, 1, 3, 4, 6 or 9 months before) to X- and gamma-irradiation (400 rads). It was found that preliminary irradiation led to the increase in the mutagenic effect of cyclophosphan as compared to that obtained for intact rats. This effect was demonstrated during 4 months after acute X-irradiation at a dose rate of 70 rads/min and during 1 month after chronic gamma-irradiation at a dose rate of 100 rads/day. Later the effect was shown to disappear in both cases. Chronic irradiation was found to be less efficient in the stimulation of chromosome damages caused by chemical mutagens. The increase of the mutagenic effect of cyclophosphan resulted in the increase of both the number of cells carrying chromosome breaks and the severity of a damage per cell. Different ways of the irradiation effect on the mutagenic action of chemicals are discussed.     

Nature of chromosome gaps induced by alkylating agents and γ-rays as revealed by caffeine treatment

In the cells of primary roots of Crepis capillaris, post-treatment with caffeine increased the frequency of gaps and chromosomal aberrations induced by the alkylating agents ethyleneimine and N-nitroso-N-methylurethane and γ-rays. The increase in the frequency of gaps was considerably lower than that observed in chromosomal aberrations, this being more strongly expressed in the case of the alkylating agents. The potentiating effect of caffeine on the γ-ray-induced chromosomal gaps was a little higher in S as compared with G₂. These results lead to the conclusion that the alkylating agents and the γ-rays might induce 2 types of chromosomal gap.     

Cytotoxicity of zinc chloride in mice in vivo

Intraperitoneal administration of zinc chloride (ZnCl2) to Swiss albino mice in vivo induced a significant (p less than or equal to 0.05) increase in the frequencies of chromosomal aberrations of the bone-marrow cells at all concentrations used following acute (7.5, 10, 15 mg/kg body weight) and chronic (2.0, 3.0 mg/kg body wt) treatment. The degree of clastogenicity was directly proportional to the concentrations (p less than or equal to 0.05, trend test) and indirectly to the period of treatment (p less than or equal to 0.05, ANOVA test). It induced a dose-dependent, statistically significant increase (Mann-Whitney U statistics, Student's t-test) in sperm-head abnormalities. The data designate ZnCl2 as a potent clastogen and as a toxic chemical at the concentrations used.     

DNA-protein cross-links in different organs of mice induced by the combined action of zinc and gamma-irradiation

Fractional whole-body gamma-irradiation of mice at total doses of 0. 5-1.5 Gy induces increased DNA-protein cross-links (DPCs) in thymus, spleen, and brain, whereas in liver no DPCs are detected. Chronic administration of zinc ions in drinking water at concentration 10 mg/liter for 20-30 days increased DPCs in thymus, spleen, brain, and liver of mice. The combined action of zinc ions and gamma-radiation produced a significantly lower amount of DPCs than was induced by the separate action of these agents.