The mechanism of action of low doses of gamma radiation connected with the development of human cell resistance to mutagens]

The human fibroblasts were gamma-irradiated with low doses (0.07-0.21 Gy). After a short time interval (3 h), a study was made of the postirradiation viability of cells (by the trypan blue dye exclusion method); post-N-methyl-N'-nitro-nitrosoguanidine-DNA synthesis (by 3H-thymidine incorporation immediately after the mutagen treatment) and postirradiation induction of DNA single-strand breaks (by alkaline elution of cells lysed on the membrane filters). The preirradiation of cells with low doses of gamma-rays was shown to render the cells resistance to induction of DNA breaks by the following exposure to gamma-radiation. The survival rate increased; DNA synthesis was resistant to alkylation damage in these cells, as compared to nonirradiated cells.     

Role of superoxide dismutase in maintaining cellular homeostasis upon exposure to gamma-rays and nickel sulfate

It was shown that the inhibition of one of the main enzymes of antioxidant system--superoxide dismutasw (SOD)--increased the level of damage in gamma-irradiated and NiSO4-treated human cells. The viability of cells pretreated with inhibitor of SOD (TRIEN) decreased in the experiments with gamma-radiation and with NiSO4. DNA repair and replication involved in cell homeostasis also were suppressed in the cells with inhibited SOD. These data are served as proof of participation of SOD in the defence of human cells against oxidative radicals.     

Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster

In view of the scarcely available information on the in vivo mutagenic and co-mutagenic activity of nickel, the genotoxic potential of two nickel-compounds, nickel chloride (NiCl(2)) and nickel sulphate (NiSO(4)), was assessed in Drosophila melanogaster by measuring two different genetic endpoints. On the one hand, we used the wing-spot assay, which is based on the principle that the loss of heterozygosity of two suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in the imaginal disks of larval cells. On the other hand, the in vivo comet assay, which detects single- and double-strand DNA breaks, was also used with larval haemocytes. These cells offer several advantages: they are highly sensitive to genotoxic agents, the sampling and processing methodologies are quite simple and the level of basal DNA damage is relatively low. No significant increases in the frequencies of the three categories of mutant spots (i.e. small single spots, large single spots, and twin spots) were observed in the wing-spot assay; however, NiSO(4) induced significant dose-dependent increases in DNA damage in the comet assay. In addition, the combined treatments with gamma-radiation and NiCl(2) and NiSO(4) showed a slight but significant increase in the frequency of the three categories of mutant spots compared with the frequency induced by gamma-radiation alone, indicating that both nickel compounds have a synergistic interaction. These results support the assumption that both nickel compounds could act as co-mutagens interfering with DNA-repair processes and that the in vivo comet assay is a sensitive and effective method for detecting the DNA damage induced by NiSO(4) in haemocytes of D. melanogaster.     

Mechanism of the antimutagenic action of interferon: the capacity to protect the human cellular repair system

It has been shown that premutagenic treatment with leukocytic interferon (10, 100 IU/ml) of human peripheral blood lymphocytes cultivated in vitro at the G1-stage of the mitotic cycle results in different cell response to gamma-radiation in doses of 0.5, 1, 2, 4 Gy according to chromosome aberration. The antimutagenic effect failed to be attained with the doses 0.5 and 1 Gy, being maximal at the dose 2 Gy. According to sister chromatid exchanges (SCE) cell pretreatment with interferon leads to a reduction in the effect of gamma-radiation at the dose 2 Gy to the level obtained in the cells after exposure to interferon. In experiments with 4-nitroquinoline-I-oxide, there was a significant decrease in the number of SCE in interferon-treated cells.     

Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation

Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10–100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low-LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.     

Haematoporphyrin derivative photosensitization and gamma-radiation damage interaction in Chinese hamster ovary fibroblasts

The interaction of haematoporphyrin derivative (HPD) photosensitization and gamma-irradiation was studied with regard to clonogenicity of Chinese hamster ovary (CHO) fibroblasts. Exposure to either treatment alone resulted in shouldered response curves. Exposure to 4.2 Gy gamma-radiation immediately before graded doses of visible light had no effect on the shape of the visible-light survival curve; similarly, exposure to 8.75 kJ/m2 light immediately before graded doses of gamma-radiation had no effect on the shape of the gamma-radiation response curve. These data indicate that damage due to gamma-radiation and HPD photosensitization did not interact, suggesting that the mechanisms of cell killing are different.     

Modification of repair DNA synthesis in mutagen-treated human fibroblasts during adaptive response and the antimutagenic effect of garlic extract

Repair DNA synthesis (RDS) in human fibroblasts during the adaptive responses (ARs) induced by cadmium chloride (CdCl2), gamma-radiation, and 4-nitroquinoline-1-oxide (4NQO) was compared in cells pretreated and not pretreated with garlic extract. The RDS was increased during the ARs induced CdCl2 and gamma-irradiation. Garlic extract stimulated RDS in cells treated by the same mutagens. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, decreased the RDS rate in cells treated with CdCl2 and gamma-irradiation but had no significant effect on cells treated with 4NQO. It was demonstrated that DNA repair was involved into cell protection in different ways in the cases of antimutagen treatment and AR.     

Dose responses for adaption to low doses of (60)Co gamma rays and (3)H beta particles in normal human fibroblasts

The dose response for adaption to radiation at low doses was compared in normal human fibroblasts (AG1522) exposed to either (60)Co gamma rays or (3)H beta particles. Cells were grown in culture to confluence and exposed at either 37 degrees C or 0 degrees C to (3)H beta-particle or (60)Co gamma-ray adapting doses ranging from 0.1 mGy to 500 mGy. These cells, and unexposed control cells, were allowed to adapt during a fixed 3-h, 37 degrees C incubation prior to a 4-Gy challenge dose of (60)Co gamma rays. Adaption was assessed by measuring micronucleus frequency in cytokinesis-blocked, binucleate cells. No adaption was detected in cells exposed to (60)Co gamma radiation at 37 degrees C after a dose of 0.1 mGy given at a low dose rate or to 500 mGy given at a high dose rate. However, low-dose-rate exposure (1-3 mGy/min) to any dose between 1 and 500 mGy from either radiation, delivered at either temperature, caused cells to adapt and reduced the micronucleus frequency that resulted from the subsequent 4-Gy exposure. Within this dose range, the magnitude of the reduction was the same, regardless of the dose or radiation type. These results demonstrate that doses as low as (on average) about one track per cell (1 mGy) produce the same maximum adaptive response as do doses that deposit many tracks per cell, and that the two radiations were not different in this regard. Exposure at a temperature where metabolic processes, including DNA repair, were inactive (0 degrees C) did not alter the result, indicating that the adaptive response is not sensitive to changes in the accumulation of DNA damage within this range. The results also show that the RBE for low doses of tritium beta-particle radiation is 1, using adaption as the end point.     

Toxic effects of heavy metal salts on intact and irradiated cultured mammalian cells]

The toxicity of solutions of K2Cr2O7 and NiSO4.8H2O for cultivated Chinese hamster fibroblasts and murine lymphoma Sp-2 cells was determined using three criteria of damage: cell death (dyeing with trypan blue), inhibition of cell proliferation and their colony-forming activity. It was shown that both salts have equal toxicity in (10(-3)-10(-2)) M interval for both culture investigated relative to inhibition of cell proliferation. The threshold of toxicity of K2Cr2O7 relative to reproductive cell death (10(-4) M is smaller than to the inhibition of cell proliferation. The nontoxic concentration of K2Cr2O7 enhanced the radiation-induced reproductive death of fibroblast culture at doses 2 and 4 Gy.     

Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells

Radiation damage incurred by nuclear DNA is well documented and interest is increasing in the properties of 'bystander' factor(s) and their ability to induce radiation-like damage in cells never exposed to radiation. 'Bystander' and direct low-LET radiation effects on the mitochondria, and more particularly the mitochondrial genome are less well understood. In this study HPV-G cells (a human keratinocyte cell line derived from human neonatal foreskin transfected with the HPV-16 virus) were exposed to either gamma-radiation doses as low as 5 mGy and up to 5 Gy from a 60Co teletherapy unit, or to growth medium taken from similarly irradiated cells, i.e. irradiated cell conditioned medium (ICCM). Mutation and deletion analysis was performed on mitochondrial DNA (mtDNA) 4-96 h after exposure. Primers flanking the so-called mitochondrial 'common deletion' were employed to assess its possible induction. Single-strand conformation polymorphism (SSCP) analysis was conducted to identify induced point mutations. The relative mitochondrial number per cell was analysed by semi-quantitative PCR (sqPCR). Results indicate the induction of a relatively novel deletion in the mitochondrial genome as early as 12 h after direct exposure to doses as low as 0.5 Gy and 24 h after exposure to 0.5-Gy ICCM. SSCP analysis identified the induction of point mutations, in a non-consistent manner, in only the D-loop region of the mitochondrial genome and only in cells exposed to 5 Gy, and neither in cells exposed to lower doses of direct radiation nor in those exposed to ICCM. SqPCR also identified an increase in the number of mitochondria per cell after both exposure to low level gamma-radiation and ICCM, indicative of a possible mechanism to respond to mitochondrial stress by increasing the number of mitochondria per cell.     

Infection of repair-deficient human cells by the vaccinia virus decreases the formation of gamma-induced DNA breaks

The alkaline elution method was used to study DNA breaks induced by the different mutagens in human cells. Normal fibroblasts and fibroblasts from patients with homocystinuria, deficient in gamma-type repair were shown to be resistant to gamma-radiation after the vaccinia virus infection. In both cell lines there was a decrease in the number of the mutagen-induced DNA breaks.     

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.     

Changes in the number of DNA-protein cross links in spleen lymphocytes of mice exposed to low intensity low dose gamma irradiation

Levels of DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in spleen lymphocytes were studied in mice exposed to low-intensity gamma-radiation (1.7 mGy/day) for 1, 4, 10, 20, and 30 days. The spleen mass and count of lymphocytes isolated from this organ also has been investigated. The significant increase in the DPC level as compared to the control occurred on the 10-th and 30-th days of irradiation at doses of 1.7 and 5.1 cGy, accordingly. The number of spleen lymphocytes normalized to organ mass significantly decreased on the 4-th and 30-th days of the experiment. No increase was found in levels of alkali-labile sites and SSB. In contrast, the increase in the amount of duplex form DNA was recorded on the 4-th and 30-th days of the experiment. Our indicate that DPC formation after irradiation at low doses represents some form of cellular response to the damaging agent.     

Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.     

HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.     

Ataxia telangiectasia: an anomaly in DNA replication after irradiation.

The effect of increasing dose of gamma-radiation on DNA synthesis in an ataxia telangiectasia lymphoblastoid cell line and a number of control lymphoblastoid cell lines was investigated. No significant inhibition of low molecular weight DNA synthesis was observed in the AT cell line at doses which resulted in considerable inhibition in the control cell lines. At higher doses, 600 to 800 rad, low molecular weight DNA synthesis and chain elongation were enhanced in the AT cell line. At time course study of DNA synthesis after 200 rads of gamma-radiation, revealed no appreciable inhibition of low and high molecular weight DNA synthesis up to 60 minutes postirradiation. However, in control cell lines, overall DNA synthesis was depressed to a level 50% of that shown by the unirradiated cells.