Martha S. Bianchi

Kinetics of human lymphocyte division and chromosomal radiosensitivity

Human blood from normal donors was irradiated with 200 R during the Go phase, and the X-ray sensitivity of early and late dividing lymphocytes in culture was expressed as percentage of induced dicentrics. Cells in first or subsequent divisions were individualized by BrdU-Giemsa techniques. Lymphocytes in the first division at 40, 44 and 72 h after the start of culture had a lower sensitivity to radiation than lymphocytes making their first division at 48, 52 and 56 h. It was observed that: (a) the combination of radiation followed by BrdU did not increase the clastogenic action of X-rays, (b) X-rays in the dose and duration used in our cultures did not increase the frequency of SCEs, and (c) minor changes in culture conditions probably influenced the frequency of SCEs.

Sister chromatid exchanges in down syndromes and normal human beings

The BrdU-Giemsa method was used to analyze the frequency of SCEs in a group of five Down syndromes and in a group of five normal human beings. In total 25 second mitoses were scored for SCEs in each individual. Although Down syndromes exhibited a tendency to have higher rates of exchange than normal human beings the analysis of variance showed that these differences were not statistically significant. On the other hand, significant differences were observed in the rates of SCEs between individuals within each group. This variability may reflect inter-individual differences in the efficiency of the mechanisms involved in the production of exchanges. The frequency of SCEs in blood cultures in probably the average of the rates exhibited by two or more lymphocyte sub-populations with different sensitivities to BrdU. Hence, the variability in the rate of exchange between different cultures of the same individual probably arises by changes in the percentage of cells in the second mitosis deriving from each lymphocyte sub-population.

Effect of bleomycin on interstitial telomeric sequences of immortalized Chinese hamster ovary cells.

The effect of the radiomimetic compound bleomycin (BLM) on interstitial telomeric sequences (ITSs) was investigated in Chinese hamster ovary (CHO) cells by using PNA-FISH with a pantelomeric probe. CHO cells were exposed to increasing concentrations of BLM and chromosomal aberrations were analyzed in the first mitosis after treatment. Cytogenetic analysis revealed that 18.1% and 9.5% of the total aberrations observed in cells exposed to BLM and harvested 18h and 3h after treatment, respectively, exhibited one or more FISH-detectable telomeric signals. Most of the chromosome breaks exhibiting telomeric signals observed in BLM-treated cells occurred in the centromeric regions of chromosomes. This observation, along with the finding of entirely labeled acentric fragments in BLM-exposed cells but not in untreated cells, shows that this antibiotic induces breakage at chromosomal sites containing ITSs. In addition, our results show that heterochromatic ITSs are involved more than expected in the formation of chromosome/chromatid breaks - and perhaps chromatid exchanges - induced by BLM, taking into account the percentage of the genome covered by telomeric sequences. On the contrary, our data strongly suggest that ITSs are not preferentially involved in the formation of dicentrics, multicentrics, centric rings, acentric fragments or chromatid deletions induced by BLM. Moreover, our results show that BLM is capable of inducing amplification and translocation of telomeric repeats, and suggest that this antibiotic produces breakage within centromeric ITSs, although chromosome regions containing these sequences are not the preferential target for BLM clastogenic action. On the other hand, our results show that BLM treatment increases the size of ITSs and that this effect is not related to the chromosomal sensitivity of the exposed cells to this compound.

Karyological conservatism in South American camelids

Llama, guanaco, vicuna and alpaca show similar diploid numbers, gross chromosomal morphology and homologous G, C and NOR banding patterns. This chromosomal homology is also found in the two-humped camel and very probably in the one-humped camel as well. These findings indicate that the camelid karyotype can probably be traced back to early Miocene times. This probably represents the most extreme case of chromosomal conservatism among mammals.

Chromosomal radiosensitivity of pig leucocytes in relation to sampling time

Pig blood cultures were used to analyse the sensitivity to X-rays (measured as frequency of induced dicentrics) of lymphocytes sampled at variable times. By using the BrdU-Giemsa method it was possible to identify the lymphocytes that were performing their first division at early (less than 30% of cells in second division), intermediate (30-50% of cells in second or subsequent divisions) and late stages (more than 50% of cells in second or subsequent divisions). No difference was found in the radiosensitivity of these 3 varieties of lymphocyte. It was also observed that: (a) the combination of radiation followed by BrdU treatment did not increase the clastogenic action of X-rays, (b) X-rays in the dose used in our cultures did not increase the frequency of SCEs, and (c) minor changes in culture conditions probably influence the basal frequency of SCEs.

Effect of Thiol Compounds on Bleomycin-Induced DNA and Chromosome Damage in Human Cells

ABSTRACT Non-protein thiols are considered radioprotectors, preventing DNA damage by ionizing radiation. As bleomycin (BLM) is a radiomimetic agent it was proposed that thiols may prevent DNA damage produced by this antibiotic. However, results obtained with thiols and BLM-combined treatments in living cells are contradictory. The goal of this work was to assess the influence of five non-protein thiols of different electrical charge and chemical composition, on the DNA damage, DNA repair, chromosomal aberrations and cell killing induced by BLM. We found that, at the chromosomal level and cell killing, Glutathione, β-Mercaptoethanol and cysteine showed a protective effect, while ditiothreitol and cysteamine increased them, whereas at the DNA level all thiols potentiated the DNA damage induced by BLM, most probably due to a reactivation of the BLM complex.

Analysis of spontaneous and bleomycin-induced chromosome damage in peripheral lymphocytes of long-haul aircrew members from Argentina

Spontaneous and bleomycin (BLM)-induced chromosomal aberrations in G0 and G2 stages of the cell cycle have been analyzed in peripheral lymphocytes of 21 long-haul aircrew members from Argentina in order to assess BLM-induced clastogenesis as a first approach to determine the DNA repair capacity and thereby the susceptibility to environmental cancers in aircrew. The possibility that occupational exposure of flight personnel to cosmic radiation can induce an adaptive response in their peripheral lymphocytes that can be detected by a subsequent in vitro treatment with BLM was also investigated. For comparison, aberrations were also scored in the lymphocytes of 15 healthy volunteers matched by age, health, sex, drinking and smoking habits to the flight personnel group. Aircrew exhibited a higher frequency of spontaneous dicentrics and ring chromosomes than the control population (p<0.05). BLM sensitivity test showed that aircrew and controls are equally sensitive to BLM G2 clastogenic effects, since both groups exhibited a similar frequency of chromatid breaks per cell (p>0.05). However, the aircrew sampled population was almost two times more sensitive to BLM G0 clastogenic effects than controls (p<0.05). Therefore, our data suggest that chronic exposure of aircrew to cosmic radiation increases the in vitro chromosomal sensitivity of their peripheral lymphocytes to BLM (at least in the G0 stage of the cell cycle), and that occupational exposure of flight personnel to cosmic radiation does not induce an adaptive response to this radiomimetic compound. Our results justify further studies aimed at determine if those aircrew members hypersensitive to BLM are more prone to develop environmental cancer than BLM-insensitive individuals.

Kinetics of division in PHA-stimulated pig lymphocytes

Our results indicate that pig lymphocytes in culture complete their 1st division at 24 h. At 36 h there are 9% of cells in 2nd division. 3rd mitosis appears at 48 h; and at 72 h there are cells engaged in the 4th division.

Telomere instability is present in the progeny of mammalian cells exposed to bleomycin.

We analyzed the chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if this antineoplastic drug induces long-term telomere instability. To this end, rat cells (ADIPO-P2 cell line, derived from adipose cells from Sprague-Dawley rat) were treated with a single concentration of BLM (2.5 μg/ml), and chromosomal aberrations were analyzed 18 h and 10 days after treatment by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18 h and 10 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations 10 days after treatment decreased about 25% compared with the one at 18 h after treatment. Moreover, the level of telomerase activity in BLM-treated cells compared with that of untreated control cells was significantly higher at 10 days after treatment, but did not differ at 18 h after treatment. These data indicate that in terms of unstable aberrations, the in vitro clastogenic effect of BLM on ADIPO-P2 cells persists for at least 10 days after exposure. In addition, our data demonstrate, for the first time, that BLM-induced telomere instability in mammalian cells (cytogenetically detectable as incomplete chromosome elements and telomere FISH signal loss and duplication) persists for several generations after exposure. Moreover, the appearance of telomere fusions in BLM-exposed cells 10 days after treatment suggests that this compound can induce delayed telomere instability. The increase in telomerase activity in BLM-exposed cells 10 days after treatment is accompanied by the presence of aberrations directly related to telomere dysfunction. This fact suggests that telomerase is not directly involved in BLM-induced telomere instability.

Relationship between heterochromatic interstitial telomeric sequences and chromosome damage induced by the radiomimetic compound streptonigrin in Chinese hamster ovary cells.

The relationship between (heterochromatic) interstitial telomeric sequences (ITSs) and the chromosome damage induced by the radiomimetic compound streptonigrin (SN) was investigated in Chinese hamster ovary (CHO) cells by using PNA- and Q-FISH techniques with a pantelomeric probe. CHO cells were exposed to increasing concentrations of SN and chromosomal aberrations were analyzed in the first mitosis after treatment. Cytogenetic analysis revealed that 16.9% and 11.7% of the total aberrations induced by SN in cells harvested 18 h and 3 h after treatment, respectively, exhibited one or more FISH-detectable telomeric signals. Although there was a significant induction by SN of chromosome breaks at centromeric regions containing ITSs, about 70% of the chromosome breaks exhibiting telomeric signals observed in SN-treated cells occurred outside the centromeric regions of chromosomes. This observation, along with the finding of entirely labeled acentric fragments in both untreated and SN-treated cells show that, although this antibiotic induces breakage at centromeric regions containing ITSs, these chromosome regions are not the preferential target for the clastogenic action of SN. In addition, our results show that heterochromatic ITSs are involved more than expected in the formation of chromatid breaks and exchanges induced by SN, and that these sequences are not preferentially involved in the formation of dicentrics, multicentrics, centric rings, chromosome breaks, acentric fragments and chromatid deletions induced by this antibiotic. These findings indicate that the involvement of heterochromatic ITSs in the chromosome damage induced by SN is not random. Moreover, our results show that SN induces telomeric repeats translocations, although this effect depends on the concentration of the drug, and that this antibiotic increases the size of ITSs, this latter effect not being related to the chromosomal sensitivity of the exposed cells to this compound. The mechanism by which SN induces amplification of heterochromatic ITSs remains to be elucidated.