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Bleomycin (BLM) is an antibiotic isolated from Streptomyces verticillus. It has radiomimetic actions on DNA thus it has been widely used in clinical chemotherapy for the treatment of different types of cancer, including head and neck tumors, lymphomas, squamous-cell carcinomas and germ-cell tumors. Because of this, the study of BLM genotoxicity is of practical interest. This antibiotic is an S-independent clastogen and an agent that generates free radicals and induces single- and double-strand breaks in DNA. In the present review, we will summarize our current knowledge concerning the DNA and chromosome damage induced by BLM in mammalian cells, with emphasis on new developments published since 1991. Fil: Bolzan, Alejandro Daniel. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Bianchi, Martha S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

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.

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 (p0.05). 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 (p0.05). However, the aircrew sampled population was almost two times more sensitive to BLM G0 clastogenic effects than controls (p0.05). 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.

In this work, we extend our previous studies concerning mutagen sensitivity in flight personnel from commercial airlines by analyzing the frequency of spontaneous and streptonigrin (SN)-induced sister chromatid exchanges (SCEs) in peripheral blood lymphocytes of 18 long-haul aircrew members from Argentina and of 18 control individuals. Statistical analysis revealed no significant differences between aircrew and controls in the background level of SCEs (p0.05), which suggests that chronic exposure to cosmic radiation and other occupational hazards does not affect SCEs frequency in peripheral lymphocytes of aircrews. The fact that almost no correlation was found between cumulative flight hours and the yield of spontaneous SCEs in aircrews adds further support to this assumption. Therefore, the background SCEs frequency cannot be use as a valid biomarker to determine the genotoxic effects of cosmic radiation or other occupational hazards exposure in aircrews. Following SN treatment, a significant increase in the mean frequency of SCEs was observed in the control group (p0.05) but not in the aircrew group (p0.05), suggesting that at the population level, aircrew are more resistant to the mutagenic effects of SN than controls. The reasons of this resistance remain to be determined. Since cosmic radiation had no effect on the background SCEs frequency and no relationship was found between cumulative flight hours and SCEs inducer effect by SN in aircrews, a direct effect of cosmic radiation on SN resistance should be discarded.

Ctenomys is the most numerous genus of South American subterranean rodents and one of the most karyotypically diverse clades of mammals known. Ctenomys magellanicus is the southernmost species of the group and the only one living in Isla Grande de Tierra del Fuego (Argentina). This species presents two chromosomal forms, i.e. 2n=34, and 2n=36 (FN=68). Recent studies suggest that genetic divergence between both karyotypic forms resulted from a chromosomal speciation process. In order to identify the chromosomal rearrangement involved in the process of karyotype evolution in this species, we used chromosome banding techniques and fluorescence in situ hybridization with a telomeric probe to metaphase chromosomes of the two chromosomal forms of Ctenomys magellanicus. Chromosome analysis of Giemsa-stained and G-banding preparations showed that Cm34 and Cm36 karyotypes differ in one rearrangement involving chromosomes A9 from Cm34 and B12 and B17 from Cm36. In addition FISH analysis showed that all of the chromosomes from both chromosomal forms exhibit a telomeric-only distribution pattern of the (TTAGGG)n sequence, indicating that none of the chromosomal forms of Ctenomys magellanicus has true telocentric chromosomes. Our results suggest that a chromosome fission event would have occurred during the process of karyotype evolution in this species.

We investigated the clastogenic effects of the methylating agent streptozotocin (STZ) on two human colon cancer cell lines. COLO320DM and COLO320HSR are cell lines derived from a human neuroendocrine colon carcinoma. STZ produced a dose-dependent increase in the frequency of chromosomal aberrations in both cell lines (p < 0.05) and induced fragmentation and/or pulverization of COLO320DM and COLO320HSR chromosomes. This effect was dose and time dependent. Severe chromosome damage was also found in cells that had progressed beyond the first metaphase, and a higher percentage of metaphases showing pulverized chromosomes were found in cells after the second than after the first division. This seems to indicate that STZ has a persistent and delayed clastogenic effect on COLO320DM and COLO320HSR cells. In addition, STZ produced a marked depression of the mitotic index in both cell lines. These results demonstrate that human colon cancer cell lines COLO320DM and COLO320HSR are highly sensitive to STZ, and suggest that this antibiotic has a good potential as a chemotherapeutic agent for colon cancer.

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the radiomimetic antibiotic streptonigrin (SN, 250 ng/ml, 20 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-45,000 IU/ml) was added to the cell cultures 30 min before SN and left in the culture medium until the end of SN treatment or until cell harvesting. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with SN (P0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs and SCEs over control values. Low rIFN-alpha-2a doses produced a reduction in the frequency of CAs and an increase in the yield of SCEs induced by SN, while high doses of the cytokine caused an increase in the yield of CAs and a reduction in the frequency of SCEs induced by the antibiotic. In addition, rIFN-alpha-2a caused a marked inhibition (around 50%) on the yield of SN-induced chromatid-type aberrations in the G(2) phase of the cell cycle. It is suggested that the inhibitory effect of rIFN-alpha-2a on the SN-induced chromosome damage is due to the stimulation of DNA synthesis and repair by the cytokine. On the other hand, our results give further support to our previous hypothesis that the induction of CAs and SCEs by SN is based on different mechanisms.

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) by the radiomimetic antibiotic bleomycin (BLM, 5 microg/ml, 30 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-180,000IU/ml) was added to the cell cultures 0.5 or 24h before BLM (and left in the culture medium until the end of treatments) or immediately after BLM treatment (and left in the culture medium until harvesting). Cells were sampled at 18 or 2.5h after the end of treatments, in order to determine, respectively, the effect of rIFN-alpha-2a on the total chromosome damage induced by BLM and on the chromosome damage induced by this antibiotic in the G(2) phase of the cell cycle. A statistically significant increase in the frequency of CAs was observed following treatment with BLM (P0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs over control values (P0.05). The yield of CAs by BLM was significantly inhibited by rIFN-alpha-2a (P0.05, 65.3% maximum inhibition). A strong inhibitory effect (around 80%) of rIFN-alpha-2a on the yield of BLM-induced CAs in the G(2) phase of the cell cycle was also observed. It is suggested that the inhibitory effect of rIFN-alpha-2a on the induction of CAs by BLM is mainly due to the stimulation of DNA synthesis and repair by the cytokine.

We tested for azoospermia factor (AZF) deletions 17 loci corresponding to AZF subintervals a–d in 17 cases of testicular tumors occurring in Finns. While DNA samples from 48 CEPH and 32 Finnish males showed no deletions, patients with testicular cancer displayed AZF deletion mosaicisms in various non-tumor tissues (13 cases) and specific deletion haplotypes in tumor tissues (10 cases). Two of the cases with AZF deletions were testicular non-Hodgkin lymphomas indicating that Y-microdeletions appear also in malignancies other than seminoma and non-seminoma tumors. In good agreement with this assumption, we detected one AZF deletion in normal cells from 1 of 5 HNPCC cases, heterozygous for an MLH1 mutation. We propose that AZF deletions occur in early embryogenesis due to mutations of TSPY , mismatch repair (MMR), or X-specific genes. Since fathers of testicular, tumor cases did not exhibit AZF deletions, we assumed they were not carriers of the mutation inducing AZF deletion-mosaicisms. Therefore, tumor cases should have received the MMR gene or X mutations via the maternal lineage, or for the case of TSPY and MMR genes via a sperm carrying a mutation occurred in the paternal germ-cell line. We consider AZF microdeletions in non-tumor cells to be part of a broader pattern of chromosome instability producing susceptibility to testicular tumors. Clonal transformation and expansion of one of these tumor-susceptible cell lineages give rise to testicular tumors showing genome anomalies characteristic of testicular cancers (i12p, LOH and genetic imbalance for various autosomal regions, Y- and autosomal MSI, specific AZF deletion haplotypes).

The behaviour of telomeric repeat sequences in Chinese hamster CHO and CHE cell lines treated with the radiomimetic drugs bleomycin (BLM) and streptonigrin (STN) and the effect of these drugs on telomerase activity was investigated. Fluorescence in situ hybridisation revealed that 18% of the scored aberrations induced by BLM and 14% of those induced by STN in CHO cells exhibited telomeric repeat signals. In CHE cells, 29% of the total aberrations induced by BLM and 45% of those induced by STN involved telomeric repeat sequences. Acentric fragments labelled along their entire length and translocations of telomeric repeat sequences were also found in both cell lines. These results suggest that telomeric repeat sequences are preferentially involved in chromosome breakage, fragility and recombination induced by radiomimetic agents. In addition, some of the damaged CHE cells exhibited one or more chromosomes with additional zones of hybridisation, indicating the possible amplification of (TTAGGG)n repeats by telomerase. However, the fact that none of the radiomimetic compounds tested produced any effect on telomerase activity suggests that this enzyme is not related to the assumed amplification events induced by BLM and STN in CHE cells.

The effect of the metal-chelating agent 1,10-phenanthroline (PNT) on the clastogenesis induced by streptonigrin (SN) in CHO cells was investigated. When CHO cells were exposed to SN, chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner (P OH* + OH(-) + Fe(3+)) may be responsible for the production of CAs by this compound. Moreover, the fact that PNT did not completely inhibit the induction of SCEs by SN suggests that this phenomenon might be attributable to a different mechanism, in which transition metals and free radicals play a minor role.

The effects of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and the hydroxyl radical scavenger mannitol (MAN) on the clastogenesis induced by STZ in Chinese hamster ovary (CHO) and mosquito cells were investigated. The addition of liposome-entrapped SOD, CAT and MAN to both cell lines caused a significant decrease in the yield of STZ-induced chromosome aberrations (p

We assessed the chromosomal response of insect (mosquito, Aedes albopictus) and mammalian (Chinese hamster ovary, CHO) cells to streptonigrin (SN). Both types of cells were pulse-treated for 20 min with increasing doses of SN and the frequency of chromosome aberrations and sister chromatid exchanges (SCEs) for each SN dose was determined. Our results show that the SN doses inducing remarkable chromosome damage (expressed as frequency of aberrations per cell and per chromosome) in CHO cells fail to produce a significant increase of aberrations in mosquito chromosomes. Moreover, CHO cells exhibited a dose-dependent increase in SCEs which was not observed in mosquito cells. Our results show that while mammalian cells are very sensitive, insect cells are very resistant to SN at the chromosome level. It is possible that variations in the chromatin fibril structure and in the intracellular antioxidant pool may be responsible for the differential response of insect and mammalian chromosomes to SN.

In order to assess the possible involvement of endocrine factors and antioxidant enzymes (AOE) in the mammary pathology typically observed in old female rats, we undertook to determine the relationship between pituitary hormones, AOE activity, and histopathological changes in the mammary gland of senescent rats carrying neoplastic and nonneoplastic mammary pathologies. Serum levels of several pituitary hormones were determined by RIA in young (five months) and senescent (33 months) Sprague-Dawley female rats. The activity of catalase (CAT) and superoxide dismutase (SOD) in mammary tissue from the senescent animals was also determined. Senescent rats showed higher levels of prolactin (PRL) (p < 0.01), thyroid-stimulating hormone (TSH) (p < 0.05) and follicle-stimulating hormone (FSH) (p < 0.01) than their young counterparts. In senescent females the main histopathological findings at mammary level were a marked hyperplasia and the presence of fibroadenomas. In this group there was a positive correlation between serum levels of PRL and the activity of mammary SOD (p < 0.05). There was also a positive correlation between serum levels of FSH and the activity of mammary CAT (p < 0.001). Young females, rendered moderately hyperprolactinemic by means of anterior pituitary grafts, showed clear proliferative changes in their mammary glands. Senescent rats carrying fibroadenomas were less hyperprolactinemic than those with mammary hyperplasia (p < 0.05). Our results provide additional support to the idea that PRL may be a physiological modulator of mammary SOD activity and suggest that FSH can possibly influence the activity of CAT in mammary gland. They also suggest that tumorigenesis but not hyperplasia in rat mammary gland may be associated with low mammary SOD and CAT activities.

We treated CHO cells with streptonigrin (SN) alone, in combination with BrdUrd or IdUrd substitution, and with or without the addition of caffeine. The cells assessed for chromosome damage by SN were in the G2 period and the magnitude of the damage was expressed as monosubstituted chromatid breaks, bisubstituted chromatid breaks and boundary regions breaks (boundary regions indicate the point of exchange of mono- and bisubstituted chromatids). We found that the combination of BrdUrd or IdUrd substitution with SN treatments produced a remarkable increase in the frequency of breaks over the frequencies observed with the halogenated compound only. The effect was more evident with IdUrd than with BrdUrd, and more dramatic in bisubstituted than in monosubstituted chromatids. The frequency of boundary breaks in cells treated with BrdUrd plus SN was similar to the frequency of breaks in monosubstituted chromatids treated similarly. Conversely, the damage in boundary regions was almost similar to that in bisubstituted chromatids in cells challenged with IdUrd plus SN. The addition of caffeine to BrdUrd-substituted chromosomes gave rise to a marked enhancement of breakages with a gradient of chromatid damage that was: bisubstituted > monosubstituted > boundary regions. A further increase of chromatin breaks maintaining the gradient indicated above was obtained when the cells were treated with BrdUrd plus SN plus caffeine. We propose that BrdUrd and IdUrd substitution alone or in combination with caffeine treatments and with SN in its capacity to bind DNA, give rise to different chromatin structures capable of modulating the DNA damage induced along the chromatin fibril by the active oxygen species liberated by SN-DNA complexes.

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 Fil: Mira, Anabela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Giménez, Esteban Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Bolzan, Alejandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Bianchi, Martha S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Lopez Larraza, Daniel Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

The correlation between serum levels of pituitary and thyroid hormones and the activity of two antioxidant enzymes (AOE), catalase (CAT) and superoxide dismutase (SOD), in different immune and nonimmune organs of young and old rats was investigated. Serum levels of growth hormone (GH), prolactin (Prl), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroxine (T4), and triiodothyronine (T3) as well as the activity of AOE in liver (CAT and SOD), spleen (CAT and SOD), thymus (SOD) and mammary tissue (SOD) were determined in 7- and 29-month-old Sprague-Dawley female rats. A marked hyperprolactinemia and lower levels of TSH, LH, FSH and GH were observed in the old rats when compared to the young ones (p < 0.05). Old animals showed a higher SOD activity in liver and thymus but a lower activity in spleen than their young counterparts (p < 0.05). In addition, CAT activity in spleen was lower in old than in young rats (p < 0.05). The Spearman's test revealed a positive correlation between: 1) serum levels of TSH and the activity of SOD in liver and 2) serum levels of Prl and GH and the activity of SOD in mammary tissue of young and old animals (p < 0.05). Likewise, CAT in liver showed a highly significant correlation with serum TSH (but not T4 and T3) levels in young (p < 0.01) but not in old rats. Our results confirm that aging is associated with alterations in the endocrine balance and the activity of AOE in lymphoid as well as in nonimmune organs. In addition, our findings suggest that Prl and GH may be physiological modulators of mammary SOD activity, and that TSH can possibly influence the activity of both CAT and SOD in liver via a thyroid-independent pathway. Furthermore, our data show that, when significant, the correlation between hormone levels and AOE activity deteriorates with age.

CHO cells were pulse-treated for 20 min with increasing doses of streptonigrin (SN). First division cells analyzed 18 h after the end of treatment showed a combination of chromosome and chromatid aberrations with a typical dose-response curve. The frequency of SCEs in second division cells (24 h harvesting time) also exhibited a positive correlation with the SN dose. A high incidence of chromatid aberrations was detected in second and third division metaphases. This indicates that SN has a persistent clastogenic action that lasts for at least three cell cycles after the end of treatment. The kinetics of DNA damage and repair was studied by the alkaline unwinding and single cell gel methods. It was found that a pulse treatment with SN elicited a triphasic response characterized by repair-damage-repair. It is proposed that SN forms stable complexes with the DNA. These complexes by a continuous cycling redox process would produced active oxygen species inducing direct damage to DNA.

The Y chromosome is known to induce testis differentiation in Eutheria and Metatheria. Recently, in Eutheria, a gene ( Sry ), which is the major candidate to be the testis-determining gene, has been identified. We present evidence indicating that DNA sequences homologous to Sry are male specific in two marsupial species. This finding leads us to conclude that Sry also is the testis-determining gene in Metatheria.

SEX chromatin in the amniotic cells of rat foetuses is useful for sex diagnosis before the development of the gonads, especially in view of the fact that sexual dimorphism is not conspicuous in other tissues1,3. To be a simple and accurate technique, however, the preparation of amnion for the examination of sex chromatin requires some special precautions. Absolute or 95 per cent alcohol has been habitually used as the fixative3, but we feel that it is troublesome because it shrinks the cells and coagulates the mucopolysaccharides. Cells of rat foetuses have several heterochromatic bodies1,3, and consequently sex chromatin is not easy to distinguish when the cells shrink. Furthermore, the amnion in young rat foetuses has a considerable content of mucopoly-saccharides which when coagulated hinder fixation and make the cells difficult to see. To overcome these difficulties we have used 50 per cent glacial acetic acid as the fixative, and have obtained with it good fixation and swelling of cells, which makes it easier to differentiate sex chromatin from chromocentres.

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.

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 18 h and 3 h 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.

COLO320DM and COLO320HSR are cell lines derived from a human colon carcinoma. Both lines have an amplification of the MYC oncogene: COLO320DM in the form of double minute chromosomes, COLO320HSR as a large marker chromosome with homogeneously staining regions. We have used pulsed field gel electrophoresis (PFGE) to analyze undigested DNA from both COLO320 cell lines. Upon blotting and hybridization with a MYC probe, the autoradiograms showed the existence of discontinuities in the amplified DNA domains. Additional evidence indicating a preferential concentration of DNA breaks in the region containing the MYC amplicons was obtained with the alkaline unwinding technique. We propose that amplicons are connected by hairpin formation or Hoogsteen pairing between free DNA ends.

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.

Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid probe was employed to analyze the induction of incomplete chromosome elements (ICE; i.e., incomplete chromosomes and terminal fragments) by bleomycin (BLM) in two mammalian cell lines. Chinese hamster embryo cells (CHE cell line, average 2n = 23) and domestic rabbit cells (CPC cell line, average 2n = 44) were treated with 2.5 micro g/ml BLM; after 18 hr of incubation, first-division metaphases were stained with the telomeric probe, and ICE and other unstable chromosomal aberrations were scored. BLM induced ICE, dicentrics, and interstitial acentric fragments in CHE cells, but only ICE in CPC cells. About 50% of the metaphases in BLM-treated CHE cells contained one or more pairs of ICE, while only 20% of treated CPC cells contained ICE. Almost 100% of the BLM-induced ICE in both cell lines consisted of pairs formed by an incomplete chromosome and a terminal fragment. Our results confirm that ICE are the most frequent type of unstable chromosomal aberration induced by BLM in mammalian cells. Moreover, the present study shows that an increase in the chromosome number does not necessarily result in an increase in the frequency of BLM-induced ICE. The results also show that the difference in the chromosomal sensitivity to BLM in CHE and CPC cells is due to differences in the absolute frequency but not in the pattern (i.e., type and proportion) of ICE.

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.

We investigated the induction of incomplete chromosome elements (ICEs; i.e., elements with a telomeric signal at only one terminal end) and interstitial fragments induced by the antibiotic streptonigrin (SN) in a Chinese hamster embryo (CHE) cell line using FISH with a telomeric peptide nucleic acid probe. CHE cells were treated with 0-250 ng/ml SN and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Exposure of CHE cells to SN resulted in a linear concentration-related increase in all of the aberration types analyzed (P < 0.05) except ring chromosomes. Depending on the SN concentration employed, 33-68% of the metaphases contained one or more pairs of ICEs (an incomplete chromosome accompanied by a terminal fragment or two incomplete chromosomes accompanied by a compound fragment). Pooled data from all SN concentrations revealed that 77.8% of the acentric fragments were terminal fragments, 18.8% interstitial fragments, and 3.4% compound fragments. Furthermore, it was estimated that about 80% of excess acentric fragments induced by SN originated from incomplete exchanges or terminal deletions and 20% from complete exchanges (interstitial deletions). These results show that incomplete chromosomes and terminal fragments are the most frequent asymmetrical chromosomal aberrations induced by SN and indicate that true incompleteness is a very common event following exposure to SN.

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid (PNA) probe was employed to analyze the induction of incomplete chromosome elements (ICE, i.e., unjoined or "open" chromosome elements with telomeric signal at only one end) and excess acentric fragments (i.e., in excess of fragments resulting from the formation of dicentric and ring chromosomes) by the methylating agent streptozotocin (STZ) in a Chinese hamster embryo (CHE) cell line. CHE cells were treated with 0-4 mM STZ and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Centric (incomplete chromosomes) and acentric (terminal fragments) ICE were the only unstable chromosome-type aberrations induced by STZ in CHE cells. The induction of these aberrations exhibited a curvilinear concentration-response relationship. About 40% of the metaphases present in cell cultures treated with STZ contained one or more pairs of ICE. In STZ-treated cells, ICE were always observed as pairs consisting of an incomplete chromosome and a terminal fragment. Moreover, all of the excess acentric fragments induced by STZ were of terminal type. These results indicate that chromosomal incompleteness is a very common event following exposure to STZ and suggest that all of the excess acentric fragments induced by STZ originate from terminal deletions.

Seventy-six percent of testicular cancers of origin in Finns have been reported to exhibit AZF deletions. We analyze here 40 testicular tumor cases from Norway and Argentina and we found that AZF deletions occur also in non-Finnish cases but at significantly lower frequency (25%) than in Finland testicular tumor cases. This frequency difference can be attributed to the condition of genetic isolate of the Finnish population and the subsequent prevalence in this ethnic group of genetic factors involved in the origin of AZF deletions associated with malignancies. The finding of a correlation between AZF deletions and a given Y haplogroup would indicate the existence of Y lineages carrying AZF deletion-enhancing gene or genes. This possibility was explored using a set of Y-DNA-markers allowing the identification of Y lineages occurring at high frequency in Finns. We characterized the Y haplogroups in 32 normal Finn males (control group) and 17 cases of testicular cancer in Finns with and without AZF deletions. We found no association between Y lineages and AZF microdeletions, nor between AZF microdeletions and a Y microdeletion (DYS7C) exhibiting high prevalence (>50%) in Finns. The lack of correlation between AZF deletions and Y haplogroups indicates that the origin of these deletions is independent from the Y chromosome genetic background. No AZF deletions were found in familial cases of testicular tumors; hence, hereditary factors inducing the appearance of testicular malignancies in certain genealogies apparently do not increase the susceptibility to AZF deficiencies. AZF deletions are de novo events occurring in prezygotic or in post-zygotic stages. We propose that most AZF deletions associated with testicular tumors are due to post-zygotic Y microdeletions, while most cases of deletions associated with infertility are due to deletions occurring in the germ cell line of proband fathers.

We investigated the clastogenic effects of the methylating agent streptozotocin (STZ) on two human colon cancer cell lines. COLO320DM and COLO320HSR are cell lines derived from a human neuroendocrine colon carcinoma. STZ produced a dose-dependent increase in the frequency of chromosomal aberrations in both cell lines (p0.05) and induced fragmentation and/or pulverization of COLO320DM and COLO320HSR chromosomes. This effect was dose and time dependent. Severe chromosome damage was also found in cells that had progressed beyond the first metaphase, and a higher percentage of metaphases showing pulverized chromosomes were found in cells after the second than after the first division. This seems to indicate that STZ has a persistent and delayed clastogenic effect on COLO320DM and COLO320HSR cells. In addition, STZ produced a marked depression of the mitotic index in both cell lines. These results demonstrate that human colon cancer cell lines COLO320DM and COLO320HSR are highly sensitive to STZ, and suggest that this antibiotic has a good potential as a chemotherapeutic agent for colon cancer.

We assessed the effect of recombinant IFN-alpha-2a (rIFN-alpha-2a) on the induction of CAs and sister-chromatid exchanges (SCEs) by the methylating compound streptozotocin (STZ), in Chinese Hamster Ovary (CHO) cells. The cytokine was added to cell cultures 30 min before STZ and left in the culture medium until the end of the treatment. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with STZ alone (p0.05) compared to control, whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs or SCEs over the control values (p0.05). Moreover, rIFN-alpha-2a had a marked inhibitory effect on the frequency of STZ-induced CAs--both chromosome- and chromatid-type--(p0.05) but was unable to prevent SCEs induced by the antibiotic (p0.05). A decrease in the replication index (RI) was observed in the combined treatments compared with STZ alone-treated cultures, indicating inhibition of DNA synthesis. It is suggested that rIFN-alpha-2a exerts its protective action against the induction of CAs by STZ by stimulating DNA repair.

The detection of incomplete chromosome elements (ICE, i.e., elements with telomeric signal at only one terminal end) and interstitial fragments induced by the radiomimetic compound bleomycin (BLM) was carried out in a Chinese hamster embryo (CHE) cell line using FISH with a telomeric peptide nucleic acid (PNA) probe. CHE cells were treated with 0, 1, 2.5, 5, and 7.5 microg/ml of BLM and chromosomal aberrations were analyzed in the first mitosis after treatment using a telomeric PNA probe. The relationship between chromosomal aberrations frequency and bleomycin concentration was of linear type (P0.05 for all type of aberrations analyzed, i.e., multicentric chromosomes, centric rings, interstitial fragments and ICE). After BLM treatment, about 20-30% of the analyzed metaphases contained one or more pairs of ICE. Acentric interstitial fragments, lacking telomeric signals, were observed with a frequency of about 4-7 times higher than the dicentric frequency. Acentric interstitial fragments and ICE were induced at similar frequencies, except for the lowest BLM concentration (1 microg/ml), where the latter ones showed a higher frequency than the former ones. Furthermore, it was estimated that about 53% of excess acentric fragments originate from complete exchanges (interstitial deletions) and 47% from incomplete exchanges or terminal deletions. These results show that interstitial fragments and ICE are the most frequent asymmetrical chromosomal aberrations induced by BLM and indicate that true incompleteness is a common event following exposure to BLM. Moreover, the comparable trend of the concentration-response relationship for the different aberrations strongly suggests that all BLM-induced asymmetrical aberrations are formed by a similar underlying mechanism.

Streptozotocin (Streptozocin, STZ, CAS No. 18883-66-4) is a monofunctional nitrosourea derivative isolated from Streptomyces achromogenes. It has broad spectrum antibiotic activity and antineoplastic properties and is often used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic beta cells. STZ is a potent alkylating agent known to directly methylate DNA and is highly genotoxic, producing DNA strand breaks, alkali-labile sites, unscheduled DNA synthesis, DNA adducts, chromosomal aberrations, micronuclei, sister chromatid exchanges, and cell death. This antibiotic was found to be mutagenic in bacterial assays and eukaryotic cells. STZ is also carcinogenic; a single administration induces tumors in rat kidney, liver, and pancreas. Several lines of evidence indicate that free radicals are involved in the production of DNA and chromosome damage by this compound. Because of the use of STZ as an antineoplastic agent, the study of its genotoxicity has considerable practical significance. The purpose of this review is to present our current knowledge regarding the genotoxicity of STZ.

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G(0)) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5-24h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) (P0.05). On the contrary, STZ produced a significant and dose-response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes (P0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.

Malfunction of mismatch repair (MMR) genes produces nuclear genome instability (NGI) and plays an important role in the origin of some hereditary and sporadic human cancers. The appearance of non-inherited microsatellite alleles in tumor cells (microsatellite instability, MSI) is one of the expressions of NGI. We present here data showing mitochondrial genome instability (mtGI) in most of the human cancers analyzed so far. The mtDNA markers used were point mutations, length-tract instability of mono- or dinucleotide repeats, mono- or dinucleotide insertions or deletions, and long deletions. Comparison of normal and tumoral tissues from the same individual reveals that mt-mutations may show as homoplasmic (all tumor cells have the same variant haplotype) or as heteroplasmic (tumor cells are a mosaic of inherited and acquired variant haplotypes). Breast, colorectal, gastric and kidney cancers exhibit mtGI with a pattern of mt-mutations specific for each tumor. No correlation between NGI and mtGI was found in breast, colorectal or kidney cancers, while a positive correlation was found in gastric cancer. Conversely, germ cell testicular cancers lack mtGI. Damage by reactive oxygen species (ROS), slipped-strand mispairing (SSM) and deficient repair are the causes explaining the appearance of mtGI. The replication and repair of mtDNA are controlled by nuclear genes. So far, there is no clear evidence linking MMR gene malfunction with mtGI. Polymerase gamma (POLgamma) carries out the mtDNA synthesis. Since this process is error-prone due to a deficiency in the proofreading activity of POLgamma, this enzyme has been assumed to be involved in the origin of mt-mutations. Somatic cells have hundreds to thousands of mtDNA molecules with a very high rate of spontaneous mutations. Accordingly, most somatic cells probably have a low frequency of randomly mutated mtDNA molecules. Most cancers are of monoclonal origin. Hence, to explain the appearance of mtGI in tumors we have to explain why a given variant mt-haplotype expands and replaces part of (heteroplasmy) or all (homoplasmy) wild mt-haplotypes in cancer cells. Selective and/or replicative advantage of some mutations combined with a severe bottleneck during the mitochondrial segregation accompanying mitosis are the mechanisms probably involved in the origin of mtGI.

Streptonigrin (SN, CAS no. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of Streptomyces flocculus. This compound is a member of a group of antitumor agents which possess the aminoquinone moiety and that includes also mitomycin C, porfiromycin, actinomycin, rifamycin and geldanamycin. Because of the potential use of SN in clinical chemotherapy, the study of its genotoxicity has considerable practical significance.SN inhibits the synthesis of DNA and RNA, causes DNA strand breaks after reduction with NADH, induces unscheduled DNA synthesis and DNA adducts and inhibits topoisomerase II. At the chromosome level, this antibiotic causes chromosome damage and increases the frequency of sister-chromatid exchanges.SN cleaves DNA in cell-free systems by a mechanism that involves complexing with metal ions and autoxidation of the quinone moiety to semiquinone in the presence of NADH with production of oxygen-derived reactive species. Recent evidence strongly suggests that the clastogenic action of this compound is partially mediated by free radicals. The present review aims at summarizing past and current knowledge concerning the genotoxic effects of SN.

The effect of the metal chelating agent 1,10-Phenanthroline (PNT) on the streptozotocin (STZ)-induced chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) and mosquito (Aedes albopictus) cells was investigated. Treatment of CHO and mosquito cells with STZ produced a significant and dose-response increase in the yield of CAs as well as SCEs (p0.05). The addition of PNT prevented the induction of CAs by STZ in both types of cells, causing a significant decrease in the frequency of STZ-induced CAs (46.5-72.5%) (p0.05). This fact indicates that intracellular transition metals are implicated in STZ-induced CAs and that the Fenton reaction (Fe(2+)+H(2)O(2)--OH degrees +OH(-)+ Fe(3+)) is partly responsible for the production of CAs by this compound. On the other hand, the addition of PNT to CHO and mosquito cell cultures did not prevent the induction of SCEs by STZ. Therefore, it is valid to assume that the induction of CAs and SCEs by STZ occurs by different mechanisms.

COLO320DM and COLO320HSR are cell lines derived from a human malignant neuroendocrine colon carcinoma. Both lines have a 30–40-fold amplification of a large DNA domain containing the MYC oncogene. By using fluorescence in situ hybridization techniques with a MYC probe, we could demonstrate that MYC amplicons are contained in a large marker chromosome in COLO320HSR cells, in double minutes (dmin) of COLO320DM cells, and in the interstitial regions of 3–4 additional chromosomes in both cell lines. Amplicons in homogeneous staining regions (HSRs) comprise normal MYC genes, while dmin chromosomes contain PVT/MYC chimeras. Although both cell lines showed similar levels of telomerase activity, the telomere length and telomere distribution in chromosomal termini were considerably lower in COLO320DM than in COLO320HSR cells. This indicates that the average telomere length in cancer cells is regulated no only by the rates of telomerase activity but also by some other non-enzymatic mechanisms. Fil: Bolzan, Alejandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Páez, Gerardo L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Bianchi, Martha S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Bianchi, Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina

An adaptive response, low doses of a mutagen rendering cells more able to subsequently cope with higher doses of that or a related challenging mutagen, enhances nucleotide excision repair in human fibroblasts. After fibroblasts were flashed with 20 J/m 2 of UVC, the cyclopyrimidine dimer frequency at any single dinucleotide position remained unchanged for several hours before abruptly displaying first order kinetics of repair. These kinetics were determined by ligation-mediated PCR along exon 9 of the human p53 gene. When a chronic dose of quinacrine mustard (QM) preceded the UVC challenge, the duration of the cyclobutane pyrimidine dimer (CPD) repair lags were reduced by a factor of three and the kinetic half-lives for CPD repair were reduced by a factor of three. The observed repair kinetics are consistent with the following model. The UVC dose required ( K m ) to generate a substrate concentration which half-saturates the cell's repair capacity is 3 J/m 2 for the high affinity (6–4) photoproducts and greater than 100 J/m 2 for the low affinity cyclobutane dimers. After 20 J/m 2 of UVC, the repair enzyme is saturated with (6–4) photoproducts; these competitively inhibit CPD repair by binding all available repair enzyme. After the (6–4)s are repaired, the CPD concentration is less than K m(CPD) and so CPD repair kinetics initiate with first order kinetics. QM-induced enhancement, by increasing the concentration, V max , of repair enzyme, shortens the duration of (6–4) saturation and increases the rate constant for cyclobutane dimer repair. The data exactly fit the expectations from Michaelis kinetics. Transcription coupled repair is less amenable to Michaelis interpretations and enhanced global repair was almost as rapid as the slightly enhanced transcription coupled repair. We infer that repair enhancement is unable to proportionally increase the number of matrix attachment sites necessary for transcription coupled repair. Understanding competitive inhibition between adduct classes and adaptive enhancement of V max is important to understanding the effects of high doses of mutagen mixtures.

The effect of several free radicals scavengers on DNA damage and clastogenesis induced by streptonigrin (SN) in CHO cells was investigated. The addition of the antioxidant enzymes superoxide dismutase and/or catalase on CHO cell cultures did not prevent the induction of DNA and chromosome damage by SN. In fact, when superoxide dismutase was added to the culture medium an increase on the frequency of SN-induced chromosome aberrations was observed. Moreover, the addition of the hydroxyl radicals scavenger mannitol caused a significant increase in DNA and chromosome damage induced by SN. On the contrary, when all the antioxidants mentioned above were added-alone or in different combinations-encapsulated into liposomes, a significant decrease in the yield of SN-induced chromosome aberrations and DNA damage was observed. These findings indicate that free radicals are involved in the production of DNA and chromosome damage by SN and that this damage can be partially inhibited through the incorporation of antioxidants by the cells.

We have characterized the mtDNA in normal and breast cancer tissues from seven patients. Both types of tissue showed point mutation heteroplasmies and extensive deletions of the mtDNA. The analysis of these polymorphisms allowed us to infer the mtDNA composition of the breast cell that underwent malignant transformation.

The sex determining region Y gene (Sry) is the strongest candidate to be the testis determining factor gene (Tdy). Several South-American Akodon species have two varieties of Y chromosome. One type transmitted via male specimens induces testis development. The second Y variety fails to induce male gonadal differentiation and gives rise to fully fertile XY females. These variant females test positive for Sry. Moreover, sequencing of a partial open reading frame of the conserved region of Sry from males and XY females shows no sequence difference. Sry is two- to sixfold amplified in six of eight akodont species tested. Since Sry amplification was found in species having and not having XY females, amplification apparently does not in itself play a primary role in the origin of sex reversal. The development of fully fertile ovaries in XY Akodon females is not due to a deletion of Sry or to mutations in the Sry segment analyzed in this report. Sex reversal may be due to abnormal expression of this gene at the stage of gonadal differentiation. Alternatively, other genes in the sex-determining pathway may be involved. Several of the Akodon species showing Sry amplification also have amplification of Zfy, which may map to the same region of the Akodon Y chromosome.

The superoxide dismutase (SOD) activities in normal and tumor breast tissues from 14 human females were determined by the epinephrine autoxidation assay. SOD levels showed a marked interindividual variability in normal and malignant cells. However, each donor had a higher SOD activity in cancer than in normal tissue samples. In three cases in which Mn- and CuZnSOD activities were determined, it was found that tumoral increases in SOD were due to increases in both enzymatic forms. Therefore, it seems reasonable to assume a similar situation for all cases in our series. The level of DNA methylation in the SOD-1 gene was assessed in the first four donors. The four cases exhibited full methylation of SOD-1 genes corresponding to normal as well as to cancer cells. It is concluded that the variability in CuZnSOD activities is not related with the state of methylation of the SOD-1 gene. MspI restriction fragment polymorphisms between DNA samples from normal and malignant cells were detected in the four DNA donors. This phenomenon may be due to point mutations changing the frequency of MspI sites or to methylation of the external C in CCGG sequences.

Zinc finger-Y (Zfy) and zinc finger-X (Zfx) genes were analyzed by Southern blotting in male and female specimens of 10 species belonging to the oryzomyne-akodontine stock of Cricetidae rodents. DNA fragments were used as characters to construct a parsimony tree of the genes. Zfx and Zfy trees in general coincide with the evolutionary history of the taxa. Both trees show Oryzomys longicaudatus genes as the outgroup whereas Akodon xanthorrhinus genes are also distant from those of the other species. Oxymycterus rufus and Bolomys obscurus share related sequences, while genes from the other six Akodon species form a group of their own. It was found that 9 out of the 10 species analyzed show Zfy amplification in a range varying from 2 to 24 copies and with a pattern that is clade specific. The estimation of the average changes per character strongly suggests that Zfy has evolved more rapidly than Zfx; our estimates of the rate of nucleotide substitution are 4.6 times higher for Zfy than for Zfx.

COLO320DM and COLO320HSR are two human cell lines with 30–40-fold c-myc amplification. Half of the c-myc gene copies in COLO320DM are truncated and expressed as the predominant mRNA, while half are normal. Most c-myc copies in COLO320HSR are normal and expressed. Truncated c-myc genes are fully methylated, while the normal ones are fully demethylated irrespective of their stage of expression. The normal transcriptionally active c-myc from fibroblast cells is fully methylated, while c-myc from ganulocytes (probably downregulated) is almost fully demethylated. These results indicate a lack of correlation between expression and the state of methylation for human c-myc oncogenes. Furthermore, exons 1 and 2 and intron 1 of c-myc are CpG-rich islands. Since these islands are constitutively demethylated, it is assumed that demethylation is the constitutive state and methylation the facultative state of the c-myc oncogene.