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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 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

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.

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.

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.

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 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.

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.

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 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.

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.

The distribution of heterochromatin in the chromosomes of the rat was determined by analysing two of its properties: late replication and differential stain with the DNA d-r method. The presence of late and non-late replicating c-heterochromatin in the genome of Rattus norvegicus indicates that this chromatin is an heterogeneous substance exhibiting different properties. Furthermore, the existence of heterochromatin formed by nonrepeated sequences or by sequences with a low degree of repetitiveness is suggested by the presence of late replicating areas which do not react with the DNA d-r method.

Duplication of sex chromosomes was studied in bone marrow cells from adult rats and in short-term cultures of rat fetus cells. Results obtained indicate that: a) G2 period takes 4 hours in cells from fetuses and 3 hours in bone marrow cells, b) S period lasts 7 hours in bone marrow cells and about 20 hours in short-term cultured cells from fetuses, c) In cells from female fetuses one X-chromosome is the last to start DNA synthesis and also one of the X's is the last to end replication, d) In cells from female adult rats both X-chromosomes start and finish DNA synthesis early, e) In both line of cells the Y-chromosome was the last to begin and the last to finish DNA synthesis. — Causes which can account for the differences between the two line of cells are discussed.

SUMMARYThe distribution of secondary constrictions, late replication and c-heterochromatin (as demonstrated by the DNA d-r method) was analysed in the chromosome complement of L. ocellatus. The finding of late and non-late replicating c-heterochromatin allow to surmise that c-heterochromatin is probably an heterogeneous substance formed by different families of repetitive DNA. The coincidence of c-heterochromatin with late replication and secondary constrictions may represent the location of the nucleolar-associated heterochromatin.Several regions of the complement showed the combination of a positive (late replication) with a negative property (lack of differential stain with the DNA d-r method). It is known that f-heterochromatin does not react with the DNA d-r method. Therefore, it is discussed whether the foregoing areas can be considered f-heterochromatin.

We have analyzed the pattern of methylation of rearranged and germ line C kappa genes in DNA samples from human B-type chronic lymphatic leukemia lymphocytes and normal fibroblasts, and from granulocytes, T-, and non-T-lymphocytes separated from normal blood. C kappa alleles are flanked by one HpaII site on each side. Leukemic B-CLL DNA from five patients showed demethylation of these two sites in the productively rearranged allele and methylation of both sites in the nonexpressed allele whether rearranged or in germ-line configuration. Non-T-lymphocytes from normal individuals also showed part of the C kappa genes to have demethylation of both HpaII sites. On the other hand, C kappa genes from normal granulocytes, T-lymphocytes, and four of five fibroblast samples were methylated in one or both HpaII sites. We propose that in B-lymphocytes, demethylation of C kappa alleles is a specific event taking place in the expressed genes and being associated with an increased transcriptional activity of the gene. In addition to this specific demethylation, there is also an unspecific one that may appear in cells with silent C kappa genes and that do not modify the expression of the gene.

Human Cμ genes show two different patterns of demethylation. The first is associated with gene expression; this pattern is restricted to productive Cμ alleles and is detected in leukemic and normal B-lymphocytes but not in T-lymphocytes, granulocytes or fibroblasts. The second pattern, not related with gene expression, is observed in B-lymphocytes, fibroblasts and part of T-lymphocytes but not in granulocytes.

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m2 there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m2 the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m2 did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.

Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the G0 period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into mono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be reparied between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.

The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G2 periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H3. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S1 to S4.