Alejandro D. Bolzán

Genotoxicity of Streptozotocin

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.

Superoxide Dismutase, Catalase and Glutathione Peroxidase Activities in Human Blood: Influence of Sex, Age and Cigarette Smoking

OBJECTIVE To obtain reference ranges for each of the main antioxidant enzymes (AOE) and analyze the influence of sex, age, and cigarette smoking on AOE activity in human blood. DESIGN AND METHODS We investigated superoxide dismutase (SOD), catalase (CAT), and seleno-dependent glutathione peroxidase (GSH-Px) activities in the whole blood from 103 healthy subjects, from 18-67 years old (51 males and 52 females). RESULTS We found a large and highly significant interindividual variability in the activity of all the AOE studied (p < 0.001). The interindividual coefficients of variation were 13.5% for SOD, 21.0% for CAT, and 36.2% for GSH-Px, indicating that GSH-Px exhibits the highest interindividual variability. Females showed higher SOD (p < 0.001) and CAT (p < 0.001) activities but lower GSH-Px (p < 0.05) activity than males. We found a significant effect of age on SOD activity (p < 0.001), showing that in human blood it decreases with age and that this decrease is not linear, beginning at 28 years of age. We also observed a linear effect of age on GSH-Px activity indicating that the activity of this enzyme increases with age (p < 0.01). No effect of age on CAT activity was observed (p > 0.05). AOE activity in smokers was found not to be significantly different from that observed in non-smokers (p > 0.05) except in the case of CAT activity in females, which was found to be lower in smokers than in non-smokers (p < 0.05). In addition, we determined reference ranges for the activity of each antioxidant enzyme studied. CONCLUSIONS Our results confirm that AOE activity in human blood exhibits a wide interindividual variability and suggest that this variability may be ascribed, at least in part, to the sex and age of the individuals. Moreover, our results suggest that cigarette smoking does not influence AOE activity in human blood. Accordingly, it is suggested that for clinical purposes it may be necessary to consider the sex and age of the subjects involved in the study.

Genotoxicity of streptonigrin: a review

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.

Hormonal modulation of antioxidant enzyme activities in young and old rats

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 Spearmans 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.

Chromosomal aberrations involving telomeres and interstitial telomeric sequences

Telomeres are specialised nucleoproteic complexes localised at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. In vertebrate chromosomes, the DNA component of telomeres is constituted by (TTAGGG)n repeats, which can be localised at the terminal regions of chromosomes (true telomeres) or at intrachromosomal sites (interstitial telomeric sequences or ITSs, located at the centromeric region or between the centromere and the telomere). In the past two decades, the use of molecular cytogenetic techniques has led to a new spectrum of spontaneous and clastogen-induced chromosomal aberrations being identified, involving telomeres and ITSs. Some aberrations involve the chromosome ends and, indirectly, the telomeric repeats located at the terminal regions of chromosomes (true telomeres). A second type of aberrations directly involves the telomeric sequences located at the chromosome ends. Finally, there is a third class of aberrations that specifically involves the ITSs. The aims of this review are to provide a detailed description of these aberrations and to summarise the available data regarding their induction by physical and chemical mutagens.

FISH analysis of telomeric repeat sequences and their involvement in chromosomal aberrations induced by radiomimetic compounds in hamster cells

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.

Superoxide dismutase activity and superoxide dismutase-1 gene methylation in normal and tumoral human breast tissues

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.

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.

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.

Chromosomal sensitivity of human lymphocytes to bleomycin: Influence of antioxidant enzyme activities in whole blood and different blood fractions

The activity of the antioxidant enzymes catalase (CAT), peroxidases (POD), and superoxide dismutases (SOD) in whole blood and different blood fractions was analyzed in 20 normal human beings and correlated with the chromosomal sensitivity of lymphocytes to bleomycin (BLM) (measured as frequency of dicentric chromosomes per BLM dose). Our results demonstrate that both the physiologic activities of the enzymes and the chromosomal sensitivity to BLM exhibit an ample and significant interindividual variability. An inverse and linear correlation between chromosomal sensitivity to BLM and the concentration of 1) CAT and POD in plasma and 2) SOD in whole blood, erythrocytes, and plasma was found. On the other hand, the chromosomal sensitivity to BLM showed a direct correlation with the concentration of SOD and POD in mononuclear leukocytes. It is suggested that a determination of antioxidant enzyme (AOE) activities in a given cell population may serve to predict the chromosomal sensitivity to BLM.