Effects of the pesticide chlorpyrifos on breast cancer disease. Implication of epigenetic mechanisms.

Chlorpyrifos (CPF) is an organophosphorus pesticide used for agricultural pest control all over the world. We have previously demonstrated that environmental concentrations of this pesticide alter mammary gland histological structure and hormonal balance in rats chronically exposed. In this work, we analyzed the effects of CPF on mammary tumors development. Our results demonstrated that CPF increases tumor incidence and reduces latency of NMU-induced mammary tumors. Although no changes were observed in tumor growth rate, we found a reduced steroid hormone receptor expression in the tumors of animals exposed to the pesticide. Moreover, we analyzed the role of epigenetic mechanisms in CPF effects. Our results indicated that CPF alters HDAC1 mRNA expression in mammary gland, although no changes were observed in DNA methylation. In summary, we demonstrate that the exposure to CPF promotes mammary tumors development with a reduced steroid receptors expression. It has also been found that CPF affects HDAC1 mRNA levels in mammary tissue pointing that CPF may act as a breast cancer risk factor.

Integrative analysis of chromosome banding, telomere localization and molecular genetics in the highly variable Ctenomys of the Corrientes group (Rodentia; Ctenomyidae).

The genus Ctenomys comprises about 70 species with great chromosome diversity. The Corrientes group is one of the most chromosomally variable lineages in the genus, where the diploid number (2n) varies from 41 to 70. In this group, three nominal species and numerous polymorphic and polytypic populations have been described. In order to get insight into the chromosomal evolution of this species complex, we applied different banding and molecular cytogenetic techniques. The results were interpreted in an evolutionary context, based on mitochondrial cytochrome b analyses. Studied samples are representative of the broad chromosomal variability in the group, including specimens with 2n = 42 to 2n = 70. Heterochromatin was scarce but concentrated in a few chromosomes. Centromeric DAPI-negative heterochromatin was observed in some autosomal pairs, which differed among populations. Location and amount of DAPI-neutral heterochromatin within the Y chromosome varied among populations. The variable distribution of heterochromatin indicates its dynamic behavior. NORs were detected in one pair of autosomes, which also differed among some populations. Telomeric FISH signals were observed in all complements only at the chromosome ends. The Corrientes group belongs to a clade that also includes C. pearsoni, C. lami, C. minutus, C. ibicuiensis and C. torquatus. Almost all of these species are variable at the chromosomal level, suggesting that this is the ancestral condition of the clade. Within the Corrientes group, the observed low genetic divergence, in contrast with its high chromosomal variability, is indicative of decoupling between the rates of chromosomal and mitochondrial evolution.

The karyotype of Alouattapigra (Primates: Platyrrhini): mitotic and meiotic analyses.

We describe for the first time the karyotype of the black howler monkey, Alouatta pigra. Conventional staining, G- and C-banding, and fluorescence in situ hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe were performed. Eight free ranging adult individuals, four males and four females, within the natural distribution of the species presented a diploid karyotype with 2n = 58. Mitotic analyses showed an autosomal complement composed of 6 submetacentric, 3 metacentric, and 19 acrocentric chromosome pairs for females, and 6 submetacentric, 3 metacentric, and 18 acrocentric pairs for males. Meiotic analyses in males revealed 27 autosomal bivalents and a quadrivalent composed of a submetacentric X(1) and acrocentric X(2), Y(1), and Y(2). The G-banded karyotype allowed us to identify pair #17 as the autosomal pair involved in the rearrangement and the morphology of the quadrivalent components. C-banding technique in metaphase I corroborated the structure of the quadrivalent showing four C+ centromeres. FISH analysis showed telomeric signals at the terminal regions of all chromosomes. No interstitial signals were detected. DNA sequence data were in accordance with those previously published for this species.

Chromosomal localization of the telomeric (TTAGGG)n sequence in eight species of New World Primates (Neotropical Primates, Platyrrhini).

Chromosomal localization of the telomeric sequence (TTAGGG)(n) in eight New World Primates (Platyrrhini) (Alouatta caraya, Alouatta palliata, Alouatta guariba clamitans, Aotus azarae, Ateles chamek, Cebus nigritus, Cebus paraguayanus, and Saimiri boliviensis) using Fluorescence In Situ Hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe and their possible relationship with the C-banding pattern were analyzed. FISH showed telomeric signals only at the terminal regions of chromosomes from all the species analyzed. Although all of them showed centromeric C+ bands and different size and location of extracentromeric C+ bands, none, except Aotus azarae exhibited (peri)centromeric interstitial telomere-like sequences (ITS). The presence of ITS in Aotus azarae was limited to one pair of submetacentric chromosomes and very likely represents telomeric sequences remaining after a fusion event of ancestral chromosomes during karyotype evolution. Therefore, our data indicate that the distribution of heterochromatin blocks do not correlate with the presence of ITS. However, we cannot rule out the possibility that simple ITS arrays with a few copies of the (TTAGGG)(n) sequence, not detectable by conventional FISH, might play a role in the karyotypic evolution of Ceboidea. Further FISH and molecular studies will be needed to confirm this hypothesis.

Chromosomal localization of the telomeric (TTAGGG)n sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra.

The distribution of the vertebrate telomeric sequence (TTAGGG)(n) in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.

Effect of recombinant interferon-alpha on streptozotocin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells.

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 (p < 0.05) compared to control, whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs or SCEs over the control values (p < 0.05). Moreover, rIFN-alpha-2a had a marked inhibitory effect on the frequency of STZ-induced CAs--both chromosome- and chromatid-type--(p < 0.05) but was unable to prevent SCEs induced by the antibiotic (p > 0.05). 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.

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.

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.

Modulation of streptonigrin's clastogenic effects in CHO cells by the metal-chelating agent 1,10-phenanthroline.

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 < 0.05). When PNT was present in the culture medium, the production of CAs by SN was strongly inhibited (inhibition range = 54.9-80.8%). Similarly, the induction of SCEs by SN was significantly decreased by the addition of PNT to CHO cultures (P < 0.05), although the effect was minor. This finding suggests that intracellular transition metals are implicated in the clastogenesis by SN, and that the Fenton reaction (Fe(2+) + H2O2 --> 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.

Analysis of telomeric repeats and telomerase activity in human colon carcinoma cells with gene amplification.

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.

The effect of 1,10-phenanthroline on the chromosome damage and sister-chromatid exchanges induced by streptozotocin in mammalian and insect cells.

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 (p<0.05). 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%) (p<0.05). 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.

Effects of antioxidants on streptozotocin-induced clastogenesis in mammalian and insect cells.

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<0.05). However, the inhibitory effect was more evident in CHO (40.6-67.5%) than in mosquito (15.2-53.6%) cells. These findings indicate that the chromosome damage induced by STZ can be partially inhibited through the incorporation of antioxidant compounds into the cells and suggest that free radicals are involved in the clastogenesis by STZ.

Chromosomal response of insect and mammalian cells to streptonigrin: a comparative study.

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.

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.

Relationship between pituitary hormones, antioxidant enzymes, and histopathological changes in the mammary gland of senescent rats.

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.

Effects of antioxidants on streptonigrin-induced DNA damage and clastogenesis in CHO cells.

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.

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 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.

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.

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.