Genotoxic Effects Induced by Cd(+2), Cr(+6), Cu(+2) in the Gill and Liver of Odontesthes bonariensis (Piscies, Atherinopsidae).

Genotoxic effects of Cd(+2), Cr(+6), and Cu(+2) on the gill and liver of the Argentinean Silverside (Odontesthes bonariensis) were studied using the comet assay and in relation with the metal tissue accumulation. Fish were exposed to three waterborne concentrations of each metal for 2 and 16 days. Genotoxicity was assessed by the single cell gel electrophoresis (comet assay). After 2 days, significant increase of the genetic damage index (GDI) was only observed in the gill of fish exposed to Cr(+6) and Cu(+2), and the LOECs were 2160 nM and 921.1 nM, respectively. The gill LOEC for Cd(+2) by 16 days was 9.4 nM. In the liver, LOECs were obtained only for Cd(+2) and Cr(+6) and were 9.4 and 2160 nM, respectively. The three metals were able to induce genotoxic effects at environmentally relevant concentrations and the gill was the most sensitive organ.

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation.

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR (192)Ir). Experiments were carried out by irradiating G(1) CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 microg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 microg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.

Differential response of two cell lines sequentially irradiated with low X-ray doses.

An experiment was designed to compare the effect of repeated low doses of X-rays in two different cell lines: one transformed, epithelial like and aneuploid Chinese hamster ovary K-1 (CHO-K1); the other originated from a human primary culture, fibroblast, diploid and non-transformed, MRC-5. CHO and MRC-5 cells were cultured for 14 or eight passages, respectively. Irradiation was performed once per passage when cells were in the quiescent state (90 - 95% in G1/G0). Cells were exposed to 10.0 mSv X-ray doses. Ionizing radiation did not induce apoptosis or necrosis in the exposed CHO cell population. Significant increases of low-level damaged cells (degrees 1 and 2) were found for the 14 cycles of radiation when compared with controls, except for the first irradiation cycle. No significant increases in the frequency of cells with severe damage were observed. The frequency of MRC-5 cells with low-level damage increased significantly when compared with controls for radiation cycles seven and eight. Significant increases of apoptosis, necrosis and severe damage were found only for the highest dose. Transformed and non-transformed cell types responded differently to direct and indirect damage using low-dose repeat exposures to ionizing radiation. Though more investigation is needed to understand the mechanisms of radiation effects in chronic low-dose-exposed cell populations, cellular type should be taken into account in the design of in vitro experiments for understanding low-dose-irradiation effects.

Association between copper deficiency and DNA damage in cattle.

Cattle hypocuprosis is the second most widespread mineral deficiency affecting grazing cattle. The consequences of hypocuprosis include a failure of copper metalloenzymes, many of which form part of the antioxidant defence system. This work focuses on the association between copper (Cu) plasma concentration and DNA damage in Aberdeen Angus cattle. Two-hundred and ninety-nine heparinized blood samples from 2-year-old Aberdeen Angus cows were obtained from different farms located in the Salado River basin, Argentina. Plasma copper level analysis was carried out in whole samples, while cytogenetic analysis and single cell gel electrophoresis assay (comet assay) were carried out in 82 and 217 samples, respectively. Cytogenetic analysis showed a significant increase in the frequency of abnormal metaphases in moderate/severe hypocupremic groups (groups B and C) in relation to the normocupremic group (group A) (4.5 and 1.5 abnormal metaphases/100 cells, respectively, P < 0.01). The Spearman correlation test showed a negative association between cupremic values and the yield of chromosomal aberrations (r = -0.708, P < 0.0001). In the comet assay greater migration was observed in cells from the hypocupremic group, from a median of 54 in the severe hypocupremic group to 31 in the normocupremic group (P < 0.01). Accordingly, the Spearman correlation test showed a significant positive relationship between copper levels and cells without DNA migration and a significant negative relationship between copper levels and cells with a tiny tail (P < 0.0001 in both cases). The results obtained show that hypocupremia in cattle is associated with an increase in the frequency of chromosomal aberrations as well as in DNA migration as assessed by the comet assay. Whereas the comet assay could differentiate copper plasma level groups, chromosomal aberrations only detected differences between normal and hypocupremic animals. The increase of DNA damage found in hypocupremic animals could be explained by higher oxidative stress suffered by these animals.

Gene frequency distribution of the BoLA-DRB3 locus in Saavedreño Creole dairy cattle.

The objective of this study is to describe the gene frequency distribution of the bovine lymphocyte antigen (BoLA)-DRB3 locus in Saavedreño Creole dairy cattle and to compare it with previously reported patterns in other cattle breeds. One hundred and twenty-five Saavedreño Creole dairy cattle were genotyped for the BoLA-DRB3.2 allele by polymerase chain reaction and restriction fragment length polymorphism. Twenty-two out of 53 previously identified BoLA-DRB3.2 alleles were detected, with gene frequencies ranging from 0.4 to 16.8%. Seventy percent of the variation corresponded to the seven most frequent alleles (BoLA-DRB3.2*7, *8, *11, *16, *27, *36, and *37). The studied population exhibits a high degree of expected heterozygosity (he = 0.919). The FIS index did not show significant deviation from Hardy-Weinberg equilibrium. However, the neutrality test showed an even gene frequency distribution. This result could be better explained assuming balancing selection instead of neutral or positive selection for one or a few alleles. In conclusion, the results of this study demonstrated that BoLA-DRB3.2 is a highly polymorphic locus in Saavedreño Creole dairy cattle, with significant variation in allele frequency among cattle breeds.

Polymorphism in the bovine BOLA-DRB3 upstream regulatory regions detected through PCR-SSCP and DNA sequencing.

In the present work, we describe through polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing the polymorphism within the URR-BoLA-DRB3 in 15 cattle breeds. In total, seven PCR-SSCP defined alleles were detected. The alignment of studied sequences showed six polymorphic sites (four transitions, one transversion and one deletion) in the interconsensus regions of the BoLA-DRB3 upstream regulatory region (URR), while the consensus boxes were invariant. Five out of six detected polymorphic sites were of one nucleotide substitution in the interconsensus regions. It is expected that these mutations do not affect significantly the level of expression. In contrast, the deletion observed in the sequence between CCAAT and TATA boxes could have some effect on affinity interactions between the promoter region and the transcription factors. The URR-BoLA-DRB3 DNA analyzed sequences showed moderate level of nucleotide diversity, high level of identity among them and were grouped in the same clade in the phylogenetic tree. In addition, the phylogenetic tree, the similarity analysis and the sequence structure confirmed that the fragment analyzed in this study corresponds to the URR-BoLA-DRB3. The functional role of the observed polymorphic sites among the regulatory motifs in bovine needs to be analyzed and confirmed by means of gene expression assays.

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays.

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.

African and European mitochondrial haplotypes in South American Creole cattle.

South American Creole cattle are direct descendants of the animals brought to the New World by the Spanish and Portuguese during the 16th century. A portion of the mitochondrial D-loop was sequenced in 36 animals from five Creole cattle populations in Argentina and four in Bolivia. Individuals belonging to the potentially ancestral Spanish breed Retinta were also analysed. Sequence comparisons revealed three main groups: two with the characteristics of European breeds and a third showing the transitions representative of the African taurine breeds. The African sequences were found in two populations from Argentina and three populations from Bolivia, whose only connections go back to colonial times. The most probable explanation for the finding is that animals could have been moved from Africa to Spain during the long-lasting Arabian occupation that started in the seventh century, and from the Iberian Peninsula to America eight centuries later. However, since African haplotypes were not found in the Spanish sample, the possibility of cattle transported directly from Africa cannot be disregarded.

Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

Patagonian Argentine Creole cattle polymorphism: comparison with North-West populations of this breed.

The relict Patagonian Argentine Creole cattle population consist of a small feral population (Los Glaciares population) that is geographically isolated in the South-West of Patagonia. In order to determine the level of genetic variability of this population, the polymorphism of eight structural genes and two microsatellites loci were studied using the polymerase chain reaction (PCR). In addition, genetic characterisation was used to compare Los Glaciares population and the ACc breed of cattle. Results obtained in this study show that the value of average heterozygosity of the studied loci for the Los Glaciares were not significantly different from the ACc. Furthermore, the data of this report were consistent with the hypothesis that Los Glaciares originated from ACc brought to the area by colonialists in the last century. Such data may be useful in formulating management plans for Feral Patagonian Creole cattle populations.

Association of human papillomavirus viral load with HPV16 and high-grade intraepithelial lesion.

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other 'high-risk' viral types (HPV -18, -31, -33 or -51) and 'low-risk' types (P < 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p< 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.

Phylogenetic relationships of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences.

South American horses constitute a direct remnant of the Iberian horses brought to the New World by the Spanish conquerors. The source of the original horses was Spain, and it is generally assumed that the animals belonged to the Andalusian, Spanish Celtic, Barb or Arabian breeds. In order to establish the relationship between Argentinean and Spanish horses, a portion of the mitochondrial D-loop of 104 animals belonging to nine South American and Spanish breeds was analysed using SSCP and DNA sequencing. The variability found both within and between breeds was very high. There were 61 polymorphic positions, representing 16% of the total sequence obtained. The mean divergence between a pair of sequences was 2.8%. Argentinean Creole horses shared two haplotypes with the Peruvian Paso from Argentina, and the commonest haplotype of the Creole horses is identical to one of the Andalusian horses. Even when there was substantial subdivision between breeds with highly significant Wright's Fixation Index (FST), the parsimony and distance-based phylogenetic analyses failed to show monophyletic groups and there was no clear relationship in the trees between the South American and any of the other horses analysed. Although this result could be interpreted as mixed ancestry of the South American breeds with respect to the Spanish breeds, it is probably indicating the retention of very ancient maternal lineages in the breeds analysed.

Malsegregation as a possible mechanism of aneuploidy induction by metal salts in MRC-5 human cells.

Many aneugenic compounds are known to affect one or more components of the mitotic apparatus leading to an erroneous migration of chromosomes. Malsegregation occurs when a chromosome (or a chromatid) fails to migrate and remains at the metaphase plate. Nondisjunction implies the lack of dissociation between sister chromatids and the migration of both together to the same pole. The aim of the present study was to provide evidence that the aneugenic effect of some metal salts is the consequence of malsegregation at anaphase and that it is not caused by nondisjunction mechanisms. The frequencies of lagging chromosomes at anaphase-telophase of mitosis, hypoploid metaphases, and kinetochore-positive micronuclei induced by cadmium chloride, potassium dichromate, and cacodilic acid (dimethylarsinic acid) in MRC-5 human cells were compared. The data indicate that all the tested compounds are able to induce aneuploidy in MRC-5 human cells. Positive, statistically significant correlations were found when kinetochore-positive micronuclei, hypoploidy, and lagging chromosome frequencies were compared. The results suggest that malsegregation is the main mechanism involved in the induction of aneuploidy by metal salts in MRC-5 cells.

Incidence of 1/29 translocation in Bolivian Creole and Brahman Yacumeño cattle.

In Bolivia, four different Creole cattle breeds can be found, as well as other European and Zebu breeds adapted to local environments. The relationship between the occurrence of the 1/29 translocation and subfertility is well known, and analysis of Y chromosome morphology is useful to determine a possible introgression with Bos indicus. The incidence of the 1/29 translocation was analyzed in four Bolivian Creole cattle breeds and the Brahman Yacumeño population, as well as on four farms with phenotypical Creole-type cattle. In 259 (164 dams and 95 sires) Bolivian Creole cattle, 10.42% of the individuals demonstrated the 1/29 translocation, with a variation from 0 to 28.20% between the breeds. In contrast, 43 (19 dams and 24 sires) Yacumeño Brahman and the Creole-type cattle did not show the centric fusion. The highly significant differences between Creole cattle breeds in relation to the incidence of 1/29 translocation could be a consequence of factors such as founder group, genetic drift, and selection. The low frequency observed in the Saavedreñio Creole dairy cattle might be explained by its breeding under a more intensive system, and selection according to milk yield and fertility traits. Finally, no relation between acrocentric Y chromosomes and 1/29 translocation was observed.

Greater genetic variability in Argentine Creole than in Thoroughbred horses based onserum protein polymorphisms

Genetic polymorphism was analyzed for five blood proteins: albumin - Al, esterase - Es, a1B-glycoprotein - Xk, transferrin - Tf and hemoglobin - Hb in 200 Thoroughbred (TB) and 124 Argentine Creole (AC) horses. Of the five systems examined, Tf and Hb were not in Hardy-Weinberg equilibrium in either breed and Es was not in equilibrium in the Creole breed. Genetic variability, estimated as average heterozygosity, was higher in AC (H = 0.585 ± 0.131) than in TB (H = 0.353 ± 0.065). The genetic differentiation between these two populations (FST) was 0.109. Thus, of the total genetic differences between breeds, the proportion of genetic variation attributable to breed differences was about 10%; the remaining 90% was due to individual variation within breeds. The high degree of genetic variability seen in Argentine Creole horses could be a consequence of natural selection. Selection of TB through the centuries has most likely modified the gene pool of the ancestral population, with a consequent reduction in variability at certain loci. Probably, different mechanisms exist for maintaining polymorphism at these loci in TB and in AC horses. Heterozygosity may have played a fundamental role in adaptation.

Greater genetic variability in Argentine Creole than in Thoroughbred horses based on serum protein polymorphisms.

Genetic polymorphism was analyzed for five blood proteins: albumin - Al, esterase - Es, alpha(1)B-glycoprotein - Xk, transferrin - Tf and hemoglobin - Hb in 200 Thoroughbred (TB) and 124 Argentine Creole (AC) horses. Of the five systems examined, Tf and Hb were not in Hardy-Weinberg equilibrium in either breed and Es was not in equilibrium in the Creole breed. Genetic variability, estimated as average heterozygosity, was higher in AC (H = 0.585 +/- 0.131) than in TB (H = 0.353 +/- 0.065). The genetic differentiation between these two populations (F(ST)) was 0.109. Thus, of the total genetic differences between breeds, the proportion of genetic variation attributable to breed differences was about 10%; the remaining 90% was due to individual variation within breeds. The high degree of genetic variability seen in Argentine Creole horses could be a consequence of natural selection. Selection of TB through the centuries has most likely modified the gene pool of the ancestral population, with a consequent reduction in variability at certain loci. Probably, different mechanisms exist for maintaining polymorphism at these loci in TB and in AC horses. Heterozygosity may have played a fundamental role in adaptation.

Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eigth variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse mitochondrial DNA

Genetic variation of the second exon of ELA-DRB genes in Argentine Creole horses.

Genetic variation in the equine leucocyte antigen-DRB (ELA-DRB) second exon was investigated using polymerase chain reaction (PCR) amplification, restriction fragment length polymorphism (RFLP) of PCR products (PCR-RFLP) and deoxyribonucleic acid (DNA) sequencing. Eight distinct PCR-RFLP patterns could be identified in the studied Argentine Creole (AC) horses. The number of observed patterns per individual ranged from four to six, thus confirming the presence of multiple DRB copies in AC horses. Three PCR-RFLP alleles and three new sequences were identified. The estimated rates of synonymous and non-synonymous substitutions among ELA-DRB exon 2 sequences were higher within the antigen recognition site (ABS) than on the non-ABS. Phylogenetic analysis showed that the nucleotide sequences clustered in two main groups, while some sequences were not included in either group. Finally, the identification of the number of alleles per animal, the phylogenetic and segregation analyses allowed us to explain the number of ELA-DRB loci. However, it was not possible to identify specific alleles with specific loci.

Detection of c-erbB-2 gene amplification in cervical scrapes positive for human papillomavirus (HPV).

c-erbB-2 gene amplification has been described in a variety of human cancers, but it has been poorly studied in noncancerous cytological samples from genital specimens positive for human papillomavirus (HPV). Furthermore, the relationship between this genetic event and the presence of high-risk and low-risk HPV types is poorly studied. Eighty-four noncancerous cytological samples from exocervical specimens that were positive for HPV types 6, 16, and 18 were analyzed for c-erbB-2 gene amplification using the genomic differential polymerase chain reaction with the single copy reference gene. An association between c-erbB-2 gene amplification and the group corresponding to HPV type 6 was found. Within the low-risk HPV group, c-erbB-2 amplification was associated to cervical intraepithelial neoplasia of grade I (CIN I). Because in the samples analyzed, most of the CIN I stage was characterized by a koilocytotic pattern, c-erbB-2 amplification could be related to this kind of cellular alteration. It would be important to study c-erbB-2 gene amplification and also gene expression in different CIN stages in order to determine its role and significance in cervical cancer.

DNA damage induced by copper deficiency in cattle assessed by the Comet assay.

Cattle hypocuprosis is a well-known endemic disease in several parts of the world. In a previous paper, the clastogenic effect of copper deficiency in cattle has been described although the occurrence of DNA damage was not directly tested. For this reason, the relation between DNA damage assessed by the Comet assay and Cu plasma concentration was studied in Aberdeen Angus cattle. Blood samples were obtained in heparinized Vacutainer tubes from 28 female Aberdeen Angus cows during pregnancy or immediately after to give birth. Each sample was divided into two aliquots for Comet assay and Cu plasma determination, respectively. From the 28 cattle sampled, 17 were normocupremic and 11 were hypocupremic. Results obtained showed that whereas the average plasma Cu level in normocupremic cattle was 67.6 microg/dl, in hypocupremic cattle it was 32.1 microg/dl. The increase of DNA damage was mostly evidenced by the decrease of comet degree 1 cells and an increase of comet degree 2 cells. Correlation analysis comparing plasma Cu levels and degree 1 cells showed a correlation coefficient 0.72 (P<0.01). The comparison between plasma Cu levels and comet degree 2 cells was -0.65 (P<0.01). The comparison between plasma Cu levels and the comet length-head diameter medians determined in 23 out of 28 animals showed a correlation coefficient of -0.54 (P<0.01). The induction of DNA damage was clearly supported by the fact that the decrease of plasma Cu levels was correlated with the increase of comet length-head diameter. These findings could be considered as a contribution to the hypothesis that DNA and chromosome damage are a consequence of the higher oxidative stress suffered by hypocupremic animals.