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Biomedical subjects

R Marcos

Publications and source records attributed to R Marcos.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of glutathione and vitamin C effects on the benzenetriol-induced DNA damage in isolated human lymphocytes.

The alkaline single-cell gel electrophoresis (or Comet) assay was applied to evaluate the eventual DNA damage induced by the triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), in isolated human lymphocytes. Prior to BT treatment, ranging from 5 to 50 microM, a supplementation with glutathione (GSH, 350 microg/ml) was carried out to assess whether GSH may have a modulating effect on the Comet response. The effect of a fixed dose of BT was also evaluated in the presence of the exogenous antioxidant vitamin C (40 and 200 microM). Additionally, we investigated whether the polymorphism of glutathione S-transferase T1 (GSTT1) gene may affect the individual level of BT-induced DNA damage in vitro. For all donors included in the present study, BT produced a significant dose-response relationship. No clear effect of GSH preincubation was seen on the BT-induced response. On the contrary, a significant reduction of DNA damage was observed in the presence of vitamin C (at least at 200 microM). Although our data suggest some individual differences according to the GSTT1 genotype in the outcome of the Comet assay, a large number of individuals should be studied in further investigations to obtain reliable conclusions.

Adult↗
PubMed2006

Cutaneous transmissible venereal tumor without genital involvement in a prepubertal female dog.

An 11-month-old prepubertal crossbreed female dog was presented with multiple nodular lesions disseminated over the cervical, back, flank, and abdominal regions. The lesions were ulcerated and cauliflowerlike, or nodular and subcutaneous, measuring up to 13 cm in diameter. Cytologic preparations of one of the lesions revealed a uniform population of round to oval cells, with lightly basophilic cytoplasm that contained multiple distinct vacuoles. Frequent mitotic figures and occasional lymphocytes were also observed. The cytologic diagnosis was cutaneous transmissible venereal tumor (TVT) in a progressing growth phase. This was confirmed by histologic and immunohistochemical findings. Vaginal TVT was diagnosed later in the dog's mother. TVT is a contagious neoplasm of sexually mature dogs that usually is transmitted by coitus and affects the genital mucosa. To our knowledge, this is the first report of naturally occurring multicentric TVT in a prepubertal female dog and also is unique in its exclusively cutaneous (no mucosal) involvement. We speculate that transmission of neoplastic cells occurred during cohabitation and social/mothering behavior between the dogs. Despite the atypical clinical presentation, response to chemotherapy with vincristine was excellent, leading to complete regression of the neoplasm without relapse after 6 months.

Animals↗
PubMed2006

Cytochemical detection of calcium in a case of calcinosis circumscripta in a dog.

A 9-month-old intact female Serra-da-Estrela dog (giant Portuguese breed) was presented with a 2-month history of lingual lesions. Two hard, nodular, nonpainful lesions were observed on the lateral border of the tongue. Fine-needle aspiration yielded a chalky white material. Hemacolor-stained smears showed an amorphous granular basophilic background with crystal-like structures and granulomatous inflammation consisting of foamy macrophages and giant cells. Cytochemical staining with von Kossa and Alizarin red S confirmed the calcareous nature of the material. The cytologic diagnosis was lingual calcinosis circumscripta; this was later confirmed by histopathologic examination. To our knowledge, this case is the first to document the use of von Kossa and Alizarin red S on cytologic smears to obtain a definitive diagnosis of lingual calcinosis circumscripta.

Animals↗
PubMed2005

Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6.

The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species.

Antibiotics, Antineoplastic↗
PubMed2005

Micronuclei assessment in buccal cells of people environmentally exposed to arsenic in northern Chile.

To determine the genotoxic risk associated to environmental arsenic exposure, the frequency of micronuclei in buccal cells (BCMN) of people drinking arsenic-contaminated water has been evaluated. A group of 105 individuals from the Antofagasta region (north Chile), and 102 individuals from the area of Concepcion, used as reference group, were included in the study. Arsenic concentration in drinking water was high (0.75 mg/L) in the Antofagasta area, 75-fold the maximum recommended level by WHO (0.01 mg/L), while the values obtained in Concepcion were significantly lower (0.002 mg/L). Individual measures of arsenic exposure were also determined in fingernails, which clearly confirm the existence of chronic exposure in the sampled populations from the Antofagasta region (10.15 microg/g versus 3.57 microg/g). The cytogenetic results indicate that, although the BCMN frequency is higher in exposed than in controls, this increase does not attain statistical significance. When the exposure biomarkers were related with the cytogenetic values, no correlations were observed between BCMN and arsenic content in water or in fingernails. In addition, the genotoxicity values do not seem to be related to the ethnic origin from people belonging to the exposed group. As a conclusion it appears that, in the studied population, the chronic ingestion of arsenic-contaminated water does not induce cytogenetic damage, measured as micronuclei, in the cells of the oral mucous in a significant extent.

Adult↗
PubMed2004

Evaluation of micronucleus induction in a Chilean population environmentally exposed to arsenic.

In the present study we have evaluated whether or not environmental exposure to arsenic in ground drinking-water results in a significant increase in the frequency of micronuclei (MN) in peripheral blood lymphocytes. Thus, 106 individuals from the Antofagasta region (North Chile), together with 111 individuals from the area of Concepción, were used in this investigation. In the Antofagasta area, arsenic levels in drinking-water as high as 0.750 mg/L were measured. In Concepción, located about 2500 km towards the south and used as reference area, arsenic levels in tap water were as low as 0.002 mg/L. The total content of arsenic in fingernails was determined as a biomarker of individual exposure. The cytogenetic results obtained in this study indicate that in the exposed group the overall frequency of binucleated micronucleated cells (BNMN) is higher than in the reference group, the difference being statistically significant. In addition, no differences were found between the exposed and the reference groups, regarding the cytokinesis-block proliferation index (CBPI). No association was observed between BNMN and arsenic content in water or arsenic in fingernails. On the other hand, when the exposed group was divided according to their Atacameno or Caucasian ethnicity, no significant differences were observed between them. In addition, as usually found in other human biomonitoring studies, sex and age are factors that modulate the frequency of MN in both exposed and reference populations.

Adult↗
PubMed2004

Estimation of the number of stellate cells in a liver with the smooth fractionator.

To better evaluate the activation and proliferative response of hepatic stellate cells (HSC) in hepatic fibrosis, it is essential to have sound quantitative data in non-pathological conditions. Our aim was to obtain the first precise and unbiased estimate of the total number of HSC in the adult rat, by combining the optical fractionator, in a smooth sampling design, with immunocytochemistry against glial fibrillary acidic protein. Moreover, we wanted to verify whether there was sufficiently relevant specimen inhomogeneity that could jeopardize the high expected estimate precision when using the smooth fractionator design for HSC. Finally, we wanted to address the question of what sampling scheme would be advisable a priori for future studies. Microscopical observations and quantitative data provided no evidence for inhomogeneity of tissue distribution of HSC. Under this scenario, we implemented a baseline sampling strategy estimating the number (N) of HSC as 207E(06) (CV = 0.17). The coefficient of error [CE(N)] was 0.04, as calculated by two formerly proposed approaches. The biological difference among animals contributed congruent with 95% to the observed variability, whereas methodological variance comprised the remaining 5%. We then carried out a half reduction of sampling effort, at the level of both sections and fields. In either occasion, the CE(N) values were low ( congruent with 0.05) and the biological variance continued to be far more important than methodological variance. We concluded that our baseline sampling (counting 650-1000 cells/rat) would be appropriate to assess the lobular distribution and the N of HSC. However, if the latter is the only parameter to be estimated, around half of our baseline sampling (counting 250-600 cells/rat) would still generate precise estimates [CE(N) < 0.1], being in this case more efficient to reduce the number of sections than to reduce the sampled fields.

Animals↗
PubMed2004

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate.

Alu Elements↗
PubMed2004

Dyspnoea at rest and at the end of different exercises in patients with near-fatal asthma.

Blunted perception of dyspnoea under resistive loading has been observed in patients with a history of near-fatal asthma (NFA). The perception of dyspnoea at rest and at the end point of various exercises was assessed in such patients. Respiratory function and exercise capacity (6-min walking distance, incremental cycloergometry and inspiratory threshold loading) were assessed in seven NFA and eight non-NFA patients. Dyspnoea (Borg scale) was measured at rest and at the end point of the various exercises. Dyspnoea at rest was significantly lower in NFA patients. Although exercise tolerance was similarly reduced in both the NFA and non-NFA groups, dyspnoea at peak cycle exercise was significantly lower in the former (2.6+/-2 versus 6.1+/-3.8 (Borg scale; mean+/-SD)), who mainly (86%) stopped because of leg discomfort. A similar trend was observed in the 6-min walking distance and inspiratory threshold loading tests. Dyspnoea at peak exercise was the best indicator of the NFA condition, with a sensitivity of 100% and specificity of 63% for a Borg scale score of < or = 6. Perception of dyspnoea is blunted in near-fatal asthma patients at both rest and the end point of various forms of exercise. Dyspnoea at peak exercise is the best indicator of the near-fatal asthma condition.

Adolescent↗
PubMed2003

Germline mutations at microsatellite loci in homozygous and heterozygous mutants for mismatch repair and PCNA genes in Drosophila.

Microsatellite instability (MSI) is a phenotype associated with the deficient repair of replication errors. Replication errors persist in defective mismatch repair (MMR) conditions, although alterations in components of the replication machinery, such as the proliferating cell nuclear antigen (PCNA) factor, could also increase the replication errors; therefore, MSI is expected in both situations. It also seems that heterozygous individuals for MMR genes have a high risk of cancer, as in the case of human non-polyposis colon carcinoma (HNPCC), characterised by MSI. Thus, here we investigate the effect of heterozygosity for a Msh2-null allele or for altered PCNA alleles, on the stability of microsatellite sequences. The study was carried out in Drosophila germ cells analysing the progeny of individual crosses. We found that one Msh2 disrupted allele is sufficient to produce MSI in germ cells. Although the MSI in Msh2(-/+) individuals was in the same order of magnitude as in Msh2(-/-) individuals, the former manifested a MSI that was four-fold lower. To a lesser extent, PCNA homozygous and heterozygous mutants also show MSI in the germline, which reveals the importance of DNA replication factors to maintain genomic stability in vivo. Furthermore, the high MSI found both in heterozygous Msh2 and PCNA mutants suggests a high degree of genomic instability in individuals bearing a mutant allele of these genes, which could have important implications in cancer susceptibility.

Animals↗
PubMed2003

Intraductal pancreatic carcinoma associated with pancreas divisum.

OBJECTIVE: we report the case of a 36 years old man with a pancreatic cancer associated to a pancreas divisum presenting as acute and relapsing recurrent pancreatitis. The coexistence of intraductal carcinoma and pancreas divisum is uncommon as let us do analyze its clinical, radiological and therapeutical features. CONCLUSIONS: 1. Pancreatic duct obstruction and pancreas divisum as congenital anomaly may be consider potential etiologies for pancreatic cancer. 2. Widespread use of magnetic resonance xholangiopancreatography and endoscopic retrograde xholangiopancreatography examinations together with brush cytology smears will allow more accurate approach to tumor diagnosis and management. 3. The use of pancreatoscopy in patients suffering from chronic pancreatitis may reveal helpful to discover intraductal neoplasms otherwise misdiagnosed.

Adenocarcinoma, Papillary↗
PubMed2002

Spontaneous and bleomycin-induced genomic alterations in the progeny of Drosophila treated males depends on the Msh2 status. DNA fingerprinting analysis.

Deficiency in DNA mismatch repair (MMR) confers instability of simple repeated sequences and increases susceptibility to cancer. Some of the MMR genes are also implicated in other repair and cellular processes related to DNA damage response. Supposedly, lack of their function can lead to a global genomic instability, besides microsatellite instability (MSI). To study the spontaneous and induced genomic instability in germ cells, related to the Msh2 status, DNA alterations in the progeny of individual crosses of Drosophila deficient in one or two copies of the Msh2 gene, were analysed by the arbitrarily primed polymerase chain reaction (AP-PCR). The results indicate that the progeny of homozygous parents for the normal Msh2 allele (+/+) presents a significantly lower frequency of genomic alterations than those from heterozygous (+/-) or mutant homozygous (-/-) parents. In addition, the DNA damage transmitted to the progeny, after the adult parental males were exposed to bleomycin, indicates that whereas the induction of mutations related to MSI depends on the lack of the Msh2 function, the induction of other mutational events may require at least one functional Msh2 allele. Thus, the results obtained with heterozygous individuals may have special relevance for cancer development since they show that a disrupted Msh2 allele is enough to generate genomic instability in germ cells, increasing the genomic damage in the progeny of heterozygous individuals. This effect is enhanced by mutagenic stress, such as occurs after bleomycin exposure.

Animals↗
PubMed2002

Spontaneous and induced genetic damage in T lymphocyte subsets evaluated by the Comet assay.

High inter- and intra-individual variability was reported in the level of DNA damage, both spontaneous and induced, when peripheral blood mononuclear leukocytes were used to perform the Comet assay. In order to find out the underlying causes for such variability, different subsets of T lymphocytes were isolated by immunomagnetic cell sorting. The level of DNA damage was evaluated with the alkaline version of the Comet assay by using three different parameters: tail moment, tail length and amount of DNA in the tail (%). Helper T cells (CD4+), cytotoxic T cells (CD8+), their negative fraction and the mixed cell population were evaluated both in untreated cells and after 10 and 20 microM H(2)O(2) treatments. Differences between cell subsets were only observed after H(2)O(2) treatment. The results indicate that, although CD4+ is the fraction with the highest induced level of genetic damage, this value is not high enough to explain the large inter- and intra-individual variability found.

Adult↗
PubMed2002

Germ cells microsatellite instability. The effect of different mutagens in a mismatch repair mutant of Drosophila (spel1).

Mismatch repair (MMR) process confers a type of genomic stability that maintains stable single repeated sequences, hence a failure of this process could deviate in cancer development. A characteristic phenotype of MMR-deficient cells is microsatellite instability (MSI) that could be modulated by mutagenic agents. The induction of MSI by the mutagens, bleomycin (BLM), hydrogen peroxide (H(2)O(2)), 2-acetylaminofluorene (2-AAF) and ethidium bromide (EB) was evaluated in vivo, by using a Drosophila melanogaster-null mutant of the msh2 mismatch repair gene (spel1). Whereas in the germ cells of the spel1 strain, we found microsatellite mutations in the five repeated sequences studied in untreated individuals, no alterations were found in the MMR-proficient strain. On the other hand, the data obtained from the treatment experiments show that BLM and 2-AAF induced a slight mutagenic effect in the MMR-deficient background but not in the normal one. These results indicate that the use of the Drosophila spel1 mutant (MMR-deficient) could be of relevant importance to identify environmental factors involved in carcinogenesis processes through genomic instability.

2-Acetylaminofluorene↗
PubMed2002

The clastogenic response of the 1q12 heterochromatic region to DNA cross-linking agents is independent of the Fanconi anaemia pathway.

Fanconi anaemia (FA) is a rare genetic syndrome of cancer susceptibility characterized by spontaneous and induced chromosome fragility, especially after treatment with cross-linking agents. Recent investigations showed interactions between FA proteins and chromatin remodelling factors. To investigate a potential uneven distribution of the FA pathway through the human genome depending on chromatin conformation, we have analysed chromosome breakage in the largest constitutively heterochromatic region in the human genome, the 1q12 band, in lymphocytes from FA patients, carriers and healthy controls after treatment with the cross-linking agents mitomycin-C (MMC) and diepoxybutane (DEB). As expected, a higher level of MMC-induced cytotoxicity and chromosome breakage was observed in cells from FA patients when compared with normal controls and carriers. However, the increase in 1q12 breakage after increasing concentrations of MMC was of a similar magnitude in FA patients, carriers and controls. Similarly, DEB induced a high level of overall genome chromosome fragility in cells from FA patients when compared with controls with no parallel increase in chromosome breaks specifically involving the heterochromatic band 1q12. We therefore conclude that, unlike the overall genome, the sensitivity of chromosome 1 constitutive heterochromatin to the chromosome breaking activity of cross-linking agents is independent of a functional FA pathway, indicating that the action of the FA pathway is unevenly distributed through the human genome.

Case-Control Studies↗
PubMed2002

A follow-up study on micronucleus frequency in Spanish agricultural workers exposed to pesticides.

To determine whether occupational exposure to a complex mixture of pesticides results in a significant increase in the level of cytogenetic damage, a follow-up study was planned on 39 greenhouse workers from Almería (southeastern Spain). Taking into account that pesticide exposure can be season-related, two blood samples were taken from each individual at different times: one in a period of high exposure (sample A, spring-summer) and the other in a period of lower exposure (sample B, autumn-winter). Using the cytokinesis block micronucleus technique the frequency of binucleated cells with micronuclei (BNMN) and the cytokinesis blocked proliferation index (CBPI) were determined in peripheral blood lymphocytes. The results obtained indicate that there were no statistically significant differences in BNMN frequencies between the two sampling periods nor between exposed and controls. ANCOVA analysis of repeated measures revealed that the age of the individuals showed a direct relation with BNMN in the first study period. With regard to CBPI, a significant and season-related effect was found.

Age Factors↗
PubMed2002

The Fanconi anaemia genome stability and tumour suppressor network.

Fanconi anaemia (FA) is a rare autosomal recessive disease characterized by increased spontaneous and DNA crosslinker-induced chromosome instability, progressive pancytopenia and cancer susceptibility. An increasing number of genes are involved in FA, including the breast cancer susceptibility gene BRCA2. Five of the FA proteins (FANCA, FANCC, FANCE, FANCF and FANCG) assemble in a complex that is required for FANCD2 activation in response to DNA crosslinks. Active FANCD2 then interacts with BRCA1 and forms discrete nuclear foci. FANCD2 is independently phosphorylated by ATM (the protein whose gene is mutated in ataxia telangiectasia) in response to ionizing radiation. In addition, the FA proteins are interconnected with other nuclear and cytoplasmic factors all related to cellular responses to carcinogenic stress and to caretaker and gatekeeper functions. In this review, the most recently published data on the molecular biology of the FA pathway and its molecular crosstalk with ATM, BRCA1 and BRCA2, proteins involved in xenobiotic and reactive oxygen species metabolism, apoptosis, cell cycle control and telomere stability, are summarized. The currently available data indicate that FA is a central node in a complex nuclear and cytoplasmic network of tumour suppressor and genome stability pathways fully committed to prevent cancer.

Apoptosis↗