Rommel Rodriguez Burbano

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 microg/l) of HgCl2 and CH3HgCl and incubated at 37 degrees C for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 microg/l alone, and at 1, 10, 100, and 1000 microg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Apolipoprotein A1 gene polymorphisms as risk factors for hypertension and obesity

Several polymorphisms in apolipoprotein A1 (APOA1) gene have been associated with metabolic diseases. Increased transcription efficiency was observed in −75A allele carriers compared to −75G allele homozygotes. +83C allele was associated with higher body mass index and waist-to-hip ratio in type II diabetes subjects. −75G/A and +83C/T polymorphisms were analyzed by RFLP-PCR in 334 individuals from a Brazilian elderly cohort. APOA1 polymorphisms were associated with age-related morbidities, as well as with triglycerides, total cholesterol, HDL, VLDL, LDL, creatinine, urea, albumin, glycated hemoglobin and fasting glucose serum levels. Allele frequencies were 0.102 and 0.21, respectively, for −75A and +83T. −75G allele showed significant association with hypertension (Pxa0=xa00.001). An association between +83C allele and obesity was observed (Pxa0=xa00.040) and this allele also showed an association with hypertension in the presence of cardiovascular disease (Pxa0=xa00.047). Moreover, +83T allele was associated with lower glycated hemoglobin values (Pxa0=xa00.026). To our knowledge, there is no data associating this polymorphism with glycated hemoglobin. Furthermore, individuals carrying AT haplotype have lower risk for developing hypertension (Pxa0=xa00.0002), while GT haplotype carriers present decreased risk to develop obesity comparing to GC haplotype (Pxa0=xa00.025). APOA1 polymorphisms analysis may be a useful tool to identify risk factors for subjects and families and clarify the physiopathological role of these polymorphisms in age-related diseases, such as hypertension and obesity.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Association of PPARα gene polymorphisms and lipid serum levels in a Brazilian elderly population

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear transcription factor strictly involved in lipid and lipoprotein metabolisms. Thus, PPARalpha gene polymorphisms have been investigated as cardiovascular risk factors. We aimed to investigate associations of L162V and intron 7G>C polymorphisms with common morbidities affecting a Brazilian elderly cohort as well as with lipid and protein serum levels. Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP), and allele frequencies were determined. In addition, we performed the linkage disequilibrium analysis. Descriptive statistics, logistic regression analysis, and Students t-test were used. Rare alleles for L162V and intron 7 G>C polymorphisms showed frequencies of 0.047 and 0.199, respectively. Our data showed that these polymorphisms were in linkage disequilibrium (p=0.0002). Intron 7 G>C polymorphism presented a tendency of association with neoplasia (p=0.053), and C allele was associated with higher HDL (p=0.010), lower triglycerides (p=0.001), and VLDL levels (p=0.003) compared to G allele. These data might suggest a protective role of intron 7 G>C polymorphism in the development of cardiovascular diseases and will help to clarify the importance of PPARalpha polymorphisms as key modulators of lipid metabolism in Brazilian population.

APOA1/A5 Variants and Haplotypes as a Risk Factor for Obesity and Better Lipid Profiles in a Brazilian Elderly Cohort

Genetic variations in the APOA1/C3/A4/A5 gene cluster have been studied and proposed to be the leading key for susceptibility to cardiovascular diseases and age-associated disorders. We aimed to investigate the associations of rs12721026 (APOA1) and rs1729408 (APOA5) polymorphisms and their haplotypes with some age-related diseases, as well as with lipids and proteins serum levels in a cohort from a Brazilian Elderly Longitudinal Study (EPIDOSO). Genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Statistical analyses were carried out using logistic regression analysis, Student’s t-test, and linkage disequilibrium (LD) analysis. Polymorphic allele frequencies were 0.095 and 0.449 for rs12721026 and rs1729408, respectively. The C-allele of rs1729408 was associated with higher high-density lipoprotein (HDL) (Pxa0=xa00.022) and glycated hemoglobin levels (Pxa0=xa00.020). We also showed that rs12721026 and rs1729408 were in LD. The GC haplotype, which is composed of the G-allele of rs12721026 and the C-allele of rs1729408, was significantly associated with obesity (Pxa0=xa00.028), with higher glycated hemoglobin (Pxa0=xa00.006), and fasting glucose (Pxa0=xa00.0003) compared to the TT haplotype, which includes the wild-type alleles of both polymorphisms. Moreover, we found an association between the TC haplotype and higher HDL levels (Pxa0=xa00.0039). This is the first time that haplotypes involving these polymorphisms were evaluated. Our results showed that these polymorphisms were involved in the development of obesity and in alterations of lipids and proteins serum levels in a Brazilian population. The present findings might also clarify the role of these polymorphisms and their haplotypes in lipids and proteins metabolism.

Frequency of Werner helicase 1367 polymorphism and age-related morbidity in an elderly Brazilian population

Werner syndrome (WS) is a premature aging disease caused by a mutation in the WRN gene. The gene was identified in 1996 and its product acts as a DNA helicase and exonuclease. Some specific WRN polymorphic variants were associated with increased risk for cardiovascular diseases. The identification of genetic polymorphisms as risk factors for complex diseases affecting older people can improve their prevention, diagnosis and prognosis. We investigated WRN codon 1367 polymorphism in 383 residents in a district of the city of São Paulo, who were enrolled in an Elderly Brazilian Longitudinal Study. Their mean age was 79.70 +/- 5.32 years, ranging from 67 to 97. This population was composed of 262 females (68.4%) and 121 males (31.6%) of European (89.2%), Japanese (3.3%), Middle Eastern (1.81%), and mixed and/or other origins (5.7%). There are no studies concerning this polymorphism in Brazilian population. These subjects were evaluated clinically every two years. The major health problems and morbidities affecting this cohort were cardiovascular diseases (21.7%), hypertension (83.7%), diabetes (63.3%), obesity (41.23%), dementia (8.0%), depression (20.0%), and neoplasia (10.8%). Their prevalence is similar to some urban elderly Brazilian samples. DNA was isolated from blood cells, amplified by PCR and digested with PmaCI. Allele frequencies were 0.788 for the cysteine and 0.211 for the arginine. Genotype distributions were within that expected for the Hardy-Weinberg equilibrium. Female gender was associated with hypertension and obesity. Logistic regression analysis did not detect significant association between the polymorphism and morbidity. These findings confirm those from Europeans and differ from Japanese population.

APOA4 polymorphism as a risk factor for unfavorable lipid serum profile and depression: a cross-sectional study.

Introduction APOA1/C3/A4/A5 gene cluster is closely involved in lipid metabolism, and its polymorphisms have been associated with coronary heart disease and lipid plasma levels. Here, we aimed to investigate associations of APOC3 (3238C>G, −482C>T, 1100C>T) and APOA4 (Gln360His, Thr347Ser) polymorphisms in 382 individuals from a cohort of a Longitudinal Brazilian Elderly Study with major age-related morbidities and with lipid and protein serum levels. Materials and Methods The whole sample was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Descriptive statistics, logistic regression analysis, Student t test, deviation from Hardy-Weinberg, Bonferroni correction for multiple testing, and haplotype analyses were performed. Results Although APOC3 1100T allele carriers presented lower triglyceride and very low density lipoprotein levels than non-T carriers, these associations disappeared after Bonferroni correction (P > 0.05). Moreover, APOA4 360His allele was associated with depression (P = 0.03), increased triglyceride (P = 0.035) and very low density lipoprotein (P = 0.035) levels, and reduced HDL levels (P = 0.0005). Haplotype analyses found an association between His/C/C haplotype (Gln360His/−482C>T/1100C>T) with depression, but this result was due to Gln360His polymorphism. Conclusions Our data suggest that 360His allele might be a risk factor for depression and unfavorable lipid profile and depression for elderly people in the Brazilian population.

Drifter Technique: a New Method to Obtain Metaphases in Hep-2 Cell Line Cultures

The Hep-2 cell line is derived from laryngeal carcinoma cells and is often utilized as a model in carcinogenesis and mutagenesis tests. To evaluate the proliferative potential of this line, we developed a cytogenetic methodology (drifter technique) to obtain metaphases from cells that loose cellular adhesion when they underwent mitosis in culture. By this procedure, 2000 cells were counted, resulting in a mitotic index (MI) of 22.2%. Although this MI was not statistically different from the one obtained using either a classical cytogenetic method or a cell synchronization technique, the drifter technique has the advantage of not requiring the use of some reagents for the obtention of metaphases and also of diminishing the consumption of maintenance reagents for this cell line.

Asociacion entre la pérdida del cromosoma x en mujeres de edad avanzada y el polimorfismo de la apolipoproteina a-iv: 360 gln/his

La perdida del cromosoma X en mujeres, asociada a la edad avanzada, es un fenomeno ya reconocido hace decadas. Los factores geneticos que llevan a esta perdida o a la no-disyuncion cromosomica, son poco conocidos. Polimorfismos de apolipoproteinas fueron asociados a la ocurrencia de sindromes cromosomicos como las trisomias del 18 y 21 y fue sugerida su participacion en la segregacion cromosomica. Polimorfismos de la apolipoproteina AIV:360 fueron asociados a molestias cardiovasculares en la poblacion brasilena.Veinticuatro mujeres entre 72 a 87 anos, fueron seleccionadas para estudiar la presencia o ausencia del alelo 2 (HIS) y analizar la perdida del cromosoma X. El analisis citogenetico fue realizado en nucleos interfasicos de cultivos de linfocitos por tecnica de hibridacion in situ con sonda fluorescente centromerica del cromosoma X. El analisis genotipico fue realizado a partir de ADN extraido de la sangre, amplificado por PCR y digerido por la enzima de restriccion Fnu4HI. Se observo una proporcion significantemente mayor de celulas con la perdida del cromosoma X, en mujeres con alelo 2 (HIS). Estos resultados indican la participacion del gen de la ApoA-IV en la no-disyuncion cromosomica. Los mecanismos de su actuacion pueden estar relacionados a danos oxidativos producto de alteraciones en la microcirculacion e induccion de muerte celular. La comprension de este mecanismo todavia necesita de mayor investigacion