Polymorphism study of alcohol dehydrogenase and aldehyde dehydrogenase (adh and aldh) genes and the alcohol dependence in a population of Goiania, GO, Brazil

Submitted by Jaqueline Silva ([email protected]) on 2017-01-04T16:37:13Z No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Jaqueline Silva ([email protected]) on 2017-01-04T16:37:24Z (GMT) No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2017-01-04T16:37:24Z (GMT). No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-28Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESAlcoholism is a disease defined as a physical and / or mental condition, which characterized the need of the individual to ingest the substance constantly in order to experience its psychic effects or stop the withdrawal symptoms of the same. Heavy alcohol consumption brings many consequences for the health and quality of life by increasing the frequency of morbidity and mortality, causing several functional limitations. Worldwide, enormous resources are being invested in campaigns against alcohol abuse. WHO, for example, establishing a standard dose contains about 10 to 12 g of pure alcohol equivalent to a can of beer (350 mL) and draft (330 mL), a glass of wine (100ml), or distillate dose (30 mL). Some individuals metabolise alcohol better than others. The main ethanol elimination pathway occurs through oxidation of acetaldehyde to the same and then acid and water. These reactions are catalyzed by the enzyme alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In this context, the scientific endeavor of this study to characterize the presence of polymorphisms of ADH and ALDH genes in alcohol users region of the state of Goiás, it is necessary to establish a possible correlation with alcoholism. This study was conducted in Goiás Alcoholics Recovery Center (GO CEREA) and Psychosocial Care Center Alcohol and Drugs (CAPS AD) both in Goiania. For the experiments, a sample of blood from each patient for DNA extraction was collected. Assays were performed by RT-PCR TaqMan SNP Genotyping Assays system. Statistical analyzes were made through GENEPOP 4.5 and Haploview software. Significant differences were found for SNPs studied in the analysis “Case vs. Control” and “Female vs. Male”. Furthermore, binding was observed moderate linkage disequilibrium between the ADH1B*2 and ADH1C*2 SNPs and the presence of haplotype by segregation dependent thereof. These results therefore suggest that these genetic variants may be associated with protection or addiction to alcoholism in the population studied in the city of Goiania, GO, Brazil.O alcoolismo é uma doença definida como um estado físico e/ou psíquico onde se caracteriza a necessidade do indivíduo de ingerir a substância constantemente, no intuito de sentir seus efeitos psíquicos ou fazer cessar os sintomas da abstinência da mesma. O consumo abusivo de álcool traz inúmeras consequências para a saúde e qualidade de vida, aumentando a frequência de morbimortalidade, causando diversas limitações funcionais.Em todo o mundo, enormes recursos estão sendo investidos em campanhas contra o abuso do álcool.A OMS, por exemplo, estabelece que uma dose padrão contenha aproximadamente de 10 a 12 g de álcool puro, o equivalente a uma lata de cerveja (350 mL), ou chope (330 mL), uma taça de vinho (100 mL), ou uma dose de destilado (30 mL). Alguns indivíduos metabolizam o álcool melhor que outros. A principal via de eliminação do etanol ocorre por meio de oxidação do mesmo à acetaldeído e posteriormente em ácido e água. Estas reações são catalisadas pelas enzimas Álcool desidrogenase (ADH) e Aldeído desidrogenase (ALDH). Neste contexto, o empenho científico deste estudo para caracterizar a presença de polimorfismos nos genes ADH e ALDH nos usuários de álcool da região do Estado de Goiás, faz-se necessário para se estabelecer uma possível correlação com o alcoolismo. Este estudo foi realizado no Centro de Recuperação de Alcoólatras de Goiás – regional Goiânia (CEREA GO) e no Centro de Atenção Psicossocial Álcool e Drogas (CAPS AD). Para a realização dos experimentos, foi coletado uma amostra de sangue de cada paciente para extração de DNA. Os ensaios foram realizados através da PCR em Tempo Real pelo sistema TaqMAn® SNP Genotyping Assays. As análises estatísticas foram feitas através dossoftware GENEPOP 4.5 e Haploview.Foram encontradas diferenças significativas para os SNPs estudados nas análises Caso versus Controle e Feminino versus Masculino. Além disso, foi observada ligação moderada de Linkage disequilibrium entre os SNPs ADH1B*2 e ADH1C*2 e a presença de haplótipos através da segregação dependente destes. Estes resultados sugerem, portanto, que estas variantes genéticas possam estar associadas à proteção ou dependência ao alcoolismo na população estudada na cidade de Goiânia, GO, Brasil

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 mu M to 50.18 mu M, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)] PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Effect of complex 2 on the mitochondrial membrane potential.

<p>The percentages of cells with depolarized mitochondrial membrane potential mitochondrial membrane potentials of cells treated with 40 µM and 60 µM complex 2 as well as control cells were assessed by flow cytometry after staining with JC-1. The data are expressed as the means ± SD of two individual experiments; ***p<0.001 compared with the control.</p

Effect of complex 2 on the activity of caspase -3, -8 and -9 in S180 cells.

<p>S180 cells were treated with the complex at the indicated concentrations for 24 h. The data are expressed as the means ± SD of two individual experiments; *p<0.05 and **p<0.01 compared with the control.</p

Effects of complex 2 on the cell cycle distribution of S180 cells after 24 h (A) and 48 h (B) of exposure.

<p>The data are the means ± SD of three experiments. Significant differences from the untreated control are indicated by ***p<0.001.</p

Effect of complex 2 on the mechanism of S180 cell death (early and late apoptosis/necrosis) evaluated by flow cytometry after 24 h (A) and 48 h (B) of incubation.

<p>The data are the means ± SD of three experiments. Significant differences from the untreated control are indicated by **p<0.01 and ***p<0.001.</p

Primer sequences used for the real time RT-qPCR assay.

<p>Primer sequences used for the real time RT-qPCR assay.</p

Viability of the S180 tumor cell line and the L929 normal cell line in the presence of ruthenium amino acid complexes and cisplatin.

<p>Viability of the S180 tumor cell line and the L929 normal cell line in the presence of ruthenium amino acid complexes and cisplatin.</p

Relative expression of <i>bax</i>, <i>caspase 3</i>, <i>caspase 8</i>, <i>caspase 9</i>, and <i>p53</i> in S180 cells treated with 40 µM of complex 2 for 6 h as determined by real-time RT-PCR analysis.

<p>The fold change is expressed relative to the respective untreated control using <i>β-actin</i> as a reference gene. The data are expressed as the means ± SD; * p<0.05, **p<0.01, and ***p<0.001 compared with the control.</p

Microanalyses data for the [Ru(AA)(dppb)(bipy)]ⁿ⁺ complexes.

<p>*calculated (found).</p><p>Microanalyses data for the [Ru(AA)(dppb)(bipy)]ⁿ⁺ complexes.</p