Resumo

BECERRIL-VILLANUEVA, Enrique et al. Preliminary study of the genetic message expression of serotonin transporter in peripheral blood mononuclear cells in patients with alcohol dependence with and without comorbid major depression. Salud Ment [online]. 2011, vol.34, n.2, pp.139-147. ISSN 0185-3325.

The 2008 National Addiction Survey demonstrated the existence of 39 million alcohol drinkers, of whom 4.2 million are excessive drinkers and 4.8 million are alcohol dependents. No reports of the comorbidity of psychiatric disorders in alcohol consumers in our country exist. Nevertheless, 40% to 50% of alcohol-dependent patients from other countries have some sort of psychiatric disorder, such as major depression. Serotonergic function is a key mediator of mood states, impulsiveness, and addictive behavior, including alcohol consumption. Several studies have noted alterations in the serotonergic system in alcoholics (as demonstrated by an increase in the shooting frequency of raphe nuclei serotonergic neurons, an increase in serotonin levels in the accumbens nuclei, and a loss in serotonergic neurons in the raphe nuclei) and depressed patients (decreases in the density of serotonin reuptake transporter [5-HTT] and serotonin levels [5-HT]). Clinical studies have documented that excessive alcohol intake reduces 5-HT levels and that this condition potentiates psychiatric disorders, such as anxiety, major depression, and alcohol dependence. These data demonstrate an association between alcoholism, psychiatric disorders, and alcohol dependence. By molecular biology techniques, genetic risk factors have been identified and candidate genes, such as 5-HTT, have been selected. This gene is associated with a greater susceptibility to onset of alcohol-dependence and major depression. The 5-HTT gene lies in the SLC6A4 locus of 1 7q1 1.1-q12 and encodes a 600-amino-acid integral membrane protein. This transporter regulates serotonergic neurotransmission through removal of 5-HT from the synaptic space. Pharmacological research has shown that selective reuptake inhibitors (5-HTT blockers) reduce alcohol intake in alcohol-dependent and major depression patients. Serotonergic system receptors, such as 5-HTT, 5-HT₁, and 5-HT₂, are expressed in nervous system and immune system cells; thus it is likely that both systems have functional similarities. Due to this property, peripheral blood mononuclear cells (PBMCs) can be used to research neurodegenerative, psychiatric, and alcohol dependence disorders. The aim of this study was to assess 5-HTT expression levels in the PBMCs from alcohol-dependent patients and patients with comorbid alcohol-dependence and major depression disorder. Materials and methods The Outpatient Consultative Service from the Centro de Ayuda a Alcohólicos y Familiares (CAAF) and the Centro de Alcohólicos y Drogadictos <<Carrasco>> screened 70 patients from February to November 2008. Twenty patients who met the criteria of alcohol dependence, according to the Spanish version of the Mini International Neuropsychiatric Interview (MINI), were accepted. Alcohol dependence was based on the Alcohol Dependence Scale (EDA) that was adapted to the Mexican population. Healthy volunteers (n = 12) were selected concurrently from the general population of Mexico City with the start of patient enrollment. A psychiatrist evaluated the mental health of these patients using MINI. Clinical and laboratory trials demonstrated normal standard reference values. Patients were included based on INPRFNC092318.1 research protocol procedures, which were approved by the Ethics Committee of the Instituto Nacional de Psiquiatría Ramón de la Fuente. All participants were given a detailed explanation concerning the study objectives and gave their signed informed consent. The patients for this study were men and women, aged 18 years and older who presented a diagnosis of alcohol dependence. Patients were grouped as follows: Group A: diagnosed alcohol dependence (n=12) and Group B: diagnosed alcohol dependence depressed patients (n=12). All patients arrived to the clinical laboratory from 8:00 to 9:00 a.m. to undergo a peripheral blood test. Blood was drawn by venous puncture and placed into anticoagulant-coated tubes. The blood was then diluted in an isotonic saline solution (SSI) (1:1, v/v) and transferred to a tube that contained Ficoll-Histopaque solution (1:2, v/v) to form a density gradient. The sample was centrifuged at 1600 rpm for 30 minutes at 4°C. The gradient interphase, which contained the mononuclear cells, was collected, SSI cells were washed, and Trizol was added. RNA isolation and RT-PCR RNA extraction from the PBMCs was performed using Trizol, wherein a monophase phenol solution and guanidine isothiocyanate were used to lyse cells and dividing the sample into 2 phases: aqueous and organic. The aqueous phase was separated with chloroform, and RNA was precipitated with isopropanol. Genetic material was washed with alcohol and allowed to dry. RNA was resuspended with RNase- and DNase-free water. The index of purity of the RNA samples ranged between 1.8 and 2.0. RNA samples were treated with DNase 1 (Invitrogen Life Technologies, CA, USA) to eliminate DNA contamination. Total RNA was used for the generation of cDNA and was performed from 1 |Ig of total RNA in a reaction volume of 20 (xL containing 5X Buffer 5|lL Retro-Transcriptase (Promega, WI, USA), 15 mM MgCl₂, 1.25 ul 10 mM dNTP mix (Promega, WI, USA), 200 U M-MLV reverse transcriptase (Promega, WI, USA) and 1 |xL of 10 mM OligodT (GE, UK). The reaction mixture was incubated for 60 minutes at 42°C in a thermocycler (Thermocycler Gradient, Eppendorf, Germany) and applied a final cycle of 90°C for 5 minutes. The reverse-transcribed product (cDNA) was amplified by PCR in a final reaction volume of 25 |xL containing 2 |xL of cDNA, 1 mM deoxyribonucleotide dNTP Mix (Promega, WI, USA), 0.75 mM MgCl₂, 2.5 mM oligonucleotide GoTaq and 1.25 units DNA polymerase (Promega, WI, USA). PCR was performed for 25 cycles, using a cycling program of 94°C for 1 minute, 60°C for 1 minute and 72°C for 1.30 minutes in a thermocycler for the amplification of 5-HTT and GAPDH. The oligo-nucleotides used for 5-HTT were: sense 5'-GAACTCCTGGAACAC TGGCAAC-3' and antisense 5'-ATGACAAATCCCGAAACGAAGC-3' (534 base pairs, [bp] product). In the case of GAPDH sequences of oligonucleotides used were: sense 5'-TGGGGAAGGTGAAGGTC GGAGTC-3' and antisense 5'-GACTTCAACAGCGACACCCACTC-3' (874 bp product). The amplicons were separated on an agarose gel and stained with ethidium bromide. The bands were analyzed by computerized densitometry. The optical density values for 5-HTT were calculated as the quotient between the values for 5-HTT and GADPH, expressed as the mean ± standard deviation. Statistic analysis was performed using the Shapiro-Wilk homogeneity test and nonparametric Mann-Whitney test. Differences were considered statistically significant when ≤ p 0.05 Results 5-HTT levels were nondetectable (ND) in patient with comorbid alcohol-dependence and major depressive disorder. By Shapiro Wilk test, healthy volunteers and alcohol-dependent patients did not differ. Healthy volunteers expressed higher levels of 5-HTT gene (0.5012 ± 0.1349) compared with alcohol-dependent patients (0.3150 ± 0.1836) (p < 0.0036, Mann-Whitney). Discussion Our PCR method allowed us to determine that 5-HTT expression is lower in the two groups of patients compared with healthy volunteers. These results are consistent with studies that have reported that 5-HTT expression declines in lymphocytes from major depression disorder patients compared with healthy volunteers. The lack of detection of 5-HTT in alcohol- dependence depressed patients suggests that this comorbidity leads to alterations in the expression of this 5-HTT. Finally, our work is the first preliminary study that characterizes 5-HTT expression in the Mexican population, comparing alcohol dependence patients and patients with comorbid alcohol-dependence and major depression.

Palavras-chave : Alcohol-dependent; major depressive disorder; 5-TT; PBMC; RT-PCR.

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