Estudio preliminar de la expresión del mensaje genético del transportador de serotonina en células mononucleares de sangre periférica en pacientes con dependencia al alcohol con y sin depresión mayor comórbida / 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

ABSTRACT

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 <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 800 to 900 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) (11, v/v) and transferred to a tube that contained Ficoll-Histopaque solution (12, 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.

RESUMEN

La Encuesta Nacional de Adicciones 2008 reportó que en México existen 39 millones de personas que consumen alcohol y 4.8 millones presentan dependencia. A nivel mundial varios estudios indican que los pacientes con dependencia al alcohol (40 a 50%) presentan comorbilidad con algún tipo de padecimiento psiquiátrico. La función serotoninérgica es un mediador clave en los estados de ánimo, la impulsividad y las conductas adictivas, entre ellas el consumo de alcohol. Se ha reportado que el consumo excesivo de alcohol etílico disminuye los niveles de serotonina, aumenta la frecuencia de disparo de las neuronas serotoninérgicas en el núcleo del rafé y aumenta los niveles de serotonina en el núcleo accumbens. Las técnicas de biología molecular han permitido identificar factores de riesgo genético y se han seleccionado genes candidatos del sistema serotoninérgico, siendo uno de ellos el gen para el transportador de serotonina (5-HTT), el cual se ha demostrado que se encuentra asociado tanto a una mayor susceptibilidad para el establecimiento de la dependencia al alcohol como a la depresión mayor. Los receptores del sistema serotoninérgico como el 5-HTT, el 5-HT1 y el 5-HT2 se expresan tanto en las células del Sistema Nervioso como en las células del sistema inmunológico, lo que sugiere una similitud funcional de ambos sistemas. Es por ello que las células mononucleares de sangre periférica (PBMC) han sido utilizadas como un modelo de estudio en los trastornos de dependencia al alcohol y en los psiquiátricos. El objetivo de este estudio fue evaluar los niveles de expresión del gen 5-HTT en células mononucleares de sangre periférica de pacientes con dependencia al alcohol con y sin depresión mayor comórbida. En el Servicio de Consulta Externa del Centro de Ayuda a Alcohólicos y Familiares (CAAF) y en el Centro de Alcohólicos y Drogadictos <<Carrasco>> se evaluaron 70 pacientes durante el periodo comprendido entre febrero y noviembre de 2008. De entre éstos se incluyeron 24 que cumplieron con los criterios diagnósticos para la dependencia al alcohol de acuerdo al DSM-IV por medio de la entrevista estructurada Mini International Neuropsychiatric Interview (MINI) en su versión en español. Cumplieron además con los siguientes criterios de inclusión a) Que fueran pacientes de nuevo ingreso, b) Mayores de 18 años, c) De sexo femenino o masculino, d) Libres de tratamiento farmacológico tres semanas antes de incluirlos al estudio y e) Firmar carta de consentimiento informado para participar en el estudio. Se incluyeron para comparación voluntarios sanos (n = 12) los cuales fueron seleccionados de la población abierta de la Ciudad de México en el periodo de febrero a noviembre de 2008 y que cumplieran con los siguientes criterios a) No tener algún diagnóstico psiquiátrico, para lo cual se aplicó el MINI, b) Mayores de 1 8 años, c) Sexo femenino o masculino, d) Libres de tratamiento farmacológico tres semanas antes de incluirlos en el estudio y e) Firmar carta de consentimiento informado para participar en el estudio. Todos los participantes asistieron de 800 hs. a 900 hs. al laboratorio clínico del Instituto Nacional de Psiquiatría Ramón de la Fuente para la obtención de la muestra de sangre periférica de la cual se obtuvieron las PBMC. Se realizó la extracción del material genético (RNA) de las muestras de PBMC con el reactivo de Trizol®. Aislamiento del RNA y RT-PCR Las muestras de RNA fueron tratadas con DNasa 1 (Invitrogen Life Technologies; CA, USA) para eliminar la contaminación con DNA. La síntesis de cDNA (DNA copia) fue equivalente para todos los casos y se realizó a partir de 1 -Xg de RNA total en un volumen de reacción de 20-lLque contenía 5¡lLde Buffer 5x para Retro-Transcriptasa (Promega; WI, USA), 15 mM de MgCl2, 1.25 -xl 10 mM de la mezcla dNTP (Promega; WI, USA), 200 U M-MLV transcriptasa reversa (Promega; WI, USA) y 1 -xL de 10 mM OligodT (GE, UK). La mezcla de reacción se incubó durante 60 minutos a 42°C en un termociclador (Gradient Thermocycler; Eppendorf, Germany) y se aplicó un ciclo final de 90°C por cinco minutos. La amplificación para el 5-HTT se realizó en un volumen final de reacción de 25 (xL que contenía 2 (xL de cDNA, 1 mM de desoxiribonucleotidos Mix dNTP (Promega; WI, USA), 0.75 mM de MgCl2, 2.5 mM de oligonucleótidos y 1.25 unidades de GoTaq DNA Polimerasa (Promega; WI, USA) con una fase de desnaturalización de 94°C/1 minuto, una fase de alineación de 60°C/1 minuto y una fase de extensión de 72°C/1.30 minutos, durante 35 ciclos. Los oligonucleótidos utilizados para el 5-HTT fueron los siguientes sentido (5'-GAACTCCTGGAACACTGGCAAC-3') y el antisentido (5'- ATGACAAATCCCGAAACGAAGC-3') con un peso de 534 pares de bases (pb). En el caso del GAPDH las secuencias de oligonucléotidos utilizadas fueron sentido (5'-TGGGGAAGGTGAAGG TCGGAGTC-3') y antisentido (5 '-GACTTCAACAGCGACACCCACTC-3') con un peso de 874 pb. Los productos de amplificación se separaron en geles de agarosa al 2% y se tiñeron con bromuro de etidio. El análisis se realizó por densitometría computarizada (QuantityOne, Bio-Rad 2008). Los voluntarios sanos presentaron una mayor expresión del gen (0.5012±0.1349) comparados con los pacientes con dependencia al alcohol (0.3150±0.1836), esta diferencia fue significativa (p<0.0036, Mann-Whitney). Los niveles de expresión del gen 5-HTT para los pacientes con dependencia al alcohol con depresión mayor comórbida fueron significativamente reducidos (no detectables). Con la técnica del PCR se determinó que la expresión del gen 5-HTT está disminuida en los dos grupos de pacientes comparados con los voluntarios sanos. Estos resultados coinciden con los estudios que han reportado que la expresión del gen 5-HTT en linfocitos de pacientes con trastorno depresivo mayor es menor comparada con los sujetos sanos. La no detección del mensaje genético del 5-HTT en los pacientes dependientes al alcohol con depresión mayor comórbida sugiere que la comorbilidad puede potenciar la reducción en la expresión del 5-HTT.