PHARMACOLOGY AND CELL METABOLISM Ethanol Reduces Zincosome Formation in Cultured Astrocytes Raúl Ballestín 1 , Asunción Molowny 1 , María Pilar Marín 2 , Guillermo Esteban-Pretel 2 , Ana María Romero 2 , Carlos Lopez-Garcia 1 , Jaime Renau-Piqueras 2 and Xavier Ponsoda 1, * 1 Biologia Cellular, Universitat de València, Avda. Dr. Moliner 50, 46100 Burjassot, Spain and 2 Biologia y Patologia Celular, Hospital La Fe, Avda. Campanar 21, 46009 Valencia, Spain *Corresponding author: Tel.: +34-96-354-3242; Fax: +34-96-354-3404; E-mail: ponsoda@uv.es (Received 20 July 2010; in revised form 21 October 2010; accepted 26 October 2010) Abstract — Aims: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency is present in many physio- logical and health problems affecting most body organs, including the brain. Among the circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. A dietary zinc supplement has been proposed as possibly being an effi- cient method to palliate zinc deficiency. Astrocytes form part of the hematoencephalic barrier, and they are apparently implicated in the homeostasis of the neuronal medium. In this work, we analyze the effect of ethanol on extracellular zinc management by rat astrocytes in culture. Methods: Intracellular levels of ‘free zinc ions’, in controls and 30 mM ethanol-treated astrocytes, were visual- ized by using the zinc fluorochrome TSQ. Cytoplasmic fluorescence and zincosome formation were measured after adding extra- cellular 50 μM ZnSO 4 to cell monolayers. Zincosomes were also observed at the electron microscopy level. Results: Exposure to ethanol for 7 days lowered the basal zinc levels of astrocytes by ~30%. This difference was consistently maintained after the zinc pulse. Zinc ions were confined to bright fluorescent particles, the ‘zincosomes’, which appeared to be formed by the endocytic pathway. Zincosomes were less abundant in alcohol-treated cells, indicating a deficit in endocytoses as the origin of low zinc intake in astrocytes after ethanol treatment. Conclusions: Ethanol reduces both intracellular ionic zinc levels and extracellular zinc uptake, resulting in poorer zincosome formation. Given the endocytic nature of zincosomes, the effect of ethanol on membrane trafficking is apparently the origin of this deficit. INTRODUCTION During pregnancy, ethanol consumption causes a neurotoxic/ dysmorphogenic syndrome that includes craniofacial malfor- mations that are associated with a variety of neurobehavioral disturbances ranging from hyperactivity/attention deficit dis- order and learning disabilities in childhood (Streissguth and O’Malley, 2000) to major depressive and psychotic disorders in adulthood. In addition, exposure to ethanol causes a variety of anomalies in the developing brain, including neuronal-glial heterotopias, cerebellar dysplasia, agenesis of the corpus callosum, hydrocephalus and microcephaly (Guerri et al., 2009; Streissguth et al., 1994). These toxic effects are indicative of aberrant migration, decreased pro- liferation and neural cell death (Goodlett et al., 2005; Guerri et al., 2009; Streissguth et al., 1994). This syndrome is known as fetal alcohol syndrome (FAS). No single mechan- ism has been sufficient to account for these varying effects; it is likely that multiple factors are involved, and a number of mechanisms have been suggested (Goodlett et al., 2005; Guerri et al., 2001; Martínez and Egea, 2007). One such mechanism indicates that ethanol perturbs the cytoskeleton and protein trafficking, thus ethanol treatment substantially impairs endocytosis, exocytosis and nucleocytoplasmic traffic in rat astrocytes in primary cultures (Marín et al., 2008; Megías et al., 2000; Tomás et al., 2002, 2004, 2005). Zinc is an essential trace element that is critical for a large number of structural proteins, enzymatic processes and tran- scription factors. The importance of the role of zinc in the growth and development, including brain development, of mammals was recognized in the first third of the 20th century (Todd et al., 1934) and was observed in humans in 1963 (Prasad, 1991). Since then, the functions of zinc and the effects of its deficiency on the brain and other organs have been continuously studied (Bitanihirwe and Cunningham, 2009). Thus, low levels of zinc, prolonged in time and at different levels, can cause fatal or mild effects. These effects appear in a wide spectrum fashion depending on the magnitude of the deficiency and, more importantly, the variety of clinical manifestations seen in zinc deficiency illustrates the numerous roles that zinc plays in the body (Tuerk and Fazel, 2009). In addition, ethanol consumption during gestation can cause zinc deficiency, as demonstrated in pregnant mothers, which was directly related to the expression of FAS (Flynn et al., 1981). These severe effects were found in rodents exposed to alcohol or to a low-zinc diet during pregnancy (Carey et al., 2000; Record et al., 1986) and, under some experimental conditions, zinc treat- ment has been found to reverse severe ethanol effects on mouse fetuses (Summers et al., 2009). Under other con- ditions, however, ethanol effects on neuronal loss did not lower with zinc supplementation (Chen et al., 2001). Therefore, the avoidance of zinc supplementation is rec- ommended in some cases as it may increase morbidity or lead to further deterioration; even very high intakes may result in the deficiency of other metals or in growth retar- dation (Tuerk and Fazel, 2009). The functions of zinc in biological systems can be divided into three main categories: structural, catalytic and regulatory. ‘Structural zinc’ in cells participates in protein folding and macromolecular stabilization, producing active molecules con- taining structures such as zinc fingers by, for example, stabiliz- ing some enzymes such as the antioxidant superoxide dismutase (Roberts et al., 2007). ‘Catalytic zinc’ is a com- ponent of the catalytic site of hundreds of metalloenzymes (Vallee and Falchuk, 1993). ‘Regulatory zinc’ acts on a number of biologic processes, including gene regulation (Cousins, 1994; Dalton et al., 1997). Zinc present in tissues is delivered between cells or cellular compartments according to cell’s needs to maintain zinc homeostasis (Sekler et al., 2007). Alcohol and Alcoholism Vol. 46, No. 1, pp. 17–25, 2011 doi: 10.1093/alcalc/agq079 Advance Access Publication 1 December 2010 © The Author 2010. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved at Servicio Valenciano de Salud on December 16, 2010 alcalc.oxfordjournals.org Downloaded from