A Longitudinal Analysis of Circulating Stress‐Related Proteins and Chronic Ethanol Self‐Administration in Cynomolgus Macaques

Allostasis is a term used to describe adaptations of biological networks beyond the optimum range to achieve physiological stability necessary for vital functions during chronic stress (McEwen 1998). Among various chronic stressors, allostasis is defined by abnormal levels of cytokines, catecholamines and the hypothalamic-pituitary-adrenal (HPA) axis hormones corticotropin-releasing hormone (CRH), adrenocorticotropic-releasing hormone (ACTH) and cortisol (McEwen and Wingfield 2003; Juster et al. 2010). Neurons in the paraventricular nucleus of the hypothalamus and median eminence release CRH onto the anterior pituitary, increasing circulating ACTH, which stimulates glucocorticoid (primarily cortisol in primates) release into circulation from the adrenal cortex. In addition, immune-related proteins, including cytokines, promote secretion of pituitary ACTH and adrenal glucocorticoids to moderate inflammation (Turnbull and Rivier 1999). Cytokines are a large class of proteins (e.g., interleukins, growth factors, tumor necrosis factors, chemokines) produced by cells of the immune system in response to infection or tissue damage to mediate inflammation, immune and allergic responses (Turnbull and Rivier 1999). Cytokines are pleiotropic, redundant (Ozaki and Leonard 2002) and interact in complex signal cascades in tissues far from their origin to coordinate responses to stressors (Turnbull and Rivier 1999). Cytokines appear to modulate complex behavioral processes (e.g., mood and memory; Reichenberg et al. 2001) and exert bidirectional modulation of the HPA axis (Turnbull and Rivier 1999). Similar to other acute physical (e.g., exercise) and psychological stress (e.g., restraint), acute ethanol influences cytokine concentrations (Deaciuc 1997; Turnbull and rivier 1999). Thus, cytokines are attractive targets in the characterization of ethanol-allostasis and treatment of alcohol-related disorders (Achur et al. 2010; Neuman 2003). Data from abstinent (Adinoff et al. 1990; Costa et al. 1996), intoxicated (Adinoff et al. 2003) or actively drinking but sober (Wand and Dobs 1991) human alcoholics suggest that chronic ethanol alters HPA axis function. For example, cortisol was suppressed (< 0.2 μg/dl) among alcoholics abstinent for about 43 days (Adinoff et al. 2003), suggesting that chronic ethanol resulted in an allostatic state in which cortisol was maintained at a new level. Adrenal hypofunction was also indicated by the failure of a low dose of synthetic ACTH (250 ng Cortrosyn) to increase plasma cortisol in alcoholics, although this dose was effective in controls (Wand and Dobs 1991). Similarly, a cross-sectional approach has been used to assess cytokines in alcoholism. For example, hospitalized alcoholics had greater serum tumor necrosis factor (TNF)-α compared to controls or moderate alcohol drinkers (Gonzalez-Quintela et al. 2008). To characterize allostasis in response to chronic ethanol, however, a longitudinal approach involving multiple dependent measures is necessary as cascading adaptive mechanisms are expected in response to cumulative stress. Furthermore, the pleiotropic actions of cytokines (Ozaki and Leonard 2002), their diverse tissues of origin (Freeman et al., 2010) and effects on microvasculature (Banks and Erickson 2010) argues for a simultaneous assessment of many cytokines in circulation. This is the approach taken in the current study using samples from non-anesthetized cynomolgus monkeys participating in an ethanol self-administration procedure (Vivian et al., 2001; Grant et al., 2008b). In this procedure, cynomolgus monkeys are trained to drink ethanol using schedule induced polydipsia, followed by 22 h/d concurrent access to ethanol and water. Mean daily intakes range from 0.60 to 4.0 g/kg/d, the equivalent of about 2-16 drinks/d, and blood ethanol concentration (BEC) is 100-400 mg/dl 7-8 h after the onset of drinking (Grant et al. 2008b; Vivian et al. 2001). Approximately 40% of monkeys become heavy drinkers, defined by an average consumption > 3.0 g/kg/d over 12 months (Grant et al. 2008a). Intake and BEC are similar to alcoholic men with 24 h/d access to ethanol for 20-60 d (Majchrowicz and Mendelson 1970; Mendelson 1970). In this model, ethanol consumption for six months results in fatty liver and after 18 months there are loci of inflammation consistent with the onset of alcoholic liver disease (Ivester et al. 2007). In a study designed to identify diagnostic biomarkers of alcohol consumption, Freeman et al. (2010) showed that plasma concentrations of 14 proteins distinguished macaque samples obtained during induction compared to chronic ethanol self-administration with 88% accuracy. Freeman et al. used a conservative approach, comparing protein levels from two samples each during ethanol-naive, induction and self-administration phases using repeated measures ANOVA. In contrast, the current study took advantage of the longitudinal data to assess changes in protein levels using linear mixed models. We characterized variation in protein concentration on an individual basis across the experiment, in relation to cumulative ethanol intake, and endocrine activity.