NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol

Abstract

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior.

Prenatal Ethanol Exposure Misregulates Genes Involved in Iron Homeostasis Promoting a Maladaptation of Iron Dependent Hippocampal Synaptic Transmission and Plasticity

“…We followed the protocol described previously by Contreras et al, 2017. Pregnant Sprague–Dawley rats were exposed to ethanol (10% v/v) and 64 mg/l of sucralose (daily).…”

“…We used the protocol described by Contreras (Contreras et al, 2017). Briefly, 100 mg of brain tissues (PFC, hippocampus, or VTA) were homogenized with 300 µl lysis buffer (20 mM MOPS/Tris pH 7, 0.3 M sucrose, 2 mM EDTA, 2 mM EGTA, 1% NP-40, and 0.1% sodium dodecyl sulfate), plus protease inhibitors.…”

“…The consumption of alcohol during pregnancy significantly alters fetal development and growth (Cuzon et al, 2008), and also affects cognitive processes throughout the child’s life (Ramsay, 2010; Skorput et al, 2015). Several clinical and preclinical studies have shown that prenatal ethanol exposure (PAE) can trigger cognitive disability and/or promote drug addiction during adolescence and adulthood (Bond and di Giusto, 1976; Phillips and Whitlock, 1976; Fabio et al, 2015) (Contreras et al, 2017). According to the World Health Organization, Chile has the highest alcohol consumption per capita within Latin America, with men consuming 13.9 L and women consuming 5.5 L of pure alcohol annually (Aros et al, 2009).…”

Pre‐ and postnatal alcohol exposure delays, in female but not in male rats, the extinction of an auditory fear conditioned memory and increases alcohol consumption

“…We followed the procedures of Contreras et al () and Fernández, Carrizo, Plaza, Haeger, and Pautassi (). Sprague‐Dawley female rats were exposed to water or alcohol (10% v/v, sweetened with 64 mg/L sucralose) throughout pregnancy and during the first 7 days of nursing (P7).…”

“…Consistent with these ethanol‐stress interactions, we recently reported greater anxiety‐like behavior in the light‐dark box paradigm (Di Doi, Fernández, Hager, & Pautassia, ), and greater isolation‐induced alcohol intake and preference, in PAE versus control adolescent rats (Fernández, Carrizo‐Largo, Plaza, Haeger, & Pautassi, ). Moreover, several clinical and pre‐clinical reports have indicated that individuals exposed to prenatal alcohol are more likely to develop drug abuse during adulthood (Baer, Sampson, Barr, Connor, & Streissguth, ; Contreras et al, ; Fabio et al, ; Malanga & Kosofsky, ; Miranda‐Morales, Nizhnikov, & Spear, ; Pautassi, Nizhnikov, Spear, & Molina, ). Comorbidity between stress and substance use disorders is more evident in patients diagnosed with PTSD (Fullerton, Ursano, & Wang, ; Gilpin & Weiner, ).…”