The results shown in Figure 2 and in Figure 3 suggest that zinc treatment can increase GM-CSF-dependent signaling (via PU.1 nuclear binding) and activation of the ARE (via Nrf2 nuclear binding) in the alveolar macrophages of alcohol-fed rats. We had already published that zinc treatment increases the expression of the GM-CSF receptor and normalizes bacterial phagocytic function in alveolar macrophages from alcohol-fed rats (Joshi et al., 2008). As both of these functions are dependent on GM-CSF signaling, and therefore on PU.1 nuclear binding, we decided to examine whether or not Nrf2-dependent functions were likewise augmented by zinc treatment. Metallothionein is a zinc storage protein that also possesses antioxidant properties and is important in the synthesis of thiol antioxidants, and whose expression is part of the ARE and therefore mediated by Nrf2. Therefore, we quantified metallothionein gene expression as a surrogate marker of Nrf2-mediated activation of the ARE to correlate with the Nrf2 binding studies in Figures 2 and 3. In parallel, we quantified Nrf2 gene expression to determine if the salutary effects of zinc treatment on Nrf2 nuclear binding could
