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 be explained at least in part through a direct induction of Nrf2. As shown in Figure 4, panel A, chronic alcohol ingestion significantly (P<0.05) decreased metallothionein gene expression in alveolar macrophages by more than 50% compared to alveolar macrophages from control-fed rats. In contrast, dietary zinc supplementation significantly increased (P<0.05) metallothionein gene expression in the alveolar macrophages of alcohol-fed rats. In fact, dietary zinc supplementation actually increased (P<0.05) alveolar macrophage metallothionein gene expression ~2-fold compared to control macrophages. In parallel, and as shown in Figure 4, panel B, chronic alcohol ingestion significantly decreased (P<0.05) the gene expression Nrf2 itself in alveolar macrophages when compared to Nrf2 expression in alveolar macrophages from control-fed rats. In contrast, and consistent with the induction of metallothionein in panel A, dietary zinc supplementation increased Nrf2 gene expression in the alveolar macrophages of alcohol-fed rats to the same level of expression (P>0.05) as in alveolar macrophages from control-fed rats.
