ZINC-mediated gene expression offers protection against H 2O 2-induced cytotoxicity

Abstract

The ability of zinc to mobilize defense against reactive oxygen species (ROS) and H 2O 2-induced apoptosis was studied using a primary culture of rainbow trout gill cells. Gill cells were pretreated for 24 h with 100 μM ZnSO 4 followed by 24-h exposure to 100 or 200 μM H 2O 2, or were subjected to 100 μM ZnSO 4 together with 100 or 200 μM H 2O 2. Metallothionein-A (MTA) and metallothionein-B (MTB) mRNA levels were increased after treatment with zinc or H 2O 2, separately or in combination. Similarly, mRNA for glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD) were increased in response to either zinc or H 2O 2, or after sequential treatments with zinc followed by H 2O 2. The stimulatory effects of zinc or H 2O 2 on MTA, MTB, GST, and G6PD mRNA levels could be blocked by addition of the membrane permeable zinc chelator, N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that H 2O 2-induced upregulation of these genes is zinc-dependent. Pretreatment with zinc protected the cells from subsequent cell damage and apoptosis, as assessed by lactate dehydrogenase leakage, mitochondrial dehydrogenase activity (MTT assay), caspase-3 activity, and DNA fragmentation. In contrast, when gill cells were coincubated with zinc and H 2O 2 at the same time, H 2O 2 toxicity was higher than after treatment with H 2O 2 alone. It is concluded that zinc had a direct pro-oxidant effect when administered together with H 2O 2, but that pretreatment of zinc inhibited cytotoxicity and apoptosis through an indirect antioxidant action. We propose that the antioxidant action is manifested through zinc-dependent expression of several genes encoding antioxidant proteins (e.g., MTA, MTB, G6PD, and GST). Furthermore, the apparent zinc-dependency of H 2O 2-induced expression of antioxidant genes suggests that zinc might act as a physiological signal to mediate the response to oxidative stress.