Approaches to define pathways of redox regulation of a eukaryotic gene: the heme oxygenase 1 example

Heme oxygenase 1 is a heme catabolic enzyme that is strongly induced in most eukaryotic cell types as a result of transcriptional activation by oxidants including UVA (320-380 nm) radiation. Three factors are clearly diagnostic for the characterization of this gene as redox regulated: (i) lowering the levels of cellular reduced glutathione using the drug buthionine (S, R)-sulfoximine enhances gene expression; (ii) iron depletion by various iron chelators suppresses activation; (iii) antioxidants suppress activation. Using chemical modulators to alter the levels of specific oxidizing intermediates, the nature of the effector species can be tentatively identified. A multiple approach is usually necessary because of the lack of specificity of most modulators. The involvement of a given species can be further implicated by generating the intermediate by an established protocol and confirming that it will activate the gene. As an example of such an approach, singlet oxygen has been implicated in the UVA radiation activation of HO-1 because the activation is enhanced by deuterium oxide (which enhances singlet oxygen lifetime) and suppressed by histidine (which scavenges the species). Singlet oxygen generated in a pure form using a photodynamic agent also strongly activates the gene. We describe a general approach designed to identify a role for singlet oxygen, superoxide anion, hydrogen peroxide, and/or hydroxyl radical in the activation of genes by oxidants.