Analysis of metallothionein expression levels in mitochondrial NADH:ubiquinone oxidoreductase deficiency

Abstract

Some well defined functions of crucial importance for cell physiology are carried out by mitochondria1 NADH:ubiquinone oxidoreductase (complex I). Deficiencies of this complex, which is one of the most frequently encountered disorders of the mitochondria, lead to multi-system disorders that includes type 2 diabetes, Parkinson's disease, Alzheimer's disease and MELAS to name only a few. The generation of reactive oxygen species (ROS) in complex I deficiency has received much attention in the last few decades. Metallothioneins (MT), which have a metal homeostasis regulating and ROS-scavenging function, were recently identified to be over expressed in complex I deficient cell lines although the cause and role of this expression remains to be investigated. The aim of this study was to investigate metallothionein gene expression in complex I deficiency in vitro and evaluate related biochemical parameters, including ROS production. For this purpose, cell cultures were treated with various concentrations of an irreversible and specific complex I inhibitor, rotenone, for different incubation periods. Results of the 24 hour incubation period indicated that with a decrease in complex I activity from 49%, the production of ROS increased approximately two fold with a 7 times increase in MT-IIA expression. Furthermore, expression of MT-IA and -IB showed baseline levels of expression, suggesting possible isoform specificity in HeLa cells. CdCI2 induction showed excessive expression of MT-IIA (49 times) with almost no production of ROS, thus suggesting possible protection against ROS production. A specific ROS inducer, t-BHP, showed a 5 times increase in both ROS and MT-IIA expression compared to baseline levels. From our results it is evident that a complex I deficiency not only results in the production of ROS, but also the expression of MTs.