Antioxidant enzyme gene expression in response to oxidative stress




Anne McDonough

Introduction

Aerobic metabolism generates reactive oxygen species as byproducts due to the incomplete reduction of oxygen; these byproducts can be involved in cell signaling such as the induction of apoptosis and in defense against pathogens, but can also lead to cell damage if present in excessive levels (Morel & Barouki 1999; Apel & Hirt 2004). Antioxidant enzymes protect aerobic prokaryotes, aerotolerant anaerobic prokaryotes, and eukaryotic cells from the detrimental effects of reactive oxygen species such as mutagenesis, DNA strand breakage, damage to membrane lipids, and damage to proteins found in connective tissue (Harris 1992; Scandalios 1993; Toyokuni et al. 1995). Three of the most critical antioxidant enzymes are catalase, glutathione peroxidase, and superoxide dismutase, which differ in their cofactors, cellular locations, and mechanisms of action (Harris 1992). Periods of oxidative stress increase cellular levels of reactive oxygen species and may lead to changes in the expression levels of genes encoding antioxidant enzymes; an appropriate balance of such enzymes is necessary to optimize their protective effects (Michiels et al. 1994).

Catalase is a heme-containing enzyme found in peroxisomes that catalyzes the dismutation of hydrogen peroxide (H2O2) to water and molecular oxygen (Harris 1992). It is a ubiquitous enzyme found in the tissues of most species, and its homotetrameric structure and the active site configurations of each subunit are highly conserved (Zámocký & Koller 1999). Glutathione peroxidase is a selenium-containing enzyme that catalyzes the oxidiation of glutathione to glutathione disulfide, the ratio of which can be used as an indicator of oxidative stress (Carey et al. 2003b), and is involved in the metabolism of lipid hydroperoxides and H2O2 (Arthur 2000). Superoxide dismutases catalyze the metabolism of superoxide anions to hydrogen peroxide and molecular oxygen using a variety of cofactors such as copper, zinc, manganese, and iron (Harris 1992). The enzyme of interest for this study, human superoxide dismutase 1, is located in the cytosol and utilizes copper and zinc. The three antioxidant enzymes described are involved in the metabolism of different oxidant substrates and work in concert to protect cells against damage if excessive levels of reactive oxygen species are present, such as when the organisms are subject to oxidative stress.

Certain mammalian species hibernate to conserve energy resources in periods of cold temperatures when food is scarce and thermoregulation is more costly. Hibernation induces oxidative stress in mammals and may damage susceptible tissues due to changes in blood flow caused by necessary periodic arousal and warming (Carey et al. 2000). Studies of urate levels in the plasma of hibernators suggest that increases in the production of reactive oxygen species may occur during arousal (Carey et al. 2003a). This, in turn, may lead to changes in the expression of antioxidant enzymes in hibernating mammals to prevent cellular damage as body temperature changes. Studies of glutathione peroxidase enzyme activity in 13-lined ground squirrels, however, did not demonstrate a significant difference despite evidence for oxidative stress in hibernators when compared with active individuals (Carey et al. 2003b).

An increase in the exposure to reactive oxygen species and subsequent cellular damage has also been demonstrated in studies of cancer (Toyokuni et al. 2005). Considering the occurrence of oxidative stress in both cancer cells and mammalian hibernators, techniques used to assay gene expression in response to oxidative stress could be applied to a variety of sample types. The objective of this study, therefore, was to examine the levels of gene expression for the three most critical human antioxidant enzymes – catalase, glutathione peroxidase 1, and superoxide dismutase 1 – in BE(2)-C cells, a neuroblastoma cell line. Neuroblastoma is the most common solid tumor found in children and tumors are often classified into subtypes based on pathogenicity and prognosis for the afflicted child (Brodeur 2003). The BE(2)-C cells used here are classified as I-type cells, which are phenotypically intermediate between neuroblastic N-type cells and substrate-adherent S-type cells; I-type cells can be induced to differentiate to either cell type and are considered highly malignant (Ross et al. 1995). The methods and primers used in this study to examine the levels of antioxidant enzyme gene expression in cancer cells could potentially be applied to tissues from mammalian hibernators.

Figures


Figure 1-Primers used in this study, including mRNA position, sequence information, and predicted band size for both mRNA products and genomic DNA products.


Figure 2-RT-PCR amplification of the genes encoding human enzymes (A) catalase, (B) glutathione peroxidase 1, and (C) superoxide dismutase 1, with GAPDH as a loading control in all instances. Treated (T) cells were exposed to 50 μM H2O2 for 6 hours, while untreated (U) cells were not exposed to H2O2.


Figure 3-Partial alignment of purified RT-PCR products and previously published sequences for human enzymes (A) catalase, (B) glutathione peroxidase 1, and (C) superoxide dismutase 1. A search using the NCBI Nucleotide BLAST database revealed high sequence homology.


• In BE(2)-C cells, the expression of catalase and superoxide dismutase 1 increased in response to oxidative stress after cells were exposed to 50 μM H2O2 for 6 h. The expression of glutathione peroxidase did not differ between treated and untreated cells.

• Sequences targeted by the primers were successfully amplified, as demonstrated by both the size of the bands produced on a 1% agarose gel and by high sequence homology when entering the amplified cDNA sequence into the NCBI Nucleotide BLAST database.

• Oxidative stress has been demonstrated to influence the expression of antioxidant enzymes, and the techniques used here may be applied to other sample types such as animal tissues.

Full Paper

Acknowledgments

I am grateful to Dr. Berish Rubin for helpful advice in the design and progression of this project, and to Bo Liu and Leleesha Samaraweera for their patience and assistance in the laboratory.


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