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).
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.
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.
|This document was last modified 05/12/2009.|
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