Antioxidant Enzyme Genes Expression in Conjunctival Epithelium of Normal Subjects

Abstract

Abstract: : Purpose:Normal levels of antioxidant enzyme genes in normal conjunctival epithelium is unknown at present. This study determines the profile and relative quantitation of 5 antioxidant enzyme genes in conjunctival impression cytology (CIC) samples from healthy subjects. Methods:Two polyethersulfone filters were applied to the superior bulbar conjunctiva of both eyes of eight healthy donors. Real time PCR was performed using isolated and retrotranscripted total RNA to quantify the expression of 5 antioxidant enzyme genes: mitochondrial Mn superoxide dismutase (MnSOD), cytosolic Cu- and Zn-containing superoxide dismutase (CuZn-SOD), extracellular superoxide dismutase (ecSOD), glutathione synthetase (GSS) and glutathione reductase (GSR). The relative quantity of this mRNAs was determined by SYBR® Green I dye detection. The housekeeping gene GAPDH was used to normalize the amount of expression levels. Conventional RT-PCR was also performed to confirm that only a single band was obtained when amplifying all cDNAs with the adecuate primers. After amplification, PCR products were electrophoresed on agarose gel and stained with ethidium bromide. Correlations on mRNA levels were performed among the different genes and between both eyes of the same donor using the Pearson's product moment correlation. Results:All transcripts were obtained in all tested samples. Amplified products by RT-PCR showed the expected amplicon size. Antioxidant enzyme expression levels (mean±SD) were: MnSOD, 0.70±0.45; CuZn-SOD, 2.10±1.37; ecSOD, 3.00±0.002; GSS, 2.55±1.51; GSR, 2.50±1.21. Correlations found were: CuZn-SOD and GSS (r=0.9429, p<0.001); CuZn-SOD and GSR (r=0.9505, p<0.001); GSS and GSR (r=0.9811, p<0.001). There was not correlation between both eyes. Conclusions: Normal human conjunctiva expresses the antioxidant enzyme genes MnSOD, CuZn-SOD, ecSOD, GSS, and GSR. The relative quantitation of these genes performed in normal conditions may allow further comparisons in pathological circunstances.

Keywords: conjunctiva • oxidation/oxidative or free radical damage • gene/expression