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J.-D. Huang, J. W. Lee, I. R. Rodriguez; Metabolism of 7-Ketocholesterol in Cultured RPE Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):497.
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In atheromatous plaques 7-ketocholesterol (7KCh) is the major oxysterol that induces cytotoxicity. The cytochrome P450’s, CYP27A1 and CYP46A1 as well as the sulfotransferase SULT2B1b are known to hydroxylate and sulfate 7KCh. The purpose of this study is to quantify the expression of these genes in the retina and to determine whether overexpression of these enzymes will protect cultured RPE cells from 7KCh cytotoxicity.
The mRNA expression of CYP27A1, CYP46A1 and SULT2B1b in human tissues and cultured RPE cells were determined by qRT-PCR. Full-length ORFs of CYP46A1, SULT2B1b, and PAPSS1 were respectively cloned into pcDNA4/HisMax TOPO expression vector. Cultured D407 cells were either transfected with CYP46A1 construct or co-transfected by SULT2B1b construct and PAPSS1 construct. Expression of these enzymes was confirmed by immunoblot. The transfected cells were treated with various concentrations of 7KCh in serum-free medium for 24 hours and the cell viability was measured using the Dojindo Cell Counting Kit.
In the retina, the per cell gene copy number for CYP27A1 (2548) is much higher than CYP46A1 (625) and SULT2B1b (3). CYP27A1 mRNA level in the retina was 1/3 of that found in the liver. CYP46A1 mRNA level in the retina was approximately 1/5 of that found in the brain. Overexpression significantly increased the levels of CYP46A1 (105-fold), SULT2B1b (100-fold), and PAPSS1 (10-fold) over the mock-transfected cells. In four independent experiments overexpression of CYP46A1 and SULT2B1b did not provide statistically significant protection from 7KCh cytotoxicity. Experiments with CYP27A1 are pending. Analyses for 7KCh metabolites, 24-hydroxy-7-ketocholesterol and 7-ketocholesterol-3-sulfate are also in progress.
Our results indicate that the retina contains significant levels of CYP27A1 but considerably lower levels of CYP46A1 mRNA. The levels of SULT2B1b are essentially nil. Overexpression of CYP46A1 and SULT2B1b do not convey any protection from 7KCh cytotoxicity. Our data suggests that these enzymes are unlikely to play a significant role in the detoxification of 7KCh in the RPE.
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