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KMID : 0603520010060010001
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Journal of Korean Association of Cancer Prevention
2001 Volume.6 No. 1 p.1 ~ p.9
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Metabolic Activation of Human Cytochrome P450 1A2 and Cytochrome P450 Reductase in Chinese Hamster Ovary Cell Using the Double Promoter System
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Park Chang-Hwan
Kim Dong-Won
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Abstract
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For the in vitro metabolic activation using cytochrome p450 (CYP) and NADPH-cytochrome p450 reductase (CYPR), dicistronic construct (CYP1A2DC-DCMV) was made by double cytomegalovirus promoters. As for mammaliam cell expression plasmid transfer was achieved in Chinese hamster ovary (CHO) cells by lipofectamine. Using neomycin and limiting dilution method, the CHO cells for protein expression were established (CHO.1A2DC-DMCV). Western blotting was carried out with the microsomes obtained after the process with sonication and ultracentrifugation. The molecular weight of recombinant human CYP1A2 was determined to be 57 kDa and that of CYPR, 76 kDa, respectively. Results of MTS assay to measure the cytotoxicity of 2-aminoanthracene (2-AA) on CHO.1A2DC-DCMV treated with 250 μM of 2-AA showed significant increase of cytotoxicity compared to the negative control (p<0.05); In vitro micronucleus test in CHO.1A2DC-DCMV cells treated with 10 μM of 2-AA showed significant increase of micronucleus formation against dimethyl sulfoxide (DMSO) treated group (p<0.05). And single cell gel electrophoresis in CHO.1A2DC-DCMV cells treated with 5 μM of 2-AA showed significant increase of Olive tail moment against DMSO treated group (p<0.01). With these results single cell gel electrophoresis is most sensitive method for detect the toxicity of 2-AA using CHO.1A2DC-DCMV cell line.
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KEYWORD
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CYP 1A2, Metabolic activation, Double promoter, CHO cell
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