Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.
The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.
Wu
Susan J
SJ
New York State Department of Health, Wadsworth Center, PO Box 509, Albany 12201-0509, USA.
Spink
David C
DC
Spink
Barbara C
BC
Kaminsky
Laurence S
LS
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D065633:Cytochrome P-450 CYP1B1; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D011083:Polycyclic Compounds / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014407:Tumor Cells, Cultured
D011083:Polycyclic Compounds; D012333:RNA, Messenger; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1