CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.
Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.
Vanden Heuvel
J P
JP
School of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907.
Clark
G C
GC
Thompson
C L
CL
McCoy
Z
Z
Miller
C R
CR
Lucier
G W
GW
Bell
D A
DA
D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004781:Environmental Exposure / Q000032:analysis*; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry* / Q000187:drug effects; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000032:analysis*; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012307:Risk Factors; D012907:Smoking / Q000097:blood
D015415:Biomarkers; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1