Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells.
In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.
Farin
F M
FM
Department of Environmental Health, School of Public Health and Community Medicine, Seattle 98195.
Pohlman
T H
TH
Omiecinski
C J
CJ
D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000502:physiology; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000032:analysis; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D014471:Umbilical Veins / Q000201:enzymology / Q000502:physiology
D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D004851:Epoxide Hydrolases