Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes.
The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription-polymerase chain reaction (StaRT-PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen-immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S-transferase M3, glutathione S-transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S-transferase M1, 2, 4, 5, glutathione S-transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S-transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT-PCR or the above methods, whereas others were barely quantifiable by StaRT-PCR, i.e., were present at 1-10 molecules/10(6) molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium.
Experimental Carcinogenesis, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Martin
M
Vondracek
David A
DA
Weaver
Zsolt
Z
Sarang
Jesper J
JJ
Hedberg
James C
JC
Willey
Lars
L
Wärngård
Roland C
RC
Grafström
D000952:Antigens, Polyomavirus Transforming; D002461:Cell Line, Transformed / Q000201:enzymology* / Q000473:pathology; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D009061:Mouth Mucosa / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015262:Xenobiotics / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases