Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen‐immortalized oral keratinocytes

Abstract

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 antigenimmortalized 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. © 2002 Wiley-Liss, Inc. Key words: oral epithelium; biotransformation; phase I and II metabolizing enzymes; mRNA expressionSeveral enzyme systems including cytochrome P450s (CYPs), conjugation enzymes and enzymes involved in detoxification of reactive oxygen species (ROS) cooperate to protect the organism against reactive agents that are potentially carcinogenic and stress inducing. 1 Oxidative metabolism (so called phase I metabolism), carried out by the CYP family of enzymes, initiate biotransformation of compounds, which do not possess functional groups suitable for conjugation and can thereby generate toxic and mutagenic/ carcinogenic intermediates. 2 Following CYP dependent oxidative activation, conjugation enzymes (involved in so called phase II metabolism) including transferases for glutathione, sulfate, acetyl moieties and glucuronic acid generally decrease the reactivity of ...

Quality-Controlled Measurement Methods for Quantification of Variations in Transcript Abundance in Whole Blood Samples from Healthy Volunteers

“…The presence of an IS in each measurement controls for analytical false positives and false negatives. Recent reports have described the successful use of StaRT-PCR to measure the TA values of several promising biomarkers in samples of blood (14 ) or other tissues (15)(16)(17)(18).…”

Unraveling gene-gene interactions regulated by ligands of the aryl hydrocarbon receptor

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Alcohol dehydrogenase 3 transcription associates with proliferation of human oral keratinocytes

“…Band intensities were analyzed by densiometry using the Gel Doc 1000 system (Bio Rad, Hemel Hempstead, UK) and NIH Image software [obtained from http://rsb.info.nih.gov/nih-image; for details, see ref. 22] The values for transcript levels at each growth condition were derived from analysis of a minimum of three separate PCR reactions. Depending on the efficiency of the PCR, including aspects related to the primer hybridizations, the sensitivity of the assay was between 1-10 molecules per 10 6 molecules of b-actin [21][22][23].…”

“…An established protocol termed 'standardized quantitative RT-PCR' (StaRT-PCR) was utilized [22,23]. The SV Total RNA Isolation System protocol (Promega, Madison, Wis.) was applied for isolation of total RNA.…”