Short Communication EXPRESSION OF CYTOCHROME P450 AND OTHER BIOTRANSFORMATION GENES IN FETAL AND ADULT HUMAN NASAL MUCOSA

Despite recent progress in the identification and characterization of numerous nasal biotransformation enzymes in laboratory animals, the expression of biotransformation genes in human nasal mucosa remains difficult to study. Given the potential role of nasal biotransformation enzymes in the metabolism of airborne chemicals, including fragrance compounds and therapeutic agents, as well as the potential interspecies differences between laboratory animals and humans, it would be highly desirable to identify those biotransformation genes that are expressed in human nasal mucosa. In this study, a global gene expression analysis was performed to compare biotransformation enzymes expressed in human fetal and adult nasal mucosa to those expressed in liver. The identities of a list of biotransformation genes with apparently nasal mucosa-selective expression were subsequently confirmed by RNA-polymerase chain reaction (PCR) and DNA sequencing of the PCR products. Further quantitative RNA-PCR experiments indicated that, in the fetus, aldehyde dehydrogenase 6 (ALDH6), CYP1B1, CYP2F1, CYP4B1, and UDP glucuronosyltransferase 2A1 are expressed preferentially in the nasal mucosa and that ALDH7, flavin-containing monooxygenase 1, and glutathione S-transferase P1 are at least as abundant in the nasal mucosa as in the liver. The nasal mucosal expression of CYP2E1 was also detected. These findings provide a basis for further explorations of the metabolic capacity of the human nasal mucosa for xenobiotic compounds. Biotransformation of xenobiotic compounds in the nasal mucosa may play important roles in chemical toxicity, drug efficacy, and, possibly, detection of odorants (see Ding and Dahl, 2003, for a recent review). Numerous biotransformation enzymes have been identified in the nasal mucosa in mammals, including cytochrome P450 (P450) monooxygenases, flavin-containing monooxygenases (FMOs), aldehyde dehydrogenases (ALDHs), alcohol dehydrogenase, carboxylesterases, epoxide hydrolases, UDP-glucuronosyltransferase (UGT), glutathione S-transferases (GSTs), sulfotransferases, and rhodanese (Ding and Dahl, 2003). However, only a few of these enzymes, such as CYP2A13, have been studied in human nasal mucosa (Ding and Dahl, 2003; Ding and Kaminsky, 2003), primarily because of the lack of sufficient quantities of human nasal tissue samples for in vitro studies. A detailed knowledge of biotransformation enzyme expression in human nasal mucosa is critical for our ability to determine the roles of human nasal biotransformation enzymes in drug metabolism, chemical toxicity, and chemoreception. Recent advances in genomic technologies have made it possible to detect and quantify gene expression using small amounts of RNA samples. In the present study, we first performed a global gene expression analysis, so as to obtain preliminary data on the biotransformation genes expressed in human fetal and adult nasal mucosa. Gene expression in fetal and adult liver was also examined and compared with that in the nasal tissues, for identification of those genes that are preferentially expressed in the nasal mucosa. The nasal expression of a list of biotransformation genes that were identified by the microarray analysis as “nasal mucosa-predominant” was subsequently confirmed by qualitative RNA-PCR and DNA sequencing. Further quantitative RNA-PCR experiments were performed to determine the relative expression levels of the apparently nasal mucosapredominant biotransformation genes, in fetal liver and nasal mucosa. Our findings provide a basis for further explorations of the metabolic capacity of the human nasal mucosa for xenobiotic compounds. Materials and Methods Total RNA Preparation. The Institutional Review Boards at the participating institutions approved the study. Human fetal liver and nasal mucosa were provided by the University of Washington Birth Defects Research Laboratory. Adult nasal biopsy samples (from the middle or upper nasal turbinate) were obtained at Shandong University, China; all subjects underwent nasal polypectomy and had normal sense of smell. The postmortem time for the fetal samples was less than 30 min, and the adult nasal biopsy samples were frozen within 30 min of dissection. Total RNA was prepared with use of TRIzol reagent (Invitrogen, Carlsbad, CA). Quality of RNA was assessed by the A260/A280 ratio and by electrophoretic analysis on denaturing gels. Autopsy liver RNA from a 27-year-old male Asian, who died suddenly of unknown causes (postmortem time unknown), was obtained from BD Biosciences Clontech (Palo Alto, CA). The RNA samples were used for RNA-PCR and microarray analysis. GeneChip Microarray Analysis. The Affymetrix GeneChip Instrument System (Affymetrix, Santa Clara, CA) was used to perform global expression analysis at the Wadsworth Center Microarray Core facility. Prior to microarray analysis, RNA samples were further purified with an RNeasy kit from QIAGEN (Valencia, CA). The purified RNA samples were used to prepare doublestranded cDNAs, which in turn were used to synthesize biotin-labeled cRNAs. This work was supported in part by grants from the National Institutes of Health (ES07462 and CA092596); a Fogarty International Research Collaboration Award (TW01177) from the Fogarty International Center, National Institutes of Health; and a grant from Givaudan Fragrance Research. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005769. ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction, ALDH, aldehyde dehydrogenase; FMO, flavin-containing monooxygenase; UGT, UDP-glucuronosyltransferase; GST, glutathione S-transferase; bp, base pair(s). 0090-9556/05/3310-1423–1428$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 10 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 5769/3052985 DMD 33:1423–1428, 2005 Printed in U.S.A. 1423 at A PE T Jornals on July 6, 2017 dm d.aspurnals.org D ow nladed from Fragmented cRNAs were hybridized to Affymetrix GeneChips (U95Av2) according to standard protocols established by the manufacturer. The U95Av2 chip contains probe sets for all full-length genes represented in Build 95 of the UniGene database. The GeneChips were washed and then stained with streptavidin-phycoerythrin using a fluidics station, and the hybridization signals were detected with a laser scanner. The data were analyzed using the Affymetrix MicroArray Suite software (version 5.0). Normalized signal intensity values were used for estimating the relative expression levels of a given transcript in differing samples. The “absolute call” (present, marginal, or absent) for the expression of an individual transcript was determined according to statistical analysis of the differences in hybridization intensity between perfect-match probe and mismatch probes for each probe set. RNA quality and labeling efficiency were confirmed by first hybridizing a portion of the labeled cRNA samples to an Affymetrix Test3 chip, prior to hybridization of the U95Av2 Chip. Qualitative RNA-PCR. RNA-PCR was performed in a PE9600 PCR machine (Applied Biosystems, Foster City, CA) with Thermoscript RNase H reverse transcriptase (Invitrogen). First-strand cDNAs were synthesized at 50°C, with the use of 2 g of total RNA, 2.5 M oligo(dT)16 primer, and 5 mM MgCl2, in a total volume of 20 l. Two microliters of the reverse transcription product were added to PCR mixtures, which contained 0.5 M concentrations each of the two primers, 0.2 mM concentrations each of deoxynucleoside-5 -triphosphates, 2 mM MgCl2, 1 PCR buffer A (Promega, Madison, WI), and 2.5 U of Thermus aquaticus polymerase, in a total reaction volume of 50 l. PCRs were performed for 35 cycles with the following conditions: denaturation at 95°C for 30 s, annealing (at the temperature indicated in Table 1) for 30 s, and elongation at 72°C for 45 s. PCR products were analyzed by electrophoresis on agarose gels, purified using the QIAquick Gel Extraction Kit (QIAGEN), and sequenced by the Molecular Genetics Core facility of the Wadsworth Center, to confirm PCR specificity. Negative control reactions (no template) were routinely included, to monitor potential contamination of reagents. For detection of CYP2E1 in human fetal nasal mucosa, the reverse transcription reaction was carried out using the SuperScript III First-Strand Synthesis System (Invitrogen). PCR amplification was carried out in a LightCycler (Roche Diagnostics, Mannheim, Germany). Each reaction mixture, in a total volume of 10 l, contained 1 l of reverse transcription product, 0.8 l of 25 mM MgCl2, a 0.4 M concentration of each primer, and 1 l of LightCycler FastStart DNA Master Reaction Mix SYBR Green I (Roche Applied Science, Indianapolis, IN). Gel-purified PCR product was sequenced for confirming the detection of CYP2E1. Quantitative PCR. Real-time PCR analyses were performed in a LightCycler according to the instructions in the LightCycler Kit (Roche Applied Science). Primers (Table 2) were used at 0.25 M in a 20l reaction volume. Two microliters of the reverse transcription product (diluted with water or used directly) were added per capillary, and 40 PCR cycles were monitored. At the end of the PCR cycles, melting curve analysis was performed by heating to 95°C, followed by cooling to 65°C, and gradual heating to 92°C at 0.2°C/s. For each primer set, a serial dilution of the reverse transcription products was made, so that amounts equivalent to 0.2 g (1/10th) to 0.2 ng (1/10,000th) of the initial total RNA used in the reverse transcription step were used to perform quantitative PCR. This procedure was followed to ensure that the amounts of PCR products detected in the experimental groups fell within the linear range of PCR amplification. For all of the biotransformation enzymes studied, an amount of reverse transcription product equivalent to 0.04 g of RNA, and a final Mg concentration of 4.0 mM, gave the best results. Therefore, these parameters were applied to each reaction. All reactions were performed in duplicate. To monitor the extent of potential contributions of contaminating genomic DNA templates to the final PCR products, additional control reactions were performed by omitting reverse transcriptase during the reverse transcription step.