Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogenand oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals ( 2 ‫؍‬ 52.91, P < 0.001). Finally, quantitative realtime reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ␤-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. Experimental Design: The ''RNA'' extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, h-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase (''no-RT'') to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA. Results: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated ''RNA'' extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, h-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non^RNA-specific primers on saliva samples yielded PCR products for 36B4, h-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the ''no-RT'' and ''+RT'' conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva. Conclusions: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RTsaliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.Large-scale human cancer studies for the assessment of functional genomics or gene-environment interactions require a reliable source of RNA that can be accessed with minimal invasiveness, and is compatible with downstream, highthroughput applications. Options that have emerged include brush-exfoliated buccal cells (1), urine (2), and saliva (3, 4). Given the potential translational importance of mRNA expression biomarkers in such noninvasively acquired biospecimens, it is imperative that the RNA specificity of gene expression studies from these specimens be confirmed.Many human transcripts have homologous sequences in nontranscribed genomic DNA sequences, remote from the structurally active coding genes, which are termed pseudogenes. Common examples include h-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal phosphoprotein P0 (36b4), hypoxanthine phosphoribosyl transferase, glutathione S-transferases M1 and P1, and glutathione peroxidase. These pseudogenes are intronless, often have poly(A) tails, and have extremely high homology wit...

We examined the effects of fencing on deer browsing on seedlings 13 years after the building the experimental fence on Mt. Ohdaigahara, central Japan, where the sika deer (Cervus nippon) population is high. There was no difference in the species number of seedlings between 1991 and 2004. The density of seedlings in the fenced plot in 2004 was three times higher than in 1991. Although the unfenced plots had no seedlings higher than 20 cm, there were seedlings up to 120 cm in the fenced plot. These results suggest that 13-year fencing promoted regeneration of seedlings in this area.

Phase II detoxification of carcinogens is reported to mediate some of the anticarcinogenesis effects of candidate chemopreventive agents. We explored the interaction between sequence variation in the GSTP1 gene promoter and candidate chemopreventive exposure in regulating human GSTP1 expression. Polymorphisms along 1.8 kb of the GSTP1 promoter were identified in leukocytes [peripheral blood mononuclear cells (PBMC)] from 40 Caucasian subjects. Ten promoter polymorphisms (9 previously unreported) displayed strong linkage disequilibrium, yielding identification of three frequently observed haplotypes [HAP1 (43%), HAP2 (36%), and HAP3 (8%)]. Each haplotype was cloned into luciferase reporter constructs and transfected into normal human bronchial epithelial cells. Basal HAP3 reporter activity was significantly elevated (1.8-fold) but decreased to the same levels as HAP2 and HAP1 with increasing concentrations of sulforaphane, benzyl isothiocyanate (BITC), and epigallocatechin gallate (EGCG). To confirm native HAP3 functionality, we quantitated mRNA expression in uncultured PBMCs and in laser microdissected normal lung epithelial cells (MNLEC) from the same patients. Basal mRNA expression was higher in HAP3 individuals [1.8-fold (PBMC) and 4-fold (MNLEC) for HAP3 heterozygotes and 2.3-fold (PBMC), and 15-fold (MNLEC) for the HAP3 homozygote] than in the other genotypes. PBMC GSTP1 mRNA expression correlated to MNLEC expression (R 2 = 0.77). After culture and in vitro exposure to sulforaphane, BITC, or EGCG, the elevated GSTP1 mRNA expression of PBMCs from HAP3 individuals decreased to common expression levels. Elevated HAP3 function was confirmed at the protein level in PBMCs (5-fold higher for HAP3 heterozygotes and 7.6-fold for the HAP3 homozygote). These data suggest a potentially protective GSTP1 promoter haplotype and unpredicted inhibitory chemopreventive agenthaplotype interactions. (Cancer Res 2006; 66(12): 6439-48)