Peroxidase Is Low in Subjects with Bronchogenic Carcinoma Transferase P1, Glutathione Transferase M3, and Glutathione Normal Bronchial Epithelial Cell Expression of Glutathione

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10 b-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST 3 GSTM3 3 GSHPx 3 GSHPxA 3 GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5–10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies. INTRODUCTION NBECs are at an increased risk for oxidative damage following inhalational exposure to reactive oxygen species in cigarette smoke (1, 2), ozone (3), possibly asbestos (4), and other particulates in the environment. NBECs also are exposed to endogenous oxidative products produced through normal cellular metabolism (5) and during inflammation (6, 7). In addition, inhaled daughters of radon-222 decay (polonium-218 and polonium-214) may deposit on NBECs and emit a particles that generate reactive oxygen products as they encounter the cells. NBECs also are exposed through inhaled cigarette smoke or urban air pollution to PAHs. These procarcinogens may be metabolically activated in the cytoplasm and subsequently damage nuclear DNA. Damage to NBECs and adjacent structures from oxidants and/or activated carcinogens may result in a variety of pulmonary disorders, including bronchogenic carcinoma, pulmonary fibrosis, chronic bronchitis, and emphysema (5, 8). NBECs express several enzymes, including GSTs and glutathione peroxidases, that are capable of preventing or reducing injury from reactive oxidants or carcinogens. The GST enzymes conjugate reactive chemical groups, including reactive oxygen species and diol-epoxide ultimate carcinogens, to glutathione and thereby prevent them from binding to and damaging DNA (9). There are several classes of GSTs, including one microsomal class (mGST) and four cytosolic classes: GSTA, GSTM, GSTP, and GSTT (10, 11). In addition, a human homologue of rat GSTK1 has been reported (12). Each GST enzyme has substrate specificity, but there is considerable overlap (for review, see Ref. 13). For example, diol-epoxides derived from PAH procarcinogens are metabolized by GSTP1 and GSTM1–3 (14). Other substrates for the cytosolic GSTs include steroids, alkenals, and quinones (for review, see Ref. 9). In contrast to the cytosolic GST enzymes, mGST has very little specificity for epoxides (15). However, mGST has activity against a broad range of other substrates, including styrene-7– 8-oxide (16), 1-chloro-2,4-dinitrobenzene, and cumene hydroperoxide (17). Further, various halogenated alkynes and alkenes are metabolized preferentially by mGST compared to the cytosolic forms (13, 18). The glutathione peroxidase enzymes catalyze the inactivation of peroxides (including hydrogen peroxide and lipid peroxides) using reduced glutathione as a cofactor (19). Several enzymes have glutathione peroxidase activity, including GSHPx (19), GSHPxA (a secreted form; Ref. 20), mGST (21), GSTA (22), and GSTM3 (23). Both intertissue and interindividual variation in the expression Received 8/18/99; accepted 1/19/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants NIEHS R01 05719, NIEHS P01 01640, NIEHS R01 02679, and NHLBI R01 HL51701 and a grant from Gene Express, Inc. 2 To whom requests for reprints should be addressed, at Medical College of Ohio, Division of Pulmonary and Critical Care Medicine, Department of Medicine, 3000 Arlington Avenue, Toledo, OH 43699-0008. Phone: (419) 383-3543; Fax: (419) 3836244; E-mail: [email protected]. 3 The abbreviations used are: NBEC, normal bronchial epithelial cell; PAH, polycyclic aromatic hydrocarbon; GST, glutathione transferase; RT-PCR, reverse transcription-PCR; COPD, chronic obstructive pulmonary disease; CT, competitive template; NT, native template.