Gene expression in normal human bronchial epithelial (NHBE) cells following in vitro exposure to cigarette smoke condensate.

Cigarettes that burn tobacco produce a complex mixture of chemicals, including mutagens and carcinogens. Cigarettes that primarily heat tobacco produce smoke with marked reductions in the amount of mutagens and carcinogens and demonstrate reduced mutagenicity and carcinogenicity in a battery of toxicological assays. Chemically induced oxidative stress, DNA damage, and inflammation may alter cell cycle regulation and are important biological events in the carcinogenic process. The objective of this study was to characterize and compare the effects of smoke condensates from cigarettes that burn tobacco and those that primarily heat tobacco on gene expression in NHBE cells. For this comparison, we used quantitative RT/PCR and further evaluated the effects on cell cycling using flow cytometry. Cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F cigarettes (a tobacco-burning product designed to represent the average full-flavor, low "tar" cigarette in the US market) and Eclipse (a cigarette that primarily heats tobacco) using FTC machine smoking conditions. The CSC from 1R4F cigarettes induced statistically significant increases in the mRNA levels of genes responsive to DNA damage (GADD45) and involved in cell cycle regulation (p21;WAF1/CIP1), compared to the CSC from Eclipse cigarettes. In addition, genes coding for cyclooxygenase-2 (COX-2) and interleukin 8 (IL-8), which are associated with oxidative stress and inflammation, respectively, were increased statistically significantly more by CSC from 1R4F than by that from Eclipse. Furthermore, a dose-dependent increase in IL-8 protein secretion into cell culture media was stimulated by 1R4F exposure, whereas minimal IL-8 protein was secreted after Eclipse treatment. The biological relevance of the differential effect on gene expression was reflected in differential cell cycle regulation, as cells exposed to 1R4F CSC exhibited more significant S phase and G2 phase accumulation than cells exposed to Eclipse CSC. These data indicate that the simplified smoke chemistry of the tobacco-heating Eclipse cigarette yields statistically significant reductions in the expression of key genes involved in DNA damage, oxidative stress, inflammatory response, and cell cycle regulation in normal human bronchial epithelial cells compared to a representative tobacco-burning cigarette.

Differential c-myc expression profiles in normal human bronchial epithelial cells following treatment with benzo[a]pyrene, benzo[a]pyrene-4,5 epoxide, and benzo[a]pyrene-7,8-9,10 diol epoxide.

Bronchial epithelial cells are often exposed to airborne mutagens that have the potential to induce genetic changes involved in the development of lung cancer. Although lung tumors often display alterations in the expression of oncogenes and/or tumor suppressor genes, the role of specific chemicals and/or metabolites in causing these alterations is not well defined. The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P), a by-product of combustion, is a prevalent airborne environmental mutagen and a constituent of cigarette smoke. The primary objective of this study was to compare the effect of B[a]P and two of its reactive metabolites, benzo[a]pyrene diol epoxide (BPDE or bay region epoxide) and benzo[a]pyrene-4,5-dihydroepoxide (BPE or K-region epoxide), on expression of the proto-oncogene c-myc in normal human bronchial epithelial (NHBE) cells using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Changes in c-myc gene expression were compared with DNA adduct formation, growth inhibition, and cell-cycle progression as determined by (32)P-postlabelling, neutral red (NR), and flow cytometric analyses, respectively. None of the three test compounds altered the levels of 18S ribosomal RNA or beta-actin at the concentrations evaluated for c-myc expression, indicating that nonspecific changes in gene expression induced by cytotoxicity, for example, were not present at the concentrations evaluated. Cells exposed to B[a]P exhibited a dose-dependent increase in c-myc expression; conversely, a dose-dependent decrease in c-myc expression was observed following BPDE exposure. A marginal but concentration-dependent increase in c-myc mRNA levels was observed following exposure to the K-region epoxide. Our results demonstrated that, although B[a]P and its metabolites alter c-myc expression, the parent compound and its metabolites produce unequal and contrasting effects on the expression of this gene.