The kinetics of transcriptomic changes induced by cigarette smoke in rat lungs

Gene expression profiling in animal models exposed to cigarette mainstream smoke (CS) shapes up as a promising tool for investigating the molecular mechanisms involved in the onset and development of CS-related disease and may aid in the identification of disease candidate genes. Here we report on differential gene expression in lungs of rats exposed for 2, 7, and 13 weeks to 300 and 600 µg total particulate matter (TPM)/l CS with sacrifice 2, 6, or 20 h after the last exposure. The results showed a distinct, mostly transient, expression pattern of differentially expressed genes encoding antioxidant and xenobiotic-metabolizing enzymes, which confirmed and extended in qualitative and quantitative terms the findings of a former study, where rats were exposed to CS either acutely (once) or short-term (3 weeks). With increasing length of exposure, there was a distinct, mostly sustained, expression of genes implicated in innate and adaptive immune responses, clearly pointing to an emerging inflammatory response. Notably, this inflammatory response included the expression of lung disease-related genes not yet linked to CS exposure, such as osteopontin, arginase 1, and chitinase, as well as genes encoding proteolytic enzymes. Finally, our experiments also revealed a CS exposure-dependent shift in the cyclical expression of genes involved in controlling the circadian rhythm. Altogether, these results provide further insight into the molecular mechanisms of CS-dependent disease onset and development and thus may also be useful for defining CS-specific molecular biomarkers of disease. Keywords: gene expression profiling, cigarette smoke-related disease Four rats per group were nose-only exposed in flow-past exposure chambers (type FPC1-132) to diluted CS (300 or 600 µg TPM/l) or to conditioned fresh air (control) for 2, 7, or 13 weeks (2 times 1 h/day with a 30 min fresh air break in between, 5 days/week) and sacrificed at 2, 6, or 20 hours after the last exposure. Whole lung tissue was used for total RNA preparation, was frozen in liquid nitrogen immediately after dissection and stored at -70°C. After quantification and checking for integrity, equal mounts of RNA from the 4 animals per group were pooled, and, for hybridization on PIQOR microarrays, subjected to linear amplification and labeling. Two micrograms of RNA was labeled by reverse transcription via Cy5-dCTP incorporation (CS-exposed tissue) and Cy3-dCTP incorporation (unexposed tissue). Each labeled sample was then divided in half and hybridized on 2 customized PIQOR microarrays.