Integration of toxicological end points with molecular measurements in a 28-day rat repeated dose inhalation study with cigarette smoke provides mechanistic understanding of smoke impact

Demonstration of reduced biological effects with a prototypic modified risk tobacco product. Towards a systems toxicology-based risk assessment, we investigated molecular perturbations accompanying histopathological changes in a 28-day rat inhalation study combining transcriptomics with classical histopathology, and demonstrated reduced biological activity of a prototypic modified risk tobacco product (pMRTP) in comparison to the reference research cigarette 3R4F. Rats were exposed to filtered air or to three concentrations of mainstream smoke (MS) from 3R4F, or to a high concentration of MS from a pMRTP. Histopathology revealed dose-dependent changes for 3R4F: irritative stress-related in nasal and bronchial epithelium and inflammation-related in the lung parenchyma. For pMRTP, significant changes were seen in the nasal epithelium only. Transcriptomics data were obtained from nasal and bronchial epithelium and lung parenchyma. Dose-dependent gene expression changes were seen for 3R4F with much smaller changes for pMRTP. A computational-modeling approach that is based on causal models of tissue-specific biological networks identified cell stress, inflammation, cell proliferation, and senescence as the most perturbed molecular mechanisms. These perturbations correlated with the histopathological observations. Only weak perturbations were observed for the pMRTP. In conclusion, a correlative evaluation of classical histopathology together with gene expression?based computational network models may facilitate a systems toxicology-based risk assessment as shown for a pMRTP. SD-rats were exposed for 28 days to filtered air (sham), or to a low, medium, or high concentration of mainstream (MS) cigarette smoke (CS) from the Reference cigarette 3R4F in addition to MS CS from a prototypic modified risk tobacco product (pMRTP) at the same nicotine concentration as the high 3R4F group, i.e., 23 ug nicotine/l. Each treatment group contains 5 biological replicates. 3 different tissue types were dissected: nasal cavity respiratory epithelium, lung parenchyma and lung bronchi. Tissues were processed and hybridized on Affymetrix Rat230_2 chips.