Using an iTRAQ Approach to Investigate the Effect of Cigarette Smoke Exposure in a 90-day Inhalation Study Followed by 42-days Recovery Period on Sprague-Dawley Rats Lung Tissues


Abstract

In order to update and advance our knowledge regarding the impact of cigarette smoke on the lungs of Sprague-Dawley rats, a system biology approach that combines state-of-the-art proteomics with transcriptomics and toxicological endpoints, was developed. A 90-day inhalation study followed by recovery period of 42 days was conducted as described in the Organization for Economic Co-operation and Development (OECD) Testing Guideline 413. A quantitative proteomics approach using isobaric tags for absolute and relative quantification (iTRAQ®) was performed on the lung tissues of Sprague-Dawley rats to detect changes in protein expression levels between control rats (exposed to air), and rats exposed to mainstream smoke (MS) of the Reference Cigarette 3R4F (2 exposure concentrations of 8, and 23 mg/L nicotine). For the recovery period, the same treatment and control groups were used. Six biological replicates were analyzed to assess reproducibility within each group. The obtained mass-spectrometry data was searched against the Uniprot rat reference proteome database. Exposure of Sprague-Dawley rats to MS induced inflammatory changes in the lungs as detected by an increase in number of differentially expressed proteins and networks perturbed (as determined by network enrichment approaches) compared to the control sample. At the end of the recovery period, the protein expression profiles of MS-exposed rats showed a decreased amount of differentially expressed proteins indicating reverting back to normal levels compared to the control rats; that was shown by monitoring at least10 of the highly regulated proteins. The obtained proteomics results complemented those obtained from other endpoints such as transcriptomics, lipidomics as well as lung histopathology, thereby providing a unique opportunity to reveal underlying molecular mechanisms of MS exposure that were captured within this study that will further establish the foundation of PMI’s systems biology approach to assess the impact of modified risk tobacco products (MRTPs) on biological systems.