An 8-Month Systems Toxicology Inhalation/Cessation Study in Apoe−/− Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared With Conventional Cigarettes

Smoking cigarettes strongly contributes to the development and progression of a number of serious diseases, including cardiovascular disease (CVD) (Office of the Surgeon General et al., 2010; Price et al., 1999; Siasos et al., 2014) and chronic obstructive pulmonary disease (COPD) (GOLD, 2014; Office of the Surgeon General et al., 2010; Pauwels and Rabe, 2004; Postma et al., 2015). Important health benefits have been associated with smoking cessation (Bakhru and Erlinger, 2005; Dhariwal et al., 2014; Gepner et al., 2011). Although quitting is preferable to reduce smoking-related health risks, modified risk tobacco products (MRTPs) are being developed to provide alternatives for smokers who do not quit. It is therefore important to assess to what extent the benefits of smoking cessation are transferrable to the switching to MRTPs, comparing the impact of cessation and switching on physiological, histological, and molecular measurements associated with biological mechanisms implicated in disease progression. The U.S. Family Smoking Prevention and Tobacco Control Act of 2009 defines an MRTP as “any tobacco product that is sold or distributed for use to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products” (Food and Drug Administration, 2009). The U.S. Food and Drug Administration published draft guidance on “MRTP Applications” (Food and Drug Administration, 2012) stating that applications must provide scientific evidence to demonstrate that the product will “significantly reduce harm and the risk of tobacco-related diseases to individual tobacco users, and benefit the health of the population as a whole, taking into account both users of tobacco products and persons who do not currently use tobacco products.” In this context, nonclinical studies play an integral role in the evaluation of MRTPs (Food and Drug Administration, 2012). In this article, the toxicological mechanisms of a candidate MRTP, the tobacco heating system (THS) 2.2, have been investigated. THS2.2 electrically heats tobacco without combustion. THS2.2 is functionally different from a recently tested prototypic candidate MRTP that uses a carbon tip as a heat source (Kogel et al., 2014; Phillips et al., 2015) and utilizes a different design from the electrically heated smoking systems described previously (Moennikes et al., 2008; Schorp et al., 2012; Terpstra et al., 2003; Werley et al., 2008). However, like the carbon tip candidate MRTP, THS2.2 generates an aerosol that mainly contains water, glycerin, nicotine, and tobacco flavors. The electronically controlled heating technology is designed to avoid tobacco combustion, and in turn, reduce the formation of harmful and potentially harmful constituents (HPHCs). Our study objectives were: (1) to assess reduced exposure effects, compared with conventional cigarette smoke (CS), for THS2.2 aerosol in a chronic inhalation toxicity study; (2) to investigate the effects of cessation or switching to THS2.2 aerosol; and (3) to investigate disease endpoints related to both CVD and COPD in one mouse model, leveraging state-of-the-art systems toxicology approaches. Apolipoprotein E-deficient (Apoe−/−) transgenic mice are commonly used as a model for atherogenesis (Veniant et al., 2001), particularly to investigate smoking-related atherosclerosis (Boue et al., 2012; Chan et al., 2012; Lietz et al., 2013; von Holt et al., 2009), as well as CS-induced lung inflammation and emphysema (Arunachalam et al., 2010; Boue et al., 2013; Han et al., 2012). They were therefore chosen in this study to investigate indicators of both COPD and CVD in the same animals exposed to either mainstream CS or to the mainstream aerosol from THS2.2 (nicotine concentration matched to CS: 29.9 mg/m3) over an 8-month period, using physiological, histological, and molecular evaluations. The effects of cessation or switching to THS2.2 aerosol after 2 months of CS exposure were also investigated (Figure 1). FIG. 1. Study design. A, Groups and exposures. B, Daily exposure schedule. For animal numbers and allocations to endpoints, see Table 1. 3R4F, standard reference cigarette 3R4F; THS2.2, tobacco heating system 2.2. The applied CS exposure regimen resulted in larger atherosclerotic plaques and increased pulmonary inflammation and emphysematous changes in Apoe−/− mice compared with controls. Exposure to the THS2.2 aerosol neither induced lung inflammation and emphysema nor did it consistently change the lipid profile or enhance the plaque area. Cessation and switching caused a reversal of the inflammatory responses and halted progression of initial emphysematous changes and the aortic plaque area. Our results confirm that Apoe−/−mice are an excellent model to evaluate both COPD and CVD disease biomarkers (Boue et al., 2012, 2013), thereby serving the “Reduce” goal of the 3Rs in animal research. The “Refine” goal was also considered by our systems toxicology approach, which combined the functional, physiological, and morphological toxicity endpoints with high-density molecular investigations and computational modeling (Hoeng et al., 2013; Sturla et al., 2014).