Journal Article 1)
Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed.
The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.
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Several studies have affirmed THS as an underappreciated public health hazard [4– 6,9,10]. Unfortunately, just as in the 1980s concerning SHS, today’s public is skeptical about these risks . Public convictions and support of THS exposure-control policies depend on biological evidence of THS toxicity. Contamination of the homes of smokers by SHS residues (THS) is high, both on surfaces and in dust, including in children’s bedrooms . Re-emission of nicotine from contaminated indoor surfaces in these households can lead to nicotine exposure levels similar to that of smoking  and similar levels of contamination are found on surfaces and dust of vehicles of smokers [13,14]. Recently, it was shown that THS remains in houses, apartments and hotel rooms after smokers move out [15,16]. Tobacco-specific nitrosamines (TSNAs) are strong carcinogens present in the THS residues deposited on indoor surfaces. In addition, nicotine (and probably other tobacco components) adsorbed in large amounts (microgram per sq meter levels) onto surfaces can react with nitrous acid (HONO) to form TSNAs [7,8,17,18]. Sources of indoor HONO and its precursors NO and NO2 include: a) smoking; b) combustion sources such as improperly ventilated gas stoves and heaters and c) infiltration of outdoor air pollution generated by vehicle exhaust or biomass burning [19,20]. Thus, THS presents toxicants similar to those present in mainstream (MS) or SHS and, in addition, also contains new toxicants due to aging and reaction with other chemicals. The exposure to tobacco smoke toxicants in THS can occur via ingestion, skin adsorption/absorption and/or inhalation . In the US alone, nearly 88 million nonsmokers ages 3 and older live in homes where they are exposed to sufficient SHS+THS to produce significant blood levels of a tobacco-specific nitrosamine and cotinine (a metabolite of nicotine) . Although the potential risks attributed to THS exposure are increasing, virtually nothing is known about the specific health implications of acute or cumulative exposure. Therefore, there is a critical need for animal experiments to evaluate biological effects of THS-exposure that will inform subsequent human epidemio- logical and clinical trials. Such studies can determine potential human health risks, design of clinical trials and potentially can contribute to policies that lead to reduction in both exposure and disease.
To address this need, we have conducted the first animal study on the effects of THS on several organ systems under conditions that simulate THS exposure of humans.
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Results and Discussion
We have developed a mouse model for THS exposure that approximates that of children and others in environments contaminated by THS (Materials and Methods). Materials commonly present in the homes and cars of smokers are exposed for specific periods of time to SHS from a smoking machine, 6 hrs/day, 5 days/wk for 24–26 wks, at a total particulate matter (TPM) of 30+/25 mg/m3, a value that falls within the range detected by the Environmental Protection Agency (EPA) in the homes of smokers (15–35 mg/m3) [22,23].
Direct comparison between biomarkers in our mice and in humans is difficult because population studies concerning THS exposure in humans are just beginning. Moreover, these comparisons are difficult to make because humans do not always comply with the needed experimental constraints.
Some of the components of SHS undergo chemical reaction with the indoor air to produce additional toxicants, at least some of which can be highly carcinogenic [1,5,7,8,24,25]. Principal among these is nicotine that adsorbs on environmental surfaces and reacts with a ubiquitous environmental contaminant, nitrous acid, leaving those surfaces with potentially dangerous levels of NNA (1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) and NNK (4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone) [7,8]. NNA is not found in SHS itself but NNK is. Furthermore, NNK is a lung-selective carcinogen and there has been considerable research on its toxicity and biomonitoring. A recent study in vitro showed that THS extracts and NNA itself are genotoxic to human cell lines .
To obtain a measure of the exposure to THS, we have measured NNAL (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol), the principal metabolite of NNK. We chose to use nitrosamines to compare between humans and mice because the half-life of the nitrosamines (days) is much longer than that of cotinine (hours) and therefore makes the comparison between our well-controlled experiments and not-as-well-controlled human data collection more reliable [27,28]. . . . .
The median NNAL level in the THS-exposed mice is 20% less than those of the SHS-exposed children (Table 1). The fact that we are exposing the materials to total particulate matter (TPM) levels similar to those detected by the EPA in the homes of smokers and the fact that we find the levels of a metabolite of NNK in the urine of the mice to be lower than those in children exposed to SHS (and inevitably also THS), gives us confidence that our THS exposure system is reliable and similar to that found in homes of smokers.
. . . . Materials and Methods THS Exposure
Using the Teague smoking apparatus , common household fabrics were placed in mouse cages and subjected to SHS. Each cage contained 10 g of curtain material (cotton) 10 g of upholstery (cotton and fiber) and two 16in2 pieces of carpet (fiber) to maintain equal exposure levels across experimental groups. Two packs of 3R4F research cigarettes were smoked each day, 5days/week and smoke was routed to a mixing compartment and distributed between two exposure chambers, each containing 8 cages with the materials.
We use the gravimetric method to determine the particulate concentrations. Whatman grade 40 quantitative cellulose filter papers are first weighed, then introduced into the filtering device and, after running the test for 15 mins, the filter is weighed again to determine the particulate mass that has accumulated during this time. This procedure is repeated with 2 more filters and the average of the 3 masses calculated gives the TPM values for each chamber. All cigarettes were smoked and stored in accordance with the Federal Trade Commission (FTC) smoking regimen . At the end of each week, cages were removed from the exposure chamber, bagged, and transported to the vivarium where mice were placed into the cages. For the next week, an identical set of cages and fabric was then prepared and exposed to smoke in the same way as described above. By using two sets of cages and material, each of which were exposed on alternating weeks, we ensured that mice inhabited cages containing fabric that had been exposed (according to the regimen described above) to fresh SHS during the previous week. Throughout the exposure period, hair was removed weekly from the backs of the mice to mimic the bare skin of humans. Control animals were tested for behavior and wound healing in a pilot study to look for possible changes related to living in the (non-exposed) materials of this project. No significant differences were observed in relation to animals living in normal conditions in the vivarium. During these experiments, in all cases the cages for THS-exposed and control mice were the same, food was the same, temperature the same, etc. Hair was not removed in this particular set of experiments but we have removed the hair in control mice in many other studies of dermal exposure to tobacco chemicals and found no differences between controls with and without removed hair.
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