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.

Funding: This work was funded by Tobacco Research Disease Related Program (TRDRP) grant #19XT-0166 to MM-G, a Porter Fellowship from the American Physiological Society to MV and a The University of California Institute for Mexico and the United States/Consejo Nacional de Ciencia y Tecnología grant to MC-C. The Analytical Chemistry work done at the University of California, San Francisco, was supported by the California Consortium on Third Hand Smoke, TRDRP 20PT-0184, and the National Institutes of Health, grants #S10 RR026437 and #P30 DA012393. Child NNAL assays were supported by a grant from Maternal & Child Health Bureau #R4O MC 00185 to MH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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. We show that significant damage occurs in liver, lung and during healing of wounds. In addition, the mice display hyperactivity. The results of our study delineate the early stages of potentially serious THS-induced damage in each of these organs and in behavior, problems that are known to be associated in humans with THS and SHS but have not previously been identified in humans exposed to THS.

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 epidemiological 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.

The exposure to tobacco smoke toxicants in THS can occur via ingestion, skin adsorption/absorption and/or inhalation [11] . 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) [21] .

Contamination of the homes of smokers by SHS residues (THS) is high, both on surfaces and in dust, including in children's bedrooms [11] . Re-emission of nicotine from contaminated indoor surfaces in these households can lead to nicotine exposure levels similar to that of smoking [12] 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 NO 2 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 first complete ban in the world on indoor smoking in all public spaces (including bars and restaurants) occurred in 1990 in San Luis Obispo, CA. That legislation, and its expansion to many countries, was achieved only because of clear scientific evidence that SHS is dangerous to non-smokers. Now, more than 20 years later, evidence is emerging that THS exposure potentially poses similar health risks, especially for children. 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 [9] . Public convictions and support of THS exposure-control policies depend on biological evidence of THS toxicity.

Despite efforts by governments and health organizations worldwide, cigarette smoking remains a serious health threat for smokers and nonsmokers alike [1] – [4] . Tobacco smoking causes significant human disease and economic burden worldwide, afflicting approximately 1.5 billion smokers while additional billions are at underappreciated health risk from exposure to cigarette smoke, with estimated annual costs of hundreds of billions of dollars. It has become clear that health threats are particularly serious for children who constitute a vulnerable population that cannot voluntarily avoid secondhand smoke (SHS) exposure. It is now well known that SHS is intrinsically more toxic than directly-inhaled firsthand smoke (FHS) [1] , [2] , [5] , [6] and recently, a new and persistent potential threat has been discovered – thirdhand smoke (THS) – the accumulation of SHS on environmental surfaces that ages with time, becoming progressively more toxic [6] – [8] .

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+/−5 µg/m3, a value that falls within the range detected by the Environmental Protection Agency (EPA) in the homes of smokers (15–35 µg/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-(methylnitrosamino)-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 [26].

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 intermittent exposure of children to cigarette smoke introduces uncertainty in cotinine data because the timing between the exposure and the urine collection for a study cannot be closely controlled. In contrast, nitrosamine data will average better over exposure time and urine collection time, producing a more reliable signal. We compared the levels in our mice with those we measured in a cohort of 50 SHS-exposed infants/toddlers aged 0.5 to 4 years [29]. 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.

At age 3 weeks, just-weaned wild-type mice (not genetically altered) are placed in standard, well-ventilated, mouse cages containing SHS-exposed materials. The animals are not constrained; they can move about the cage normally and the cages are kept in a large room with normal ventilation. Therefore, the environmental conditions also closely mimic those of THS exposure of children in the homes of smokers. These mice live in this environment for 6 months; materials exposed to THS are refreshed periodically as explained in the Materials and Methods. Control mice live in the same conditions but without THS exposure. At the end of 6 months, the animals are sacrificed and analyses performed.

In liver, THS stimulates accumulation of fat in the hepatocytes (steatosis), giving the liver a pale red color compared to the deep red in normal liver (Fig. 1A,B). This occurs in 30% of the animals. Sections of liver tissue show small lipid droplets (red staining) in the control (Fig. 1 C) whereas in THS-exposed animals (Fig. 1 D), the ∼2.5-fold greater amount of lipid (Fig. 1E) coalesces into much larger droplets. There is also a greater increase of triglycerides (∼4-fold; Fig. 1F). Lipid elevation of more than 5% above normal fat indicates that steatosis has progressed to non-alcoholic fatty liver disease (NAFLD), a condition that, with prolonged exposure in humans, can lead to fibrosis, cirrhosis, and cancer. The blood of the animals exposed to THS shows significantly elevated levels of triglycerides and low-density lipoprotein (LDL, bad cholesterol) whereas high-density lipoprotein (HDL, good cholesterol) is significantly decreased (Fig. 1 G–I). These changes in liver metabolism have potential implications for cardiovascular disease and stroke [30]–[35].

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. THS exposure results in non-alcohol fatty liver disease (NAFLD) with concomitant dyslipidemia. (A,B) Livers from mice exposed to THS for 24 weeks are a much paler pink than that of the control, an indication of fatty liver. (C,D) Liver cryosections from mice exposed to THS and stained with Oil-Red-O show increased lipid content and greater lipid aggregation than in the control. (E) Quantification of total hepatic lipid content shows lipid levels ∼2.5 times that of the control, with the majority of this increase being triglyceride accumulation (F). (G) Plasma triglyceride levels of mice exposed to THS were significantly increased. (H,I) Mice exposed to THS showed increased plasma LDL-Cholesterol and decreased HDL-Cholesterol levels. * p<0.05, ** p<0.01, *** p<0.001. Scale bars = 100 µm. https://doi.org/10.1371/journal.pone.0086391.g001

The discovery that these mice have NAFLD, with abnormal hepatocyte function, suggests that they are susceptible to developing an impaired inflammatory response that could lead to fibrosis. This situation may aggravate drug-induced damage (e.g. by acetaminophen) at doses that normally would not be damaging. This is of particular concern for children because they are frequently treated with acetaminophen for fever and pain.

A related important function of liver is regulation of insulin metabolism. We find that THS-exposed animals have fasting glucose levels that indicate they are pre-diabetic (Fig. 2A) and are significantly less efficient than control animals at using insulin to bring down blood glucose levels when an insulin tolerance test is performed (Fig. 2B). Moreover, a glucose tolerance test showed that THS-exposed mice handle the introduced glucose much less effectively than controls (Fig. 2C). The elevated triglycerides, increased LDL, decreased HDL and defects in insulin metabolism show that these animals are prone to developing “metabolic syndrome”, a condition that predisposes humans to stroke, coronary artery disease and type 2 diabetes [36]–[37]. These results are consistent with findings that show that tobacco smoke exposure and active smoking contribute to insulin resistance and could be associated with metabolic syndrome in US adolescent children [37].

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. THS exposure results in hyperglycemia and decrease in insulin sensitivity. (A) Fasting glucose levels of mice exposed to THS were significantly increased in comparison to control. (B) Intraperitoneal Insulin Tolerance Test (IITT) time course and calculated area under the curve reveal that THS-exposed mice have decreased sensitivity to insulin which is highly correlated with both fatty liver disease and smoke exposure. (C) Intraperitoneal Glucose Tolerance Test (IGTT) time course shows impaired glucose clearance following glucose administration. *p<0.05, ** p<0.01, *** p<0.001. https://doi.org/10.1371/journal.pone.0086391.g002

In lung, we observe that in the region of the alveolar sacs in THS-exposed mice the walls of the alveoli are disrupted significantly more often than in the controls (Fig. 3A,B) whereas the walls of the alveoli are thicker than those of the controls in the alveoli of the terminal respiratory bronchioles and some alveoli appear to contain secretions (Fig. 3 C,D). In the exposed mice, collagen fibers are disorganized but remain fibrillar (Fig. 3E–H) and the levels of collagen are higher (Fig. 3I). We also find that in some areas of the respiratory bronchioles the alveoli of the THS-exposed mice show cellular infiltration (Fig. 4A) whereas the controls do not (not shown). These observations suggest that the lung tissue could be producing pro-inflammatory cytokines. Indeed, we find that the pro-inflammatory cytokines/chemokines IP10, KC, MCP1, MCSF, MIG, MIP1β, MIP2, Eotaxin, LIX, VEGF are elevated whereas the anti-inflammatory cytokines/chemokines IL1α, IL9, IL10 are down-regulated (Fig. 4B,C), suggesting that there is a pro-inflammatory environment in the lungs. Indeed, we found significant numbers of macrophages and their presence was primarily in groups in the walls of the alveoli in the region of the respiratory bronchioles (Fig. 4D).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. THS exposure results in excess deposition of collagen in lungs. Cross-sections through the lungs show that in THS-exposed animals, the alveoli in the region of the alveolar sacs are disrupted in comparison to the control animals (A,B). In the terminal respiratory bronchioles of the lung, however, the walls of the alveoli in the THS-exposed animals are thicker and appear to contain secretions (C,D). (E–F) Masson-trichrome staining for fibrillar collagen (blue) shows that the level of collagen in normal lung is low but THS-exposed animals show higher levels of fibrillar collagen with disrupted structure between alveoli (*). (G,H) Second-harmonic imaging microscopy (SHIM) confirms that collagen between alveoli (bright white) remains fibrillar in THS-exposed animals. (I) Hydroxyproline (an amino acid that is highly present in fibrillar collagen) is much higher in lung tissue of THS-exposed animals than in the control. Alveoli in E–H marked by *. Scale bar in A,B is 100 µm, in C,D is 50 µm, in E–H is 20 µm. In A–H and in I. *** p<0.001. https://doi.org/10.1371/journal.pone.0086391.g003

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. THS exposure results in inflammation and excess production of pro-inflammatory cytokines/chemokines in lung tissue. (A) Cross-section through the alveoli in the region of the terminal respiratory bronchioles shows that in THS-exposed animals there is significant inflammation in the tissue. (B,C) A multiplex cytokine array shows that many pro-inflammatory cytokines are elevated in THS-exposed animals (red) compared to control (blue) whereas some anti-inflammatory cytokines are decreased. (D) Lung tissue staining with an antibody for the F4/80 antigen that labels mouse macrophages. n = 3 for control; n = 5 for THS. Stars in B,C indicate * p<0.05, ** p<0.01, *** p<0.001. Scale bar in A = 50 µm and in D = 20 µm. https://doi.org/10.1371/journal.pone.0086391.g004

Our reproducible observation that the damage to the alveoli is different depending on the region of the lung is difficult to explain. We speculate that it could be due to the effects of oxidative stress induced by the THS toxins in regions with different cellular microenvironments. In the alveoli of the alveolar sacs, the oxidative stress may cause cell death whereas in the alveoli of the respiratory bronchioles it may stimulate fibroblasts to produce collagen. The elevated level of interstitial collagen, the thickened walls of some alveoli, the presence of macrophages in the walls of those alveoli and the increase in pro-inflammatory cytokines suggest an increased risk for development of fibrosis in people who have been exposed to THS for prolonged periods of time. Fibrosis in lung ensues when fibrotic tissue replaces the parenchymal tissue. The consequence is scar tissue formation and decreased oxygen diffusion rate. It is possible that THS-exposed people have a lower tolerance for drugs that induce lung fibrosis, given that they already have a propensity for development of this condition [38]–[40]. Doctors should take this into consideration when designing treatments for individuals who have been living in environments in which THS is a contaminant (e.g. spouses and, potentially, elderly parents of smokers).

In skin, wounds take longer to heal in THS-exposed mice (Fig. 5A) and show characteristics that are conducive to reopening, such as heavy keratinization of the epithelium. The expression of numerous genes for keratins and keratin-associated proteins that are normally produced for hair and nails is increased (Fig. 5B, upper panel, C). Moreover, the expression of genes that are important in the inflammatory response and response to wounding is decreased (Fig. 5B, lower panel). In the healing tissue, the level of fibrillar collagen (blue) is greatly decreased in THS-exposed animals (Fig. 5D,E). The majority of collagen is not fibrillar and appears to be degraded (Fig. 5F,G), an observation consistent with gene array analysis showing a decrease in expression of tissue-inhibitor metalloproteinase 1 (TIMP1), an inhibitor of matrix metalloproteinases. This is in contrast to the lung where the fibrils of collagen are still intact, albeit disorganized. These observations could explain why, in the case of the lung, fibrosis develops whereas, in the skin, the lack of fibrillar collagen could lead to poor healing. These differences would be due to the fact that the lung suffers a chemical injury and the skin a chemical and mechanical injury.

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 5. THS exposure delays closure of and weakens cutaneous wounds. (A) Representative excision wounds performed on the backs of mice after hair removal show that THS exposure results in keratinization of the epithelium (crusty appearance) and delayed wound closure. (B) At day 14, when control wounds are closed, gene expression evaluated by Affirmetrix Arrays shows that THS exposure upregulates keratin genes and genes involved in epithelial migration and contractile function of wound tissue and downregulates genes involved in inflammatory and immune responses. (C) The upregulated keratin genes are primarily associated with hair and nail production, resulting in stiffening of the wound, making it crusty and potentially more fragile. “Ker.-Assoc. Prot.” = Keratin-Associated Protein. (D–G) In cross sections through the healing tissue, Masson-trichrome staining shows great decrease of interstitial collagen (blue) in THS-exposed tissue; second-harmonic imaging microscopy (SHIM) shows that the collagen is strongly fibrillar in the control (arrow) but not at all fibrillar in THS-exposed mice. For A; for B,C, for D–G. Scale bar for D,E = 100 µm and for F,G = 20 µm. White * labels a hair follicle. https://doi.org/10.1371/journal.pone.0086391.g005

It has long been known that smokers' wounds heal poorly [41]. This is particularly important when they undergo surgery. As a consequence, surgeons commonly recommend or require cessation of smoking for at least four weeks prior to surgery. Evidence shows that the early effects of smoking on blood-vessel constriction are reversible in less than an hour after smoking whereas the deficiencies in the inflammatory response do not return to normal until ∼4 weeks after cessation [41] and it is not known how long it takes for the damage to cells to be reversed. Indeed, many times surgical wounds reopen even if the patient stopped smoking well before surgery. It is not yet clear why this occurs. Our work shows that not only smokers but also those exposed to SHS and THS may suffer from these wound-healing risks. The delay in wound closure accompanied by the presence of decreased fibrillar collagen (Fig. 5D–G) in the healing tissue results in marked reduction of strength of wound tissue. This, in conjunction with the presence of keratins that convey rigidity to the epithelium and cells rich in contractile filaments, could be the cause for reopening of surgical wounds in smokers and, potentially, for those exposed to SHS and THS.

In behavior, THS-exposed animals showed behavior that seemed anxious or hyperactive. Therefore, we performed a standard test that is designed to examine anxiety [42] – the “Elevated Plus” T-maze (Fig. S1A). Time spent in the closed arms represents anxious behavior; rodents generally spend much more time in the closed arms. THS-exposed mice and controls display the same level of anxious behavior, as illustrated by their similar preference for closed over open arms of the maze (Fig. 6A). In contrast, when the frequency of entry into the open and closed arms was scored, the THS-exposed mice showed a significantly higher frequency of entries into the closed arms than did the control (Fig. 6B), suggesting that THS-exposed mice may be hyperactive. To test this possibility, we used the Open Field test (Fig. S1B). Individual mice were placed in the Open Field; walking, stationary and rearing behaviors were assessed, as well as the frequency of transition from one of these behaviors to another. THS-exposed mice spent significantly more time walking, much less time standing still and more time rearing than control mice (Fig. 6C). The frequency of transitions between these behaviors shows a similar pattern (Fig. 6D). In particular, THS-exposed mice were almost constantly in motion whereas control mice were stationary for a considerable fraction of the time. We illustrate this comparison with two movies, one of a control mouse (Video S1) and the other of a THS-exposed mouse during the first 10 minutes of the videotaped hour (Video S2). These movies are sped up by a factor of two to better illustrate the differences in activity during this period (the movies are found in the Video S1 & S2).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 6. Effects of THS-exposure on behavior of mice. (A,B) Testing for anxiety. Control and THS mice were tested using an Elevated Plus T-maze (Fig. S1A). (A) Total time spent in the open and closed arms was measured. The two groups both spent much more time in closed than in open arms of the maze, indicating normal anxious behavior. (B) The frequency of entries into the closed arms was significantly greater for THS-exposed as compared to control mice, indicating more locomotor activity. n = 6 controls and 6 THS-exposed mice. (C,D) Testing for hyperactivity. The Open Field test (Fig. S1B) was used to perform these experiments. The behavior of control and THS-exposed mice was observed for 1 hr. (C) The THS mice spent much more time walking, much less time stationary, and more time rearing than the controls. (D) The same general pattern was observed for the frequency of transition between these behaviors; n = 12 controls and 12 THS-exposed mice. https://doi.org/10.1371/journal.pone.0086391.g006

For confirmation, a second set of mice was tested in the Open Field and using Ethovision 7.1 video tracking software, we tracked mice individually for an hour. Again, it was seen that the THS-exposed mice covered longer distances (Fig. 7A) at higher velocities (Fig. 7B) and spent significantly more time in the periphery of the field (Fig. 7C). The difference in behavior between the two groups was particularly striking in the first two minutes during which the THS-exposed mice moved on average at high but decreasing velocity (Fig. S2) and the last 10 minutes of the hour in which the control mice showed on average little activity whereas the THS-exposed mice remained very active (Fig. 7D). We conclude that THS-exposed mice are hyperactive.

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 7. Effects of THS-exposure on hyperactive behavior. (A) Distance and (B) velocity of travel during the first and last 10 minutes of the 1 hr test. (C) Time spent in center of the field vs. the periphery during the first 10 minutes. (D) Raster plots showing the integrated paths that a control mouse (top) and a THS-exposed mouse (bottom) traveled in the first 10 mins and last 10 mins of the hour. The plots were chosen based on the average distance traveled by each cohort of control and THS exposed mice. The Ethnovision software produced the raster images for each individual mouse along with an Excel sheet containing the distance traveled by each mouse as represented by the raster image. The mean distance traveled for each cohort was then calculated and, based on that mean, the raster image representing the path traveled by the single mouse closest to the cohort mean distance traveled is shown. See also video s1 showing the behavior of the control and video s2 showing the behavior of the THS-exposed mice. n = 12 control and 12 THS-exposed mice. * p<0.05, ** p<0.01. https://doi.org/10.1371/journal.pone.0086391.g007

These data are consistent with previous findings in humans that link hyperactivity to tobacco smoke exposure [43], [44]. Examination of the 2007 National Survey on Children's Health of 55,358 children under the age of 12, found that 5.6% had attention-deficit/hyperactivity disorder (ADHD), 8.6% had learning disabilities and 3.6% had behavior and other conduct disorders but children who were exposed to SHS at home (and therefore unavoidably also exposed to THS) had a 50% greater chance of having 2 or more neurobehavioral disorders than children not exposed. It was also found that children 9–11 living in poverty were at even higher risk of tobacco-smoke associated neurobehavioral disorders [45], [46]. Even very low-level exposure is associated with cognitive deficits in children [47].