Tobacco Documents Online

CR900213R3 In press Circulation PASSIVE SMOKIr1G AND HEART DISEASE: EPIDEMIOLOGY, PHYSIOLOGY, AND BIOCHEMISTRY Stanton A. Glantz, PhD William W. Parmley, MD Division of Cardiology, Department of Medicine Cardiovascular Research Institute University of California San Francisco, CA 94143 Short title: Passive Smoking and Heart Disease This manuscript is based on a background paper prepared for the U.S. Environmental Protection Agency. It was also presented at the Seventh World Conference on Tobacco and Health, Perth, Australia, April 1-5, 1990, and the World Conference on Lung Health, Boston, MA, May 20-24, 1990. Address for Correspondence and Reprints: Stanton A. Glantz, Ph.D. Professor of Medicine Division of Cardiology ~ Box 0124 M1186 University of California San Francisco, CA 94143-0124 (415) 476-3893 FAX: (415) 476-0424

CR900213R3 ABSTRACT The evidence that environmental tobacco smoke (ETS) increases risk of death from heart disease is similar to that which existed in 1986 when the Surgeon General concluded that ETS caused lung cancer in healthy nonsmokers. Eleven epidemiological studies, done in a variety of locations, reflect a 3096 increase in risk of death from ischemic heart disease or myocardial infarction in nonsmokers living with smokers. The larger studies demonstrate a dose-response effect. The epidemiological studies are complemented by a variety of physiological and biochemical data from human studies which show that ETS adversely affects platelet function and damages arterial andothelium, increasing the risk of heart disease. ETS also exerts adverse effects on exercise capability of healthy people and those with heart disease by reducing the body's ability to deliver and use oxygen. In animal experiments, ETS also depresses cellular respiration at the level of mitochondria. The polycyclic aromatic hydrocarbons in ETS also accelerate, and may initiate, the development of itherosclerotic plaque. Nonsmokers. appear to be more sensitive to ETS than smokers. In terms of cardiovascular disease, it is incorrect to compute "cigarette equivalents" for passive exposure to ETS, then try to extrapolate the effects of this exposure on nonsmokers from the effects of direct smoking on smokers. ETS causes heart disease, and ETS-induced heart disease may account for about ten times as many deaths as ETS-induced lung cancer. ETS is the third leading preventable cause of death, after primary smoking and alcohol. CONDENSED ABSTRACT The evidence that environmental tobacco smoke (ETS) increases risk of death from heart disease is similar to that available when the Surgeon General concluded that ETS caused lung cancer in healthy nonsmokers. ETS increases risk of death from heart disease by 3096 among nonsmokers living with smokers. ETS adversely affects platelet function and damages arterial endothelium. ETS significantly reduces exercise capability of healthy people and those with heart disease, as well as mitochondrial respiration. The polycyclic aromatic hydrocarbons in ETS accelerate the development of atherosclerotic plaque. ETS causes heart disease and is the third leading preventable cause of death, after active smoking and alcohol. c:\Siana\maau.cribuheart.doc

CR900213R3 Key Words artherosclerosis myocardial infarction bentio(a)pyrene passive smoking carbon monoxide platelets environmental tobacco smoke polycyclic aromatic hydrocarbons epidemiology secondhand smoke mitochrondia tobacco smoke pollution

CR900213 R3 The first disease linked definitively to active smoking was lung cancer. It is, therefore, not surprising that the first disease identified as caused by passive smoking was also lung cancer'. Before the advent of mass marketed cigarettes, lung cancer was a rare disease. Since smoking is the primary cause of lung cancer, identication of this link - for both active2 and passive smoking' - was relatively straightforward. This situation contrasts with heart disease, which has many risk factors. It is not surprising that it took longer for the scientific community to conclude that active smoking caused heart disease. Once the link between smoking and heart disease was established, it became clear that smoking killed more people by causing or aggravating heart disease than did the lung cancer. In fact, smoking is the most important preventable cause of coronary disease. Exposure to environmental tobacco smoke (ETS) has now been linked to heart disease in nonsmokersxa. Much of the evidence for this link has appeared since the US Surgeon General' and National Academy of. Sciences7 reviewed the evidence on the health effects of ETS in 1986. Based on the information available then, both reports concluded that the evidence linking ETS and heart disease was equivocal and that more research was necessary before any definitive statements could be made. These conclusions were reasonable in 1986. In the four years since these reports were written, considerable information on both the epidemiology and biological mechanisms by which ETS causes heart disease has accumulated. Most of the results presented here were published after the 1986 Surgeon General and National Academy of Science reports. There are now eleven epidemiological studies on the relationship between exposure to environmental tobacco smoke in the home and the risk of heart disease in the nonsmoking spouse of a smoker. All but one of these studies yielded relative risks or odds ratios greater than 1.0. There are several lines of biological evidence which make this association plausible. There is evidence that exposure to ETS reduces exercise tolerance of both healthy individuals as well as people with existing coronary artery disease. Such reduced exercise capability is one of the landmarks of acute compromises to the coronary circulation. There is good evidence, from both human and animal studies, that exposure to tobacco smoke, including passive smoking, increases aggregation of blood platelets. Such increases in platelet aggregation are an important step in the genesis of atherosclerosis. In addition, increasing platelet aggregation contributes to risk of coronary thrombosis, i cause of acute myocardial infarction. Finally, carcinogenic agents in ETS, including benzo(a)pyrene have been shown to produce injuries to the endothelial cells which line ¢ajlaMZ\aarnuacrikaheut.doc I

CR900213R3 arteries. Such injuries are the first step in the development of atherosclerosis. Thus, exposure to ETS can contribute to both short term and long term insults to the coronary circulation and the heart. It is not surprising, therefore, that epidemiological studies have identified an increase in the risk of coronary artery disease in nonsmokers living with smokers. Effects of Primary Smokina Before reviewing the evidence linking ETS with coronary artery disease, it is worth summarizing the evidence linking active smoking with coronary artery disease. This evidence was summarized in the 1983 Surgeon General's Report, which was devoted entirely to cardiovascular disease; it concluded that cigarette smoking is one of the three major independent heart disease risk factors. It also concluded that the magnitude of the risk associated with cigarette smoking is similar to that associated with the other two major heart disease risk factors, hypertension and hypercholesterolemia; however, because cigarette smoking is present in a larger percentage of the U.S. population than either hypertension or hypercholesterolemia, cigarette smoking ranks as the largest preventable cause of heart disease in the United States. Since 1983, evidence has also mounted that the polycyclic aromatic hydrocarbons in cigarette smoke can injure the arterial endothelium and initiate the atherosclerotic process. All the compounds implicated as damaging to the cardiovascular system of smokers have been identified in ETS'.'. Epidemiological Studies on ETS and Heart Disease Since 1984, the epidemiological evidence linking exposure to ETS with heart disease has rapidly accumulated. The results of the eleven published studies'''= are summarized in Table 1 and Figure 1; four studies present data on men, nine on women, and one on both sexes combined. Despite minor differences in methodology or end points (some used death from ischemic heart disease of any origin and some were limited to death from myocardial infarction), the results of these studies are remarkablly consistent. All the studies on men yielded relative risks of death from heart disease exceeding 1.0 when a nonsmoking man was married to a woman who smoked, with a median risk of 1.2. All but one of the studies on women' yielded relative risks exceeding 1, with a median relative risk of 1.4. Several studies'a"•"a0 have also suggested an increase in the risk of nonfatal coronary cA;lantzl=nuscrikt.heart.doc 2

CR900213R3 symptoms. Consistency of an observation across different studies increases the confidence one can have that an association is causal. Several authors also observed a dose-response relationship between increasing amounts of smoking by the spouse and the risk of heart disease in the nonsmoking spouse'"13•`5'", which in most cases was statistically significant. The presence of such dose-response effects across multiple studies, done in different locations with different criteria, supports the hypothesis that ETS causes heart disease in nonsmokers. While all but one of the studies in Table 1 and Figure 1 yielded relative risks greater than 1.0, the fact remains that 3 of the studies in men and 5 of the studies in women had 95 96 confidence intervals for the relative risk of passive smoking for heart disease that included 1.0, meaning that the risk was not statistically significantly elevated above 1.0 (with P <.05). It is important to note that the 95 96 confidence intervals do not lie symmetrically about 1.0, but rather are skewed towards higher risks. Exn*+,ining the confidence intervals leads to the conclusion that exposure to ETS elevates the risk of heart disease (Figure 1). It is also possible to combine the results of these studies in a formal analysis to derive a global estimate of the relative risk and associated 95 96 confidence interval. By combining the studies, the sample size and so the power to detect an effect increases. Wells' used several of the studies in Table 1''''"' to compute a pooled relative risk of 1.3 (95% confidence interval 1.1 to 1.6) for men and and 1.2 (95 96 confidence interval 1.1 to 1.4) for women. A similar analysis using all the studies in Table 1 yields a relative risk of 1.3 (with a 95 96 confidence interval from 1.1 to 1.6). When interpreting the results of such epidemiological studies, it is always important to consider biological plausibility and potential confounding variables which could explain the results. Aside from noting that the compounds in mainstream smoke that have been implicated in heart disease are in ETS, we will defer the discussion of biological plausibility until we discuss the effects of ETS on platelets and the atherogenic agents in ETS. For now, we will concentrate on potential confounding variables, such as the possible confounding effect of a correlation of spouses' poor health behaviors (e.g., diet high in animal fat). These confounders are particularly important in a disease like heart disease, because it is known to be caused by multiple risk factors. All the studies controlled for the most important confounding variable, age, and several"'`" controlled for known risk factors for coronary artery disease, in particular levels of serum or plasma cholesterol, blood pressure and body mass. Most of the studies also included one or more measures of socioeconomic status, such as the nature of the housing or amount of education. Indeed, studies that estimated the relative risk both with and c:4lantzlmanu.criletaheart.doc 3

CR900213R3 without taking these confounding variables into account found an increase in risk associated with ETS after taking the confounding variables into account'a's. Lee='" has suggested that the elevated risk of heart (and other) disease with passive smoking could be due to misclassification of nonsmokers who are really smokers. In addition, Wale has noted that some people who say they live with nonsmokers have detectable levels of the nicotine metabolite cotinine in their blood, indicating that they are actually exposed to ETS, either at work or at home. The former type of misclassification would tend to lead to an overesdmate of the risks associated with ETS and the latter would lead to an underestimate of the risk. Careful analysis of the question of misclassification - which applies generally to studies of ETS - have demonstrated that the observed risks cannot be explained by this problems-2`a. There is always the possibility that there is some other confounding variable relating to cultural factors, such as the nature of housing or employment or the nature of time spent outside the home. The fact that results from all over the world in widely varying cultural settings - including several regions in the United States, the United. Kingdom, Japan, and China - argues against this concern. One can assess formally how confident one can be in reaching a negative conclusion by computing the power of the study to detect an effect of specified siuP. Table 1 shows estimates of the power of each of the studies to detect a 20% increase in risk of heart disease (i.e., a relative risk of 1.2) with the available samples. The power was computed as described in Muhm and Olshan30, using a two-sided test for the relative risk with a Type I risk of 5% (i.e., requiring the 95% confidence interval for the relative risk to exclude 1.0 before concluding a statistically significant elevation in risk in an individual study). Most of the studies have low to moderate power. All fourl','4,'5," that have power above 4096 identified significant increases of heart disease risk with ETS exposure. Examining Table 1 reveals that the greater the power of the study to detect an effect, the more likely it was to find a significant adverse effect of ETS. Finally, it is worth noting that all these studies are based on the smoking habits of the nonsmoker's sQousc, and so exposure to ETS at home. Household exposures to ETS at home are generally much smaller than exposures at work, where the density of smokers is generally higher"X. As a result, these studies generally underestimate the risk and attendant public health burden due to ETS-induced heart disease. Kawachi at alm have adjusted Wells's relative risks to account for workplace exposures to ETS and found that the relative risks increase to 2.3 (95 96 Cl 1.4 - 3.4) for men and 1.9 (95% Cl 1.4 - 2.5) for women. Thus, any potential confounding of the results due to e:ijlaatzlmanuacciktaheart.doc 4

CR900213R3 exposure to ETS outside the home will tend to produce underestimates rather than overestimates of the effect of ETS. Likewise, estimates of public health impact based on risks computed from household exposures' will be lower than the true public health impact. In addition, Wellss and Kawachi et al-" indicate that the number of heart disease deaths due to passive smoking is an order of magnitude greater than then number of lung cancer deaths due to passive smoking. Even though the relative risks for heart disease and lung cancer caused by ETS are similar (about 1.3 for both diseases), the attributable deaths for heart disease is greater since heart diease is much more common than lung cancer. Of 53,000 annual deaths in the U.S. attributed to passive smokings, 37,000 are attributed to heart disease, compared with 3,700 for lung cancer (Figure 2). These epidemiological studies demonstrate a connection between ETS exposure and death from heart disease. We now turn our attention to possible physiological and biochemical mechanisms which could explain these observations. Acute Effects of ETS Exposure Chronic exposure to ETS exerts carcinogenic effects by increasing the cumulative risk of a molecule of one of the carcinogens in the ETS damaging a cell and initiating or promoting the carcinogenic process. The situation with heart disease is different. In heart disease there are both important chronic changes (i.e., the development of atherosclerotic lesions) and acute changes. The latter include an increase in myocardial oxygen demand which may outstrip the oxygen supply and produce ischemia, and increased platelet aggregation which can lead to coronary thrombosis and acute myocardial infarction. When the coronary circulation cannot provide enough oxygen to the myocardium to meet the demand, the result is ischemia which can be silent or result in aaginal chest pain. Earlier onset of angina or hypotension during exercise is a reflection of more severe heart disease. Oxygen supply can be reduced by atherosclerotic narrowing or vasoconstriction of the coronaries or by reducing the oxygen carrying capacity of the blood by forming carboxyhemoglobin. Khalfen and Klochkov" confirmed earlier work by Aronow" demonstrating that exposure to ETS significantly reduced exercise ability in patients with coronary artery disease and the rate pressure product (heart rate times systolic blood pressure). In both studies, patients were exposed to realistic levels of ETS by simply sitting in a waiting room while someone was smoking. '1'hese effects were present in both smokers and nonsmokers" and regardless of whether or not the room was ventilated"-'s. Exposure to ETS also increased resting c:4laatzlmuw.crikUheait.doc 5

CR900213 R3 heart rate and systolic and diastolic blood pressure, and resulted in a lower heart rate at the onset of angina's. Blood carboxyhemoglobin was increased by about 196 after exposure to ETS'3. Thus, acute exposure to ETS leads to an imbalance between myocardial oxygen supply and demand during exercise in patients with coronary artery disease. While this discussion has concentrated on the carbon monoxide in ETS as the active agent, it is possible that some other component of the ETS is causing or contributing to this effect. The effects of ETS on cardiac performance are, in fact, severe enough to affect exercise performance in young healthy subjects with no evidence of heart disease. McMurray et al" exposed young healthy women to pure air and air contaminated with ETS while they exercised on a treadmill. The results were similar to those observed in patients with coronary artery disease. Resting heart rate was increased during exposure to ETS, which increased blood carboxyhemoglobin by about 196. Exposure to ETS significantly reduced maximum oxygen uptake (by 0.251/min and time to exhaustion (by 2.1 min). Exposure to ETS also increased the perceived level of exertion during exercise, maximum heart rate, and CO2 output. It also significantly increased levels of lactate in venous blood (from a mean of 5.5 mM during control period to 6.8 mM after exposure to ETS). This greater lactate at a lower oxygen consumption during the passive smoking trials indicates a greater reliance on anaerobic metabolism. The combined effect of the reduced oxygen carrying capacity and increased lactate resulted in a reduction in maximal aerobic power and the duration of exercise. Thus, even in healthy subjects, exposure to ETS adversely affects exercise performance. Lamb" has suggested that at maximal exertion levels, up to 90% of the oxygen carrying capacity of the blood may be needed. Probably because of carbon monoxide, ETS reduces this capacity, so the muscle cannot maintain its high rate of aerobic metabolism unless cardiac output is further increased; people with heart disease and reduced ventricular reserve have difficulty meeting this demand. In sum, exposure to ETS increases the demands on the heart during exercise and reduces the capacity of the heart to respond. This imbalance increases the ischemic stress of exercise in patients with existing coronary artery disease and can acutely precipitate symptoms. Moskowitz et al" found evidence that adolescent children of parents who smoked may suffer from chronic tissue hypoxia such as that observed in anemia, chronic pulmonary disease, cyanotic heart disease or high altitude. These children had significantly elevated levels of 2,3-diphosphoglycerate (DPG), even after correcting for age, weight, height and sex. 2,3-DPG acts as a physiologic modulator of hemoglobin oxygen affinity. It binds to specific amino acid sites and increases the P., (lowers the oxygen affinity), thus making more oxygen available to c:1=lamzlmanu.crikuheact.doc 6

CR900213R3 peripheral tissues. This observatiion suggests that the body is attempting to compensate for hypoxia by increasing DPG level in blood to meet tissue oxygen requirements, The changes were dose-dependent; the greater the exposure to ETS (measured both in terms of parental smoking and serum thiocyanate in the children), the greater the increase in DPG. There is also evidence that acute exposure to ETS directly affects respiration of the myocardium at a cellular level. GvotdjQkovli et al" exposed rabbits in a 50 liter child's incubator to the smoke of three burning cigarettes smoked over a 30 minute period and measured several variables related to the metabolism of cardiac mitochondria. They had three groups of rabbits: one group exposed to a single dose of ETS, one group exposed to 30 min of ETS twice daily for two weeks, and one group exposed to 30 minutes of ETS twice daily for eight weeks. They measured mitochondrial respiration as the consumption of oxygen after adding ADP to a vessel containing mitochondrial fragments. Using pyruvate as a substrate, mitochondrial respiration was reduced significantly compared to control (pure air) for all doses of ETS, by even a single exposure; to about half the control value. The oxidative phosphorylation rate was also reduced significantly at all exposures by about one-third. There were no significant changes in the coefficient of oxidative phosphorylation with ETS exposure. Gvozdjikovi et al" concluded that pyruvate as a substrate was a sensitive indicator of the toxic action of the ETS on the oxidative process. Later, to further isolate where in the process of mitochondrial respiration, the ETS acted, GvozdjQk et al'0." reported data on succinate, NADH, and cytochrome oxidase activity in the mitochondria in the four groups of rabbits. Exposure to ETS affects the activity of NADH oxidase, succinate oxidase and cytochrome oxidase of myocardial mitochondria. The activity of the first two oxidases exhibited no changes compared with the control group - neither after a single exposure to ETS or following exposures up to 2 weeks. Cytochrome oxidase activity decreased both after a single exposure to ETS and over time, with increasing effects as the duration of exposure to ETS is extended. The observation that cytochrome oxidase and not NADH or succinate oxidase activity was affected by ETS suggests that the deleterious effects of ETS on myocardial mitochondrial respiration occur at the terminal segment of the mitochondrial respiration process. Prolonged exposure to carbon monoxide has been shown to induce ultrastructural changes in myocardium4z'' and may account for the adverse effects of ETS exposure on mitochondrial function. c:\gtuuz\m.nuactAeuheaR.doc 7

CR900213R3 Thus, acute exposure to ETS not only increases the demand and compromises the supply of oxygen to the heart, but also reduces the myocardium's ability to use the oxygen to create ATP to provide energy to support the heart's pumping activity. Effects on Platelets The action of ETS to increase platelet aggregation is another way in which ETS can acutely increase the risk of a coronary event. Platelets are important for the normal process of hemostasis, to prevent blood loss after an injury. When blood platelets aggregate inappropriately and form a thrombus in the coronary circulation, they can precipitate a myocardial infarction. Hemostasis depends on complex interactions among the dynamics of blood flow, components of the vessel wall, platelets and plasma proteins. Definitive evidence has confirmed that platelets play a major role in thrombus formation and embolization, especially in the arterial system. In addition, increasing evidence has shown that platelet deposition and thrombus formation can contribute to the growth and progressioa of atherosclerotic plaques". An arterial thrombus appears to develop in three phases: platelet adhesion, platelet aggregation, and activating of clotting mechanisms. Passive smoking increases platelet aggregation and so increases the likelihood of thrombus formation and myocardial infarction. Table 2sum*^9rizes the results of several studies by Davis et al"" on the effects of cigarette smoke on platelet aggregation and damage to the arterial endothelium. Davis et alsl also measured platelet aggregate ratios and eadothelial cell counts in nonsmokers before and after being exposed to 20 minutes of ETS while sitting in a hospital atrium. The platelet aggregate ratio in these studies is the ratio of the platelet count of platelet rich plasma prepared from blood mixed immediately with EDTA and formaldehyde to the same mixture without formaldehyde. This method assumes that platelet aggregates circulating in blood are fixed in the EDTA-formaldehyde solution, and break apart in the EDTA solution. Thus, a decrease in the platelet aggregate ratio reflects an increased formation of platelet aggregates. Mean values before and after passive smoking were 0.87 and 0.78 (P=.002) for platelet aggregate ratios and 2.8 and 3.7 (P=.002) for counts of anuclear endothelial cell carcasses in venous blood. These changes are intermediate between the effects observed after nonsmokers smoked two tobacco cigarettes and the effects observed after smoking two non-tobacco cigarettes'r and similar to the values observed in nonsmokers who smoked two cigarettes while trying not to inhale°. These effects were not correlated with the level of nicotine in the blood of the experimental subjects in any of these or other'°'"; related studies on how drugs modify platelet c:1=taatz\manuscri\etsheart.doc 8

CR900213R3 aggregation and endothelial cell counts. In particular, the effects observed in nonsmokers smoking without inhaiing were similar to the effects on smokers smoking two cigarettes, despite the fact that the plasma nicotine levels in the nonsmokers were a factor of 5 smaller than those observed in the smokers". Other work in the same laboratory comparing smoking with snuff use revealed similar changes in platelet function in response to these two forms of tobacco useP. This result, combined with the finding that smoking non-tobacco cigareites" failed to produce changes in platelet function as large as observed with tobacco cigarettes, suggests that nicotine is an important active agent. Since non-tobacxo cigarettes also affected platelet aggregation somewhat, however, it is possible that carbon monoxide or other combustion products are also influencing the platelets. Sinzinger and KefalidesA measured platelet sensitivity to antiaggregatory prostaglandins (E„ 12, and D2) before, during and after 15 minutes of exposure to ETS in healthy nonsmokers and smokers. Passive smoking reduced platelet sensitivity to the antiaggregatory prostaglandins IZ and E, significantly (P <.01) by a factor of about 2 by the end of 15 minutes exposure to ETS among nonsmokers. This effect persisted at 20 minutes after the end of exposure, and was gone by 40 minutes. Platelet response to prostaglandin D2 changed modestly in a similar pattern, but did not reach statistical significance. Among smokers, the control level of platelet aggregation was higher (P <.01) and the prostaglandins had no significant effects on platelet aggregation over time during or following exposure to ETS. Sinzinger and Virgolini-4 also showed that repeated exposure to ETS for one hour per day for ten days produced lasting changes in platelet function in nonsmokers similar to that observed in smokers. Thus, nonsmokers' platelets seem much more sensitive to a single exposure to ETS than do smokers' platelets, with platelet sensitivity to disaggregating prostaglandins having similar effects in nonsmokers acutely exposed to ETS as it does on the chronic levels of platelet aggregation observed chronically in smokers. Further evidence from the same laboratory that passive smoking increases platelet aggregation comes from work by Burghuber at a1", who had smokers and nonsmokers smoke two cigarettes and also exposed a different group of smokers and nonsmokers to ETS in an 18 m3 room in which 30 cigarettes had been smoked just before exposing the nonsmokers. They measured the sensitivity of platelets to the disaggregating substance prostaglandin 12 which is released by endothelium and inhibits platelet aggregation. Figure 3 shows the results of this experiment. In smokers, neither smoking nor passive smoking affected the sensitivity of the platelets to the disaggregating effect of prostaglandin 12. The sensitivity of platelets in smokers. was also significantly lower than nonsmokers. In contrast, platelets were more sensitive to prostaglandin 12 in nonsmokers, with both smoking and passive smoking e:%Slantr\muwsori%euheatt.doc 9

CR900213R3 producing similar reduction in platelet sensitivity to prostaglandin 12. These results suggest that the platelets of smokers are already desensitized to the anti-aggregatory substance prostaglandin 12, so that no further decrease in aggregation is seen. The significant decrease in platelet sensitivity to prostaglandin after acute exposure to ETS suggests that after ETS exposure platelets are more likely to aggregate, with adverse consequences. Earlier work by Saba and Mason" also indicated that nicotine increased a variety of measures of platelet aggregation in nonsmokers and smokers. While the in vitro effects of nicotine on platelets from smokers was greater than in nonsmokers, the effect generally did not vary with dose (between 2z10'9 and 2z10'4 molar), suggesting that the effects of nicotine on platelets occur at low doses and that the system saturates quickly. This observation may explain why passive and active smoking have such similar effects on plateletss'~. The probable link between nicotine and adverse physiologic effects is nicotine-induced release of catecholamines. Catecholamines are then responsible for increased platelet aggregation. This reasoning suggests that beta blockers might provide some protection in smokers. This premise is borne out by a trial comparing the effects of the beta blocker metoprolol to a thiazide diuretic in the control of moderate hypertension., For the same reduction in blood pressure, the metoprolol treated group had a significantly lower mortality rate than the thiazide treated group. Virtually all of this reduction in mortality, however, was seen in smokers, and not nonsmokers. This study provides evidence that blocking the effects of catecholamines (released by nicotine) was the cause of the reduced mortality in smokers who were receiving metoprolol. In sum, passive smoking increases platelet aggregation, with a magnitude similar to that observed in active smoking. Moreover, the response of nonsmokers to both active and passive smoking appears to be different from smokers, with nonsmokers being more sensitive to low exposures to cigarette smoke than smokers. This observation suggests that the pharmacology of ETS in nonsmokers may be different than in smokers, with nonsmokers being more sensitive to low doses of ETS. In particular, it invalidates attempts to estimate "cigarette equivalent' doses of ETS in nonsmokers or extrapolating from risks of smoking in smokers to effects of ETS on nonsmokers". The resulting increase in platelet aggregation can contribute to acute thrombus formation and myocardial infarction. In addition to the role of platelets in acute thrombus formation, platelets are also important in the development of atherosclerosis's. Once there is damage to the arterial endothelium, either through mechanical or chemical factors, platelets interact with or adhere to subeadothelial connective tissue and initiate a sequence which leads to atherosclerotic plaque. When platelets interact with or adhere to subendocardial connective tissue, they are c:\aamz4nuwsceikt.heut.doc 10

CR900213R3 stimulated to release their granule contents. Endothelial cells normally prevent platelet adherence because of the nonthrombogenic character of their surface and their capacity to form antithrombotic substances such as prostacyclin. Once the endothelial cells have been damaged, the platelets can stick to them. Once the platelets are bound to the endothelium, they release mitogens such as platelet-derived growth factor (PDGF), which encourage migration and proliferation of smooth muscle cells in the region of the endothelial injury". If platelet aggregation is increased because of exposure to ETS, the chances of platelets building up at an endothelial injury will be increased. Thus, in addition to contributing to acute effects through increasing the likelihood of thrombus formation, the effects of ETS on platelets also increase the chances that endothelial injury will lead to arterial plaque. ETS also plays a role in causing damage to the endothelium and initiating the atherosclerotic process. As discussed above, Davis et al found that acute exposure to ETSs', like active smoking'l-" and use of chewing tobacco-'2, lead to a significant increase (P <.002) in the appearance of anuclear endothelial cell carcasses in the blood of people exposed to ETS (or tobacco or tobacco smoke) constituents. The appearance of these cell carcasses indicates damage to the endothelium, which is the initiating step in the atherosclerotic process. As noted above, the appearance of endothelial cells following passive smoking is almost as great as following primary smoking ~ (Table 2). Exposure to ETS has been shown to produce injuries similar to those oliserved with exposure to primary smoke and also affects platelets in a way that increases the chances that they will bind to the injured area and promote growth of smooth muscle cells'6. The Role of the Polvcvclic Aromatic Hydrocarbons in ETS Many atherosclerotic plaques in humans are either monoclonal or possess a predominantly monoclonal component°D, which indicates that the smooth muscle cells of each plaque have a predominant cell type. Several aaimal studies have also shown that injections of polycyclic aromatic hydrocarbons (PAHs), in particular 7,12- dimethylbenz(a,h)anthracene (DMBA), benzo(a)pyrene°i-" accelerate the development of atherosclerosis. Benzo(a)pyrene is an important element in ETS'. The effects of PAHs or other carcinogenic or mutagenic elements in ETS66 relate directly to the response .to injury theory of atherogenesis discussed above. Changes in the underlying smooth muscle stimulated by these agents could then initiate the "injury" that leads to platelet aggregation and plaque formation. Thus, chronic exposure to ETS could have effects on plaque formation through mechanisms similar to that by which long term exposures produce cancer in other organs. c:\glantz\manu.crilehheart.doc 11

CR900213R3 Albert et ai6t gave chickens weekly intramuscular injections of DBMA and benzo(a)pyrene for up to 22 weeks, then killed the chickens at various times beginning after 13 weeks and measured the plaque volume in the chickens' aortas. They found that both DBMA and benzo(a)pyrene significantly increased the volume of plaque compared to control chickens who had just received injections of the solvent used to carry these agents. This study provided the first evidence that known carcinogenic chemicals could be atherogenic as well. Penn et al°' extended this result in a similar experiment by showing that the effects of DBMA on the extent of plaque buildup in chickens was dose-dependent. The median cross-sectional area of plaques on individual aortic segments and the plaque volume index (an approximate measure of the total volume of placjue per aorta) increased in a nearly linear fashion with DBMA dose. In contrast to the marked increase in plaque area in the DBMA-treated aaimals, there was only a slight increase in the percentage of aortic sections with plaques in carcinogen-treated animals than in controls. Plaques with a small cross sectional area were present in all animals. Lesions of widely differing cross sectional areas appeared to be similar histologically under the 'light microscope. Together, these data suggest strongly that a major effect of chronic DBMA exposure is to increase the size of spontaneous aortic lesions. Rather than inducing some sort of cancer-like change in an individual cell that begins the process which ultimately leads to formation of a plaque, Penn et al suggested that chronic DBMA exposure causes preferential division of individual cells or patches of cells within the preexisting spontaneous lesions. From this perspective, DBMA and other exogenous compounds would be acting as a mitogen, similar to that released by activated platelets, to stimulate division of aortic smooth muscle. Revis et al'2 found similar results in White Carneau pigeons injected with DMBA and benzo(a)pyrene weekly for 6 months, beginning when the pigeons were 3 months old. Compared with the work described above, they found a greater effect on atherogenesis of benzo(a)pyrene than DBMA, and also failed to observe a dose-response relationship between the dose given and the amount of aortic plaque. These differences from the work just described may be related to species differences, differences in the carrier used to inject the PAHs (DMSO in the previous studies vs. corn oil in this one) or differences in the age of the pigeons or dosing schedule. They also found an increase in aortic plaques in pigeons treated with the PAH 3-methylcholanthrene, but not the carcinogen 2,4,6- trichlorophenol or the PAH benzo(e)pyrene, which is not considered a carcinogen. This result suggests that carcinogenic PAHs, rather than carcinogens or PAHs in general, are implicated in the atherosclerotic process. c:\alana\musu.cribtahcart.doc 12

CR900213R3 Revis et al°2 also studied the distribution of these compounds after they had been radiolabelled. Forty-eight hours after the injection of PAHs, radioactivity in the liver, aorta and lung accounted for 7596 of the injected dose, whereas in animals injected with 2,4,6-trichlorophenol, radioactivity in the liver and kidney accounted for 8096 of the dose. In addition, 80% of the radioactivity observed in the plasma immediately following injection of radiolabelled PAHs was associated with the LDL and HDL cholesterol fractions, compared with only 24% of the 2,3,6-trichlorophenol, suggesting that plasma lipoproteins are an important vehicle for transporting PAHs to their sites of activation in the arteries. There is also evidence that ETS directly affects plasma lipoproteins. Moskowitz et al" showed that adolescent children whose parents smoked had elevated levels of cholesterol and depressed levels of HDL, even after correcting for age, weight, height and sex. These effects were dose dependent; the greater the exposure to ETS, the greater the changes in these variables. Pomrehn et al .7 observed similar effects of ETS on HDL in children whose parents smoked, even in children who smoked or crewed tobacco themselves. High cholesterol and low HDL are important for the development of plaque. Data on cholesterol and HDL from adults married to smokers is mixed`a's. To further elucidate the possible mechanisms by which PAHs induce atherosclerotic changes, Majesky et alo gave White Carneau and Show Racer pigeons a single injection of benzo(a)pyrene, then looked for metabolites of the benzo(a)pyrene in aortic and hepatic tissues 48 hours later. White Carneau pigeons develop severe atherosclerosis by the time they are 3 years old, whereas Show Racer pigeons are relatively resistant to aortic atherosclerosis. Aortic preparations of the White Carneau strain exhibited a much greater inducibility of the microsomal monooxygenase system than did those of the Show Racer strain, particularly in young pigeons. Aortic tissues from White Carneau pigeons aged 6-12 months exhibited a 3-12 fold inducibility whereas aortic tissues from the same strain at 2-5 years of age exhibited only minor (maximum of 3.3 fold) and, for the most part, statistically insignificant increases. No age differences in inducibility could be detected in the Show Racer strain. Interestingly, the differences in inducibility manifest in aortic tissues were greater in aortic tissues than in hepatic tissues from the same birds. Thus, the PAHs seem to accelerate any preexisting tendency to develop atherosclerosis. Regardless of the ultimate mechanism by which PAHs exhibit atherogenic effects, it seems logical to suppose that the reactive intermediary metabolites of these chemicals are the proximate atherogenic or co-atherogenic agents since the parent compounds are relatively inert both chemically and biologically. Thus, bioactivation and c:\flantz\rtunu*caktaheart.doc 13

CR900213R3 inactivation (and regulatory control of these processes) may be presumed to play extremely important roles in their atherogenic properties. Bioactivated chemicals vary in their stability and reactivity according to four general categories: (i) those which are extremely unstable and persist only at the immediate site (enzyme) of bioactivation, (ii) those which persist only within cells in which bioactivation occurs, (iii) those which persist primarily only within tissues in which bioactivation occurs, and (iv) those capable of being transferred in the circulation from one organ to another. For the first three of these four categories, biotransformation in the aorta per se (target tissue activation) would be of prime interest and importance. Thus, it appears that PAHs could be playing either a mutagenic or mitogenic role in beginning the atherosclerotic process in susceptible cells or individuals, depending on how the PAHs in ETS are metabolized in the aorta. The finding that enzymes that metabolize DMBA and benzo(a)pyrene are in the artery wall led Penn et ah` to search for specific molecular events in plaque cells that would lead to DNA changes similar to those previously found in tumors. Identification of such processes would be supportive of the monoclonal hypothesis of atherogenesis. They obtained human DNA samples from coronary artery plaques as well as DNA from normal sections of the coronary arteries at surgery to remove the plaque. These DNA samples were tested with the NIH 3T3 cell transection assay. Foci arose in cells transfected with each of the DNA samples obtained from the human coronary plaque, with an efficiency (number of foci per ug of DNA) ranging from 0.016 to 0.060 (mean 0.036). The transfection efficiencies for DNA's from normal coronary artery, liver, spleen, lung, kidney and trachea were all below 0.008. The transformed cells were also injected into the scalps of nude mice, where they developed tumors. These results provide direct evidence for similarities on the molecular level in the development of plaques and tumors. Human coronary artery plaque DNA contains sequences capable of transforming NIH 3T3 cells and these transformed cells can cause tumors after injection into nude mice. Control experiments verified that the transforming cells did indeed contain human DNA and that the tumorigenic (or transforming) activity was not due to the as oncogene family. Although these results clearly demonstrate that human plaque DNA has transforming ability, the temporal expression of this activity in vivo is not known. The plaques were taken from adult patients in late stages of vascular disease. Thus, we cannot determine from these samples whether the manifestation of transformation is a relatively late event in plaque development or an early but stable event. Oncogene activation and expression is an important early event in transformation and tumor genesis. These results identify specific molecular events that may underlie the proliferation of smooth muscle cells that is a hallmark of atherosclerotic c:1`lantzlmanuscriletaheut.doc 14

CR900213 R3 plaque development and demonstrates that plaque cells exhibit molecular alterations that had previously only been thought to be present in cancer-cell transformation and tumorigenesis. These results provide direct support for the monoclonal hypothesis. Randerath et e also demonstrated that constituents of cigarette "tar," including benzo(a)pyrene, are preferentially attracted to the heart and damage DNA there. They studied molecular mechanisms of smoking-related carcinogenesis by examining the induction and distribution of covalent DNA damage in internal organs of the mouse following topic application of cigarette smoke condensate daily for 1, 3, or 6 days then killed 24 hr later. DNA samples were obtained from skin, lung, heart, kidney, liver, and spleen. Adducts containing benzo(a)pyrene-derived moieties were identified, together with others. At all three times, the number of adducts in heart and lung DNA was about five times higher than that in liver and slightly higher than that in skin. Covalent DNA damage was estimated to be 6.2, 5.7, 3.9, and 1.9 times higher, respectively, in lung, heart, skin and kidney than in liver, ranging from approximately 1 adduct in 5.4x106 DNA nucleotides in lung to 1' adduct in 3.3x10' DNA nucleotides in liver. Spleen DNA was virtually adduct free. While the DNA adduct profiles resembled each other qualitatively among the different tissues, there were major quantitative differences between the different tissues, with the highest DNA binding occurring in the lung and heart. The reasons for the high incidence of DNA adducts in the heart are not known, but may be related to the role of plasma lipids in transporting PAHs such as benzo(a)pyrene and binding of these lipids to coronaries arteries. In sum, there is a growing body of evidence at a molecular level supporting the nonoclonal hypothesis of atherogenesis, with compounds in tobacco smoke and ETS strongly implicated as agents which stimulate the development of coronary lesions. Regardless of whether the monoclonal hypothesis proves to be true (or, more likely, one of several initiators of the atherosclerotic process), the fact is that there is clear evidence that components of ETS, in particular PAHs such as benzo(a)pyrene, initiate or accelerate the development of plaque. These biochemical findings are consistent with the epidemiological finding that chimney sweeps, which are exposed to high levels of PAHs in soot, have an increased risk of heart disease (as well as cancer) and tend to develop these diseases younger than other, comparable, occupations which avoid exposure to PAHe. The PAHs in ETS are clearly implicated at epidemiological, physiological and biochemical levels in the genesis of heart disease. cASlauiCtlmanu.cektuheart.doc 15

CR900213R3 The evidence that ETS increases risk of death from heart disease is similar to that which existed in 1986 when the Surgeon General concluded that ETS caused lung cancer in healthy nonsmokers`. There are eleven epidemiological studies, done in a variety of locations, which reflect about a 30% increase in risk of death from ischemic heart disease or myocardial infarction among nonsmokers living with smokers. The larger studies also demonstrate a statistically significant dose-response effect, with larger exposure to ETS being associated with greater risks of death from heart disease. These epidemiological studies are complemented by a variety of physiological and biochemical data which show that ETS adversely affects platelet function and damages arterial endothelium in a way that increases the risk of heart disease. Moreover, ETS, in realistic exposures, also exerts significant adverse effects on exercise capability of both healthy people and those with heart disease by reducing the body's ability to deliver and utilize oxygen. In animal experiments, ETS also depresses cellular respiration at the level of mitochondria. The polycyclic aromatic hydrocarbons in ETS also accelerate, and may initiate, the development of atherosclerotic plaque. It is also important to note that the cardiovascular effects of ETS appear to be different in nonsmokers and smokers. Nonsmokers appear to be more sensitive to ETS than smokers, perhaps because some of the affected systems are sensitive to low doses of the compounds in ETS, then saturate and also perhaps because of physiological adaptions smokers undergo as a result of chronic exposure to the toxins in cigarette smoke. In any event, these findings indicate that, in terms of cardiovascular disease, it is incorrect to compute "cigarette equivalents' for passive exposure to ETS, then try to extrapolate the effects of this exposure on nonsmokers from the effects of direct smoking on smokers. These results combine to suggest that heart disease is an important consequence of exposure to ETS. The combination of epidemiological studies with demonstration of physiological changes with exposure to ETS, together with biochemical evidence that elements of ETS have significant adverse effects on the cardiovascular system, lead to the conclusion that ETS causes heart disease. This increase in risk translates into about 10 times as many deaths from ETS-induced heart disease as lung cancer; these deaths contribute greatly to the estimated 53,000 deaths annually from passive smokings. This toll makes passive smoking the third leading preventable cause of death in the United States today, behind active smoking70 and alcohol't c:\Saana\manuacrl\etaheat.doe 16

CR900213R3 AclrnowledQements We thank James Stoughton for assistance in library work, A. Judson Wells, Donald Shopland, James Repace, Neil Benowitz, Takeshi Hirayama and the Tobacco Institute for their comments on drafts of the manuscript, Voij tech Licho and Art Sussnman for translating foreign language articles, and Jerry Simnitt for typing.

CR900213R3 References 1. USPHS: The Health Consequences of Involuntary Smoking: A report of the SurQeon General. 1986; DHS (CDC) 87-8398. 2. Doll R, Hill AB: A study of the aetiology of carcinoma of the lung. Br Med J 1952; 2:1271-1286. 3. Hirayama T: Nonsmoking wives of heavy smokers have a higher risk of lung cancer: a study from Japan. Br Med J 1981; 282 (6273):183-185. 4. USPHS: The Health Conseauences of Smoking: Cardiovascular Disease: A Mort of the Surgeon General. 1983; DHHS(PHS) 84-50204. 5. Wells A: An estimate of adult mortality in the United States from passive smoking. Environ. Int. 1988; 14:249-265. 6. Kristensen T: Cardiovascular diseases and the work environment: A'critical review of the epidemiologic literature on chemical factors. Scand J Work Environ Health 1989; 15:245-264. 7. National Research Council: Environmental Tobacco Smoke: Measuring Exposure and Assessing Health Effects. Washington DC: National Academy Press, 1986. 8. Gillis C, Hole D, Hawthorne V, Boyle P: The effect of environmental tobacco smoke in two urban communities in the west of Scotland. Eur. J. Resp. Dis. 1984; 65 (suppl 133):121-126. 9. Lee P, Chamberlain J, Alderson M: Relationship of passive smoking to risk of lung cancer and other smoking-associated diseases. Br. J. Cancer 1986; 54:97-105. 10. Svendsen K, Kuller L, Martin M, Ockene J: Effects of Passive Smoking in the Multiple Risk Factor Intervention Trial. Atn. J. Eoidemiol. 1987; 126:783-795. 11. Helsing K, Sandler D, Comstock G, Chee E: Heart disease mortality in nonsmokers living with smokers. Am. J. Enidemiol. 1988; 127:915-922. 12. Hirayama T: Lung cancer in Japan: Effects of nutrition and passive smoking. In Lung Cancer: Causes and ven 'o M. Mizell and P Correa ads, New York, Verlag Chemie International, 1984, pp 175-195. 13. Garland C, Barrett-Connor E, Suarez L, Criqui M, Wingard D: Effects of passive smoking on ischemic heart disease mortality of nonsmokers. Am J. Enidemiol. 1985; 121:645-650. c:1=1analmanuscriktaheut.doc 18

CR900213R3 14. Martin M, Hunt S, Williams R: Increased incidence of heart attacks in nonsmoking women married to smokers. Paper presented at annual meeting of American Public Health Association, October, 1986. 15. He Y: Women's passive smoking and coronary heart disease. Chun4-Hua-Yu-Fang-I-Hsueh-Tsa-Chin 1989; 23:19-22. 16. Humble C, Croft J, Gerber A, Casper M, Hames C, Tyroler H: Passive smoking and twenty year cardiovascular disease mortality among nonsmoking wives in Evans County, Georgia. Arn. J. Pub. Health 1990; (in press). 17. Hole D, Gillis C, Chopra C, Hawthorne V: Passive smoking and cardiorespiratory health in a general population in the west of Scotland. Br. Med. J. 1989; 299: 423-427. 18. Butler T: The relationship of passive smoking to various health outcomes among Seventh-Day Adventists in California. 1990; Seventh World Conference on Tobacco and Health, 316 (abstract). 19. Palmer J, Rosenberg L, Shapiro S: Passive smoking and myocardial' infarction. CVD Eflid. Newsletter, 1988; 43:29 (abstract). 20. Dobson A, Heller R, Alexander H, LIoyd D: Passive smoking and the risk of heart attack. 1990; Seventh World Conference on Tobacco and Health, 102 (abstract). 21. Lee P: Misclassification of smoking habits and passive smoking. A review of the evidence. International Archives of Occupational and Environmental Health. Berlin. Springer-Verlag. 1988. 22. Lee P: Deaths from lung cancer and ischaemic heart disease due to passive smoking in New Zealand (letter). N. Zeal. Med. J. 1989; 102:448. 23. Lee P: An estimate of adult mortality in the United States from passive smoking: A response (letter). Environ. Int. 1990; 16:179-181. 24. Wald N: Does breathing other people's tobacco smoke cause lung cancer? Br. Med. J. 1986; 293:1217- 1222. 25. Wells A: Misclassification as a factor in passive smoking risk. Lancet 1986; ii:638. 26. Wells A: An estimate of adult mortality in the United States from passive smoking: A response to criticism. Environ. Int. 1990; 16:187-193. 27. Kawachi I, Pearce N: Passive smoking in New Zealand (letter). N. ?.eal. Med. J. 1989; 102:479. 28. Reinken J(1989) Passive smoking in New Zealand (letter). N. Zeal. Med. J. 1989; 102:515. c:\Xlantz\manuacri\ebheart.doc 19

CR900213R3 29. Friedman J, Chalmers T, Smith H Jr, Keubler R: The importance of beta, the Type II error, and sample size in the design and interpretation of the randomized controlled trial: Survey of 71 "negative" trials. ~i Engl. J. Med. 1978; 299:690-694. 30. Muhm J, Olshan A: A program to calculate sample size, power, and least detectable relative risk using a programmable calculator. Am. J. Enidemiol. 1989; 129:205-11. 31. Repace J, Lowrey A: A quantitative estimate of nonsmokers' lung cancer risk from passive smoking. Environ. Int. 1985; 11:3-22. 32. Repace J, Lowrey A: Predicting the lung cancer risk of domestic passive smoking. Am. Rev. Resp. Dis. 1987; 136:1308. 33. Kawachi 1, Pearce N, Jackson R: Deaths from lung cancer and ischaemic heart disease due to passive smoking in New Zealand. N. Zeal. Med. J. 1989; 102:337-340. 34. Khalfen E, Klochkov V: Effect of passive smoking on physical tolerance of ischemic heart disease patients. Ter. Arkh. 1987; 59:112-115. 35. Aronow W: Effect of passive smoking on angina pectoris. N. Engl. J. Med. 1978; 299:21-24. 36. McMurray R, Hicks L, Thompson D: The effects of passive inhalation of cigarette smoke on exercise performance. Eur. J. Appl. Phvsiol. 1985; 54:196-200. 37. Lamb D: Physiology of exercise: Resoonses and adaptation. New York. MacMillan Publishing Company. 1984. 38. Moskowitz W, Mosteller M, Schieken R, Bossano R, Hewitt J, Bodurtha J, Segrest J: Lipoprotein and oxygen transport alterations in passive smoking preadolescent children: The MCV twin study. Circulation 1990; 81:586-592. 39. Gvozdjs{kovi A, Bada V. Siny L, Kucharskst J, Krutjr F, Bolek, Tr§tanksf L, Gvozdjik J: Smoke cardiomyopathy: Disturbance of oxidative process in myocardial mitochondria. Cardiovasc. Res. 1984; 18:229-232. 40. Gvozdjikovi; A, Kucharski J, SQny L, Bada V, Bolek, Gvozdjdk J: Effect of smoking on the cytochrome and oxidase system of the myocardium. Bratisl- lek. Listy. 1985; 83:10-15. 41. Gvozdjik J, Gvozdj[kovi A, Kucharski, Bada V: The effect of smoking on myocardial metabolism. Czech. Mgd,, 1987; 10:47-53. cA&mz\muwscri\otaheart.doc 20

CR900213R3 42. Kjeldsen K, Thomsen H, Astrup P: Effects of carbon monoxide on myocardium: Ultrastructural changes in rabbits after moderate, chronic exposure. Circ. Res. 1974; 34:399-348. 43. Thomsen H, Kjeldsen K: Threshold limit for carbon monoxide-induced myocardial damage. Arch. Environ. Hwlth 1974; 29:73-78. 44. Lough J: Cardiomyopathy produced by cigarette smoke. Arch. Pathol. Lab. Med. 1978; 102:377-380. 45. Fuster V, Chesebro J: Antithrombotic therapy: Role of platelet-inhibitor drugs: 1. Current Concepts of Thrombogenesis: Role of Platelets. Mayo Clin. Proc. 1981; 56:102-112. 46. Ross R: The pathology of atherosclerosis - An Update. N. E20. J. Med. 1986; 314:488-500. 47. Davis J, Shelton L, Eigenberg D, Hignite C, Watanabe I: Effects of tobacco and non-tobacco cigarette smoking on endothelium and platelets. Clin. Pharmacol. Ther. 1985; 37:529-533. 48. Davis J, Hartman C, Lewis H Jr, Shelton L, Eigenberg D, Hassanein K, Hignite C, Ruttinger H: Cigarette smoking-induced enhancement of platelet function: lack of prevention by aspirin in men with coronary artery disease. j. Lab. Clin. Med. 1985; 105:479-483. 49. Davis J, Shelton L, Eigenberg D, Hignite C: Lack of effect of aspirin on cigarette smoke-induced increase in circulating endothelial cells. Haemostasis 1987; 7:66-69. 50. Davis J, Shelton L, Hartman C, Eigenberg D, Ruttinger H: Smoking-induced changes in endothelium and platelets are not affected by hydroxyethylrutosides. Br. J. Exp. Path. 1986; 67:765-771. 51. Davis J, Shelton L, Watanabe 1, Arnold J: Passive smoking affects endothelium and platelets. Arch. Intern. Med. 1989; 149: 386-389. 52. Davis J, Shelton L, Zucker M: A comparison of some acute effects of smoking and smokeless tobacco on platelets and endothelium. 1990; (submitted) 53. Sinzinger H, Kefalides A: Passive smoking severely decreases platelet sensitivity to antiaggreatory prostaglandins. jj= 1982; 2(8294):392-393. 54. Sinzinger H, Virgolini I: Are passive smokers at greater risk of thrombosis? Wiener klinische Wochenschrift 1989; 20: 694-698. 55. Burghuber 0, Punzengruber C, Sinzinger H, Haber P, Silberbauer: Platelet sensitivity to prostacyclin in smokers and non-smokers. Chest 1986; 90: 34-38. c:\=Iantz\manu.cri\euheait.doc 21

CR900213R3 56. Wikstrand J, Warnold 1, Olsson G, , Tvomilehto J, Elmfeldt D, Berglund G: Primary prevention with metoprolol in patients with hypertension: Mortality results from the MAPHY trial. JAMA 1988; 259:1976- 1982. 57. Saba S, Mason R: Some effects of nicotine on platelets. Throm. Res. 1975; 7:819-824. 58. Wu J, Ecobichon D: Environmental tobacco smoke: nroceedings of the international svmnosium ffunded bv the tobacco industrvl at McGill University. 1989. Toronto: Lexington Books, 1989, p. 370. 59. Fox P, DiCorleto P: Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells. J. Cell. Physiol. 1984; 121:298-208. 60. Benditt E, Benditt J: Evidence for a monoclonal origin of human atherosclerotic plaques. Proc. Nat. Acad. &L 1973; 70:1753-1756. 61. Albert R, Vanderlaan F, Nishizumi M: Effect of carcinogens on chicken atherosclerosis. Cancer Res. 1977; 37:2232-2235. 62. Revis N, Bull R, Laurie D, Schiller C: The effectiveness of chemical carcinogens to induce atherosclerosis in the white carneau pigeon. ox'col 1984; 32:215-227. 63. Penn A, Batastini G, Soloman J, Burns F, Albert R: Dose-dependent size increases of aortic lesions following chronic exposure to 7,12-Dimethylbenz(a)anthracene. Cancer Res. 1981; 41:588-592. 64. Penn A, Garte S, Warren L, Nesta D, Mindich B: Transforming gene is human atherosclerotic plaque DNA. Poc. Nat. Acad. Sci. 1986; 83:7951-7955. 65. Majesky M, Yang H, Benditt E: Carcinogenesis and atherogenesis: Differences in monoxygenase inducibility and bioactivation of benzo[a]pyrene in aortic and hepatic tissues of atherosclerosis-susceptible versus resistant pigeons. CarcinoQenesis 1983; 4:647-652. 66. Remmer H: Passively inhaled tobacco smoke: A challenge to toxicology and preventive medicine. Arch. Toxicol. 1987; 61:89-104. 67. Pomerehn P, Hollarbush J, Clarke W, Lauer R: Childrens' HDL-chol: The effects of tobacco; Smoking, smokeless and parental smoking. Circulation 1990; 81:720 (abstract). 68. Randerath E, Mittal D, Randerath K: Tissue distribution of covalent DNA damage in mice treated dermally with cigarette 'tar': preference for lung and heart DNA. Carcinogenesis 1988; 9:75-80. c:\=lantz\manuacnlauheart.doc 22

CR900213R3 69. Hansen E: Mortality from cancer and ischemic heart disease in Danish chimney sweeps: A five-year follow-up. Am. J. Epidemiol. 1983; 117:160-164. 70. USPHS: I3educinQ the health conseauences of smoking: 25 vears o grqg ,ss. A report of the Surgeon General. 1989; DHHS(CDC) 89-8411. 71. NIAAA: Sixth ispport to the U.S. ConQress on alcohol and health from the Secretary of Health and Human Services. U.S. Dept. of Health and Human Services, Public Health Service, Alcohol, Drug Abuse, and Mental Health Administration, National Institute on Alcohol Abuse and Alcoholism, 1987; DHHS(ADM) 87-1519. o:\;l.ntz\,n.nusctilsdheut.doo 23

CR900213R3 Table 1 Epidemiological Studies of Environmental Tobacco Smoke and Coronary Heart Disease Death Author Type' Location Deaths Relative 95% Dose Power` Controlling for: or Risk Confidence Response?b Cases Intervat MENEM Males Gillis at ats P Scotland 32 1.3 0.7 - 2.6 - 4% age (1984) Lee et al9 C United 41 1.2 0.5 - 2.6 - 10% age, marital status (1986). Kingdom Svendsen et P United States 13 2.1 0.7 - 6.5 Yes 18% age, blood pressure, at10 (1987)° serum cholesterol, weight, education, alcohol Helsing et P Maryland 370 1.3 1.1 - 1.6 No 27% age, marital status, al~~ (1988) housing, education Ferles Hirayama12 P Japan 494 1.2 0.9 - 1.4 Yes 32% age, diet (1984) Gillis et ata P Scotland 21 3.6 0.9 -13.8 - 15% age (1984) Garland et P California 19 2.7 0.9 -13.6 - 23% age, blood pressure, att3 (1985) plasma cholesterol, weight, years of marriage Lee et als C United 77 0.9 0.7 - 1.3 - 3% age, marital status (1986) Kingdom Martin et a114 C Utah 23 2.6 1.2 - 5.7 - 44% age, family history of (1986) CHD, hypertension, diabetes, weight, alcohol exercise Helsing at P Maryland 988 1.2 1.1 - 1.4 Yes 98% age, housing, marital at" (1988) status, education He (1989) is C China 34 1.5 1.2 - 1.8 Yes 74% age, race, residence, occupation, hypertension, family history of hypertension or CHD, alcohol, exercise, h rli idemia Humble et atls P Geor9ia 76 1.6 1.0 - 2.6 Yes 30% age, serum cholesterol, (1990) blood pressure, weight Butler~i P California 64 1.4 0.5 - 3.8 - 4% age (1990) ioth sexes ca.bined Hote et al17 P Scotland 84 2.0 1.2 - 3.4 - 87% age, sex, social class, C1989)' blood pressure, cholesterol, weight 'P = Prospective cohort, C= Case control bHo entry in this column indicates no coanknt on the presence or absence of dose-response relationship `Power to detect relative risk of 1.2 with 95% confidence dHigh risk population; members of MRFIT trial 'This report is a tater follow-up of the population reported in Gittis et als 50778 2005 c:lfiantzlmanuacrileu heart.doc 24

CR900213R3 Table 2 Effect of Passive and Active Smoking on Platelet Aggregation and Endothelial Cell Damage Platelet A re ate Ratio Endothelial Cell Count n Before After Change Before After Change Passive Smoking .87 .78 -.09 2.8 3.7 0.9 10 (nonsmoker) Tobaccoa(nonsmoker) .80 .65 -.15 2.3 4.8 2.5 20 vs. Non-tobacco .81 .78 -.03 2.5 3.0 0.5 cigarette (nonsmoker) Inhale cigarette .81 .68 -.13 4.0 5.4 1.4 24 (smoker) vs. Not inhale cigarette .82 .73 -.09 3.3 4.7 1.4 22 (nonsmoker) Smoke (smoker) vs. .85 .70 -.15 4.4 6.4 2.0 17 Snuff (smoker) .82 .76 -.06 J 1 3.9 4.7 0.8 Notes: All studies are paired and reflect significant differences (P<.005). Platelet aggregate ratio is the ratio of platelet count of platelet-rich plasaw, prepared immediately after venipuncture with a sotution containing edetic acid and formaldehyde, to that of pLateLet-rich plasma prepared in the same manner, except for the absence of formaldehyde. A decrease in the platelet aggregate ratio reflects an increased formation of platelet aggregates. Endoth ~ti a ~~ ell count is mean number of anuctear cell ~ ~ carcasses in 0.9 µl chambers. Source: Davis et al c:\flantzlmanuscriletsheait.doc 25

CR900213R3 FIGURE LEGENDS Figure 1: Relative risk in epidemiological studies of the risk of death from coronary heart disease or myocardial infarction among nonsmokers living with smokers compared with nonsmokers living with nonsmokers. Lines indicate 95 96 confidence intervals. (Note that two studies have upper bounds to the 95 96 confidence interval off the scale of the graph.) Figure 2: Heart disease accounts for the majority of annual deaths attributed to environmental tobacco smoke. Source: Wells26. Figure 3: Effect of active (left) and passive (right) smoking on platelet aggregation in smokers and nonsmokers. The sensitivity index. SIpGu, is defined as the inverse of the concentration of *prostaglandin I; necessary to inhibit ADP-induced platelet aggregation by 50%. Lower values of SI,,u indicate greater platelet aggregation. Source: Burghuber et al-" Figures 3 and 4. c:\glantz\rtunu.crikuheart.doc 26

Relative Risk N) W .A UT d) --1 i ~ Gillis (1984) I Lee (1988) . Svendsen (1987) J 1 Aft I -}- Helsing (1988) T~ Hirayama (1984) I .: Gillis (1984) ~ Garland (1985) I I ' -{-!- Lee (1988) ~-- Martin (1986) -}- Helsing (1988) -}~- He (1989) Butler (1990) -}- Humble (1990) Hole (1989)

. s N ~ ~ ~D

, . PGh I PcAI 0.5 1PcA3 SMOKEft 0 8EF0RE AFTER 0 BEFORE Pc.01 AFTER