Tobacco Documents Online

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.