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I Clinical Progress Series Passive Smoking and Heart Disease Epidemiology, Physiology, and Biochemistry Stanton A. Glantz, PhD„and William W. Parmley, MD he first disease linked definitively to active smoking was lung cancer. lt~ is, therefore, not surprising that the firsn disease identified as causcd by passive smoking was also lung cancer.t Before the advent of mass-marketed cigarettes, lung cancer was a rare disease. Because smoking is the primary cause of lung cancer, identification of this link-for both active2 and passive smoking'-was relatively straightforward. This situation contrasts with heart disease, which has many risk factors„and unsurprisingly, the scientific community was longer in concluding that active smoking caused heart disease! Once the link between smoking and heart disease was established, smoking was found to kill more people by causing or aggravating heart disease than lung cancer. In fact, smoking is the most important, preventable cause of coronary disease. Exposure to environmental tobacco smoke (ETS) has now been linkedito heart disease in nonsmokers.'M, Much of the evidence for this link has appeared since 1986, when the US Surgeon Generalt and the National Academy of Sciencesl reviewed the evi- dence on the health effects of ETS. Based on the information available then, both report6 concluded that the evidence linking ETS and heart disease was equivocall and that more research was necessary, before any definitive statements coul& be made. These conclusions were reasonable in 1986. How- ever, in the 4 years since publication of these reports, considerable information on both the epidemiologyand biological mechanisms by which, ETS causes heart disease has accumulated: Most of the results presented here were published after the 1986 Sur- geon General and National Academy of Sciences reports. There are now 10 epidemiological studies on the relation between exposure to environmental tobacco From the Divisitm of Ca-diobgy Depanment of Medicine, CardilWVascular Rcaearch Institutc. University of California, San Francisco. This manuscript is based'on a bachground'paper prepared for the US Environmcntal Protenion,Agenry. It was also presented at the Seventh Worltl Conferencc on Tobaceo and Health, Perth, Auctralia; April 1-5. 171011, and the Wurld Conference on Lung ttcaltft, &xton• May, 20-24. l990'~ Funded in part with it gift from Pyramid Film and Video. Address for conespondencc: Stanton A. Glantz. PhD. Professor of Medicine. Division of Cardiology; Box 0124 M1186; Universiry uf California: San Franciscn, CA 94 1 43-01 24. smoke in the home and the risk of heart disease death in the nonsmoking spouse of a smoker and five epidemiological studies that examine nonfatal car- diac events. All but one of these studies yielded relative risks or odds ratios greater than 1.0: There are several lines of biological evidence that make this association piausiblc. There is evidence that expo- sure to ETS reduces exercise tolerance of healthy individuals and people with existing coronary artcry disease. Such reduced exercise capability is one of the landmarks of acute compromises to the coronary, circulation. There is good evidence, from both hu. man and animali studies, that exposure to tobacco smoke, including passive smoking, increases aggrega- tion of blood platelets. Such increases in platelet aggregation are an important step in the genesis of atherosclerosis. In addition, increasing platelet ag- gregation contributes to risk of coronary thrombosis, a cause of acute myocardial, infarction. Last,,carcino- genic agents in ETS, including benzo(a)pyrene, have been shown to injure the endothelial cells that, line arteries. Such injpries are the first step in the devel, opment of atherosclerosis. Thus, exposure to ETS can contribute to short- and, long-term insults to the coronary circulation and the heart. It is not surpris- ing, therefore, that epidemiological studies have identified an increase in the risk of coronary artery disease in nonsmokers living with smokcrs. Effects of Primary Smoking Before reviewing the evidence linking ETS with eoronary , artery disease, summarizing the evidence that links active smoking with coronary artery disease is worthwhile. This evidence was summarized in the 1983 Surgeon General's R'eport,4 which was devoted entirely, to eardiovascular disease; it concluded that cigarette smoking is one of the three major indepen- dent 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 ciga• rette smoking is present in,a larger percentage of the US population than either hypertension or hypercho- lesterolemia, cigarette smoking ranks as the largest preventable cause of heart disease in the United States. Since 1983, an increasing body of evidence has shown that the polycyclic aromatic hydrocarbons !

2 Circulation Vd 83, No 1. January 1991 TAatt 1. Epidemiobgkal Studies ot Farir...eotal' Tob.cco Smokc and'Genoaary Heart Disea.e Dntb Author Males Gillis et al" (1984) lse eV at' (1986) Svendsen e1 all" (I 9g7)# Helsing et all, (1988) Poofedi Females Hirayamau (1984) Gi1Vis et al" (1984)' Garland et alts ();9g5) Lee et at (1986) ~ Htlsingtt all I (1988) He (1989)" Humble et aP! (1990): ButlerN (1990) Pooled Both sexes combined Hole a al" (1989) d Pooled9 Deaths 95% ' Type lroeation or cases (n) Relative Confidence Dose' Powcrt risk interval response?, (5r). Controlling for P Scotland 32 13 0.7-2.6 - 5 Age C United tGngdom 41 1.2' 05-26 - 4 Age, marital status P United States 13 2:1 0.7-6.5 Yes 3 Age, blood pressure, P arrlsnd 70 3 .1-1.6 o 0, serum cholesterol, .veight„education4 alcohol Age, marital status, 1.3 1J-1.6 trousing, education P Japan 494 1.2 0.9-1.1 Yes 40 Age, diet P Sootland 21 3.6 0.9-13.8 - 2 Age P Califorttia 119 27 0.9-13.6 - 2 Age, btood pressure, C nited Kingdom 7 .9 5-1.6 - 6 plasma choluterol, weight, years of marriage Age, marital status P Maryland 988 1.2 1.1-1.4 Yes 2 .,ge, housing, marital' C China 34 15 13-1.g Yes 3 status, education Age. race, residence, P eorgia 6 !6 .0L26 es 8 occupation, hypertension, f'amily history of hypertension or CHD, alcohol, exertise, hyperlipidemia Age, serum cholesterol, P California 64 1.4 0.5-3.8 - 4 blood pressure, weight Age 1,.3 1.2-1.4 P Seotdand 84 2.0 1.2=3.41 - ]0 Age, aex, social class, I L3 1.2-1.4 blood pressure, eholestero4, weight P. Prospective cohort; C, Case control; CHD, coronary heart disease. 'Notmry in this column indicates no comment on the presence or absence of dose-esponse relation. tPower to detect relative risk of 1.2 with 95% confidence. tHigh-risk population; members of Multiple Risk Factor Intervention Trial. ;Poo6ed relative risk computed as R=exp (I w, In, RJfw,), where w,-(Xlln R;)r. I This repon is a laterfollow-up of the population reported in Gillis et al." UtII studies combined without regard for sez, with Gillis et a!' excluded because Hole et allr report later follow-up on the same people. in cigarette smoke can injure the arterial endothe- hum and' iniaiate the atherosclerotic process. All the compounds from cigarette smoke that have been implicate& as damaging to the cardiovascular system of active smokers have been identified in bTS.t•' Epidemioiogical 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 10 published stud- ies"-t7 that use dcathas an end point are summarized in Table I and Figure 1; four studies present data on men, eight on women, and one on both sexes com- bined. 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 remarkably consistent! All the studies on menyielded relative risks of death from heart disease exceeding 1.0 when a nonsmoking man was married to a woman who smoked, with an overall risk of 1.3. All but one of the studies on women" 'yielde&relativc risks exceeding 1, with an overall'relative risk of 13. Five studiestu•t7-19-w have also suggested an increase in the risk of nonfatal coronary symptoms, incfudingg angina and myocardial infarction, Consistency of an observation across different studies increases the eonfidence that a particular association is causal. Several investigative teams also observed' a dose- response relation between increasing amounts of

I 7 6 . Q 5 4 2 1 0 m rn J FwmN f I m a 7----~- ~ R © ww smoking by the spouse and the risk of heart disease in the . nonsmoking spouse, "-'s•" which in most cases was statistically significant. The presence of such dose-responsc effects across multiple studies,, eon- ducted in different locations with different criteria, supports the hypothesis that ETS causes heart dis- ease in nonsmokers. While all but one of the studies in Table I and Figure 1 yielded' relative risks greater than 1.0, the fact remains that~ three of the studies in men and five of the studies in women had 95% confidence inter- vals 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<0.05). Of note, the 95% confidence intervals do not lie symmetrically about 1.0 but are skewed toward higher risks. By examining the eonfidence intervals, the conclusion is reached that exposure to ETS elevates the risk of heart disease (Figure 1). Also, the results of these studies may, be combined in a formal analysis to derive a global estimate of the relative risk and associated 95% confidence Interval. By combining the studies, the sample size and, there- fore, the power to detect an effect increases. Wellss used then-availablc studics"•9•13-t3•" to compute a pooled relative risk of 13 (95% confidence interval, 1.1-1.6) for men and 1.2 (95% confidence interval, 1.2-1.4) for women. Our analysis on all the studies in Table I yields a combined relative risk of 13 (95% confidence intervall 1.2-1.4). When interpreting the results of such epidemiolog- ical studies, it is always important to consider biolog- ical plausibility and potential confounding variables that can explain the results. Aside from noting that the hydrocarbons in mainstream smoke already, im- plicated in heart disease are also 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 concen- trate on potentiat confounding variables, which are particularly important in a disease like heart disease i Glana and Parmley, Passire Smoking and Heart Disease 3 . . , Both r..a FIGURE 1. Graph of relative rssk in epi- demioJogical studies of the risk of death from coronary hean disease or myocardial infarction among' nocsmokers living with smokers compared with nonsmokers living with nonsmokers. Lines indicate 95%a can- fidenee intervalr. Note that two studies have upper bounds to the 95% confidence ituerval ofJthe scale of the graph. because it is known to be caused by multiple risk factors. All the studies controlled' for the most important confounding variable, age, and several'u•1.1-1y17 eon• trolled for known risk factors for coronary aneryy disease, ut patticular 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 housing or education. Ind'eed; studies that estimated the relative risk both with and without taking these confounding variables into account found an increase in risk associated'with ETS after taking the confounding variables into atxount.1u.u Lee21-u suggested that the elevated risk of hean. (and other) disease with passive smoking may be due to misclassification of nonsmokers who are really smokers. In ad'dition, Waldz• 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 tends to lead to overestimating the risks associated with ETS an& the latter leads to underestimating the risk. Careful analysis of the question of misclassification, which applies generally to studies of ETS, has demonstrated that the ob- served risk cannot be explained by this problem s-36-2x The possibility always exists that some other'eon- founding variable relates to cultural factors, such as the nature of housing or employment or the nature of time spent outside the home. Also, it is possible that there are other confounders, such as a correlation of spouses' poor health behaviors (e.g., diet), which are not controlled for in analysis. The fact that results art from all over the world in widely varying cultural settings-including several regions in the United States, the United Kingdom, lapan+, and China- argues against this concern. One can assess fortnally the confidence in reaching a negative conclusion by computing the power of the study to detect an effect of specified size.2" Table l

0 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 Ol'shan,-'41 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 power. This low power of the individual studies argues against drawing an overall negative conclusion concerning the link between ETS expo- surc and risk of death from heart disease, based on the individual studies taken one at a time. Last, and of note, all these studies are based on the smoking habits of: the nonsmoker's spouse and, therefore, the 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.31-« As a result, these studies generally underestimate the risk and~atten• dant public health burden due to ETS-induced heart disease. Kawachi:et al" adjusted Wells'S relative risks to account for workplace exposures to ETS and found that the relative risks increase to 2.3 (95% CI, 1.4-3.4) for men and 1.9 (95% Cl, 1.4-2.5) for women. Thus, any potential confounding of the re- sults because of exposure to ETS outside the home will! tend to produce underestimates rather than overestimates of the effect of ETS. Likewise„ esti+ mates of public health impact base& om risks comr puted from household exposuress will be lower than the true public health impact. In addition, Wellss and Kawachi ct al" indicate that the number of heart disease deaths due to passive smoking i's an order of magnitude greater than the 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 Is3 for both diseas- es); the attributable deaths for heart disease is greater because heart disease is much more common than, lung cancer. Of 53,000 annual deaths in the United States attributed to passive smoking,s 37,000 arc attributed to heart disease compared with 3,700 for lung cancer (Figure 2). These epidemiological studies demonstrate a con- nection between ETS exposure and death from heart disease. We now turn our attention to possible physiological and biochemical mechanisms that ex- plain these observations. Short-term Effects of ETS Exposure Long-term exposure to ETS exerts carcinogenic effcets by increasing the cumulative risk that a carci- nogcnic molecule from f'TS will damage a cell and then initiate or promott the carcinogenic process. The situation with heart disease is different. In heart disease, important long-term changes (i.e., the devel= opment of atherosclerotic lesions) and shon-term changes occur. The latter include an increased myo- Deaths from Passive Smoking Total Deaths: 53,000 t+...t tDi..as. $7000 o+n« c.rlo.. 12000 w.V c.no« 2700 FtGUAE 2. Pic charr of US dearhs from environmenml tobacco smoke. The majority ojannual deaths arr atrribused !o hcan direase. Modified from Wtlis.'" cardial oxygen demand that may outstrip the oxygen supply and produce ischemia and an increased plate- let aggregation that may lead to coronary thrombosis and acute myocardial, infarction: When the coronary circulation eannot, provide enough oxygen to the myocardium to meet the de- mand, the result is ischemia„which can, be a silent or an anginal episode. Earlier onset of angina or hypo- tension during exercise is a reflection of more severe heart disease. Oxygen supply can be reduced by atherosclerotic narrowing or, vasoconstriction of the coronary aneries or by reducing the oxygen-carrying capacity of the blood because the carbon monoxide in the ETS forms carboxyhemoglobin, which, in turn, reduces the blood's oxygen-carrying capacity. Khal- fen and Klochkov*A confirmed earlier work by Flronowu demonstrating that exposure to ETS sig- nificantly reduced both the exercise ability in patients with coronary artery disease and the rate-pressure product (heart rate multiplied by systolic blood pres- sure). In both studies, patients were exposed to realistic levels of ETS by sitting in, a waiting room while someone was smoking. These effects were present in smokers and nonsmokers" and regardless of whether the room was ventilated! 3'-35 Exposure to ETS also increased resting heart rate and systolic and diastolic blood pressure and resulted in a lower, heart rate at the onset of angina." Blood carboxyhemoglo- bin was increased by about 1% after exposure to ETS:ys Thus, short-term exposure to ETS leads to an imbalance between myocardial oxygem supply and demand during exercise in patients with coronary artery, ddisease. While this discussion has concen- trated on the carbon monoxide in ETS as the active agent, some other component of the ETS may be causing,or contributing to this effec[. The effects of ETS on cardiac performance art, in fact, severe enough to affect exercise performance in

I young healthy subjects with no evidence of heart • discase, 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 wi'th~ coronary artery disease. Resting heart rate was increased' during cxposure to ETS, which increased blood car- boxyhemoglobin by, about 1%. Exposure to ETS significantly reduced maximum oxygen uptake (by 0.25 11min) and time to exhaustion (by 2.1 minutes). Exposure to ETS also increased the perceived level of exertion during exercise, maximum heart rate, and' carbon dioxide output. It also significantly increased' levels of lactate in venous blood (from a mean of 5.5 mM during the control period to 6.8 mM after exposure to ETS). This greater lactate at a lower oxygen consumption during the passive smoking tri- als indicates a greater reliance on ancrobic metabo- lism: The combined effects 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-17 suggested'that at maximal exertion levels,,up to 90% of the oxygen-carrying capacity of the blood may be needed. Probably because of carbon monox- ide, 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 diffi- culty meeting this demand. Imsum, 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 exer- cise in patients with existing coronary artery disease and' can quickly 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 signifi- cantly elevated levels of 2;3-diphosphoglycerate (DPG), even after correcting for age, weight, height, and sex. DPG acts as a physiological modulator of'; hemoglobin oxygen affinity. It binds to specific amino acid sites and increases the Pso (lowets the oxygen affinity), thus making more oxygen available to pe- ripheral tissues. This observation suggests that the body is attempting to compensate for hypoxia by increasing the DPG level in blood to meet tissue oxygen requirements. The changes were dose depen- dent; the greater the exposure to ETS (measured both in terms of parental smoking and serum thiocy- anate levels in the children), the greater the increase in D~PG.. There is also evidence that short-term exposure to ETS directly, affects respiration of the myocardium at a cellular Icvel: Gvozdjakova er al'y exposed rabbits im a 50 I child's incubator to the smoke of three burning cigarettes smoked during a 30-minute pe- riod, an6they measured several variables related to 11- Gana and Parmlty Passive Smoking and 1Neart Disease 5 the metabolism of cardiac mitochondria. They had three groups of rabbits: one group was exposed to a single dose of ETS, one group was exposed to 30 minutes of ETS twice daily for~ 2 weeks, and one group was exposed to 30 minutes of ETS twice daily for 8 weeks. They measured mitochondrial respira- tiomas the consumption of oxygen after adding ADP to a vcsscl containing mitochondrial' fragments. Us- ing pyruvate as a substrate,,mitochondrial respiration was reduced significantly compared with control (pure air) for all doses of ETS, by, even a single exposure, to about half the controlvalue. The oxida- tive phosphorylation rate was also reduced signifi- cantly at all exposures by about one third. There were no significant changes in the coefficient of oxidative phosphorylation with ETS exposure. Gvozdjakava 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, Gvozdja- kova et a1w and Gvozdjak et al41 reported data om succinate, NADR ; and cytochrome oxidase activity in the mitochondria in the four groups of rabbits. Exposure to ETS affects the activity of NADH oxi- dasc, succinate oxidase, and cytochrome oxidase of myocardial mitochond'ria. The activiry, of the first twoo oxidases exhibited no changes compared with the control group;,neither after a single exposure to ETS or after exposures to 2 weeks. tytochromc oxidase activity decreased both after a single exposure to ETS and over time, with greater decreases as the duration of exposure to ETS was 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 myo- cardial mitochondrial respiration occur at the termi- nal segment of the mitochondrial respiration process. Prolonged exposure to carbom monoxide has been shown to induce ultrastructural changes in myocar- dium42-" and may account for the adverse effects of ETS exposure on mitochondrial function. Thus, short-term exposure to ETS not only in- creases the demand and compromises the supply of oxygen to the heart, but also reduces the myocardi- um's ability to use the oxygen to create ATP to provide energy to support the heart's pumping activity. Effects on Ptateliets The action of ETS to increase platelet ag,gregationn is another way in which ETS can increase the risk of a coronary event. Platelets arc important for the normal process of hemostasis, to prevent blood loss after an injury. When blood platelets aggregate inap- propriately and form a thrombus in the coronary circulation, they can precipitate a myocardial infarc- tion. Hemostasis depends on complex interactions among the dynamics of~ blood flow; components of the vessel wall; platelets, and plasma protcins. De- finitive evidence has confirmed that platelets play a major role in thrombus formation and emholization; f

. . 6 Ciesi+latioa Yo183, No 1, JonuarI 1991 TaatE 2. EQect of Pasatie and Aeti.c StaoiUnB ea Ptatelet ASVeptioo and EadotbetLl Crll' Damage Platelet aggregate ratio EndotheUalicell onunt' Before After Change t3efore After Change n Passive smoking (nonsmoker) ~ 0.87 0.78' -0.09 2:8 3.7 0.9 10 Tobacco (nonsmoker) 0 8I 0.65 -0.15 23 4.8 2.5 . 20 Nbntobaooo cigarette (t;xutQnoker) 0.81 0:7b -0.03 2-5 3.0 0.5 Inhale cigarette (smokor) 0.81 0.68 -0.13 4.0 5.4 1.4 24 Not inhale cigarette (nonsnroker)' 0.82 0.73 -0.09 33 4.7 1.4 22 Smoke (smoker) 0.85 0.70 -0.15 4.4 6.4 2.01 17 Snufl.(smokcr)' 0.82 0.76 -0.06 3.9 4.7 0.8 Alt studies are paired and reflect significant differences (p<0.005). Platelet aggregate ratio is the ratio of platelet oount of piateletrrich plasma; prepared immediately after venipuneture with a aolution oontaining edctic acid and formaldehyde, to that of platelet-rich plasma prepared in the same manner4 except for the absence of formaldehyde. A decrease in the platelet aggregate ratio reflects an increased formation of plateleraggregrtes: Endothelial cell oount is mean number of anuetur eell carcasses in 0.9-µL ehamtxrs. Modified from Davis et at4rA11.51.3= especially in the arterial system. In addition, increas- ing evidence has shown that platelet deposition and thrombus formation can contribute to the growth and progression of atherosclerotic plaques,4s•'d An arte- rial thrombus appears to develop in three phases; platelet adhesion, platelet aggregation, and activat- ing of clotting mechanisms. Passive smoking in- creases platelet aggregation and, thus, increases the likelihood of thrombus formation and myocardial infarction. Table 2 summarizes the results of several studies by Davisat al*1-w on the effects of cigarette smoke on platelet aggregation and damage to the arterial en- dothelium. Davis et al;t, also measured platelet ag- gregate ratios and endothelial cell' counts in non- smokers before and' after exposure 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 formalde- hyde to the same mixture without formaldehyde. This method assumes that platelet aggregates circulating in blood are fixed in the EDTA-fotzrtaldehyde solu- tion and'that they break apart in the EDTA solution. Thus, a decrease in the platelet aggregate ratio reflects an increased formation of platelet aggre- gates. Mean values before and after passive smokingg were 0.87 and 0:78 (p=0:002) for platelet aggregate ratios and 2.8 and 3.7 (p=0:002) for counts of anuclear endothelial cell carcasses in venotu blood. These changes are intermediate between the effects observed after nonsmokers smoked two tobacco cig- arettes an& the effects observed after smoking two nontobacco cigarettes'7 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 otherw-w relatedIstudies on how drugs modify platelet aggre- gation and endothelialicell counts. In particular, the effects observed in nonsmokers who smoked without inhaling were similar to the effects on smokers who smoked two cigarettes even though the plasma nico- tine levels in the nonsmokers were five times lower than those observed in the smokers.SO 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 use.52 7-his result, combined with the finding that smoking non- tobacco cigarettes" failed to produce changes in platelet function as large as observed with tobacco cigarettes, suggests that nicotine is an important active agent. Because nontobacco cigarettes also affected platelet aggregation somewhat, however, carbon monoxide or other combustion products may also influence the platelets. Sinzinger and Kefalidess3 measured platelet sensi- tivity to antiaggregatory prostaglandins (E„ Iz, and D2) before, during, and after 15 minutes of exposure to ETS in healthy nonsmokers an&smokers. Passive smoking reduced platelet sensitivity to the antiaggre- gatory prostaglandins lz and E, significantly (p<0.01) by a factor of about 2 by the end of 15 minutes of exposure to ETS among nonsmokers. This effect persisted at 201minutes after the end of exposure and ceased by, 40, minutes. Platelet response to pros- taglandin D2 ehanged modestly in a similar pattern but was not, significant. Among smokers, the control level of platelet aggregation was higher (p<0;01), and the prostaglandins had no significant effects on platelet aggregation over time during or after expo- sure to E'TS. Sinzingcr and Virgolinix also showed that repeated exposure to ETS for I hr/day for 10 days produced lasting changes in platelet function in nonsmokers similar to those observed in smokers. Thus, nonsmokers' platelets seem much more sensi- trve to a single exposure to ETS than do smokers' platelets, and change in platelet sensitivity to disag- gregating prostaglandins in nonsmokers exposed to ETS' for short periods is similar to that observed in smokers. Further evidence from the same laboratory that passive smoking increases platelet aggregation comes from work by Burghuber et al?s' who studied smokers and nonsmokers who smoked two cigarettes and also exposed a different group of smokers and' nonsmok-

Glanu and Pamdcy Passive Smoking and Heart Diseasc 7 SI LO PGtz 0.5 MC1r'typ~fR { Psll1 sf10KE/1 }'Ks O+ ethRf AFTER s, I PC* 0 FIGURE 3: Plots of effect of active (lcft) and passivr (right) smoking on platelet aggregarlon in smokers and ntMsmokers. The sensitiviry inrlet; S1 PGl,, isdcfnrd as the inmse of the conctntrction oJPnutaglandin It necessary to inhibit ADP-induced platekt ss aggtegarion by S(l%. Lower vaGres of SI 'PG1 y indicate grcateu platelet agg+egation. Adapted from Figures 3 and 4 of Bwgliuber tt al5 ers to ETS in an 18 m' room in whi& 30 cigarettes had been smoked just before exposing the nonsmok- ers. They measured the sensitivity of platelets to the disaggregating substance prostaglandin 12 that is re- leased by endothelium and inhibits platelet aggrega- tion. Figure 3 shows the results of this experiment. ln smokers, neither smoking nor passive smoking af- fected the sensitivity of the platelets to the disaggre- gating effect of prostaglandin 12. The sensitivity, of platelets in-smokers was also significantly lower than that of nonsmokers. In contrast, platelets were more sensitive to prostaglandin 12 in nonsmokers, with both smoking an& passive smoking producing a similar reduction in platelet sensitivity to prostaglandin 1.. These results suggest that the platelets of smokers are already desensitized to the antiaggregatory sub- stance prostaglandin 12 so that no further decrease in aggregation is seen. The significant decrease in plate- let sensitivity to prostaglandin after short-term expo- sure to ETS suggests that after ETS exposure plate- lets are more likely to aggregate with adverse consequences. Earlier work by Saba and Mason% also indicated that nicotine increased a variety of ineasures of platelet aggregation in nonsmokers and smokers. Although the in vitro effects of nicotine on platelets from smokers was greater than that in nonsmokers, the effect generally did not vary with dose (between 2x lU"9 and 2x 1Q-' M), suggesting that the effects of nicotine on platelets occur at low doses and that the system saturates quickly. This observation may ex- plain why passive and active smoking have such similar effects on platelets.s1-s2-t Tlne probable link between nicotine and adverse physiological, effects is nicotine-indutxd release of catecholatnines. Catecholarrtines are then responsi- blc for increased platelet aggregation. This reasoning suggests that 0-adrenergic receptor blockers may provide some protection in smokers. This premise is borne out by a trial comparing the effects of the A-blocker metoprolol to a thiazide diuretic in the control of moderate hypertension.s'' For the same reduction in blood pressure, the metoprolol-treated group had a significantly lower mortality rate than did the thiazide-treated group. Practically 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 aggre- gation, with a magnitude similar to that observed~ in active smoking. Moreover, the response of nonsmok- ers to both active and passive smoking appears to be different from smokers, with nonsmokers being more sensitive to lower exposures to cigarette smoke thann are smokers. This observation indicates that the pharrnacology, of ETS in nonsmokers may be dif- ferent than in smokers, with nonsmokers being more sensitive to low doses of ETS. In particular, it inval= idates attempts to estimate "cigarette equivalent" doses of ETS in nonsmokers or extrapolating from ri'sks of smoking in smokers to effects of ETS on nonsmokers.t" The resulting increase in platelet ag- gregation can contribute to acute thrombus forma- tion and rnyocardial, infarction. Imaddition to the role of platelets in acute throm- bus formation„ platelets are also important in the development of atherosclerosis,'" Once there is dam- age to the arterial endothelium, either through me- chanical or chemical factors„platelets interact with or adhere to subendotheliall connective tissue and ini- tiate a sequence that leads to atherosclerotic plaque. When platelets interact with or adhere to suben- docardial connective tissue, they are stimulated to release their granule contents. Endothelial cells nor- mally prevent platelet adherence because of the nonthrombogenic character of their surface and their eapacity to form antithrombotic substances such as prostacyclin, Once the endothelial cells have been damaged, the platelets can stick to them. Once the platelets arc bound to the endothelium, they release mitogcns such as platelet-derived growth factor, which encourage migration and proiiferation, 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 short-term effects through increasing the likelihood of thrombus formation, the BEFORE AFTER . "'

Y ' 8 Circulation Vol 83, No 1', January 1991 effects of ETS on platelets also increase the chances , that cndothclial injury will lead to arterial plaque. ETS also plays a role in causing damage to the endothclium and initiating the atherosclerotic pro- cesa: As discussed above, Davis et als' found that short-term exposure to ETS, like active smoking"-3°' and use of chewing tobaoco,52 leads to a significant increase (p<0.002) in the appearance of anutdear endothelial cell carcasses in the blood of people exposed to ETS (or tobacco product) constituents. The appearance of these cell carcasses indicates dam. age to the endothelium, which ~ is the initiating step in the atherosclerotic process. As noted above, the ap- pearance of endotticlial cells after passive smoking is almost as great as after primary smoking (Table 2). Exposure to ETS has been shown to produce injuries similar to those observed with exposure to primary smoke and also affects platelets in a way that increases the chances that theywill bind'to the injured area and promote growth of smooth muscle cells!° Role of the Polycyd'ic Aromatic Hydrocarbons in ETS Many atherosclt'rotic plaques in humans are either monoclonal or possess a predominantly monoclonal component;a" which~indicates that the smooth muscle cells of each plaque have a predominant cell type. Several animal studies have also shown that injections of polyryclic aromatic hydrocarbons (PAHs), in par- ticular 7,12-dimethylbenz(a,h)anthracene (I7MBA)) and benzo(a)pyrene!1-65 accelerate the development of atherosclerosis. Benzo(a)pyrene is an important element in E"I'S' The effects of PAHS or other carcinogenic or mutagenic elements in E'T'S°6 relate directly to the response to injury theory of atherogen- esis discussed above!" Changes in the undertyirt& smooth muscle stimulated by these agents can thcn initiate the "injury^'that leads to platelet aggregation and plaque formation: Thus, long-term exposure to £TS can affect plaque formation through mechanisnts similar to those by which long-term exposures produce cancer in other organs. Albert et al61 gave chickens weekly intramuscular injections of DMBA 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. Thcy found thatboth DMBA and benzo(a)pyrene significantly increased the volume of plaque compared with 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 can be atherogenic as welli Penm et alO extended this result in a similar expcriiment by showing that the effects of DMBA on the extent of plaque buildup iri chiFkens 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 plaque per aorta) increased in a nearly linear fashion with DMBA dose. In contrast to the marked' increase in plaque area in the DMBA- treated animals, the percentage of aonic sections with plaques in carcinogen-treated animals was only slightly higher than in controls. Plaques with a small cross-sectional area were present in all animals. Lesions of widely differing cross-sectional areas ap- peared to be similar histologically under the light microscope. Together, these data suggest strongly that a major effect of long-term DMBA exposure is to~increase the size of spontaneous aortic lesions. Rather than induc- ing a eaneerlike change in an individual cell that begins the process that ultimately leads to plaque formation, Penn et al63 suggested that long-tertn DMBA exposure causes preferential division of indi- vidual, cells or patches of cells within the preexisting spontaneous lesions. From this perspective, DMBA 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 a102 found similar results in White Carneau pigeons injected with, DMBA and ben- zo(a)pyrene weekly for 6 months, beginning when the pigeons were 3 months old. Compared with the work described above, they found that benzo(a)pyrene had a greater effect on atherogenesis than did DMBA,, and they also failed to observe a dose-response relation 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 (dimethyl sulfoxide in the previous studies compared with 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 PAN benzo(e)pyrene, which is not considered a carcinogen. This result suggests that carcinogenic PAHs„rather than carcin- ogens or PAHs in general„ are implicated in the atherosclerotic process. Revis et al62 also studied the distribution of these compounds after they had been radiolabeled. Forty, eight hours after the injection of PANs, radioactivity in the liver, aorta, and lung accounted for 75% of'the injected dose, whereas in animals injected with 2,4,6- trichlorophenol, radioactivity in the liver and kidney accounted for 80% of the dose. In addition; 80% of the radioactivity observed in the plasma immediately after injection of radiolabeled PAHs was associated with the low density and high densiry, lipoprotein cholesterol fractions compared with only 24% of the 2,3,6-trichlorophenol, suggesting that plasma lipo- proteins are an important vehicle for transporting PAl-Is 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 e1e- vated levels of cholesterol and depressed levels of high density lipoproteins, even after correcting for age, weight„height, and sex. These effects were dose dependent; the greater the exposure to ETS, the

I greater were the changes in these variables. Pomerehn et all,' observed similar effects of ETS on high density hpoprotcin in children whose parents smoked and in children who smoked or chewed tobacco themselves. High levels of total cholesterol and low levels of high densiry'lipoprotein are impor- tant for the development of plaque. Data on total cholesterol and high density lipoprotein from non- smokers marricd to smokers are inconclusive.M14 To further elucidate the possible mechanisms by which PAHs induce atherosclerotic changes, Majesky et al"s administered a single injection of benzo(a)py- rene to White Carneau and Show Racer pigeons„then looked for metabolites of the benzo(a)pyrene in aortic and hepatic tissues 48 hours later. White Carneau pigeons typically develop severe atherosclerosis by 3 years of age, whereas Show Racer pigeons are rela- tively resistant to aortic atherosclerosis. Aortic prep- arations 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 Carneaulpigeons aged'6-12 months exhibited a threefold to 12-fold inducibility, whereas aortic tissues from the same strain at 2-5 years of age exhibited only minor (maximum; 3.3-fold) and, for the most part, statistically insignificant increases. No age differences in inducibility couid be detected in the Show Racer strain. Interestingly, the differences in inducibility manifest in aortic tissues were grcater 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 coatherogenic agents because the parent compounds are relatively inert both chemically and biologically. Thus bioactivation and inactivation (an& regulatoryy control of these processes) may be presumed to play extremely important roles in their atherogenic prop- erties. Bioactivated chemicals vary in their stability and reactivity according to four generali categories: 1) those that are extremely unstable and persist only at the immediate site (enzyme) of bioactivation, 2) those that persist only within cells inwhich bioac- tivation occurs, 3) those that persist primarily only, within tissues in which bioactivation occurs, and 4) 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 suscepti- ble 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 ev all,' to search for specific molecular events in plaque GJanu and Pannky, P'assive Smoking and Heart Disease 9 cells that would lead' to DNA changes similar to those previously found in tumors. Identification of such processes would be supportive of the monoclo- nal hypothesis of atherogenesis. They obtained hu- man DNA samples from coronary artery plaques ass 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 transsec- tion assay. Foci'arose in cells transfected'with each of the DNA samples obtained from the human coronary plaque, with an efficiency (number of foci/µg of DNA) ranging from 0.016 to 0.060 (mean, 0.036). The transfection efficiencies for DNA from normal coronary artery, liver, spleen, lung, kidney, and tra- chca were alli less than 0.008. The transformed cells were also idjected into the scalps of nude mice, where they developed tumors. These results provide directt evidence for similarities on the molecular level in the development of plaques and! tumors. Human coro- nary artery plaque DNA contains sequences capable of transforming NIH 3T3 cells, and these trans- forme& cells can cause tumors after injection into nude mice. Control experiments verified that the transforming cells did' indeed contain humam DNA and that the tumorigcnic (or transforming) activity was not due to the ras oncogene family: Although these results clearly demonstrate that human plaque DNA has transforming ability, the temporall expres- sion of this activity in vivo is not known. The plaques were taken from adult patients in late stages of vascular disease. Thus, we eannot' determine from these samples whether the manifestation of transfor- mation is a relatively late event, in plaque develop, ment or an early but stable event. Oncogene activa- tion 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 plaque development and demonstratcs that plaque cells exhibit molecular alterations that had previously only been thought to be present in cancer-cell transformation and turnori- genesis. These results provide direct support for the monoclonal 1 hypothesis. Randerath et ald" also demonstrated that onnstit- uents 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 in- duction and distribution of covalent DNA damage in internal organs of the mouse after topical application of eigarette smoke condensate daily, for 1„3, or 6 days then killed 24 hours later. DNA samples were ob- tained from skin, lung, heart, kidney; liver, and spleem 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 frve times higher than that in liver and slightly higher tham 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 I

10 Circulat"ion fWol 83, No 1', lanuary 1991 kidney than in liver, ranging from approximately I adduct/5.4 x l0" DNA nucleotides in lung to 1 adduct/ 3.3x 10' DNA nucleotides in liver. Spleen DNA was practically adduct free. Although 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 adductss 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 coro- nary arteries. In sum, there is a growing body of evidenee at a molecular level supporting the monoclonal hypothesis of atherogenesis, with compounds in tobaceo smoke and ETS strongly implicated as agents that stimulate the development of coronary lesions. Regardless of whether the monoclonal hypothesis proves to be true (or, more likely, one of several initiatiors of the atherosclerotic process), there is clear evidence that components of ETS, in particular PAHs such as benzo(a)pyrene, initiate or accelerate the develop- ment of plaque. These biochemical findings are con- sistent with the epidemiological finding that chimney sweeps,,who 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 earlier than do members of other, comparable, occupations that are not exposed to PAHs.69 The PAHs in ETS are clearly implicated at epidemiological, physiological, and biochemical levels in the genesis of heart disease. Summary The evidence that ETS increases risk of death from heart disease is similar to that which existed in 1986 when the US Surgeon General concluded that ETS caused lung cancer in healthy nonsmokers.r There are 10 epidemiologicalistudies, conducted in a variety of locations, that 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 sig- nificant dose-response effect, with greater exposure to ETS associated with greater risk of' death from bearY disease. These epidemiological studies are complemented by a variety of physiological and biochemical data that 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 abiliry to deliver and utilize oxygen. in animal exper- iments, ETS also depresses cellular respiration at the level of mitochondria. The polycyclic aromatic hydro- carbons in ET5 also accelerate, and may initiate, the development of atherosclerotic plaque. Of note, the cardiovascular effects of ETS appear to be different in nonsmokers and smokers. Non- smokers appear to be more sensitive to ETS than do : smokers, perhaps because some of the affected phys- iological systems are sensitive to low doses of the compounds in ET'S, then saturate, and also~perhaps because of physiological adaptions smokers undergo as a result of l'ong-tcrm exposure to the toxins in cigarette smoke. In any event, these findings indicate that, for cardiovascular disease, it is incorrect to compute "cigarette equivalents"'for passive exposure to ETS and then to extrapolate the effects of this exposure on nonsmokers from the effects of direct smoking on smokers. These results suggest that heart disease is an important consequence of exposure to ETS. The combination of epidemiological studies with demon- stration of physiological changes with exposure to ETS, together with biochemical evidence that ele- ments of ETS have significant adverse effects on the cardiovascular system, leads 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 eancer, these deaths contribute greatly to the estimated 53,000 deaths annually from passive smoking.s This toll makes passive smoking the third leading preventable cause of death in the United States today, behind active smoking10 and alcohol.'r AcknowCedgmsnts We thank James Stoughton for assistance in library work;, A. Judson 1Welis, Donald Shopland, James Repace, Neil Benowitz, Takeshi Hirayama, and the Tobacco Institute for their comments on drafts of the manuscript;, Peter Lee for carefully checking the power calculations; Voijtech licko, Bo-Oing 2tiu; and Art Sussman for translation of foreign language anicles; and Jerry Simnitt for typing. References 1. US Public Health Service: The Healrh Coruequences of'ln.nl- uruary Smolang: A Report of the SurEron Geneml.' DHS (CDC) 87-li198; 1986 2: Doll R; Hill AB: A study otthe actiobgy of eareinoma of the lung. Br Med I 1952:2:1271-1286 3. Hirayama T:, Nonsmoking wives of heavy smokers have a higher risk of lung eane.er. A study fromJapan. Br Med J 19a 1:2[i2( 6273 ):183 -1 g5 4. US Public Hulth Service: TJu Heakh Canxqurncri ofSmok- ing. Cardiow.scular Uiuare: A Rtporr of the Suraron General. DHHS (PHS) 84-50204, 1983 5. Wtlls A: An estimate of adult mortality in the United States from passivc smoking. Enwon Inr 1988;14:249-265 6. Kristensen T: Cardiovascular diseases and the work environ• rnent: A ailiwl review of the epidemiologic literature on~ chemical f'actms. Scand ! Work' Eriviron Hralrh' 19A9:15: 245-264. 7. National Resureti Couneili Enriroarnenipl Tobaccv Smukc: Measunng E.iposun: and itssersing Health EfJectr. Wuhingion. DC„Natbnal Academy Press, 1986 8. Gillis C, Hole 1D, Hawthorne V. Bvyle P: The effect of environmental utbatcnsmok'e in two urban communities in the wesrof Scotland. Eurl Rap Di.r 19K4:65(suppl 133):12J,126 9. l.ee P; Chamherlain J. Alderson M: Relatwnship of pac.ive smoking to risk of lunguncer and other r:moking-assncuted diseases. BrJ Cancer 1986:54:97-105