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I BIOLOGICAL MECHANISMS ACCOUNTING FOR THE PURPORTED RELATIONSHIP BETWEEN ENVIRONMENTAL TOBACCO SMOKE EXPOSURE AND ADVERSE CARDIOVASCULAR EFFECTS: A REPONSE TO DR. GLANTZ By Joseph M. Wu, Ph.D. ''I I I I I I I. INTRODUCTION Recognized as the primary cause of death in all developed countries and in many developing countries, coronary heart disease (°CHD") is believed to have a complex and multifaceted etiology. Numerous risk factors for CHD have been identified. These risk factors include both modifiable lifestyle characteristics such as diet and weight, as well as non- modifiable personal characteristics such as age, sex and family history. Risk factors are largely identified by means of observational, epidemiological studies. Because such studies lack the rigorous scientific controls characterizing many experiments performed in the laboratory, these studies typically cannot distinguish between the impact of one particular risk factor and the confounding effects of other risk factors. This is particularly true with respect to epidemiological studies focusing on CHD because of the large number of suspected risk factors for this disease. Accordingly, results of epidemiological studies identifying CHD risk factors should only be seriously considered when they are supported by biologically plausible mechanisms that adequately explain the relationship between the purported risk factor and the onset of CHD. ,.

'I , I I ''I 1 I I I I I I I I Recognizing the importance of establishing a biologica'lly plausible mechanism before labeling a particular agent as a risk factor for CHD, Dr. Glantz has attempted to identify biologically plausible mechanisms that account for the purported association between environmental tobacco smoke ("ETS") exposure and adverse cardiovascular effects, including CHD. The biological explanations offered by Dr. Glantz, however, are based on unsound and selective interpretations of existing data that are lacking in scientific validity. As demonstrated below, when the existing data are analyzed as a whole, it is clear that any biological association between ETS exposure and adverse cardiovascular effects is equivocal and remains to be scientifically established. II. DISCOBSION . Dr. Glantz attempts to explain the biological mechanisms for three distinct, adverse cardiovascular effects purportedly associated_with exposure to ETS: 1) reduced delivery of oxygen to the heart; 2) increased reperfusion injury following myocardial infarction; and, 3) increased development of atherosclerosis. None of the explanations offered by Dr. Glantz finds compelling support based on current scientific data. A. The Delivery of Oxygen to the Heart Dr. Glantz claims that individuals have a reduced ability to exercise after exposure to ETS and that this provides evidence that ETS reduces the delivery of oxygen to the heart. He asserts that ETS hampers the flow of oxygen to the heart by I

I I I I I I I I 11 increasing the amount of carbon monoxide in the body. Dr. Glantz offers absolutely no substantive support for this claim, however, and it amounts to little more than sheer speculation. Dr. Glantz correctly notes that carbon monoxide competes with oxygen for binding sites on red blood cells. It would be predicted, therefore, that when carbon monoxide in the body reaches a critical level, the delivery of oxygen to the heart may be impaired. Dr. Glantz provides no evidence, however, that exposure to ETS is associated with carbon monoxide concentrations even remotely reaching such critical levels. Lacking such data, Dr. Glantz has absolutely no empirical basis for his assertion that carbon monoxide resulting from ETS causes decreased oxygen flow to the heart. Moreover, Dr. Glantz has completely ignored the data on- vascular relaxation associated with greater reliance on anaerobic metabolism that results from decreased oxygen flow during exercise.- When the body relies on anaerobic metabolism, it produces significant levels of a chemical called lactate, which exhibits the potential to relax blood vessels. As early as 1880, Gaskell already reported that lactate caused relaxation of the arteries of the mylohyoid muscle of the frog (1). Other studies have subsequently demonstrated relaxation by lactate in the coronary vasculature (2, 3). Lactate levels in umbilical arterial and venous blood have been reported to increase under both physiologic and patholog'lcal conditions (4-7). The manner by which lactate causes relaxation in human blood vessels has 3

recently been determined to involve an oxygen and cGMP-dependent mechanism, through the generation of hydrogen peroxide (8, 9). This tendency for lactate to act as a blood vessel relaxant means that an increase in blood lactate levels may actually inhibit constriction of blood vessels, thereby decreasing the prospect of a heart attack resulting from a complete blockage of these vessels. I I I I I ' I I I 1 B. Production of ATP Dr. Glantz' claim that exposure to ETS compromises the production of ATP in cardiac cells via a free radical-mediated mechanism, particularly those damaged by reperfusion-induced arrhythmias and by episodes of myocardial infarction is equally vague and speculative. The underlying mechanisms of reperfusion-induced arrhythmias are not well understood. Indeed, multiple factors are known to influehce the vulnerability of the heart to reperfusion-induced arrhythmias. These include: (1) the duration of the preceding period of ischemia, (2) the degree of ion distribution, (3) the metabolic patterns of metabolites such as fatty acids, (4) the activation level of adrenergic receptors and the content of tissue cyclic AMP, and (5) the concentrations of free radicals. Additionally, although metabolic changes during ischemia-reperfusion are known to be heterogeneous, they have been shown to be stabilized (i.e., prevented from excessive fluctuation) by substances such as adenosine whose release from 4

1 i I I 1 I I I the heart is actually increased by nicotine (10). Similarly, as discussed below, existing data suggest that ETS exposure may actually reduce free radical concentrations. . Finally, although energy has been proposed to play a role in the ability of cells and tissues to defend against oxidative stress, the ultimate antioxidant capacity of a tissue is determined by the supply of reducing eqtiivalents. The pathways involved in supplying reducing equivalents in response to an oxidative stress remain unclear, although some data suggest that energy is not a factor in the mechanisms by which reducing equivalents are made available to neutralize exogenous oxidants. The supply of reducing equivalents is not entirely oxygen dependent. Glutathione (GSH), a major component of cellular antioxidant systems, is maintained in the reduced form by glutathione reductase. Although this enzyme is specific for NADPH, the ability of intact cells, isolated mitochondria (which are a major source of free radicals and contain antioxidant systems independent of the rest of the cell), and whole tissue to supply reducing equivalents and maintain normal levels of GSH appear to involve NADH. NADH can be generated both by aerobic and anaerobic biochemical reactions and hence are not entirely dependent on the delivery of oxygen to tissues in the cardiovascular system. .. in sum, it is clear that additional research is needed to gain a clear understanding of the mechanisms and the dynamics 5

I I , , I I I of energy change in response to oxidative stress and the extent to which these mechanisms are influenced by exposure to ETS. C. Reperfusion Iniury Dr. Glantz' argument that ETS exposure increases reperfusion injury relies largely an the work of Van Jaarsveld et al., who recently reported that rats exposed to ETS showed decreased mitochondrial oxidative function and increased myocardial sensitivity to ischemia/reperfusion (11). Van Jaarsveld et. al. hypothesized that the impairment of the mitochondrial oxidative function associated with ETS exposure contributed to increased free radical concentrations, based on elevated concentrations of low molecular weight iron (LMWI) and reduced concentrations of a-tocopherol, which in turn caused the enhanced reperfusion injury. . The Van Jaarsveld et. al. data are of questionable validity. The mitochondrial oxidative function was inadequately measured using a glutamate substrate. Other substrates (e.g., succinate) should have been studied at different concentrations (and in the presence of varying concentrations of ADP) to determine more accurately the extent to which mitochondrial oxidative function was impaired, if at all. Furthermore, no attempt was made to measure the overall free radical scavenging capacity of the ETS exposed rats. without knowing the overall .. capacity of the rats' systems to neutralize free radicals, Van Jaarvseld et. al. had no basis for concluding that increased free 6

1 ' , 1 .t 1 1 I I t I I radical concentrations were related to their observations following reperfusion. Finally, without data on concentrations of superoxide dismutase, glutathione peroxidase and catalase, the meaning of the change in a-tocopherol is really unclear, particularly when coupled with the fact that the LMWI content of the ETS-exposed rats was actually statistically lower than that of the non- exposed group. A lower LMWI level implies that the capacity for generating potentially deleterious free radicals has been reduced; which is an effect opposite to that claimed by Dr. Glantz. D. Atherosclerosis Dr. Glantz attempted to demonstrate a biological connection between ETS exposure and the development of atherosclerosis. Atherosclerosis refers to the formation of fatty, cholesterol-laden atheromas in the tunica intima and media of large and medium sized arteries, which are most commonly encountered in areas of high blood flow such as coronary arteries. Over time, the thickened vascular wall compromises the vessel lumen, causing a reduction in cross-sectional blood vessel size and hence decreased blood flow that may not take on physiological significance until maximum flow is needed. This "narrowing of the arteries" may eventually show itself in the form of clinical symptoms. Dr. Glantz asserts that ETS exposure contributes to atherosclerosis in three ways; l) it damages the endothelium; 7 L

2) it increases the rate of cholesterol induced lipid accumulation; and, 3) it promotes platelet aggregation and thrombus formation. Each of these purported mechanisms will be discussed in turn. 1. Endothelial damage The endothelium consists of a thin layer of cells that line the blood vessels. Through intensive study in the past decade, it has been established that the endothelium plays an important role in modulating blood vessel constriction, in addition to serving as an antithrombcgenic surface (12). It is also generally thought that structural damage to the endothelium contributes to the accumulation of lipid deposits and is among the earliest events in the atherosclerotic process (13). It has been reported that smoking is associated both with damaged endothelium (14-16) and ultrastructural changes of the endothelium (17-19). Although studies in animals provide data supporting an association of smoking with endothelial cell changes (16, 17), the results of studies in humans are more conflicting. One study found ultrastructural changes to the endothelium in the umbilical artery of smoking mothers (20). By contrast, another study failed to detect endothelial changes in the iliac artery of smokers (16). Similarly, several in vitro studies using human blood vessels and endothelial cell cultures have demonstrated reduced levels of prostacyclin, a substance produced by the endothelium. However, both reduced (21) and increased (22) urinary levels of the prostacyclin metabolite,

f I I I prostaglandin FlQ, have been reported in smokers in vivo, suggesting that ETS does not have any definitive impact on the production of prostacyclin, or by extension, the function and structure of the endothelium. Jacobs et al. (23) recently studied ETS exposure in relation to the functioning of the endothelium by observing endothelium-dependent vasodilation in the forearm of habitual smokers and non-smokers. Jacobs et. al. used intraarterial infusion of methacholine to cause vasodilatation. Methacholine is a muscarinic-receptor agonist known to cause release of endothelium dependent relaxant factors ("EDRF") from endothelial cells (24). In the same study, endothelium-independent vasodilatation was also investigated by intraarterial infusion of sodium nitroprusside, a-chemical known to cause vasodilatation by directly stimulating guanylate cyclase of vascular smooth muscle cells. By measuring changes in bilateral forearm blood flow, arterial blood pressure and forearm vascular resistance, no difference in endothelium dependent vasodilation of the forearm was observed between habitual smokers and non-smokers. These experiments provide strong evidence that habitual smoking does not result in permanent endothelial dysfunction in humans. It seems therefore most unreasonable to expect that exposure to ETS could elicit endothelial damage leading to CHD. 2. Cholesterol and lipid accumulation Dr. Glantz asserts that ETS exposure also contributes to atherosclerosis by promoting the rate of cholesterol induced 9 I

lipid accumulation. However, neither the relationship between I cholesterol and CHD, on the one hand, nor the relationship between ETS and cholesterol, on the other hand, is completely understood. The influences of diet on cholesterol and lipoprotein changes have been amply illustrated by population studies showing that a high intake of antioxidant vitamins (a-tocopherol, 0- carotene, vitamin C) may be associated with a decreased CHD risk (25-30). Similarly, age has been shown to have a significant impact on the observed association between cholesterol and CHD. A recent analysis of 2544 white men, aged 25-84 years, who were entered in the Milwaukee Cardiovascular Data Registry from 1977 to 1986 following coronary angiography (31), found that although plasma cholesterol for all men was associated with an increase in coronary artery occlusion, the association actually applied only to the younger men. when stratified by age, the association diminished to near zero in the oldest age group. Indeed, a multivariate analysis of the negative association between cholesterol and age in predicting CHD proved to be highly significant. The foregoing data demonstrate that the specific relationship between cholesterol and CHD is highly complex. Moreover, in recent years it is been discovered that what has traditionally been regarded as the danger of cholesterol appears to Fie, more precisely, the danger of low-density lipoproteins ("LDL") relative to high-density lipoproteins ("HDL"). When high total cholesterol reflects a 10

disproportionately high LDL, then high total cholesterol is associated with increased heart disease risk. If, on the other hand, total cholesterol is high because HDL is high, then it does not have this association with increased heart disease risk. The process by which LDL contributes to atherosclerosis is not entirely clear. It is believed that LDLs are oxidatively modified during the development of atherosclerosis, resulting in alteration of their gross physical structure and chemical properties (32). This may be caused in vivo by free radical attack of the polyunsaturated free fatty acids which proceeds via a chain reaction (33). The extent of oxidation appears to be• influenced by the ratio of lipid components and antioxidant levels in the LDL of the individual and is thought to occur in three phases: an initial lag phase when endogenous LDL antioxidants such as vitamin E are consumed; a propagation phase with rapid oxidation of unsaturated fatty acids to lipid hydroperoxides; and a decomposition phase, when hydroperoxides are converted to reactive aldehydes (e.g., malondialdehyde and 4- hydroxynonenal) (34). Interaction of these aldehydes with positively charged epsilon-amino groups of lysine residues in the apolipoprotein B-100 (apo B-100) moiety renders the LDL more negatively charged, resulting in decreased affinity for LDL receptors and increased affinity for scavenger receptors (35), which in turn allows delivery of an excess of cholesteryl esters to target cells via a receptor-independent mechanism. This process, coupled with the fact that oxidized LDLs are cytotoxic ~ ~ 11 ~ 05 ~

,I .I as well as chemotactic for monocytes, probably explains to some degree how L'DL contributes to atherosclerosis (36-38). Although it has been suggested that cigarette smoking contributes to oxidation of LDL by releasing free radicals (39- 42), a contrary result has recently been demonstrated experimentally. Specifically, an aqueous cigarette smoke extract was reported to have antioxidant properties that actually inhibited the oxidative modification of LDL resulting from incubation with either copper or 2,21-azo-bis(2-amidinopropane) hydrochloride (43). These data are in the opposite direction from that predicted on the basis of the claim that ETS exposure contributes to lipid formation and atherosclerosis resulting from oxidation of LDL. Before any definitive conclusions can be drawn about whether ETS exposure is related to LDL, however, further analysis is required of other LDL subgroups as well as the influence of external factors on LDL levels. Many studies report greater , proportional elevations of inean apolipoprotein B (apo B) than LDL cholesterol ("LDL-C") in patients with clinical coronary heart disease (44, 45). This suggests that specific apolipoproteins, such as apo B, may be more strongly associated with atherosclerosis than LDL-C. Additionally, numerous studies have reported that HDL and LDL levels can be affected by diet, alcohol consumption, and physical activity (46-49). In one recent study, treatment of hypercholesteremic rats with ascorbate was associated with reductions in both HDL-C and LDL-C (50). 12

i1 I Accompanying the decrease in the cholesterol, triglycerides, and protein content of all plasma lipoproteins in ascorbate-treated rats was a marked modification of the apoprotein pattern of all lipoprotein classes, with an increase in apo E in LDL and a decrease of C, AI and B in VLDL-IDL and of apo C in LDL. By contrast, it was found that ascorbate induces an increase in C apoproteins and a decrease of E.and B apoprotein in HDL fractions. The need for further study is highlighted by the animal studies that Dr. Glantz invokes in support of his claim that ETS :. _ .. exposure contributes to atherosclerosis by promoting cholesterol induced lipid accumulation. Unrealistic and stressful exposure conditions in these animal studies introduce numerous confounding problems. Thus, these studies provide no insight into the biological connection, if any, between ETS exposure and lipid accumulation. Furthermore, these studies used either fresh sidestream smoke or aged mainstream smoke rather than ETS. As noted below, different forms of cigarette smoke are not comparable, particularly with regard to potential associations with effects on the cardiovascular system. Finally, these studies used unrealistically high doses of smoke. This is particularly significant given that CHD is a chronic disease with long latency periods and the studies investigated only short term exposure. The significance of cardiovascular measurements and/or changes as part of a short-term response to unrealistic 13

environmental exposures in relation to the eventual manifestation of CHD is open to serious question. In sum, there is little question that additional research needs to be performed before any definitive conclusions can be drawn about the relationship, if any, between ETS exposure and cholesterol in promoting atherosclerosis. Accordingly, Dr. Glantzf claim that ETS contributes to atherosclerosis by promoting cholesterol induced lipid accumulation is, at best, premature. 3. Platelet aggregation and thrombus formation 11 I Finally, Dr..Glantz contends that ETS exposure contributes to CHD by increasing platelet aggregation and thrombus formation. However, although it has been suggested that spontaneous and induced increases in platelet aggregability may - contribute to CHD, attempts to relate the effects of smoking to changes in platelet function have produced only conflicting results. Some investigators have reported a positive association between smoking and platelet adhesiveness and aggregability (51, 52) while others have failed to demonstrate any differences between smokers and non-smokers (53, 54). Moreover, those studies reporting positive associations suffer from experimental defects which render their results highly suspect. First, the studies did not control for diet. Plasma or serum lipids have been associated with changes in platelet aggregability. Vitamin E has an inhibiting effect an the platelet release reaction (55) and may also play an indirect 14

i ! I role in platelet function by influencing prostacyclin (56) and thromboxane (57) production. Foo et al. (54) recently studied habitual smoking in relation to whole blood platelet aggregation and production of prostacyclin and thromboxane AZ in young adult males under controlled dietary conditions. According to their data, the mean platelet aggregation was significantly lower in smokers than non-smokers. These results suggest that smoking does not directly enhance aggregation and may be associated with a reduction in platelet aggregability when diet is taken into account. Second, the few studies reporting positive associations between ETS exposure and platel:et aggregability were generally performed in vitro. Substantial differences have been observed in platelet aggregability, however, depending on whether reactions were performed in vitro or in vivo. Larsson et al. (58) studied platelet aggregability in healthy volunteers during mental stress and low- and high-dose..adrenaline infusion using ex vivo (filtragometry) and conventional in vitro (aggregometry) methods. Results of their experiments show that the conventional in vitro techniques are not representative of platelet aggregability in vivo. This difference between the results of in vitro and in vivo studies is likely due, in part, to the selection of less sensitive platelets in in vitro'studies, owing to greater loss or artifactual activation of platelets during blood sample preparation. Additionally, the difference in experimental 15

1 I I I results may also be related to the failure of in vitro studies to account for all of the complex processes that regulate platelet aggregability in vivo. For example, as noted earlier, it has been claimed that ETS exposure may impair the oxidation of LDL in the body (43). Recent studies suggest that oxidation of LDL may be one of the primary mechanisms contributing to platelet aggregation (59). Secause the in vitro studies fail to account for any influences on platelet aggregability by indirect mechanisms such as that involving LDL, they do not accurately reflect platelet function in vivo. When properly analyzed by using in vivo studies that control for dietary effects, an association between ETS exposure and platelet aggregability has not been reported. Finally, Dr. Glantz completely omits any discussion of the potential vasodilatory effects of ETS that may completely offset any vascular constriction resulting from increased platelet aggregation. NO, a cigarette smoke constituent (60), has recently been established as a key EDRF having a pivotal role in endothelial cell function_and in signal transduction. It is thus of interest that the inhaled gas phase of cigarette smoke has been reported to relax the pulmonary circulation in pigs in almost an identical fashion as NO (61-63). To a lesser extent, vasodilatory responses have also been associated with the particulate phase of cigarette smoke. In a recent study, the vasodilation was assessed during/following continuously administered cigarette smoke in concentrations 16

''I I I I 1 II relevant to normal smoking (64). The relative importance of nicotine versus other particulate phase constituents of cigarette smoke in counteracting the gas-phase induced pulmonary vasodilation was also examined. The study reported that unfiltered cigarette smoke induced variable responses in the pulmonary circulation whereas inhalation of filtered smoke was consistently associated with pulmonary vasodilation. The major part of the vasodilatory response was attributed to NO. This apparent effect of NO was partially opposed in the unfiltered smoke by the particulate phase (but not by nicotine) presumably through a mechanism involving the induction of sympathetic reflexes. In a somewhat related study, Murohara et al. (65) studied stable contraction of pig coronary artery rings, incubated in organ chambers with prostaglandin FZa, in the absence or presence of cigarette smoke extracts (CSE). They reported that CSE induced an initial contraction followed by a relaxation of the coronary artery rings. They proposed that the initial contraction may be, at least in part, mediated through the degradation of basally released EDRFs by superoxide anions derived from CSE. Taken as a whole, these studies suggest that if ETS exposure has any relationship with vascular tone, it is highly complex and poorly understood. Dr. Glantz' uncritical and oversimplified claim regarding a vasoconstrictive effect of ETS exposure, therefore, reflects a clear bias and lack of scientific candor that is incompatible with accepted scientific procedure. 17 I

III. CONCLUSION I I It has been suggested on the basis of epidemiological studies that prolonged exposure to ETS may increase the risk of CHD. Nonetheless, studies focusing on this issue fall far short of providing conclusive evidence for a causal association. Potential confounders may account for spurious positive results and the multifactorial nature of CHD often makes it difficult to determine which confounders are likely to be the most important. For this reason, examining and identifying the biological mechanisms that could account for the association, if any, between ETS exposure and CHD is of critical importance. Only in this way can scientists gain an accurate understanding of the potential significance of ETS exposure as a risk factor for this disease. As the discussion in this comment demonstrates, researchers are beginning to explore the biological processes which might be relevant to an association between ETS exposure and the cardiovascular system. Such studies may eventually enable us to appropriately evaluate whether there is a biological relationship between ETS exposure and adverse cardiovascular effects. At present, however, the results of these studies'are equivocal at best, with the findings varying both in direction and magnitude of association. Accordingly, whether ETS exposure has an adverse impact an the cardiovascular system and contributes to CHD is a question that can not be scientifically resolved on the basis of the data currently available. 18 I

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