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I I I I I i I I I I I I I I I I I I COMMENTS ON ENVIRONMENTAL TOBACCO SMOKE A Compendium of Technical Information CHAPTER ii PASSIVE SMOKING AND HEART DISEASE EPIDEMIOLOGYr PHYSIOLOGY, AND BIOCHEMISTRY Prepared by Joseph M. Wu, Ph.D. I am a Professor in the Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York. I received my Ph.D. in biological sciences from Florida State University in 1975, spent two years as a post-doctoral fellow in the Department of Biochemistry, Temple University, and joined New York Medical College in 1978. My research interests are in the following areas: (i) developmental and hormonal regulation of enzyme synthesis and degradation, (2) control of eukazyotic cellular proliferation and differentiation, (3) biochemistry of 2', 5' -oligoadenylate synthesis and expression in normal and interferon-treated mammalian cells, (4) studies of biochemical changes in Alzheimer's Disease cells, and (5) modulation of gene expression by environmental agents. I have been the recipient of I

I I I I I I I I I I I I I I I I I ! I numerous research awards from governmental agencies and private foundations. Currently I have one research grant award from the National Institutes of Health and two research grants from private foundations. My curriculum vitae is attached. I have been asked to review "Passive Smoking and Heart Disease: Epldemiology, Physiology, and Biochemistry," which is Chapter Eleven of an EPA draft compendium of technical literature on environmental tobacco smoke (ETS). The authors for this chapter are Stanton A. Glantz, Ph.D., and William W. Parmley, M.D. In this chapter, the authors give a superficial review of the data from ten epidemiological studies concerning incidences of heart diseases and exposure to environmental tobacco smoke (ETg), then proceed to offer some discussion of physiological and biochemical mechanisms in an effort to show how ETS may conceivably contribute to increasing the risk of heart disease. Changes in platelet functions, alterations in the pattern of blood flow resulting from chemicals present in ETS, the suppression of mitochondrial activity based on animal studies, and the presence of polycyclic aromatic hydrocarbons in ETS, are all cited by these authors as purported evidence to link ETS exposure to weakened heart function, leading ultimately to the initiation and establishment of atherosclerotic lesions. Alternate mechanisms unrelated to ETS exposure which would lead to the same set of physiological and biochemical changes are not - 2 -

I I I I I I I I I I I I I I I I I I I considered or eliminated by these authors. Moreover, based on circumstantial information, they postulate the existence of a different platelet sensitivity to ETS between smokers and nonsmokers, and imply that the latter group is at greater risk because of a lower threshold characteristic of their platelets. The authors also briefly discuss several animal studies involving the use of benzo(a]pyrene. Some recent experiments showing that DNA extracted from human atherosclerotic plaques is able to induce transformation in transferred mouse 3T3 cells are used to support the concept that plaque-derived human cells possess the unique ability to trigger arterial smooth muscle cell proliferation, a key event associated with the initiation, progression, and establishment of atherosclerotic plaques. The chapter closes with a brief description of a report showing the selective localization of adducts containing benzo(a)pyrene-derived moieties in heart and lung DNA. In their opening paragraph, the authors acknowledge the multi-factorial nature of heart disease. On the question of possible ETS contributions to heart disease in nonsmokers, there now appear to be altogether ten epidemiological studies considering whether ETS of heart disease in the epidemiology is outside exposure is related to an increased risk nonsmoking spouse Of a smoker. Although the area of expertise of this reviewer, there are several general scientific principles regarding the validity and/or plausibility of epldemiological findings which warrant brief mention. For example, systematic error may distort - 3 -

I I I I I I I I I I I I I I I l I I I the study base if it is selectivs in nature. It should also be recognized that the etiological link between environmental variations and the endpoints for chronic diseases such as diabetes, coronary heart disease, arthritis, asthma, and cancer is complex and is characterized by (i) continuous variations in clinical, physiological, and biochemical phenotypes that are measures of health, (2) environmental modification of the biological predisposition of an individual to disease, and (3) multiple confounding factors giving rise to the same disease endpoint. Accordingly, increased or decreased incidences of a certain disease in a group can be attributed to a specific environmental factor, e.g., ETS exposure, only where that factor is the single factor that is free to vary. that expanded knowledge base in biochemical measurements. epidemiological studies is Another complication in the analysis of chronic disease is the definition of the endpoint may change as a result of an the clinical, physiological, and A further issue regarding the character of the reference population. For example, an industrial population other than the one under study could have some totally different exposure causing the same dlsorder(s) as the exposure at issue. Hence, for example, the choice of a group of copper smelter workers as a reference population for miners would fail to reveal fully the excess risk of lung cancer due to radon exposure in the mine, since the copper smelter could also suffer from lung cancer due - 4 -

I I i ! I I l l l i I I I I l I I I I to arsenic exposure. Nor should the reference 9roup be from an urbanized area, if the index population is rural. The authors next discuss the issue of the effects of primary cigarette smoking on coronary heart diseases. They correctly point out that cigarette smoking is but one of the three reported major independent risk factors for coronary heart disease (CHD), the other two being hypercholesferolemia and hypertension. A wealth of evidence indicates that cholesterol is causally related to atheroslcerosis (McGilI, Jr., 1984; Nilsson, 1986; Kaunitz, 1988). By definition, cholesterol is present in all atherosclerotic plaques. Moreover, atherosclerosis cannot develop in animals unless they are first made hypercholesterolemic (Mcgill, Jr., 1984). Indeed, studies in nonhuman primates have shown that regression of atherosclerosis is possible if the plasma cholesterol level is sufficiently reduced. In humans, genetic studies have indicated a striking relationship between early, severe coronary disease and the presence of either elevated plasma levels of low density lipoproteins or reduced levels of high density lipoprotein. The evidence is overwhelming that reducing plasma cholesterol levels suppresses or reverses the progression of coronary artery plaque and lowers the cases of clinical coronary artery disease mortality and morbidity (Brown and Goldstein, 1986). Similarly, hypertension has been known as a risk factor for coronary heart disease as long as serum - 8 -

I I I I ! I ! I I I ! ! i I I I I I I cholesterol concentration. It is associated with accelerated atherogenesis in adult humans and in experimental animals. A strong relationship has been reported between blood pressure and arterials lesions for men in certain age groups (Holme et al., 1981). Notwithstanding their failure to emphasize hyperllpidemia and hypertension as equally important risk factors for CDH, the authors proceed to describe the salient features of atherosclerosls, and make reference to the fact that there is a lack of full understanding of the pathogenesis of the disease. Areas where cigarette smoking could theoretically influence cardiovascular efficiency and capacity are highlighted. The authors also note in this regard that components in cigarette smoke which have been claimed to have an adverse effect on the cardiovascular system of smokers have also been identified in ETS. Yet, they do not seem to recognize that ETS is not the same as the mainstream tobacco smoke inhaled by a smoker to begin with, and that there are complex, as yet poorly defined physical and chemical changes occurring during the aging of ETS in an indoor environment (Eatougb et al, 1989). Accordingly, it is misleading to imply, as the authors do, that data about the former can be employed in studying the effects Of the latter. Discussion of this sort should not be included in a compendium of technical literature on ETS. - 6 -

I I I ! I I I I I I I I i I I I I i The authors next review the data of ten epidemiological studies in several pages. AS stated above, this scientific discipline is outside the scope of expertise of this reviewer. ACUTE EFFECTS OF ETS EXPOSURE Following their limited analysis of data from epidemiological studies, Glantz and Parmley go on to review several published reports describing selective physiological reactions observed in human subjects exposed to ETS in an artificial laboratory setting. The first paper examined is the work of Aronow (1978). The scientific artefacts of Aronow's study have been repeatedly addressed in the past. The Surgeon General's Report of 1986 summarized its findings as follows: "This study was criticized because the endpoint angina was based on subjective evaluation, and because other factors such as stress were not controlled for .... More important, the validity of Aronow's work has been questioned." (USPBS, 1986, P.106). Because of the lack of control for "stress," the reported "increased resting heart rate and systolic and diastolic blood pressure" observed by Aronow may be due to stress-lnduced release of catecholamines which would likewise influence the subjects to respond in the manner described. - 7 -

I ! I I i I II I I ! I I I I i l I ,I I Glantz and Parmley then refer to the observation of McMurray et al. (1985) as the basis for arguing that ETS exposure "significantly reduced maximum oxygen uptake and time to exhaustion, . . . increased the perceived level of exertion during exercise," and "significantly increased levels of lactate in venous blood," and they suggest that "the combined effect of reduced oxygen carrying capacity and increased lactate resulted in a reduction in maximal aerobic power and duration of exercise" in "blindly exposed young healthy women." To this reviewer, there are a number of difficulties in interpreting the data of this rather small-scale study in this manner. First, only eight females of a narrowly defined age group (21.8 +/- 2.4 years) were entered into the study. Although each subject was "screened by a medical history," the details of the screening protocol were not provided. Four of the subjects were smokers while the other four described themselves as nonsmokers. NO verification of their smoking status by measuring cigarette smoke-specific products in the biological fluid of the subjects was provided. Regarding the "blind" nature of the study, it is interesting to note that the authors pointed out that "all of the smokers could tell when they were breathing the smoke but none of the nonsmokers knew for certain." It is equally intriguin@ to note the observation by McMurray et al., that "the presence of smoke raised the carboxyhemoglobin levels of the nonsmokers from a pre-level of 1.1% to 2.2% at the - 8 -

I I I I I I ! I I I I I i I i I a I I end of the exercise." This pre-level of 1.1% is some 80% higher than that found in a more recent study in which the mean carboxyhemoglobin level of the subjects was 0.6% +/- 0.02% (Allred et al., i989). It is also important to mention that the manner in which ETS was delivered to the subjects would represent an extreme, arbitrary, and unrealistic form of ETS exposure since there is virtually no dilution by ventilation nor is the normal modification of ETS in an ordinary indoor environment allowed. By far the most significant increase in the study by McMurray et al. is in the concentration of post exercise venous blood lactate, which "averaged 6.8 mM during the smoke trials, significantly greater than the controls (5.5 mM)." From a biochemical viewpoint, it is well established that lactate is generated from pyruvate by the enzyme lactate dehydrogenase. The heart and skeletal muscles, however, exhibit marked differences in their ability to oxidize glucose anaerobically. Lactate dehydrogenase in the heart muscle, because of its unique structural composition (H4), is allosterically inhibited by pyruvate and is thus unable to convert pyruvate to lactate. In contrast, the same enzyme in the skeletal muscle having a structure of M4 effectively catalyzes the enzymatic conversion of pyruvate to lactate. Lactate is expelled into the bloodstream, where it is taken up by the liver to be resynthesized into glucose via the enzymes in the gluconeogenic pathway. Since exercising muscles typically oxidize glucose anaerobically to generate ATP during periods of severe exercise, and because - 9 -

I I ! ! ! ! ! lactate is released into the blood stream, concentration of lactate in venous blood post exercise may be viewed as a biochemical marker for the severity of exercise to which the skeletal muscles were subjected. Accordingly, the 24% rise in lactate concentration during the smoke trials (6.8 r~4 versus 5.5 mM) is a strong indication that the subjects (for some unknown reason) are "exercising" harder during the "smoke trial" than the "control" periods. It may then be deduced that the increased level of exercise by the "smoke-trial" group could conceivably account for the "significant reduction in time to exhaustion," as well as the "increased perceived level of exertion during exercise." I I I i! I I I I I In short, the McMurray study is handicapped by the fact that the "control group" and the "experimental group" are not identical since the degree of exercise in the treadmill test is apparently different. It would be important in future studies tc have the "treadmill exercise output" recorded in some fashion to ensure that the same amount of effort is spent during the experimental "smoke-trial" periods as during the control "non-smoked" periods. The authors then discuss the findings of Moskowitz et al. (1990) and assert that these investigators "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 - l0 -

I ! ! II i n I m m ! ! ! ! im ! | m m ! had significantly elevated levels of 2,3-diphosphoglycerate (DPG), which suggests that the body is attempting to compensate for hypoxia by increasing DPG level in blood to meet tissue oxygen requirements." These sweeping remarks require a close examination of the data reported by Moskowitz etal. First of all, as noted by these investigators, the bematocrit values for the ETS-exposed and non-ETS exposed children were identical, raising doubt as to whether these children are anemic. Certainly, the weight and height of the ETS-exposed group do not support such a conclusion. With respect to the argument that the DPG increase supports the conclusion that the "body is attempting to compensate for hypoxia by increasing DPG," it must first be noted that the method used by Moskowitz etal. is one already described 65 years ago. The method of Fiske and SubbaRow which Moskowitz etal. applied for determining DPG is actually designed for the colorimetric measurement of phosphorus and is not specific at all for DPG. The principle of the method takes advantage Of the fact that sugar phosphates show quite different stabilities in acid; those which are hydrolyzed completely in 1 N sulfuric acid at 100 C during a 7-minute incubation are referred to as labile, while those which are resistant to hydrolysis under the same conditions are referred to as stable. A third class of sugar phosphates including DPG, ribose 1-phosphate, etc. show extra lability toward acid and may be estimated like inorganic phosphate (Leloir and Cardine, 1957). Concentrations of 2,3-DPG are more - ii -

Ii a I itI i I I a I I I I a ! il a ! specifically measured by the stoichiometric cleavage of 2,3-DPG to 3-phosphoglyceric acid by diphosphoglycerate kinase coupled with the sequential enzymatic conversion of the monophosphoglycerate to glycerol-3-phosphate and the Oxidation of NADH to NAD (Michal, 1974). The ETS-exposed twin group showed significant reduction in cholesterol level, a decrease in LDL concentration (especially in girls), and an excellent correlation of serum thiocyanate with the number of cigarettes smoked. Because serum tbiocyanate levels are poorly correlated with the measured cotinine levels and since thiocyanate is also known to be present in certain foods, especially leafy vegetables and some nuts (USPH8, 1986), it is possible that this group had significantly different nutritional and dietary habits as compared with the nonsmoking twin group. Such a possibility should be further evaluated as one of the confounding parameters in the future. Nutrient intake is expected to influence overall protein synthesis (Castro, 1987) and could regulate the synthesis of lipoproteins and other polypeptides involved in oxygen transport, which could give the same results as shown. Glantz and Parmley next review a number of animal studies dealing with mitochondrial ultrastructural and biochemical changes of rabbits and guinea pigs upon exposure to carbon monoxide. The carbon monoxide is administered singly for short (less than one hour), intermediate (2 weeks) and long periods - 12 -

I I I iI I i I I i I I I I i i I i I specifically measured by the stoichiometric cleavage of 2,3-DPG to 3-phosphoglyceric acid by diphosphoglycerate kinase coupled with the sequential enzymatic conversion of the monophosphoglycerate to glycerol-3-phosphate and the oxidation of NADN to NAD (Michal, 1974). The ETS-exposed twin group showed significant reduction in cholesterol level, a decrease in LDL concentration (especially in girls), and an excellent correlation of serum thiocyanate with the number of cigarettes smoked. Because serum thiocyanate levels are poorly correlated with the measured cotinine levels and since thiocyanate is also known to be present in certain foods, especially leafy vegetables and some nuts (USPHS, 1986), it is possible that this group had significantly different nutritional and dietary habits as compared with the nonsmoking twin group. Such a possibility should be further evaluated as one of the confounding parameters in the future. Nutrient intake is expected to influence OVerall protein synthesis (Castro, 1987) and could regulate the synthesis of lipoproteins and other polypeptides involved in Oxygen transport, which could give the same results as shown. Glantz and Parmley next review a number of animal studies dealing with mitochondrial ultrastructural and biochemical changes of rabbits and guinea pigs upon exposure to carbon monoxide. The carbon monoxide is administered singly for short (less than one hour), intermediate (2 weeks) and long periods - 12-

I I I i ! I ! I I I II i I i iI I I (more than 2 weeks). The structuEal changes in these animals appear real and significant. However, it is often difficult to extrapolate results of animal studies directly to humans, since different species may not react in an identical fashion to the same external challenge as hypoxia. For example, Bischoff et al. (1969) investigated myocardial ultrastructure in dogs, rabbits, and rats maintained at an altitude of 4,300 m for 5 months; the structural derangements in dogs and rabbits were similar to those found in cattle with high mountain disease, whereas the rats appeared to be only marginally affected. Even among the same animal species, mitochondria isolated from different organs may show remarkably different sensitivity toward mitochondria active chemicals. Muscatello and Carafoli (1969) demonstrated a large stimulation by the nonionic detergent Lubrol on the ability of mitochondria isolated from heart and skeletal muscles of rats to oxidize endogenous and exogenous cytochrcme c by cytochrome c oxidase, while the liver mitochondria failed to respond to the same concentration of Lubrol. PLATELET FUNCTION The next area surveyed by Glantz and Parmley concerns the asserted action of ETS on platelet function. The authors maintain that ETS exposure promotes platelet hyperaggregability and "so increases the likelihood of thrombus formation." In - 13 -

I I I I I I I I I I I I I I I I I I i contrast to this assertion, there is quite an extensive literature to show that smoking actually has no influence on the development of venous thrombosis and in certain situations appears to exert a protective effect. In their study of mortality in relation to smoking in British doctors, Doll and Peto (1976) found no association between mortality from venous thromboembolism and smoking. Additionally, a protective effect of cigarette smoking on venous thromboembolic disease has been noted after myocardial infarction and surgery. For example, Handley and Teather (1974) found that the incidence of thrombosis in the patients with a history of regular smoking within the month before admission was significantly lower than that of nonsmokers. Also, Pollock and Evans (1978) noted in patients undergoing emergency or elective laparotomy for benign or malignant disease or retropubic prostatectomy that cigarette smokers had a significantly lower incidence of deep venous thrombosis than pipe smokers or nonsmokers. The relationship between the effects of cigarette smoking on platelet aggregation and the appearance of non-hematological endothelial cells in circulating blood was next assessed by the authors. Table 2 of Chapter Eleven summarizes the reported effect of ETE exposure and smoking on platelet aggregation ratio and endothelial cell count based on the published results of Davis et al. (1985, 1986, 1989, 1990). It is worth noting that previously Davis and Davis (1979) claimed that a fall in platelet - 14 -

I l I I I I I I I I I ! I I I I I I i aggregate ratio occurred after smoking cigarettes, but this could not be confirmed by others (Rang et al., 1983). In any case, the significant though small reduction in platelet aggregation ratio and the rise in endothelial cell count could not be correlated with "the level of nicotine in the blood of the experimental subjects in any of these or other related studies." To this reviewer, the failure in this correlation would tend to support the lack of importance of nicotine as an active agent in promoting platelet aggregation and increase in endothelial cell count, though the exact opposite conclusion was reached by Glantz and Parmley, who believe the data suggest that "nicotine is an important active agent." Their rationale in arriving such a conclusion simply escapes this reviewer. The authors proceed to state that "since non-tobacco cigarettes also affected platelet aggregation somewhat, however, it is possible that carbon monoxide or some other combustion products are also influencing the platelets." Indeed, platelet aggregation is known to be extremely sensitive and variable. Factors contributing to the variability include: venipuncture technique; the effects of anticoagulants; sodium citrate concentrations; platelet concentration; time interval after venipuncture; pH changes (Triplett, 1978). In addition, the estimation of platelet aggregation ratio can also be rather subtle and requires consistent and precise manipulations at all stages of the platelet aggregation study in order to avoid - 15 -

! ! ! I I I I I I I ! I I I I I ! I I artefacts. Similarly, while circulating endothelial cells may be isolated together with platelets by the method of leucoconcentration, the demonstration of the origin of circulating endothelial cells is not definite (Hladovec and Rossman, 1973). The question of whether platelets derived from smokers versus non-smokers display differential sensitivity toward chemicals affecting platelet aggregatability is also addressed by the authors. The data of Sinzinger and Kefalides (1982) showing that ETS exposure reduced platelet sensitivity to prostacyclin (PGI2) by nearly a factor of 2 in nonsmokers, but only by 20% in smokers, was suggested to reflect that "nonsmokers' platelets seem much more sensitive to a single exposure than do smokers' platelets." However, the authors fail to mention that in the original report of Sinzinger and Kefalides, it was stated that "passive smoking reduced platelet sensitivity to the antiaggregatory PGs, being much more severe in nonsmokers than in smokers. 20 min after passive smoking, platelet sensitivity started to return to basal values and this happened more quickly in nonsmokers." Thus, the quick return to basal sensitivity toward prostacyclin by platelets of nonsmokers, an effect unmatched by platelets from smokers, suggests that the effects of ETS are only transitory and are not expected to cause permanent platelet hyperaggregability. That being the case, the statement that "the resulting increase in platelet aggregation can - 16 -

I l I I I I I I I I ! I I I I I ! I I contribute to acute thrombus formation and myocardial infarction" seems subjective, elusive and speculative. In connection with this discussion, I would point out that there have been numerous studies on the influence of habitual smoking on hemostatic function. Mustard and Murphy [1963) reported that platelet survival was significantly shorter in smokers than in nonsmokers, but they were unable to detect significant differences between the smoking and nonsmoking groups in respect of the whole-blood clotting time, one-stage prothrombin time or the partial thromboplastin time. Older smokers were found by Hawkins (1972) to have platelets which aggregated to a greater extent in response to ADP, but White et al (1983] reported that heavy smokers did not differ from control subjects in respect of platelet aggregation in response to ADP or malondialdehyde production. ATHEROSCLEROSIS The last area covered by Glantz and Parmley in this chapter is the etiology and pathogenesis of atheroscletosis. By way of background, it should be appreciated that despite almost a century of scientific study, the etiology and pathogenesis of atherosclerosis remain unknown. In humans, clinical complications and sequelae occur when the lesions have evolved to produce the fibrous plaque. The main histologic features of this stage are lipid accumulation and fibrobelastic and fibromuscular - 17 -

! ! ! ! ! ! ! ! ! I l I I I I I I I I thickening, which initially are present in a patchy distribution but later become more diffuse as individual plaques coalesce. A fatty streak stage, characterized by lipid accumulation within intimal cells, either of monocyte/macrophage or arterial smooth muscle cell origin, giving them a "foam cell" appearance in histologic sections, is considered by some to represent an intermediate step in the development of the final lesion (McGill, Jr., 1984). Because of the characteristic features of lipid accumulation and intimal thickening, it is generally believed that in humans some form of endothelial injury contributes strongly to the pathogenesis (BOSS, 1986). Much of the endothelial biology today is an attempt to probe the more subtle forms of endothelial dysfunction, since obvious evidence of damage, such as morphologic stigmata at the microscopic level, is not easily found in human observations or animal experiments. In humans, however, clinical and epidemiological studies have uncovered statistically significant risk factors which are associated with advanced disease (Keys, 1970; Doll and Peto, 1976). ~espite these associations however, relatively little light has been shed on the dark shadows of pathogenesis. Moreover, even if the major risk factors are taken into account, collectively they are unable to predict the majority of new cases of the disease (Eliot, 1987). - 18 -

I I I I i I I I i I I I I I I I I I I Over the last decade, two formal hypotheses of atherogenesls have been formulated. The first hypothesis discussed by Glantz and Parmley, starting on page ii of Chapter ii, is what has been termed the response to injury hypothesis of atherosclerosis (ROSS, 1986). This hypothesis suggests that various systemic and local changes occur in the arterial network in association with the different risk factors commonly shown to be related to increased incidence of atherosclerosis, and that these changes result in various forms of injury to the endothelial cell lining the arterial tree. This injury to the endothelium may take several forms because the endcthelium is not only a blood container but a source of vasoactive substance, a permeability barrier, and a nonthrombogenic surface. The results of these various forms of injury may culminate in a series of changes in the endothelium that at one end of the spectrum may lead to minor alterations in functional capacities of the endothelial cells with no apparent morphological alteration, whereas at the other end of the spectrum the changes may result in endothelial cell-cell detachment and cell-connective detachment, leading to oportunities at particular anatomical sites (such as branches and bifurcations) for endothelial cell detachment, desquamation into the blood stream, and exposure of denuded areas of the artery wall. The hypothesis suggests that these denuded areas lead to interactions between elements from the blood (including plasma constituents, platelets, and monocytes) and the artery wall at these sites. - 19 -

i. I i I I I I I i I I I i I I I ! I I This would provide opportunities for mitogenic substances such as platelet-derived growth factor to interact, bind to the tissues, and attract smooth muscle cells from the media into the intima, where they subsequently proliferate and develop into a proliferative, preatberosclerotic lesion. This hypothesis suggests that if the injury is self-limited, the response may be reversible. Thus, a most important feature of the stated hypothesis is endothelial cell injury, at specific anatomical sites, resulting in alteration of its permeability and subsequent detachment of endothelial cells. Although Davis et al. (1986, 1987, 1989, 1990) found that acute exposure to ETS increases the number of anuclear endothelial cell remnants in circulation, the origin of such remnants is unknown (Hladovec and Rossman, 1973). It is possible that there are loosely attached endothelial cell carcasses normally present in various anatomical sites which become dislodged and subsequently detach because of changes in hemodynamic forces in the blood circulation, brought on by nicotine-induced vasoconstriction. In short, ETS exposure does not promote "endothelial cell injury," but may simply play a role in the harmless "shedding" Of endothelial cell fragments which are continuously generated in vivo as a result of the normal arterial network turnover mechanism. Another model of atherogenesis, referred to as the "monoclonal smooth muscle cell hypothesis" (Benditt and Benditt, - 20 -

I I I I I I I I I I I I I I I I I I I 1973) is more focused on th~ nature of the smooth muscle cells located in the atheroclerotic lesion. This hypothesis suggests that plaques are benign smooth muscle cell tumors of the artery wall, and that, through viral or mutational events, the cells are activated to proliferate uncontrollably. Glantz and Parmley seek to link ETS exposure with smooth muscle cell proliferation by citing results from several animal studies in which injection of polycycllc aromatic hydrocarbons was reported to accelerate the development oE atherosclerosis. It should be noted that in a number of these animal studies, the animal species used, namely the chicken, is prone spontaneously to develop large atheroclerotic plaques by one year o~ age [Albert et al., 1977; Penn et al., 1981). Since many chemical carcinogens exhibit species/cell/organ specificity in vivo, and because factors that determine the susceptibility Of a particular cell type to a given carcinogen remain largely unknown, it is unclear whether results obtained from these animal studies may be extrapolated to the human species. Moreover, only 7,12-dimethylbenz(a,h)anthracene (DMBA] is able to increase significantly the number of atherosclerotic plaques. Benzo(a)pyrene, by contrast, is rather ineffective in accelerating the development of atherosclerosis (Albert et al., 1977). Another technical point worth noting is the manner by which these aromatic hydrocarbons were administered, namely, as bolus intramuscularly. It is known that polycyclic aromatic hydrocarbons are metabolized by a complex series of microsomal - 21 -

I I I I I I I I I I I I I I I I I I I and cytoplasmic enzymes that appear to be the major detoxification pathway for xenobiotics. Highly reactive electrophile intermediates of these compounds such as epoxides and diol-epoxides, generated during the detoxification process, react with cellular nucleophiles, including macromolecular targets in the cell. Although the mechanism of action by which the carcinogen/macromolecular interaction initiates the cell toward malignant transformation is uncertain, it is generally accepted that metabolism plays a critical role in the carcinogenic and mutaqenic effects of the polycyclic aromatic hydrocarbons including benzo(a)pyrene. Since ETS is not expected to interact with potential target sites via the intramuscular route, it remains to be determined whether data generated from the animal studies may be applicable in linking ETS with atherosclerosis. The authors next state that "there is also some evidence that ETS directly affects plasma lipoproteins. Moskowitz et al. (1990) 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." This assertion appears to be a gross misrepresentation of the data of Moskowitz et al. For example, ETS exposure clearly reduced the cholesterol concentration from 172.2 to 164.1 for all twins (Moskowitz et al., Table 2), caused no change in boys, and led to a significant lowering in the ETS-exposed group of girls. The LDL/HDL ratio also was lower in the ETS-exposed group of girls. - 22 -

I I I I i I I I ! I I I I I I I I ! I The LDL/HDL ratio was not altered after adjustments were made for age, weight, height and sex. Some decrease in total HDL was noted at a slight significance of p less than 0.05. Perhaps the most interesting paper cited by the authors is the work of Penn et al. (1986), reporting that human coronary artery plaque DNA samples transfected into 3T3 cells gave rise to transformed loci, especially when DNA from cloned loci were used successfully in a second round of transfection. Clearly these results "provide direct evidence for similarities On the molecular level in the development of plaques and tumors." However, Penn et al. (1986) also pointed out that "the three transfection assays we used had three major limitations (i) it selects only dominant genes; (ii) most of the genes identified with this assay to date have been members of the ras complementation group; and (iii) in the case of human samples only 20% of all tumors of a type that would he expected to be positive in this assay (e.g. bladder carcinoma) actually give rise to loci." In addition, the plaques were taken from adult patients in late stages of vascular disease. It should also be noted that although the 3T3 cell is often spoken of as a "normal" cell by those studying transformation (Todaro and Green, 1964), 3T3 cells are different from the fihrohlasts in any mouse in several respects. 3T3 cells divide essentially forever {certainly longer than the lifetime of any mouse) and have lost the perfect diploid assortment of chromosomes. Furthermore, the maximum density to which 3T3 cells will grow is lower than that of - 23 -

I i i I i I I I i I I I ! ! ! I I I I primary mouse embryo fibroblasts (Stoker, 1967) and 3T3 cells are somewhat less readily agglutinated by the lectins concanavalin A and wheat germ agglutinin than are cells of most mouse tissues. Consequently, extrapolation of these data to patbogenesis of diseased states must be approached with great caution. It remains to be determined whether the same DNA would successfully promote proliferation of smooth muscle cells should it prove feasible to incorporate this DNA into the genome of arterial smooth muscle cells. In conclusion, while the urgency to reach an understanding of the etiology and pathogenesis of atherosclerosis, and to obtain a means oE identifying individuals at risk, is indisputable, the existing evidence is inadequate to establish a causal link between ETS and cardiovascular disease. In terms of providing technical information on biologically plausible models associating ETS exposure and atherosclerosis, Chapter Ii has presented only a limited view of what is currently known about this disease. Moreover, the interpretations of many of the findings cited therein are quite subjective and give the appearance of being seriously biased. - 24 -

I I I I I I I I I I iI I I I I I I I ADDITIONAL REFERENCES {NOT LISTED IN THE EPA DOCUMENT) Allred, E.N., Bleecker, E.R., Chaitman, B.R. et al (1989) Short-term effects of carbon monoxide exposure on the exercise performance of subjects with coronary artery disease. New Engl.J.Med. 321, 1426-1432. Bischoff, M.B. Dean, W.D., Bucci, T.J. and Fries, L.A. (1969). Ultrastructural changes in the myocardium of animals after five months at 14,110 feet. Fed. Proc. 2[3], 1268-1273. Brown, M.S. and Goldstein, J.L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34-47. Castro, C.E. (1987). Nutritional Influences on ehromatin: Toxicological implications. In: Nutritional Toxicology (Ed. Hathcock, J.N.) Vol. 2, 129-155. Academic Press, New York. Davis, J.W. and David, R.F. (1979). Acute effects OE tobacco cigarette smoking on the platelet aggregation ratio. Am. J. of Med. Sci. 278, 139-143. Doll, R. and Peto, R. (1976). Mortality in relation to smoking: 20 years' observations on male British doctors. Br. Med. J. 2, 1525-1536. Eatough, D.J., Hansen, L.D. and Lewis, E.A. (~90). The chemical - 28 -

I I I I I I I I I I I I I I I I I I I characterization of ETS. In Environmental tobacco smoke: Proceedings of the International Symposium at McGill University 1989 (Ed. Ecobichon and Wu). D.C. Heath and Co. Lexington, MA. Eliot, R.S. (1987). Coronary artery heart disease : Biobehavioral factors, Overview, in Circulation, Suppl. part 2, vol. 76) J.T. Shepherd, and S.M. Weiss, eds. Fiske, C.H. and SubbaRow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375-378. Handley, A.1. and Teather, D. (1974). Influence Of smoking on deep vein thrombus after myocardial infarction. Br. Med. J. 230-231. 3, Hawkins, R.J. (1972). Smoking, platelets and thrombosis. Nature 236, 450-452. Hladovec, J. and Rossman, P. (1973). Circulating endothelial cells isolated together with platelets and the experimental modification of their counts in rats. Thrombosis Res__.__~. 3, 665-674. Holme, I., Enger, S.C., Helgeland, A. et al (1985). Risk factors - 26 -

I I I I I I I I I I I I I I I I I I I and raised atherosclerotic lesions in coronary and cerebral arteries. Statistical analysis from the Oslo study. Arteriosclerosis i, 250-256. Kanunitz, H. (1988) Adaptive changes in aging and arteriosclerosis: role of cholesterol. Mechanism of A~einq Development 43, 35-43. Keys, A. (1970). Coronary heart disease in seven countries. Circulation 41, I-I. and Leloiur, L.P. and Cardine, C.E. (1957). Characterization of phosphorus compounds by acid lability. Methods in Enzymology 3, 840-850. Michal, G. (1974). D-glycerate-2,3-diphosphate. In : Methods of enzymatic analysis (Ed. Bergmeyer, H.U.) 3, 1432-1438. MoGilI, H.C., Jr. (1984). Persistent problems in the pathogenesis of atherosclerosis. Arteriosclerosis 4, 443-451. Mustard, J.F. and Murphy, E.A. (1963). Effects of smoking on blood coagulation and platelet survival in man. Br. Med. J. i, 846-849. Nilsson, J. (1985). Growth factors and the pathogenesis of atherosclerosis. Atherosclerosis 62, 185-199. - 27 -

I I I i I I ! I I I I I i I I I I I I Muscatello, J. and Carafoli, E. (1969). The oxidation of exogenous and endogenous cytochrome c in mitochondria. Cell. Biol. 40, 602-621. J_.~. Pollack, A.V. and Evans, M. (1978). Cigarette smoking and postoperative deep vein thrombosis. Br. Med. d. 3, 637. Ring, T., Kristensen, S.D., Jenson, P.N., MouritsAndersen, T., Madsen, H. and Dyerberg, J. (1983). Cigarette smoke shortens the bleeding time. Thrombosis Res. 32, 531-536. Stoker, M. (1967). Contact and short-range interactions affecting growth of animal cells in culture. In: Current Topics in Dev. Biol. (ed. A.A. Moscona and A. Monroy) Vol. 2, P.IOS. Academic Press, New York. Todaro, G., Green, H. and Goldberg, B. (1964). Transformation properties of an established cell line by SV40 and polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 51, 66-69. Triplett, D.A. (1978) Platelet function laboratory evaluation and clinical application. Am. Soc. of Clin. Pathologists. White, P., Blackman, B., Tsou, W. and Finkel, D. (1983). Platelet function in chronic cigarette smokers and in insulin dependent diabetics. Thrombosis and Homeostasis 58, 449-453. - 28-