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Designing in vitro assay systems for hazard characterization. Basic strategies and related technical issues
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 57 (2005) 227–232
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Designing in vitro assay systems for hazard characterization. Basic strategies and related technical issues Yuzo Hayashi Biological Safety Research Center, National Institute of Health Sciences, Japan Received 25 April 2005
Abstract Adverse effects of chemicals on humans are typically assessed following four steps: hazard identification, hazard characterization, exposure assessment and risk characterization. Hazard characterization is defined as the qualitative and/or quantitative evaluation of the nature of the adverse effects associated with biological, chemical and physical agents of interest. For chemical agents, hazard characterization is based on a series of in vitro and/or in vivo data obtained from mechanistic, kinetic and dose–response studies on the agent of concern, which are analyzed and integrated for extrapolation to eventually match human conditions. Thus, an accurate experimental design and the development of test methods capable of generating data relevant to hazard characterization are essential for the useful risk assessment of chemicals, including inhaled materials. It should, however, be stressed that hazard characterization has widely been limited to single chemicals. The hazard characterization of airborne mixtures therefore poses a new problem in toxicology, which calls for a novel approach to its scientific assessment. During the last three decades, a number of epidemiological and experimental studies have been conducted focusing on two kinds of inhaled complex mixtures, namely cigarette smoke and diesel exhausts. A new approach to the assessment of airborne complex mixtures may be elaborated through the appropriate, combined use of the findings of such studies. In this context, the present review article is intended to illustrate some basic strategies for and technical issues related to the hazard characterization of inhaled complex mixtures, thereby taking up representative epidemiological and experimental data from published papers on tobacco smoke. r 2005 Elsevier GmbH. All rights reserved. Keywords: In vitro assay systems; Hazard characterization; Review; Epidemiological studies; Experimental studies
Review of existing information Central Laboratory of Experimental Animals, Unomori 1-30-2-
711, Sagamihara, 228-0801 Kanagawa, Japan. Fax: 0081 42 746 3591. E-mail address: [email protected]. 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.05.012
Epidemiological studies indicate a significant association between exposure to tobacco smoke and occurrence or promotion of cancers, chronic obstructive pulmonary disease, cardiovascular disease and low birth weight. A
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Y. Hayashi / Experimental and Toxicologic Pathology 57 (2005) 227–232
number of relevant publications on these tobaccorelated diseases and adverse health conditions have been reviewed during the present study. The results are used as a basis for the designing of in vitro tests for the toxicological evaluation of inhaled mixtures.
Cancer It is estimated that 20% of all cancers worldwide are attributable to smoking (Parkin et al., 1999). Epidemiological studies indicate that the most common cancers related to tobacco smoking occur in the lung and oropharyngeal area. This observation is consistent with the physiological consideration that tobacco smoke exposes the entire respiratory mucosa and the upper gastrointestinal mucosa to carcinogens. Lung cancer can be classified into four major histological types: squamous cell carcinoma, small-cell carcinoma, adenocarcinoma and large-cell carcinoma. It has long been assumed that squamous cell carcinoma of the lung is related to tobacco smoke, but there has been a shift in the prevalence of histological types over time, in which adenocarcinoma has been increasing relative to squamous cell carcinoma (Charloux et al., 1997). Squamous cell carcinoma of the lung is preceded by a series of histopathological changes of bronchial/bronchiolar epithelia, i.e. hyperplasia, metaplasia, dysplasia and carcinoma in situ (Auerbach et al., 1961). These histopathological changes occur far less frequently in never-smokers than in cigarette smokers. Their occurrence increases with the amount of cigarettes smoked (Auerbach et al., 1979). Oropharyngeal cancers including cancers arising in the oral cavity, pharynx and larynx are also preceded by sequential occurrence of preneoplastic lesions, such as keratosis, dysplasia and carcinoma in situ. Smoking cessation does not remove the potential for progression of this type of cancer (Gillis et al., 1983). Tobacco smoke is known to contain more than 100 carcinogens and mutagens with different potencies and effects. Therefore, strategies of risk assessment for tobacco smoke should include the prioritized identification of carcinogens. A recent review of cigarette smoke constituent data suggested that tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) are the two classes of compounds with the highest human cancer-risk potential. This proposal is mainly derived from experimental and epidemiological studies on individual chemical compounds. However, tobacco smoke contains a number of compounds with different activities and synergies. It is well known that carcinogenesis is a multifactorial and multistage process involving activation of oncogenes, inactivation of cancer suppressor genes, modification of hormonal and immunological function, or triggering of inflammation. Therefore, the evaluation of
all carcinogenic compounds would be required for a comprehensive risk assessment of tobacco smoke.
Chronic obstructive pulmonary disease (COPD) COPD is characterized by airflow limitation that is not fully reversible. The airflow limitation is in most cases both progressive and associated with an abnormal inflammatory response of the lungs to either noxious particles or gases (Pauwels et al., 2001). Cigarette smoking is regarded as the main but not exclusive cause of COPD. Exposure to indoor pollution or biomass fuels can produce identical health problems (PerezPadilla et al., 1996). Cigarette smoking is estimated to contribute to 80–90% of cases of COPD (Novotny and Giovino, 1998). Epidemiological studies on cigarette smoking in women have indicated that the prevalence of COPD appears to follow the prevalence of smoking by 20–30 years (Tanoue, 2000). The term COPD encompasses chronic obstructive bronchitis with obstruction of small airways, emphysema with enlargement of air spaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways (Barnes, 2000). COPD in the form of either emphysema, bronchitis or both is a well-recognized consequence of cigarette smoking (Vial, 1986). In chronic bronchitis, an inflammatory response caused by chronic exposure to airborne toxins (tobacco smoke, air pollutants, dust) is the central pathogenic mechanism (Barnes, 2000). Inflammation leads to edema, cellular infiltration, fibrosis, smooth muscle hypertrophy and mucous hypersecretion that narrow the bronchioles. The inflammatory basis of COPD is now well established. Studies with induced sputum and bronchoalveolar lavage have shown that, compared with smokers without airflow obstruction, COPD patients have higher amounts of macrophages and neutrophils contained in these fluids (Calverley and Walker, 2003). Evidence from human and laboratory studies suggests that an oxidant–antioxidant imbalance in favor of oxidants occurs in COPD. Oxidants in patients of COPD are presumably derived from oxygen free radicals generated in both the gas–vapor phase and the particle phase of tobacco smoke (Zang et al., 1995). Inflammation itself induces oxidant stress in the lung of smokers through a greater generation of oxygen free radicals by leucocytes (Morrison et al., 1999). The pathological examination of the alveolar regions of smokers’ lungs has shown increases in the number and percentage of leucocytes, as compared to nonsmokers (Hunninghake and Crystal, 1983). Considerable evidence supports the protease–antiprotease theory, which holds that pulmonary emphysema is caused by excessive exposure to elastolytic enzymes in connection with inhibitors of these enzymes (Barnes,
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2000). Tobacco smoke promotes injury of the lungs by increasing the proteolytic burden and compromising the antiproteolytic defenses, thus leading to a breakdown of the lung structure. Most attention has been given to neutrophil elastase and proteinase 3, which can produce emphysema in laboratory animals. Evidence of elastin degradation in patients of COPD is provided by the greater excretion of desmosine, derived from elastin cross-links (Gottlieb et al., 1996). The major antiproteolytic protein in the lower respiratory tract is a1antitrypsin (a1-AT), which inhibits neutrophil elastase (Senior and Shapiro, 1998). There is increasing evidence that matrix metalloproteinases derived from macrophages and neutrophils are involved (Shapiro and Senior, 1999). An increase in the activity of matrix metalloproteinase-1 and matrix metalloproteinase-2 in the lung parenchyma has been observed in patients of emphysema (Ohnishi et al., 1998). Sustained airflow obstruction associated with an accelerated decline in lung function occurs in only a minority of tobacco smokers (approximately 15% of whites and 5% of Asians) (Barnes, 2000). Genetic factors are likely to be major determinants of susceptibility to COPD, and genes implicated in the pathogenesis of COPD are divided into four categories according to their function: (1) antiproteolysis, (2) xenobiotic metabolism, (3) inflammation and (4) mucociliary clearance (Sandford and Pare, 2000).
Cardiovascular disease It has been well documented that smoking significantly increases the risk for many types of cardiovascular morbidity including myocardial infarction, sudden cardiac death and peripheral vascular disease (Green et al., 1993). There is a dose–response relationship between the number of cigarettes smoked and the incidence of cardiovascular events (Thun et al., 1997). The risk of cardiac ischemic events is rapidly reversible upon smoking cessation (USDHHS, 1983). The World Health Organization (WHO) has reported that 1 year after quitting, the risk of coronary heart diseases (CHD) decreases by 50%, and with 15 years, the relative risk of health from CHD for an ex-smoker reaches that of long-time nonsmokers. A prospective autopsy study indicates that there is a strong dose-related association of smoking with atherosclerosis of the coronary arteries (Reed et al., 1987). Prospective epidemiological studies have consistently demonstrated a substantial increase in acute ischemic events in individuals who smoke (USDHHS, 1983). The mechanisms that associate tobacco smoking with coronary heart disease are divided into two kinds, i.e. (i) mechanisms by which smoking accelerates atherosclerosis, and (ii) mechanisms by which smoking promotes acute ischemic events including myocardial infarction and sudden death.
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Vascular endothelial dysfunction plays a central role in the acceleration of atherosclerosis in chronic smokers (Heitzer et al., 1996). Impaired endothelium-dependent vasodilation in both forearm and coronary vascular beds has been observed even in young smokers. Structural endothelial damage may either be caused by toxic tobacco smoke constituents or result from smoking-induced oxidative stress (Heitzer et al., 1996). Smoking is associated with oxidized abnormalities, such as increased levels of oxidized low-density lipoprotein, an increase of triglyceride or a reduction of high-density lipoprotein levels that may contribute to the development or acceleration of atherosclerosis (Duthie et al., 1993). Smoking has been shown to increase monocyte adhesion to endothelial cells (Adams et al., 1997). Inhaled carbon monoxide (CO) in tobacco smoke bonds with hemoglobin, displacing the oxygen and thus reducing oxygen delivery to myocardial cells. Therefore, CO decreases anginal threshold (Allred et al., 1989). It should also be known that CO has a deleterious effect on vascular endothelium (Thun et al., 1997).
Low birth weight The adverse effect of smoking on birth weight (fetal tobacco syndrome) has been extensively studied (Nieburg et al., 1985). Among pregnant smokers the risk of low birth weight babies is doubled, as compared to nonsmokers, and about 20% of all low birth weight babies are attributable to smoking (USDHHS, 1983). The effect of smoking is particularly pronounced if exposure occurs after the third month of pregnancy. A prospective intervention study indicated that smoking reduction or cessation as determined by serum cotinine levels leads to significantly increased birth weight, as compared to women who continue smoking (Li et al., 1993). It has been further shown that women who stop smoking no later than 20 weeks before gestation have the lowest risk of developing fetal tobacco syndrome (Secker-Walker et al., 1998). The pathogenesis of fetal tobacco syndrome is assumed to be multifactorial, and there is evidence that CO plays a major role in growth retardation through increased tissue hypoxia (Benowitz et al., 2000). An animal study suggests a possible role of nicotine in reducing placental blood flow-through by causing vasoconstriction (Bassi et al., 1984).
Strategies and technical issues Preparation and application of test materials Tobacco smoke is a complex mixture comprising more than 4000 chemical constituents, and many kinds of chemicals contained in either the gas–vapor phase or
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the particle phase of tobacco smoke are known to cause lung injuries. Oxidants and aldehydes, two major classes of compounds found in the gas–vapor phase of tobacco smoke, are known to injure alveolar epithelial cells and macrophages. Therefore, a number of chemical compounds found in either phase of cigarette mainstream smoke need to be tested in order to assess the risk of cigarette smoking. The technical issues related to the proper design and execution of these tests include the standardization of methods for the preparation of test materials and their application to the test systems.
Designing test systems In vitro toxicity assays are used to determine the general toxic properties of a chemical or a chemical mixture, usually focusing on their cytotoxicity. The cytotoxicity of a compound can be predictive of its ability to induce inflammation; the genotoxicity of a compound is an indicator of its potential to induce cancer. Thus, the main technical issues in designing test systems include the selection of appropriate cell lines and the development of methods to evaluate the effects of test materials on the cells. Cytotoxicity The cells that line alveoli, bronchioli or bronchi are directly exposed to high concentrations of tobacco smoke. Therefore, cell lines derived from human alveolar epithelial cells such as A-549 cells (Alveolar type II cell-derived cell line) are considered to be appropriate targets. Alveolar epithelial cells are known to have multiple functions, i.e. the production of surfactant proteins to reduce alveolar surface tension, the release of cytokines and growth factors to regulate inflammation and cell growth and the release of matrix protein, proteinases and proteinase inhibitors to regulate turnover of alveolar structure. Therefore, damage to alveolar epithelial cells may induce inflammation, increase epithelial permeability, decrease surfactant synthesis and cause inappropriate production of cytokines, ultimately leading to pulmonary edema, alveolar collapse or destruction of alveolar structures. Injury of the alveolar epithelial cells by tobacco smoke presumably constitutes an important process involved in the pathogenesis of tobacco-related pulmonary diseases (Hoshino et al., 2001). Cytotoxic effects are evaluated by means of the following assays: (1) cell viability assays (e.g. neutral red/trypanblue exclusion method); (2) morphological examination (counting of living cells, apoptotic cells and necrotic cells); (3) qualitative or quantitative DNA fragmentation assays to confirm apoptosis; and (4) intracellular oxidative activity assays. An early response to inhaled materials including tobacco smoke leads to the mobilization of macro-
phages to the lung for phagocytosis of the materials. Phagocytosis of tobacco smoke-derived particles is a defense mechanism of toxic particulate fraction clearance. Pathological alterations of the lung have been observed to grow with the amount and duration of smoking. Examinations of the lungs of young smokers have revealed the accumulation of macrophages in the bronchiolar regions in association with bronchiolitis (Jeffery, 1998). Macrophages appear more pronounced in the lungs of smokers and patients of COPD, as they accumulate in the alveoli, bronchioli and in the small airways. There is a positive association between the amount of macrophages and the occurrence of mild to moderate emphysema in the patients of COPD (Jeffery, 1998). In addition to phagocytosis, macrophages have many other diverse functions in maintaining pulmonary homeostasis, including the production of multiple chemical mediators such as cytokines and growth factors (Tetley, 2002). Thus, either activation of or damage to macrophages may alter homeostasis of the lung. Macrophages may be activated by tobacco smoke and other irritants as to release neutrophil chemotactic factors such as leukotrien B4 and interleulkin-8. Neutrophils and macrophages release multiple proteinases that break down the connective tissue in the lung parenchyma, thus causing emphysema (Barners, 2000). Macrophages are long-lived effector cells located within the lung. The life span is known to increase in smokers (Tetley, 2002). Taking the long life span into consideration, together with various physiological functions and pathogenetical roles in COPD, macrophages can be selected as target cells for in vitro systems applied to tobacco smoke. Genotoxicity Carcinogenesis is considered to be a multistage process that includes initiation, promotion and progression. The first and last stages of neoplastic development, i.e. initiation and progression are assumed to involve direct structural changes in the genome. At the intermittent stage, promotion does not appear to involve direct structural changes in the genome but depends on the altered expression of genes (Boyd and Barrett, 1990). Thus, it is assumed that the biological effects of initiation and progression are irreversible, while those of promotion are operationally reversible. In view of this consensus, an appropriate strategy seems to be to focus on genotoxicity assays for the human cancerrisk evaluation of inhaled mixtures, including tobacco smoke. Practically, genetic toxicity assays generally used for pharmaceuticals may be applied for tobacco smoke and other inhaled mixtures, provided that appropriate methods are developed for the preparation and application of test materials. Tobacco-related cancers are known to most frequently originate from epithelial cells of respiratory mucosa or oropharyngeal mucosa, and
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therefore, it is recommendable to use some of these epithelial cells in mammalian cytogenetic assays and DNA damage or repair assays.
Effects on endothelial cells The effects of smoking on the cardiovascular system have been studied mainly in terms of the interaction of nicotine and CO with the myocardium, but there still remains a possibility that some other smoke constituents are involved in originating smoking-induced cardiovascular disease. To study the effects of tobacco smoke on vascular endothelial cells can be proposed as an important project in near future.
In vitro assays and hazard characterization Risk characterization is the last step of risk assessment that combines information from exposure assessment and hazard characterization in order to create a suitable basis for decision-making for risk management (Renwick et al., 2003). As mentioned before, hazard characterization is based on a series of information from mechanistic, toxico-kinetic and dose–response studies. In vitro tests were formerly used merely as screening tools to determine the toxic properties of chemicals. Recent advances in molecular biology enable however the application of in vitro assays in the mechanistic analysis of the toxicological effects of chemicals. At the same time, it is necessary to understand the limitations of in vitro assays, as the results are based on the response of single cell types and do not include the influence of the whole-body system on the response. Thus, a proper combination of data from well-designed in vitro, in vivo and toxico-kinetic studies is necessary to obtain useful information for hazard characterization.
References Adams MR, Jessup W, Celermajer DS. Cigarette smoking is associated with increased human monocyte adhesion to endothelial cells: reversibility with oral L-arginine but not vitamin C. J Am Coll Cardial 1997;29. Allred EN, Bleecker ER, Chaitman BR, et al. Short-term effects by carbon monoxide exposure on the exercise performance of subjects with coronary artery disease. New Engl J Med 1989;321:1426. Auerbach O, Stout AP, Hammond EC. Changes in bronchial epithelium in relation to cigarette smoking. New Engl J Med 1961:252–6. Auerbach O, Hammond EC, Garfinkel L. Changes in bronchial epithelium in relation to cigarette smoking, 1955–1960 vs. 1970–1977. New Engl J Med 1979;300:381–5. Barnes PJ. Chronic obstructive pulmonary disease. New Engl J Med 2000;343:269–80.
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Bassi JA, Rosso P, Mocssinger AC, et al. Fetal growth retardation due to maternal tobacco smoke exposure in the rat. Pediatr Res 1984;18:127–30. Benowitz NL, Dempsey DA, Goldenberg RL, et al. The use of pharmacotherapies for smoking cessation during pregnancy. Tob Control 9 Suppl 2000;3:91–4. Boyd JA, Barrett JC. Genetic and cellular basis of multi step carcinogenesis. Pharmacol Ther 1990;46:469–86. Calverley PMA, Walker P. Chronic obstructive pulmonary disease. Lancet 2003;362:1053–61. Charloux QE, Wolkove N, et al. The increasing incidence of lung adenocarcinoma: reality on artifact? A review of the epidemiology of lung adenocarcinoma. Int J Epidemiol 1997;26:14–23. Duthie GG, Arthus JT, Beattie JA, et al. Cigarette smoking, antioxidants, lipid peroxidation, and coronary heart disease. Ann NY Acad Sci 1993;686. Gillis TM, Incze J, Strong MS, et al. Natural history and management of keratosis, atypia, carcinoma in situ, and microinvasive cancer of the larynx. Am J Surg 1983;146: 512–6. Gottlieb DJ, Stone PJ, Sparrow D, et al. Urinary desmosine excretion in smokers with and without rapid decline of lung function: the Normative Aging Study. Am J Respir Crit Care Med 1996;154:1290–5. Green F, Hamsten A, Blomback M, et al. The role of betafibrinogen genotype in determining plasma fibrinogen levels I young survivors of myocardial infarction and healthy controls from Sweden. Thromb Haemost 1993;70:915–20. Heitzer T, Just H, Munzel T. Antioxidant vitamin C improves endothelial dysfunction in chronic smokers. Circulation 1996;94. Hoshino Y, Mio T, Nagai S, et al. Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line. Am J Physiol Lung Cell Mol Physiol 2001;281: L509–16. Hunninghake GW, Crystal RG. Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 1983;128:833–8. Jeffery PK. Structural and inflammatory changes in COPD: a comparison with asthma. Thorax 1998;53:129–36. Li CQ, Windsor RA, Perkins L, et al. The impact on infant birth weight and gestational age of cotinine validated smoking reduction during pregnancy. J Am Med Assoc 1993;269:1519–24. Morrison D, Rahman I, Lannan S, et al. Epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers. Am Respir Crit Care Med 1999;159:473–9. Nieburg P, Marks JS, Mclaren NM, et al. The fetal tobacco syndrome. J Am Med Assoc 1985;253:2998–9. Novotny TE, Giovino GE. Tobacco Use. In: Brownson RE, Renington PL, Davis JT, editors. Chronic disease epidemiology and control. 2nd ed. Washington, DC: American Public Health Association Press; 1998. p. 117–48. Ohnishi K, Takagi M, Kurokawa Y, et al. Matrix metalloproteinase mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 1998;78:1077–87. Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin 1999;49:33–64.
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Pauwels RA, Buist AS, Calverley PMA, et al. Global strategy for diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHIBI/WHO Global Initiative Workshop Summary. Am J Respir Crit Care Med 2001;163:1256–76. Perez-Padilla R, Regaldo J, Vedak S, et al. Exposure to biomass smoke and chronic airway disease in Mexican women: a case control study. Am J Respir Crit Care Med 1996;154:701–6. Reed DM, MacLean CJ, Hayashi T. Predictors of atherosclerosis in the Honolulu Heart Program: I. biologic, dietary and lifestyle characteristics. Am J Respir Crit Care Med 1987;126:214. Renwick AG, Barlow SM, Hertz-Picciotto L, et al. Risk characterization of chemicals in food and diet. Food Chem Toxicol 2003:1221–71. Sandford Aj, Pare PD. Genetic risk for chronic obstructive pulmonary disease. Clin Chest Med 2000;21:633–43. Secker-Walker RH, Vacek PM, Flyun BS, et al. Estimated gains in birth weight associated with reductions in smoking during pregnancy. J Reprod Med 1998;43:967–74. Senior RM, Shapiro SD. Chronic obstructive lung disease: epidemiology, pathophysiology, and pathogenesis. In: Fishman A, Elias J, Hishman J, et al. editors. Fishman’s
pulmonary diseases and disorders. 3rd ed. New York: Mcgraw-Hill; 1998 p. 680. Shapiro SD, Senior RM. Matrix metalloproteinases: matrix degradation. Am J Respir Cell Mol Biol 1999;20:1100–2. Tanoue LT. Cigarette smoking and women’s respiratory health. Clin Chest Med 2000;21:47–65. Tetley TD. Macrophages and the pathogenesis of COPD. Chest 2002;121:156–9. Thun MJ, Myers DG, Day-Lally C, et al. Age and exposure–response relationships between cigarette smoking and premature death in cancer prevention study II. National Cancer Institute. Changes in cigarette-related disease risk and their implication for prevention and control. NCI Smoking and Tobacco Control Monograph, vol. 8. Washington, DC, NCI, 1997. p. 383–475. USDHHS (US Department of Health and Human Services). The health consequences of smoking; cardiovascular disease; a report of the Surgeon General 1983. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention. Vial WC. Cigarette smoking and lung disease. Am J Med Sci 1986;291:130–42. Zang LY, Stone K, Pryor WA. Detection of free radicals in aqueous extracts of cigarette tar by electron spin resonance. Free Radic Biol Med 1995;146:888–94.
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EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Designing in vitro assay systems for hazard characterization. Basic strategies and related technical issues Yuzo Hayashi Biological Safety Research Center, National Institute of Health Sciences, Japan Received 25 April 2005
Abstract Adverse effects of chemicals on humans are typically assessed following four steps: hazard identification, hazard characterization, exposure assessment and risk characterization. Hazard characterization is defined as the qualitative and/or quantitative evaluation of the nature of the adverse effects associated with biological, chemical and physical agents of interest. For chemical agents, hazard characterization is based on a series of in vitro and/or in vivo data obtained from mechanistic, kinetic and dose–response studies on the agent of concern, which are analyzed and integrated for extrapolation to eventually match human conditions. Thus, an accurate experimental design and the development of test methods capable of generating data relevant to hazard characterization are essential for the useful risk assessment of chemicals, including inhaled materials. It should, however, be stressed that hazard characterization has widely been limited to single chemicals. The hazard characterization of airborne mixtures therefore poses a new problem in toxicology, which calls for a novel approach to its scientific assessment. During the last three decades, a number of epidemiological and experimental studies have been conducted focusing on two kinds of inhaled complex mixtures, namely cigarette smoke and diesel exhausts. A new approach to the assessment of airborne complex mixtures may be elaborated through the appropriate, combined use of the findings of such studies. In this context, the present review article is intended to illustrate some basic strategies for and technical issues related to the hazard characterization of inhaled complex mixtures, thereby taking up representative epidemiological and experimental data from published papers on tobacco smoke. r 2005 Elsevier GmbH. All rights reserved. Keywords: In vitro assay systems; Hazard characterization; Review; Epidemiological studies; Experimental studies
Review of existing information Central Laboratory of Experimental Animals, Unomori 1-30-2-
711, Sagamihara, 228-0801 Kanagawa, Japan. Fax: 0081 42 746 3591. E-mail address: [email protected]. 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.05.012
Epidemiological studies indicate a significant association between exposure to tobacco smoke and occurrence or promotion of cancers, chronic obstructive pulmonary disease, cardiovascular disease and low birth weight. A
ARTICLE IN PRESS 228
Y. Hayashi / Experimental and Toxicologic Pathology 57 (2005) 227–232
number of relevant publications on these tobaccorelated diseases and adverse health conditions have been reviewed during the present study. The results are used as a basis for the designing of in vitro tests for the toxicological evaluation of inhaled mixtures.
Cancer It is estimated that 20% of all cancers worldwide are attributable to smoking (Parkin et al., 1999). Epidemiological studies indicate that the most common cancers related to tobacco smoking occur in the lung and oropharyngeal area. This observation is consistent with the physiological consideration that tobacco smoke exposes the entire respiratory mucosa and the upper gastrointestinal mucosa to carcinogens. Lung cancer can be classified into four major histological types: squamous cell carcinoma, small-cell carcinoma, adenocarcinoma and large-cell carcinoma. It has long been assumed that squamous cell carcinoma of the lung is related to tobacco smoke, but there has been a shift in the prevalence of histological types over time, in which adenocarcinoma has been increasing relative to squamous cell carcinoma (Charloux et al., 1997). Squamous cell carcinoma of the lung is preceded by a series of histopathological changes of bronchial/bronchiolar epithelia, i.e. hyperplasia, metaplasia, dysplasia and carcinoma in situ (Auerbach et al., 1961). These histopathological changes occur far less frequently in never-smokers than in cigarette smokers. Their occurrence increases with the amount of cigarettes smoked (Auerbach et al., 1979). Oropharyngeal cancers including cancers arising in the oral cavity, pharynx and larynx are also preceded by sequential occurrence of preneoplastic lesions, such as keratosis, dysplasia and carcinoma in situ. Smoking cessation does not remove the potential for progression of this type of cancer (Gillis et al., 1983). Tobacco smoke is known to contain more than 100 carcinogens and mutagens with different potencies and effects. Therefore, strategies of risk assessment for tobacco smoke should include the prioritized identification of carcinogens. A recent review of cigarette smoke constituent data suggested that tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) are the two classes of compounds with the highest human cancer-risk potential. This proposal is mainly derived from experimental and epidemiological studies on individual chemical compounds. However, tobacco smoke contains a number of compounds with different activities and synergies. It is well known that carcinogenesis is a multifactorial and multistage process involving activation of oncogenes, inactivation of cancer suppressor genes, modification of hormonal and immunological function, or triggering of inflammation. Therefore, the evaluation of
all carcinogenic compounds would be required for a comprehensive risk assessment of tobacco smoke.
Chronic obstructive pulmonary disease (COPD) COPD is characterized by airflow limitation that is not fully reversible. The airflow limitation is in most cases both progressive and associated with an abnormal inflammatory response of the lungs to either noxious particles or gases (Pauwels et al., 2001). Cigarette smoking is regarded as the main but not exclusive cause of COPD. Exposure to indoor pollution or biomass fuels can produce identical health problems (PerezPadilla et al., 1996). Cigarette smoking is estimated to contribute to 80–90% of cases of COPD (Novotny and Giovino, 1998). Epidemiological studies on cigarette smoking in women have indicated that the prevalence of COPD appears to follow the prevalence of smoking by 20–30 years (Tanoue, 2000). The term COPD encompasses chronic obstructive bronchitis with obstruction of small airways, emphysema with enlargement of air spaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways (Barnes, 2000). COPD in the form of either emphysema, bronchitis or both is a well-recognized consequence of cigarette smoking (Vial, 1986). In chronic bronchitis, an inflammatory response caused by chronic exposure to airborne toxins (tobacco smoke, air pollutants, dust) is the central pathogenic mechanism (Barnes, 2000). Inflammation leads to edema, cellular infiltration, fibrosis, smooth muscle hypertrophy and mucous hypersecretion that narrow the bronchioles. The inflammatory basis of COPD is now well established. Studies with induced sputum and bronchoalveolar lavage have shown that, compared with smokers without airflow obstruction, COPD patients have higher amounts of macrophages and neutrophils contained in these fluids (Calverley and Walker, 2003). Evidence from human and laboratory studies suggests that an oxidant–antioxidant imbalance in favor of oxidants occurs in COPD. Oxidants in patients of COPD are presumably derived from oxygen free radicals generated in both the gas–vapor phase and the particle phase of tobacco smoke (Zang et al., 1995). Inflammation itself induces oxidant stress in the lung of smokers through a greater generation of oxygen free radicals by leucocytes (Morrison et al., 1999). The pathological examination of the alveolar regions of smokers’ lungs has shown increases in the number and percentage of leucocytes, as compared to nonsmokers (Hunninghake and Crystal, 1983). Considerable evidence supports the protease–antiprotease theory, which holds that pulmonary emphysema is caused by excessive exposure to elastolytic enzymes in connection with inhibitors of these enzymes (Barnes,
ARTICLE IN PRESS Y. Hayashi / Experimental and Toxicologic Pathology 57 (2005) 227–232
2000). Tobacco smoke promotes injury of the lungs by increasing the proteolytic burden and compromising the antiproteolytic defenses, thus leading to a breakdown of the lung structure. Most attention has been given to neutrophil elastase and proteinase 3, which can produce emphysema in laboratory animals. Evidence of elastin degradation in patients of COPD is provided by the greater excretion of desmosine, derived from elastin cross-links (Gottlieb et al., 1996). The major antiproteolytic protein in the lower respiratory tract is a1antitrypsin (a1-AT), which inhibits neutrophil elastase (Senior and Shapiro, 1998). There is increasing evidence that matrix metalloproteinases derived from macrophages and neutrophils are involved (Shapiro and Senior, 1999). An increase in the activity of matrix metalloproteinase-1 and matrix metalloproteinase-2 in the lung parenchyma has been observed in patients of emphysema (Ohnishi et al., 1998). Sustained airflow obstruction associated with an accelerated decline in lung function occurs in only a minority of tobacco smokers (approximately 15% of whites and 5% of Asians) (Barnes, 2000). Genetic factors are likely to be major determinants of susceptibility to COPD, and genes implicated in the pathogenesis of COPD are divided into four categories according to their function: (1) antiproteolysis, (2) xenobiotic metabolism, (3) inflammation and (4) mucociliary clearance (Sandford and Pare, 2000).
Cardiovascular disease It has been well documented that smoking significantly increases the risk for many types of cardiovascular morbidity including myocardial infarction, sudden cardiac death and peripheral vascular disease (Green et al., 1993). There is a dose–response relationship between the number of cigarettes smoked and the incidence of cardiovascular events (Thun et al., 1997). The risk of cardiac ischemic events is rapidly reversible upon smoking cessation (USDHHS, 1983). The World Health Organization (WHO) has reported that 1 year after quitting, the risk of coronary heart diseases (CHD) decreases by 50%, and with 15 years, the relative risk of health from CHD for an ex-smoker reaches that of long-time nonsmokers. A prospective autopsy study indicates that there is a strong dose-related association of smoking with atherosclerosis of the coronary arteries (Reed et al., 1987). Prospective epidemiological studies have consistently demonstrated a substantial increase in acute ischemic events in individuals who smoke (USDHHS, 1983). The mechanisms that associate tobacco smoking with coronary heart disease are divided into two kinds, i.e. (i) mechanisms by which smoking accelerates atherosclerosis, and (ii) mechanisms by which smoking promotes acute ischemic events including myocardial infarction and sudden death.
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Vascular endothelial dysfunction plays a central role in the acceleration of atherosclerosis in chronic smokers (Heitzer et al., 1996). Impaired endothelium-dependent vasodilation in both forearm and coronary vascular beds has been observed even in young smokers. Structural endothelial damage may either be caused by toxic tobacco smoke constituents or result from smoking-induced oxidative stress (Heitzer et al., 1996). Smoking is associated with oxidized abnormalities, such as increased levels of oxidized low-density lipoprotein, an increase of triglyceride or a reduction of high-density lipoprotein levels that may contribute to the development or acceleration of atherosclerosis (Duthie et al., 1993). Smoking has been shown to increase monocyte adhesion to endothelial cells (Adams et al., 1997). Inhaled carbon monoxide (CO) in tobacco smoke bonds with hemoglobin, displacing the oxygen and thus reducing oxygen delivery to myocardial cells. Therefore, CO decreases anginal threshold (Allred et al., 1989). It should also be known that CO has a deleterious effect on vascular endothelium (Thun et al., 1997).
Low birth weight The adverse effect of smoking on birth weight (fetal tobacco syndrome) has been extensively studied (Nieburg et al., 1985). Among pregnant smokers the risk of low birth weight babies is doubled, as compared to nonsmokers, and about 20% of all low birth weight babies are attributable to smoking (USDHHS, 1983). The effect of smoking is particularly pronounced if exposure occurs after the third month of pregnancy. A prospective intervention study indicated that smoking reduction or cessation as determined by serum cotinine levels leads to significantly increased birth weight, as compared to women who continue smoking (Li et al., 1993). It has been further shown that women who stop smoking no later than 20 weeks before gestation have the lowest risk of developing fetal tobacco syndrome (Secker-Walker et al., 1998). The pathogenesis of fetal tobacco syndrome is assumed to be multifactorial, and there is evidence that CO plays a major role in growth retardation through increased tissue hypoxia (Benowitz et al., 2000). An animal study suggests a possible role of nicotine in reducing placental blood flow-through by causing vasoconstriction (Bassi et al., 1984).
Strategies and technical issues Preparation and application of test materials Tobacco smoke is a complex mixture comprising more than 4000 chemical constituents, and many kinds of chemicals contained in either the gas–vapor phase or
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the particle phase of tobacco smoke are known to cause lung injuries. Oxidants and aldehydes, two major classes of compounds found in the gas–vapor phase of tobacco smoke, are known to injure alveolar epithelial cells and macrophages. Therefore, a number of chemical compounds found in either phase of cigarette mainstream smoke need to be tested in order to assess the risk of cigarette smoking. The technical issues related to the proper design and execution of these tests include the standardization of methods for the preparation of test materials and their application to the test systems.
Designing test systems In vitro toxicity assays are used to determine the general toxic properties of a chemical or a chemical mixture, usually focusing on their cytotoxicity. The cytotoxicity of a compound can be predictive of its ability to induce inflammation; the genotoxicity of a compound is an indicator of its potential to induce cancer. Thus, the main technical issues in designing test systems include the selection of appropriate cell lines and the development of methods to evaluate the effects of test materials on the cells. Cytotoxicity The cells that line alveoli, bronchioli or bronchi are directly exposed to high concentrations of tobacco smoke. Therefore, cell lines derived from human alveolar epithelial cells such as A-549 cells (Alveolar type II cell-derived cell line) are considered to be appropriate targets. Alveolar epithelial cells are known to have multiple functions, i.e. the production of surfactant proteins to reduce alveolar surface tension, the release of cytokines and growth factors to regulate inflammation and cell growth and the release of matrix protein, proteinases and proteinase inhibitors to regulate turnover of alveolar structure. Therefore, damage to alveolar epithelial cells may induce inflammation, increase epithelial permeability, decrease surfactant synthesis and cause inappropriate production of cytokines, ultimately leading to pulmonary edema, alveolar collapse or destruction of alveolar structures. Injury of the alveolar epithelial cells by tobacco smoke presumably constitutes an important process involved in the pathogenesis of tobacco-related pulmonary diseases (Hoshino et al., 2001). Cytotoxic effects are evaluated by means of the following assays: (1) cell viability assays (e.g. neutral red/trypanblue exclusion method); (2) morphological examination (counting of living cells, apoptotic cells and necrotic cells); (3) qualitative or quantitative DNA fragmentation assays to confirm apoptosis; and (4) intracellular oxidative activity assays. An early response to inhaled materials including tobacco smoke leads to the mobilization of macro-
phages to the lung for phagocytosis of the materials. Phagocytosis of tobacco smoke-derived particles is a defense mechanism of toxic particulate fraction clearance. Pathological alterations of the lung have been observed to grow with the amount and duration of smoking. Examinations of the lungs of young smokers have revealed the accumulation of macrophages in the bronchiolar regions in association with bronchiolitis (Jeffery, 1998). Macrophages appear more pronounced in the lungs of smokers and patients of COPD, as they accumulate in the alveoli, bronchioli and in the small airways. There is a positive association between the amount of macrophages and the occurrence of mild to moderate emphysema in the patients of COPD (Jeffery, 1998). In addition to phagocytosis, macrophages have many other diverse functions in maintaining pulmonary homeostasis, including the production of multiple chemical mediators such as cytokines and growth factors (Tetley, 2002). Thus, either activation of or damage to macrophages may alter homeostasis of the lung. Macrophages may be activated by tobacco smoke and other irritants as to release neutrophil chemotactic factors such as leukotrien B4 and interleulkin-8. Neutrophils and macrophages release multiple proteinases that break down the connective tissue in the lung parenchyma, thus causing emphysema (Barners, 2000). Macrophages are long-lived effector cells located within the lung. The life span is known to increase in smokers (Tetley, 2002). Taking the long life span into consideration, together with various physiological functions and pathogenetical roles in COPD, macrophages can be selected as target cells for in vitro systems applied to tobacco smoke. Genotoxicity Carcinogenesis is considered to be a multistage process that includes initiation, promotion and progression. The first and last stages of neoplastic development, i.e. initiation and progression are assumed to involve direct structural changes in the genome. At the intermittent stage, promotion does not appear to involve direct structural changes in the genome but depends on the altered expression of genes (Boyd and Barrett, 1990). Thus, it is assumed that the biological effects of initiation and progression are irreversible, while those of promotion are operationally reversible. In view of this consensus, an appropriate strategy seems to be to focus on genotoxicity assays for the human cancerrisk evaluation of inhaled mixtures, including tobacco smoke. Practically, genetic toxicity assays generally used for pharmaceuticals may be applied for tobacco smoke and other inhaled mixtures, provided that appropriate methods are developed for the preparation and application of test materials. Tobacco-related cancers are known to most frequently originate from epithelial cells of respiratory mucosa or oropharyngeal mucosa, and
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therefore, it is recommendable to use some of these epithelial cells in mammalian cytogenetic assays and DNA damage or repair assays.
Effects on endothelial cells The effects of smoking on the cardiovascular system have been studied mainly in terms of the interaction of nicotine and CO with the myocardium, but there still remains a possibility that some other smoke constituents are involved in originating smoking-induced cardiovascular disease. To study the effects of tobacco smoke on vascular endothelial cells can be proposed as an important project in near future.
In vitro assays and hazard characterization Risk characterization is the last step of risk assessment that combines information from exposure assessment and hazard characterization in order to create a suitable basis for decision-making for risk management (Renwick et al., 2003). As mentioned before, hazard characterization is based on a series of information from mechanistic, toxico-kinetic and dose–response studies. In vitro tests were formerly used merely as screening tools to determine the toxic properties of chemicals. Recent advances in molecular biology enable however the application of in vitro assays in the mechanistic analysis of the toxicological effects of chemicals. At the same time, it is necessary to understand the limitations of in vitro assays, as the results are based on the response of single cell types and do not include the influence of the whole-body system on the response. Thus, a proper combination of data from well-designed in vitro, in vivo and toxico-kinetic studies is necessary to obtain useful information for hazard characterization.
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