Chapter I

GENERAL INTRODUCTION

1. INTRODUCTION

     The techniques of growing animal cells in culture were originally attempted more than 80 years ago (Harrison, 1907). Different in vitro techniques have since been developed for maintaining animal cells in culture, including organ cultures, explant cultures, outgrowth cultures, primary cell cultures and the development of stable cell lines. Other important methods include co-culture models and systems utilizing cell cultures growing on porous membranes.

     Particle toxicity has been studied using in vivo and in vitro systems. Both systems have their limitations, such as cost, sensitivity, relevance to human health, while each provide an important component of the continuously growing repository of toxicological information. One relevant area with respect to particle toxicology is the effects of ambient airborne particles towards respiratory tissue. The cellular interaction of different resident and transitory cell types (macrophages, neutrophils, epithelial and structural cells) can mediate the potential toxicity of these particles in the alveoli. In an attempt to model the dynamics of these effects, experiments have been carried out to generate a model of particle/cell dynamics in the mammalian alveoli. This thesis examines the use of cell culture models in studying the potential effects of exposure of the alveoli to ambient airborne particles.

2. TOXICOLOGICAL RELEVANCE OF AMBIENT AIRBORNE PARTICLES

     The toxic potential of ambient airborne particles is presently gaining much interest in the field of pulmonary research. Ambient airborne particles are carbon or silica-based materials present in the ambient atmosphere which are mainly produced by natural or man-made processes such as combustion, e.g. from automobiles, industry, residential heating (Sideropoulos and Specht, 1994; Stevens et al., 1990). This material can range from visible to the naked eye, to sub-micronic in size. Upon inhalation, this material has the potential to deposit in the airways and alveoli (Vincent, 1990).

     Exposure to silica based particles (e.g. quartz, asbestos) is most prevalent in occupational exposure either as chronic or single acute particle exposures, while other non-fibrous particles present in ambient air may play a more important role in environmental pulmonary toxicology due to the presence of other chemical and elemental material. These particles contain polycyclic aromatic hydrocarbons (PAH's) and metals which may become bioavailable upon deposition in the lungs (Menichini, 1992). This bioavailability may also be elevated following phagocytosis by the alveolar macrophage. As well, chemical compounds may be biotransformed into mutagenic or carcinogenic species by the cytochrome P450 enzymes present in the pulmonary cells. These reactive chemical species can cause DNA damage which may lead to the production of pulmonary tumours (Hext, 1994).

     The duration of exposure to airborne pollutants can be in the order of seconds (e.g. gases) to days (e.g. particles deposited in the alveoli). Different exposure patterns related to time and dose can therefore lead to different levels of structural and functional damage. It is generally accepted that particles with a diameter of less than 3-5 µm have the ability to become deposited in the alveoli (Menichini, 1992; Wheeler, 1990; Seemayer et al., 1988). Once deposited, particles are phagocytosed by the wandering macrophages and transported out of the lungs via the mucociliary escalator. However, this mechanism of removal usually takes 2 to 3 days (Vincent, 1990). As well, some particles can remain embedded in the alveoli for several hundred days (Vincent, 1990). This long term exposure can result in toxicity towards the resident epithelial cells, endothelial cells and fibroblasts and transitory macrophage populations of the alveoli. Due to the presence of potentially toxic particles in the atmosphere, it is important to develop methodologies for assessing the toxicity of this material.

3. OVERVIEW OF PARTICLE TOXICITY IN THE MAMMALIAN LUNG

3.1. Structural and Functional Alterations Caused by Airborne Particles

3.1.1. In Vivo Studies

     The study of particle toxicity in the mammalian alveoli is relevant to human health due to the potential for environmental exposure. Inhalation of hazardous respirable particles has been the focus of several animal studies with a substantial portion of work aimed at identifying the detrimental effects of silica based particles. This particulate material has been shown to cause structural damage (Arden and Adamson, 1992; Donaldson et al., 1992; Cantin et al., 1988) and increased cell product synthesis such as cytokines and phospholipids (Li et al., 1992; Panos et al., 1992; Suabe et al., 1991). Brief inhalation of asbestos fibres has also been shown to cause increased epithelial and interstitial cell proliferation in mice (Brody and Overby, 1989; Brody et al., 1989).

     Other work has also been carried out to characterize the responses of the lungs to carbon-based materials such as coal-fly ash and diesel exhaust particles. Different indicators such as increased macrophage recruitment (Geiser et al., 1994) and DNA adduct formation (Gallagher et al., 1994; Bond et al., 1990) have been examined. It has also been shown that homogenates of rat and mouse lungs can bioactivate extracts of collected particulate material (van Houdt et al., 1988). This has important implications with respect to the biotransformation of adsorbed particle components to mutagenic and carcinogenic species. Although important, these studies do not allow for repeatable examination of the effects of collected particles on the mammalian lung. It is difficult to carry out representative in vivo studies with collected airborne particles due to the normally small quantity collected (milligrams).

3.1.2. In Vitro Studies

3.1.2.1. Macrophages

     Many in vitro studies have examined the cytotoxicity of particulate material towards alveolar macrophages. Several studies have demonstrated that asbestos and silicate particles are cytotoxic towards the alveolar macrophage (Schimmelpfeng and Seidel, 1992; Adamis and Krass, 1991; Kandaswami et al., 1988; Nadeau et al., 1987; Tasat and de Rey, 1987) and have the ability to elicit superoxide production by macrophages (Vallyathan et al., 1992; Mossman et al., 1989) and bronchoalveolar leukocytes (Vallyathan et al., 1992; Donaldson et al., 1988). Other studies have focussed on non-mineral particles which are closely related to ambient airborne particles. It has been determined that particles produced from combustion processes (Mumford et al., 1986) and coal-fly ash (Kondo et al., 1993; Hatch et al., 1985) are cytotoxic towards alveolar macrophages. As well, particulate material collected as road dust caused a decrease in expression of the macrophage Fc receptor (Ziegler et al., 1994) which has implications in immune response and antigen presentation.

3.1.2.2. Fibroblasts

     Fibroblasts have also been used to study the toxic effects of particles. Rat lung fibroblasts have been shown to undergo lipid peroxidation and disintegration of the cell membrane, and to experience reduced growth and increased cytotoxicity when treated with certain types of asbestos (Iguchi et al., 1993; Wydler et al., 1988). Lipid peroxidation appears to be associated with the structure and the iron content of the fibre (Wydler et al., 1988; Brown et al., 1986). It has also been shown that the interaction of mineral fibres with the membrane of fibroblasts (V79-4 cells) results in the production of free radicals causing peroxidative damage (Brown et al., 1986).

3.1.2.3. Epithelial Cells of the Tracheal and Alveoli

     The effects of environmental pollutants have also been studied using monolayer cultures of airway epithelial cells. Exposure of a tracheal epithelial cell line to asbestos has revealed some important mechanisms of fibre toxicity to the airways (Hesterberg et al., 1987). It was shown that fibres cause little membrane damage to tracheal epithelial cells, but induce damage through a process of binding, phagocytosis and disruption of the normal activities of the cell (Hesterberg et al., 1987). This study also determined that particle dimensions and surface charge are important factors in particle toxicity.

     Alveolar type II cells in culture have been used to study the effects of airborne particles towards epithelial cells. These cells comprise 5% of the total epithelial population in the alveoli, are responsible for the production of pulmonary surfactant, and differentiate into type I cells (remaining 95% of epithelium) following alveolar damage (Chevalier and Collet, 1972; Adamson and Bowden, 1974). Following direct exposure to low levels of silica, there was an initiation of cellular repair and growth while high doses resulted in direct cytotoxicity (Lesur et al., 1992). It appears that the surface properties of silica dictate this cytotoxicity as aluminum coated silica had much lower effects on normal cellular processes (Lesur et al., 1992). Silica has also been shown to cause increases in the permeability of type II cells following exposure in vitro (Merchant et al., 1990). This may indicate one of the mechanisms by which silicosis occurs. As well, cultures of type II cells have been shown to sequester large quantities of synthesized protein into the extracellular matrix following exposure to coal and mine dusts (Lee et al., 1994).

3.2. Chemical Messengers Involved in Particle Toxicity

3.2.1. Overview of Cytokine Activity

     Cytokines are extracellular polypeptides secreted by specific effector cells which act to cause changes in growth and biosynthetic activity in nearby cells (Figure 1.1). The concept of cytokines and the study of their role in mammalian systems has been developing since the early 1950's (Levi-Montalcini and Hamburger, 1951). In the last 20 years, an increasing amount of research has been carried out to understand the mechanisms by which cytokines exert their influences on cellular processes. The term cytokine includes interleukins, interferons, colony-stimulating factors and peptide growth factors (Nathan and Sporn, 1991). It should be noted that the cytokines differ from the endocrinehormones in that cytokines are secreted at very low concentrations and act upon local cell populations (Kelley, 1990).

     The homeostatic balance in the lung is most likely controlled by the presence of local acting cytokines. These bioreactive peptides are produced by different cells of the lung including epithelial and endothelial cells, fibroblasts and macrophages (Karmiol et al., 1993; Boylan et al., 1992; Kelley 1990). These cytokines are necessary for proper cell growth, tissue maintenance, remodelling and repair. Their expression must be under tight control to maintain normal cellular function.
  

Figure 1.1:
Cytokine Pathways

     Cytokines secreted from a specific effector cell can have several different modes of action, as represented in Figure 1.1. Their primary modes of action are through either paracrine or autocrine mechanisms. Paracrine activity is the process in which a secreted cytokine acts upon target cells which are different from the effector cell and autocrine activity is where a secreted cytokine acts upon cells identical to the effector cell. Juxtacrine cell signalling is when an effector cell expresses the cytokine on its surface, and the target cell must come into direct contact with the effector cell to undergo stimulation by the surface bound molecule. A final important pathway is one in which the cytokine is never released from the effector cell but interacts with a specific receptor inside the original cell. This process is termed intracrine signalling.

     The growth state of the target cell population can play an important role in determining the mode of action of some circulating cytokines. For example, IFN-g can stimulate quiescent fibroblasts to proliferate, while proliferating cultures of the same cell type are inhibited to grow and divide in the presence of IFN-g (Elias et al., 1987). The maturational state of the cells, the regulation of cytokine receptors (Sporn and Roberts, 1988), cytokine synergism or antagonism, and the regulation of secondary cytokine production pathways by the target cells can alter their response to cytokines (Kelley, 1990).

3.2.2. Directed Research

     The macrophage produces growth factors for both type II and fibroblast cells and may be important in modulating fibroproliferative response following particulate exposure (Driscoll et al., 1993; Perkins et al., 1993; Kumar et al., 1992; Adamson et al., 1991a). As well, the type II cells appear to stimulate fibroblast growth when the cells are in direct contact, but produce inhibitory factors for fibroblasts when compartmentalized using tissue culture inserts (Adamson et al., 1991b). It was shown that fibroblasts can stimulate growth in type II cells (Adamson et al., 1991b).

     Cytokines from pulmonary cells can be important in the orchestration of an inflammatory response (for a review of the leukocyte-endothelial cell adhesion cascade see Albelda et al., 1994 & Pilewski and Albelda, 1993). Following particulate exposure, the alveolar macrophage can produce large quantities of soluble cytokines which are released into the surrounding alveolar space (Bauman et al., 1990; Driscoll et al., 1993; Perkins et al., 1993; Schapira et al., 1991). It is possible that these cytokines could play a role in recruitment of other inflammatory cells in the leukocyte- endothelial cell adhesion cascade, stimulate the induction of epithelial cell proliferation for repair of alveolar damage or signal the onset of alveolar fibrosis by stimulation of the fibroblasts. In recent studies, the information regarding the ability of particle-stimulated macrophages to alter fibroblast growth has been contradictory (summarized in Kumar et al., 1992). It is believed that differences in the growth state of the target cells (fibroblasts) may be responsible for some of the discrepancies in these results (Kumar et al., 1992; Adamson et al., 1991a).

4. POTENTIAL TECHNIQUES FOR STUDYING PARTICLE DYNAMICS

4.1. Direct Co-Culture Systems

     Co-culture systems are important in vitro tools as they allow for the study of a single stimulus on several different cell types. One of the primary focuses of co-culture research in pulmonary toxicology has been the study of the effects of activated neutrophils on other pulmonary cells. Activated neutrophils have been shown to cause inhibition of normal phosphatidylcholine synthesis in type II cell cultures (Zimmerman and Lewandoski, 1991). Mineral fibres have also been shown to cause activation of neutrophils. Neutrophils from the lungs of animals exposed to silica were shown to induce macrophages to produce a fibroblast growth factor (Li et al., 1992). Quartz and asbestos can also stimulate neutrophils to cause detachment injury and direct cytotoxicity to type II cells in co-culture (Donaldson et al., 1988). This detachment injury was attributed to the breakdown of fibronectin, laminin and collagen by the activated neutrophils (Donaldson et al., 1988). These results may give some insight into possible mechanisms of induction of silicosis and pronounced fibrosis of the lungs following silica exposure.

     Alterations in arachidonic acid synthesis and metabolism have also been studied using cells maintained in co- culture. Addition of activated neutrophils to cultures of type II cells resulted in increased production of certain leukotrienes by the type II cells due to the production of neutrophil-derived leukotriene A₄ (Grimminger et al., 1992). Some mechanisms of normal arachidonic acid synthesis have also been studied using type II cells and macrophages in co-culture (Peters-Golden and Feyssa, 1993).

4.2. Transmembrane Culture Systems

     Mangum and colleagues (1990) have presented an in vitro model of the alveolar system. Primary type II cells and early passage lung fibroblasts were plated on opposite sides of a collagen-coated polycarbonate filter. The epithelial layer produced a tight barrier on the upper surface of the membrane, preventing the flux of albumin, trypan blue, PDGF and alpha₂-macroglobulin (Mangum et al., 1990). The model was subjected to taurine chloramine, which has been previously shown to alter endothelial and epithelial cell permeability (Shasby and Hampson, 1989). Exposure to taurine chloramine caused a dose-dependent flux in albumin across the membrane indicating disruption of the epithelial monolayer (Mangum et al., 1990). Macrophages were also added to the epithelial compartment and monitored for morphological characteristics. The macrophages were seen to remain viable in this system with some of the macrophages becoming spread out on the epithelial monolayer (Mangum et al., 1990). This model allows the study of epithelial integrity, but also studies regarding macrophage phagocytosis and the production of factors that have activities towards type II cells. If an applied stimulus activates macrophages and compromises the epithelial barrier, this model could be used to study interstitial fibrosis under controlled conditions in vitro.

     Another study was carried out to determine the reciprocal effects of fibroblasts and type II cells isolated from developing fetal and adult lungs and cultured in a transmembrane model (Adamson et al., 1991b). Using tissue-culture inserts, direct epithelial/fibroblast cell interactions and supernatant interactions were studied using both fetal and adult cells. It was found that supernatants from fibroblast cultures caused increased proliferation of type II cells, while type II cell supernatants caused a decrease in fibroblast proliferation. When the two cell types were placed in direct co- culture, fibroblast growth rates increased indicating some mechanism requiring direct cellular contact. Alteration in phosphatidylcholine synthesis was also observed in the type II cells. Production of phosphatidylcholine was increased when type II cells were exposed to fibroblasts that had been previously exposed to hydrocortisone, and lamellar bodies were detected in areas of direct epithelial/fibroblast cell contact. These results give insight into the cell-mediated control mechanisms present in the mammalian lung and demonstrate the differences in direct and indirect cell/cell interactions. These interactions are believed to play a role in fetal lung development and in the response of the lung to epithelial damage (Adamson et al., 1991b).

4.3. Transgenic Cell Lines

4.3.1. Introduction of Foreign DNA into Mammalian Cells

     Gene transfer is a methodology that is routinely used to study gene structure and function, and to identify the regulatory sequences for gene expression (Watson et al., 1992). Several different methodologies have been developed for the transient transfection of different cell types. One of the simplest methods is microinjection, consisting of quantitative delivery of DNA into the nucleus of the target cell. Due to the small size of cells in culture, this technique is almost exclusively used for introducing DNA into the large, fertilized ova (DePamphilis et al., 1988). Another method for introducing foreign DNA into cells is the use of DEAE (diethylaminoethyl) coated-dextran particles (McCutchan and Pagano, 1968). When DNA and these particles are incubated together, the negatively-charged DNA associates with the positively-charged DEAE. Application of the DNA-DEAE complex to cultured cells results in its binding to the cell membrane and uptake of the DNA-DEAE complex by endocytosis (Watson et al., 1992).

     As well, it has been shown that DNA precipitated with calcium phosphate is readily taken up by mammalian cells (Chen and Okayama, 1988). Another method, electroporation, consists of brief electrical stimulation of cellsuspensions which results in the production of leaky cell membranes and the transient uptake of DNA (Andreason and Evans, 1988; Shigekawa and Dower, 1988). Liposomes have also been used to deliver DNA to target cells: DNA is placed inside lipid membranes which fuse with the cell surface, delivering the DNA into the cytoplasm (Mannino and Gould-Fogerite, 1988). The technique of lipofection allows the targeting of the DNA to specific cell types and has potential uses in gene therapy. While these methods are useful for the transient expression of foreign genes, they do not generally lead to the production of cell lines which stably maintain the transfected DNA in their genome (Watson et al., 1992).

     One of the most useful techniques for permanently introducing foreign DNA into mammalian cells is the use of retroviruses (Eglitis and Anderson, 1988). Retroviruses are RNA viruses that convert their RNA genome to DNA by reverse transcription and permanently insert this DNA into the genome of the host cell. The inserted DNA or provirus is then reproduced along with the genomic DNA of the host cell resulting in the production of more virus particles (Watson et al., 1992). Using this technology, retroviral vectors have been produced which allow insertion of a particular DNA sequence as well as a marker sequence (CAT, lacZ) into an engineered viral vector. This technique is very useful as it provides a means to introduce stable DNA sequences into the genome of many different types of mammalian cells. When this DNA is linked to a marker enzyme (CAT, lacZ), the cells containing the marker sequences can be detected using different immunological and biochemical analyses.

4.3.2. Directed Research Using CAT Cell Lines

     Transgenic cell lines containing exogenous gene regulatory sequences linked to the reporter enzyme chloramphenicol acetyltransferase (CAT) have been used to study several different induction processes. CAT is a bacterial enzyme not produced in mammalian cells. By linking mammalian promoter or enhancer sequences to this CAT gene and introducing the constructs into mammalian cells, in vitro and in vivo models of gene induction have been developed. Due to the mammalian cells not producing endogenous CAT protein, any increase in intracellular levels of CAT mRNA or protein indicates activation of the transgenic promoter or enhancer sequences.

     The activity of the cytochrome P4501A1 (CYP1A1) promoter sequence has been studied by linking the 5' upstream DNA sequence from this gene to a reporter sequence coding for CAT (Xu et al., 1993). Following assembly of this construct, it was transfected into the rat hepatoma cell line, H4IIE. This methodology was also compared to the standard biochemical analysis of cytochrome P450 activity, 7-ethoxyresorufin-O-deethylase (EROD). Both methods showed high levels of homology when used to study the ability of different chemical standards (benzo(a)pyrene, 3- methylcholanthrene, tetrachlorodibenzofuran) to induce CYP1A1 activity in these cells (Xu et al., 1993). Promoter- CAT constructs have also been generated which incorporate the xenobiotic responsive element (XRE) found within the 5' upstream region of the cytochrome P450 gene (Kubota et al., 1991). These constructs were also found to be responsive to methylcholanthrene exposure when tested in the Hepa-1 cell line (Kubota et al., 1991).

     CAT transgenes have also been assembled to study the inducibility of the glutathione-S-transferase (GSTYa) gene. Phenobarbital, an inducer of cytochrome P450, can induce constructs containing the GST responsive element, EpRE, in hepatoma cells (Pinkus et al., 1993). As well, this GST responsive element was inducible following exposure to planar aromatic compounds, phenolic antioxidants and hydrogen peroxide (Nguyen et al., 1993). Transfection of this responsive element-CAT construct into HepG2 cells resulted in the production of an inducible gene construct as demonstrated following 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure (Nguyen et al., 1994).

     CAT transgenes have also been used to study the inducibility of several other different genes including nuclear factor kappa B (NFKb) (Daffada et al., 1994; Finch and Baldwin, 1993; Ray and Ray, 1993), the c-fos oncogene (Uberall et al., 1994; Osei-Frimpong et al., 1994) and heat shock protein 70 (HSP70) (Perry et al., 1994). All of these CAT transgenes are useful tools for studying the inducibility and steady state expression of particular genes when working with either in vivo or in vitro systems. For in vivo systems, it is also possible to generate transgenic animals for the study of site-specific gene induction, site-specific levels of basal gene expression, as well as the levels of gene expression during development. From a toxicological perspective, the transgenic cell lines can be useful in vitro tools for studying gene induction following toxicant exposure. These cell lines provide a method for determining the potential of chemical compounds to induce specific mammalian genes (CYP1A1, GSTYa, etc) and can be used to rank different environmental samples with respect to xenobiotic or stress response in the cell lines.

5. THESIS OBJECTIVES

5.1. Working Hypothesis

     Phagocytosis of ambient airborne particles by bronchoalveolar macrophages can produce alterations in macrophage activity as well as enhance the availability of adsorbed particle components to other adjacent cells present in the alveoli.

5.2. Rationale

     The lung is a complex organ that requires the maintenance of a homeostatic balance to maintain proper function. A key to understanding the potential hazardous nature of airborne particles should be through the use of in vitro co- culture models which incorporate bronchoalveolar macrophages as effector cells and suitable target cells. This type of model attempts to recreate the alveolar environment and measures alterations in an interacting cell system, rather than in individual cells.

     Ambient airborne particles contain several different components that can have adverse effects on cells of the lungs. Once deposited in the lungs, chemicals present in the particle can solubilize in the extracellular lining fluid. This can result in direct chemical toxicity towards the cells of the airways and alveoli. As well, trace elements such as metals can be directly cytotoxic or catalyze Fenton reactions resulting in the production of reactive oxygen species in the lung. Also, biological material such as endotoxin or bacterial spores present in the air may play an important role in modulating cellular activity.

     As mentioned, the key cell involved in the engulfment and removal of deposited material in the lungs is the alveolar macrophage. Removal of deposited material by the macrophages is carried out by 1) initial membrane contact, 2) engulfment and 3) digestion. Deposited particles have been shown to exert different effects on macrophage activity. Engulfment and attempted digestion, in the case of silica-based particles can be directly cytotoxic. The attempted digestion by the macrophages can produce large amounts of oxygen-based reactive molecules (H₂O₂, O₂^-, ^.OH) which can be cytotoxic towards the effector cell, towards surrounding macrophages, as well as to cells of the alveoli, e.g. epithelial cells, endothelial cells, fibroblasts. In the case of airborne particulate material, the presence of endotoxins, metals and adsorbed chemicals can cause a poisoning of the macrophage resulting in reduced particle clearance. All of these effects can cause a reduction in the removal of particulate material from the lung, which may contribute to lung damage.

     A more plausible explanation for alterations in lung function following particle exposure is a shift in this homeostatic balance in the lungs. Particle toxicity can proceed by several different mechanisms including direct particle toxicity, production of damaging molecules (reactive oxygen species) or growth factors (inhibitory/stimulatory), liberation of adsorbed or soluble particulate material by the macrophage as well as possible bioactivation of adsorbed chemicals by the macrophage or other resident cell types. At levels not causing macrophage cytotoxicity, a situation may develop in which large amounts of particles are phagocytozed by the resident and infiltrating population of alveolar macrophages. In its attempt to enzymatically destroy the foreign agent, the macrophages could solubilize chemicals (PAHs, other organics) and metals from the particles, making them more bioavailable to the cells of the lung. These processes could potentially lead to increased levels of free metals and organic chemicals in the lung resulting in increased chemical toxicity and free radical production. Therefore, with the use of a co-culture model incorporating bronchoalveolar macrophages as effector cells and suitable target cells that can be potentially monitored for induction following particulate exposure, it may be possible to rank different particles with respect to their ability to induce the target cells.

     What is proposed is the use of CAT-transgenic cell lines as target cells following exposure to culture medium conditioned by macrophages which have been treated with particles. This assay system has been developed using a commercial panel of human liver cell lines (HepG2) that have had several different gene promoter sequences inserted into their genome, resulting in the production of fourteen transgenic cell lines (Cat-Tox assay, Xenometrix). The cell lines respond to different stimuli including metals, PAHs, antioxidants, cytokines as well as indicators of altered cellular processes including DNA damage and changes in intracellular cAMP levels (MacGregor et al., 1995). Following exposure to particles, and depending on their chemical composition (hydrocarbon content, metals, endotoxin), the transgenic cell lines will respond differently, giving a characteristic fingerprint of the biological reactivity of the test material. This will not only give an indication of whether or not there is a response, but it may also help to identify what the cells are responding to (cytokines, antioxidants, increased free metals or PAHs).

     To model this activity in the lung, particles were incubated with bronchoalveolar macrophages in cell culture inserts and the conditioned culture medium outside the insert was then collected. The exposed macrophages were assayed for cellular viability (alamarBlue). As well, particles or macrophages were incubated alone in the inserts to serve as controls. The collected supernatants were frozen until applied to the transgenic cell lines. Increases in transgene expression following exposure of HepG2 transgenic cell lines to culture medium conditioned by macrophages incubated with particles, as compared to those from macrophages or particles alone, were attributed to modulation of particle toxicity, element availability or macrophage induction/suppression. These results may give insights into possible mechanisms of particle toxicity in the lungs in vivo.

5.3. Specific Objectives

1. To use a series of cell culture models to determine the cytotoxicity of total particulate preparations as well as soluble and insoluble particulate fractions.

2. To rank a series of particles with respect to their ability to cause induction of CAT transgene cell lines using particulate standards (NIST 1648, NIST 1649, EHC-93, TiO₂) and known chemical inducers (benzo(a)pyrene, cadmium sulfate).

3. To use a sequential exposure model to compare the pattern of gene induction of different particle preparations before and after incubation with alveolar macrophages to study the ability of the macrophage to alter the availability of adsorbed particle components such as metals and PAHs.