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Direct exposure methods for testing native atmospheres
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Experimental and Toxicologic Pathology 57 (2005) 213–226
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Direct exposure methods for testing native atmospheres Michaela Aufderheide Department of In Vitro Toxicology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany Received 25 April 2005
Abstract In vitro studies of adverse cellular effects induced by inhalable substances face a number of problems due to the difficulties in exposing cultured cells of the respiratory tract directly to test atmospheres composed of complex gases and particulate compounds. This paper discusses the characteristics of in vitro work and summarizes the use of different in vitro technologies to determine the adverse effects of inhaled pollutants. The exposure of cells to test atmospheres requires accurate control of the pollutant levels, as well as the close contact of cells and gas without interfering with the medium. Systems which rely on the solution of the gas in the medium overlay do not resemble the exposure conditions in vivo, and may not be suitable for studying, for example, the effects of poorly soluble gases. Exposure to gases or complex mixtures can be performed with roller bottles or flasks on rotating and rocking platforms and, using these techniques, the cells are periodically exposed to the test atmosphere. However, the most promising approach is based on a biphasic cell culture technique, where cells are grown on microporous membranes at an air–liquid interface. Here the cells are nutrified from the basal side of the membrane whilst the apical part with the cultivated cells is in direct contact with the test atmosphere. Based on this culture technique, different exposure systems have been developed and these are described and discussed. Exposure of cells from the respiratory tract to gases or particles is responsible for cell injury or cell activation associated with an overexpression of mRNA and the release of bioactive mediators. Therefore, in vitro studies using such a strategy, in combination with relevant and efficient exposure devices, open up new ways to test native complex gases and aerosols. Furthermore, such an experimental approach is not only suitable for cultivated cells, but it can also be used for exposing bacteria to inhalable test compounds. It is possible to analyze the mutagenic potency of in- and outdoor pollutants and several attempts have been made to determine the induction of revertants in a modified Ames assay after exposure to single gases or complex mixtures. r 2005 Elsevier GmbH. All rights reserved. Keywords: Air/liquid interface; Direct exposure methods; Review; CULTEXs; Quantitative data; Modified ames assay
Introduction
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E-mail address: [email protected]. 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.05.019
The analysis of complex mixtures like ambient air, diesel exhaust, environmental tobacco smoke or cigarette smoke itself represents a multifactorial problem due to the complexity of the test atmosphere. In most cases,
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they consist of a particulate and gas phase, including hundreds of substances, short- and long-living radicals, which undergo chemical reactions after generation. It is well known that the mixture changes both qualitatively and quantitatively in a short time (Pryor, 1992, 1997). Therefore, toxicological investigations should be carried out under conditions resembling the in vivo situation as far as possible. The exposure of lung cells to complex atmospheres or single components like ozone is responsible for a cascade of cellular reactions, initiating the development of chronic lung disorders or even cancer. Ozone for example, an atmospheric photochemical of major concern because of its deleterious health effects upon inhalation, has been shown to result in alterations in lung function, development of bronchial hyperresponsiveness, loss of alveolar Type I cells, an influx of inflammatory neutrophils into the airways, increased epithelial and vascular permeability, a decrease in the mitogen-induced T-cell proliferation, and increased cell mortality resulting from aerolized microbial agents delivered after exposure (Coffin and Gardner, 1972; Evans, 1984; Koren et al., 1989; Orlando et al., 1989; Seltzer et al., 1986). The responses of lung cells to toxic gases are difficult to explain. To understand the functional and pathological disorders induced in the respiratory tract requires the investigation of the direct effects of pollutants on the state and activity of lung cells. So far, three approaches have been used: (1) animal experiments, (2) ex vivo studies of cells of bronchial lavage or biopsies and (3) in vitro systems of exposure of lung cells to pollutants under controlled conditions (Rasmussen, 1984; Wallaert et al., 2000). This paper emphasizes the evolution of the in vitro approaches which offer the possibility to expose cells of the respiratory tract directly to native atmospheres.
In vitro exposure systems for inhalable compounds Requirements The development of in vitro methods for the exposure of lung cells has to follow some principle requirements (Rasmussen, 1984): 1. Controlled generation and monitoring of the test atmosphere to study dose-dependent effects. 2. The contact between the cells and the test compounds should be as close as possible to avoid interactions, for example of oxidant gases with medium components, and to realize particle deposition and, in the case of complex mixtures, direct contact of cells and gas phase components.
3. The exposure systems should be designed to allow significant exposure times. In general, the exposure of cultivated cells to ambient atmospheres rapidly results in their inactivation due to drying. Therefore, methods had to be developed to maintain a humidified atmosphere or to moisten the cells in a specific way. 4. After exposure, representative endpoints considered to be predictive, as shown by in vivo studies or human evidence, should be analyzed as indicators of the toxicological effect.
Exposure using roller bottles and rocking or rotating platforms The simplest method is to bubble the gas through a cell suspension or to flush the culture flask with the pollutant. This technique was used, for example, to expose alveolar macrophages to NO2 (Voisin et al., 1974) or ozone (Cardile et al., 1995). However, under these conditions the cells are not in direct contact with the test gas. The importance of the diffusion barrier for gas phase toxicants was described by Pace et al. (1961, 1969). They observed that the reduction or removal of the media overlay facilitates contact of cell monolayers with gas phase oxidants and reduces the oxidant concentration to induce cell injury. Accordingly, several studies using various techniques to reduce the diffusion barrier for gas phase toxicants have been described incorporating roller bottles and rocking or rotating platforms (Baker and Tumasonis, 1971; Bolton et al., 1982; Fischer and Placke, 1987; Friedman et al., 1992; Guerrero et al., 1979a, b; Madden et al., 1991; Samuelsen et al., 1978; Valentine, 1985; Wenzel et al., 1979). Here, the culture medium is periodically based over the apical surface of the cells, forming a variable diffusion barrier. These concepts were mainly used to expose cells to gases like ozone or NO2 (Guerrero et al., 1979a, b; Pace et al., 1969; Samuelsen et al., 1978; Wenzel et al., 1979) and volatile compounds (Muckter et al., 1998). It became clear that the technical effort necessary to establish stable, reproducible and efficient conditions with regard to the generation of the test atmosphere and the exposure itself is of particular importance, especially when studying dose–effect relationships. Bolton et al. (1982) described an in vitro exposure system for exposing mammalian cell cultures or viruses to ozone (Fig. 1). They used borosilicate glass roller culture bottles equipped with specially designed caps to permit humidified gas to flow through the rotating vessel and to react with the cells with only a thin film of fluid over most of the surface of the bottle. Ozone concentrations were measured immediately before entering and after
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Fig. 1. In vitro cell culture exposure system. Ozone at about 2 ppm was introduced into the system and mixed by two regulating valves with compressed air entering the system through a pressure regulator, to adjust the ozone concentrations. The main flow of the gas was directed into a 37 1C incubator, housing the humidifying bubblers, the roller apparatus and the sequential sampler. The gas flow rate through the exposure vessel was held constant by complementary solenoid valves (Bolton et al., 1982).
exiting the exposure vessel, thus permitting the calculation of reaction rates, the total amount of ozone reacted over any time period and giving some idea of the total exposure dose actually received by the biological material. This technique was not only used for gases but also for studying complex atmospheres like diesel exhaust. Morin and coworkers (Le Prieur et al., 2000; Morin et al., 1999) adapted the method and exposed precise-cut rat lung slices placed in roller bottles to a continuous flow of diluted diesel exhaust. The design of such an exposure chamber is shown in Fig. 2. The exposure chamber consists of two concentric cylinders, placed over a continuous Wheaton rolling system, in an incubator at 37 1C. To guarantee an adequate hygrometry (85–90%), the external cylinder contains a solution of 1% CuSO4 in water. The internal
cylinder has slits on its periphery, allowing the passage of humidity from the external to the internal cylinder where a constant and controlled flow of atmosphere is applied. Freshly prepared lung slices are positioned on a titanium grid of a Teflon rolling insert. They are placed into scintillation vials with opened caps, allowing free access to the complex test atmosphere (gas and particulate phase). The gas phase was tested after the integration of filter-bearing caps, preventing the penetration of particles. The vials were then placed into the specially designed flow-through humidified cylindrical chambers for exposure. The whole exposure system is placed in a slight vacuum to ensure a continuous flow of the particles without physical obstruction. After an exposure time of 1 h, different biological endpoints were measured like histopathological examination, the ATP content, and intracellular glutathione or anti-oxidant
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Double jacket constant flow through chamber
Culture vial Lung slices on Teflon inserts Culture medium Water for chamber humidification Rotation device
Fig. 2. Cross-section of the flow through the exposure chamber. The two external concentric circles delimit the humidification compartment. The culture vial rotates freely on the internal wall of the chamber and the lung slices are alternately fed by culture medium and exposed to the flow of the complex atmosphere (Morin et al., 1999).
activities. So far, only few attempts have been made to design a dynamic flow-through system for the representative in vitro exposure of mammalian cells to volatile compounds. Such a device would allow the exposure of organotypic cultures to a controlled flow of the test atmosphere for the determination of dose-dependent cellular reactions.
Fig. 3. Scheme of a method for exposing CHO cells on collagen gels to volatile compounds by using the static technique: (A) Cells were grown on collagen gels in glass bottles; (B) the medium was aspirated and the bottles inverted; (C) test substance was introduced into the bottle by a syringe and the chemical was volatilized by briefly heating the bottle (Zamora et al., 1983).
Exposure of the cells on collagen gels or microporous membranes To attain more realistic and efficient exposure conditions without interfering medium components above the cells, alternative studies concentrated on the cultivation and exposure of the cells on collagen gels or microporous membranes (Rasmussen and Crocker, 1981; Voisin et al., 1977a, b; Zamora et al., 1986). Zamora et al. (1983, 1986) described procedures for the static and dynamic exposure of cells to volatile compounds to investigate the mutagenic activity of highly volatile compounds. In the first case, cells were grown on hydrated collagen gels under submersed conditions. Prior to use, the vessels were inverted and the medium aspirated, leaving cells attached at an air/ collagen interface (Fig. 3). The test compound was then injected via a silicone rubber stopper into the vessel and incubated for 1 h and subsequently analyzed for cell survival (Li and Brooks, 1981) and mutations at the HGPRT gene locus (Li, 1982). For dynamic exposures, for example to ethylene oxide, CHO cells were placed in Petri dishes on collagen gels. Immediately prior to exposure, medium was aspirated, leaving the cells attached on the gel at an
Fig. 4. Schematic drawing of the dynamic exposure system. In a modular incubation chamber, cells were exposed to 0, 0.8, 2.0, 3.3 or 6 ppm of NO2 (Zamora et al., 1986). Exposure atmospheres were generated by metering 500 ppm of the gas in air through a rotameter into a stream of 95% air/5% CO2. The system supplied the desired NO2 concentrations to the cells while maintaining environmental conditions necessary for cell survival (Zamora et al., 1986).
air/collagen interface. The cultures were then placed in an incubator and a dynamic flow of the test substances was introduced into the chamber at a flow rate of 2.6 and 2.8 l/min and continuously monitored during the exposure phase of 1 h (Fig. 4). After exposure, cell survival and mutation frequencies were analyzed in comparison to the static exposure. This system was also used for exposing an epithelial cell line from the lung (Li and Brooks, 1981) to different concentrations of NO2 up to 6 ppm for 15–60 min (Zamora et al., 1986). In consequence, a time- and dosedependent increase in cell death could be established
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under these experimental conditions, thus indicating the efficiency of such a direct exposure method without interfering medium components above the cells. Other techniques using microporous membranes were described by several authors (Alink et al., 1979; Rasmussen and Crocker, 1981; Rasmussen, 1984; Samuelsen et al., 1978; Voisin et al., 1977a, b). Alink et al. (1979) presented a specially designed culture dish in which the bottom consisted of 25 mm thick Teflon membranes (Fig. 5). After seeding the cells, the dishes were placed in a chamber through which an O3containing atmosphere was directed. The O3 dose was calculated by measuring the concentration in the medium above the cells. The actual dose to the cells is difficult to specify in this system, since the cells on the membrane encounter the O3 almost immediately after its penetration through the membrane. Furthermore, relatively high external concentrations (170 and 290 ppm
Fig. 5. Culture disk with gas-permeable film bottom: (A) glass dish cover, (B) top plate, (C) base plate, (D) Teflon ring, (E) gas-permeable Teflon film, (F) culture medium (Alink et al., 1979).
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O3) are needed to produce significant levels of O3 in the cell culture medium (0.1970.08 and 0.4370.05 mg/4 ml). To overcome this problem, Gabridge and Gladd (1984) presented a modified exposure method to evaluate the gaseous oxide toxicity with cell monolayers on collagen-coated, gas-permeable Teflon membranes (Fig. 6). Human lung fibroblasts seeded on FEP-Teflon membranes coated with collagen were placed directly over holes in a Plexiglas shelf, inserted into a Plexiglas chamber and placed inside an incubator. Immediately below the shelf, a propeller-type gas mixer distributed the test gases NO2, CO2 and SO2 entering the Plexiglas chamber through a warming coil. Gases passed under the chamber/dishes with monolayers on the membranes, and left through a liquid trap above the incubator. Measurements of the ATP content as an index of actual cell viability showed an increased effect of SO2 at concentrations above 0.005% after an exposure period of 30 min, whereas NO2 was less toxic and CO2 up to levels of 20% did not affect the cells. It becomes clear that a close contact between cells and test atmosphere favors the induction of dose-dependent reactions, although we have to deal with uncontrolled exposure conditions with regard to the real amount of test compound coming into contact with the biological material. None of these systems really resembles the respiratory epithelium in vivo because bronchiolar and epithelial cells are maintained by nutrient and fluids from underlying capillaries, while the luminal surface is exposed directly to the airstream, except for a layer of mucus or surfactant of varying thickness. The ideal in vitro system would be a culture in which in vivo relationships were maintained, and which was accessible to experimental manipulation.
Fig. 6. Diagram of the system used to expose monolayers to toxic gases: (A) flow meters, (B) incubator, (C) warming coil for incoming gas mixtures, (D) Plexiglas chamber, (E) gas inlet, (F) mixing propeller, (G) magnetic stirrer, (H) shelf, (I) chamber/dish with Teflon membrane cell substrate. Details of the chamber/dish: a: gas-permeable Teflon membrane, b: collagen coating on membrane, c: cell monolayer, d: metal base plate, e: chamber/dish core unit of solid Teflon, f: plastic Petri dish (35 mm) cover (Gabridge and Gladd, 1984).
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Exposure at the air/liquid interface Voisin et al. (1975, 1977a, b) were the first to achieve a reasonable concept for exposing cells at the air–liquid interface. They planted pulmonary alveolar macrophages on cellulose acetate filters and then floated the filters on top with nutrient medium in a dish. Diffusion of the medium through the filter was sufficient to maintain cell viability during exposure to NO2, while not submerging the cells (Fig. 7). The exposed cells showed morphological changes, a decrease in bactericidal activity and a reduction in the ATP content. The cultivation and exposure method at the air/liquid interface was favored for the development of exposure strategies to analyze the biological effects of inhalable pollutants on cells from the respiratory tract. Based on this concept, Samuelsen et al. (1978) and Rasmussen and Crocker (1981) worked on a system employing lung fibroblasts planted on cellulose acetate filters (0.45 mm pore size). The filters are placed in a specially designed holder that permits the diffusion of the medium through the filter from below the cells. The device is assembled with the filter in place and the lower chamber filled with culture medium. The cells are seeded in the upper well and allowed to settle and attach to the filter. After being placed in a gas exposure chamber, exposure to O3 and NO2 starts after removing the medium in the upper well. When exposure has been
Fig. 7. (A) Petri dish, (B) plastic ring, (C) medium reservoir, (D) microporous membrane, (E) zone for the cultivation of the macrophages (Voisin et al., 1977a, b).
completed, the process is reversed and the cells can be recovered for the analysis of different endpoints. Studies on the cytotoxic effects of O3 and NO2 in this system have shown that most cell types are extremely sensitive to low concentrations of these gases. In experiments with V79 fibroblasts, exposure to 0.15 ppm NO2 or 0.05 ppm O3 for 6 h resulted in a loss of about 90% of the cells from the filters, while clean air had little effect over the same period (Rasmussen and Crocker, 1981; Samuelsen et al., 1978). A further development in establishing the experimental conditions for direct cell exposure at the air/liquid interface was the design of a biphasic chamber (Whitcutt chamber) for maintaining differentiated respiratory epithelial cells from the guinea pig between air and liquid phases (Adler et al., 1987; Whitcutt et al., 1988). The chamber consists of three parts (1) a plastic tissue culture dish, 35 mm for a single chamber, larger for groups of chambers, (2) the chamber itself, consisting of a polycarbonate ring glued on one side to a 100 nm pore size polycarbonate or nitrocellulose membrane, crosslinked to gelatin, and (3) a plastic spacer (2–5 mm thick) to raise the chamber when basal feeding only is desired (Whitcutt et al., 1988). Based on this technique, Tarkington et al. (1994) designed a new in vitro exposure system for the direct exposure of cultivated airway epithelial cells and of explants from the trachea in several replicate vessels to ozone. Cells could be exposed simultaneously to an atmosphere without ozone as control (Fig. 8). This device was designed to generate and monitor consistent, reproducible levels of ozone over a fairly wide range of concentrations in a humidified atmosphere, thereby allowing exposure durations greater than those reported by others (Rasmussen, 1984; Eisenberg et al., 1984). As shown in Fig. 8, one vessel for ozone and one control vessel were used. Medical grade liquid oxygen is vaporized and passed through three stages of pressure regulation to be introduced into the system at a constant pressure (0.21 kg/cm2). The oxygen stream is diverted to the ozonizer and a through flow of 5 l/min of oxygen resulted in a maximum production of about 60 ppm of ozone. To establish the desired concentrations, ozone was mixed at different flow rates into a stream of oxygen supplemented with carbon dioxide. Except for the lack of provision for ozone introduction, the atmosphere stream for the control vessel was produced in the same way. Lines passed through a port in the incubator and conveyed the gas stream to the exposure and control vessels (Fig. 9). The gas streams were humidified by bubbling through temperature-controlled bottles containing distilled water and directed to the exposure vessels with an appropriate volume to support 5 culture vials. Inside each vessel, the gases enter at the top through a jet in such a way that the atmosphere is injected tangentially to the wall and swirls across the top
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Fig. 8. Schematic drawing of the ozone exposure system: (A) ozonizer, (B) ozone bypass valve, (C) incubator, (D) humidification bottles, (E) exposure vessels, (F) control vessels, (G) pressure gauges, (H) ozone analyser, (I) thermometer, (J) heating tape, (K) thermometer, (L) thermal insulation (Tarkington et al., 1994).
Fig. 9. Ozone exposure system. View inside the incubator: (A) inlet exposure atmosphere, (B) inlet control atmosphere, (C) exhaust exposure atmosphere, (D) exhaust control atmosphere, (E) humidified exposure atmosphere, (F) humidified control atmosphere, (G) humidifier bottle, exposure, (H) humidifier bottle, control, (I) distilled water, (J) heating mat, (K) motorized rocker, (L) thermal insulation, (M) exposure vessel, (N) control vessel (Tarkington et al., 1994).
of the culture vials. Exhaust from each vessel was taken from the bottom in the center. Exhaust lines with integrated tees exit from the vessels. A pressure line is connected to the tee for measuring the pressure in each vessel with a remotely mounted gauge. The exposure vessel exhaust line passes through a port in the incubator and is connected to the ozone analyzer. All exhaust, pressure and sample lines as well as valves and gauges are wrapped with heating tape and thermal insulation to prevent condensation in the system.
Using this construction, Tarkington and coworkers (1994) exposed bronchial epithelial cells and explant cultures from the trachea to different ozone concentrations. Here, for the first time, the gas stream is guided in a vertical stream directly to the cells to realize an optimal contact with the gas phase. Furthermore, this system presents an overall controlled exposure device for establishing stable and reproducible exposure conditions. Based on the observations of Pace et al. (1961, 1969), Tarkington also studied the protective influence of a medium overlay on the cells. A major conclusion from the studies of Pace was that thin layers of liquid are sufficient to protect the cells from NO2 and, therefore, studies on the effects of gas must employ systems that allow more direct contact between the gases and cells. Consequently, Tarkington and his group analyzed the effects of culture surface fluid volume on ozone toxicity. As shown in Fig. 10, without any fluid on top of the surface, ozone caused substantial damage to the airway epithelial cells. Cell viability was in the range of 20% in comparison to control cultures without any exposure. In contrast, in the control vessel exposed to filtered air, the viability was almost 85%. The addition of medium on the top of the cells reduced the damage caused by ozone. These results demonstrate the importance of direct exposure of cells to assess the toxicity in vitro (Fig. 10). The exposure studies conducted by Tarkington et al. (1994) clarified the preconditions necessary for running efficient in vitro toxicity experiments: (1) the generation and monitoring of reproducible levels of the test gas, (2) a stable and reproducible delivery of the gas to replicate vials, (3) multiple exposure at the same concentration, even when performed on different days with different test batches should produce similar results. Encouraged by the results gained under the exposure conditions at the air/liquid interface, Sun et al. (1995)
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120
100
Viability %
80
60
40
20
0
Controls
0
0.2
0.6
1.0
Volume of Medium on Top of Cells (ml)
Fig. 10. Effects of culture surface fluid volume on ozone toxicity. A human bronchial epithelial cell line was exposed to 1 ppm ozone or to filtered air for 2 h. Prior to exposure, various volumes of cell culture medium were added on top of the cells (Tarkington et al., 1994).
studied the effects of sidestream cigarette smoke as an example for complex mixtures, ozone and a combination of both on bronchial epithelial cells (BEAS-2B). The exposure of the cells to ozone was carried out using the technique described by Tarkington et al. (1994) whilst exposure to sidestream smoke was conducted using a different system developed by Teague et al. (1994). BEAS-2b cells were cultured in the biphasic Millicell-CM system (30 mm diameter, 0.4 mm pore size; Millipore, Bedford, MA, USA) and transferred into a cell incubator (37 1C) with apical exposure to a welldefined atmosphere (filtered air and 5% carbon dioxide saturated with water vapor) with the addition of sidestream cigarette smoke generated from an ADL/II smoking machine (Arthur D. Little, Inc.) according to ISO norms. Both ozone and sidestream cigarette smoke induced a dose-dependent decrease of cell viability, inhibition of cellular metabolism and replication, as well as a compromise of external cell membrane integrity. Interestingly, the combined exposure had a less than additive cytotoxic effect on all the dose groups tested. For the exposure of cells from the respiratory tract to reactive gases and complex mixtures, exposure at the air/liquid interface was favored and different experimental setups were described in the following years based on a biphasic culture technique. More attention was directed towards improving a controlled gas generation and delivery to the cells to run dose-related studies. The knowledge of in vitro exposure techniques was based mainly on experiments with single gases like ozone or NO2. Only a limited number of studies
(Aufderheide et al., 2002; Massey et al., 1998; Morin et al., 1999; Sun et al., 1995;) dealt with native complex atmospheres like cigarette smoke or diesel exhaust, probable due to difficulties in creating stable and reproducible atmospheres. A particular challenge to such aerosols is posed by the particulate phase, which has to be brought into contact with the cells. Especially when dealing with complex atmospheres, the biological activity of the particulate and the gas phases is important to characterize the toxicologic activity of such test compounds. Consequently, a development was started to construct exposure devices to expose cells of the respiratory tract to complex aerosols like cigarette smoke (Aufderheide, 1997; Massey et al., 1998). A special exposure chamber based on the biphasic cultivation of cells on microporous membranes was designed to carry out static exposure of cultured cells to fresh diluted mainstream smoke (Fig. 11). Here, the exposure chamber contains six positions to house transwell inserts with a membrane of 24 mm diameter and 3.0 mm pore size. The cells were supplied with a continuous flow of medium from the bottom (base plate). On this plate, a support frame was located to provide an exact and reproducible positioning of the transwell and membranes in the culture medium. On the support frame, a smoke distribution panel ensured homogeneous distribution of the smoke to the transwells. From a central inlet, the smoke is guided radially via six channels to six cylindrical holes above the inserts. Here, a stainless steel mesh screen is located for homogenous distribution of the smoke to all parts of the membrane. A coverplate of stainless steel with a 3 mm central inlet for the smoke completes the exposure chamber and was connected via a Quickfit adapter to the smoke generation and dilution system. The exposure chamber was placed in a commercial incubation chamber to provide a suitable environment (37 1C, 95% relative humidity and 10% CO2) to maintain cell viability during smoke exposure. Cigarette puffs (35 ml in 2 s) were taken via a step motor-driven glass syringe and further diluted. After 2 s, the smoke was expelled from the syringe and diluted with preconditioned air drawn from the incubator. After the exposure chamber was filled with smoke, valves were closed and static exposure commenced (10–40 s). Once completed, the smoke was cleared from the chamber and replaced with incubator-conditioned air before starting the next puff. In such a way, V79 cells were not only exposed to whole cigarette smoke, but also to the gas vapor phase by inserting a Cambridge filter over a total period of 3 h. It could be shown that, by increasing the smoke concentrations (457–840 mg/cm3 TPM), there was a decrease in cell proliferation. Analysis of genotoxic effects showed an increase in micronuclei induction with decreasing smoke concentration, also for
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Fig. 11. Schematic drawing of smoke exposure device detailing arrangements of the exposure chamber, smoking and dilution system, control system and medium supply (Massey et al., 1998).
the gas vapor phase. Exposure to whole smoke resulted in a 3-fold induction of micronuclei and a 2.4-fold induction when the cells were exposed to the vapor phase only. With regard to the design of the exposure chamber, the smoke was introduced into the system at the top of the transwell inserts, resulting in an inhomogenous distribution of the particles on the membrane, which could be minimized by inserting the described steel mesh screen. These results demonstrated that direct contact between the cells and the test atmosphere might be a critical point for realizing enhanced particle deposition on the membrane. Therefore, the idea of Tarkington and his coworkers (1994), to guide the atmosphere via a vertical dynamic stream directly to the biphasic cultivated cells was taken up again in another exposure system, called CULTEXs (Vitrocell, Germany) (Fig. 12). Aufderheide and coworkers (Aufderheide and Mohr, 1999, 2000; Aufderheide et al., 2002, 2003) used a specially designed exposure unit in which cultivated human cells of the respiratory tract were exposed at the air/liquid interface to mainstream cigarette smoke as well as to its vapor phase
without heating, humidification, or addition of carbon dioxide. This strategy has also been successfully used for the in vitro testing of native gaseous compounds (Ritter et al., 2001), complex mixtures like sidestream and whole cigarette smoke (Aufderheide et al., 2001; Ritter et al., 2003, 2004; Wolz et al., 2002), and automobile exhaust (Knebel et al., 2002). The components of the module are shown in Fig. 12. The lower part of the module houses three vessels for transwell inserts (growth area 1 cm2). These vessels are insulated against the inner space of the module, which can be floated in temperature-controlled water (37 1C). The vessels are filled with medium, thus providing humidification and supply of the cells with nutrients. The exposure top of the module has three specially designed inlet tubes, which guarantee the homogenous distribution of the particles of a test atmosphere, which is sucked via negative pressure through the module, entering the system via the inlets and leaving them through the corresponding outlets. The inlet tube starts with a small pipe, ending in a hyperboloid-shaped air distribution section (subsequently referred to as a ‘‘trumpet’’) that allows the
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Fig. 12. The CULTEX exposure module consisting of a lower part for maintenance of cell viability and the upper exposure top for reproducible delivery of the test atmosphere.
Fig. 13. Vector diagram of the velocity field in the central plane (Aufderheide and Mohr, 2004).
uniform deposition of particles on the surface of cell cultures (Prof. Durst, Institute of Fluid Mechanics, LSTM-Erlangen, University of Erlangen-Nu¨rnberg, Germany). The flow remains separation-free, ensures a continuous delivery of the aerosol towards the cells and constant particle deposition (Figs. 13 and 14; Aufderheide and Mohr, 2004). Using the CULTEXs system, A549 cells, which are described to share several fundamental characteristics with human lung alveolar type II cells (Lieber et al., 1976; Nardone and Andrews, 1979; Mendelson and Boggaram, 1991; Smith, 1977; Smith et al., 1982; Young and Mendelson, 1997) and competence of xenobiotic metabolism (Hukkanen et al., 2000; Iwanari et al., 2002; Urani et al., 1998), were exposed to mainstream smoke and the gas phase of a number of different cigarettes in the non-toxic range. Fresh cigarette smoke, generated
according to ISO norms in a specially designed smoking robot VC10 for in vitro exposure (Vitrocell, Germany), was sucked at different concentrations above the cells for 32 min. To characterize the biological activity of smoke, protein content and, as a sensitive marker for cell damage, intracellular reduced glutathione were analyzed directly after exposure. Cellular protein revealed no statistically relevant changes in comparison to control cultures exposed to synthetic air. However, throughout all tested cigarettes, dilutions and exposures to whole and filtered smoke, a significant dose-dependent depletion of reduced glutathione was observed under such non-toxic experimental conditions (Fig. 15). In addition, it could be demonstrated that the quantitative effects of whole smoke and the gas phase were statistically distinguishable for the research cigarette. The susceptibility and flexibility of the system indicate its suitability for analyzing the biological effects of airborne pollutants in the environment. Owing to the fact that cell viability can be maintained independently of an incubator, it can be transported directly to the source of pollution. The integration of inlet tubes manufactured from surface treated stainless steel (Fig. 16) represents a further advantage, because the deposition efficiency for nano-sized particles reached 80% of the exposed mass. This system has also been adapted to expose bacteria of Salmonella typhimurium strains to complex mixtures like mainstream cigarette smoke in a modified Ames assay (Aufderheide and Mohr, 2004). In this case, the basal part of the module houses Petri dishes (35 mm diameter) which are exposed, in the same way as described above, to different numbers of Kentucky research cigarettes K2R4F (tar content 9 mg/cigarette) and a commercially available one with a tar content of 1 mg per cigarette. The bacteria were seeded according to the spread culture technique without an agar overlay and exposed for 30 min to mainstream smoke of up to
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Fig. 14. Concentration of the smoke in the central plane (A) and at the surface of the insert (B). The numbers indicate the smoke concentration relative to air (Aufderheide and Mohr, 2004).
100 3 cigarette s
GSH [% Control]
80
y = 117.18e -0.0506x R2 = 0.9509
60
40
y = 111.18e -0.0753x R2 = 0.9583
20 Gas Gas & particles
1.0
10.0
100.0
Puffs / dilution
Fig. 15. Dose–response curves from exposures of A549 cells to fresh filtered or whole smoke from K1R4F. Intracellular glutathione contents were plotted as a percentage of control values against exposure dose calculated as the ratio of number of smoked cigarette puffs and dilution rate used. Each dot represents the result of a single exposure experiment (Ritter et al., 2004).
seven cigarettes at different dilution rates by using the smoking robot VC10 (Vitrocell, Germany). As shown in Fig. 17, it is possible to bring about a dose-dependent induction of revertants with this exposure procedure. The analysis of the mutagenic activity of two cigarettes with different tar content showed significant differences in the induction of revertants correlated with the tar content of the cigarettes. A modified version of the CULTEXs system has also been used by other research groups to expose cells to
Fig. 16. Inlet tubes with a hyperboloid-shaped air distribution section manufactured from (A) Teflon and (B) surface treated stainless steel. 14
Quotient [smoke/synthetic air]
0
12 10 8 6 9 mg tar/cigarette 1 mg tar/cigarette
4 2 0 0.1
1
10
100
Dosage (cig./dil.)
Fig. 17. Exposure of Salmonella typhimurium strain TA98 to mainstream cigarette smoke of K2R4F cigarettes (tar content 9 mg/cigarette, violet squares) and a commercial brand (tar content 1 mg/cigarette, magenta squares) using the CULTEXs system. Each dot represents the result of a single exposure experiment.
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complex mixtures like mainstream smoke (Fukano et al., 2004) based on the principle of sedimentation (Fig. 18). The cells were maintained in the exposure module under submersed conditions, i.e. covered by a layer of medium. Each culture module was then filled with freshly generated smoke, smoke supply was shut off and cells were exposed for 24 s per puff. The medium was discharged, allowing the particles to drop down onto the cells by sedimentation. Fresh medium was supplied until the inserts were floated again. Finally, the system was purged with clean air to remove residual smoke. Cells were maintained in the liquid medium except during the exposure period to maintain culture conditions and protect the cells from initial puff pressure and the final cleansing air flow. This cycle was repeated and cells were exposed to freshly generated cigarette smoke once every minute. The membrane in culture inserts was pierced with five holes by needles to control the speed of liquid medium exchange. This modified exposure procedure also resulted in the determination of dose-dependent effects after exposure of human epithelial lung cells to cigarette smoke.
Conclusion Taking into consideration all the presented exposure procedures, it can be concluded that, dependent on the hypothesis to be tested, a variety of experimental approaches can be realized. The most important aspect might be the susceptibility of the system, that means a sensitive biological test system and an optimal exposure device: (1) realizing an optimal contact between cells and the test atmosphere, (2) avoiding interactions of compounds of the test atmosphere with medium components and (3) guiding it without minimal chemical and physical changes to the cells. In summary, the biphasic growth of cells of the respiratory tract at the air/liquid interface in combination with relevant exposure methods offers new possibilities to analyze the biological activity not only of synthetically generated complex mixtures in the laboratory, but also under in- and outdoor conditions in the future. The same might be true for analyzing the genotoxic potency of such atmospheres by using a modified and mobile Ames assay.
Fig. 18. Exposure of human epithelial lung cells (A549) to mainstream cigarette smoke (K2R4F) using a modified CULTEXs system (Vitrocell, Germany) (Fukano et al., 2004).
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EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Direct exposure methods for testing native atmospheres Michaela Aufderheide Department of In Vitro Toxicology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany Received 25 April 2005
Abstract In vitro studies of adverse cellular effects induced by inhalable substances face a number of problems due to the difficulties in exposing cultured cells of the respiratory tract directly to test atmospheres composed of complex gases and particulate compounds. This paper discusses the characteristics of in vitro work and summarizes the use of different in vitro technologies to determine the adverse effects of inhaled pollutants. The exposure of cells to test atmospheres requires accurate control of the pollutant levels, as well as the close contact of cells and gas without interfering with the medium. Systems which rely on the solution of the gas in the medium overlay do not resemble the exposure conditions in vivo, and may not be suitable for studying, for example, the effects of poorly soluble gases. Exposure to gases or complex mixtures can be performed with roller bottles or flasks on rotating and rocking platforms and, using these techniques, the cells are periodically exposed to the test atmosphere. However, the most promising approach is based on a biphasic cell culture technique, where cells are grown on microporous membranes at an air–liquid interface. Here the cells are nutrified from the basal side of the membrane whilst the apical part with the cultivated cells is in direct contact with the test atmosphere. Based on this culture technique, different exposure systems have been developed and these are described and discussed. Exposure of cells from the respiratory tract to gases or particles is responsible for cell injury or cell activation associated with an overexpression of mRNA and the release of bioactive mediators. Therefore, in vitro studies using such a strategy, in combination with relevant and efficient exposure devices, open up new ways to test native complex gases and aerosols. Furthermore, such an experimental approach is not only suitable for cultivated cells, but it can also be used for exposing bacteria to inhalable test compounds. It is possible to analyze the mutagenic potency of in- and outdoor pollutants and several attempts have been made to determine the induction of revertants in a modified Ames assay after exposure to single gases or complex mixtures. r 2005 Elsevier GmbH. All rights reserved. Keywords: Air/liquid interface; Direct exposure methods; Review; CULTEXs; Quantitative data; Modified ames assay
Introduction
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E-mail address: [email protected]. 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.05.019
The analysis of complex mixtures like ambient air, diesel exhaust, environmental tobacco smoke or cigarette smoke itself represents a multifactorial problem due to the complexity of the test atmosphere. In most cases,
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they consist of a particulate and gas phase, including hundreds of substances, short- and long-living radicals, which undergo chemical reactions after generation. It is well known that the mixture changes both qualitatively and quantitatively in a short time (Pryor, 1992, 1997). Therefore, toxicological investigations should be carried out under conditions resembling the in vivo situation as far as possible. The exposure of lung cells to complex atmospheres or single components like ozone is responsible for a cascade of cellular reactions, initiating the development of chronic lung disorders or even cancer. Ozone for example, an atmospheric photochemical of major concern because of its deleterious health effects upon inhalation, has been shown to result in alterations in lung function, development of bronchial hyperresponsiveness, loss of alveolar Type I cells, an influx of inflammatory neutrophils into the airways, increased epithelial and vascular permeability, a decrease in the mitogen-induced T-cell proliferation, and increased cell mortality resulting from aerolized microbial agents delivered after exposure (Coffin and Gardner, 1972; Evans, 1984; Koren et al., 1989; Orlando et al., 1989; Seltzer et al., 1986). The responses of lung cells to toxic gases are difficult to explain. To understand the functional and pathological disorders induced in the respiratory tract requires the investigation of the direct effects of pollutants on the state and activity of lung cells. So far, three approaches have been used: (1) animal experiments, (2) ex vivo studies of cells of bronchial lavage or biopsies and (3) in vitro systems of exposure of lung cells to pollutants under controlled conditions (Rasmussen, 1984; Wallaert et al., 2000). This paper emphasizes the evolution of the in vitro approaches which offer the possibility to expose cells of the respiratory tract directly to native atmospheres.
In vitro exposure systems for inhalable compounds Requirements The development of in vitro methods for the exposure of lung cells has to follow some principle requirements (Rasmussen, 1984): 1. Controlled generation and monitoring of the test atmosphere to study dose-dependent effects. 2. The contact between the cells and the test compounds should be as close as possible to avoid interactions, for example of oxidant gases with medium components, and to realize particle deposition and, in the case of complex mixtures, direct contact of cells and gas phase components.
3. The exposure systems should be designed to allow significant exposure times. In general, the exposure of cultivated cells to ambient atmospheres rapidly results in their inactivation due to drying. Therefore, methods had to be developed to maintain a humidified atmosphere or to moisten the cells in a specific way. 4. After exposure, representative endpoints considered to be predictive, as shown by in vivo studies or human evidence, should be analyzed as indicators of the toxicological effect.
Exposure using roller bottles and rocking or rotating platforms The simplest method is to bubble the gas through a cell suspension or to flush the culture flask with the pollutant. This technique was used, for example, to expose alveolar macrophages to NO2 (Voisin et al., 1974) or ozone (Cardile et al., 1995). However, under these conditions the cells are not in direct contact with the test gas. The importance of the diffusion barrier for gas phase toxicants was described by Pace et al. (1961, 1969). They observed that the reduction or removal of the media overlay facilitates contact of cell monolayers with gas phase oxidants and reduces the oxidant concentration to induce cell injury. Accordingly, several studies using various techniques to reduce the diffusion barrier for gas phase toxicants have been described incorporating roller bottles and rocking or rotating platforms (Baker and Tumasonis, 1971; Bolton et al., 1982; Fischer and Placke, 1987; Friedman et al., 1992; Guerrero et al., 1979a, b; Madden et al., 1991; Samuelsen et al., 1978; Valentine, 1985; Wenzel et al., 1979). Here, the culture medium is periodically based over the apical surface of the cells, forming a variable diffusion barrier. These concepts were mainly used to expose cells to gases like ozone or NO2 (Guerrero et al., 1979a, b; Pace et al., 1969; Samuelsen et al., 1978; Wenzel et al., 1979) and volatile compounds (Muckter et al., 1998). It became clear that the technical effort necessary to establish stable, reproducible and efficient conditions with regard to the generation of the test atmosphere and the exposure itself is of particular importance, especially when studying dose–effect relationships. Bolton et al. (1982) described an in vitro exposure system for exposing mammalian cell cultures or viruses to ozone (Fig. 1). They used borosilicate glass roller culture bottles equipped with specially designed caps to permit humidified gas to flow through the rotating vessel and to react with the cells with only a thin film of fluid over most of the surface of the bottle. Ozone concentrations were measured immediately before entering and after
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Fig. 1. In vitro cell culture exposure system. Ozone at about 2 ppm was introduced into the system and mixed by two regulating valves with compressed air entering the system through a pressure regulator, to adjust the ozone concentrations. The main flow of the gas was directed into a 37 1C incubator, housing the humidifying bubblers, the roller apparatus and the sequential sampler. The gas flow rate through the exposure vessel was held constant by complementary solenoid valves (Bolton et al., 1982).
exiting the exposure vessel, thus permitting the calculation of reaction rates, the total amount of ozone reacted over any time period and giving some idea of the total exposure dose actually received by the biological material. This technique was not only used for gases but also for studying complex atmospheres like diesel exhaust. Morin and coworkers (Le Prieur et al., 2000; Morin et al., 1999) adapted the method and exposed precise-cut rat lung slices placed in roller bottles to a continuous flow of diluted diesel exhaust. The design of such an exposure chamber is shown in Fig. 2. The exposure chamber consists of two concentric cylinders, placed over a continuous Wheaton rolling system, in an incubator at 37 1C. To guarantee an adequate hygrometry (85–90%), the external cylinder contains a solution of 1% CuSO4 in water. The internal
cylinder has slits on its periphery, allowing the passage of humidity from the external to the internal cylinder where a constant and controlled flow of atmosphere is applied. Freshly prepared lung slices are positioned on a titanium grid of a Teflon rolling insert. They are placed into scintillation vials with opened caps, allowing free access to the complex test atmosphere (gas and particulate phase). The gas phase was tested after the integration of filter-bearing caps, preventing the penetration of particles. The vials were then placed into the specially designed flow-through humidified cylindrical chambers for exposure. The whole exposure system is placed in a slight vacuum to ensure a continuous flow of the particles without physical obstruction. After an exposure time of 1 h, different biological endpoints were measured like histopathological examination, the ATP content, and intracellular glutathione or anti-oxidant
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Double jacket constant flow through chamber
Culture vial Lung slices on Teflon inserts Culture medium Water for chamber humidification Rotation device
Fig. 2. Cross-section of the flow through the exposure chamber. The two external concentric circles delimit the humidification compartment. The culture vial rotates freely on the internal wall of the chamber and the lung slices are alternately fed by culture medium and exposed to the flow of the complex atmosphere (Morin et al., 1999).
activities. So far, only few attempts have been made to design a dynamic flow-through system for the representative in vitro exposure of mammalian cells to volatile compounds. Such a device would allow the exposure of organotypic cultures to a controlled flow of the test atmosphere for the determination of dose-dependent cellular reactions.
Fig. 3. Scheme of a method for exposing CHO cells on collagen gels to volatile compounds by using the static technique: (A) Cells were grown on collagen gels in glass bottles; (B) the medium was aspirated and the bottles inverted; (C) test substance was introduced into the bottle by a syringe and the chemical was volatilized by briefly heating the bottle (Zamora et al., 1983).
Exposure of the cells on collagen gels or microporous membranes To attain more realistic and efficient exposure conditions without interfering medium components above the cells, alternative studies concentrated on the cultivation and exposure of the cells on collagen gels or microporous membranes (Rasmussen and Crocker, 1981; Voisin et al., 1977a, b; Zamora et al., 1986). Zamora et al. (1983, 1986) described procedures for the static and dynamic exposure of cells to volatile compounds to investigate the mutagenic activity of highly volatile compounds. In the first case, cells were grown on hydrated collagen gels under submersed conditions. Prior to use, the vessels were inverted and the medium aspirated, leaving cells attached at an air/ collagen interface (Fig. 3). The test compound was then injected via a silicone rubber stopper into the vessel and incubated for 1 h and subsequently analyzed for cell survival (Li and Brooks, 1981) and mutations at the HGPRT gene locus (Li, 1982). For dynamic exposures, for example to ethylene oxide, CHO cells were placed in Petri dishes on collagen gels. Immediately prior to exposure, medium was aspirated, leaving the cells attached on the gel at an
Fig. 4. Schematic drawing of the dynamic exposure system. In a modular incubation chamber, cells were exposed to 0, 0.8, 2.0, 3.3 or 6 ppm of NO2 (Zamora et al., 1986). Exposure atmospheres were generated by metering 500 ppm of the gas in air through a rotameter into a stream of 95% air/5% CO2. The system supplied the desired NO2 concentrations to the cells while maintaining environmental conditions necessary for cell survival (Zamora et al., 1986).
air/collagen interface. The cultures were then placed in an incubator and a dynamic flow of the test substances was introduced into the chamber at a flow rate of 2.6 and 2.8 l/min and continuously monitored during the exposure phase of 1 h (Fig. 4). After exposure, cell survival and mutation frequencies were analyzed in comparison to the static exposure. This system was also used for exposing an epithelial cell line from the lung (Li and Brooks, 1981) to different concentrations of NO2 up to 6 ppm for 15–60 min (Zamora et al., 1986). In consequence, a time- and dosedependent increase in cell death could be established
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under these experimental conditions, thus indicating the efficiency of such a direct exposure method without interfering medium components above the cells. Other techniques using microporous membranes were described by several authors (Alink et al., 1979; Rasmussen and Crocker, 1981; Rasmussen, 1984; Samuelsen et al., 1978; Voisin et al., 1977a, b). Alink et al. (1979) presented a specially designed culture dish in which the bottom consisted of 25 mm thick Teflon membranes (Fig. 5). After seeding the cells, the dishes were placed in a chamber through which an O3containing atmosphere was directed. The O3 dose was calculated by measuring the concentration in the medium above the cells. The actual dose to the cells is difficult to specify in this system, since the cells on the membrane encounter the O3 almost immediately after its penetration through the membrane. Furthermore, relatively high external concentrations (170 and 290 ppm
Fig. 5. Culture disk with gas-permeable film bottom: (A) glass dish cover, (B) top plate, (C) base plate, (D) Teflon ring, (E) gas-permeable Teflon film, (F) culture medium (Alink et al., 1979).
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O3) are needed to produce significant levels of O3 in the cell culture medium (0.1970.08 and 0.4370.05 mg/4 ml). To overcome this problem, Gabridge and Gladd (1984) presented a modified exposure method to evaluate the gaseous oxide toxicity with cell monolayers on collagen-coated, gas-permeable Teflon membranes (Fig. 6). Human lung fibroblasts seeded on FEP-Teflon membranes coated with collagen were placed directly over holes in a Plexiglas shelf, inserted into a Plexiglas chamber and placed inside an incubator. Immediately below the shelf, a propeller-type gas mixer distributed the test gases NO2, CO2 and SO2 entering the Plexiglas chamber through a warming coil. Gases passed under the chamber/dishes with monolayers on the membranes, and left through a liquid trap above the incubator. Measurements of the ATP content as an index of actual cell viability showed an increased effect of SO2 at concentrations above 0.005% after an exposure period of 30 min, whereas NO2 was less toxic and CO2 up to levels of 20% did not affect the cells. It becomes clear that a close contact between cells and test atmosphere favors the induction of dose-dependent reactions, although we have to deal with uncontrolled exposure conditions with regard to the real amount of test compound coming into contact with the biological material. None of these systems really resembles the respiratory epithelium in vivo because bronchiolar and epithelial cells are maintained by nutrient and fluids from underlying capillaries, while the luminal surface is exposed directly to the airstream, except for a layer of mucus or surfactant of varying thickness. The ideal in vitro system would be a culture in which in vivo relationships were maintained, and which was accessible to experimental manipulation.
Fig. 6. Diagram of the system used to expose monolayers to toxic gases: (A) flow meters, (B) incubator, (C) warming coil for incoming gas mixtures, (D) Plexiglas chamber, (E) gas inlet, (F) mixing propeller, (G) magnetic stirrer, (H) shelf, (I) chamber/dish with Teflon membrane cell substrate. Details of the chamber/dish: a: gas-permeable Teflon membrane, b: collagen coating on membrane, c: cell monolayer, d: metal base plate, e: chamber/dish core unit of solid Teflon, f: plastic Petri dish (35 mm) cover (Gabridge and Gladd, 1984).
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Exposure at the air/liquid interface Voisin et al. (1975, 1977a, b) were the first to achieve a reasonable concept for exposing cells at the air–liquid interface. They planted pulmonary alveolar macrophages on cellulose acetate filters and then floated the filters on top with nutrient medium in a dish. Diffusion of the medium through the filter was sufficient to maintain cell viability during exposure to NO2, while not submerging the cells (Fig. 7). The exposed cells showed morphological changes, a decrease in bactericidal activity and a reduction in the ATP content. The cultivation and exposure method at the air/liquid interface was favored for the development of exposure strategies to analyze the biological effects of inhalable pollutants on cells from the respiratory tract. Based on this concept, Samuelsen et al. (1978) and Rasmussen and Crocker (1981) worked on a system employing lung fibroblasts planted on cellulose acetate filters (0.45 mm pore size). The filters are placed in a specially designed holder that permits the diffusion of the medium through the filter from below the cells. The device is assembled with the filter in place and the lower chamber filled with culture medium. The cells are seeded in the upper well and allowed to settle and attach to the filter. After being placed in a gas exposure chamber, exposure to O3 and NO2 starts after removing the medium in the upper well. When exposure has been
Fig. 7. (A) Petri dish, (B) plastic ring, (C) medium reservoir, (D) microporous membrane, (E) zone for the cultivation of the macrophages (Voisin et al., 1977a, b).
completed, the process is reversed and the cells can be recovered for the analysis of different endpoints. Studies on the cytotoxic effects of O3 and NO2 in this system have shown that most cell types are extremely sensitive to low concentrations of these gases. In experiments with V79 fibroblasts, exposure to 0.15 ppm NO2 or 0.05 ppm O3 for 6 h resulted in a loss of about 90% of the cells from the filters, while clean air had little effect over the same period (Rasmussen and Crocker, 1981; Samuelsen et al., 1978). A further development in establishing the experimental conditions for direct cell exposure at the air/liquid interface was the design of a biphasic chamber (Whitcutt chamber) for maintaining differentiated respiratory epithelial cells from the guinea pig between air and liquid phases (Adler et al., 1987; Whitcutt et al., 1988). The chamber consists of three parts (1) a plastic tissue culture dish, 35 mm for a single chamber, larger for groups of chambers, (2) the chamber itself, consisting of a polycarbonate ring glued on one side to a 100 nm pore size polycarbonate or nitrocellulose membrane, crosslinked to gelatin, and (3) a plastic spacer (2–5 mm thick) to raise the chamber when basal feeding only is desired (Whitcutt et al., 1988). Based on this technique, Tarkington et al. (1994) designed a new in vitro exposure system for the direct exposure of cultivated airway epithelial cells and of explants from the trachea in several replicate vessels to ozone. Cells could be exposed simultaneously to an atmosphere without ozone as control (Fig. 8). This device was designed to generate and monitor consistent, reproducible levels of ozone over a fairly wide range of concentrations in a humidified atmosphere, thereby allowing exposure durations greater than those reported by others (Rasmussen, 1984; Eisenberg et al., 1984). As shown in Fig. 8, one vessel for ozone and one control vessel were used. Medical grade liquid oxygen is vaporized and passed through three stages of pressure regulation to be introduced into the system at a constant pressure (0.21 kg/cm2). The oxygen stream is diverted to the ozonizer and a through flow of 5 l/min of oxygen resulted in a maximum production of about 60 ppm of ozone. To establish the desired concentrations, ozone was mixed at different flow rates into a stream of oxygen supplemented with carbon dioxide. Except for the lack of provision for ozone introduction, the atmosphere stream for the control vessel was produced in the same way. Lines passed through a port in the incubator and conveyed the gas stream to the exposure and control vessels (Fig. 9). The gas streams were humidified by bubbling through temperature-controlled bottles containing distilled water and directed to the exposure vessels with an appropriate volume to support 5 culture vials. Inside each vessel, the gases enter at the top through a jet in such a way that the atmosphere is injected tangentially to the wall and swirls across the top
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Fig. 8. Schematic drawing of the ozone exposure system: (A) ozonizer, (B) ozone bypass valve, (C) incubator, (D) humidification bottles, (E) exposure vessels, (F) control vessels, (G) pressure gauges, (H) ozone analyser, (I) thermometer, (J) heating tape, (K) thermometer, (L) thermal insulation (Tarkington et al., 1994).
Fig. 9. Ozone exposure system. View inside the incubator: (A) inlet exposure atmosphere, (B) inlet control atmosphere, (C) exhaust exposure atmosphere, (D) exhaust control atmosphere, (E) humidified exposure atmosphere, (F) humidified control atmosphere, (G) humidifier bottle, exposure, (H) humidifier bottle, control, (I) distilled water, (J) heating mat, (K) motorized rocker, (L) thermal insulation, (M) exposure vessel, (N) control vessel (Tarkington et al., 1994).
of the culture vials. Exhaust from each vessel was taken from the bottom in the center. Exhaust lines with integrated tees exit from the vessels. A pressure line is connected to the tee for measuring the pressure in each vessel with a remotely mounted gauge. The exposure vessel exhaust line passes through a port in the incubator and is connected to the ozone analyzer. All exhaust, pressure and sample lines as well as valves and gauges are wrapped with heating tape and thermal insulation to prevent condensation in the system.
Using this construction, Tarkington and coworkers (1994) exposed bronchial epithelial cells and explant cultures from the trachea to different ozone concentrations. Here, for the first time, the gas stream is guided in a vertical stream directly to the cells to realize an optimal contact with the gas phase. Furthermore, this system presents an overall controlled exposure device for establishing stable and reproducible exposure conditions. Based on the observations of Pace et al. (1961, 1969), Tarkington also studied the protective influence of a medium overlay on the cells. A major conclusion from the studies of Pace was that thin layers of liquid are sufficient to protect the cells from NO2 and, therefore, studies on the effects of gas must employ systems that allow more direct contact between the gases and cells. Consequently, Tarkington and his group analyzed the effects of culture surface fluid volume on ozone toxicity. As shown in Fig. 10, without any fluid on top of the surface, ozone caused substantial damage to the airway epithelial cells. Cell viability was in the range of 20% in comparison to control cultures without any exposure. In contrast, in the control vessel exposed to filtered air, the viability was almost 85%. The addition of medium on the top of the cells reduced the damage caused by ozone. These results demonstrate the importance of direct exposure of cells to assess the toxicity in vitro (Fig. 10). The exposure studies conducted by Tarkington et al. (1994) clarified the preconditions necessary for running efficient in vitro toxicity experiments: (1) the generation and monitoring of reproducible levels of the test gas, (2) a stable and reproducible delivery of the gas to replicate vials, (3) multiple exposure at the same concentration, even when performed on different days with different test batches should produce similar results. Encouraged by the results gained under the exposure conditions at the air/liquid interface, Sun et al. (1995)
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120
100
Viability %
80
60
40
20
0
Controls
0
0.2
0.6
1.0
Volume of Medium on Top of Cells (ml)
Fig. 10. Effects of culture surface fluid volume on ozone toxicity. A human bronchial epithelial cell line was exposed to 1 ppm ozone or to filtered air for 2 h. Prior to exposure, various volumes of cell culture medium were added on top of the cells (Tarkington et al., 1994).
studied the effects of sidestream cigarette smoke as an example for complex mixtures, ozone and a combination of both on bronchial epithelial cells (BEAS-2B). The exposure of the cells to ozone was carried out using the technique described by Tarkington et al. (1994) whilst exposure to sidestream smoke was conducted using a different system developed by Teague et al. (1994). BEAS-2b cells were cultured in the biphasic Millicell-CM system (30 mm diameter, 0.4 mm pore size; Millipore, Bedford, MA, USA) and transferred into a cell incubator (37 1C) with apical exposure to a welldefined atmosphere (filtered air and 5% carbon dioxide saturated with water vapor) with the addition of sidestream cigarette smoke generated from an ADL/II smoking machine (Arthur D. Little, Inc.) according to ISO norms. Both ozone and sidestream cigarette smoke induced a dose-dependent decrease of cell viability, inhibition of cellular metabolism and replication, as well as a compromise of external cell membrane integrity. Interestingly, the combined exposure had a less than additive cytotoxic effect on all the dose groups tested. For the exposure of cells from the respiratory tract to reactive gases and complex mixtures, exposure at the air/liquid interface was favored and different experimental setups were described in the following years based on a biphasic culture technique. More attention was directed towards improving a controlled gas generation and delivery to the cells to run dose-related studies. The knowledge of in vitro exposure techniques was based mainly on experiments with single gases like ozone or NO2. Only a limited number of studies
(Aufderheide et al., 2002; Massey et al., 1998; Morin et al., 1999; Sun et al., 1995;) dealt with native complex atmospheres like cigarette smoke or diesel exhaust, probable due to difficulties in creating stable and reproducible atmospheres. A particular challenge to such aerosols is posed by the particulate phase, which has to be brought into contact with the cells. Especially when dealing with complex atmospheres, the biological activity of the particulate and the gas phases is important to characterize the toxicologic activity of such test compounds. Consequently, a development was started to construct exposure devices to expose cells of the respiratory tract to complex aerosols like cigarette smoke (Aufderheide, 1997; Massey et al., 1998). A special exposure chamber based on the biphasic cultivation of cells on microporous membranes was designed to carry out static exposure of cultured cells to fresh diluted mainstream smoke (Fig. 11). Here, the exposure chamber contains six positions to house transwell inserts with a membrane of 24 mm diameter and 3.0 mm pore size. The cells were supplied with a continuous flow of medium from the bottom (base plate). On this plate, a support frame was located to provide an exact and reproducible positioning of the transwell and membranes in the culture medium. On the support frame, a smoke distribution panel ensured homogeneous distribution of the smoke to the transwells. From a central inlet, the smoke is guided radially via six channels to six cylindrical holes above the inserts. Here, a stainless steel mesh screen is located for homogenous distribution of the smoke to all parts of the membrane. A coverplate of stainless steel with a 3 mm central inlet for the smoke completes the exposure chamber and was connected via a Quickfit adapter to the smoke generation and dilution system. The exposure chamber was placed in a commercial incubation chamber to provide a suitable environment (37 1C, 95% relative humidity and 10% CO2) to maintain cell viability during smoke exposure. Cigarette puffs (35 ml in 2 s) were taken via a step motor-driven glass syringe and further diluted. After 2 s, the smoke was expelled from the syringe and diluted with preconditioned air drawn from the incubator. After the exposure chamber was filled with smoke, valves were closed and static exposure commenced (10–40 s). Once completed, the smoke was cleared from the chamber and replaced with incubator-conditioned air before starting the next puff. In such a way, V79 cells were not only exposed to whole cigarette smoke, but also to the gas vapor phase by inserting a Cambridge filter over a total period of 3 h. It could be shown that, by increasing the smoke concentrations (457–840 mg/cm3 TPM), there was a decrease in cell proliferation. Analysis of genotoxic effects showed an increase in micronuclei induction with decreasing smoke concentration, also for
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Fig. 11. Schematic drawing of smoke exposure device detailing arrangements of the exposure chamber, smoking and dilution system, control system and medium supply (Massey et al., 1998).
the gas vapor phase. Exposure to whole smoke resulted in a 3-fold induction of micronuclei and a 2.4-fold induction when the cells were exposed to the vapor phase only. With regard to the design of the exposure chamber, the smoke was introduced into the system at the top of the transwell inserts, resulting in an inhomogenous distribution of the particles on the membrane, which could be minimized by inserting the described steel mesh screen. These results demonstrated that direct contact between the cells and the test atmosphere might be a critical point for realizing enhanced particle deposition on the membrane. Therefore, the idea of Tarkington and his coworkers (1994), to guide the atmosphere via a vertical dynamic stream directly to the biphasic cultivated cells was taken up again in another exposure system, called CULTEXs (Vitrocell, Germany) (Fig. 12). Aufderheide and coworkers (Aufderheide and Mohr, 1999, 2000; Aufderheide et al., 2002, 2003) used a specially designed exposure unit in which cultivated human cells of the respiratory tract were exposed at the air/liquid interface to mainstream cigarette smoke as well as to its vapor phase
without heating, humidification, or addition of carbon dioxide. This strategy has also been successfully used for the in vitro testing of native gaseous compounds (Ritter et al., 2001), complex mixtures like sidestream and whole cigarette smoke (Aufderheide et al., 2001; Ritter et al., 2003, 2004; Wolz et al., 2002), and automobile exhaust (Knebel et al., 2002). The components of the module are shown in Fig. 12. The lower part of the module houses three vessels for transwell inserts (growth area 1 cm2). These vessels are insulated against the inner space of the module, which can be floated in temperature-controlled water (37 1C). The vessels are filled with medium, thus providing humidification and supply of the cells with nutrients. The exposure top of the module has three specially designed inlet tubes, which guarantee the homogenous distribution of the particles of a test atmosphere, which is sucked via negative pressure through the module, entering the system via the inlets and leaving them through the corresponding outlets. The inlet tube starts with a small pipe, ending in a hyperboloid-shaped air distribution section (subsequently referred to as a ‘‘trumpet’’) that allows the
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Fig. 12. The CULTEX exposure module consisting of a lower part for maintenance of cell viability and the upper exposure top for reproducible delivery of the test atmosphere.
Fig. 13. Vector diagram of the velocity field in the central plane (Aufderheide and Mohr, 2004).
uniform deposition of particles on the surface of cell cultures (Prof. Durst, Institute of Fluid Mechanics, LSTM-Erlangen, University of Erlangen-Nu¨rnberg, Germany). The flow remains separation-free, ensures a continuous delivery of the aerosol towards the cells and constant particle deposition (Figs. 13 and 14; Aufderheide and Mohr, 2004). Using the CULTEXs system, A549 cells, which are described to share several fundamental characteristics with human lung alveolar type II cells (Lieber et al., 1976; Nardone and Andrews, 1979; Mendelson and Boggaram, 1991; Smith, 1977; Smith et al., 1982; Young and Mendelson, 1997) and competence of xenobiotic metabolism (Hukkanen et al., 2000; Iwanari et al., 2002; Urani et al., 1998), were exposed to mainstream smoke and the gas phase of a number of different cigarettes in the non-toxic range. Fresh cigarette smoke, generated
according to ISO norms in a specially designed smoking robot VC10 for in vitro exposure (Vitrocell, Germany), was sucked at different concentrations above the cells for 32 min. To characterize the biological activity of smoke, protein content and, as a sensitive marker for cell damage, intracellular reduced glutathione were analyzed directly after exposure. Cellular protein revealed no statistically relevant changes in comparison to control cultures exposed to synthetic air. However, throughout all tested cigarettes, dilutions and exposures to whole and filtered smoke, a significant dose-dependent depletion of reduced glutathione was observed under such non-toxic experimental conditions (Fig. 15). In addition, it could be demonstrated that the quantitative effects of whole smoke and the gas phase were statistically distinguishable for the research cigarette. The susceptibility and flexibility of the system indicate its suitability for analyzing the biological effects of airborne pollutants in the environment. Owing to the fact that cell viability can be maintained independently of an incubator, it can be transported directly to the source of pollution. The integration of inlet tubes manufactured from surface treated stainless steel (Fig. 16) represents a further advantage, because the deposition efficiency for nano-sized particles reached 80% of the exposed mass. This system has also been adapted to expose bacteria of Salmonella typhimurium strains to complex mixtures like mainstream cigarette smoke in a modified Ames assay (Aufderheide and Mohr, 2004). In this case, the basal part of the module houses Petri dishes (35 mm diameter) which are exposed, in the same way as described above, to different numbers of Kentucky research cigarettes K2R4F (tar content 9 mg/cigarette) and a commercially available one with a tar content of 1 mg per cigarette. The bacteria were seeded according to the spread culture technique without an agar overlay and exposed for 30 min to mainstream smoke of up to
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Fig. 14. Concentration of the smoke in the central plane (A) and at the surface of the insert (B). The numbers indicate the smoke concentration relative to air (Aufderheide and Mohr, 2004).
100 3 cigarette s
GSH [% Control]
80
y = 117.18e -0.0506x R2 = 0.9509
60
40
y = 111.18e -0.0753x R2 = 0.9583
20 Gas Gas & particles
1.0
10.0
100.0
Puffs / dilution
Fig. 15. Dose–response curves from exposures of A549 cells to fresh filtered or whole smoke from K1R4F. Intracellular glutathione contents were plotted as a percentage of control values against exposure dose calculated as the ratio of number of smoked cigarette puffs and dilution rate used. Each dot represents the result of a single exposure experiment (Ritter et al., 2004).
seven cigarettes at different dilution rates by using the smoking robot VC10 (Vitrocell, Germany). As shown in Fig. 17, it is possible to bring about a dose-dependent induction of revertants with this exposure procedure. The analysis of the mutagenic activity of two cigarettes with different tar content showed significant differences in the induction of revertants correlated with the tar content of the cigarettes. A modified version of the CULTEXs system has also been used by other research groups to expose cells to
Fig. 16. Inlet tubes with a hyperboloid-shaped air distribution section manufactured from (A) Teflon and (B) surface treated stainless steel. 14
Quotient [smoke/synthetic air]
0
12 10 8 6 9 mg tar/cigarette 1 mg tar/cigarette
4 2 0 0.1
1
10
100
Dosage (cig./dil.)
Fig. 17. Exposure of Salmonella typhimurium strain TA98 to mainstream cigarette smoke of K2R4F cigarettes (tar content 9 mg/cigarette, violet squares) and a commercial brand (tar content 1 mg/cigarette, magenta squares) using the CULTEXs system. Each dot represents the result of a single exposure experiment.
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complex mixtures like mainstream smoke (Fukano et al., 2004) based on the principle of sedimentation (Fig. 18). The cells were maintained in the exposure module under submersed conditions, i.e. covered by a layer of medium. Each culture module was then filled with freshly generated smoke, smoke supply was shut off and cells were exposed for 24 s per puff. The medium was discharged, allowing the particles to drop down onto the cells by sedimentation. Fresh medium was supplied until the inserts were floated again. Finally, the system was purged with clean air to remove residual smoke. Cells were maintained in the liquid medium except during the exposure period to maintain culture conditions and protect the cells from initial puff pressure and the final cleansing air flow. This cycle was repeated and cells were exposed to freshly generated cigarette smoke once every minute. The membrane in culture inserts was pierced with five holes by needles to control the speed of liquid medium exchange. This modified exposure procedure also resulted in the determination of dose-dependent effects after exposure of human epithelial lung cells to cigarette smoke.
Conclusion Taking into consideration all the presented exposure procedures, it can be concluded that, dependent on the hypothesis to be tested, a variety of experimental approaches can be realized. The most important aspect might be the susceptibility of the system, that means a sensitive biological test system and an optimal exposure device: (1) realizing an optimal contact between cells and the test atmosphere, (2) avoiding interactions of compounds of the test atmosphere with medium components and (3) guiding it without minimal chemical and physical changes to the cells. In summary, the biphasic growth of cells of the respiratory tract at the air/liquid interface in combination with relevant exposure methods offers new possibilities to analyze the biological activity not only of synthetically generated complex mixtures in the laboratory, but also under in- and outdoor conditions in the future. The same might be true for analyzing the genotoxic potency of such atmospheres by using a modified and mobile Ames assay.
Fig. 18. Exposure of human epithelial lung cells (A549) to mainstream cigarette smoke (K2R4F) using a modified CULTEXs system (Vitrocell, Germany) (Fukano et al., 2004).
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