Direct Methods

Several direct in-vitro models have also been developed to deal with gas-phase exposure of airborne contaminants, using different exposure techniques. The different features of these techniques have been discussed in terms of their relevance, advantages, and limitations [83, 84]. In principle, these methods include the exposure of cells under submerged conditions, intermittent exposure procedures and, more recently, direct exposure techniques at the air-liquid interface.

Exposure by bubbling test gaseous compounds through cells suspended in media can easily be achieved using variations of standard laboratory processes (Fig. 4.2). One such example was an investigation of the in-vitro toxic effects of ozone on human hematic mononucleated cells (HHMC), with exposure to ozone being effected by single injection of the gas into cell suspensions in vacuum test

Fig. 4.2 Exposure by bubbling test gaseous compounds through cells suspended in media.

tubes [85]. Exposure patterns in vivo may not be closely simulated by this method, however, as only a very small interface between the test gas and the target cells can be provided when using the submerged exposure technique.

Variations of laboratory techniques have been developed that allow the intermittent exposure of cultures to gaseous contaminants. Cell culture dishes held on chambers or platforms rounded, stacked or tilted at certain angles, were exposed periodically to gaseous compounds [86-88]. Cell culture flasks were also tilted at regular intervals to expose the cell cultures to volatile anesthetics [89], while rolling culture bottles on roller drums were set up for in-vitro gas exposure [38, 90]. Lung slices were alternatively fed by culture medium and exposed to diesel exhausts by rotating the culture vial on the internal wall of a flow-through chamber [91]. Tissue culture flasks on a rocking platform were used to expose the cells to mainstream cigarette smoke, followed by intermittent immersion in culture media [92].

A micro-roller bottle system has been developed for cytotoxicity screening of volatile compounds in which primary hepatocytes are attached to a collagen-coated nylon mesh (Fig. 4.3). The primary hepatocytes were exposed to volatile compounds injected into the roller bottle, which was placed on a roller apparatus in an incubator at 37 °C, with the hepatocytes being exposed alternately to the medium and the test atmosphere. Medium samples were then taken to measure cellular LDH and aspartate aminotransferase activities [38]. Compared to submerged exposure, the intermittent exposure technique provides a larger interface between the gaseous compound and the target cells. Nevertheless, under such exposure conditions, cells are always covered by an intervening layer of medium that may influence both the accuracy and reproducibility of the results.

During the 1990s, the technology became available that would allow cells to be cultured on permeable porous membranes in commercially available transwell or snapwell inserts (Fig. 4.4). Once the cells have become established on the membrane, the upper layer of culture medium can be removed, and the cells exposed directly to air contaminants. In a direct exposure technique, at the

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Fig. 4.3 A micro roller bottle system (modified from [39]).

Fig. 4.4 The culture of human cells on porous membranes.

(a) Culture of cells on microporous membranes.

(b) Exposure of cells to airborne contaminants following removal of the media in the upper compartment.

Fig. 4.4 The culture of human cells on porous membranes.

(a) Culture of cells on microporous membranes.

(b) Exposure of cells to airborne contaminants following removal of the media in the upper compartment.

air-liquid interface the target cells can be exposed to airborne contaminants continuously on their apical side, while receiving nourishment via their basolateral side. The direct exposure of cells to airborne contaminants was initially achieved by growing cells on collagen-coated membrane located on special platforms [93], and more recently on porous membranes in transwell inserts [94-96], or snap well inserts [97, 98]. Both static and dynamic direct exposure methods were established for exposure purposes.

Although exposure to volatile chemicals represents a significant contribution to human health problems, the toxicity testing of volatile compounds has always faced major technological problems [43, 47, 48, 99]. Apart from high volatility, many volatile organic compounds (VOCs) are less water-soluble, or insoluble in water, and these physico-chemical properties may lead to technical challenges during the course of in-vitro experiments. Static direct exposure methods have been developed for the toxicity assessment of VOCs, in which test atmospheres of selected chemicals were generated in sealed glass chambers with known volumes [97].

Human cells, including A549-pulmonary type Il-like cell lines, HepG2-hepatoma cell lines and skin fibroblasts, were exposed to airborne toxicants at different concentrations directly at the air-liquid interface. Post-exposure cytotoxicity was investigated using the tetrazolium salt (MTS) and neutral red uptake (NRU) in-vitro assays. Using the static direct exposure method enabled the establishment of airborne IC50 (50% inhibitory concentration) values for selected VOCs such as xylene (IC50 = 5350-8200 ppm) and toluene (IC50 = 10 500-16 600 ppm) after 1 h exposure. Indeed, the static direct exposure method proved to be both practical and reproducible for in-vitro inhalation studies with volatile chemicals.

A typical experimental set-up for dynamic direct exposure at the air-liquid interface requires the use of appropriate exposure chambers. Standard tissue culture incubators were used for exposure purposes [100], while dynamic delivery and direct exposure of human cells to airborne contaminants was achieved with specific exposure chambers [101] or horizontal diffusion chamber systems [97]. The toxic effects of single airborne chemicals such as ozone and nitrogen dioxide [97, 102], and complex mixtures such as diesel motor exhaust [94], cigarette smoke [96] and combustion products [103, 104], were studied using cultured human lung cells on porous membranes permeable to culture media. The dynamic direct exposure technique at the air-liquid interface offers a reproducible contact between chemically and physically unmodified airborne contaminants and target cells and, from a technically standpoint, more closely reflects inhalation exposure in vivo [83, 95-97].

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