Chapter III

TOXICITY OF ATMOSPHERIC PARTICLES TOWARDS PULMONARY CELL CULTURES

INTRODUCTION

     Ambient airborne particles contain material (chemicals, metals, etc) which can be damaging to the cells of the lungs if retained for extended periods of time. Following deposition in the lungs, this material may solubilize in the extracellular lining fluid of the alveoli and reach the septal cells, resulting in chemical toxicity. As well, insoluble material may also maintain sufficiently high levels of material such as complexed metals to cause cellular toxicity. In an attempt to characterize the toxicity of these different particle fractions, work was carried out using a cell monolayer model. Total particulate preparations, as well as water soluble and insoluble fractions, were applied to several cell lines derived from pulmonary tissue. Using the viability indicator alamarBlue, the cytotoxic effects of the different particle fractions towards low density cell cultures were monitored over a two day period. This provided a system in which the toxicity of several particulate preparations could be compared. As well, fractionation of the particulate material allowed for identification of the fractions (soluble vs insoluble) containing cytotoxic material.

MATERIALS AND METHODS

1. Materials and Reagents

1.1. Labware

     Multi-well plates (96-well) were purchased from Costar (Cambridge, MA). AlamarBlueTM was from Alamar Biosciences (Sacramento, CA). M199 culture medium (with and without phenol red), heat inactivated fetal bovine serum (FBS), sodium bicarbonate, HEPES, L-glutamine, phosphate buffered saline (PBS), microcentrifuge filters (1000 MW cutoff) and Tween-80 were from Sigma Co. (Mississauga, ON). Gentamycin was from Gibco (Grand Island, NY). SRM1648 (St. Louis Dust, urban particulate matter), SRM1649 (Washington dust, "organics") and TiO2 (SRM154b, titanium dioxide) were obtained from the National Institute of Standards and Technology (NIST; Gaithersberg, MD). The EHC-93 preparation consists of urban air particles collected from bag filters at the Environmental Health Centre (Tunney's Pasture in Ottawa, Ontario). The EHC-93 particles are similar in composition to SRM1648 and SRM1649.

2. Particle Stock Preparation

     Stocks of total particulate material for SRM1648, SRM1649, EHC-93 and TiO2 were prepared as outlined in chapter II. Aliquots of this particulate material were also fractionated in water to produce stocks of soluble and insoluble material (Fig 3.1). Particle stocks (6 mg in 600 無 NaCl/Tween-80) were prepared in eppendorf tubes, sonicated for 10 minutes and centrifuged at 13000 rpm for 30 minutes in a desktop microcentrifuge (IEC/MicroMax; Needham Hts, MA). The supernatants (600 無) were kept, the pellets resuspended in 600 無 of NaCl/Tween-80, sonicated for 10 minutes and centrifuged at 13000 rpm for 30 minutes. The supernatants were pooled with the first wash. The supernatants were filtered through a microcentrifuge filter (1000 MW cutoff) producing particle-free, aqueous extracts (final volume: 1200 無; 5 mg/mL). The particle pellets were also resuspended in 1200 無 of NaCl/Tween-80 (5 mg/mL) and constitute the washed, insoluble matrix.

Figure 3.1:
Preparation of insoluble and soluble (aqueous) fractions of ambient air particle preparations



            EHC-93 and SRM1649 particles were also subjected to quantitative water extraction. The particles were placed in pre-weighed vials (1 gram/vial), subjected to three successive washs (5 mL each wash) with water and the washings collected into a second pre-weighed vial. These washings were then filtered through a nylon 0.2 痠 filter to remove any particulate material. The insoluble fraction was resuspended in 15 mL of water and, along with the water soluble fraction, the samples were frozen in liquid nitrogen, lyophilized, and weighed.

3. Effects of Particles on Low Density RFL-6, AK-D and R10M Cell Cultures

     The RFL-6, AK-D and R10M cell lines were used to assay the cytotoxicity of several different particle types. The cell lines were seeded into 96-well plates at low density (1000 cells/well) in M199 medium (without phenol red) containing 10% FBS and allowed to attach for 24 hours. The medium was then removed and replaced with 100 無 of fresh M199 containing 10% FBS. The cells were then exposed to either the total particulate material or the soluble/insoluble particulate fractions. Total particulate material (SRM1648, SRM1649, EHC-93 and TiO2) was applied at 0 to 100 痢/well. Fractionated material (soluble and insoluble fractions) was applied at doses that were equivalent to 0 to 50 痢/well of total particulate material. The final volume in all culture wells was 200 無. AlamarBlue was added to each well (10 無/well) and an initial fluorescence reading was taken using the Cytofluor 2300 (Goegan et al., 1995). Plates were then placed in the CO2 incubator and readings were taken over a period of 48 hours.

4. Statistics

     The data were analyzed by using one-way analysis of variance. Group means were compared to controls using the Dunnett's test.

RESULTS

1. Effects of Particles on Cell Growth

     The effects of the total particle preparations towards the RFL-6, AK-D and R10M cell lines, as measured by the alamarBlue assay, are presented in figures 3.2 to 3.4. The response of the cell lines following particulate exposure was calculated as the change in fluorescence (F), providing estimates of the rates of reduction for the 0 to 24 hour and 24 to 48 hour periods. The SRM1648, SRM1649 and EHC-93 preparations showed similar dose-dependent depression of cellular growth for both the RFL-6 (Fig 3.2) and AK-D (Fig 3.3) cell lines. At the 100 痢/well level, there was still some cellular activity in both cell lines, as indicated by the reduction of alamarBlue, although this dose is quite cytotoxic. The R10M cell line demonstrated a different response following exposure to these particulate materials (Fig 3.4). There was not a smooth dose-dependent decrease in cellular viability following exposure but rather a sharp drop at the highest particle dose for SRM1648, SRM1649 and EHC-93 (Fig 3.4). As well, SRM1649 stimulated cellular activity at 1.2, 4 and 11 痢/well while exposure to 100 痢 caused high levels of cytotoxicity (Fig 3.4).
Figure 3.2:
Cellular viability of low density RFL-6 cell cultures following exposure to total particulate preparations
Figure 3.3:
Cellular viability of low density AK-D cell cultures following exposure to total particulate preparations
Figure 3.4:
Cellular viability of low density R10M cell cultures following exposure to total particulate preparations



            TiO2, which should have been relatively non-toxic, produced a low level of cytotoxicity towards all of the cell lines (Fig 3.2 to 3.4). However, we have later found that SRM154b from NIST was contaminated with naphthalene (analyses done by Dr. P. Kumarathasan, Health Canada). This can explain why this relatively non-toxic particle caused cytotoxicity towards the pulmonary cell lines.

     To determine the cytotoxic effects of different particle fractions, the cell lines were exposed to preparations consisting of the aqueous extract and the insoluble residue. Different responses were measured in the three cell lines following exposure to solubilized particulate material (Fig 3.5 to 3.7). Cytotoxic effects were seen in the exposure period from 24 to 48 hours. A decrease in cellular viability was observed in the RFL-6 (20 to 25%) and R10M (10 to 20%) following exposure to SRM1648 extracts (50 痢 equivalent/well). Soluble SRM1649 material was cytotoxic in all cases, although to a different extent. The SRM1649 extracts were cytotoxic towards the RFL-6 cell line (50% depression) while lesser effects were measured in the AK-D and R10M cell lines (20 to 25% depression). EHC-93 was cytotoxic towards the RFL-6 and R10M cell lines at the highest dose (20 to 25% decrease in viability).
Figure 3.5:
Cellular viability of low density RFL-6 cell cultures following exposure to aqueous particulate extracts
Figure 3.6:
Cellular viability of low density AK-D cell cultures following exposure to aqueous particulate extracts
Figure 3.7:
Cellular viability of low density R10M cell cultures following exposure to aqueous particulate extracts



            Responses of the RFL-6, AK-D and R10M cell lines to the insoluble particulate materials were also examined (Fig 3.8 to 3.10). After 48 hours, there was a dose-dependent depression in cellular viability for the RFL-6 cell line following exposure to all of the particle types (Fig 3.8). At the maximum dose (50 痢 equivalent/well), all particles caused a 30 to 50% drop in viability as compared to controls. This was also observed with the TiO2 preparation. The AK-D cell line responded in a similar fashion with a 30 to 50% depression in viability at the maximum doses (Fig 3.9). The R10M cell line demonstrated an increase in cellular activity for several of the doses at the 24 and 48 hour time points (Fig 3.10). The highest concentration (50 痢 equivalent/well) of the insoluble SRM1648, SRM1649 and EHC- 93 material caused low levels of cytotoxicity (10 to 20% depression). However, exposure to TiO2 did not stimulate reduction of alamarBlue, but caused a dose-dependent depression in cellular growth.
Figure 3.8:
Cellular viability of low density RFL-6 cell cultures following exposure to insoluble particulate material
Figure 3.9:
Cellular viability of low density AK-D cell cultures following exposure to insoluble particulate material
Figure 3.10:
Cellular viability of low density R10M cell cultures following exposure to insoluble particulate material



DISCUSSION

     Using the alamarBlue assay, it was verified that preparations of SRM1648, SRM1649 and EHC-93 were toxic towards the RFL-6 and AK-D cell lines (Fig 3.2 and 3.3). This cytotoxic response was dose-dependent for both cell lines. As well, at the highest dose (100 痢/well), these preparations were also very toxic towards the R10M cell line (Fig 3.4). Slight increases in cellular activity were also measured in the R10M cell line following SRM1649 exposure (Fig 3.4b).

     In an attempt to determine the toxic potential of different particle components, particles were fractionated into water soluble and insoluble fractions. The soluble material present in the SRM1648, SRM1649 and EHC-93 preparations was toxic towards all of the cell lines. At the highest dose (50 痢 equivalent/well) the SRM1648, SRM1649 and EHC-93 material was toxic towards the RFL-6 and R10M cell lines producing a 25 to 50% decrease in cellular viability. Only the SRM1649 material was cytotoxic towards the AK-D cell line. Aqueous extracts of TiO2 did not cause cytotoxicity in any case. These results demonstrate that the ambient airborne particles contain materials that are cytotoxic towards several different pulmonary cells. It has also been determined that preparations of SRM1649 and EHC-93 are composed of approximately 15% water soluble material. Upon deposition in the lung, this material will solubilize in the surfactant and mucus layers and may cause cytotoxicity to nearby cell populations. In the case of the alveoli, the surfactant layer is very thin (microns) resulting in high, local concentrations of solubilized materials. This will cause a chemical poisoning towards the pulmonary epithelial cells and resident macrophages which may in turn have deleterious effects on normal lung function.

     The insoluble matrix of the different particle preparations were also assayed for cytotoxic effects. These insolubleparticle preparations were cytotoxic towards the RFL-6 and AK-D cell lines demonstrating the presence of toxic materials. The responses were primarily dose-dependent with decreases in viability of up to 50%. Exposure of the R10M cell line, however, resulted in increases (10 to 50%) in cellular activity as measured by the alamarBlue assay. Again, this activation of the R10M cells following particle exposure has important implications warranting further study. It is interesting to note that the insoluble fractions of TiO2 caused depressions in cellular viability at levels comparable to the ambient airborne particle standards. As mentioned, we later realized that the TiO2 standard reference material NIST154b was contaminated with naphthalene. This may explain the cytotoxicity that is seen following exposure of the pulmonary cell lines to this TiO2 preparation.

     The results demonstrate that the ambient airborne particles are complex mixtures and that differences in cytotoxic potential may be attributable to different chemical and elemental components. The results indicate that fractionation of the particles can provide insight into the compartmentalization of the toxic components. Particulate materials which solubilize in the lung may be important in causing toxicity to nearby cell populations which do not directly contact the deposited particles. However, the insoluble matrix may exert its cytotoxic effect by direct contact with epithelial cells or following phagocytosis by the alveolar macrophages. Assembly of a cell culture model incorporating alveolar macrophages and test cells is an important next step in characterizing the cytotoxicity of ambient airborne particles. This type of a system would allow for the determination of cytotoxic effects towards the alveolar macrophage and would allow for the study of its ability to modulate the toxicity of deposited particulate material.


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