Chapter IV

DEVELOPMENT OF A SEQUENTIAL CELL CULTURE MODEL FOR STUDYING THE DYNAMICS OF PARTICLE TOXICITY

INTRODUCTION

     Following assembly of an initial co-culture system and characterization of the responses of pulmonary cells to particulate materials (total particles, soluble and insoluble fractions), work was continued to study the phenomenon of cellular interaction in the dynamics of particle toxicity. To begin to assess the potential activity of ambient airborne particles in the alveoli, a sequential cell culture model was assembled using transgenic cell lines containing xenobiotic-, metal- and stress-responsive gene promoter regions linked to the gene sequence for chloramphenicol acetyltransferase (CAT). These CAT transgenic cell lines serve as a panel of reporter cells which specifically respond to a series of different stimuli and can be used to functionally identify classes of materials present in complex mixtures. The responsiveness of the cell lines was validated with chemical compounds known to induce specific promoter-CAT constructs (cadmium sulfate: metal response; benzo(a)pyrene: xenobiotic response) and also tested for their ability to respond following direct exposure to ambient airborne particles. After characterization of the response to particles added directly to the culture medium, the cells were exposed to macrophage-particle conditioned culture medium to determine the potential of bronchoalveolar macrophages to enhance the availability of chemical and elemental materials present in the particulate preparations.

MATERIALS AND METHODS

1. Materials and Reagents

1.1. Labware

     The Cat-Tox assay from Xenometrix Inc. (Boulder, Colorado) was used (described below: section 2.1). Transwell- COL inserts and 24-well plates were from Costar (Cambridge, MA). M199 culture medium (without phenol red), heat inactivated fetal bovine serum (FBS), sodium bicarbonate, HEPES, L-glutamine and phosphate buffered saline (PBS) were from Sigma Co. (Mississauga, ON). Gentamycin was from Gibco (Grand Island, NY). SRM1648, SRM1649 and EHC-93 particles were obtained as outlined in chapters II and III. TiO₂ particles (SRM154b) were washed repeatedly with methanol to remove aromatic contaminants (naphthalene) and then rinsed in Tween-saline. The washed particles were resuspended in Tween-NaCl and prepared as described previously.

1.2. Animals

     Fisher 344, specific-pathogen-free rats were used as a source of bronchoalveolar macrophages (appendix II).

2. Xenobiotic-, Metal- and Stress-Responsive CAT Transgenes
  
2.1. HepG2-Transgenic Cell Lines

     The panel of Cat-Tox HepG2 cells consisted of fourteen different cell lines carrying xenobiotic-, metal- and stress- responsive promoter-CAT gene constructs on two 96-well plates. As well, a single row of wild-type HepG2 cells wasprovided for use in determining the cytotoxicity of the agent under test. The cell lines are outlined in Table 1.


Table 1:  Cell Lines of the Cat-Tox Assay

2.2. Cat-Tox Assay Protocol

     Upon arrival, the cells were fed with fresh culture medium and the plates were placed in the CO₂ incubator for 24 hours. The volume in the wells was then adjusted to 100 無 with fresh culture medium and the wells were dosed with the test agents. The final volume in all of the wells was 200 無. Following a 24-36 hour exposure to the particular test agents, the medium was aspirated out of the culture wells (except for the wild-type HepG2 cells) and was replaced with 100 無 of lysis buffer. After a 30 minute incubation, the cell lysate from the duplicate wells (for each dose) was pooled. Total cellular protein was measured by mixing a 10 無 aliquot of this lysate with 190 無 of protein reagent (Coomassie Blue) and reading at 600 nm using a multiplate spectrophotometer (Molecular Devices; Menlo Park, CA). The cell lysate was transferred to the wells of pre-coated ELISA strips coated with antibodies against CAT protein and incubated for 2 hours. This lysate was then discarded, the wells were washed 3 times with buffer and incubated with a biotinylated antibody against CAT for 1 hour. The wells were rinsed 3 times with buffer and incubated for 30 minutes with strepavidin alkaline phosphatase. The ELISA wells were rinsed and a solution of p-nitrophenol phosphate in diethanolamine was added to each well (200 無) and incubated at 37蚓 for 45 minutes. The absorbance was measured at 405 nm. Levels of expression of CAT for treated and control cells was calculated as the absorbance of the ELISA wells (405 nm) normalized over the absorbance of the protein wells (600 nm). Induction of CAT synthesis following application of the test agent (i.e. chemical standards, particles, macrophage conditioned culture medium) was calculated as response of the treated cells (CAT synthesis divided by total protein) relative to response of the control cells (CAT synthesis divided by total protein).

     As well, the wild-type HepG2 cells were assayed for cellular viability by adding 50 無 of MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution (5 mg/mL in PBS) to the wells, incubating for 30 minutes, solubilizing in DMSO (200 無) and reading at 562 nm.

3. Responses of Cell Lines Following Exposure to Known Chemical Inducers

     Cadmium sulfate was prepared to a final stock concentration of 50 然 in sterile particle solution. Benzo(a)pyrene was prepared by first dissolving 1 mg of benzo(a)pyrene in absolute ethanol and then diluting to a final concentration of 100 然 in sterile particle solution containing 1 mg/mL bovine serum albumin. Stocks were stored at -80蚓.

     Cadmium sulfate and benzo(a)pyrene were diluted 1:5 in culture medium and then diluted in the culture wells to produce appropriate final concentrations. Cadmium sulfate was applied at a final concentration of 1, 2.5 and 5 然 per well. Benzo(a)pyrene was applied at 2.5, 5 and 10 然 per well. Final volume in all wells was 200 無 and the final concentration of particle solution was 10%. Cells were exposed to chemical standards for 24-36 hours. All chemical concentrations were dosed in duplicate, pooled and assayed by ELISA for CAT protein (section 2.2.). Cells (wild-type HepG2) were also exposed to the same doses of chemicals and were assayed for cellular viability(MTT assay).

4. Responses of the Cells Following Exposure to Particulate Preparations

     SRM1648, SRM1649 and EHC-93 stocks were prepared as outlined previously (Chapter II). TiO₂ was washed in methanol and then Tween-saline prior to preparation of stock solutions. The HepG2 cells (transgenic cell lines and wild-type) were dosed at final concentrations of 0, 1.2, 4, 11, 33 and 100 痢/well. The final volume in each culture well was 200 無 and the final concentration of particle solution was 10%. Cells were exposed to particles for 24 hours. All particle concentrations were dosed in duplicate, pooled and assayed by ELISA for CAT protein (section 2.2.). Cells (wild-type HepG2) were also exposed to the identical particulate preparations and were assayed for cellular viability (MTT assay).

5. Cellular Interaction (Sequential Cell Culture) Model

5.1. Macrophage/Particle Exposure

     Transwell-COL inserts were prepared as outlined in chapter II. In brief, the inserts were placed in 24-well plates, inner and outer wells filled with PBS and allowed to incubate for 24 hours. The PBS was then removed and the outer well was filled with 1 mL of complete M199 containing 10% FBS.

     In an attempt to reduce variability associated with harvesting macrophages from different test animals, the pooled macrophages from three rats were used for each experiment. Rats were anesthetized and lavaged for alveolar macrophages as outlined in appendix II. Following harvesting, the macrophages were pooled, counted and resuspended to 6.0 x 10⁵ cells/mL in complete M199 containing 10% FBS. Macrophages were aliquotted into half of the prepared inserts (100 無/insert: 6.0 x 10⁴ cells/insert) and the remaining inserts were filled with 100 無 of complete M199 with 10% FBS (Fig 4.1)

¹ Data kindly provided by P. Kumarathasan and R. Vincent (unpublished)

     Elemental components were also identified for preparations of SRM1648, SRM1649 and EHC-93 (Table 3). Examination of figures 4.5 to 4.7 for levels of metal-mediated transgene induction shows a high level of HMTIIA activity for EHC-93, a lower level of induction for SRM1648 and very little induction following SRM1649 exposure. Comparison of these results with the data presented in table 3 demonstrates a correlation betweenHMTIIA responses with the levels of copper and zinc present in the samples.


Table 3:  Analysis of SRM1648, SRM1649 and EHC-93 for Selected Elements (痢/g or %)

¹ NIST Data for SRM1648
² R. Vincent, unpublished


3. Cytotoxicity of Ambient Airborne Particles Towards Macrophages

     Following incubation of the bronchoalveolar macrophages in tissue culture inserts (6.0 x 10⁴ insert) with different particulate preparations (0, 100 and 200 痢/insert) and collection of the conditioned culture medium in the bottom well, the viability of the macrophages was measured using the alamarBlue assay. As shown in figure 4.9, EHC-93 proved to be the most toxic particle type (two-way ANOVA), although resulting in only a 40% decrease in measured viability. The SRM1648 and SRM1649 particles showed similar cytotoxic effects (20-30% decrease in viability) and TiO₂ was essentially non-toxic as expected (0-10% decrease in viability).