Study of the cell-transforming ability of amosite and crocidolite asbestos and the ability to induce changes in the metabolism and macromolecular binding of benzo(a)pyrene in C3H10T1/2 cells.

The cell transforming ability of asbestos dusts was investigated using C3H10T1/2 murine fibroblasts. In a series of experiments, crocidolite and amosite caused no increase in the number of transformed colonies over that seen in cultures from untreated cells. The dusts were, however, capable of augmenting the oncogenic effect of benzo(a)pyrene. This synergistic effect was evident when fibers and chemicals were added to cultures as simple mixtures and when benzo(a)pyrene was adsorbed to the surface of fibers. Asbestos dust did not, however, appear to exert its oncogenic enhancing effect by modifying the metabolism of benzo(a)pyrene in C3H10T1/2 cells.


Introduction
It is well established that exposure to amphibole asbestos leads to an increased incidence of lung cancer and mesotheliomata (1) but the mechanism by which it exerts its effect is largely unknown. On the basis of results obtained from chromosomal aberration studies (2) and the induction of point mutations (3) in Chinese hamster cells, it has been suggested that crocidolite may behave as a "conventional" carcinogen causing genetic damage. The apparent inactivity of asbestos dusts in bacterial mutation tests (4,5) and negative results in sister chromatid exchange analysis (6) would, however, suggest that, at best, mineral fibers are only weakly genotoxic.
Epidemiological evidence linking asbestos exposure and cigarette smoking in the etiology of lung cancer (7)(8)(9)(10) has provoked many experiments based on the supposition that these fibers might exert their biological effect by enhancing the tumorigenicity of some chemical carcinogens. These studies have recently been reviewed (11). *MRC Pneumoconiosis Unit, Llandough Hospital, Penarth, S. Glam., CF6 IXW, UK.
The aim of the work presented here was (a) to determine whether amphibole asbestos fibers possessed the ability to cause the in vitro transformation of C3H1OT1/2, (b) to explore the possibility that they might enhance the cell-transforming activity of the polycyclic aromatic hydrocarbon benzo(a)pyrene (BaP) and (c) to ascertain whether they can cause any change in the ability of C3H1OT1/2 cells to metabolize BaP.

Asbestos
The UICC standard samples of crocidolite and amosite (12) were used. Milled amosite was prepared as reported previously (13) and was milled for 4 hr.

Chemicals
Benzo(a)pyrene (BaP) was -obtained from the Sigma Chemical Co., Poole, Dorset, UK. Fetal calf serum and other tissue culture media and reagents were obtained from Flow Laboratories, Irvine, Scotland.
[3H]-Benzo(a)pyrene (20 mCi/mmole) was oh-tained from the Radiochemical Centre, Amersham, England. Both [3H]-labeled and nonradioactive BaP were purified before use by eluting through a Sep Pak silica gel cartridge with petroleum ether. BaP was added to cultures in one of two ways: as a solution in acetone final concentration in medium (<0.5%) or adsorbed to the surface of the dust. This adsorption was carried out by suspending the dust in ethanol, adding the appropriate concentration of BaP solution (in acetone), and drying down at 800C in a stream of nitrogen.
Dusts were sterilized by autoclaving (15 psi for 15 min) and then suspended in complete medium and added to the cultures to give the desired concentrations.
All solutions were prepared immediately prior to use.

Cell Culture
The 10T /2 Cl 8 cell line derived from C3H mouse embryo fibroblasts (14) was received in this laboratory at passage 9 as the kind gift of Dr. W. J. Harris, Inveresk International Ltd. All experiments were carried out on cells from passage 10-13. The cells were grown in Dulbecco's modification of Eagle's Medium with a concentration of sodium bicarbonate of 3.6 g/L to permit equilibration with a gas phase of 8% CO2 in air. It was supplemented with twice the normal concentration of glutamine, plus heat-inactivated fetal calf serum (10% v/v) and contained penicillin (100 units/mL) and streptomycin (100 pg/mL). Transformation Studies Two methods based on the basic protocol of Reznikoff et al. (15) were used for transformation assays. Studies in 6 cm Peti Dishe& Cells were seeded together with the agent under tests such that approximately 1000 colony forming units could be expected to survive on each plate. The cells were incubated at 370C in an atmosphere of 8% CO2 in air in the presence of the test agent. The medium was changed 48 hr later and thereafter twice weekly for 3 weeks, at which time the serum concentration was reduced to 5%. This medium was then changed weekly for 3 weeks more. The plates were fixed in formalin (10°h), stained in aqueous methylene blue (1°h) and, after washing and drying, the number of type III transformed foci were counted by using the criteria described by Reznikoff et al. (15). Toxicity was assessed in this method by plating 400 cells into each of a series of 9 cm Petri dishes together with a range of concentrations of the agent under study. After 10 days these survival plates were fixed in formalin (10%), stained in methylene blue (1%), and the colonies counted. The toxicity of the agent at the concentration used in the transformation assay was estimated from the resulting survival curve.
Studies in 25 cm' Tissue Cultre Flasks. Samples of C3H1OT1/2 cells (5 mL, 200 cells/mL) from subconfluent cultures of C3H10T1/2 cells (passage 10) were distributed among 25 cm2 tissue culture flasks (Nunc) which were placed in a 370C humidified incubator with an atmosphere of 5% CO2 in air. The flasks were incubated overnight with caps screwed on lightly to allow for equilibration of gas. At 24 hr after plating the cultures were treated with the test agents or positive control chemical dissolved in acetone (final concentration 0.5%).
The cultures were exposed to the chemical for 48 hr at 370C, after which time a medium change was made. The culture medium was then changed twice weekly until the cells reached confluence; thereafter medium changes were once weekly. After 6 weeks the cultures were fixed, stained and scored for transformed foci as described by Reznikoff et al. (15).

Metabolism Studies
Purified [3H]-labeled and unlabeled benzo(a)pyrene in acetone were added to confluent monolayers of treated or untreated of C3H10T1/2 cells (Table 3) growing in 175 cm2 tissue culture flasks to give final concentrations of 10 jA Ci/mL, 0.5 Mg/mL. After a 24 hr incubation at 370C in an atmosphere of 8% CO2 in air, the medium and cells were separated and stored at -600C under an atmosphere of nitrogen until analyzed.
The medium was extracted three times with ether (2 vol), and aliquots were examined for organic-soluble metabolites by thin-layer chromatography TLC in benzene: petroleum ether (1:1). The organic-soluble metabolites were quantified following cutting of the chromatogram and liquid scintillation counting in a Intertechnique SL 4000. The radioactivity in the water-soluble conjugate metabolites remaining in the extracted medium was determined by liquid scintillation counting.
Estimation of Radioactivity Bound to Cellular Material Cellular macromolecules were precipitated and extracted by using published methods (16).

Transformation Assays
Amosite and crocidolite were toxic to lOT /2 cells at similar or lower concentrations to those reported for other cell lines (17). The results of the cytotoxicity studies showed that a coincident exposure of lOTh/2 cells to dust and BaP resulted in greater cell death than when cells were treated with either of the agents alone. This increase in toxicity following coincident exposure, although variable, suggested that the effect was synergistic rather than cumulative ( Fig. 1 and Table 1). The cytotoxicity of BaP adsorbed to the dust, although greater than that of the separate toxicities, was, however, less than that of the simple mixture (Fig. 1). The milled amosite sample was less toxic than the UICC sample, which is in agreement with results reported with V794 cells (13).
The results of the four transformation assays are shown in Tables 1 and 2 Table 2. These results showed that BaP at concentrations of 0.1 and 0.3,g/mL produced only very few transformed foci, while none of the dusts examined produced any significant increase in transformed cultures as compared to the negative controls. The coincubation of crocidolite or amosite and BaP with lOT /2 cells, however, caused a significant increase in numbers of transformed foci, i.e., a 2.3fold increase in experiment 4 over the number of transformed foci produced by BaP alone. BaP adsorbed onto dust also had a greater transforming ability than that of BaP or dust alone, producing 1.8, 2 and 6.1 times the number of foci in experiments 2, 3 and 4, respectively.
In addition to the observed increases in absolute numbers of transformed foci, there was also a parallel increase in transformation frequency (

Metabolism Studies
[3H]-BaP was metabolized to organic solvent-and water-soluble products by both crocidolite-treated and untreated cultures of C3H1OT1/2 cells (Table 3). There was no significant difference in the amounts of organic-soluble metabolites produced by treated and untreated cultures in any of the three separate experiments. A significant reduction in the proportion of [3H]-BaP metabolized to water-soluble products was observed in cultures treated with the hydrocarbon adsorbed to the surface of the crocidolite (expt. 3). This alteration in the production of watersoluble metabolites was not, however, accompanied by changes in other measured parameters, i.e., protein-and DNA-bound metabolites.
The levels of [3H]-BaP bound to cellular macromolecules of the various treatment groups are shown in Table 3. The results of the experiments were variable. In experiment 1 there was significantly higher binding of the [3H]-BaP to the DNA of

Discussion
The data presented here clearly show that crocidolite and amosite, at the concentrations tested, possessed no cell-transforming capability, but at similar doses were able to augment the oncogenic effect of benzo(a)pyrene. This effect was evident whether the agents were added as mixtures or with the BaP adsorbed to the surface of the fibers.
It would appear, therefore, that one effect of the coincident exposure of 1OTh/2 cells to crocidolite and benzo(a)pyrene, at individually subeffective or at best slightly effective doses, is to cause a significant increase in the production of transformed foci. This in vitro synergistic effect is considered to parallel the in vivo situation where enhanced production of lung tumors is seen in asbestos workers who also smoke cigarettes (7).
These results are also consistent with the results of animal experiments in which the synergism of polycyclic aromatic hydrocarbons (PAH) and particulates for tumor production has been demonstrated (18)(19)(20).
As the way in which the neoplastic response of tissues and cells to BaP is augmented by asbestos is still to be established, a series of experiments was undertaken to discover if crocidolite exerted an effect by modifying the ability of C3H1OT1/2 cells to metabolize polycyclic aromatic hydrocarbons (PAH). In the first of three experiments, the co-exposure of cells to asbestos (100 j.g/mL) and J3H]-BaP resulted in a reduction of the amount of BaP bound to DNA as compared to the nondusted controls. As the toxicity of crocidolite at 100 lg/mL was very high, lower concentrations (10 lg/mL) were used in subsequent experiments. In these latter studies crocidolite treatment, either simultaneously with [3H]-BaP or with a 24 hr pre-exposure of cells to the dust followed by addition of the isotope, resulted in an increase in the amount of [3H]-BaP binding to DNA (p> 0.05). The enhanced binding of BaP to deoxynucleotide in cells exposed to asbestos 24 hr before PAH treatment has also been reported by other investigators (21).
In experiment 2, the exposure of cells in mixtures of [3H]-BaP and crocidolite caused a reduction in the production of water-soluble metabolites. Although a repeat study (experiment 3) failed to confirm these findings, it was found in the same experiment that exposure of cultures to [3H]-BaP bound to crocidolite fibers resulted in a significant reduction of water-soluble metabolites (p > 0.05).
The variability of the data generated by the metabolism experiments was such that no firm conclusions can be formulated as to whether or not asbestos dusts induced any significant, reproducible changes in BaP metabolism.
In conclusion, these data show that exposure to asbestos dusts alone resulted in no significant increase in transformed foci but that the dusts were capable of augmenting the oncogenic effect of benzo(a)pyrene. The results of the studies undertaken to investigate the possibility that asbestos may exert an effect through modifying the ability of cells to metabolize benzo(a)pyrene, although equivocal, do suggest this area should be the subject of future study.