Dependence of asbestos- and mineral dust-induced transformation of mammalian cells in culture on fiber dimension.

The abilities of chrysotile and crocidolite asbestos, glass fibers of differing dimensions, and nonfibrous mineral particulates to induce morphological transformation of Syrian hamster embryo cells in culture were compared. Chrysotile and crocidolite asbestos induced morphologically transformed colonies which were indistinguishable from transformed colonies observed following treatment with known chemical carcinogens. A linear, dose-dependent increase in the frequency of transformed colonies was observed. The slope of the dose-response curve on a log-log scale was approximately 1, which is consistent with a one-hit mechanism for their induction. The transforming potency of chrysotile asbestos was decreased by milling of the fibers but not by extraction with an organic solvent. Chrysotile asbestos was nearly twice as potent in inducing morphological transformation as crocidolite asbestos. Glass fibers were also very active in this assay. Thin glass fibers with an average diameter of 0.1 to 0.2 micrometer were as active as asbestos. In contrast, two nonfibrous particulates, alpha-quartz and Min-U-Sil, were inactive over the same concentration range used for the fibrous dusts; however, both were active at higher doses. The effect of varying fiber dimension on induction of morphological transformation was examined with glass fibers. When compared on a per-weight basis, thick glass fibers [average diameter, 0.8 plus/minus 0.06 micrometer (S.E.)] were 20-fold less potent than thin fibers [average diameter, 0.13 plus/minus 0.005 micrometer] in inducing cell transformation. When the average fiber length of thin glass fibers was reduced from 9.5 to 1.7 micrometer by milling the fibers in a mortar and pestle, a 10-fold decrease in transforming activity resulted. When the average fiber length was reduced to 0.95 micrometer, transforming ability was totally absent. The cytotoxic potencies of the various mineral dusts correlated with their transforming potencies. The varying abilities of the mineral dusts to induce cell transformation in vitro are similar to their abilities to induce mesotheliomas in vivo. Thus, this system provides a unique model for studying the mechanism of mineral fiber tumorigenesis and for comparing the relative risks of mineral dusts.

Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture.

The cytogenetic effects of chrysotile asbestos on Syrian hamster embryo cells in vitro were investigated at doses which induced morphological and neoplastic transformation but which failed to induce measurable gene mutations in the cells at two genetic loci. Chrysotile asbestos treatment of the cells significantly induced chromosome changes in a dose-dependent manner. Up to 50% of the cells had chromosome abnormalities in number or structure following treatment with asbestos (2.0 micrograms/sq cm) for 48 hr. Numerical chromosome changes were the most pronounced abnormalities although significant increases in metaphases with other chromosome aberrations (breaks, fragments, exchanges, and/or dicentrics) and cells with binuclei or micronuclei were also observed. A linear relationship was observed between the incidences of cells with tetraploid metaphases and binucleated cells, suggesting that binucleation and tetraploidy are related. Cytogenetic effects of other mineral dusts were also tested 48 hr following treatment at a concentration of 2.0 micrograms/sq cm. Crocidolite asbestos was less potent than chrysotile asbestos in its ability to induce cell transformation and cytogenetic damage. Treatment of the cells with thin glass fibers (Code 100) was also able to induce cell transformation and cytogenetic effects, but thick glass fibers (Code 110) were much less potent for both endpoints. Milling of the thin glass fibers decreased the length of the fibers and abolished their ability to induce cell transformation and cytogenetic effects. Nonfibrous alpha-quartz induced neither cell transformation nor cytogenetic effects at the dose of 2.0 micrograms/sq cm. The results indicate that the physical characteristics of the fibers determine their ability to induce cell transformation and their ability to induce chromosome mutations, suggesting a possible mechanistic relationship.

Role of phagocytosis in Syrian hamster cell transformation and cytogenetic effects induced by asbestos and short and long glass fibers.

We have shown previously that asbestos and other mineral dusts, including glass fibers, induce cell transformation and chromosomal mutations in Syrian hamster embryo cells in culture. In the present study, we observed that both asbestos and glass fibers were phagocytized by these cells and accumulated in the perinuclear region of the cytoplasm. In order to understand the mechanism of fiber length-dependent cellular effects, we examined the phagocytosis and intracellular distribution of glass fibers of differing lengths in cells at various times after treatment. Glass fiber length was decreased by milling with a mortar and pestle. Cells treated with an equal dose of milled glass fibers (on a weight per surface area basis) were exposed to 7-fold more fibers since milling of glass fibers resulted in a 7-fold decrease in length with little change in diameter. However, cells exposed to milled glass fibers phagocytized a similar number of fibers as cells exposed to an equal mass of unmilled glass fibers, indicating that milled fibers were less readily phagocytized. In cells treated with either unmilled or milled glass fibers, the length of the intracellular fibers was more than 2-fold greater than the length of the fibers on the surface, suggesting that cells selectively internalized longer fibers. Fiber length, however, did not appear to affect the migration of intracellular fibers to the perinuclear region of the cytoplasm. Even though cells treated with milled glass fibers contained a similar number of fibers as those treated with unmilled glass fibers, the resulting cytotoxicity, transformation frequency, and frequency of micronuclei were greatly reduced in the cultures treated with milled glass fibers. Thus, fiber length appears to affect the phagocytosis of fibers as well as the ability of intracellular fibers to induce cytogenetic damage and the resultant transformation.

Role of biopersistence in the pathogenicity of man-made fibers and methods for evaluating biopersistence: a summary of two round-table discussions.

This paper summarizes two roundtable discussions held at the conclusion of the International Conference on Biopersistence of Respirable Synthetic Fibres and Minerals. The first round table addressed the role of biopersistence in the pathogenicity of fiber-induced disease. The panel included T. W. Hesterberg (Chairman), J.M.G. Davis, K. Donaldson, B. Fubini, N.F. Johnson, G. Oberdoerster, P. Sébastien, and D. Warheit. The second panel addressed the issue of methods for assessing biopersistence. It included R.O. McClellan (Chairman), J. Brain, A. Langer, A. Morgan, C. Morscheidt, H. Muhle, and R. Musselman. The two chairmen acknowledge the excellent contributions of all the members of the panels, whose comments formed the basis of this summary. Nonetheless, the authors assume full responsibility for the written text, recognizing that it was not reviewed by the discussants of the two panels.-Environ Health Perspect 102(Suppl 5):277-283 (1994)

Dual pH durability studies of man-made vitreous fiber (MMVF).

Dissolution of fibers in the deep lung may involve both extracellular and intracellular mechanisms. This process was modeled in vitro for each environment using an experimental flow-through system to characterize both total dissolution and specific chemical changes for three representative MMVF's: a glasswool, a slagwool, and a refractory ceramic fiber (RCF). Synthetic physiological fluids at pH 4 and at pH 7.6 were used to simulate macrophage intraphagolysosomal, and extracellular environments, respectively. Actual commercial fiber, sized to rat-respirable dimension, having an average fiber diameter of 1 micron and an average length between 15 and 25 microns, was used in the experiments. Fiber dissolution was monitored through change in chemistry of the fluid collected after percolation at a constant rate through a thin bed of sample. There are great differences in total fiber dissolution rates for the different fibers. Slagwool and RCF dissolve more rapidly at pH 4 than at pH 7.6, while the reverse is true for glasswool. Dissolution is sometimes accompanied by a noticeable change in fiber morphology or dimension, and sometimes by no change. There is strong dependency on pH, which affects not only total fiber dissolution, but also the leaching of specific chemical components. This effect is different for each type of fiber, indicating that specific fiber chemistry largely controls whether a fiber dissolves or leaches more rapidly under acidic or neutral conditions. Both total dissolution rates and calculated fiber composition changes are valuable guides to interpreting in vivo behavior of man-made vitreous fibers, and demonstrate the usefulness of in vitro acellular experiments in understanding overall fiber persistence.

Characterization of exposure and dose of man made vitreous fiber in experimental studies.

The use of fibrous test materials in in vivo experiments introduces a number of significant problems not associated with nonfibrous particulates. The key to all aspects of the experiment is the accurate characterization of the test material in terms of fiber length, diameter, particulate content, and chemistry. All data related to fiber properties must be collected in a statistically sound manner to eliminate potential bias. Procedures similar to those outlined by the National Institute of Occupational Safety and Health (NIOSH) or the World Health Organization (WHO) must be the basis of any fiber characterization. The test material to which the animal is exposed must be processed to maximize the amount of respirable fiber and to minimize particulate content. The complex relationship among the characteristics of the test material, the properties of the delivery system, and the actual dose that reaches the target tissue in the lung makes verification of dose essential. In the case of man-made vitreous fibers (MMVF), dose verification through recovery of fiber from exposed animals is a complex task. The potential for high fiber solubility makes many of the conventional techniques for tissue preservation and digestion inappropriate. Processes based on the minimum use of aggressive chemicals, such as cold storage and low temperature ashing, are potentially useful for a wide range of inorganic fibers. Any processes used to assess fiber exposure and dose must be carefully validated to establish that the chemical and physical characteristics of the fibers have not been changed and that the dose to the target tissue is completely and accurately described.

Relationship between lung biopersistence and biological effects of man-made vitreous fibers after chronic inhalation in rats.

This article describes the relationship between fiber biopersistence and the chronic toxicity of different chemical compositions of man-made vitreous fibers (MMVF) in the lung. Rats were exposed in "nose-only" inhalation chambers, 6 hr/day, 5 days/week, for 24 months to aerosol concentrations of 30 mg/m3 containing comparable fiber numbers and similar dimensions of fibrous glass (FG) or refractory ceramic fiber (RCF). Interim sacrifices were performed periodically to monitor fiber number and dimensions in the lung and the progression of pulmonary alterations. At each interim sacrifice, three to six recovery animals were removed from each exposure group and held until two years to determine the biopersistence of fibers after different exposure times. Fibers were recovered from the ashed lungs, counted, and measured using optical and scanning electron microscopy (SEM). Fiber chemistry was assessed in 91-week recovery lungs using energy dispersive spectroscopy (EDS) analysis. RCF induced lung fibrosis and an elevation in lung tumors and pleural mesotheliomas. FG exposure resulted in no lung fibrosis, no statistically significant increase in the lung tumor incidence, and no mesotheliomas. After two years of continuous exposure, the number of World Health Organization fibers per milligram dry lung recovered from RCF and FG exposed lungs was comparable. EDS analysis of recovery lungs showed that most of the alkalis and alkaline earths had leached from the FG fibers over time. A slight change in RCF chemistry was observed. These findings indicate that the change in the chemical composition of fibers may be an important determinant of the chronic toxicity of MMVFs.

An experimental approach to the evaluation of the biopersistence of respirable synthetic fibers and minerals.

The biopersistence of fibers and minerals in the respiratory tract is an important parameter in the toxicity of those materials. The biopersistence of respirable synthetic fibers and minerals in man can be most closely evaluated in an animal model. While acellular and in vitro systems are important for initial evaluation of solubility and durability, they cannot simulate the dynamics of inhalation deposition and clearance and the subsequent systemic reaction to fibers and minerals that occurs in the animal. To evaluate the biopersistence of synthetic fibers, male rats were exposed to a well defined rat respirable aerosol of man-made vitreous fibers (MMVF), 6 hr/day for 5 days. Following exposure, subgroups were sacrificed at intervals ranging from 1 hr to 52 weeks. Following sacrifice, the lungs were removed, weighed, and immediately frozen at 20 degrees C for subsequent digestion by low temperature plasma ashing. The number, size distribution, and chemical composition of the fibers in the aerosol and lung were determined. With this animal model the role of biopersistence in altering the geometry and clearance of fibers can be systematically evaluated. The model also can be applied for the evaluation of the biopersistence of nonfibrous minerals.

Biopersistences of man-made vitreous fibers and crocidolite fibers in rat lungs following short-term exposures.

Biopersistence of commercial man-made vitreous fibers (MMVF) and crocidolite were studied in Fischer 344 rats. MMVF used were size-selected to be rat-respirable, and rats were exposed nose-only 6 h/day for 5 days to gravimetric concentrations (30 mg/m3) of two fiber glass compositions--a rockwool, and a slagwool--or to 10 mg/m3 of long-fibered crocidolite, or to filtered air. Animals were sacrificed at 1 hr, 1, 5, 31, 90, 180, 270, 365, and 545 days after exposure stopped. Fibers were recovered from digested lung tissue to determine changes in concentrations (fibers/mg dry lung) and fiber retentions (expressed as percent of day 1 retention [PR]) for selected dimension categories. One-day average concentrations of lung-retained MMVF and crocidolite fibers, of diameter > or = 0.5 micron or > 20 microns in length, were nearly equal, permitting direct comparisons between MMVF and crocidolite. At 270 days average PR for MMVF > or = 0.5 micron in diameter were from 3 to 6 +/- 2% and 27 +/- 9% for crocidolite. For fibers > 20 microns, PR were 1 to 4 +/- 4% for MMVF and 37 +/- 20% for crocidolite. At 545 days, MMVF > 20 microns in length were at background level while concentration of crocidolite fibers > 20 microns in length remained at 2000 +/- 400 f/mg DL (dry lung), or 38 +/- 9% of day-1 retention. These results suggest strongly that MMVF dissolved or fractured in vivo whereas crocidolite fibers did not change.

Chronic inhalation and biopersistence of refractory ceramic fiber in rats and hamsters.

Lifetime "nose-only" inhalation studies were conducted in rats using four types of refractory ceramic fibers (FCF), 1 micron in diameter x 22 to 26 microns length: High Purity, Kaolin, Zirconia, and After-Service; and on hamsters using Kaolin RCF. For comparison, animals also were exposed to chrysotile fibers. Rats were exposed 6 hr/day, 5 days/week for 24 months to concentrations ranging between 3 and 30 mg/m3. Time- and dose-dependent lesions in the rat included the development of interstitial fibrosis, pleural fibrosis, pulmonary tumors, and mesothelioma. Exposure to 3, 9 or 16 mg/m3 produced no excess lung tumors; no fibrosis was seen at 3 mg/m3. A significant increase in lung tumors and interstitial fibrosis was observed at 30 mg/m3. A single mesothelioma was observed in rats exposed to 9 mg/m3, while two occurred at 30 mg/m3. Hamsters were similarly exposed to 30 mg/m3 Kaolin RCF for 18 months; no lung tumors were induced, but pulmonary and pleural fibrosis were observed and there was a 42% incidence of mesothelioma. Multiple interim sacrifices together with recovery animals allowed detailed assessment of the lung burden of RCF, which was found to be dose related and, at the high doses, exceeded 10(5) fibers/mg of dry lung. During the various recovery periods there was a clear reduction in fiber burden. Mathematical modeling of these data for deposition, clearance, and retention and for species is currently underway.

Chronic inhalation study of fiber glass and amosite asbestos in hamsters: twelve-month preliminary results.

The effects of chronic inhalation of glass fibers and amosite asbestos are currently under study in hamsters. The study includes 18 months of inhalation exposure followed by lifetime recovery. Syrian golden hamsters are exposed, nose only, for 6 hr/day, 5 day/week to size-selected test fibers: MMVF10a (Schuller 901 insulation glass); MMVF33 (Schuller 475 durable glass); amosite asbestos (three doses); or to filtered air (controls). Here we report interim results on airborne fiber characterization, lung fiber burden, and pathology (preliminary) through 12 months. Aerosolized test fibers averaged 15 to 20 microns in length and 0.5 to 1 micron in diameter. Target aerosol concentrations of World Health Organization (WHO) fibers (longer than 5 microns) were 250 fibers/cc for MMVF10a and MMVF33, and 25, 125, or 250 fibers/cc for amosite. WHO fiber lung burdens showed time-dependent and (for amosite) dose-dependent increases. After a 12-month exposure, lung burdens of fibers longer than 20 microns were greatest with amosite high and mid doses, similar for low-dose amosite and MMVF33, and smaller for MMVF10a. Biological responses of animals exposed for 12 months to MMVF10a were limited to nonspecific pulmonary inflammation. However, exposures to MMVF33 and each of three doses of amosite were associated with lung fibrosis and possible mesotheliomas (1 with MMVF33 and 2, 3, and 1 with amosite low, mid, and high doses, respectively). Pulmonary and pleural changes associated with amosite were qualitatively and quantitatively more severe than those associated with MMVF33. As of the 12-month time point, this study demonstrates that two different fiber glass compositions with similar fiber dimensions but different durabilities can have distinctly different effects on the hamster lung and pleura after inhalation exposure. (Preliminary tumor data through 18 months of exposure and 6 weeks of postexposure recovery became available as this manuscript went to press: No tumors were observed in the control or MMVF10a groups, and no additional tumors were observed in the MMVF33 group; however, a number of additional mesotheliomas were observed in the amosite groups.

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Role of gene and chromosomal mutations in cell transformation.

To understand the role of mutagenesis in carcinogenesis fully, we must consider all types of mutations including gene, chromosomal, and gene-number mutations and all changes involved in the progressive development of neoplastic cells. We have found that certain known human carcinogens (i.e., DES and asbestos), which were classified as epigenetic carcinogens based on gene-mutation assays, have mutational activity at the chromosomal level that correlates with their ability to induce cell transformation. This should caution against classification of chemicals as genotoxic or epigenetic without a complete understanding of their mechanism of action. Furthermore, our studies indicate that more than gene-mutation assays is needed for carcinogen testing. In particular, chromosomal changes induced by chemicals, both aberrations and aneuploidy, need to be carefully assessed. In addition, the role of all types of mutation in the overall process of neoplastic transformation needs to be determined. This can only be examined by studying each individual change involved in neoplastic progression. Thus, any attempt to assess a chemical's carcinogenic potential should consider not only all types of mutational changes but both early and late changes involved in neoplastic transformation.

An early, nonrandom karyotypic change in immortal Syrian hamster cell lines transformed by asbestos: trisomy of chromosome 11.

Cytogenetic studies were performed on eight early passage Syrian hamster embryo cell lines independently derived following asbestos exposure. The modal chromosome number of all the immortal cell lines was near-diploid. At the earliest passage examined, six of eight cell lines had only numerical chromosome changes. Cells in each of these six cell lines had an extra chromosome #11, either as a sole karyotypic change or with other numerical changes. The remaining two cell lines displayed both numerical and structural chromosome changes, but without involvement of chromosome #11. Common abnormalities were -X or -Y, +3, and 8p- in one cell line, and -13 and t(13;21) in the other cell line. A nonrandom gain of chromosome #8 was also found in four cell lines. In three of the four cell lines, trisomy of chromosome #8 seems to have occurred during karyotypic progression. The observation that nonrandom changes in chromosome number are an early karyotypic change after carcinogen treatment supports our hypothesis that induction of aneuploidy by asbestos is mechanistically important in the transformation of Syrian hamster embryo cells in culture and, further, suggests that trisomy 11 plays a major role in the early steps of immortalization and neoplastic progression.

The use of tracheal implants in toxicology and carcinogenesis research.

Tracheal implants have served as an important experimental pathology tool with which to study the toxic and/or carcinogenic effects of chemicals upon upper respiratory tract epithelium. Initial studies with this method utilized heterotopic rat tracheal transplants which were exposed to compounds of interest, and assessed for toxic and/or carcinogenic endpoints. Grafts containing rodent tissue have proved useful for studying the cellular and biochemical features of neoplastic progression at different time intervals following in vivo exposure to carcinogens. More recent studies have utilized epithelial denuded tracheal implants inoculated with respiratory cell populations, and xenografted into immunodeficient nu/nu mice. This technique permits the study of airway epithelium from a variety of species, including man. The advent of molecular pathology techniques such as in situ hybridization will further expand the uses of tracheal implant technology for studies with xenografted human tissues. Such implants should prove useful for the examination of species- and tissue-specific characteristics of growth and differentiation by providing a bridge between cell culture and whole animal studies.

In vitro toxicity of respirable-size particles of diatomaceous earth and crystalline silica compared with asbestos and titanium dioxide.

The relationship between particle characteristics and in vitro toxicity was investigated using Chinese hamster ovary cells. Test dusts included respirable natural (Nat) and flux-calcined (FC) diatomaceous earth (DE), quartz, cristobalite, TiO2, and chrysotile and crocidolite asbestos. All dusts elicited a qualitatively similar, concentration-dependent response: particle uptake, induction of micro- and polynuclei, and reduction in cell proliferation. However, similar mass concentrations of the dusts yielded a 35-fold range of toxicity: chrysotile > crocidolite > Nat DE > FC DE > quartz > Cristobalite > TiO2. In vitro toxicity did not correlate with crystalline silica content, surface area, composition, volume, particles/cm2, or fibrous geometry. Toxicity was closely associated with the number of particles/cm2 culture surface that had at least one dimension > 7.5 mu. Thus particle size but not shape could be a determinant of in vitro toxicity. Particle size might also impact in vivo pathogenesis.

Mechanisms of cytotoxicity of asbestos fibres in rat tracheal epithelial cells in culture.

Little is known about the mechanism(s) of asbestos toxicity, especially in respiratory epithelium. Studies were carried out to elucidate some important aspects of the cytotoxic effects of asbestos, using a tracheal epithelial cell line in culture. Chrysotile and crocidolite asbestos with similar aspect ratios were used. Both induced in 2C5 cells a concentration-dependent inhibition of colony-forming ability, a measure of proliferative capacity. In this respect, chrysotile (LC(50), 0.95 mug/cm(2)) was about six times more toxic than crocidolite (LC(50), 5.8 mug/cm(2)). Both types of asbestos caused only minor changes in membrane permeability, measured by trypan blue exclusion and by [(75)Se]selenomethionine release, even at concentrations of asbestos that caused > 90% inhibition in colony formation. Thus membrane damage was only a minor component of the toxicity produced by the fibres. Chrysotile was phagocytized much more readily than crocidolite and produced an approximately threefold greater increase in binucleated cells and micronuclei than crocidolite, suggesting that phagocytosis was the rate-limiting step in fibre toxicity. Our studies suggest a potentially important pathway of fibre toxicity involving binding, phagocytosis, nuclear damage, disruption of mitosis, inhibition of proliferation and/or cell death. In this process, the fibre aspect ratio is not the only determinant of fibre toxicity, since chrysotile and crocidolite fibres of similar length and diameter exhibit very different degrees of toxicity. It appears that other fibre characteristics, such as fibre surface charge, are of equal importance.

Differential effects of tumour promoters on the growth of normal human bronchial epithelial cells and human lung tumour cell lines.

The effects of the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the colony-forming efficiency and growth of normal human bronchial epithelial (NHBE) cells and five lung squamous carcinoma cell lines were compared in medium containing 1% foetal bovine serum. TPA (0.1-5.0 ng/ml) inhibited the growth of NHBE cells and one carcinoma cell line, while four of the five carcinoma lines were less sensitive to the growth inhibitory properties of TPA but were slightly inhibited at higher TPA concentrations. The responses of NHBE cells and carcinoma cells to TPA, and the related compounds, mezerein, 4-O-methyl TPA, and phorbol were then compared in serum-free medium. In general, the removal of serum from the medium increased the differences in the responses to TPA between normal and tumour cells. Two carcinoma lines inhibited by TPA in 1% serum were stimulated by TPA in the absence of serum. Mezerein and, to a lesser extent, 4-O-methyl TPA also produced differential responses in colony-forming efficiencies between tumour lines and NHBE cells. Phorbol had no effect on either NHBE cells or on carcinoma cell lines. The relative insensitivity of carcinoma cell lines to the growth inhibitory effects of tumour promoters is consistent with the hypothesis that tumour promotion involves selection against normal cells to permit clonal expansion of preneoplastic or neoplastic cell types.

Cytotoxicity of refractory ceramic fibres to Chinese hamster ovary cells in culture.

The toxicity/oncogenicity of refractory ceramic fibres have been tested in chronic inhalation studies in rodents. Because these studies are time consuming and expensive, there is a need to develop and validate short-term models to screen fibres for their toxicological potential. In the present study, the toxic effects of four different compositions of refractory ceramic fibres were determined using Chinese hamster ovary cells grown in culture. These refractory ceramic fibres were the same size-selected fibres that had been used in animal inhalation studies, thus facilitating a direct comparison of findings in the two systems. Chinese hamster ovary cells were treated with refractory ceramic fibres 24 hr after seeding into 60-mm culture dishes in Ham's F12 medium with 10% serum. Inhibition of cell proliferation and colony formation were determined after 3-5 days of fibre exposure. Crocidolite and chrysotile asbestos were used as positive controls. Concentration-dependent inhibition of both cell proliferation and colony formation was observed after treatment with refractory ceramic fibres. The LC(50) for the different refractory ceramic fibres ranged from 10 to 30 mug/cm(2). The LC(50)s for crocidolite and chrysotile were 5 mug/cm(2) and 1 mug/cm(2), respectively. To assess the genotoxic potential of these fibres, fibre-exposed Chinese hamster ovary cell cultures were stained with acridine orange and scored for the incidence of micronuclei and other nuclear abnormalities. The incidence of nuclear abnormalities for refractory ceramic fibres at 20 mug/cm(2) ranged from 20 to 40%. Toxic endpoints of the in vitro studies were compared with those of the chronic animal inhalation studies. The latter included induction of lung fibrosis and pleural and airway tumours. A correlation was observed between the in vitro and in vivo toxicological potencies of the respective four refractory ceramic fibres: the fibres that were most toxic in vitro were also the most toxic in the chronic animal inhalation studies. A direct relationship was also observed, both in vitro and in vivo, between average fibre length and the severity of the toxic effect.