- Email: [email protected]
Tracheal epithelial cells in vitro as a model to study genotoxicity of airborne particulates
0887-2333(9!5)00025-9
Toxic.in VirroVol. 9, No. 4, PP. 397-402,1995 Copyright 0 1995Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95$9.50+ 0.00
Tracheal Epithelial Cells In VzIro as a Model to Study Genotoxicity of Airborne Particulates C. HORNBERG
and N. H. SEEMAYER
Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, Diisseldorf, Germany
Gurlittstrasse 53, D-40223
Abstract-The major target site of airborne particulates is the tracheobronchial epithelium of the respiratory tract. It is also the origin of the most common cancer in man, bronchogenic carcinoma. Rodent tracheal epithelial cells in culture can be used to study the genotoxic activity of airborne particulates leading to mutation and cancer. Airborne particulates were collected in the heavily industrialized Rhine-Ruhr region using a high volume sampler HVS 150 (Striihlein Instruments) equipped with glass fibre filters. Chemical substances were extracted with di-chloromethane or methanol and quantitatively transferred to dimethyl sulfoxide for tissue culture experiments. Tracheal epithelial cells of the Syrian golden hamster and the Wistar rat were dissociated by pronase treatment and cultivated in a ‘complex’ medium. The induction of sister chromatid exchanges was used as a sensitive bioassay for detection of genotoxic activity of airborne particulates. Extracts of airborne particulates led to a dose-related highly significant induction of sister chromatid exchanges in cell cultures of tracheal epithelial cells of the hamster and the rat. Even quantities of chemical substances equivalent to airborne particulates from less than 1rn’ air were markedly genotoxic.
Introduction It is widely believed that the development genie
carcinoma
is a multistep
process
of bronchoinvolving
complex biological events (Mass et al., 1985). Different models have been used successfully to investigate these steps and to identify chemicals causing neoplastic progression of normal cells to malignant cells (Steele and Mass, 1985). Cultures of various cell types isolated from the respiratory system have been established, but particular attention has focused on airway epithelial cells, Clara cells, type II alveolar cells, alveolar macrophages and vascular endothelial cells (Balls and Fentem, 1994). Tracheal epithelial cells in culture have also been used for investigating the potential respiratory toxicity of various compounds. Steele et al. (1977) used young rat tracheal explants to develop an in vitro cell transformation system assay for chemical carcinogens. Thomassen et al. (1983) developed another transformation assay, which involved isolating rat tracheal epithelial cells by enzymatic dissociation and then plating them on a feeder layer of irradiated fibroblasts (according to the method of Wu and Smith, 1982). Steele and Mass (1985) subsequently modified this approach by eliminating the need for the feeder layer. Other studies have focused on the effects of carcinogens and tumour promoters on cell proliferation in tracheal Abbreoiations: DMSO = dimethyl sulfoxide; GEX = global extract; PBS = phosphate buffered saline; SCE = sister chromatid exchanges. TI”
9,bD
epithelium (Chopra and Cooney, 1985; Lasntizki and Bollag, 1987). Zhu et al. (1991 and 1992) have used rat tracheal epithelial cell cultures to study the toxicity and carcinogenicity of chemical and physical agents. The rodent tracheal epithelial culture system has a number of features that make it particularly useful as an in vitro model to study the effects of toxic and carcinogenic agents on airway epithelium (Nettesheim and Barrett, 1984; Nettesheim and Marchok, 1983; Zhu et al., 1992). Cultures of various cell types isolated from the respiratory system have been used in assays involving a number of indicators of micronucleus formation genotoxicity, including (Whong et al., 1990) and sister chromatic exchanges (SCE) (Hornberg et al., 1993 and 1994; Seemayer et al., 1994; Shimizu et al., 1984). For detection of genotoxicity of extracts of airborne particulates we utilized as a sensitive bioassay the induction of SCE in cultures of tracheal epithelial cells of the rat and the Syrian golden hamster. As a criterion of genotoxicity we determined dose-dependently the induction of SCE in both test systems. Materials aud Methods Collection and extraction
of airborne particulates
Airborne particulates no. 69 and no. 70 were collected in spring 1991 with a High-Volume Sampler (HVS 150, Strijhlein Instruments) on glass fibre filters in the highly industrialized Rhine-Ruhr area. Global extract (=GEX) no. 69 represents a sample of a 391
398
C.
Hornberg and N. H. Seemayer
location polluted mainly by industrial sources; GEX no. 70 is a sample that was collected at a site with a high traffic density. The filters were extracted with dichloromethane as described previously (Hadnagy and Seemayer, 1994; Seemayer et al., 1984 and 1990). For biological testing, GEX were quantitatively transferred to dimethyl sulfoxide (DMSO) for tissue culture experiments. The final concentration of DMSO in medium did not exceed 0.5%. Air volumes of collection of airborne particulates were measured and extractable substances are presented as cubic metres (m3)/ml medium. Furthermore, the benzo[a]pyrene content of the extracts was determined as previously described in detail (Tomingas et al., 1977). GEX no. 69 containing extractable substances of airborne particulates collected from 1557 m3 air, revealed a benzo[u]pyrene concentration of 1.6 pg DMSO/ml. GEX no. 70 with extractable substances of airborne particulates from 2252m3 air showed a benzo[a]pyrene content of 1.2pg DMSO/ml. Establishment of tracheal epithelial cells
Syrian golden hamsters and Wistar rats were killed by CO, asphyxia and their tracheas aseptically removed proximal to the bifurcation and distal to the larynx. Excised tracheas were placed in a petri dish and longitudinally opened along the cartilaginous part. They were then spread out and tracheal epithelial cells were isolated by pronase digestion (type 14, Sigma) of tracheal tissue from normal 68-wk-old hamsters or from Wistar rats. After enzyme treatment for 16 hr at 37°C or 4°C respectively, dissociated airway epithelial cells were flushed out of the tracheas, washed and suspended in a complex mixture of media (Complete Medium) containing Ham’s F12, CMRL 1066 and conditioned medium of confluent NIH 3T3 cells supplemented with insulin, hydrocortisone, transferrin, bovine serum albumin and epidermal growth factor. Penicillin and streptomycin were added to all media. Cultivation of tracheal epithelial cells was performed on fibronectin-precoated plastic flasks. Tracheal epithelial cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO, in air (Hornberg et al., 1993). Detection of SCE in tracheal epithelial cells
For detection of SCE, tracheal epithelial cells at passage l-4 were detached with a trypsin-versen solution, resuspended in fresh Complete Medium and seeded into plastic dishes (Quadriperm, Heraeus) on fibronectin-precoated sterile glass slides at a cell concentration of 1.25 x lo5 cells/5 ml. After a cultivation period of 24 hr, GEX no. 69 and no. 70 were added to cell cultures in various concentrations. Incubation was continued for 48 hr in the presence of bromodeoxyuridine (15 pg/ml), the last 3 hr exposed to demecolcine (Colcemid 10 pg/ml, Sigma) or nocodazole (20 ng/ml, Janssen, Belgium). After hypotonic treatment and fixation of cells, chromosomes were stained by a fluorescent plus Giemsa (FPG) technique
as reported earlier (Hadnagy et al., 1986; Hornberg et al., 1993; Seemayer et al., 1994). Morphological analysis and cell proliferation
For morphological analysis, tracheal epithelial cell cultures were fixed in Bouin’s solution and stained with haematoxylin and eosin. Cell proliferation of tracheal epithelial cells was determined by enumeration of cells in cultures utilizing an electronic counter (Coulter Electronics; Model ZB). Immunojluorescence detection of cytokeratin
Cells were grown to near confluence on coverslips in Leighton tubes and then rinsed with phosphate buffered saline (PBS). Fixation was achieved in 2% formaldehyde solution at 4°C for 1 hr. Thereafter, coverslips were rinsed in PBS and covered with the monoclonal mouse antibody Anti-Cytokeratin-pan (Boehringer Mannheim) at 1: 10 dilution and incubated for 30 min at 37°C. Coverslips were then rinsed three times with PBS and covered with a fluoresceinconjugated goat anti-mouse antibody (GAM-FITC; Nordic Immunological Laboratories) at 1: 10 dilution for 30 min at 37°C. After rinsing in PBS, cells were analysed under a fluorescence microscope. Statistical analysis
Experimental data were computerized and mean values, limits of confidence and standard deviations determined. Bartlett’s test for equal variances, oneway analysis of variance and Student’s t-test were performed. Rer&S The rodent tracheal epithelial cells used in this study show major features of epithelial cells. Under the above-mentioned standard conditions, cells grew as a typical epithelial monolayer. Plate 1 shows a growing culture of Syrian golden hamster tracheal epithelial cells, revealing their epithelial nature. These cells are characterized by cytokeratin, as shown by immunocytochemistry. The dose-dependent induction of SCE by GEX no. 69 (Duisburg 1991) in tracheal epithelial cell cultures of the rat and of the Syrian golden hamster is depicted in Figs 1 and 2. On the abscissa, increasing concentrations of GEX no. 69 are outlined corresponding to air volumes of particulate collection in the range of 0.25 up to 15.57 m3 air. On the ordinate, SCE/chromosome are shown. Rodent tracheal epithelial cells reveal a strong dose-related increase of SCE in the presence of GEX no. 69. Up to 2.5-fold values of SCE can be seen in the presence of the extract from 15 m3 air; substances from 0.25 m3 air are also effective. The concentration of benzo[a]pyrene in the experiment with GEX no. 69 was in the range of 0.25 to 16 ng/ml medium. The lowest effective dose leading to a significant increase of SCE in the presence of GEX no. 69 was observed with rat tracheal epithelial cells at a concentration of
Plate. I. Tracheal
epithelial
cells of the Syrian golden hamster in culture. magnification x 240.
399
Haematoxylin
and eosin. Original
Genotoxicity testing on tracheal epithelial cells in vitro
401
1.2 r
2
E si z e .c 23 VJ
1.0
0.8
2
0.8
0.6
0.6
0.4
! 2 2
0.2
fs
0.2
’ DMSO
0.25
0.49 Air
0.98
1.95
volume
3.90
7.80
15.57
0.4
’ D&O
0.35
O.;O Air
(m3)
Fig. I. Dose-related induction of SCE in tracheal epithelial cells of the rat in vitro in the presence of various concentrations of GEX no. 69 of airborne particulates. Values are
means, and limits of confidence are presented. 0.49 m3/ml medium (P < 0.001) and with hamster tracheal epithelial cells at 0.98 m3/ml medium (P < 0.001). Quantitative data on the induction of SCE by GEX no. 70 of airborne particulates in cell cultures of tracheal epithelial cells of the rat and of the golden Syrian hamster are presented in Figs 3 and 4. Sample no. 70 of airborne particulates was collected in spring 1991 in the city of Dusseldorf, Germany, at a site with a high traffic density. On the abscissa increasing concentrations of GEX no. 70 are outlined corresponding to air volumes of particulate collection in the range from 0.35 to 22.52 m3 air; on the ordinate, SCE/chromosome are shown. Tracheal epithelial cells from the rat show a strongly dose-dependent increase in SCE chromatid exchanges in the presence of GEX no. 70. Up to two-fold increases in SCE can be seen in the presence of 22 m3 air, but substances from 0.35 m3 air are also effective. Tracheal epithelial cells from the hamster also demonstrate a highly significant induction of SCE by GEX no. 70. The benzo[a]pyrene concentration of GEX no. 70 was in the range 0.18 to 12 ng/ml medium. In contrast, GEX no. 70 induced a significant increase of SCE in cultures of rat tracheal epithelial cells at a concentration as low as 1.4 m3/ml medium (P (: 0.001) and of hamster tracheal epithelial cells at concentration of 0.35 m3/ml (P < 0.01) and 0.70 m3/ml medium (P < 0.001).
I.bl
2.82
volume
5.63
II:26
22:52
(m3)
Fig. 3. Dose-related induction of SCE in tracheal epithelial cells of the rat in vitro in the presence of various concentrations of GEX no. 70 of airborne particulates. Values are means, and limits of confidence are presented.
Discussion Inhaled airborne particulates pose a health risk for humans by their mutagenic and carcinogenic activity. The tracheobronchial epithelium is an important target of respirable aerosols and the origin of the most common cancer in man, bronchogenic carcinoma (Tomatis, 1990). Tracheal epithelial cells in culture appear to be a meaningful alternative to other human and rodent cell cultures that have been used for genotoxicity testing of airborne particulates (Alink et al., 1983; Courtois et al., 1988; Hadnagy et al., 1989; Seemayer et al., 1984). Explants of rabbit tracheal epithelial cells have been used for evaluation of respiratory toxicity of acrolein, mechlorethamin, parathion and paraoxon (Blanquart et al., 1991) as well as for demonstration of ciliotoxic effects (Romet et al., 1990). Unscheduled DNA synthesis has been demonstrated in primary cultures of tracheal epithelial cells of the Syrian golden hamster by benzo[u]pyrene and methylmethanesulfonate and of corresponding rat cells by methylmethanesulfonate (Kuper and Benford, 1991). Induction of SCE is considered to be a sensitive cytogenetic endpoint for evaluation of genotoxic potential of mutagens and carcinogens (WHO, 1993). Earlier reports (Hornberg et al., 1993 and 1994;
1.2
B si ; 2 c 2z m
I .o 0.8 0.6 0.4 0.2 ” DMSO ’ DMSO
0.25
0.49
Air
0.98 volume
1.95
3.90
7.80
15.57
(m3)
Fig. 2. Dose-related induction of SCE in tracheal epithelial cells of the Syrian golden hamster in vitro in the presence of variousconcentrationsofGEXno.69ofairbomeoarticulates. Values are means, and limits of confidence a& presented.
0.35
0.70
1.41
2.82
5.63
11.26 22.52
Air volume (m3) Fig. 4. Dose-related induction of SCE in tracheal epithelial cells of the Syrian golden hamster in vitro in the presence of various concentrations of GEX no. 70 of airborne particulates. Values are means, and limits of confidence are presented.
402
C. Hornberg
Seemayer
et al.,
demonstrate
that
1994) rodent
and
results
tracheal
of
epithelial
this
and N. H. Seemayer
study
cells offer
in oitro model for genotoxicity testing of inhaled toxins. Extracts of airborne particulates are able to induce a significant dose-related increase of SCE in cultures of tracheal epithelial cells of the Syrian golden hamster and the rat. Medical health risk evaluation of airborne particulates must consider a daily respiratory ventilation in man of 12-15 m’ air at rest, as well as the lowest a reliable
effective
genotoxic
dose,
as was
demonstrated
GEX no. 69, of 0.49 m’ air, and with GEX
with no. 70. of
0.70 m3 air.
REFERENCES
Ahnk G. M., Smit H. S.. van Houdt J. J.. Kolkman J. R. and Boleii J. S. (1983) Mutagenic activitv of airborne particulates at non-industrial locations. Mutation Research 116, 21-34. Balls M. and Fentem J. H. (1994) In vitro test systems and respiratory toxicology. In Respira/iq Toxicology and Risk Assessment. Edited by P. G. Jenkins. pp. 291 -314. Wissenschaftliche Verlagsgesellschaft, Stuttgart. Blanquart C., Romet S., Baeza A., Guennou C. and Marano P. (1991) Primary cultures of tracheal epithelial cells for the evaluation ofrespiratory toxicity. Toxicology in Vitro 5. 4999522. Chdpra D. P. and Cooney R. A. (1985) Histogenesis of benzo[a]pyrene-induced _lesions in tracheal -explants. Virchow Archit:. B. Cell Pathalorrr, .>. 48. 299- 315. Courtois Y. A., Min S., Lachenal C.. Jacquot-Deschamps J. M., Callais F. and Festy B. (1988) Genotoxicity of organic extracts from atmospheric particles. Annals of Ihe New York Academy of Sciences 534, 724-740. Hadnagy W. and Seemayer N. H. (1994) Inhibition of phag&ytosis of human macrophages induced by airborne uarticulates. Toxicoloav Lefrers 72. 23 3 I. Hadnagy W., Seemayer’N. H. and’Tomingas R. (1986) Cytogenetic effects of airborne particulate matter in human lymphocytes in vitro. Muturion Research 175, 977101. Hadnagy W., Seemayer N. H., Tomingas R. and Ivanfy K. (1989) Comparative study of sister-chromatid exchanges and chromosomal aberrations induced by airborne particmates from an urban and a highly industrialized location in human lvmohocvte cultures. Mutaiion Research 225. 27732. + ’ ’ Hornberg C., Seemayer N. H., Hadnagy W. and lvanfy K. (1993) Tracheal epithelial cells of the Golden Syrian hamster in vitro as a tool for detection of genotoxic activity of airborne particulates. Journal af Aerosol Science 24 (Suppl. 1). 91 92. Hornberg C., Seemayer N. H. and Hadnagy W. (1994) Airborne particulates and their genotoxic activity on rodent tracheal epithelial cells and human lymphocytes in vitro. First International Congress on Environmental Medicine, Duisburg, Germany, 23-26 February, 1994. Kuoer. A. and Benford. D. J. (1991) Unscheduled DNA synthesis in tracheal epithelial cell cultures. Toxicology in Vifro 5, 51 I-513. Lasnitzki I. and Bollag W. (1987) Prevention and reversal by a non-polar carotinoid (Ro 15-0778) of 3,4-benzpyrene- and cigarette smoke condensate-induced hyperplasia and metaplasia of rodent respiratory epithelia grown in vitro. Europeon Journal qf Cancer and Clinical Oncology 23, 861-865. Mass M., Nettesheim P., Gray T. and Barrett J. C. (1985) The effect of 12-0-tetradeconylphorbol-13-acetate and
other tumor promoters on colony formation of rat tracheal epithelial cells in culture. Carcinogenesis 5, 159771601. Nettesheim P. and Marchok A. (1983) Neoplastic development in airway epithelium. In Advances in Cancer Research, Vol. 39. Edited by G. Klein and S. Weinhouse. pp. 53-54. Academic Press, New York.
Nettesheim P. and Barrett J. C. (1984) Tracheal epithelial cell transformation: a model system for studies on neoplastic progression. In CRC Critical Reviews in Toxicology Vol. 12. Edited by L. Goldberg. pp. 2155239. CRC Press, Boca Raton, FL. Romet S., Dubreuil A., Baeza A., Moreau A., Schoevart D. and Marano F. (1990) Respiratory tract epithelium in primary culture: effects of ciliotoxic compounds. Toxicology in Vitro 4, 399-402. Shimizu R. W.. Sun J. D., Li A. P., Newton G. J. and Brooks A. L. (1984) The use of sister-chromatid exchange in Chinese primary lung cell cultures to measure genotoxicity. Mutation Research 130, 333-342. Seemayer N. H., Manojlovic N. and Schiirer C. C. (1984) Cell cultures as a tool for detection of cytotoxic, mutagenic and carcinogenic activity of -airborne particulate matter. Journal of Aerosol Science 15, 426430. Seemayer N. H., Hadnagy W. and Tomingas R. (1990) Evaluation of health risks by airborne particulates from in vitro cyto- and genotoxicity testing on human and rodent tissue culture cells: a longitudinal study from 1975 until now. Journal af Aerosol Science 21 (Suppl. I), 501-504. Seemayer N. H., Hornberg C. and Hadnagy H. (1994) Comparative genotoxicitytesting of airborne particulates using rodent tracheal epithelial cells and human lymphocytes in vitro. Toxicology Letters 72, 955103. Steele V. E. and Mass M. _I-( 1985) A rat tracheal cell culture transformation system for assessment of environmental agents as carcinogens and promoters. Environment Imernational 11, 3233329. Steele V. E., Marchok A. C. and Nettesheim P. (1977) Transformation of tracheal epithelium exposed in vitro to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). InternationaIJournal of Cancer 26, 234-238. Thomassen D. G., Gray T. E., Mass M. J. and Barrett J. C. (1983) High frequency of carcinogen-induced early, preneoplastic changes in rat tracheal epithelial cells in culture. Cancer Research 43, 595665963. Tomatis L. (1990) Air pollution and cancer: an old and new problem. In: Air Pollution and Human Cancer. Edited by L. Tomatis. pp. 1-7. Springer Vet-lag, Berlin. Tomingas R.. Voitmer G. and Bednarik H. (1977) Direct fluorimetric analysis of aromatic polycyclic hydrocarbons on thin-layer chromatograms. Science qf the Total Enaironmem 7, 216-267. WHO (1993) IPCS Environmental Health Criteria 155: Biomarkers and Risk Assessment-Concepts and Principles. pp. 82. WHO, Geneva. Whong W. Z., Stewart J. D. and Ong T. (1990) The use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays. Mutation Research 24, 7-13. Wu R. and Smith D. (1982) Continuous multiplication of rabbit tracheal eoithelial cells in a defined. hormonesupplemented medium In Vitro 18, 800-812. Zhu S., Cunningham M. L., Gray T. E. and Nettesheim P. (1991) Cytotoxicity, genotoxicity and transformin activity of 4-(methylnitrosamino)I-(3-pyridyl)-I-butanone (NNK) in rat tracheal epithelial cells. Murafion Research 261, 249-259. Zhu S., Gray T. E. and Nettesheim P. (1992) The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells. Carcinogenesis 13, 172551729.
Toxic.in VirroVol. 9, No. 4, PP. 397-402,1995 Copyright 0 1995Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95$9.50+ 0.00
Tracheal Epithelial Cells In VzIro as a Model to Study Genotoxicity of Airborne Particulates C. HORNBERG
and N. H. SEEMAYER
Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, Diisseldorf, Germany
Gurlittstrasse 53, D-40223
Abstract-The major target site of airborne particulates is the tracheobronchial epithelium of the respiratory tract. It is also the origin of the most common cancer in man, bronchogenic carcinoma. Rodent tracheal epithelial cells in culture can be used to study the genotoxic activity of airborne particulates leading to mutation and cancer. Airborne particulates were collected in the heavily industrialized Rhine-Ruhr region using a high volume sampler HVS 150 (Striihlein Instruments) equipped with glass fibre filters. Chemical substances were extracted with di-chloromethane or methanol and quantitatively transferred to dimethyl sulfoxide for tissue culture experiments. Tracheal epithelial cells of the Syrian golden hamster and the Wistar rat were dissociated by pronase treatment and cultivated in a ‘complex’ medium. The induction of sister chromatid exchanges was used as a sensitive bioassay for detection of genotoxic activity of airborne particulates. Extracts of airborne particulates led to a dose-related highly significant induction of sister chromatid exchanges in cell cultures of tracheal epithelial cells of the hamster and the rat. Even quantities of chemical substances equivalent to airborne particulates from less than 1rn’ air were markedly genotoxic.
Introduction It is widely believed that the development genie
carcinoma
is a multistep
process
of bronchoinvolving
complex biological events (Mass et al., 1985). Different models have been used successfully to investigate these steps and to identify chemicals causing neoplastic progression of normal cells to malignant cells (Steele and Mass, 1985). Cultures of various cell types isolated from the respiratory system have been established, but particular attention has focused on airway epithelial cells, Clara cells, type II alveolar cells, alveolar macrophages and vascular endothelial cells (Balls and Fentem, 1994). Tracheal epithelial cells in culture have also been used for investigating the potential respiratory toxicity of various compounds. Steele et al. (1977) used young rat tracheal explants to develop an in vitro cell transformation system assay for chemical carcinogens. Thomassen et al. (1983) developed another transformation assay, which involved isolating rat tracheal epithelial cells by enzymatic dissociation and then plating them on a feeder layer of irradiated fibroblasts (according to the method of Wu and Smith, 1982). Steele and Mass (1985) subsequently modified this approach by eliminating the need for the feeder layer. Other studies have focused on the effects of carcinogens and tumour promoters on cell proliferation in tracheal Abbreoiations: DMSO = dimethyl sulfoxide; GEX = global extract; PBS = phosphate buffered saline; SCE = sister chromatid exchanges. TI”
9,bD
epithelium (Chopra and Cooney, 1985; Lasntizki and Bollag, 1987). Zhu et al. (1991 and 1992) have used rat tracheal epithelial cell cultures to study the toxicity and carcinogenicity of chemical and physical agents. The rodent tracheal epithelial culture system has a number of features that make it particularly useful as an in vitro model to study the effects of toxic and carcinogenic agents on airway epithelium (Nettesheim and Barrett, 1984; Nettesheim and Marchok, 1983; Zhu et al., 1992). Cultures of various cell types isolated from the respiratory system have been used in assays involving a number of indicators of micronucleus formation genotoxicity, including (Whong et al., 1990) and sister chromatic exchanges (SCE) (Hornberg et al., 1993 and 1994; Seemayer et al., 1994; Shimizu et al., 1984). For detection of genotoxicity of extracts of airborne particulates we utilized as a sensitive bioassay the induction of SCE in cultures of tracheal epithelial cells of the rat and the Syrian golden hamster. As a criterion of genotoxicity we determined dose-dependently the induction of SCE in both test systems. Materials aud Methods Collection and extraction
of airborne particulates
Airborne particulates no. 69 and no. 70 were collected in spring 1991 with a High-Volume Sampler (HVS 150, Strijhlein Instruments) on glass fibre filters in the highly industrialized Rhine-Ruhr area. Global extract (=GEX) no. 69 represents a sample of a 391
398
C.
Hornberg and N. H. Seemayer
location polluted mainly by industrial sources; GEX no. 70 is a sample that was collected at a site with a high traffic density. The filters were extracted with dichloromethane as described previously (Hadnagy and Seemayer, 1994; Seemayer et al., 1984 and 1990). For biological testing, GEX were quantitatively transferred to dimethyl sulfoxide (DMSO) for tissue culture experiments. The final concentration of DMSO in medium did not exceed 0.5%. Air volumes of collection of airborne particulates were measured and extractable substances are presented as cubic metres (m3)/ml medium. Furthermore, the benzo[a]pyrene content of the extracts was determined as previously described in detail (Tomingas et al., 1977). GEX no. 69 containing extractable substances of airborne particulates collected from 1557 m3 air, revealed a benzo[u]pyrene concentration of 1.6 pg DMSO/ml. GEX no. 70 with extractable substances of airborne particulates from 2252m3 air showed a benzo[a]pyrene content of 1.2pg DMSO/ml. Establishment of tracheal epithelial cells
Syrian golden hamsters and Wistar rats were killed by CO, asphyxia and their tracheas aseptically removed proximal to the bifurcation and distal to the larynx. Excised tracheas were placed in a petri dish and longitudinally opened along the cartilaginous part. They were then spread out and tracheal epithelial cells were isolated by pronase digestion (type 14, Sigma) of tracheal tissue from normal 68-wk-old hamsters or from Wistar rats. After enzyme treatment for 16 hr at 37°C or 4°C respectively, dissociated airway epithelial cells were flushed out of the tracheas, washed and suspended in a complex mixture of media (Complete Medium) containing Ham’s F12, CMRL 1066 and conditioned medium of confluent NIH 3T3 cells supplemented with insulin, hydrocortisone, transferrin, bovine serum albumin and epidermal growth factor. Penicillin and streptomycin were added to all media. Cultivation of tracheal epithelial cells was performed on fibronectin-precoated plastic flasks. Tracheal epithelial cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO, in air (Hornberg et al., 1993). Detection of SCE in tracheal epithelial cells
For detection of SCE, tracheal epithelial cells at passage l-4 were detached with a trypsin-versen solution, resuspended in fresh Complete Medium and seeded into plastic dishes (Quadriperm, Heraeus) on fibronectin-precoated sterile glass slides at a cell concentration of 1.25 x lo5 cells/5 ml. After a cultivation period of 24 hr, GEX no. 69 and no. 70 were added to cell cultures in various concentrations. Incubation was continued for 48 hr in the presence of bromodeoxyuridine (15 pg/ml), the last 3 hr exposed to demecolcine (Colcemid 10 pg/ml, Sigma) or nocodazole (20 ng/ml, Janssen, Belgium). After hypotonic treatment and fixation of cells, chromosomes were stained by a fluorescent plus Giemsa (FPG) technique
as reported earlier (Hadnagy et al., 1986; Hornberg et al., 1993; Seemayer et al., 1994). Morphological analysis and cell proliferation
For morphological analysis, tracheal epithelial cell cultures were fixed in Bouin’s solution and stained with haematoxylin and eosin. Cell proliferation of tracheal epithelial cells was determined by enumeration of cells in cultures utilizing an electronic counter (Coulter Electronics; Model ZB). Immunojluorescence detection of cytokeratin
Cells were grown to near confluence on coverslips in Leighton tubes and then rinsed with phosphate buffered saline (PBS). Fixation was achieved in 2% formaldehyde solution at 4°C for 1 hr. Thereafter, coverslips were rinsed in PBS and covered with the monoclonal mouse antibody Anti-Cytokeratin-pan (Boehringer Mannheim) at 1: 10 dilution and incubated for 30 min at 37°C. Coverslips were then rinsed three times with PBS and covered with a fluoresceinconjugated goat anti-mouse antibody (GAM-FITC; Nordic Immunological Laboratories) at 1: 10 dilution for 30 min at 37°C. After rinsing in PBS, cells were analysed under a fluorescence microscope. Statistical analysis
Experimental data were computerized and mean values, limits of confidence and standard deviations determined. Bartlett’s test for equal variances, oneway analysis of variance and Student’s t-test were performed. Rer&S The rodent tracheal epithelial cells used in this study show major features of epithelial cells. Under the above-mentioned standard conditions, cells grew as a typical epithelial monolayer. Plate 1 shows a growing culture of Syrian golden hamster tracheal epithelial cells, revealing their epithelial nature. These cells are characterized by cytokeratin, as shown by immunocytochemistry. The dose-dependent induction of SCE by GEX no. 69 (Duisburg 1991) in tracheal epithelial cell cultures of the rat and of the Syrian golden hamster is depicted in Figs 1 and 2. On the abscissa, increasing concentrations of GEX no. 69 are outlined corresponding to air volumes of particulate collection in the range of 0.25 up to 15.57 m3 air. On the ordinate, SCE/chromosome are shown. Rodent tracheal epithelial cells reveal a strong dose-related increase of SCE in the presence of GEX no. 69. Up to 2.5-fold values of SCE can be seen in the presence of the extract from 15 m3 air; substances from 0.25 m3 air are also effective. The concentration of benzo[a]pyrene in the experiment with GEX no. 69 was in the range of 0.25 to 16 ng/ml medium. The lowest effective dose leading to a significant increase of SCE in the presence of GEX no. 69 was observed with rat tracheal epithelial cells at a concentration of
Plate. I. Tracheal
epithelial
cells of the Syrian golden hamster in culture. magnification x 240.
399
Haematoxylin
and eosin. Original
Genotoxicity testing on tracheal epithelial cells in vitro
401
1.2 r
2
E si z e .c 23 VJ
1.0
0.8
2
0.8
0.6
0.6
0.4
! 2 2
0.2
fs
0.2
’ DMSO
0.25
0.49 Air
0.98
1.95
volume
3.90
7.80
15.57
0.4
’ D&O
0.35
O.;O Air
(m3)
Fig. I. Dose-related induction of SCE in tracheal epithelial cells of the rat in vitro in the presence of various concentrations of GEX no. 69 of airborne particulates. Values are
means, and limits of confidence are presented. 0.49 m3/ml medium (P < 0.001) and with hamster tracheal epithelial cells at 0.98 m3/ml medium (P < 0.001). Quantitative data on the induction of SCE by GEX no. 70 of airborne particulates in cell cultures of tracheal epithelial cells of the rat and of the golden Syrian hamster are presented in Figs 3 and 4. Sample no. 70 of airborne particulates was collected in spring 1991 in the city of Dusseldorf, Germany, at a site with a high traffic density. On the abscissa increasing concentrations of GEX no. 70 are outlined corresponding to air volumes of particulate collection in the range from 0.35 to 22.52 m3 air; on the ordinate, SCE/chromosome are shown. Tracheal epithelial cells from the rat show a strongly dose-dependent increase in SCE chromatid exchanges in the presence of GEX no. 70. Up to two-fold increases in SCE can be seen in the presence of 22 m3 air, but substances from 0.35 m3 air are also effective. Tracheal epithelial cells from the hamster also demonstrate a highly significant induction of SCE by GEX no. 70. The benzo[a]pyrene concentration of GEX no. 70 was in the range 0.18 to 12 ng/ml medium. In contrast, GEX no. 70 induced a significant increase of SCE in cultures of rat tracheal epithelial cells at a concentration as low as 1.4 m3/ml medium (P (: 0.001) and of hamster tracheal epithelial cells at concentration of 0.35 m3/ml (P < 0.01) and 0.70 m3/ml medium (P < 0.001).
I.bl
2.82
volume
5.63
II:26
22:52
(m3)
Fig. 3. Dose-related induction of SCE in tracheal epithelial cells of the rat in vitro in the presence of various concentrations of GEX no. 70 of airborne particulates. Values are means, and limits of confidence are presented.
Discussion Inhaled airborne particulates pose a health risk for humans by their mutagenic and carcinogenic activity. The tracheobronchial epithelium is an important target of respirable aerosols and the origin of the most common cancer in man, bronchogenic carcinoma (Tomatis, 1990). Tracheal epithelial cells in culture appear to be a meaningful alternative to other human and rodent cell cultures that have been used for genotoxicity testing of airborne particulates (Alink et al., 1983; Courtois et al., 1988; Hadnagy et al., 1989; Seemayer et al., 1984). Explants of rabbit tracheal epithelial cells have been used for evaluation of respiratory toxicity of acrolein, mechlorethamin, parathion and paraoxon (Blanquart et al., 1991) as well as for demonstration of ciliotoxic effects (Romet et al., 1990). Unscheduled DNA synthesis has been demonstrated in primary cultures of tracheal epithelial cells of the Syrian golden hamster by benzo[u]pyrene and methylmethanesulfonate and of corresponding rat cells by methylmethanesulfonate (Kuper and Benford, 1991). Induction of SCE is considered to be a sensitive cytogenetic endpoint for evaluation of genotoxic potential of mutagens and carcinogens (WHO, 1993). Earlier reports (Hornberg et al., 1993 and 1994;
1.2
B si ; 2 c 2z m
I .o 0.8 0.6 0.4 0.2 ” DMSO ’ DMSO
0.25
0.49
Air
0.98 volume
1.95
3.90
7.80
15.57
(m3)
Fig. 2. Dose-related induction of SCE in tracheal epithelial cells of the Syrian golden hamster in vitro in the presence of variousconcentrationsofGEXno.69ofairbomeoarticulates. Values are means, and limits of confidence a& presented.
0.35
0.70
1.41
2.82
5.63
11.26 22.52
Air volume (m3) Fig. 4. Dose-related induction of SCE in tracheal epithelial cells of the Syrian golden hamster in vitro in the presence of various concentrations of GEX no. 70 of airborne particulates. Values are means, and limits of confidence are presented.
402
C. Hornberg
Seemayer
et al.,
demonstrate
that
1994) rodent
and
results
tracheal
of
epithelial
this
and N. H. Seemayer
study
cells offer
in oitro model for genotoxicity testing of inhaled toxins. Extracts of airborne particulates are able to induce a significant dose-related increase of SCE in cultures of tracheal epithelial cells of the Syrian golden hamster and the rat. Medical health risk evaluation of airborne particulates must consider a daily respiratory ventilation in man of 12-15 m’ air at rest, as well as the lowest a reliable
effective
genotoxic
dose,
as was
demonstrated
GEX no. 69, of 0.49 m’ air, and with GEX
with no. 70. of
0.70 m3 air.
REFERENCES
Ahnk G. M., Smit H. S.. van Houdt J. J.. Kolkman J. R. and Boleii J. S. (1983) Mutagenic activitv of airborne particulates at non-industrial locations. Mutation Research 116, 21-34. Balls M. and Fentem J. H. (1994) In vitro test systems and respiratory toxicology. In Respira/iq Toxicology and Risk Assessment. Edited by P. G. Jenkins. pp. 291 -314. Wissenschaftliche Verlagsgesellschaft, Stuttgart. Blanquart C., Romet S., Baeza A., Guennou C. and Marano P. (1991) Primary cultures of tracheal epithelial cells for the evaluation ofrespiratory toxicity. Toxicology in Vitro 5. 4999522. Chdpra D. P. and Cooney R. A. (1985) Histogenesis of benzo[a]pyrene-induced _lesions in tracheal -explants. Virchow Archit:. B. Cell Pathalorrr, .>. 48. 299- 315. Courtois Y. A., Min S., Lachenal C.. Jacquot-Deschamps J. M., Callais F. and Festy B. (1988) Genotoxicity of organic extracts from atmospheric particles. Annals of Ihe New York Academy of Sciences 534, 724-740. Hadnagy W. and Seemayer N. H. (1994) Inhibition of phag&ytosis of human macrophages induced by airborne uarticulates. Toxicoloav Lefrers 72. 23 3 I. Hadnagy W., Seemayer’N. H. and’Tomingas R. (1986) Cytogenetic effects of airborne particulate matter in human lymphocytes in vitro. Muturion Research 175, 977101. Hadnagy W., Seemayer N. H., Tomingas R. and Ivanfy K. (1989) Comparative study of sister-chromatid exchanges and chromosomal aberrations induced by airborne particmates from an urban and a highly industrialized location in human lvmohocvte cultures. Mutaiion Research 225. 27732. + ’ ’ Hornberg C., Seemayer N. H., Hadnagy W. and lvanfy K. (1993) Tracheal epithelial cells of the Golden Syrian hamster in vitro as a tool for detection of genotoxic activity of airborne particulates. Journal af Aerosol Science 24 (Suppl. 1). 91 92. Hornberg C., Seemayer N. H. and Hadnagy W. (1994) Airborne particulates and their genotoxic activity on rodent tracheal epithelial cells and human lymphocytes in vitro. First International Congress on Environmental Medicine, Duisburg, Germany, 23-26 February, 1994. Kuoer. A. and Benford. D. J. (1991) Unscheduled DNA synthesis in tracheal epithelial cell cultures. Toxicology in Vifro 5, 51 I-513. Lasnitzki I. and Bollag W. (1987) Prevention and reversal by a non-polar carotinoid (Ro 15-0778) of 3,4-benzpyrene- and cigarette smoke condensate-induced hyperplasia and metaplasia of rodent respiratory epithelia grown in vitro. Europeon Journal qf Cancer and Clinical Oncology 23, 861-865. Mass M., Nettesheim P., Gray T. and Barrett J. C. (1985) The effect of 12-0-tetradeconylphorbol-13-acetate and
other tumor promoters on colony formation of rat tracheal epithelial cells in culture. Carcinogenesis 5, 159771601. Nettesheim P. and Marchok A. (1983) Neoplastic development in airway epithelium. In Advances in Cancer Research, Vol. 39. Edited by G. Klein and S. Weinhouse. pp. 53-54. Academic Press, New York.
Nettesheim P. and Barrett J. C. (1984) Tracheal epithelial cell transformation: a model system for studies on neoplastic progression. In CRC Critical Reviews in Toxicology Vol. 12. Edited by L. Goldberg. pp. 2155239. CRC Press, Boca Raton, FL. Romet S., Dubreuil A., Baeza A., Moreau A., Schoevart D. and Marano F. (1990) Respiratory tract epithelium in primary culture: effects of ciliotoxic compounds. Toxicology in Vitro 4, 399-402. Shimizu R. W.. Sun J. D., Li A. P., Newton G. J. and Brooks A. L. (1984) The use of sister-chromatid exchange in Chinese primary lung cell cultures to measure genotoxicity. Mutation Research 130, 333-342. Seemayer N. H., Manojlovic N. and Schiirer C. C. (1984) Cell cultures as a tool for detection of cytotoxic, mutagenic and carcinogenic activity of -airborne particulate matter. Journal of Aerosol Science 15, 426430. Seemayer N. H., Hadnagy W. and Tomingas R. (1990) Evaluation of health risks by airborne particulates from in vitro cyto- and genotoxicity testing on human and rodent tissue culture cells: a longitudinal study from 1975 until now. Journal af Aerosol Science 21 (Suppl. I), 501-504. Seemayer N. H., Hornberg C. and Hadnagy H. (1994) Comparative genotoxicitytesting of airborne particulates using rodent tracheal epithelial cells and human lymphocytes in vitro. Toxicology Letters 72, 955103. Steele V. E. and Mass M. _I-( 1985) A rat tracheal cell culture transformation system for assessment of environmental agents as carcinogens and promoters. Environment Imernational 11, 3233329. Steele V. E., Marchok A. C. and Nettesheim P. (1977) Transformation of tracheal epithelium exposed in vitro to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). InternationaIJournal of Cancer 26, 234-238. Thomassen D. G., Gray T. E., Mass M. J. and Barrett J. C. (1983) High frequency of carcinogen-induced early, preneoplastic changes in rat tracheal epithelial cells in culture. Cancer Research 43, 595665963. Tomatis L. (1990) Air pollution and cancer: an old and new problem. In: Air Pollution and Human Cancer. Edited by L. Tomatis. pp. 1-7. Springer Vet-lag, Berlin. Tomingas R.. Voitmer G. and Bednarik H. (1977) Direct fluorimetric analysis of aromatic polycyclic hydrocarbons on thin-layer chromatograms. Science qf the Total Enaironmem 7, 216-267. WHO (1993) IPCS Environmental Health Criteria 155: Biomarkers and Risk Assessment-Concepts and Principles. pp. 82. WHO, Geneva. Whong W. Z., Stewart J. D. and Ong T. (1990) The use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays. Mutation Research 24, 7-13. Wu R. and Smith D. (1982) Continuous multiplication of rabbit tracheal eoithelial cells in a defined. hormonesupplemented medium In Vitro 18, 800-812. Zhu S., Cunningham M. L., Gray T. E. and Nettesheim P. (1991) Cytotoxicity, genotoxicity and transformin activity of 4-(methylnitrosamino)I-(3-pyridyl)-I-butanone (NNK) in rat tracheal epithelial cells. Murafion Research 261, 249-259. Zhu S., Gray T. E. and Nettesheim P. (1992) The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells. Carcinogenesis 13, 172551729.