Characterization of cigarette smoke-induced micronuclei in vitro

Exp Toxic Patholl996; 48: 548-550 Gustav Fischer Verlag Jena

INBIFO Institut fur Biologische Forschung GmbH, Koln, Germany

Characterization of cigarette smoke-induced micronuclei in vitro* D. VELTEL and A. HOHENEDER With 4 figures and 1 table Received: December 1,1995; Accepted: December 20, 1995 Address for correspondence: Dr. D. VELTEL, INBIFO Institut fur biologische Forschung, Fuggerstr. 3, D-51149 Koln, Germany. Key words: Cigarette smoke; Micronuclei; Smoke cigarette.

Introduction Micronuclei arise from either acentric chromosome fragments or whole chromosomes that are not incorporated in the daughter nuclei following nuclear division. They are induced by either clastogenic agents or aneugenic agents that affect the spindle apparatus. It has been reported that cigarette smoke can affect the micronucleus rate in human lymphocytes (Di GIORGIO 1994), expectorated sputum cells (FONTHAM 1986), or bronchial brushings (LIPPMANN 1990). In the present study, a modified micronucleus assay using an antikinetochore antibody has been employed in cytokinesis-blocked Chinese hamster lung cells as a simple microscopic method to characterize cigarette smokeinduced micronuclei in vitro. Using immunocytochemical antikinetochore staining, a differentiation between the clastogenic or aneugenic action of cigarette smoke is easily recognized.

The cells were treated with 0 to 0.055 puffs/ml smokebubbled PBS (mean age < 1 h) or with the positive control substances (mitomycin C, colchicine), the respective medium being supplemented with 2.5 ug/ml cytochalasin B to block cytokinesis during the following 16 h of incubation. Following treatment, cells were prepared for cytosmears and fixed with methanol abs. prior to acridine orange (AO) or kinetochore detection staining. For the latter, cytosmears were treated with antibodies to kinetochores derived from the serum of scleroderma patients (CREST syndrome, antibodies obtained from Kal-

Material and methods V79 Chinese hamster lung cells were grown under standard conditions. Mainstream cigarette smoke was generated from the Kentucky standard reference cigarette 2Rl and bubbled through PBS (smoke-bubbled PBS). I

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Poster, presented at the 5th International Inhalation Symposium, Hannover, Germany, 20-24 February 1995. 548

Exp Toxic Pathol 48 (1996) 6

Fig. 1. Number of binucleated cells with micronuclei relative to the total number of binucleated cells, mitomycin C treatment.

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Fig. 2. Number of binucleated cells with micronuclei relative to the total number ofbinucleated cells, colchicine treatment.

Fig. 3. Number of binucleated cells with micronuclei relative to the total number ofbinucleated cells, smoke-bubbled PBS treatment.

Table 1. Microscopic Analysis of Micronucleated Binucleated Cells for Kinetochores (data based on scoring ~ 300 binucleated cells for each sample).

lestad Diagnostics). Cytosmears were then incubated with a secondary FITC-conjugated goat antihuman antibody. The DNA of nuclei and micronuclei was counterstained with propidium iodide (PI). At least 300 binucleated cells were scored for micronuclei in each sample, and the fraction of kinetochore positive and kinetochore negative micronucleated binucleated cells was determined.

Test Substance (Cone.) colchicine (0.07 umol/l)

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(% binucleated cells with micronuclei)

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mitomycin C (0.5 ug/ml)

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smoke-bubbled PBS (0.05 puffs/ml)

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Results The positive control substances mitomycin C and colchicine as well as smoke-bubbled PBS induced micronuclei in a dose-dependent manner (figs. 1 to 3), the EC so

Fig. 4. Examples of micronuclei with (a) or without (b) positive kinetochore staining. Exp Toxic Patho1 48 (1996) 6

549

value of cytotoxicity for smoke-bubbled PBS being approximately 0.04 puffs/ml. The fraction of binucleated cells with positive kinetochore staining was approximately 92 % for smokebubbled PBS at a concentration of 0.05 puffs/ml, 100 % for colchicine at a concentration of 0.07 umol/l, and approximately 54 % for mitomycin C at a concentration of 0.5 ug/ml (tab. 1).

Conclusion The results of this study show that the smoke-bubbled PBS-related formation of micronuclei is aneugenic rather than clastogenicas evidenced by the evaluation of kinetochore staining.

References DEGRASSI F, TANZARELLA C: Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detectionof aneuploidy. Mutat Res 1988; 203: 339-345. DI GIORGIO C, DE MEO MP, LAGET M, et a1.: The micronucleus assayin humanlymphocytes; screening for interindividual variability and application to biomonitoring. Carcinogenesis 1994; 15: 313-317. FONTHAM ET, CORREA P, RODRIGUEZ E, et a1.: Validation of smoking history withthe micronuclei test.In: HOFFMANN D, HARRIS CC (Eds.): Mechanisms in Tobacco Carcinogenesis. Cold Spring Harbor New York. Cold Spring HarborLaboratory, Banbury Report 23, 1986; 113-118. LIPPMAN SM, PETERS EJ, WARGOVICH MJ, et al.: Bronchial micronuclei as a markerof an early stageof carcinogenesis in the human tracheobronchial epithelium. Int J Cancer 1990; 45: 811-815.

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