Mutation Research, 241 (1990) 361-367

361

Elsevier MUTGEN 01572

Unscheduled D N A synthesis in rat pleural mesothelial cells treated with mineral fibres A. R e n i e r 1, F. L 6 v y 2, F. Pilli~re 1 a n d M . C . J a u r a n d 1 1 I N S E R M U 139, C H U Henri Mondor, 94010 Cr~teil (France) and 2 Laboratoire de Biologie Cellulaire, UniversitO Ren~ Descartes, 75006 Paris (France)

(Received 18 November 1989) (Revision received 5 March 1990) (Accepted 12 March 1990)

Keywords: Attapulgite; Asbestos fibres; DNA repair; Rat pleural mesothelial cells; Unscheduled DNA synthesis

Summary Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G 1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[ a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 # g / c m 2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 / ~ g / m l of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 /~g/ml benzo[a]pyrene or 10-50 J / m 2 UV light resulted in an approximate 3 doubling of [ H]dThd inco3rporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [ H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

Correspondence: M.C. Jaurand, INSERM U 139, CHU Henri Mondor, 94010 Cr~teil (France). Ara C, arabinofuranosyl cytosine; B[a]P, benzo[a]pyrene; CAs, chromosomal aberrations; Chr, chrysotile; crocidolite; FCS, fetal calf serum; HU, liydroxyurea;

Abbreviations:

LSC, liquid scintillation counting; [3H]dThd, [3H]methylthymidine; nal ac, nalidixic acid; NG, net grains; PBS, phosphate-buffered saline; RMPCs, rat pleural mesothelial cells; SCEs, sister-chromatid exchanges; UV, ultraviolet; UDS, unscheduled DNA synthesis.

0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

362 Epidemiological and experimental data in animals have demonstrated the carcinogenicity of asbestos fibres (Wagner, 1980; Antman and Aisner, 1986). The mechanisms involved are now under investigation. Several experiments have focused on the genetic damage resulting from the in vitro exposure of mammalian cells to asbestos. Enhancement of chromosomal aberrations (CAs) has been reported in several experiments, and of sister-chromatid exchanges (SCEs) in some cell systems including rat pleural mesothelial cells (RPMCs). However, the results seem to depend on the cell type (see Jaurand, 1989, for a review). In previous studies, we have demonstrated that chrysotile fibres enhance CAs and that crocidolite increases SCEs in cultured RPMCs (Jaurand et al., 1986; Achard et al., 1987). The aim of the present work was to determine if RPMCs treated with asbestos fibres such as chrysotile or crocidolite exhibit D N A repair reflected by unscheduled D N A synthesis (UDS). Another fibre type, attapulgite, was also tested. In addition the effects of ultraviolet light (UV) and benzo[a]pyrene (B[a]P) were determined as references. It has been demonstrated elsewhere that RPMCs metabolise B[a]P (Bastir-Sigeac et al., 1984). Materials and methods

Materials and reagents Rhodesian chrysotile (Chr) and crocidolite fibres (Cr) were obtained from the Union Internationale contre le Cancer. Attapulgite was from Mormoiron (France). Attapulgite (mean fibre length 0.77/~m) was not found to be carcinogenic in rats following intrapleural inoculation (Jaurand et al., 1987). B[a]P, hydroxyurea (HU) and arabinofuranosyl cytosine (ara C) were purchased from Sigma and nalidixic acid (nal ac) from Winthrop (France). [3H]Methylthymidine ([3H]dThd, specific activity 28 Ci/mmole) was acquired from CEA (France). The basic culture medium was Ham's F10 and the test medium was RPMI 1640, both from Flow Laboratories. RPMI 1640 was used for UDS because it does not contain thymidine. Fetal calf serum (FCS) was purchased from Boehringer, phosphate-buffered saline (Ca 2+and Mg2+-free), p H 7.3 (PBS) from Eurobio, streptomycin and penicillin from Flow Laborato-

ries and N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES) was obtained from Seromed (France). Ultraviolet (UV) radiation was delivered by means of a 254-nm lamp (Vilbert Lourmat, France). Mineral fibres were dispersed in RPMI medium containing 1% FCS, by ultrasonic treatment for 5 min (Vibra cell 20 kHz, model 40 W, operating at 3 W, Sonics and Materials Inc., U.S.A.). B[a]P was solubilized in acetone at 2 mg/ml. UV radiation was delivered at an incident dose of 10-50 J / m 2.

Cells and growth medium Cell cultures were established from 6-week-old Sprague-Dawley rats according to a method previously described (Jaurand et al., 1981). Cultures were initiated from frozen stocks and the experiments were performed between passages 6 and 13. Cells were cultured at 3 7 ° C in Ham's F10 supplemented with 10% FCS, 10 mM HEPES, 100 U / m l penicillin and 50 t~g/ml streptomycin ('complete medium'). Autoradiographic assay of UDS Two-chamber tissue culture slides (Lab Tek) containing complete medium were preincubated for 24 h, then 1.6 x 105 cells per chamber were seeded in fresh complete medium. The cells reached confluence after 4 days of incubation. The medium was then replaced with RPMI containing 1% FCS, 5 mM HU, 100 U / m l penicillin and 50 /~g/ml streptomycin. The cells were further incubated for 24 h in a humidified atmosphere of 5% CO 2 in air at 3 7 ° C and then treated with 4 ~ g / c m 2 of chrysotile in RPMI containing 1% FCS, 5 m M H U and 1/~Ci/ml [3H]dThd. One chamber of each slide contained untreated cells and the other treated cells; 3 or 6 slides were analysed for each treatment. The cells were then washed 3 times with PBS and fixed in methanol (3 x 10 rain). Slides were air-dried and dipped in Kodak NTB 3 emulsion. After 8 days of exposure, autoradiographs were developed with Dektol (Kodak) and stained with Giemsa. UDS was quantified by counting nuclear grains in about 200 cells per chamber. Background was calculated as the mean count in 2 nucleus-sized areas adjacent to each nucleus. Net grains (NG) were obtained by subtraction of the background

363

from the nuclear count according to Butterworth et al. (1987). Analysis of variance was used to compare data. 20-

Preparation of RPMCs for UDS detection by fiquid scintillation counting (LSC) RPMCs were cultured in 24-well cluster culture dishes (Costar). 8 x 105 cells were seeded per well in complete medium. Six wells were used for each treatment. The cells reached confluence after 4 days of incubation and the medium was then replaced with R P M I containing 1% FCS, 5 m M HU, 100 U / m l penicillin and 50 # g / m l streptomycin. The cells were incubated for 24 h at 3 7 ° C in a humidified atmosphere of 5% CO 2 in air then treated for 24 h with the indicated dose of B[a]P or fibres in R P M I containing 1% FCS, 5 m M H U and 4 / L C i / m l [3H]dThd. The [3H]dThd concentration used for UV irradiation was 10 /~Ci/ml and the 'treatment lasted 4 h. In some experiments, 5 or 10 /~g/ml ara C or 150/~g/ml nal ac was added to the culture medium at the same time as chrysotile fibres. To test the effect of asbestos fibres on unblocked RPMCs, further experiments were performed in the absence of HU. After treatment for 24 h the cells were washed 3 times with PBS. Acid-soluble material was removed by rinsing with 10% cold trichloroacetic acid for 10 rain and incubated in a mixture of 0.2 M N a O H and 1% sodium dodecyl sulphate. Aliquots of 200/tl were placed in vials and scintillation fluid was added (Picofluor, Packard). Radioactivity was determined with an LKB Rack Beta model 1620. D N A cell content was determined according to West et al. (1985). Statistical analysis was performed using Student's t test.

E cm 10-

-10

5

0

5

10

15

net grains per cell

Fig. 1. D i s t r i b u t i o n of frequencies of the n u m b e r of net g r a i n s per nucleus, o, u n t r e a t e d R P M C s ; [], R P M C s treated with 4

/Lg/cm2 of chrysotile fibres for 20 h. and polyploidy (Jaurand et al., 1983; Hesterberg et al., 1987). This effect is only observed with R P M C s when cells are treated with chrysotile during exponential growth. The background level was not significantly different: 6.6 + 3.1 and 5.4 + 2.6 grains in the control and treated cells respectively. Fig. 1 shows the frequencies of N G per nucleus. The median n u m b e r of N G in chrysotile-treated cells was higher than in untreated controls ( + 0 . 1 and - 3.1, respectively). Similarly, the mean N G in control cells was - 1.1 ___0.6 ( + standard deviation) versus 1.5 + 0.5 in cells treated with 4 /~g/cm 2 of chrysotile. The difference was significant ( p < 0.005). It should be noted that the percentage of S cells ( N G > 60) was similar in control and chrysotiletreated cells (3.4 + 1.1% and 3.5 + 1.0%, respectively). In the absence of HU, the corresponding value in untreated cells was 5.8 + 0.7%.

LSC measurement of UDS in mineral fibre-treated RPMCs [3H]dThd incorporation in the absence of HU.

Results

Autoradiographic detection of UDS in chrysotiletreated RPMCs Since autoradiographic data are expressed as the number of net grains per nucleus, it should be noted that R P M C s do not form polynucleated cells in the conditions used here. In effect, chrysotile fibres are known to induce binucleated cells

3

•

•

[ H]dThd incorporation was reduced by treatment with chrysotile fibres compared to untreated controis (Table 1). Similar results were obtained with crocidolite, whereas no modification was detected with attapulgite.

Effect of chrysotile fibres on HU-treated RPMCs. Fig. 2 shows the effect of 2 doses of chrysotile on [3H]dThd incorporation in several identical ex-

364 TABLE 1

7,000 ]

[3H]dThd I N C O R P O R A T I O N IN C h r - T R E A T E D R P M C s IN T H E ABSENCE OF H U (dpm/~tg O F D N A )

6,000 4

* .

5,000

Chrysotile (~g/cm2)

Incubation (h) 7 17

24

0 2 4 10

3411+354 27485:148 2013 5:574 25885:245

64585:822 29835:339 1406 5:205 3935:141

a

60455:1248 27565: 608 1577 5: 42 6805: 314

4,000 3,OO0

-o

2,000 1,000

0 0

periments. When compared to untreated cultures a significant enhancement of [3H]dThd incorporation was constantly observed with 10/~g/cm 2 and in 5 out of 6 experiments with 4 / x g / c m 2 chrysotile. Use of DNA-repair inhibitors showed that [3H]dThd incorporation was reduced by ara C in both control and treated cultures (Fig. 3), although it remained significantly enhanced in the treated cultures compared to untreated controls. Nal ac also inhibited [3H]dThd incorporation (Fig. 4) but the difference between control and chrysotile-treated cells was no longer significant.

5

10

ara C i.tg/ml

Fig. 3. [3H]dThd incorporation in R P M C s treated with the indicated doses of ara C in the absence (B) or presence of chrysotile fibres at a concentration of 4 f f g / c m 2 (©) or 10 f f g / c m 2 (IN).

2,000 1" I

i 1,500 i

c',

1000

500

Effect of crocidofite and attapulgite fibres on HU-treated RPMCs. After treatment with crocidolite fibres a significant enhancement of [3H]dThd incorporation was observed at all experimental doses (Table 2). In contrast, attapulgite did not change [3H]dThd incorporation compared to untreated cultures (Table 2).

0

0

150 200 natidixic acid ~_g/ml

Fig. 4. [3H]dThd incorporation in R P M C s treated with the indicated doses of nal ac in the absence (~) or presence of chrysotile fibres at a concentration of 4 ~ g / c m 2 (©) or 10 ~tg/cm 2 (~).

TABLE 2 3,000

< z c~

U D S S T I M U L A T I O N IN M I N E R A L R P M C s (dpm/btg O F D N A )

2,000

o_

1,000

1

±i 2

3

4

Fibre type

: 5

: 6

experiment number

Fig. 2. [3H]dThd incorporation in R P M C s either untreated (~) or treated with 4 ~tg/cm 2 (~]) or 1 0 / L g / c m 2 (~) of chrysotile fibres.

Expt. Dose ( / l g / c m 2) No. 0 4

FIBRE-TREATED

10

Cro1 cidolite 2 3

7 5 0 + 29 885 5: 2 0 " * 1933+157 3029+528"** 1194+329 1571+286"**

861 5: 3 7 " * * 25575:400"** 1671+314"**

Attapulgite

886+ 57 9 4 3 + 1 5 7 1 9 1 4 + 1 8 5 1600-t-343 1314+257 15145:157" 1929+314 2114+400

1042+186 1857+314 1386+286 24575:528

1 2 3 4

* p