Chemical induction of somatic gene mutations and chromosomal aberrations in lung fibroblasts of rats

257

Mutation Research, 263 (1991) 257-262 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100068K MUTLET 0519

Chemical induction of somatic gene mutations and chromosomal aberrations in lung fibroblasts of rats Mohammed

A. Khan and John A. Heddle

Department of Biology, York University, Toronto, Ont. M3J 1P3 (Canada) (Received 8 January 1991) (Revision received 19 March 1991) (Accepted 25 March 1991)

Keywords: Micronuclei; Gene mutations, somatic; Lung fibroblasts; Fibroblasts, lung, rat; Methyl methanesulphonate; Ethyl nitrosourea

Summary Rats can be used to detect both somatic gene mutations and chromosomal aberrations induced in vivo. The technique adapted from Chinese hamsters for the isolation and analysis of lung fibroblasts confirm the surprising results obtained in hamsters; namely, methyl methanesulphonate induces many micronuclei but no significant increase in gene mutations. In contrast ethylnitrosourea induces both micronuclei and gene mutations.

It is reasonably well established that most carcinogens are mutagens and that a large number of physical and chemical agents induce cancer through mutations. Although the intricate mechanisms of cancer induction are not clearly understood, current theories on the origin of human cancer suggest that some types of cancer arise as a result of genetic alterations in a single cell. These alterations include both gene mutations and chromosomal aberrations. In some cancers, oncogenes are commonly activated by gene mutations, whereas in others chromosomal aberrations Correspondence: Dr. Mohammed A. Khan, Department of Biology, Farq. Bldg., York University, 4700 Keele Street, North York, Ont. M3J IP3 (Canada).

are involved (Land et al., 1983). Extensive studies on retinoblastoma (Murphy and Benedict, 1984; Knudson, 1985; Hansen and Cavenee, 1987) and colorectal carcinoma (Vogelstein et al., 1988) have provided convincing evidence that both gene mutations and chromosomal aberrations can be important in a single type of cancer. In view of these findings, it seemed of interest to explore the possibility o f measuring induced gene mutations in a single mammalian somatic cell in vivo. Chinese hamsters were utilized in our first attempt to analyze the ability of different classes of mutagens in inducing the two types of mutations (Heddle et al., 1990). We wished to extend our studies to rats both to confirm the results obtained in hamsters, and because rats are often used for many other tox-

258 icological tests, making them a desirable choice for genetic toxicology. Fischer (F344) rats were chosen for our present experiments because they are the standard strain used in the cancer bioassay as well as toxicology, pharmacology, biochemistry and physiology experiments. Materials and methods

Male Fischer rats (F344), aged 4-6 weeks, were obtained from Charles River Laboratories, Wilmington, MA. They were housed in standard wire cages with cotton pads placed beneath the cages in trays, and were provided with purina mouse chow and water ad libitum.

sin digestion was finally stopped by adding 10 mi Eagle's MEM with 12.5% fetal bovine serum (Bocknek). DNAase (10/,g/ml) was added per 10 ml digest and mixed well. 2 min later, the sample was centrifuged at 1500 r.p.m. The cell pellet was resuspended in 2 ml complete medium and plated in 100-mm culture dishes (Nunc). Cultures were maintained at 5% CO~ in air at 37.5°C in humidified atmosphere. The following day, the culture medium together with tissue debris and cells that failed to attach, was discarded and the attached cells (almost all fibroblasts) were trypsinized and used for both mutation and micronucleus assays.

Mutagens

Micronucleus assay

Ethylnitrosourea (ENU) was obtained from Sigma Chemicals. Methyl methanesulphonate (MMS, > 9 9 % pure) was obtained from Eastman K o d a k company. The mutagen was freshly prepared and injected into the animals within 45 min. The cell isolation procedure was carried out after 4.5h.

Flame sterilized glass slides were placed in sterile square plastic dishes (3 slides/dish). Each dish received a mixture of 15 ml complete medium (Eagle's MEM with 12.5% fetal bovine serum) and cytochalasin B (3 #g/ml). Approximately 50000 cells that came from treated animals and 30000 cells that came from untreated animals were plated onto slides in the dishes with the mixture and incubated for 72 h. Cells that divided during this period of time were rendered binucleate because of the ability of cytochalasin B to permit nuclear division but inhibit cytoplasmic division. The slides were then immersed in hypotonic solution (0.075 M KC1) for 10 rain and fixed in methanol for 2 rain. The cells were stained with acridine orange prior to scoring for micronuclei using our scoring criteria (Heddle et al., 1990).

Isolation o f primary lung cells Cells were isolated from the lungs of rats, by a modification of the method used in Chinese hamsters (Heddle et al., 1990) which was, itself, an adaptation of Dean and Senner (1977). The lungs were removed together with the trachea and the heart. Eagle's M E M (Gibco) was injected through the right ventricle, thus perfusing the lungs free of blood via the pulmonary artery. The lungs were then separated from the rest of the tissue and minced to pieces, less than 1 m m 3, with a pair of curved scissors. A partial vacuum device was used to remove small amounts of air trapped in the lung fragments. A thorough degassing process is an important step for efficient digestion of the tissue and results in a better yield of fibroblasts. The lung tissue was then rinsed once with Eagle's MEM (Gibco) and once with 0.25% trypsin (Difco 1:250) solution in H a n k s ' Balanced Salt Solution (HBSS) before it was digested by trypsin for 80 min at 38°C. The tissue was well agitated with sterile plastic transfer pipette at intervals of 10 min. Tryp-

Mutation assay Primary fibroblasts obtained from mutagen treated and untreated animals were grown in culture dishes using standard procedure. They were allowed to grow for a total of 10 days by subculturing once on the 5th, 6th or 7th day depending on the degree of confluency. The cells were harvested on the 10th day, and 100000 cells were plated in each of 10 culture dishes containing complete medium with selective agent 6-thioguanine (TG). TG selects cells deficient in the salvage enzyme hypoxanthine guanine phosphoribosyl transferase

259

TABLE 1 M U T A T I O N S A N D M I C R O N U C L E I I N D U C E D BY E T H Y L N I T R O S O U R E A (ENU) IN R A T L U N G CELLS IN VIVO Concentration (mg/kg)

Total number of cells plated

Mutants/ 100000 cells plated

Survivors (°70)

Expt. 1 Expt. 2 Expt. 3

1.5 × 106 1.0× 106 1.0× 106

0.0 0.2 0.0

3.8 27.0 10.2

0 7 0

13.0 21.5 22.0

100

Expt. 1 Expt. 2 Expt. 3

1.5× l06 1.0× 106 1.0× 10 6

2.2 2.6 4.2

5.2 13.0 14.8

423 200 283

18.0 60.0 46.0

200

Expt. 1 Expt. 2 Expt. 3

1.5 X 10 6 1.0× 106 1.0× 106

2.8 6.6 8.0

1.8 9.0 7.2

1555 733 1111

65.0 104.0 89.0

(HGPRT). Plating efficiency was determined by seeding 500 ceils in each of 5 dishes containing complete medium without TG. Colonies of more than 50 cells were scored after staining selection and non-selection (plating efficiency) dishes 2 weeks later.

Mutants/ 1 000000 survivors

Micronuclei/ 1000 cells

ing 5" by 1.5", with a small hole in the bottom, was used to inject the animal. The animal was allowed to enter the bottle, head first, while the hind limbs and the tail remained outside. This device ensured maximum safety and speed in injecting the animal. Three animals were pooled per dose.

Experimental design Male animals weighing between 120 and 150 g were used in these experiments. The animals were injected intraperitoneally and were sacrificed after 4.5 h for lung cell isolation. A glass bottle measur-

Results The frequency of ENU- or MMS-induced thioguanine resistant colonies and micronuclei is

TABLE 2 M U T A T I O N S A N D M I C R O N U C L E I I N D U C E D BY M E T H Y L M E T H A N E S U L F O N A T E (MMS) IN RAT LUNG CELLS IN VIVO Concentration (mg/kg)

Total number of cells plated

Mutants/ 100000 cells plated

Survivors (070)

Mutants/ 1 000000 survivors

Micronuclei/ 1000 ceils

0

Expt. 1 Expt. 2

1.0x 106 1.0x 106

0.3 0.2

22.0 24.0

13.6 8.3

20.0 24.5

25

Expt. 1 Expt. 2

1.0x 106 1.0x 10 6

0,2 0,0

17.7 32.0

11.2 0.0

32.0 43.5

50

Expt. 1 Expt. 2

1.0X 106 1.0X 106

0.5 0.2

23.8 22.0

21.0 9.0

49.0 54.0

100

Expt. 1 Expt. 2

1.0X 106 1.0 X 106

0.4 0.3

22.4 29.0

17.8 10.3

140.0 114.5

260

shown in Tables 1 and 2. Three independent experiments using E N U (Table 1), and two independent experiments using MMS (Table 2) were carried out. As with any experiment, variation in the number of mutations and micronuclei was observed a m o n g individuals, for each dose point. There was clear indication, in each experiment, o f a dosedependent response - - E N U producing an increase in the number of both mutations and micronuclei, and MMS producing a noticeable increase in the number o f micronuclei. The results o f all the experiments conducted to assess the mutagenic effects o f E N U or MMS are illustrated in Figs. 1-4. Each figure illustrates the results of all individual experiments and their mean value. Figs. 1 and 2 represent the ENU-induced frequency of mutations and micronuclei, and Figs. 3 and 4 represent the MMS-induced frequency of the two end-points. The mean value curves in Figs. 1 and 2 indicate that E N U is a potent inducer of both gene mutations and c h r o m o s o m a l aberrations. M M S does not show any significant increase in the number of gene mutations (Fig. 3). A strong

clastogenic property of MMS is indicated by a dose-dependent increase in micronuclei (Fig. 4). Discussion Our selection of the mutagens ENU and MMS was deliberate, because they produce different mutational spectra. Both E N U and MMS are alkylating agents but are known to react differently with the nucleophilic sites on the D N A molecule. This can be explained in terms of reaction mechanisms and the dependence of reaction rates on the nucleophilic strength of the receptor site (Osterman-Golkar et al., 1970). The Swain-Scott substrate constant 'S', used as an expression of the reactivity, is a measure of the sensitivity of the alkylating agent to changes in the strength of the nucleophile with which it reacts (Swain-Scott, 1953). Alkylating agents with low 'S' value are k n o w n to be efficient inducers of both gene mutations and c h r o m o s o m a l aberrations due to their ability to react indiscriminately with sites of low (oxygen) as well as high (nitrogen) nucleophilicity.

150

2000 • 0 x 0

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=/#= 1 1,2,3 ~ 3

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120

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m 1500

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0 ° o 0 0 0

c o

'1000

/¢

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i/

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,S iI

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90

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Fig. 1. Frequency of TG' colonies observed in cells isolated from the lungs of rats injected with ethyl nitrosourea 4.5 h earlier. The results of three independent experiments and the mean o f three values are shown.

0

I 0

I 100 ENU

dose

I 200 m g / kg)

Fig. 2. Frequency of micronuclei observed in binucleate cells isolated from lungs of rats injected with ethyl nitrosourea 4.5 h earlier. The results of three independent experiments and the mean of three values are shown.

261 Alkylating agents with high 'S' value are efficient inducers of chromosomal aberrations and poor inducers of gene mutations since they discriminate between oxygen and nitrogen atoms (OstermanGolkar et al., 1970; Vogel and Natarajan, 1981). Our results with ENU ('S' value--0.26) and MMS ('S' v a l u e = 0 . 8 6 ) agree with these findings and hopefully our present studies may offer further insight into in vivo m a m m a l i a n mutagenesis studies. M a m m a l i a n mutagenesis studies involving somatic mutations include the method of Dean and Senner (1977), the mouse spot test (Russell, 1983), the detection of thioguanine-resistant lymphocytes in vivo (Jones et al., 1985), the granuloma pouch assay (Meier, 1983), the measurement of the loss of cytologically detectable marker - - intracellular melanin distribution in mouse embryo (Stephenson and Searle, 1986) and the loss of lectin binding (Winton et al., 1988). The mouse spot test and the test for measuring the loss of cytological marker for intracellular melanin distribution require that pregnant mice be treated at a precise time. Moreover, in the mouse spot test, the nature of the

genetic event being detected is not clear. The two assays based on the loss of a cytologically detectable marker, cannot detect the number of cells at risk. The thioguanine-resistant lymphocyte assay has potential, provided enough lymphocytes can be obtained and reliably cloned. A number of mutagens have been tested using the granuloma pouch assay but dimethylnitrosamine, a potent mutagen in Drosophila (Fahmy et al., 1966) and in m a m m a l s (Legator and Mailing, 1971) was shown to be negative. Our experimental results show that both gene mutations and chromosomal aberrations can be induced in a single m a m m a l i a n cell type in vivo. The frequency of ENU- and MMS-induced gene mutations and chromosomal aberrations in rats was similar to that we obtained earlier in the Chinese hamsters. Some differences were noticed in the plating efficiency and spontaneous mutant frequency in the two animals. Both were higher in rats than in hamsters. The large size of rats is an additional advantage that should permit measurement of individual animals.

150 0

Experiment

o x

Mean value=#=1 & 2 Experiment -7~2

n Experlment ~ 1 M e a n value =~(:1 & 2 <> E x p e r l m e n t - ~ 2

-~1

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1.5

_/ 8 I'* /I /t

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/ st t /

0 0 0 6O u E o L "~ ig

0.5

- - I

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I .......... 25 dose

ID . . . . . . . . . 50

100

(mg/kg)

Fig. 3. Frequency of TGr colonies observed in cells isolated from the lungs of rats injected with methyl methanesulfonate 4.5 h earlier. The results of two independent experiments and the mean of three values are shown.

30

I

I

o

2~ MMS

dose

5'o

18o

(mg/kg)

Fig. 4. Frequency of micronuclei observed in binucleated cells isolated from the lungs of rats injected with methyl methanesulfonate 4.5 h earlier. The results of two independent experiments and the mean of two values are shown.

262

Conclusion N o w a simple quantitative assay for a native gene in a normal somatic cell is available in the rat. Our present assay may find useful ramifications in the investigation of tissue specificity, in genetic toxicology, as well as in inhalation mutagenesis. Acknowledgement We wish to thank Cesare Urlando and Marco Pagura o f Bio-Mutatech Inc., for their technical assistance with some of our experiments. References Dean, B.J., and K. Senner (1977) Detection of chemically induced somatic mutations in Chinese hamsters. Fahmy, O.G., M.J. Fahmy, J. Massasso and M. Ondrej (1966) Differential rnutagenicity of the amine and amide derivatives of nitro compounds in Drosophila melanogaster, Mutation Res., 3, 201-217. Fenech, M., and A.A. Morley (1985) Measurement of micronuclei in lymphocytes, Mutation Res., 147, 29-36. Hansen, M.F., and W.B. Cavenee (1987) Genetics of cancer predisposition, Cancer Res., 47, 5518-5527. Health and Welfare Canada/Environment Canada (1988) DNH and W/DOE Advisory Committee on Mutagenicity tests in the Toxicological Evaluation of Chemicals, Environ. Mol. Mutagen., 11, 261-304. Heddle, J.A., A. Bouch, M.A. Khan and J.D. Gingerich (1990) Concurrent detection of gene mutations and chromosomal aberrations induced in vivo in somatic cells, Mutagenesis, 5(2), 179-184. Jones, I.M., K. Burkhart-Shultz and A.V. Carrano (1985) A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes, Mutation Res., 147, 97-105. Knudson, A.G. (1985) Hereditary cancer, oncogenes and antioncogenes, Cancer Res., 45, 1437-1443. Land, H., L.F. Parada and R.A. Weinberg (1983) Cellular oncogenes and multistep carcinogenesis, Science, 222,771-778.

I.egator, M.S., and H.V. Mailing (19711 The host-mediated assay, a practical procedure lor evaluating potential mutagenic agents in nlamnlals, ill: A. Hollaender (Ed.), Chemical Mutagens - - Principles and Methods for their Detection, Vol. 2, Plenum, New York, pp. 569-589. Maier, P. (1983) The granuloma pouch assay, in: F.J. de Serres (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 8, Plenum, New York, pp. 233-260. Murphree, A.L., and W.I=. Benedict (1984) Retinoblastoma: Clues to human oncogenesis, Science, 223, 1028-I033. Osterman-Golkar, S., L. Ehrenberg and C.A. Wachtmeister (1970) Reaction kinetics and biological action, in barley, of monofunctional methanesulfonic esters, Radial. Bot., 10, 303 325. Russell, I..B. (1983) The mouse spot test as a predictor of heritable genetic damage and other endpoints, in: F.J. de Serres (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 8, Plenum, New York, pp. 95-110. Stephenson, D.A., and A.G. Searl (1986) The effects of X-rays on the induction of somatic mutations and growth in the retinal pigmented epithelium during development of the mouse eye, Mutagenesis, 1, 135-141. Swain, C.G., and C.B. Scott (1953) Quantitative correlation of relative rates: Comparison of hydroxide ion with other nucleophilic reagents towards alkylhallides, esters, epoxides and acylhallides, J. Am. Chem. Sot., 75, 141-147. Vogel, E., and A.T. Natarajan (1981) The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems, lnterspecies comparisons, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Vol. 7, Plcnmn, New York, pp. 295-336. Vogelstein, B., E.R. Fearson, S.R. Hamilton, S.C. Kern, A.C. Preisinger, M. Leppert, Y. Nakamura, R. White, A.M.M. Smith and J.L. Bos (1988) Genetic alterations during colorectal-tumour development, N. Engl. J. Med., 319, 525-532. Winton, D.J., M.A. Blount and B.A.J. Ponder (1988) A cloned marker induced by mutation in mouse intestinal epithelium, Nature (London), 333, 463-466. Communicated by B.W. Glickman