Murine bone marrow culture system for cytogenetic analysis

Mutation Research, 164 (1986) 91-99 Elsevier

91

MTR 08600

M u r i n e b o n e m a r r o w culture system for cytogenetic analysis G. Krishna 1, j. Nath 2 and T. Ong 1,2 I National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, W V 26505-2888, and 2 Division of Plant Sciences, West Virginia University, Morgantown, WV26506-6108 (U.S.A.)

(Received 1 April 1985) (Revision received 9 September 1985) (Accepted 23 September 1985)

Summary A mouse bone marrow culture system for examining genotoxicity of agents by first exposing animals in vivo then growing cells in vitro is presented. This assay can also be used for in vitro a n d / o r for the in vivo and in vitro comparative cytogenetic studies. The protocol involves culturing of approximately 1000000 nucleated cells obtained from mice tibia and femora in 5 ml of Ham's F-12 medium containing 20% fetal bovine serum, 10% whole uterus extract from pregnant mice and 1% penicillin-streptomycin. The use of flasks and mouse uterus extract for culturing are important steps for higher mitotic yield. The addition of 20/~M BrdU for 24 h helps in the differentiation of sister chromatids for sister-chromatid exchange (SCE) analysis. Cyclophosphamide, given to mice through intraperitoneal injection, induced significant dose-related SCEs in culture. Trinitrofluorenone, a direct-acting mutagen, caused dose-related SCEs in in vitro bone marrow cell culture.

Bone marrow forms an important component of the immunity system in higher organisms and consists of multipotent hemopoietic stem cells. Bone marrow has been employed to analyze chromosomes for clinical/clastogenic purposes. Cytogenetic analysis, the only method that permits direct visual analysis of chromosomes, is widely used in the evaluation of chemically induced chromosomal damage. Although numerous investigators have successfully cultured human bone marrow cells for clinical chromosome analysis (Levin and Garson, 1984; Teyssier, 1984), relatively few studies have been done in animals. The animal bone marrow culture was initiated by Bradley and Metcalf (1966) as a Address correspondence to: Dr. Gopala Krishna, NIOSH, Microbiology Section, 944 Chestnut Ridge Road, Morgantown, WV 26505-2888, U.S.A.

semisolid assay system using colony-forming units on agar as a criterion for cell growth. These studies were extended to the culturing of bone marrow in tubes for sister-chromatid exchange (SCE) analysis in mice (Roberts and Allen, 1980; Henderson et al., 1984). Bone marrow is an excellent source of potentially dividing cells and yields high mitotic index for cytogenetic analysis in vivo. It has been difficult to assess the yield of mitotic figures or efficiency of some of the previous in vitro culture methods because of the scarcity of data. The paucity of cytogenetic studies in mouse bone marrow cultures may be indicative of methodological problems (Kram et al., 1979). The SCE analysis is a sensitive indicator of chemically induced damage to DNA (Latt, 1974; Perry and Evans, 1975; Carrano et al., 1978) and has gained increasing acceptance as a suitable cytogenetic technique for the detection of environ-

0165-1161/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

92 mental mutagens. In addition, the differential staining methodology allows accurate investigation of disturbances in cell replication that usually accompany D N A damage (Craig-Holmes and Shaw, 1976; Morimoto and Wolff, 1980; Schneider et al., 1976). The BrdU differential staining techniques have recently been adapted for the detection of SCEs induced in vivo and in vitro in a variety of mammalian cells (Allen and Latt, 1976; Conner et al., 1978, 1979; Erexson et al., 1983; Goon and Conner, 1984; Kligerman et al., 1981, 1982; Stetka and Wolff, 1976). Mouse bone marrow culture assay for cytogenetic analysis is useful for several reasons: (1) since the laboratory mouse has the best defined genotype of any mammal, it is widely used as a model for toxicological investigations; (2) bone marrow is extensively used as a target tissue in in vivo studies, both for chromosomal aberration and SCE analyses, thus it would be ideal if the same tissue can be used for the in vitro and in vivo comparative studies; (3) the animals exposed in vivo can be studied under in vitro conditions, for the persistence of genotoxicity over a period of

Whole Uteri Homogenize Pellet Spin (10000× g 20 min) (Discard) Supernatant (1) Heat, stirring to 60°C for l0 min (2) Cool to 10°C Pellet Spin (10000× g 20 min) (Discard) Supernatant Filter through 0.20-/~m filter Filtrate Dilute to 1 ml to 1 g wet weight of tissue. Use 10% of this for cell stimulation

Fig. 1. The simplified protocol for pregnant mouse uterus extract preparation. This procedure is derived from that of Bradley et al. (1971). Several purification steps have been omitted.

time; (4) in vitro bone marrow culture can itself be used for genotoxicity studies; and (5) since bone marrow is one of the few tissues available from exposed human populations, the mouse and the rat can serve as models for comparative studies. This paper describes culturing of mouse bone marrow cells for examining cytogenetic damage as evidenced by SCEs following in vivo exposure of mice and in vitro treatment of mouse bone marrow cells to genotoxic agents, cyclophosphamide (CPA) and trinitrofluorenone (TNF), respectively. Materials and methods

Preparation of uterus extract Whole uteri were isolated aseptically from 17day pregnant CD 1 mice (Charles River Breeding Lab., Inc., Wilmington, MA). The method of Bradley et al. (1971) and a simplified procedure presented in Fig. 1 were used for uterus extract preparation. In the simplified procedure, the purification steps have been omitted, but the final cell-stimulating factor is retained. Standard protocol Bone marrow removal and culture. Pathogenfree CD 1 male 5-6-week-old mice, weighing 20-25 g, were also purchased from Charles River Breeding Laboratories, Inc. Femora and tibia were removed from mice killed by cervical disolation. These bones were freed of adherent muscle and cleaned with 70% ethyl alcohol. The tips of the bones were then removed with scissors. The marrow was flushed out with Ham's F-12 medium (Flow Laboratories, McLean, VA) into a 15-ml centrifuge tube. Following the removal of debris, the tubes were centrifuged at 285 × g for 6 min. The supernatant was removed and the pellet resuspended with the remaining solution. Cells were stained with crystal violet and counted using a hemocytometer. Approximately 1 000 000 cells were incubated in the following complete medium: 3.45 ml Ham's F-12, 1 ml fetal bovine serum (20%), 0.05 ml penicillin-streptomycin (1%), and 0.5 ml uterus extract (10%). Cells were cultured in the dark at 37°C. At 16 h, 20 ~M BrdU (Sigma Chemical Co., St. Louis, MO) was added to the flask. Cultures were then covered with aluminum foil and returned to the incubator. Colchicine

93 (Gibco, 33 ~M final concentration) was added 2.5 h prior to harvest (i.e., at 21.5 h after BrdU addition) and the cultures were harvested after a total incubation time of 40 h. Cell harvest, slide preparation, and staining. The contents of the flasks were decanted into 15-ml Falcon centrifuge tubes. The flasks were rinsed with 3 ml of Hank's balanced salt solution which was transferred to the centrifuge tubes. The tubes were centrifuged at 285 x g for 6 min. The supernatant was aspirated and the cell pellet resuspended in 5 ml of hypotonic solution (0.075 M KC1 at 37°C) for 20 min and recentrifuged. The cells were fixed twice, each time with 5 ml of freshly prepared 3:1, methanol:acetic acid and centrifuged. Finally, the cell suspension was diluted with a few drops of fixative and dropped on precleaned chilled wet slides which were air dried for 24 h. Staining for SCE analysis was performed according to a modified technique of Perry and Wolff (1974) and Goto et al. (1978). Slides were stained for 15 min with Hoechst 33258 (5 # g / m l ) and exposed to 'black' light at 55-60°C for 15 min at a distance of 1 cm while immersed in Sorenson's buffer (phosphate buffer pH 6.8). The slides were then rinsed in distilled water and stained with 5% Giemsa (in Sorenson's buffer) for 10-15 min. The slides were coded and analyzed for the experimental end points.

medium at 16 h of incubation. Harvesting was done at 6-h intervals starting with 18 h after BrdU addition. Slides were scored for cell-cycle kinetics. To evaluate cellular replication, the frequencies of the first, second, third and subsequent metaphases were determined in 100 consecutive metaphase cells from each of the 4 cultures. Those cells whose D N A had replicated exclusively before the addition of BrdU could not be distinguished from cells at first metaphase, and those that had gone through 3 or more cell cycles were ranked as third mitoses. The replicative index ( R I ) was calculated as follows: RI

1M 1 + 2 M 2 + 3M 3 100

where M1, M2, and M 3 represent proportions of first-, second-, and third-plus-subsequent-division metaphases, respectively (Krishna et al., 1985; Ivett and Tice, 1982).

Compar&on of mitotic indices in media with and without uterus extract and tube versus flask cultures Bone marrow was cultured in duplicate sets as follows: (1) complete medium with purified uterus extract (prepared according to the procedure of Bradley et al. (1971), (2) complete medium with unpurified uterus extract (prepared according to the simplified procedure, Fig. 1), and (3) complete medium with no uterus extract; 1, 2 and 3 were cultured in Falcon 30-ml flasks and in Falcon 15-ml centrifuge tubes for 24 h. Cells were harvested 2.5 h after colchicine treatment. The mitotic index was recorded from 1000 nuclei in each treatment.

Effect on S C E frequency of mouse bone marrow following in vivo exposure to CPA and then culturing in flasks and tubes Three groups of 4 male mice each weighing 25-30 g were injected intraperitoneally with 5, 10, and 20 mg C P A / k g dissolved in sterile distilled water. The total volume injected was 0.25-0.30 ml per animal depending on weight. A fourth group was injected only with carrier solution i.e., sterile distilled water, and served as control. The animals were sacrificed by cervical dislocation at 6 h after CPA injection. Bone marrow was obtained and prepared as described above. In one set of experiments, the cells were dispersed in 30-ml Falcon (25-cm 2) tissue culture flasks and incubated horizontally in the incubator at 37°C. In the other set, the cells were dispersed in 15-ml Falcon tubes and incubated in a slanting position. Cultures were harvested 27 h after the addition of BrdU, in anticipation of CPA-induced cell-cycle delay. Slides were coded and 100 cells per dose (with at least 38 chromosomes per cell) were scored for SCE frequencies in both flask and tube cultures.

Analysis of cell-cycle kinetics in relation to time Cultures were established using standard protocol except that 20 /~M BrdU was added in the

Effect of in vitro exposure of mouse bone marrow cells to T N F on S C E frequency Four groups of 4 male mice were sacrificed by

94 cervical dislocation and bone marrow preparations for culture were made as described previously. For the first 3 groups of cultures 1.0, 1.5, and 2.0 /~g/ml of T N F was added (4 cultures in each group) along with BrdU after 16 h of incubation. The fourth group received an appropriate amount of carrier compound dimethyl sulfoxide and served as control. The cells were harvested after 27 h as previously described. Slides were coded and SCEs scored from 100 cells in each treatment as in CPA experiments.

TABLE 1 COMPARISON O F M I T O T I C I N D I C E S IN C U L T U R E S O F M O U S E B O N E M A R R O W C E L L S G R O W N IN F L A S K S AND TUBES, WITH AND WITHOUT MOUSE UTERUS E X T R A C T ~' Treatment

M i t o t i c index ( % ) + S . D .

F l a s k s h + Purified e x t r a c t + Unpurified extract Extract

4.00 + 1.63 ~ 3.90 + 1.45 ~ 2.30 _+0.85

Tubes

1.30 + 0 . 9 5 1.40 + 0.70 Occasional metaphases observed

Statistical analysis

The mitotic index was analyzed by a 2-way analysis of variance (container and extract) with the arc' sin transformation. The data on CPA (Table 2) were analyzed in a 3-way analysis of variance procedure (container, test compound and animal). A test was also made to compare the slopes of lines for flask and tube cultures. Since there were no significant differences among cultures within a treatment group, the T N F data (Table 3) were pooled and analyzed in a 2-way analysis of variance (test compound and culture). The general linear regression lines were constructed for these data. A chi-square test was utilized to analyze SCE frequency distribution in vivo/in vitro and in vitro following CPA and T N F treatments, respectively.

'~ 1000 nuclei were scored in e a c h t r e a t m e n t for mitotic index. S i g n i f i c a n t at p < 0.01, over tubes. S i g n i f i c a n t at p < 0.01, over n o extract.

Cell-cycle kinetics in relation to time

The data on cell-cycle analysis are presented in Fig. 2. 18 h following BrdU incorporation, a higher proportion of dividing cells was at M t stage and a lower proportion had reached M 2. At 24 h there was a higher proportion of M 2, a lower proportion of M] and a few M 3 metaphases. At 30 h, the longest time tested, M 3 metaphases comprised a

Results 'm-

Bone marrow culture conditions

These experiments were designed to define the culture conditions necessary for bone marrow cell multiplication. Addition of mouse uterus extract (purified or unpurified) proved critical in obtaining significant numbers of dividing ceils (Table 1). When bone marrow cells were grown in the purified or unpurified mouse uterus extract, the cultures yielded significantly higher mitotic indices in comparison with those grown without extract ( p < 0.01). Flask cultures had significantly higher proportion of dividing cells than did the tube cultures ( p < 0.01). Since flask cultures with uterus extract consistently yielded higher mitotic activity in all cultures and since purified and unpurified extracts yielded comparable mitotic indices, most of the subsequent experiments were carried out using unpurified extract with flask culture.

+ Purified e x t r a c t + Unpurified extract - Extract

• MI CELLS o M2 CELLS o M3 CELLS

IQ6@ J w

50-

w w

_

°12

b

/

15 T8 ~0 ~2 ~4 ~6 ~8 10 12 14 TIME (HOUFR$)

Fig. 2. R e p l i c a t i o n kinetics of u t e r u s - e x t r a c t - s t i m u l a t e d m o u s e b o n e m a r r o w c e l l s . ' F i r s t - m e t a p h a s e (M ~) (O), s e c o n d - m e t a p h a s e (M 2 ) ( © ) , t h i r d - a n d - s u b s e q u e n t - m e t a p h a s e (M 3) (O) cells as a f u n c t i o n of t i m e a f t e r the a d d i t i o n of B r d U into the cultures. D a t a r e p r e s e n t a v e r a g e s of 4 c u l t u r e s a n d b a r s r e p r e s e n t s t a n dard deviation from means.

36

95 TABLE 2

55

SISTER-CHROMATID EXCHANGES IN BONE MARROW CELLS C U L T U R E D IN FLASKS AND TUBES FOLLOWING IN VIVO E X P O S U R E O F M I C E TO CYCLOPHOSPHAMIDE a

50-

r---

r---q C O N T R O L 1 5rag I ~ tOmg

I

45>_ c_) 40z LU 35-

1

20mg

C~

Concentration of cyclophosphamide (mg/kg)

Animal

Control b

1 2 3 4 Mean

9.32+0.63 8.80+0.59 9.48+-0.64 8.44+-0.62 9.01 +- 0.24

1 2 3 4 Mean

11.20+-1.02 11.20+-0.97 ~0.72+0.82 10.60 +- 0.86 10.93+-0.16 c

10.52+-0.75 10.08+-0.66 10.00+-0.82 10.04 +- 0.81 10.16+-0.12 c

1 2 3 4 Mean

13.68+-0.97 12.64+-0.78 13.64+-0.99 11.20+-0.88 12.79+-0.58 c

14.00+-1.37 11.88+-0.64 11.60+0.98 10.68+-0.80 12.045:0.70 c

1 2 3 4 Mean

14.92 +- 1.32 11.92+-0.95 15.24+-1.05 17.44+-0.97 14.88 +- 1.13 c

14.12 +- 1.05 13.20+-1.18 13.32+-0.97 15.40+-1.07 14.01 +-0.51 c

5

10

20

LU rr U_

SCEs/cell +- S.E. Flask

Tube

30-

25-

8.36+0.65 7.64+0.59 10.56+-0.81 9.16+-0.46 8.93 +- 0.63

t0-

°t5-

I

i]

i

5-

1-5

6-10

1 1 - 1 5 16-20

21-25

I

26-30

i

31-35

No. of SCEs/Cell Fig. 4. Sister-chromatid exchange frequency distribution in bone marrow cultured cells from mice treated with cyclophosphamide. Each histogram represents 100 M 2 metaphases.

majority of the cells. The R I was 1.27, 1.79 and 2.55 respectively for 18, 24 and 30 h of cultivation of cells in BrdU. Since 24-h cell culture yielded a higher proportion of second-division metaphases, this time was used in subsequent experiments involving SCE analysis. However, in anticipation of cell-cycle delay, the cultures from chemically treated animals were incubated for 3 additional hours.

a 25 second-division metaphases were scored per animal. Animal-to-animal variation was separated and analyzed. b Sterile distilled water ( - 0.3 ml/animal). c Significant at p < 0.01, over controls.

B

A • SCESPER CELL

a SeEs Ivdl CS,.L • StEs t ~ OIRQIIISO~

• SCEs~R O41101IOSOIIE 15-

.4

o

tt~ o

@

10u~

I0.3 5-

0 CONCENTRATION

OF CYCLOPHOSPHAMIDE (mg/kg body wt.)

CONCENTRATION

OF TRINI

LUOflE

"

(ug/.m~ c u l t . )

Fig. 3. (A) Regression lines of SCE data following in vivo treatment of mice with cyclophosphamide and then in vitro culture of bone marrow cells. (B) Regression lines of SCE data following in vitro treatment of mouse bone marrow cells with trinitrofluorenone.

96 TABLE 3 S I S T E R - C H R O M A T I D E X C H A N G E S IN MOUSE BONE M A R R O W CELLS F O L L O W I N G IN VITRO EXPOSURE OF CELLS TO T R I N I T R O F L U O R E N O N E ~' Concentration of S C E s / c h r o m o s o m e Mean SCEs/cell + S.E. trinitrofluorenone ( / t g / m l culture) Control b

0.19

7.33+0.19

1.0 1.5 2.0

0.38 0.44 0.52

15.00_+0.56 c 17.55 _+0.13 ~ 20.29 + 0.36 "

" 4 animals were used for each treatment and 100 second-division metaphases were scored for SCE in each treatment. The data were pooled from 4 cultures in each treatment and anayzed (as there was no significant difference among cultures within a treatment). b Dimethyl sulfoxide (0.1 ml/culture). Significant at p < 0.01, over controls.

Effect of in vivo CPA exposures and subsequent culturing on SCE frequencies The results on SCE induction following in vivo CPA exposure and subsequent culturing of bone marrow are shown in Table 2 for both flask and tube cultures. The data show a clear linear dose response with all doses tested (Fig. 3A). The distri-

50

r 45-4 ~LZ 40~

~

~ ~ ] i

CONTROL

I lug ~ t .Sug I2.0ug

~J 35k~ 30-

kL25--

i

20-

i

t0-

0

I.-5

6-10

~1-~5

16-20

21-25

26-30

31-35

No. of SCEs/Cell Fig. 5. Sister-chromatid exchange frequency distribution in mouse bone marrow cells treated with trinitrofluorenone. Each histogram represents 100 M 2 metaphases.

bution of SCE frequency in controls and CPAtreated flask cultures are presented in Fig. 4. In control and cultures treated with lower doses, a higher proportion of cells had SCE frequency between 6 and 10. However, in cultures treated with medium and high doses, the frequency distribution was over a wider range with a high proportion falling between 6 and 15 SCEs/cell. Similar SCE frequency distribution was observed in tube cultures (data not shown).

Effect of in vitro TNF treatment and SCE frequencies The in vitro TNF-treated mouse bone marrow cultures also showed a clear dose response in SCE increase at all doses tested (Table 3 and Fig. 3B). At the highest concentration, 2.0 /~g/ml culture, approximately a 3-fold increase in SCE level over controls was noticed. The SCE frequency distribution following in vitro treatment of T N F is shown in Fig. 5. In controls a higher proportion of SCE frequency was between 6 and 10/cell. In the TNF-treated cultures, a peak of SCE distribution was observed for each concentration tested. Higher proportions of cells with SCEs 11-15, 16-20, and 21-25 were observed respecively for low, medium, and high doses tested in this experiment. Discussion Cytogenetic research is highly labor-intensive. Much time is needed to set up and harvest cultures, and stain enough slides for analysis of an adequate number of spreads. Thus, it is always desirable to efficiently produce more and better spreads. With this in mind, we have established a simple mouse bone marrow cell culture technique for the study of in vivo/in vitro and in vitro chemically induced genotoxicity as evidenced by SCE induction. The technique may also be used for chromosomal aberration analysis with shorter culture time. This system employs unpurified mouse uterus extract in suspension culture to yield high mitotic index. The use of unpurified extract eliminates purifcation steps of uterus extract preparation as described by Bradley et al. (1971). The important steps in obtaining successful cultures are the use of flasks and uterus extract. The role of uterus and embryo extract in stimulation of bone

97 marrow cells has been described by Bradley et al. (1971) in semisolid agar colony conditions. A higher mitotic index of cells in flasks both with and without uterus extract, in comparison with that in tubes, indicates the importance of surface area. For any cell growth, there should be cell-to-cell contact as well as more surface area for further division and spread of cells. Even though there is greater cell-to-cell contact in tubes, the tube culture lacks the surface area for further growth and spread. The importance of surface area for cell growth in cell cultures has been previously established (Freshney, 1983). Also, flasks offer additional opportunity for diffusion exchange of gases between media and the atmosphere. BrdU differential staining technique was used to assess the effect of time on cell replication. Cells which have replicated once, twice or 3 or more times in the presence of BrdU can be unequivocally identified. In order to find the optimum time for higher proportion of second-division metaphases for SCE analysis and to find the cellcycle duration, time course experiments were carried out. The data suggested 24 h of culture after BrdU addition as the optimum time for a higher proportion of M 2 cells and the cell-cycle duration of 10-12 h under these conditions. Since a wide time interval (6 h) was employed in the study, a refined estimate of cell-cycle duration could not be made. The increase in R I with time indicates a rapid multiplication of cells. CPA was administered to mice to monitor genetic damage and to examine the persistence of D N A lesions that lead to SCEs when cultured in vitro after in vivo exposure. The mouse bone marrow culture system helped detect dose-related cytogenetic damage induced after in vivo administration of CPA, a promutagen, a carcinogen and a potent SCE inducer which requires metabolic activation. This study indicated that CPA reached the bone marrow in 6 h and caused subsequent dose-related SCEs in vitro. These results paralleled those of Goon and Conner (1984), Takeshita and Conner (1984) and Wilmer et al. (1984) in mouse blood culture following in vivo CPA exposure. Although the culturing method (flask versus tube) affected the multiplication of cells, the SCE frequencies induced by CPA were not significantly

different between cells cultured in flasks and in tubes. The dose-response regression lines, each representing SCE data from both flask and tube cultures, are shown in Fig. 3A. Because a higher proportion of cells with less than 2n chromosomes were counted as the dose increased, the regression lines of SCEs per cell shown in Fig. 3 are not exactly parallel to those of SCEs per chromosome. The dose-response findings of CPA as measured by SCE are in agreement with earlier studies (King et al., 1982; Nakanishi and Schneider, 1979; Stetka and Wolff, 1976). However, the number of SCEs were lower than those reported by others for similar doses in mouse bone marrow system. This could be due to different protocols used in these studies i.e., in vivo versus in vivo/in vitro. In the in vivo/in vitro study reported here, BrdU was added 16 h after culture initiation. By this time, some of the lesions might have been lost due to cell cycling (Bochkov et al., 1984). In fact, our preliminary studies indicate that SCE frequency is higher if BrdU is added at the beginning of cell culture rather than 16 h later. The in vitro induction of SCEs following T N F treatment in -this study was comparable to that of Tucker and Ong (1984) in human lymphocytes in the absence of metabolic activation. This indicates that the mouse bone marrow culture system can be used under both partial in vivo (exposure of animals to chemical in vivo and growing cells in vitro) and in vitro genotoxicity studies of chemicals or other potentially toxic substances. The distribution of SCE frequency under partial in vivo conditions yielded a wider spread than that of in vitro conditions. Since two different chemicals were used, valid comparisons cannot be made. However, less spread and unique dose peaks in the in vitro culture conditions may be attributed to the more controlled conditions in vitro than in vivo. The amount and distribution of chemical in the target tissue and its metabolism under in vivo conditions can influence SCE frequency distribution. Unlike in vivo situations, in in vitro conditions the chemical in the culture uniformally surrounds the cells (dose of chemical per cell is more uniform), thus causing relatively uniform damage to cells in culture. A wider SCE frequency distribution in partial in vivo than in vitro in rabbit lymphocytes treated with streptonigrin has been

98

reported (DuFrain, 1983). This mouse bone marrow culture system described can be a valuable tool for in vivo and in vitro comparative studies and for the investigation of cytogenetic effects caused by in vivo exposure of mice to genotoxic agents and subsequently culturing cells in vitro to detect persistence of D N A lesions. It can also be used for in vitro treatment of chemicals in the genotoxicity studies.

Acknowledgments This research was conducted while the senior author held an N A S / N R C Post-doctoral Research Associateship. The authors wish to thank Dr. W.-Z. Whong for his technical suggestions during this study and Dr. M. Petersen for the statistical analysis.

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