HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar

Mutation Research, 182 (1987) 99-111 Elsevier

99

MTR 08640

A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar T.J. Oberly, B.J. B e w s e y a n d G.S. P r o b s t Toxicology Division, Lilly Research Laboratories, Division of Eli Lilly and Company, Greenfield, IN 46140 (U.S.A.) (Received 4 March 1986) (Revision received 26 September 1986) (Accepted 9 October 1986)

Keywords: (Chinese hamster ovary cells); 6-Thioguanine resistance; HGPRT mutation assay; Suspension culture; Soft-agar.

Summary A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 + 3.2 × 10 -6 and cloning efficiencies of 91 + 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-l-oxide, methylnitrosourea and N-methylN'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the C H O / H G P R T mutation assay.

The Chinese hamster ovary (CHO) cell hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene mutation assay (Hsie et al., 1979a; O'Neill and Hsie, 1979) is a well characterized and useful test for the identification of chemical mutagens. Although this test has found widespread application as a component of genetic toxicology

Correspondence: Mr. T.J. Oberly, Toxicology Division, Lilly Research Laboratories, Division of Eli Lilly and Company, Greendfield, IN 46140 (U.S.A.).

test batteries, a shortcoming of the system has been that monolayer cultures are used for chemical exposure, mutant expression and cloning of 6-thioguanine-resistant (6TG r) mutants. Technical disadvantages of the monolayer procedure have been: (1) the need for trypsinization following treatment with the test chemical and for subculturing during the mutant expression period; and (2) the phenomenon of metabolic cooperation, which results from cell-to-cell contact, has limited the plating of only 2 × 105 cells/100-mm plate for mutant selection.

0165-1161/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

100

Modifications to the C H O / H G P R T assay have been aimed at reducing the labor intensiveness of the procedures, thereby improving the system as a screening test. Li (1981) reported that CHO cells can be grown as "unattached" cultures during the expression period and thus avoid repeated trypsinization during subculturing. A further modification of Li and Shimizu (1983) has involved the selection of 6TG r mutants in soft-agar medium which has permitted the plating of 10 6 cells/100mm dish without loss of mutants due to metabolic cooperation. Similarly, Nishi et al. (1984) have reported that the use of soft-agar medium in the selection of V79 cell HGPRT mutants eliminated the effects of metabolic cooperation. In spite of these improvements, these modified procedures still included an initial trypsinization step since monolayer cultures were used for exposure to the test chemical. The present manuscript describes a further modification to the C H O / H G P R T assay which includes exposure of CHO suspension cultures to the test chemical followed by expression of mutation in suspension culture and selection of 6TG r mutants in soft-agar medium. In this procedure the adaptation of CHO cells to grow in suspension culture eliminates the need for trypsinization, allows a larger number of cells (6 × 10 6) t o be exposed to the test article and provides a convenient means of subculturing during mutant expression. Furthermore, the preliminary assessment of cloning efficiency, which is essential for assessing cell survival in the monolayer procedure, is not required. The use of soft-agar medium for 6TG r mutant selection permits the plating of 10 6 cells/ dish without the risk of metabolic cooperation and allows for the automated counting of colonies. Collectively, these modifications facilitate the performance of the C H O / H G P R T assay without compromising the response to mutagens. Materials and methods

Reagents Methylnitrosourea (MNU), methotrexate (MTX) and 6-thioguanine (6TG) were purchased from Sigma Chemical Co. (St. Louis, MO). Ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) were obtained from Eastman

(Rochester, NY). 4-Nitroquinoline-l-oxide was from K and K Laboratories (Cleveland, OH) and Pluronic F68 was from BASF Wyandotte (Wyandotte, MI). Thymidine, hypoxanthine, glycine, sodium pyruvate, glutamine, neomycin, trypsin, Ham's F-12 medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). Dimethyl sulfoxide (DMSO) and sodium chloride were obtained from Fisher Scientific (Pittsburg, PA). Sodium dodecylsulfate (SDS) was from BioRad (Richmond, CO). [3H]Hypoxanthine was from New England Nuclear (Boston, M A ) a n d Noble agar was obtained from Difco (Detroit, MI). Cell culture CHO cells, strain K1-BH4, were kindly supplied by Dr. A.W. Hsie of Oak Ridge National Laboratory. Monolayer cultures of CHO cells were maintained in logarithmic growth in 75-cm2 flasks (Coming No. 25110) using Ham's F12 medium supplemented with 10% FBS (heat-inactivated at 56°C for 30 min), sodium pyruvate (50 #g/ml), neomycin (150/~g/ml) and glutamine (150 #g/ml). This medium is hereafter identified as H10. Suspension cultures of CHO cells were prepared by trypsinization (0.25% trypsin) of the monolayer culture followed by inoculating the cells into 250ml polycarbonate erlenmeyer flasks (Coming No. 25600) containing 100 ml of H10P (H10 supplemented with 1 mg/ml Pluronic F68). Suspension cultures were maintained at 37 ° in a New Brunswick (Model G-25) gyratory incubator set to 125 oscillations/min. Normal doubling time for the CHO cells in suspension was approximately 13 h. When cell densities reached 1 x 10 6 to 1.5 × 10 6 cells/ml, the cultures were passed to new flasks containing H10P such that cell densities were approximately 5 x 104 to 4 x 105 cells/ml. Suspension cultures were discarded after approximately 50 generations. Every other week cultures (1 x 105 cells/ml) were cleansed of spontaneous HGPRT- cells by a 24-h incubation in growth medium containing 4.5 /~g/ml methotrexate (Sigma), 0.7 /Lg/ml thymidine (Gibco), 4/~g/ml hypoxanthine (Gibco) and 7.5 ~g/ml glycine (THMG). Following incubation, cultures were washed twice to remove residual methotrexate, adjusted to 5 x 104 cells/ml and

101 then incubated for 72 h in similar medium without methotrexate (THG). H G P R T mutation assay Suspension cultures were adjusted to 3 x 105 cells/ml the day prior to testing tO yield approximately 1 x 106 cells/rnl in 24 h. On the day of chemical treatment (Day 0), 6 x 106 cells were suspended in 10 ml H3P (Hx0P with FBS reduced to 3%) in 50-ml polypropylene centrifuge tubes (Coming No: 25330). Test chemicals were prepared (100 x ) in DMSO or water, and 0.1 ml of the stock solution was added to the 10-ml culture. Cultures were incubated at 37 ° for 5 h on a roller drum (New Brunswick Scientific, Model TC-8) revolving at 40 rpm. The chemical treatment was terminated by washing the cells twice with H10P by centrifugation (10 min at 1400 rpm, D a m o n / IEC Model CRU-5000) and retained in the 50-ml polypropylene centrifuge tubes. The cells were resuspended in 20 ml HxoP thus reducing the cell density to 3 x 105 cells/ml to begin the expression period. Cell counts were performed at least every other day throughout the expression period. To maintain log phase growth during this period, cultures were readjusted to 4 x 105 cells/ml daily or 1.5 x 105 cells/ml every other day. Cultures maintained over a weekend were adjusted to 5 x 104 cells/ml on Friday to yield 1.0-1.5 x 106 cells/ml on Monday. After an expression period of 7 days, each culture was counted and then adjusted with H10P to 0.5 x 106 cells/ml in polystyrene tubes (Coming No. ~s~00) and these were identified as the selective ("S") cultures. Cultures which did not reach at least 0.5 x 106 cells/ml were concentrated by centrifugation. Cultures were vortexed and a 1.0-ml aliquot of each "S" culture was serially diluted using H10P through three 1:10 dilutions and one 1:5 dilution to render 100 cells/ml. These diluted cultures were used to determine cloning efficiency in non-selective media and were identified as the non-selective ("NS") cultures. 1 ml of each " N S " culture (100 cells) were added to each of three 100-mm non-tissue culture dishes (Optilux Petri Dish, Falcon No. 1001) followed immediately by the addition of 25 ml of non-selective cloning media (Hlo adjusted to 20% FBS, 100 /xg/ml sodium pyruvate and con-

taining 0.4% molten nobel agar, 65°C). For the selection of H G PRT mutants, 2 ml of each "S" culture (1 x 106 cells) were added to each of three 100-mm non-tissue culture dishes (Falcon No. 1001) followed immediately by the addition of 25 ml selective cloning medium (nonselective cloning medium containing 40 ~tM 6TG). Following the addition of the appropriate cloning medium, the plates were gently swirled to ensure even cell dispersal and were allowed to gel at room temperature for 15 min after which they were incubated at 37 °C undisturbed for 14 days in a humidified 95% : 5%/air : CO 2 environment. Colonies were counted using a Biotran II Automated Colony Counter (Model Cl11, New Brunswick Scientific, Farmingdale, NY). Cloning of all of the "N S" cultures was always performed first in order to optimize cloning efficiency (CE). Mutant frequency (MF), determined from the "S" clones, was defined as the number of 6TG-resistant colonies (6TG r) per 106 colony-forming cells (i.e. [6TG r colonies/CE]@106). Relative total growth (RTG) values were calculated for each culture based both on the suspension growth over the 7-day expression period and on the cloning efficiency relative to the controls (RCE). An explanation for the calculations of RTG is provided in Appendix A. Assessment of metabofic cooperation A reconstruction experiment was performed in order to determine if the growth of 6TG r cells in soft agar was inhibited through metabolic cooperation by wild-type CHO cells. A 6TG r cell line was obtained by 6TG selection of CHO cells exposed to EMS. This 6TG r cell line was confirmed as a H G P R T - mutant by resistance to 6TG, sensitivity to MTX and inability to incorporate [3H]hypoxanthine into DNA. For the reconstruction experiment, 200 6TG ~ cells and varying densities of the wild-type CHO cells (0-1 × 106) were mixed and clones were selected using 100-ram non-tissue culture plates in selective cloning medium as described above. After incubation for 14 days the 6TG r colonies were counted. Characterization of 6TG r clones Clones of 6TG r cells were characterized as H G P R T - mutants on the basis of heritable resis-

102 tance to 6TG, sensitivity to MTX and inability to incorporate [3 H]hypoxanthine into an acid-insoluble product. 6TG r clones were isolated from selective cloning medium with sterile Pasteur pipets, dispersed into 5 ml H10P in polystyrene tubes (Coming No. 25200) and incubated at 37 ° on a roller drum as described above. When at least 1 x 105 cells/ml were obtained, then 100 cells from each culture were replated in 100-mm nontissue culture plates in: (1) non-selective cloning medium to measure CE, (2) selective cloning medium to measure 6TG resistance and (3) T H M G cloning medium (non-selective cloning medium containing T H M G to measure MTX sensitivity. The plates were incubated for 14 days and the colonies were counted. H y p o x a n t h i n e - g u a n i n e phosphoribosyltransferase (HGPRTase) activity in C H O cells was measured in monolayer cultures by the incorporation of [3H]hypoxanthine into a trichloroacetic acid (TCA) insoluble product. Monolayer cultures of 6TG r and wild-type C H O cells were prepared by plating 5 x 105 cells in 100-mm tissue dishes (Coming No. 3100) in H~0. After 48 h the cultures were examined microscopically for confluency, and the m e d i u m was then replaced with hypoxanthine-free H 5 medium (H10 but substituting 5% dialyzed FBS) with 0.5 ~ C i / m l [3H]hypoxanthine (10 Ci/mmole). After incubation for 4 h the medium was removed and the cells were washed 5 times with 20 ml Isoton II (Coulter, Houston, TX). The cells were dissolved in 1.5 ml of 0.1% SDS, and 1.0 ml of each SDS lysate was placed into 13 x 100 mm glass tubes followed by the addition of 0.1 ml of 50% TCA. The tubes were maintained at 0 ° C for 30 min after which the precipitate was collected on 2.3-cm DE81 filter paper discs (Whatman) by vacuum filtration. Acid-soluble radioactivity was removed from the discs by successive washes at 4 ° C with 10% TCA, 5% TCA, 70% ethanol and 95% ethanol. The filters were dried and the radioactivity was determined in 10 ml of Ready-Solv ® MP cocktail (Beckman) using a Beckman LS9000 scintillation counter.

Results

6TG concentration for selection in soft agar Initial soft-agar cloning studies were conducted

using 10 /~M 6TG as reported elsewhere (Li and Simizu, 1983, Nishi et al., 1984); however, this 6TG concentration resulted in the appearance of microcolonies in the agar medium (Fig. 1A) which were thought to be clones of wild-type cells that had escaped 6TG selection. Consequently, concentrations of 10, 20 and 40 /*M 6TG were evaluated for effects on CE and mutant selection using control and EMS-treated cultures. Control cultures showed a slight decrease in spontaneous M F as the 6TG concentration increased, whereas the recovery of EMS-induced 6TG r colonies was slightly increased with the higher concentrations of 6TG (Table 1). More importantly, microcolonies present in 10 /tM 6TG medium were less evident in 20/*M 6TG medium and were absent in 40 ffM medium (Fig. 1B). As a result of these findings, 40 ffM 6TG was chosen for the selection of 6TG r clones.

Serum requirements for cloning in soft agar The use of dialyzed serum has been reported to be an essential requirement for the selection of C H O 6TG r clones (Hsie et al., 1979b; O'Neill et al., 1977). Therefore, effects of serum dialysis and serum concentration on CE and spontaneous MF in soft agar were evaluated using C H O wild-type cells. The use of non-dialyzed serum in soft-agar cloning medium resulted in lower spontaneous M F and slightly higher CE when compared to

TABLE 1 THE EFFECT OF 6-THIOGUANINE CONCENTRATION ON SELECTION OF 6TG ~ MUTANTS IN SOFT-AGAR MEDIUM a 6-TG concentration (/,M)

Colony counts Solvent control b

EMS-treatedc

10

10.3 + 3.8 (n = 8)

148 + 16 ( n = 8)

20

8.6_+3.7 (n = 8)

164+35 (n = 6)

40

8.4_+3.2 (n = 8)

2 0 7 + 4 3(n = 8)

Experiments performed over 7-month period. b Spontaneous colonies from 1 X 1 0 6 cells; values are mean+ S.D. from at least 6 cultures. c Induced colonies from a culture treated with 400 /tg/ml ethyl methanesulfonate.

103

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~

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Fig. 1. (A) The presence of microcolonies with a 10-~M concentration of 6TG in the selective cloning medium. (B) The absence of microcolonies with a 40-~M concentration of 6TG in the selective cloning medium,

104 TABLE 2 EFFECT OF SERUM DIALYSIS ON SPONTANEOUS MUTANT FREQUENCY AND CLONING EFFICIENCY OF CHO CELLS IN SOFT-AGAR M E D I U M Serum type

Serum Number concen- of tests tration (%)

Mutant Cloning frequency ~ efficiency ~'

Non-dialyzed ~

5 10 20

6 6 16

4.5 _+ 1.8 5.2_+2.3 6.6+3.2

89 + 11 84+15 91_+18

Dialyzed ~'

5 10 20

8 9 ll

12.8 + 1.0 8.6 :~ 4.6 8.5+5.8

67 -+ 24 79+21 84-+12

Mutants/106 colony-forming cells+ S:I). h Absolute cloning efficiency. • Serum purchased from Gibco, 3 lots of each type were evaluated.

ever, recovery of 6TG r clones was consistently higher with non-dialyzed serum, especially from cultures treated with 400 ~ g / m l EMS (Fig. 2). It is important to note that 6TG r clones were considerably larger in size when non-dialyzed serum was used in the cloning medium. As a result of these findings, non-dialyzed serum was chosen for all subsequent cloning experiments. The optimal serum concentration for mutant selection was determined by treating cultures with EMS followed by selection of 6TG r clones in soft agar containing 5, 10 or 20% non-dialyzed serum. Cloning efficiency was unaffected by increasing the serum concentration to 20% (Fig. 3a); however, the use of 20% serum resulted in the recovery of a larger number of 6TG r clones especially at the higher EMS treatment levels (Fig. 3b).

medium containing dialyzed serum (Table 2). Increasing the non-dialyzed serum concentration from 5 to 20% had no appreciable effect on CE or MF. Effects of serum dialysis on recovery of mutagen-induced 6TG ~ colonies were evaluated by treating cultures with EMS followed by mutant selection in soft agar containing either 20% dialyzed or 20% non-dialyzed serum. Cloning efficiency was unaffected by serum dialysis; how-

~ E

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Fig. 2. The effects of 20% dialyzed fetal calf serum ( I ) versus 20% non-dialyzed fetal calf serum (O) on mutant frequency (solid lines) and % cloning efficiency (broken lines) from EMS-treated cultures cloned in soft-agar medium. Mutant frequency is expressed as mutants per 106 survivors. Percent cloning efficiency is relative to solvent controls.

2bo

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#g/ml Fig. 3. (A) The effect of 5% (O), I0% (4) and 20% (m) non-dialyzed fetal calf serum on the cloning efficiency of EMS-treated CHO cells cloned in soft-agar medium. (B) The effect of 5% (O), 10% (*), and 20% ( I ) non-dialyzed fetal calf serum on the mutant frequency (mutants/106 survivors) of EMS-treated CHO cells cloned in soft-agar medium (mean values for 3 tests).

105

Expression period

TABLE 3

I n order determine the expression period that provided optimal recovery of 6 T G ~ clones as well as a low spontaneous MF, C H O cultures were treated with E M S and were then maintained in suspension cultures for either 7, 8 or 9 days after which 6 T G ~ clones were selected in soft agar medium. Increasing the expression period from 7 to 9 days did not affect C E (Fig. 4a) or M F (Fig.

RECONSTRUCTION EXPERIMENT TO ASSESS THE EFFECT OF METABOLIC COOPERATION ON RECOVERY OF 6TGr MUTANTS IN SOFT-AGAR MEDIUM

4b). Assessment of metabolic cooperation Metabolic cooperation was assessed in a reconstruction experiment designed to determine the effect of cell density on selection of 6 T G r clones in soft-agar medium. A clonal population of 6 T G ~ cells was isolated after treatment with E M S and 200 of these cells were mixed with increasing numbers of wild-type cells and plated in selective

Cells per plate Wild-type 6TG r cells cells

Clones observeda

Relative cloning efficiency (%)

0 1 × 105 2.5 × 105 5 × 10 s 1N106

159 + 13 143 _+10 159+_15 153 + 14 178+ 8

100 90 100 96 112

200 200 200 200 200

a Selective media with 40/~M 6TG; values are mean + S.D. of 4 plates.

soft-agar medium. Recovery of 6 T G ~ clones was not affected by the presence of up to 1 × 106 wild-type cells in the cloning m e d i u m (Table 3).

Characterization of 6TG" clones

/00t

=/ ,~:.m-----o... "-.::~. ........... ,-.,,

80

Z'

z:..

21 spontaneous and 70 chemical-induced 6 T G r clones were harvested from soft-agar selective m e d i u m and grown in suspension culture. Each of these cultures showed continued resistance to 6 T G when re-cloned in soft-agar selection m e d i u m whereas cloning of these cultures in m e d i u m containing 4.5 # g / m l M T X resulted in complete cell kill. M e a n H G P R T a s e activity measured in 22 spontaneous and 62 chemical-induced 6 T G r clones was 0.1% + 0.02 and 0.25% + 0.3, respectively, of the activity present in wild-type cells. Results for 6 T G resistance, M T X sensitivity and H G P R T a s e activity for 20 representative 6 T G r clones are shown in Table 4.

",'..

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Chemical-induced mutation

I00O" .

,

.

, 400

tzg/m/ Fig. 4. (A) The

effect

of expression

.

, 600

time

• ~0

on cloning

efficiency

of EMS-treated CHO cells cloned in soft-agar medium, day 7 (I), day 8 (A), day 9 (e). (B) The effect of expression time on mutant frequency (mutants/106 survivors) of EMS-treated CHO cells cloned in soft-agar medium, day 7 (m), day 8 (A), day 9 (o) (mean values for 3 tests).

The mutagens M N N G , MMS, M N U and 4 N Q O were each tested for the induction of m u t a tion at the H G P R T locus in suspension cultures of C H O cells followed by selection of 6 T G r mutants in soft-agar medium. In addition, NaC1 was also tested to determine if severe toxicity or high osmolality affected m u t a n t frequency in these cells. A dose-related positive response for the induction of 6 T G r mutants occurred following treatment with M N N G , MMS, M N U and 4 N Q O (Table 5). Each of these positive responses was clearly

106 TABLE 4 6 - T H I O G U A N I N E RESISTANCE, METHOTREXATE SENSITIVITY A N D HGPRTase ACTIVITY A M O N G SPONTANEOUS A N D M U T A G E N - I N D U C E D 6TG r CLONES a Culture type

Cells plated

Cloning efficiency (%) b

6TG medium; colonies observed

MTX medium; colonies observed

HGPRTase activity ( d p m / 5 × l0 s cells)

HGPRTase activity (% control)

Spontaneous 6TG r

1 2 3 4 5 6 7 8 9 10

100 100 100 100 100 100 100 100 100 100

123 114 162 126 123 120 108 77 85 162

146 114 153 109 123 118 91 92 96 153

0 0 0 0 0 0 0 0 0 0

149 116 144 169 188 154 198 106 173 144

0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.2 0.1

0.1 1.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.4

Chemical-induced " 6TG r

1 (MMS) 2 (4NQO) 3 (4NQO) 4 (4NQO) 5 (EMS) 6 (4NQO) 7 (EMS) 8 (EMS) 9 (MMS) 10 (EMS)

100 100 100 100 100 100 100 100 100 100

99 90 93 131 68 141 60 115 88 115

92 94 109 144 86 138 72 133 91 132

0 0 0 0 0 0 0 0 0 0

127 1764 130 101 119 164 120 100 92 422

Wild-Type Wild-Type

100 100

93 98

0 0

96 113

113 118 73 671

a The results shown include the extreme ranges of values determined from analysis of 21 spontaneous and 70 chemical-induced 6TG r clones. b Absolute cloning efficiency. c MMS, methyl methanesulfonate; 4NQO, 4-nitroquinoline-l-oxide; EMS, ethyl methanesulfonate.

manifested by test chemical concentrations of less than 30 /~g/ml. NaC1 concentrations as high as 15000 /~g/ml resulted in severe cytotoxicity but did not produce a dose-related induction of 6TGr mutants (Table 5).

Discussion This manuscript describes modifications to the C H O / H G P R T assay which reduce the labor intensiveness of the test and thus make the assay more attractive as a screening tool for mammalian cell mutagens. The chemical treatment of C H O cells adapted to grow in suspension culture, the selection of 6TG r mutants in soft-agar medium

and the use of non-dialyzed serum for mutant cloning are the key elements which facilitate the conduct of the C H O / H G P R T assay. Compared to the monolayer procedure (O'Neill et al., 1977; Hsie et al., 1979a), the use of suspension cultures of CHO cells described here permits the treatment of 6 × 10 6 cells in a single culture as compared to 1 x 106 cells/plate in monolayer culture. The use of suspension cultures eliminates the need for trypsinization during the expression period and avoids the additional stress of trypsinization to cells already debilitated by cytotoxic concentrations of the test article. Consequently, it is expected that the suspension procedure would favor the growth of these cells and ultimately

107 TABLE 5 CHEMICAL-INDUCED MUTATION AT THE HGPRT LOCUS IN SUSPENSION CULTURES OF CHO CELLS Chemical treatment

MNNG h

MMS

4NQO

MNU

NaC1

Concentrations tested (~g/ml) 0.5 0.25 0.1 0.05 0.01 0 30 20 10 7.5 5 0 0.6 0.5 0.4 0.3 0.2 0.1 0.05 0 20 16 12 8 4 2 1 0.5 0.1 0 15,000 14,000 13,000 12,000 11,000 10,000 5,000 1,000 0

Relative suspension growth (%)

Relative cloning efficiency (%)

Relative total growth (%)

Mutant frequency ( × 10 6)

Mutation ~ index

<1 1 3 5 42 100

27 31 62 47 56 100

<1 <1 2 2 24 100

800.0 621.4 187.5 93.0 54.9 6.6

121.2 94.2 28.4 14.1 8.3 1.0

4 29 62 102 126 100

81 102 74 89 79 100

3 30 46 91 100 100

25.2 18.8 12.4 11.9 8.0 4.6

5.5 4.1 2.7 2.6 1.7 1.0

<1 2 3 15 25 52 54 100

107 107 97 104 93 109 114 100

<1 2 3 16 23 57 62 100

140.8 155.2 158.5 129.0 130.9 56.5 30.6 11.1

12.7 14.0 14.3 11.6 11.7 5.1 2.8 1.0

4 6 9 11 45 75 105 77 74 100

56 54 59 98 97 97 87 128 114 100

2 3 5 10 44 73 91 99 84 100

90.0 53.2 68.6 30.0 33.9 16.2 15.6 11.1 10.4 4.5

20.1 11.8 15.2 6.7 7.5 3.6 3.5 2.5 2.3 1.0

2 6 16 24 38 57 60 65 100

77 71 63 89 86 57 92 70 100

2 4 10 21 33 32 55 46 100

16.1 8.6 11.1 15.8 3.1 6.2 6.7 11.4 6.1

2.6 1.4 1.8 2.6 0.5 1.0 1.1 1.9 1.0

a Mutation Index, MF of treated culture/MF of control. b A prolonged and severe cytotoxic effect resulting from treatment with MNNG necessitated an 8-day expression period.

would provide a more representative population o f cells f o r t h e s e l e c t i o n o f m u t a n t s . T h e s u s p e n sion procedure

a l s o a l l o w s t h e k i n e t i c s o f cell

growth

to b e m o n i t o r e d

during

the expression

period. By c o u p l i n g d a t a for g r o w t h in s u s p e n s i o n culture with values for C E at the time of m u t a n t

108 selection, an overall measure of toxicity expressed as relative total growth, can be ascertained. In contrast, an assessment of toxicity in the monolayer assay is limited to the measurement of CE immediately following chemical treatment (Hsie, 1980) which does not take into account either delayed toxicity or cell recovery that may occur during the expression period. A shortcoming of the C H O or V 7 9 / H G P R T assay (O'Neill et al., 1977; Nishi et al., 1984) has been the phenomenon of metabolic cooperation (Van Zeeland et al., 1972) whereby the number of observed 6 T G r mutants is decreased as the number of co-cultivated wild-type cells is increased (Chu and Malting, 1968; Myhr and Di Paolo, 1975). In order to avoid metabolic cooperation and achieve an accurate determination of induced mutation, only 2 × 105 cells can be plated per selection plate when 6 T G r mutants are cloned using the monolayer procedure (O'Neill et al., 1977). The soft-agar procedure described here for the cloning of 6 T G r mutants is an adaptation of the cloning procedure used in the L5178Y T K assay (Oberly et al., 1984) and allows up to 1 × 10 6 cells to be plated in a single selection plate without risk of metabolic cooperation. The results of this procedure are consistent with those of Li and Shimizu (1983) and Nishi et al. (1984) who have formerly shown that the soft-agar cloning techniques avoid metabolic cooperation during the selection of 6 T G r mutants of C H O and V79 cells, respectively. Since the soft agar selection method allows the plating of up to 1 × 10 6 cells in a single dish, this should facilitate the identification of chemicals that produce low mutant yield. In addition, the use of soft agar permits the quantification of mutant colonies using automatic colony counting and precludes the need for fixation, staining and hand counting of colonies which is normally done when mutants are scored using monolayer cultures (O'Neill et al., 1977). In the monolayer cloning procedure, the use of dialyzed serum has been reported to be essential for the effective selection of 6 T G r mutants (Hsie et al., 1979b). It is thought that serum dialysis reduces the concentration of hypoxanthine present in serum which otherwise would compete with 6 T G for cellular uptake and metabolism and, therefore, lessen the effectiveness of 6 T G for

mutant selection. The results presented here show that non-dialyzed serum can be used for the selection of mutants in soft-agar medium provided that the concentration of 6 T G is increased from 10/~M to 40/~M. Presumably the increased concentration of 6 T G effectively competes against hypoxanthine that may be present in non-dialyzed serum. The use of non-dialyzed serum resulted in improved CE and lower spontaneous M F relative to dialyzed serum (Table 2). It is reasonable to expect that non-dialyzed serum would be more effective for cloning since dialysis is known to lessen the concentration of serum components essential for optimal cell growth (Patterson and Maxwell, 1973). 5 compounds were tested using the suspension c u l t u r e / s o f t - a g a r cloning modification C H O / H G P R T mutation assay to determine if these procedures permitted the adequate detection of mutagens together with an accurate assessment of compound toxicity. Dose-related decreases in values for relative suspension growth were evident following treatment with M N N G , MMS, 4NQO, M N U and NaC1 (Table 5), and were considered to be a manifestation of the acute cytotoxic effects of these treatments. A dose-related decrease in values for CE was noted with M N N G and was considered to be an expression of persistent toxic effects of these treatments. The outcome of the mutagenicity testing showed M N N G to induce the highest MF; however, this was due in part to low values for CE and was associated with extremely low values for RTG. On the other hand, values for M F in MMS-, 4NQOand MNU-treated cultures were not appreciably affected by CE, although dose-related decreases in R T G were evident. Concentrations of NaCl as high as 15000 /~g/ml produced an acute toxic effect, and values for R T G were comparable to those noted in cultures treated with M N N G , MMS, 4 N Q O or M N U . Although a dose-related increase in M F was not evident following NaC1 treatment, several cultures showed values for MF that were 2-fold greater than the control. These anomalous findings for NaC1 in C H O cells may be similar to those noted in the L5178Y mutation assay (Brian Myhr, personal communication) and the C H O chromosome aberration assay (Galloway et al., 1985) where high concentrations of NaC1 induced L5178Y T K mutants and chromosome

109 aberrations, respectively. The findings for NaC1 suggest that the outcome of the C H O / H G P R T assay may be affected by severe perturbation of osmolality; therefore, results obtained with extremely high concentrations of a test article should be interpreted cautiously. An expression period of 7 days was used in testing the 5 compounds mentioned above. This period was chosen as a result of the findings with EMS (Figs. 4A and 4b), and also was consistent with the expression period reported by Hsie et al. (1979a). However, results for toxicity obtained from testing M N N G in the CHO assay (Table 5) indicated that an expression period of 7 days was insufficient. More recent evidence (Brian Myhr, personal communication) indicates that for a majority of compounds a 9-day expression period should be used following suspension treatment of CHO cells. For this reason, a 9-day expression period will be used in future studies. An attempt was made to compare results for the compounds tested in this report with other published results of the monolayer assay. Most references to the monolayer assay describe tests conducted using a 16-h treatment a n d / o r 9-days expression period. Since this differs considerably from the 5-h treatment and 7-day expression used in this report, a direct comparison between the two methods could not be made. However, in the recent publication by Li and Shimizu (1983) a comparison was made between the monolayer and soft-agar protocols in which a 3-hour treatment and 7-day expression period was used for both methods. Since the time factors were nearly compatible to those used in this report, a comparison of results was made and is shown in Fig. 5. Based on this limited comparison, it appears that the sensitivity is compatible between the 3 protocols. Further test validation with additional compounds should confirm this observation. The collective findings reported here show that the application of techniques for suspension culture and soft-agar cloning with CHO cells are attractive alternatives to the conventional monolayer procedure for the C H O / H G P R T assay. Although only 5 chemicals were tested, the results support the conclusion that the modified procedure was sensitive for the detection of direct-acting mutagens and that the 6TG r mutants observed

°t 250

150

,~o

~

35o

~o

Fig. 5. Mutagenicityof EMS (50-400 #g/ml) in CHO cells as determined by the suspension treatment/soft-agar procedure (D), the soft-agarprocedure (zx)(Li and Shimizu,1983) and the original monolayerprocedure (O) (Li and Shimizu, 1983).

displayed phenotypic characteristics consistent with forward mutation at the HGPRT locus. These modifications have also contributed to an overall improvement in the cost effectiveness for the assay since (1) trypsinization has been eliminated; (2) serum dialysis is not required; (3) non-tissue culture plates are used in cloning; (4) only one CE assay is required; and (5) it is not necessary to fix and stain colonies, since enumeration is possible with an automated colony counter. Additional studies with the CHO suspension culture H G P R T assay are currently underway to evaluate conditions for metabolic activation and to further confirm the sensitivity of the system.

Appendix A Calculation of growth index and relative total growth Examples 1 and 2 below show values for suspension-culture densities throughout the expression period for a hypothetical control and chemical-treated culture, respectively. On day 0, at the termination of chemical treatment, each culture is adjusted to 3 × 10 5 cells/ml to begin the 7-day expression period; therefore, the "adjusted cell density" is equivalent to the "initial density" for the ensuing 24-h period. Throughout the expression period downward adjustments to cell densities were made only when final cell densities exceeded 4 × 10 5 cells/ml (excepting day 3 when cultures were adjusted to 5 × 10 4 cells/ml to grow over the weekend).

110 DETERMINATION OF GROWTH INDEX Example No. 1." Control Culture

Calender day Experiment day Initial density a Final density ~ Adjusted density c Growth index (GI)

Tuesday Day 0

Wednesday Day 1

Thursday Day 2

Friday Day 3

Saturday Day 4

0.3 -

0.3 0.9 0.4 3

0.4 1.2 0.4 3

0.4 1.2 0.05 3

No counts taken; therefore, no adjust-; ments made

Sunday Day 5

Monday Day 6

Tuesday Day 7

0.05 1.2 0.4 24

0.4 1.2

Monday Day 6

Tuesday Day 7

0.05 t.2 0.4 24

0.4 1.2 3

3

Example No. 2." Treated-Culture

Calendar day Experiment day

Tuesday Day 0

Wednesday Day 1

Thursday Day 2

Friday Day 3

Saturday Day 4

Initial density a Final density b Adjusted density c Growth index (GI)

_

0.3 0.2 0.2 0.67

0.2 0.8 0.4 4.0

0.4 1.2 0.05 3.0

No counts taken; therefore no adjustments made

_

0.3 -

E x a m p l e 1 s h o w s t h a t a f t e r t h e first 24-h p e r i o d o f e x p r e s s i o n ( e n d o f d a y 1) t h e c o n t r o l c u l t u r e has g r o w n to a d e n s i t y o f 9 x 10 5 c e l l s / m l ; t h e r e fore, t h e d e n s i t y o f this c u l t u r e is a d j u s t e d d o w n w a r d to 4 x 10 s c e l l s / m l to b e g i n t h e n e x t 24 h o f e x p r e s s i o n ( b e g i n n i n g o f d a y 2). T h i s p r o c e s s is repeated throughout the expression period; however, o n d a y 3 the c u l t u r e was a d j u s t e d to o n l y 5 x 10 4 c e l l s / m l in o r d e r to m a i n t a i n e x p o n e n t i a l g r o w t h o v e r the w e e k e n d ( d a y s 4 a n d 5). F o l l o w i n g the cell c o u n t o n d a y 7 the a p p r o p r i a t e n u m b e r o f cells are c l o n e d in soft a g a r to d e t e r m i n e CE and MF.

Sunday Day 5

F o r the c h e m i c a l - t r e a t e d c u l t u r e ( E x a m p l e 2), the final d e n s i t y at the e n d o f d a y 1 was 2 x 10 5 c e l l s / m l . T h i s v a l u e was l o w e r t h a n the initial cell d e n s i t y d u e to c y t o t o x i c i t y f r o m c h e m i c a l treatm e n t o n d a y 0. C o n s e q u e n t l y , n o a d j u s t m e n t was m a d e to cell d e n s i t y ; thus, the initial cell d e n s i t y to b e g i n the d a y - 2 e x p r e s s i o n p e r i o d was 2 x l 0 s c e l l s / m l . By the e n d o f d a y 2 the t r e a t e d c u l t u r e s h o w e d g r o w t h c h a r a c t e r i s t i c s s i m i l a r to the c o n trol a n d f u r t h e r a d j u s t m e n t s to cell d e n s i t i e s w e r e as d e s c r i b e d a b o v e for the c o n t r o l culture.

DETERMINATION OF RELATIVE TOTAL GROWTH

Cumulative GI (CGI) Suspension growth (%) Absolute cloning efficiency (%) d Relative cloning efficiency (%) ¢ Relative total growth (%)

Example No. 1

Example No. 2

1944 100% 80% 100% 100%

579 30% 60% 75% 23%

All cell densities are Xl06/ml b Final density is density of culture after 24 h of growth. Cell density to begin the next period of expression (never greater than 4 x l0 s cells/ml). d Absolute cloning efficiency: number of colonies on NS plates ~ m b e r o.f cells plated OlOnaes ooserveo Example No. 1 negative control = = 80% 100 cells olated 60 colonies observed Example No. 2 treated control = = 60% 100 cells plated Relative cloning efficiency: Treated cultures = absolute cloning efficiency of treated cultures x 100 absolute cloning efficiency of negative control Example: treated/control 60%/80% = 75%.

111 Growth index (GI) was calculated each time a cell c o u n t w a s p e r f o r m e d b y d i v i d i n g t h e final cell d e n s i t y b y t h e initial cell d e n s i t y . T h e c u m u l a t i v e g r o w t h i n d e x ( C G I ) w a s d e t e r m i n e d b y the c o n s e c u t i v e m u l t i p l i c a t i o n o f e a c h s u c c e s s i v e G I value. R e l a t i v e s u s p e n s i o n g r o w t h ( e x p r e s s e d as a p e r c e n t ) was c a l c u l a t e d b y d i v i d i n g the C G I o f the t r e a t e d c u l t u r e b y the C G I of t h e s o l v e n t c o n t r o l . R e l a t i v e t o t a l g r o w t h was c a l c u l a t e d b y m u l t i p l y ing the relative suspension growth by the relative c l o n i n g efficiency.

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