Locus specificity of mutagenicity of 2,4-diaminotoluene in both L5178Y mouse lymphoma and AT3-2 Chinese hamster ovary cells

Mutation Research, 135 (1984) 115-123 Elsevier

115

MTR 00842

Locus specificity of mutagenicity of 2,4-diaminotoluene in both L5178Y mouse lymphoma and AT3-2 Chinese hamster ovary cells W.J. Coppinger

1,, S.A. B r e n n a n

1, J . H . C a r v e r 2** a n d E . D . T h o m p s o n

1

i Human and Environmental Safety Division, The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 39175, Cincinnati, Ohio," and 2 Biological Sciences Department, Battelle Columbus Laboratories, 505 King A venue, Columbus', Ohio 43201

~U.S.A.) (Received 24 October 1982) (Revision received 31 August 1983) (Accepted 27 September 1983)

Summary 2,4-Diaminotoluene, a hepatocarcinogenic aromatic amine, was tested for mutagenic potential at both the autosomal tk locus and the sex-linked hgprt locus of both L5178Y 3.7.2C mouse lymphoma cells and Chinese hamster ovary (CHO) AT3-2 cells. This compound was mutagenic in both cell types at the tk locus but not at the hgprt locus. Mutagenic activity was observed in L5178Y cells only in the absence of exogenous metabolic activation, but was observed in CHO-AT3-2 cells both with and without activation.

2,4-Diaminotoluene is a chemical of some industrial importance as an intermediate in the production of toluene diisocyanate, a polyurethane foam cross-linking agent. It is also used as an intermediate in dye production and until 1971 was a component of some oxidative hair dyes. Ito et al. (1969) have found that 2,4-DAT is a hepatocarcinogen when fed to rats. However, it was not carcinogenic in skin-painting studies in mice under conditions reported by Giles and Chung (1976). Numerous short-term tests for mutagenicity * To whom all correspondence should be addressed. ** Current address, Standard Oil Company of California, Environmental Health Center, Richmond, CA (U.S.A.). Abbreviations: 2AAF, 2-acetylaminofluorene; B(a)p, benzo[a]pyrene; BSA, bovine serum albumin; 2,4-DAT, 2,4-diaminotoluene; DMN, dimethylnitrosamine; EMS, ethyl methanesulfonate; FBS, fetal bovine serum; FUdR, 5-fluorodeoxyuridine; hgprt, hypoxanthine-guanine phosphoribosyl transferase; MEM, minimal essential medium; NADP, nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; TFT, trifluorothymidine; tk, thymidine kinase.

0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.

a n d / o r carcinogenicity have been performed with this material since the discovery of its carcinogenic properties. Ames et al. (1975), Pienta et al. (1977b), and Aune et al. (1979) have shown that it can induce reverse mutations in Salmonella typhimurium strains TA1538 and TA98 in the presence of Aroclor- or phenobarbital-induced rat liver $9 fraction. Pienta et al. (1977) have also shown that 2,4-DAT can induce morphological transformation of secondary cultures of Syrian hamster embryo cells. 2,4-DAT enhances the transformation of hamster embryo cells by Simian adenovirus 7 (Greene and Friedman, 1980). In addition, it is apparently capable of inducing unscheduled D N A synthesis in primary cultures of rat hepatocytes (Bermudez et al., 1979). However, Soares and Lock (1980) and Topham (1980) were unable to demonstrate an increase in sperm-head abnormalities in male mice treated with 2,4-DAT. 2,4-DAT was also negative for the induction of dominant lethal mutations in mice (Soares and Lock, 1980). Aune et al. (1979) were able to demonstrate irreversible

116

binding of 2,4-DAT or one of its metabolites to rat liver and kidney protein and rat liver ribosomal RNA. However, they were unable to demonstrate irreversible binding of 2,4-DAT to D N A either in vivo or in vitro. To our knowledge, there are no published reports on the mutagenicity of 2,4-DAT in cultured mammalian cells. Partially to fill this void and partially to compare the responses of different genetic loci in different standard screening tests to a known hepatocarcinogen, we have studied the mutagenicity of 2,4-DAT in cultured mouse lymphoma and Chinese hamster ovary cells. Both L5178Y 3.7.2C mouse cells (Clive and Spector, 1975, and Clive et al., 1979) and Chinese hamster ovary (CHO) AT3-2 cells (Adair et al., 1980, and Carver et al., 1980) have been used to detect mutagenic potential of chemicals at the hgprt and tk loci. Clive et al. (1979) have presented data for 13 chemicals which indicate that in mouse lymphoma cells the tk locus is more mutable than is the hgprt locus. Based on observed spontaneous mutation frequencies and responses induced by D M N , B(a)p, and EMS, Adair et al. (1980) and Carver et al. (1980) arrived at a similar conclusion regarding the tk locus in heterozygous CHO-AT3-2 cells. We have tested 2,4-diaminotoluene for the induction of mutations in both L5178Y 3.7.2C cells and CHO-AT3-2 cells. The assays for mutations were performed at both the hgprt and tk loci in both cell lines. We have found that 2,4-DAT is mutagenic in both cell types. However, the mutagenic properties observed seem to be limited to the tk locus. Moreover, a mutagenic response was observed in the mouse lymphoma cells only in the absence of rat liver $9 metabolic activation. Materials and methods

Cells L5178Y tk +/ 3.7.2C mouse lymphoma cells were obtained from Dr. Donald Clive, BurroughsWellcome Company, Research Triangle Park, NC. Cells were maintained as suspension cultures in Fischer's medium for leukemic cells of mice (Gibco) supplemented with 10% ( v / v ) horse serum (K.C. Biological), sodium pyruvate, penicillin streptomycin, and Pluronic F68 as described

by Clive et al. (1979). To reduce the incidence of spontaneous tk ,' mutants, cells were cleansed biweekly by growth in medium containing thymidine, hypoxanthine, methotrexate, and glycine (Clive et al., 1979). Cell stocks were maintained frozen in liquid nitrogen. Cultures were periodically screened for the presence of mycoplasma by growth in agar, staining with fluorescent antibody against M. hyorhinis, and fluorochrome staining of D N A (Bioassay Systems, Woburn, MA). Results were negative. The origins of the tk heterozygous CHO-AT3-2 cells used in these experiments has been described (Adair et al., 1980). Cultures were maintained in alpha minimal essential medium (MEM, K.C. Biological) suppelemented with 5% ( v / v ) fetal bovine serum (K.C. Biological) and 100 U / m l penicillin and 100 /*g/ml streptomycin. Cell stocks were maintained frozen in liquid nitrogen. Cultures used k,r preliminary cytotoxicity evaluations were maintained a maximum of 4 - 6 weeks. Cultures used in mutagenicity studies were initiated from frozen stocks 7 - 8 days before the beginning of each experiment. L5178Y tk 4 / 3. Z2C mutagenicitv assay L5178Y tk +/ 3.7.2C cells were treated with 2,4-DAT, DMSO (solvent control), or positive control chemical for 4 h as describing by Clive et al. (1979). A total of 3 x 10 6 cells were used per treatment group in 10 ml of Fischer's medium containing approximately 3% ( v / v ) horse serum of 3% horse serum and $9 fraction and cofactors. Following treatment, the cells were washed by two rounds of centrifugation, resuspended at 3 × l0 s cells/ml, and maintained as suspension cultures during the expression period prior to mutant selection. A 2-day expression period was used for selection at the tk locus. A 6-day expression period was used for selection at the hgprt locus. Cultures were counted daily during the expression period using a Particle Data Electrozone Celloscope. Dilutions were made, if necessary, in order to maintain cells in active growth. After expression, mutation induction was quantitated by plating 10 6 cells in soft agar medium containing 20% ( v / v ) horse serum, 1 ~ g / m l T F T (tk locus), or 5 / * g / m l 6-thioguanine (hgprt locus). Cell viability was determined by concurrently plat-

117

ing 200 cells in non-selective soft agar medium. After a 10-12-day growth period, colonies were counted using a New Brunswick Scientific Biotran II automatic colony counter.

CHO-A T3-2 mutagenicity assay Mutation experiments with CHO-AT3-2 cells were carried out essentially as described by Carver et al. (1980). Test cultures containing 106 cells were initiated in T25 flasks on the day before treatment. For treatment, the growth medium was replaced with alpha-MEM containing 2,4-DAT, DMSO (solvent control) or positive control chemical at the indicated concentrations. Medium from cultures to be treated in the presence of exogenous metabolic activation also contained $9 fraction and cofactors as discussed below. Following a 4-h exposure at 37.0°C, the cultures were washed twice with calcium- and magnesium-free PBS and detached with 0.25% trypsin. A portion of the cells in each culture were then diluted and plated for estimation of cell survival (six 60-mm plates each containing 200 cells). Another portion of the cells from each culture were carried for expression of mutation. 1-4 T75 flasks were seeded with at least 10 6 cells each for each treatment group. The number of flasks and the number of cells per flasks were varied according to expected toxicity. At least 106 viable cells were carried per treatment group. After 3 days of expression, the cells from each treatment group were detached with trypsin, and approximately 3 x 10 6 cells (3 x 10 4 cells/ml) were seeded into suspension culture for further expression at the hgprt locus. Another portion was used to initiate six 60-mm petri dish cultures containing 200 cells/dish for estimation of viability. The medium used was alpha MEM containing 2% FBS and 1% BSA (Reheis). An additional six 100-mm petri dish cultures containing 3 x 10 4 cells/dish were initiated in alpha MEM containing 2% FBS, 1% BSA, 20 /~g/ml deoxycytidine, 10 btg/ml uridine, and 2 / ~ g / m l F U d R for mutant selection at the tk locus. The suspension cultures initiated for mutation selection at the hgprt locus were maintained for 4 additional days of expression. On day 6 the cultures were diluted as necessary for continued exponential growth. On day 7, the cells were

centrifuged, resuspended at 5 x 10 ~ cells/ml and plated for quantitation of both viability and mutation induction. For viability estimation, six 60-mm petri dish cultures containing 200 cells/dish were initiated as discussed above. For mutation estimation, twelve 100-mm petri dish cultures containing 2 x 105 cells were initiated in alpha MEM containing 2% FBS, 1% BSA, and 4/~g/ml 6-thioguanine. All viability plates were incubated 7-9 days. Mutation induction plates were incubated undisturbed for 10-16 days. After the incubation period, cultures were fixed in methanol, stained with 0.05% crystal violet, and colonies containing at least 50 cells were counted manually using a stereomicroscope.

Preparation of rat fiver $9 fraction and cofactor mix For the L5178Y mouse lymphoma assay, rat liver $9 fraction was prepared as described by Clive et al. (1979). Male Sprague-Dawley rats (200-300 g, Charles River) were injected 5 days prior to sacrifice with a 2 : 1 mixture of Aroclors 1242:1254 in corn oil (500 mg Aroclor/kg body weight). This Aroclor mixture was used since the major data base for interpretation of results from this assay was developed using a similar procedure (Clive et al., 1979). $9 fraction was aseptically prepared in 0.25 M sucrose and stored at - 8 0 ° C for 6 months or less. The final concentration used in test cultures was 10% (v/v) $9 fraction in the presence of 2.4 m g / m l NADP and 4.5 m g / m l sodium isocitrate. For the CHO-AT3-2 assay, $9 fraction was prepared from male Sprague-Dawley rats weighing approximately 200 g as described by Carver et al. (1980). 5 days prior to sacrifice, rats were given an intraperitoneal injection of 200 m g / m l Aroclor 1254 in corn oil (500 mg/kg). $9 fraction was aseptically prepared in 0.15 M KC1 and stored less than 6 months at - 8 0 ° C . The final concentrations of $9 fraction and cofactors used in test cultures were 1.5 m g / m l $9 protein, 308 # g / m l NADPH, 660 /~g/ml NADH, 666 ffg/ml NADP, and 1.85 m g / m l glucose-6-phosphate. Chemicals 2,4-Diaminotoluene, dimethylnitrosamine and 2-acetylaminofluorene were purchased from the Aldrich Chemical Company, Milwaukee, WI. Ethyl

118 methanesulfonate

was

Chemical Company, obtained

obtained

from

the

Sigma

St. L o u i s , M O . A r o c l o r s w e r e

from Analabs,

Inc., New Haven, CT.

proposed

b y C l i v e et al. ( 1 9 7 9 ) .

considered

positive a chemical

i n c r e a s e in t h e m u t a t i o n

In order

should

frequency

to be

induce

an

at l e a s t t w i c e

t h a t o f t h e s o l v e n t c o n t r o l s at a cell s u r v i v a l r a t e a t l e a s t 10% o f t h a t o b s e r v e d in t h e s o l v e n t c o n t r o l s .

Results

In addition, Results of the L5178Y g e n i c i t y a s s a y s at b o t h

mouse lymphoma the

tk

hgprt

and

muta-

there should

loci are

According

tk

to t h e s e c r i t e r i a 2 , 4 - D A T

s h o w n in T a b l e 1 a n d 2, r e s p e c t i v e l y . W e i n t e r p r e t

tive at the

results from this assay in a manner

nous metabolic

similar to that

be some evidence of a

dose response. was posi-

l o c u s o n l y in t h e a b s e n c e o f e x o g e a c t i v a t i o n . I n t h e p r e s e n c e o f $9,

TABLE 1 RESULTS OF THE L5178Y (CLONE 3.7.2C) MOUSE LYMPHOMA M U T A G E N I C I T Y ASSAY OF 2,4-DIAMINOTOLUENE AT THE tk LOCUS Exposure concentration (/zg/ml)

Aroclor-induced rat liver $9 fraction ( + or - )

Relative suspension growth a (per cent)

Relative cloning efficiency (per cent)

Relative total growth (per cent)

Solvent control 1 c Solvent control 2 "

_ -

100 f 122

100 66

100 81

66 90

-

93 67 29 39

90 71 67 62

83 47 19 24

70 102 144 184

0.9 1.3 1.9 2.4

58.5 87.8 f 32.0 f 98.0

Mutation frequency per 106 cells

= 78

Relative increase b in mutation frequency

-

296.0 444.0 667.0 1 000.0

-

30 26 22 18

56 56 51 51

17 15 11 9

283 328 343 322

3.6 4.2 4.4 4.1

Positive control d

_

56

41

23

749

9.6

Solvent control 1 c Solvent control 2 c

+ +

100 110

100 g 79

100 87

73 67

58.5 87.8 132.0 198.0

+ + + +

85 71 52 36

99 91 77 78

84 64 40 28

65 94 _ h 109

0.9 1.3

296.0 444.0 667.0

+ + +

30 30 13

71 80 66

21 18 8

111 89 99

1.6 1.3 1.4

1 000.0

+

10

54

5

120

1.7

Positive control e

+

60

42

25

390

5.6

= 70

During 2-day expression period. b Relative increase = mutation frequency of sample/mean mutation frequency of solvent controls. " 1% dimethylsulfoxide. d 615 ~ g / m l ethyl methanesulfonate. 100/~g/ml dimethylnitrosamine. f Absolute cloning efficiencies: Solvent control 1 - 140%; Solvent control 2 95%. g Absolute cloning efficiencies: Solvent control 1 - 113%; Solvent control 2 = 95%. h Sample lost due to contamination.

1.6

119

TABLE 2 R E S U L T S O F T H E L 5 1 7 8 Y ( C L O N E 3.7.2C) M O U S E L Y M P H O M A A T T H E hgprt L O C U S

MUTAGENICITY

Exposure concentration ( ~ g/ml)

Aroclor-induced r a t liver $9 f r a c t i o n ( + or - )

Relative suspension growth a (per cent)

Relative cloning efficiency ( p e r cent)

Relative total growth ( p e r cent)

Solvent c o n t r o l 1 c Solvent c o n t r o l 2 c

_

100 g 124

100 97

100 120

250 250 400 400

-

63 67 60 67

104 96 79 78

500 500 640 640

-

47 49 49 51

800 800

-

ASSAY OF 2,4-DIAMINOTOLUENE

Mutation frequency p e r 106 cells

Relative i n c r e a s e b in m u t a t i o n f r e q u e n c y

11.0 19.1 ~ = 15.0

_

66 65 47 52

16.4 17.7 16.8 22.6

1.1 1.2 1.1 1.5

89 85 91 79

42 42 45 40

25.7 28.0 13.5 23.3

1.7 1.9 0.9 1.6

76 85 83 86

27 30 39 41

19.7 15.5 8.5 20.3

1.3 1.0 0.6 1.4

94 74

85 39

266.4 264.3

17.8 17.6

1 000

-

1 000

-

36 35 48 47

Positive c o n t r o l 1 d Positive c o n t r o l 2 d

_ _

90 53

Solvent c o n t r o l

+

100.0 h

100.0

100.0

10.0

-

250 400 400 500

+ + + +

57.8 30.4 37.3 26.7

121.5 86.5 91.0 106.0

70.2 26.3 33.9 28.3

6.6 15.0 13.2 7.6

0.7 1.5 1.3 0.8

500 640 640 800

+ + + +

27.2 27.3 21.9 21.2

95.0 101.0 99.5 89.0

25.9 22.1 27.2 17.6

8.4 15.8 5.0 14.6

0.8 1.6 0.5 1.5

800 1000 1000

+ + +

19.7 22.0 14.5

92.0 84.0 96.0

19.5 18.5 13.9

23.9 29.8 5.2

2.4 3.0 0.5

Positive c o n t r o l 1 c Positive c o n t r o l 2 c Positive c o n t r o l 3 f

+ + +

31.8 27,0 29.2

87.5 69.0 60

27.8 18.7 17.5

51.4 66.7 38.3

5.1 6.7 3.8

a b c d e f

During 6-day expression period. R e l a t i v e i n c r e a s e = m u t a t i o n f r e q u e n c y o f s a m p l e / m e a n m u t a t i o n f r e q u e n c y of solvent c o n t r o l s . 1% d i m e t h y l s u l f o × i d e . 615/~g/ml ethyl methanesulfonate. 100 ~ g / m l d i m e t h y l n i t r o s a m i n e . 50/~g/ml 2-acetylaminofluorene. A b s o l u t e c l o n i n g efficiencies: Solvent c o n t r o l 1 = 114%; Solvent c o n t r o l 2 = 110%. h A b s o l u t e c l o n i n g efficiency: 106%.

120 n o s i g n i f i c a n t i n c r e a s e in the m u t a t i o n f r e q u e n c y

concentrations

was

mutagenic

observed

DMN-positive

for

any

culture

control. A

other

repeat

than

assay

the

using a

failed

activity.

to

show

Toxicity

any levels

evidence of induced

by

d i f f e r e n t lot o f 2 , 4 - D A T g a v e r e s u l t s w h i c h w e r e

d u p l i c a t e e x p o s u r e s w e r e similar. P o s i t i v e c o n t r o l s , i n c l u d i n g a n o t h e r a r o m a t i c a m i n e , 2 A A F , all in-

qualitatively and quantitatively similar (data not

d u c e d s i g n i f i c a n t i n c r e a s e s in m u t a t i o n f r e q u e n c y .

shown).

Results of CHO-AT3-2

W h e n a s s a y e d f o r t h e i n d u c t i o n o f m u t a t i o n at

both the

m u t a g e n i c i t y a s s a y s at

tk a n d hgprt loci are s h o w n in T a b l e s 3

hgprt locus, 2 , 4 - D A T f a i l e d to i n d u c e a c o n -

a n d 4, r e s p e c t i v e l y . A g a i n , w e h a v e r e l i e d o n m e t h -

v i n c i n g i n c r e a s e in t h e o b s e r v e d m u t a t i o n f r e q u e n -

o d s for i n t e r p r e t a t i o n o f d a t a w h i c h w e r e d e v e l -

the

c y e i t h e r in t h e p r e s e n c e o r a b s e n c e o f r a t liver $9

o p e d d u r i n g v e l i d a t i o n o f this m u t a g e n i c i t y assay.

m e t a b o l i c a c t i v a t i o n . I n t h e p r e s e n c e o f $9, ex-

A c c o r d i n g to t h e c r i t e r i a u s e d b y C a r v e r et al.

posure

/~g/ml

(1980, 1983), a n i n d i v i d u a l r e s p o n s e w a s j u d g e d

i n d u c e d a p p a r e n t s i g n i f i c a n t i n c r e a s e s in m u t a t i o n

c o n c e n t r a t i o n s o f 800

and

1000

p o s i t i v e if t h e m e a n o f t h e r e s p o n s e - 1 S . E . M . o f

f r e q u e n c y . H o w e v e r , d u p l i c a t e s a m p l e s at t h e s e

the treated culture exceeded the m e a n plus 1 S.E.M.

TABLE 3 RESULTS OF THE CHlNESE HAMSTER OVARY AT3-2 CELL MUTAGEN1CITY ASSAY OF 2,4-DIAMINOTOLUENE AT THE tk LOCUS Exposure concentration (/Lg/ml)

Aroclor-induced rat liver $9 fraction (+ or - )

Mean survival after treatment ~ (per cent of mean solvent control plating efficiency)

Plating efficiency at time of mutant selection (per cent)

Mutation frequency per 10 3 cells b

Solvent control 1 c Solvent control 2 "

_ -

103 97

77 78

1.56 + 0.13 1.58 + 0.12

2000 3000 4000

-

60 51 50

72 74 69

1.77_+0.17 1.98_+0.14 f 2.03 +0.21 f

5000 6000

-

63 64

73 78

2.16+0.16 f 1.61 +0.19

850 ~tg/ml EMS d 1 050/xg/ml EMS

_ -

46 41

53 39

3.85 -+0.31 f 4.02_+0.30 f

Solvent control 1 c Solvent control 2 c

+ +

99 103

76 72

1.78 -+0.14 1.68 + 0.13

2 000 3 000 4000

+ + +

68 56 50

72 65 71

2.47 + 0.16 f 2.21 + 0.17 f 2.33 + 0.20 f

5000 6.000

+ +

51 48

62 70

2.34+0.26 f 2.23 _+0.17 f

500 ,ug/ml DMN e 1 500/~g/ml DMN

+ +

83 71

60 53

3.81 _+0.50 f 5.63-+0.37 f

" b c d e r

Absolute plating efficiencies of solvent controls ranged from 74 to 81%. Indicated limit of error is standard error of the mean or counting error whichever is greater. 1% dimethylsulfoxide. EMS = ethyl methanesulfonate. DMN = dimethylnitrosamine. Positive response.

x - 1.57 + 0.08

= 1.73 + 0.09

121 TABLE 4 RESULTS OF THE CHINESE HAMSTER OVARY AT3-2 CELL MUTAGENICITY ASSAY OF 2,4-DIAMINOTOLUENE AT THE hgprt LOCUS Exposure concentration (~g/ml)

Aroclor-induced rat liver $9 fraction ( + or - )

Mean survival after treatment a (per cent of mean solvent control plating efficiency)

Plating efficiency at time of mutant selection (per cent)

Solvent control 1 c Solvent control 2 c

_ _

97 103

73 68

1.88 + 0.33 2.55 + 0.41

1 000 3 000 5 000

-

88 67 64

80 78 68

1.49 + 0.27 0.83 + 0.21 1.29 + 0.27

7 000 9 000 10000

-

61 62 40

63 73 63

2.48 ___0.45 2.50 _+0.41 4.08 _+0.83

_

75

64

78.70 + 4.50

+ +

96 104

66 65

1.32 + 0.29 1.90 _+0.36

1 000 3 000 5 000

+ + +

100 75 59

70 66 73

2.15 _+0.37 2.89 + 0.47 f 1.72 + 0.32

7 000 9000 10000

+ + +

61 48 42

76 77 72

1.79 _+0.42 2.10+0.38 2.49 _+0.40

+

83

68

7.34+0.74

850/~g/ml EMS d Solvent control 1 c Solvent control 2 c

600 ~g/ml DMN e a b c d ¢ f

Mutation frequency per 105 cells b

= 2.22 _+0.34

f f

= 1.61 _+0.29

f

Absolute plating efficiencies of solvent controls ranged from 69 to 77%. Indicated limit of error is standard error of the mean or counting error whichever is greater. 1% dimethylsulfoxide. EMS = ethyl methanesulfonate. DMN = dimethylnitrosamine. Positive response.

o f t h e n e g a t i v e c o n t r o l s . T h e S . E . M . is d e f i n e d as the standard error of the plate counts or counting

o b t a i n e d i n m o u s e l y m p h o m a cells w i t h o u t $9. 2 , 4 - D A T could not be c o n s i d e r e d positive for

hgprt

e r r o r , w h i c h e v e r is g r e a t e r . F o r a n a s s a y t o b e

t h e i n d u c t i o n o f m u t a t i o n at t h e

considered positive, positive responses must

be

a b s e n c e o f $9 a c t i v a t i o n , a p o s i t i v e r e s p o n s e w a s

o b s e r v e d at t w o o r m o r e c o n s e c u t i v e d o s e p o i n t s .

o b t a i n e d i n c u l t u r e s e x p o s e d t o 10 000 # g / m l 2,4DAT. However, this was the only positive con-

Based on these criteria, 2 , 4 - D A T was positive for t h e i n d u c t i o n o f m u t a t i o n at t h e tk l o c u s o f C H O -

locus. I n t h e

A T 3 - 2 cells b o t h in t h e p r e s e n c e a n d a b s e n c e o f

c e n t r a t i o n i n t h i s series. H i g h e r e x p o s u r e c o n c e n t r a t i o n s were n o t feasible due to limited solu-

exogenous

con-

b i l i t y o f t h e test c h e m i c a l u n d e r e x p e r i m e n t a l c o n -

c e n t r a t i o n s n e c e s s a r y a n d level o f t h e r e s p o n s e s obtained were of the same order of magnitude for

d i t i o n s . I n t h e p r e s e n c e o f $9 f r a c t i o n , a s i n g l e p o s i t i v e r e s p o n s e w a s o b t a i n e d at 3000 ~ t g / m l .

b o t h c o n d i t i o n s . I n e a c h case, h o w e v e r , t h e d o s e r e q u i r e d w a s h i g h e r a n d r e l a t i v e m u t a g e n i c re-

H o w e v e r , t h e m u t a t i o n f r e q u e n c i e s o b t a i n e d at h i g h e r e x p o s u r e c o n c e n t r a t i o n s w e r e n o t signifi-

s p o n s e (ratio of o b s e r v e d m u t a t i o n f r e q u e n c y to mutation frequency of solvent control) lower than

cantly different from solvent controls.

metabolic

activation.

Exposure

122

Discussion

This study has revealed several interesting results regarding the mutagenic properties of 2,4D A T and the utility of these test systems to detect genotoxic aromatic amines. First, 2,4-DAT was mutagenic at the tk locus in both L5178Y mouse lymphoma and CHO cells. Moreover, in the mouse lymphoma assay, 2,4-DAT was positive only in the absence of rat liver $9 fraction. No mutagenic activity was observed when $9 fraction was present during treatment. This type of response is the opposite of what has been observed in the Ames/Salmonella assay (Ames et al., 1975; Pienta et al., 1977b; Aune et al., 1979). In this system 2,4-DAT was mutagenic only in the presence of $9. Our results imply that either 2,4D A T is mutagenic in mammalian cells without activation or that the hamster and mouse cells used have retained the capacity to metabolize this aromatic amine to a mutagenic form. 2,4-DAT also has induced transformation of secondary cultures of Syrian hamster embryo cells (Pienta et al., 1977b) and enhanced the transformation of primary cultures of hamster embryo cells by Simian adenovirus 7. Secondary and primary cultures of hamster embryo cells, however, are known to retain a high metabolic capacity (Pienta et al., 1977a). Evidence against direct genotoxic activity of 2,4-DAT is also found in the report that this compound does not detectably covalently bind to calf thymus D N A in vivo or in vitro (Aune et al., 1979). Non-covalent mutagenic interaction with D N A by intercalation does not seem likely due to the small size of this molecule. Our results also indicated that there might be potentially interesting differences in the rat liver $9 fractions prepared for these assays. In order to more easily compare our results with the validation studies of Clive et al. (1979) we prepared our $9 fraction for the mouse lymphoma assay in 0.25 M sucrose. In addition, the rats used were induced 5 days prior to sacrifice with an intraperitoneal injection of a 2 : 1 mixture of Aroclors 1242 : 1254. In order to compare our CHO-AT3-2 results with those of Carver et al. (1980), we prepared $9 fraction for this assay in 0.15 M KC1 from rats induced with Aroclor 1254 only. It appeared, then, that either the concentration of $9, cofactors, the

nature of the microsomal enzyme inducer, or the medium used has affected the balance of activation/detoxification in the two $9 fractions used. At first, we felt differences in the $9 solvent system may be the more likely source of variation. Based on measurements of cytochrome C oxidase activity, $9 fraction prepared in 0.25 M sucrose contains high levels of mitochondria relative to $9 prepared in 0.15 M KC1 (E.D. Thompson, unpublished data). Detoxification of 2,4-DAT by mitochondrial enzymes may, then, have been responsible for the lack of mutagenic activity in the mouse lymphoma assay. To address this possibility we repeated the mouse lymphoma assay with Aroclor 1254-induced $9 fraction prepared in 0.15 M KC1 as was done for the C H O assay. We again obtained results which were similar to those shown in Tables 1 and 2 for the $9 induced by the 2 : 1 Aroclor 1242:1254 mixture and prepared in 0.25 M sucrose (data not shown). At present, we have no evidence to conclusively indicate which of the remaining variables is responsible for the different responses observed in the C H O and mouse lymphoma cells. A third interesting result was that the mutagenicity of 2,4-DAT was restricted to the tk locus in both mouse lymphoma and CHO cells. There was some indication of mutagenic activity when 2,4D A T was tested at the hgprt locus in CHO-AT3-2 cells. However, as previously discussed, the results obtained could not be interpreted as a valid positive response. Locus-specific induction of mutation and the higher sensitivity of the tk locus in both L5178Y and CHO cells has been discussed previously by Clive et al. (1979) and Carver et al. (1980). Our results support the conclusion of these authors that the tk locus is, in general, a more sensitive indicator of mutagenic activity. However, our results differ from those of Clive et al. (1979) in that we obtained a positive response with 2AAF, another aromatic amine, when this compound was used as a positive control for mutation induction at the hgprt locus in the presence of the $9 fraction. In general, the L5178Y mouse lymphoma cells were more sensitive to both the toxic and mutagenic effects of 2,4-DAT. The positive response obtained at the tk locus (Table 1) was clearly related to dose of the test chemical and of an order

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of magnitude that was easy to confidently consider a positive response. In summary, our results confirm previously published reports regarding the in vitro genotoxic potential of 2,4-DAT. Moreover, for this compound, the autosomal tk locus is the more sensitive indicator of potential genotoxicity of the two assay systems used. The L5178Y mouse lymphoma mutagenicity assay gave a higher level response with results that were somewhat easier to interpret. It has been suggested that the tk locus may be useful for detecting both point mutations and chromosome damage (Clive et al., 1980; Hozier et al., 1981). If so, our data may indicate that 2,4DAT is not an effective inducer of point mutations in mammalian cells since it did not induce mutations at the hgprt locus. However, since mutation was observed at the tk locus, 2,4-DAT may induce chromosome aberrations. Additional experiments involving mutation at other gene loci (e.g. ouabain, aprt) and induction of visible chromosome damage would be helpful in addressing this problem.

Acknowledgements We thank Drs. J.A. Skare, M.J. Winrow and J.A. McDermott for critical review of this manuscript. We also thank Ms. L. Flora for her assistance in its preparation.

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