Induction of genetic tandem duplications in Salmonella by polycyclic aromatic hydrocarbon and amine carcinogens

Mutation Research, 182 (1987) 5-13

5

Elsevier MTR08634

Induction of genetic tandem duplications in Salmonella by polycyclic aromatic hydrocarbon and amine carcinogens M a r t i n L. Pall a n d Beverly J. H u n t e r Program in Geneticsand Cell Biology, Program in Biochemistry/Biophysics, Washington State Unioersity, Pullman, WA 99164-4350 (U.S.A.)

(Received4 August1986) (Accepted13 August1986)

Key words: Tandemduplications;(Salmonella typhimurium); Polycyclicaromatichydrocarbons;Aminecarcinogens.

Summary 6 polycyclic aromatic hydrocarbon or similar amine carcinogens were tested as inducers of genetic tandem duplications in a rough strain of Salmonella typhimurium. When metabolically activated by rat-liver microsomes, all 6 were active in inducing genetic tandem duplications, yielding from over 3 times to almost 14 times as many tandem duplicants per viable bacterium as did concurrent uninduced control cultures. These results extend the number and chemical diversity of carcinogens shown to induce genetic duplications in bacterial tester systems. We suggest that polycyclic hydrocarbon carcinogens may act in carcinogenesis by inducing genetic duplications or other genetic rearrangements. Duplication induction may be a useful genetic endpoint for screening potential carcinogens.

A variety of polycyclic aromatic hydrocarbons and their derivatives have been shown to be carcinogenic in several different animal species (Freudenthal and Jones, 1976). As with other carcinogens, attempts have been made to demonstrate induction of genetic changes by polycyclic aromatic hydrocarbon carcinogens. These compounds have been found to be active in inducing frameshift mutations but generally appear to be much less active in inducing base-pair substitutions in bacterial tester systems (McCann et al., 1975). However, frameshift mutations have not been demonstrated to have a role in carcinogenesis (such as in the activation of proto-oncogenes), Correspondence: Dr. M.L. Pall, Programin Geneticsand Cell Biology, Program in Biochemistry/Biophysics,Washington State University,Pullman,WA99164-4350; tel. (509) 335-5641.

raising the question as to whether polycyclic aromatic hydrocarbon carcinogens may act by some other mechanism in carcinogenesis. We have suggested that induction of genetic tandem duplications may have an important role in carcinogenesis (Pall, 1981). We and others have reported that a variety of carcinogens are active in inducing genetic tandem duplications in bacterial tester systems (Hill and Combriato, 1973; Hoffmann et al., 1978, 1985; Pall and Hunter, 1985, 1986; Straus, 1974). We report here that several polycyclic aromatic hydrocarbon carcinogens, when metabolically activated, induce genetic tandem duplications. These results suggest that such compounds may act to induce tandem duplications during carcinogenesis. Systems allowing the detection of genetic duplication induction may have a role in the

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detection of potential carcinogens as suggested earlier (Pall, 1981; Hoffmann, 1985; Pall and Hunter, 1985). Materials and methods

Bacterial s[rains and bacteriophage The Salmonella typhimurium strains T R 4 1 7 9 ( srl-201, his02355) and TT1984 ( srl-201, his02355, hisG8575: :Tnl0) (see Anderson and Roth, 1978) were provided by J.R. Roth (University of Utah, Salt Lake City, UT). Salmonella typhimurium strain TA1535 (hisG46, AuvrB, rfa) and bacteriophage ES18.hl were provided by the Salmonella Genetics Stock Centre (Calgary, Alberta, Canada). Strain MP352 (his02355, AuvrB, rfa) was constructed by transducing the his02355 mutation into TA1535 using a ES18.hl lysate of TR4179. His +, biotin-requiring isolates were screened for 3-amino-l,2,4-triazole sensitivity to detect the his02355 mutation, for crystal violet sensitivity to detect the rfa mutation and for UV light sensitivity to detect the uvrB deletion. Those isolates that were his02355, rfa, AuvrB were used in an AT-resistance assay (see below) with 10 /~1 of minimal media used in place of the test chemical. The resultant spontaneous AT-resistant colonies (potative tandem duplicants) were checked for tandem duplication using the tandem duplication assay (see below) and by tandem duplication assay B (Pall and Hunter, 1985) using a ES18.hl lysate of TT1984 in place of the P22ht lysate. Isolate MP352 was screened and found to produce spontaneous tandem duplications at a low rate.

Media Minimal medium (ME) is Vogel and Bonnet E medium (1956) containing 2% glucose. MEB medium is ME medium supplemented with biotin (0.05 mM). ATB medium is MEB medium supplemented with 3-amino-l,2,4-triazole (1.4 mM), adenine (0.4 mM) and thiamine (0.05 mM). Difco nutrient broth (NB) was used as a nonselective growth medium. Solid media contained 1.5% Difco agar.

Chemicals 3-amino-l,2,4-triazole, adenine-HCI, thiamine HC1, biotin, glucose 6-phosphate, glucose-6-

phosphate dehydrogenase, N A D P + and all chemicals tested for tandem duplication induction were purchased from Sigma Chemical Company. Aroclor 1254 induced rat-liver microsomal $9 fraction was purchased from Litton Bionetics in 2-ml aliquots and stored at - 7 0 ° C until it was used.

A T resistance assay Where used, rat-liver microsomal $9 fraction was used together with an NADPH-generating system following a modification of the method of Skopek et al. (1978). A 2-ml aliquot of $9 was thawed on ice, diluted in 15 ml MEB and sterilized by passage through a sterile 0.45-#m millipore filter (25-mm size) with a Gelman glass filter type A-E prefilter. For the AT-resistance induction with $9 activation, 50 /~1 of a stationary phase, overnight (16-18 h) NB culture of MP352 or TR4179 was added to 830-838/tl of the above described MEB, $9 mixture in 16 × 120 test tubes. The compounds to be tested were dissolved in DMSO or ethanol and delivered in 2-10 #1 volumes. Each test tube then received 0.4 units of glucose-6-P dehydrogenase in 10 /tl of 5 mM citrate and 100/~1 MEB containing 1 mg glucose6-P, 1 mg N A D P ÷ and 6.7 mg MgC12 for a final volume of 1 ml. Controls received 10/~1 of solvent in place of the test chemical. The tubes were vigorously shaken in a rotary bath shaker at 37°C for 4 h. After incubation 0.1-ml aliquots of a 10 2 dilution (in ME without glucose) were plated onto each of six 100-mm ATB plates. Viable cell counts were obtained by plating 0.1-ml aliquots of a 1 0 - 6 dilution onto each of three nutrient agar plates. The plates were incubated 60 h (48 h for TR4179) at 37°C prior to counting. For the AT-resistance assay without $9 activation 940-948 /~1 of MEB was substituted for the MEB with $9. The test compounds were delivered, the tubes placed in the water bath shaker and treated as described above.

Tandem duplication assay AT-resistant colonies were screened for the genetic instability characteristic of tandem duplicants (Anderson and Roth, 1977, 1978; Pall and Hunter, 1985). Individual AT-resistant colonies were cloned by streaking on ATB medium. After 60 h (48 h TR4179) incubation at 37°C isolated

sistance, little or no induction was seen in a strain TR4179 containing a wild-type cell surface (Table 1). However, when a rough mutant MP352 was tested for induction of AT-resistance, 2AAF was an active inducer (Table 2). We use, as a minimum criterion of induction, at least a doubling of AT resistance. 2AAF was only active when it was metabolically activated using rat-liver $9 fraction. It is not clear why unactivated 2AAF appears to lower the frequency of AT resistance. These results parallel previous findings using 2AAF and other polycyclic aromatic hydrocarbon carcinogens to induce frameshift mutations in bacteria. Such induction required the use of a rough mutant strain to increase bacterial permeability and also required metabolic activation to produce the active mutagen (Ames et al., 1975). When several concentrations of 2AAF were used to induce AT resistance, in separate experiments from those listed above, induction showed a progressive increase with increasing concentration of 2AAF from 8.95 to 447.5 nmoles/ml (Table 3).

colonies were selected and grown in NB overnight (16-18 h) 0.1 ml of a 10 -6 dilution was plated onto ATB medium and incubated 60 h (48 h for TR4179) at 37°C. The plates were examined under 30 × magnification for the presence of large and small colonies expected from rapid reversion of tandem duplications. This assay was repeated to insure the accuracy of the test results. Duplicants were scored as clones producing at least 10-15% small colonies, whereas nonduplicants generally produced < 2% small colonies. Results and discussion

6 polycyclic aromatic hydrocarbon and amine carcinogens were tested for induction of genetic tandem duplications using two Salmonella strains carrying the his02355 mutation. Genetic tandem duplication of the histidine operon carrying the his02355 mutation confers resistance to 3-amino1,2,4-triazole (AT). When 2-acetyl-aminofluorene (2AAF) was tested as a possible inducer of AT-re-

TABLE 1 L A C K OF I N D U C T I O N O F A T R E S I S T A N C E IN S A L M O N E L L A S T R A I N A C E T Y L A M I N O F L U O R E N E (2AAF) W I T H A N D W I T H O U T $9 A C T I V A T I O N $9 activation

a. Viable c o u n t / m l of culture ( × 1 0 - 7) b. AT-resistant colonies/ml of culture plated ( 1 0 - 3) c. N u m b e r of AT-resisiant colonies counted d. N u m b e r of A T plates counted

TR4179

AT

89.5

nmoles/ml

N o activation

89.5 n m o l e s / m l 2AAF

Concurrent control

89.5 n m o l e s / m l 2AAF

Concurrent control

117.8 + 7.51

115.6 + 8.54

91.0 _+6.18

92.2 + 9.69

59.5 + 2.44

46.9 + 5.94

40.5 + 4.38

46.1 + 6.70

1606

703

1216

829

27

15

30

18

e. AT-resistant colonies/105 bacteria plated ( b / a )

5.05

4.05

4.45

5.00

f. Ratio of AT-resistant colonies/10 s bacteria plated (treated/uninduced control)

1.25

-

0.89

-

Rows a and b are expressed as mean + standard error.

2-

8 TABLE 2 INDUCTION OF AT RESISTANCE IN SALMONELLA STRAIN MP352 AT 89.5 nmoles/ml 2-ACETYLAMINOFLUORENE (2AAF) WITH AND WITHOUT $9 ACTIVATION $9 activation 89.5 nmoles/ml 2AAF a. Viable count/ml of culture ( × 10 -7 • b. AT-resistant colonies/ml of plated (10 -3 ) c. Number of AT-resastant colonies counted

No activation Concurrent control

89.5 nmoles/ml 2AAF

Concurrent control

39.3 + 2.14

60.8 _+4.24

34.5 _+2.28

36.6 + 3.56

166.2 + 8.36

44.5 __+2.59

16.9 + 2.52

44.5 + 4.24

10971

2535

305

748

d. Number of AT plates counted

66

57

18

18

e. AT-resistant colonies/10 s bacteria plated (b/a)

42.25

f. Ratio of AT-resistant colonies/10 s bacteria plated (treated/uninduced control)

5.84

7.30

4.91

-

0.43

11.36

Rows a and b are expressed as mean _+standard error.

However, c o n c e n t r a t i o n s a b o v e 89.5 n m o l e s / m l showed n o higher i n d u c t i o n ( T a b l e 3), p o s s i b l y b e c a u s e 89.5 n m o l e s / m l was sufficient to p r o d u c e a s a t u r a t e d solution. When 5 other polycyclic aromatic hydrocarbon c a r c i n o g e n s i n c l u d i n g two a m i n e derivatives, were tested as i n d u c e r s of A T resistance, each of the 5 was also f o u n d to be active in such i n d u c t i o n ( T a b l e 4). T h e i n d u c e d level of A T resistance was 2.27-10.51 times the level in the u n i n d u c e d conc u r r e n t c o n t r o l s ( T a b l e 4, row f). T o d e t e r m i n e w h e t h e r these c o m p o u n d s are active in i n d u c i n g genetic t a n d e m d u p l i c a t i o n s , it is necessary to m e a s u r e n o t o n l y the frequency of A T - r e s i s t a n t colonies b u t also to d e t e r m i n e w h a t f r a c t i o n of these A T - r e s i s t a n t colonies c o n t a i n genetic t a n d e m d u p l i c a t i o n s . I n previous studies, two tests were p e r f o r m e d for d u p l i c a t i o n b a s e d o n the genetic i n s t a b i l i t y of d u p l i c a t i o n s a n d on the a b i l i t y of d u p l i c a t i o n s to be m a d e h e t e r o z y g o u s b y t r a n s d u c t i o n ( A n d e r s o n a n d R o t h , 1978; Pall a n d H u n t e r , 1985, 1986). These two tests were c o n c o r -

d a n t in over 99.9% of the tests of specific colonies. It m a y b e inferred that a single test will a l m o s t always a c c u r a t e l y score A T - r e s i s t a n t d u p l i c a t i o n s or AT-resistant nonduplications. I n the tests r e p o r t e d here, o n l y the stability of the A T resistance was tested due to the difficulty in t r a n s d u c t i n g r o u g h m u t a n t strains. As shown in T a b l e 5, row b, a m a j o r i t y of the A T - r e s i s t a n t colonies of all g r o u p s tested positive for t a n d e m duplications. W h e n the f r e q u e n c y of t a n d e m d u p l i c a t i o n s is calculated, it is clear that the polycyclic h y d r o c a r b o n or a m i n e - t r e a t e d cultures h a d increased levels of d u p l i c a t i o n s ( T a b l e 5, row c). U s i n g a d o u b l i n g of d u p l i c a t i o n frequency as our minim u m criterion for i n d u c t i o n , we c o n c l u d e that each of the 6 c o m p o u n d s are active in i n d u c i n g genetic t a n d e m duplications. T h e agents tested p r o d u c e d f r o m 3.34 to 13.96 times as m a n y duplic a t i o n s / 1 0 5 viable b a c t e r i a as d i d c o n c u r r e n t control cultures ( T a b l e 5, row d). O n a m o l a r basis, 6 - a m i n o c h r y s e n e a p p e a r s to b e the most active

9

TABLE 3 INDUCTION OF AT RESISTANCE IN SALMONELLA MP352 BY 2-ACETYLAMINOFLUORENE WITH $9 ACTIVATION Concentration: (nmoles/ml)

0

8.95

22.39

35.82

44.75

89.5

223.9

358.2

447.5

a. Viable count/ml of culture (x10 -7)

6.08 _+4.25

77.0 _+3.20

66.5 _+6.84

68.3 -+6.36

50.1 -+3.42

39.3 +2.14

40.3 -+2.43

33.2 _+2.62

36.6 +2.65

b. AT-resistant colonies/ml of culture plated (10 -3 )

44.5 _+2.59

66.2 _+7.06

81.4 +6.53

94.3 _+7.09

104.2 _+7.18

166.2 _+8.37

134.4 +_10.37

138.6 -+7.27

157.7 -+15.19

c. Number of ATresistant colonies counted d. Number of AT plates counted

2535

1191

1710

1697

3 438

10 971

6 050

5 821

6149

57

18

21

18

33

66

45

42

39

13.8

20.81

42.25

33.34

41.70

43.10

2.84

5.77

4.56

5.70

5.89

e. AT-resistant colonies/10 s bacteria plated b/a

7.3

8.59

12.24

f. Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

-

1.14

1.67

1.89

Rows a and b are expressed as mean + standard error. The lowest 3 concentrations were not tested in all experiments. Because the level of AT resistance found at higher concentrations of 2-acetylaminofluorene did not differ significantly (P > 0.05) between experiments testing and not testing in lowest 3 concentrations, all data were pooled in this table.

agent a n d b e n z [ a ] p y r e n e a p p e a r s to b e the least active ( T a b l e 5, r o w f). I n u n i n d u c e d (control) cultures of MP352 subs t a n t i a l n u m b e r s of A T - r e s i s t a n t n o n d u p l i c a t i o n s were f o u n d ( T a b l e 5, row b). This is in c o n t r a s t to p r e v i o u s studies with T R 4179 where s p o n t a n e o u s A T - r e s i s t a n t colonies are only rarely n o n d u p l i c a tions ( A n d e r s o n a n d Roth, 1978; Pall a n d H u n t e r , 1985, 1986). It has b e e n p r o p o s e d t h a t d u p l i c a t i o n i n d u c t i o n m a y b e a useful e n d p o i n t t o score in screening for p o t e n t i a l c a r c i n o g e n s (Pall, 1981; H o f f m a n n , 1985; Pall a n d H u n t e r , 1985). T h e r e a r e n o w 18 different carcinogens a n d several c o m p o u n d s of u n d e t e r m i n e d c a r c i n o g e n i c i t y which have b e e n shown to i n d u c e d u p l i c a t i o n s in b a c t e r i a l systems (Hill a n d C o m b r i a t o , 1973; H o f f m a n n et al., 1978, 1985;

Pall a n d H u n t e r , 1985, 1986; Straus, 1974). T h e s e 18 carcinogens are chemically a n d p h y s i c a l l y very diverse p r o v i d i n g evidence t h a t diverse carcinogens c a n b e d e t e c t e d in this way. W h a t is u n c l e a r at this p o i n t is w h e t h e r m a n y n o n c a r c i n o g e n s also test positive in a d u p l i c a t i o n test. Because the A T - r e s i s t a n c e selection selects b o t h d u p l i c a t i o n s a n d A T - r e s i s t a n t n o n d u p l i c a t i o n s , it is p r o b a b l y n o t the test of choice to b e u s e d to screen for t a n d e m d u p l i c a t i o n i n d u c t i o n . W e have scored n o t o n l y the n u m b e r o f A T - r e s i s t a n t colonies, b u t also tested m a n y i n d i v i d u a l colonies to d e t e r m i n e w h e t h e r or n o t they c o n t a i n duplications. C l e a r l y m a s s screening requires a select i o n system w h i c h is m o r e specific for duplioations. P e r h a p s the t r y p t o p h a n p r o t o t r o p h y selection system o f H o f f m a n et al. (1985) will

10 TABLE 4 INDUCTION OF AT RESISTANCE IN SALMONELLA STRAIN MP352 BY 5 POLYCYCLIC HYDROCARBONS AND AROMATIC AMINES WITH $9 ACTIVATION 1,2-Benzanthracene(BA)

1,2,3,4- Dibenzanthracene(DBA)

2-Aminoan thracene(2AA)

438 nmoles/ml BA

179.6 nmoles/ml DBA

5.17 nmoles/ml 2AA

Concurrent control

Concurrent control

Concurrent control

a. Viable c o u n t / m l of culture ( × 10- 7)

71.1 + 5.72

70.8 _+7.12

44.0 + 2.91

69.3 + 4.67

30.0 +_4.43

97.3 + 6.24

b. AT-resistant colonies/ml of culture plated (10 3)

108.7 +5.04

39.2 _+2.75

121.5 + 4.71

44.6 _+2.78

140.7 + 6.29

43.4 + 4.55

c. Number of AT-resistant colonies counted

3 587

1295

8384

2409

3 800

782

d. Number of AT plates counted

33

33

69

54

27

18

e. AT-resistant colonies/10 s bacteria plated ( b / a )

15.29

f. Ratio of AT-resistant colonies/10 s bacteria plated (treated/uninduced control)

5.54

27.61

2.76

6.64

46.9

4.29

4.46

10.51

Benz[ a ]pyrene (BP)

6-Aminochrysene(6AC)

39.64 nmoles/ml BP

Concurrent control

2.1 nmoles/ml 6AC

Concurrent control

a. Viable c o u n t / m l of culture ( x 10 7)

56.3 + 3.01

88.9 _+6.14

49.9 _+2.10

58.5 _+3.33

b. AT-resistant eolonies/ml of culture plated (10 3)

80.5 _+6.33

48.9 +_5 84

81.2 +_4.70

42.0 _+2.46

c. Number of AT-resistant colonies counted

2 656

1028

4 629

2 643

d. Number of AT plates counted

33

21

57

63

e. AT-resistant colonies/10- s bacteria plated (b/a)

14.29

f. Ratio of AT-resistant colonies/10 5 bacteria plated (treated/uninduced control)

5.50

2.60

16.3

-

3.35

2.27

-

almost

all s p o n t a n e o u s

Rows a and b are expressed as mean +_standard error.

prove

to be

more

specific

and

thus

more

propriate for mass screening. In previous studies of AT-resistance,

ap-

it was

shown

that

AT-resistant

colonies contained tandem duplications (Anders o n a n d R o t h , 1 9 7 8 ; P a l l a n d H u n t e r , 1 9 8 5 , 1986).

11 TABLE 5 I N D U C T I O N OF T A N D E M D U P L I C A T I O N IN SALMONELLA STRAIN MP352 BY 6 POLYCYCLIC H Y D R O C A R B O N S A N D AMINES W I T H $9 ACTIVATION

a. AT-resistant colonies/10 5 bacteria plated a

2-Acetylaminofluorene(2AAF)

Benz[a]pyrene(BP)

6-Aminochrysene(6AC)

89.5 nmoles/ml 2AAF

39.64 nmoles BP

2.1 n m o l e s / m l 6AC

Concurrent control

Concurrent control

Concurrent control

42.25

7.30

15.29

5.54

21.17

6.44

b. Tandem duplications/ total AT-resistant colonies

0.85 (70/82)

0.75 (133/178)

0.85 (46/54)

0.75 (133/178)

0.96 (53/55)

0.75 (133/178)

c. Tandem duplications/105 bacteria plated (a/b)

35.91

4.77

12.23

3.66

20.32

4.83

d. Ratio of tandem duplication/10 s bacteria (row c treated/row c untreated control)

7.53

-

3.34

-

4.21

-

e. Tandem duplications induced/105 bacteria plated (column 1 row c minus column 2 row c)

31.14

-

8.57

-

15.49

-

f. Tandem duplication~ induced/105 bacteria plated/nmole/ml

0.35

-

1.96 × 10- 2

_

7.38

-

g. AT-resistant nonduplicants/105 bacteria plated (a-c)

6.34

2.46

3.06

1.88

0.85

1.61

h. AT-resistant nonduplicants induced/10 s bacteria plated/nmole/ml minus column 2 row f)

3.88

1.18

-

0.76

a From Tables 2 and 4.

In the rough mutant strain used here, MP352, 25% of the spontaneous AT-resistant colonies scored were nonduplicants. It is not clear whether this

higher spontaneous frequency of nonduplicants is due to the rough mutation or due to some other difference between strains.

12 TABLE 5 (continued)

a. AT-resistant colonies/105 bacteria plated "

2-Aminoanthracene(2AA)

1,2-Benzanthracene(BA)

1,2,3,4-Dibenzanthracene(DBA)

5.17 nmole/ml 2AA

430 nmoles/ml BA

179.6nmoles DBA

46.9

Concurrent control

Concurrent control

Concurrent control

4.46

14.29

5.50

16.30

3.35

b. Tandem duplications/ total AT-resistant colonies

0.88 (35/40)

0.75 (133/178)

0.80 (44/55)

0.75 (133/178)

0.91 (20/22)

0.75 (133/178)

c. Tandem duplications/105 bacteria plated (a/b)

41.04

2.94

12.14

3.63

14.83

2.51

d. Ratio of tandem duplication/10 ~ bacteria (row c treated/ row c untreated control)

13.96

3.34

5.91

e. Tandem duplications induced/105 bacteria plated (column 1 row c minus column 2 row c)

38.1

8.52

12.32

f. Tandem duplications induced/10 5 bacteria plated/nmole/ml

7.36

-

0.21

9. AT-resistant nonduplications/105 bacteria plated (a-c)

5.86

1.52

2.15

h. AT-resistant nonduplications induced/105 bacteria plated/nmole/ml minus column 2 row f

4.34

0.28

6.86x 10

1.87

1.47

2

0.84

0.63

" From Tables 2 and 4.

Acknowledgements

References

We thank Drs. John Roth, Michael Kahn and Lawrence Mueller for helpful discussions. This study was supported by PHS grant CA33503 awarded by the National Cancer Institute.

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