Mutation Research, 267 (1992) !-17 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

1

MUTGEN 01752

Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: Role of DNA breaks David M. D e M a r i n i a n d B. Kay L a w r e n c e Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.4.) (Received 4 February 1991) (Revision received 16 May 1991) (Accepted 16 September 1991)

Keywords: DNA damage; SOS induction; Microscreen assay; Free radicals DNA toposiomerase !I; Reactive-oxygen species; DNA breaks, role of

Summary Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coil WP2s(A) microscreen assay. The inability of a DNA 8yrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the iigation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.

Mammalian DNA topoisomerase II and bacterial DNA topoisomerase il (DNA gyrase) play critical roles in DNA metabolism and, conse-

Correspondence:Dr. David M. DeMarini, U.S. EPA (MD.68A), Research Triangle Park, NC 27711 (U.S.A.).

quently, are the primary target at which certain antitumor agents and antibiotics, respectively, are aimed (reviewed by Geilert, 1981; Ross, 1985; Vosberg, 1985; Wang, 1985, 1987; Liu, 1989; Drlica, 1990). These drugs, referred to as topoisomerase II poisons (Liu, 1989), have also become useful probes for examining the role of these

enzymes in mediating mutagenesis induced by various agents (Pommier et al., 1985; Ripley et al., 1988; Andersson and Kihlman, 1989; Evans et ai., 1989; Holden et al., 1989; Backer et al., 1990). In general, DNA topoisomerase II poisons form a DNA-drug-enzyme complex that may lead to the induction of frameshift mutations in prokaryotes and chromosomal mutations in mammalian cells (Gaulden, 1987;Ripley et al., 1988; DeMarini and Lawrence, 1988; Pienta et al., 1989; Caldecott et al., 1990; Ferguson and Denny, 1991). A primary type of DNA damage induced by DNA topoisomerase II poisons is DNA-strand breakage (see reviews above), which is also a major type of DNA damage induced by ionizing radiation and reactive-oxygen species (Meneghini, 1988; Wallace and Painter, 1990). DNA-strand breaks are potent inducers of prophage lambda and of the SOS response in general (Elespuru, 1984, 1987). Recent work suggests that singlestranded DNA gaps may be the signal that activates the RecA protein and initiates the SOS response (Battista et al., 1990). Additional studies reviewed by Sommer et al. (1991) indicate that single-stranded DNA, which results from the processing of DNA lesions, is the inducing signal for the SOS response, Although some DNA topoisomerase I! poisons and reactive-oxygen species have been examined for their ability to induce the SOS response (Elespuru and White, 1983; Mamber et al., 1986; Nakamura et al., 1987), some important members of these classes of agents, such as oxolinic acid, m-AMSA teniposide, and potassium superoxide (KOz), have not been evaluated. Such information could help to elucidate further the role of DNA ~rase in mediating the production of SOS-inducing DNA damage. Thus, we have ex.. amined 2 DNA ~rase ,poisons (nalidixic acid and oxolinic acid), a DNA gyrase inhibitor (novobiocin), and 5 mammalian DNA topoisomerase II poisons (actinomycin D, adriamycin, m-AMSA ellipticine and teniposide) in the microscreen prophage-induction assay developed by Rossman et al. (1984). Because some topoisomerase poisons, such as adriamycin, also produce reactive, oxygen species (Bachur et al., 1979), we have evaluated 3 agents (hydrogen peroxide [H202] , paraquat, and potassium superoxide [KO2]) that

are not DNA topoisomerase II poisons but that are capable of generating reactive-oxygen species. Over 200 chemicals have been evaluated in the microscreen prophage-induction assay, including metal salts (Rossman et al., 1984), chlorinated pesticides (Houk and DeMarini, 1987), solvents (DeMarini et ai., 1991), and agents from a wide variety of chemical classes (Rossman et al., 1991). In particular, the assay has been shown to detect an oxidized nucleoside that is a product of ionizing radiation (Shirname-More et al., 1987) and pentachlorophenol, which produces free radicals that induce DNA-strand breaks (DeMarini et al., 1990). The results produced in this assay by DNA topoisomerase II poisons and reactive-oxygen species are compared to those produced by these agents in other prokaryotic and mammalian cell assays. Likely mechanisms by which these agents induce their genotoxic effects are discussed. Materials and methods Chemicals Actinomycin D (MW 1255.47, CAS No. 50-760), adriamycin (MW 580.03, CAS No. 23214-92-8), m-AMSA (MW 393.49, CAS No. 51264-14-3), ellipticine (MW 246.33, CAS No. 519-23.3), and teniposide (MW 656.67, CAS No. 29767-20-2) were gifts from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD. Nalidixic acid (MW 254.2, CAS No. 389-08-2), novobiocin (MW 612.65, CAS No. 303-81-1), oxolinic acid (MW 261.2, CAS No. 14698-29-4),KOz (MW 71, CAS No. 12030-88-5), paraquat (MW 186.25, CAS No. 1910-42-5), HzO z (MW 34, CAS No. 7722-84-1), sodium azide (NaN 3, MW 65.02, CAS No. 26628-22-8), 2nitrofluorene (2-NF, MW 211, CAS No. 607-5%8), and 2-aminoanthracene (2-AA, MW 193, CAS No. 613-13-8) were purchased from Sigma Chemical Co., St. Louis, Me. Acetone (67-64-1) was purchased from Burdick and Jackson Laboratories, Muskegon, Ml. Stock solutions of actinomycin D (10 mg/ml), m-AMSA (10 mg/ml), ellipticine (2.5 mg/ml), oxolinic acid (0.5 mg/ml), and teniposide (1 mg/ml) were prepared in acetone. Nalidixic acid (0.5 mg/ml) was prepared in acetone diluted 1 : 1

3

in Vogel-Bonner minimal medium supplemented with 0.2% glucose and 2 0 / z g / m l of tryptophan (DeMarini et al., 1990). Adriamycin (0.5 mg/ml), novobiocin (0.1 mg/ml), KO 2 (1 mg/ml), paraquat (0.25 mg/ml), H20 2 (3%), and NaN 3 (1 mg/ml) were prepared in supplemented VogelBonner minimal medium,

Microscreen assay The lambda lysogen WP2s(A) (lonH, sub4m, trpE65, uvrAl55, lamB+), was derived from Escherichia coli B / r and was obtained from Dr. Evelyn M. Witkin via Dr. Anne C. Frazer, Department of Microbiology, New York University Medical Center, New York, NY. The indicator strain TH-008 (streptomycin r) was derived from E. coli C and was obtained from Dr. A.C. Frazer. The microscreen prophage-induction assay was performed as described by DeMarini et al. (1990). 2-NF ( - $ 9 ) and 2-AA ( + $9) were the positive cont/ols. Briefly, the first well in a dilution series of a 96-well microtiter plate (Coming Laboratory Sciences Co.) received 250 ttl of supplemented minimal medium (Vogel-Bonnet salts containing 0.2% glucose and 20/~g/ml of tryptophan) and 50 ~! of either the test compound or the medium control. The remaining wells received 150 ~1 of the medium. 2-fold serial dilutions of the cornpounds or controls (150 t~l: 150 ~!) were made down the columns of each plate. Dilutions were extended into a second plate to assure identification of the dose range. Each well was inoculated with 75 ttl ( ~ 2 × 106 cells) of a resuspended log-phase culture of WP2s(A) and 25 ~! of medium or $9 mix (2.5%), prepared as described by Houk and DeMarini (1987) using rat-liver homogenate from male Sprague-Dawley rats. The contents of each microtiter plate were mixed using a Minimixer (Fisher Scientific), the plates were covered with four layers of plastic wrap (Saran Wrap, Dow Chemical Co.), the lid was placed tightly on each plate, and the plates were incubated overnight at 37°C. After incubation, the wells were scored for turbidity, with turbid wells indicating cell growth and clear wells indicating cytotoxicity and/or inhibition of cell growth. The concentration of lambda phage was determined by sampling at least the first five turbid wells adjacent to a clear

well. A sample (50 ~!) from the well was diluted in 5 ml of supplemented minimal medium, and 100 ttl of this dilution was added to ~ 2.5 ml of top agar (0.65% Bacto agar and 10 mM MgSO 4) along with 200 /zl of log-phase indicator cells (TH-008), which had been grown in Oxoid No. 2 nutrient broth. The content of each tube was poured onto bottom agar made of tryptone medium (10 g of Bacto tryptone, 5 g of NaCI, and 12 g of Bacto agar per liter of glass-distilled, deionized H20) supplemented with streptomycin (100 ttg/ml) to select against the lysogen. Plates were incubated overnight at 37°C, and plaques were counted by hand. The dilution tubes were sampled in duplicate, and all experiments were repeated at least twice. Previous studies have shown that an induced PFU/plate that reaches the upper limit of the 99% confidence interval represents an approximate 3-fold increase over the background PFU/plate (Houk and DeMarini, 1987, 1988; DeMarini et al., 1990). Consequently, a dose-related increase of induced PFU/plate that reached or exceeded a three-fold increase in PFU/plate was considered a positive response. If a compound's response reached or exceeded this fold increase at only one dose, the result was scored as a weak positive (w+). A summary response was given to each compound based on the reproducibility of the results from two independent experiments.

Results and discussion The induction of prophage lambda by the selected agents is shown in Table 1, and dose-response curves for the genotoxic agents are shown in Fig. 1. All of the compounds, except for H 202 and KO2 (+$9), were tested to cytotoxicity as indicated by the occurrence of clear wells, which are noted as "Toxic" in Table 1. As observed in previous studies (Rossman et al., 1984; Houk and DeMarini, 1987, 1988; DeMarini et al., 1990, 1991), those concentrations of the agent that produced positive responses were generally those just below toxicity. The reason for this is most likely due to the biological consequences of prophage induction, i.e., cell lysis ~Roberts and Devoret, 1983).

Ellipticine

Response

m-AMSA

Response

Adriamycin

Response

Actinomycin D

37

39

4

5

0.12 23 0~25 38

17 17

36 44 52 68 88 100

58 58 58 58 58

42

9

100 304 673 349 406

31

9

6 21

30 136 156 62 687 588 +

+

42 246 615 291 348 Toxic

Toxic .

0

0

0

0

Compd. Contl. P F U / plate

1.4 2.2

!.8 4.1 4.0 1.9 9.0 6.9

!.7 5.2 11.6 6.0 7.0

0.1

0.1

0.2

0.3

26 37

51 45 65 61 84

19 58 121 128 132

.

7

2

6

9

19 15

18 25 25 31 58

23 23 23 23 23

26

32

34

35

7 22

-

33 20 40 30 26 Toxic

0 36 93 92 96 Toxic +

Toxic .

0

0

0

0

PFO/plate

+$9

Expt. 2

1.4 2.4

1.8 1.8 2.6 1.9 1.5

0.8 2.6 4.3 3.5 3.6

0.3

0.1

0.2

0.3

36 42

48 60 85 90 264 468

50 71 120 413 294 195

.

6

7

9

19 20 19 13 10

19 19

24 26 26 26 36 58

23 23 23 23 23 23

58

58

55

23 22 29 42 47

17 23

24 34 59 68 228 410 Toxic +

+

27 48 97 390 271 172 Toxic

.

Toxic

0

0

0

0 0 0 0 0

PFU/plate

-$9

1.9 2.2

2.0 2.3 3.3 4. ! 7.3 8.1

2.2 3.1 5.2 17.0 12.8 8.5

0.1

0.1

0.2

0.8 0.9 0.7 0.3 0.2

19 16

34 32 40 58 76

33 35 35 44 85 94 23

3

21 21 17 9 9

.

12 12

20 27 23 24 44

16 16 16 16 16 16 16

21

24 28 31 19 20

7 4

14 5 17 34 32 Toxic

17 19 19 28 69 78 7 Toxic +

Toxic

0

0 0 0 0 0

1.6 1.3

1.7 1.2 1.7 2.4 1.7

2.1 2.2 2.2 2.7 5.3 5.8 1.4

0.1

0.9 0.7 0.6 0.5 0.4

increase

Induced Fold

inCompd. Contl. PFU crease plate

Induced Fold

inCompd. ContL PWO/ crease plate

Induced Fold

inCompd. Confl. P F U / crease plate

PFO/plate

PFU/plate

induced Fold

- S t)

+S9

Expt. 1

198.54 397.09 66 794.18 180 1588.35 208 3176.70 130 6353.40 775 12706.80 688

0.34 0.67 1.35 2.69 5.39 10.78 21.55 43.10

0.26 0.52 1.03 2.07 4.14 5.97 8.28 I 1.94 16.57 24.68 33.13 4936 66.27 98.72

(~M)

Compound Dose

INDUCTION O F PROPHAGE LAMBDA BY SELECTED COMPOUNDS

TABLE 1

+

+

-

+

+$9 - $ 9

response

Summary

Response

Teniposide

Response

Oxolinic acid

Response

Novobiocin

Response

Nalidixic acid

Response

73 70 I11 260 227 293 335 360 358 336 366

50 34 68 66 302 604

25 29 29 19 42 43

9.52 55 19.04 50 38.07 57 76.14 143 i 52.28

0.05 0.09 0.19 0.37 0.75 1.49 2.99

0.13 0.26 0.51 !.02 2.04 4.08 8.16

0.10 54 0.19 68 0.38 117 0.77 162 !.53 311 3.07

0.50 0.99 1.98 3.96 7.93 15.85 31.72 63.40 126.86 253.72 507.45 1014.90

44 52 68 86

33 37 25 39 42 31

33 33 33 33 33 33

37 25 39 42 31

17 21 32 29 23 24 26 26 22 36 58

!! 0 0 57 Toxic .

17 0 43 27 260 573 Toxic +

0 0 0 0 9 10 Toxic .

17 43 78 120 280 Toxic +

56 49 79 231 204 269 309 334 336 300 308 Toxic +

1.3 1.0 0.8 1.7

1.5 0.9 2.7 1.7 7.2 19.5

0.8 0.9 0.9 0.6 !.3 1.3

1.5 2.7 3.0 3.9 10.0

4.3 3.3 3.5 9.0 10.0 12.2 12.9 13.8 16.3 9.3 6.3

.

43 46 70 40

41 144 177 210

.

22 17 17 13 3

16 22 27 52 112

41 55 52 52 54 63 175 234 507 7

25 31 58 59

24 22 16 22

36 36 36 36 36

28 20 21 32 35

15 19 21 22 20 20 27 23 24 44

18 15 12 0 Toxic .

+

17 122 161 188 Toxic

.

0 0 0 0 0 Toxic

0 2 6 20 77 Toxic w+

+

26 36 31 30 34 43 148 211 483 0 Toxic

1.7 !.5 1.2 0.7

1.7 6.5 I 1.0 9.6

0.6 0.5 0.5 0.4 0.1

0.6 1.1 1.3 !.6 3.2

2.7 2.9 2.5 2.4 2.7 3.1 6.5 10.2 20.1 0.2

58 95 104 191 207 .

35 52 126 252 323 461

.

23 27 30 33 70

45 79 108 222 452

62 135 200 321 308 467 261 274 231 199 471 628

60 57 57 74 80

25 43 45 47 42 22

28 28 28 28 28

20 43 45 47 42

19 19 19 19 19 13 22 19 23 32 33 60

0 38 47 117 127 .

10 9 81 205 281 439 Toxic +

0 0 2 5 42 Toxic .

25 36 63 175 410 Toxic -I-

44 116 181 302 289 454 239 254 208 167 438 568 +

1.0 !.7 !.8 2.6 2.6

1.4 1.2 2.8 5.4 7.7 21.0

0.8 1.0 1.0 1.2 2.5

1.8 1.8 2.4 4.7 10.8

3.3 7.1 10.6 16.9 16.2 35.0 11.8 14.4 10.4 6.2 14.3 10.5

58 74 85 91 99

36 52 88 148 300

16 20 18 I1

25 38 51 74 142

27 23 22 58 76 131 163 146 112 2

.

.

55 47 56 50 49

21 19 24 28 31

19 19 19 19

25 43 45 47 42

12 12 12 12 12 18 11 14 20 31

3 27 29 41 50

+

15 33 64 120 269 Toxic

0 1 0 0 Toxic

0 9 6 27 100 Toxic w+

+

15 11 10 46 64 113 152 132 92 0 Toxic

i.0 1.6 1.5 1.8 2.(~,

0.8 2.7 3.7 5.3 9.7

0.8 1.0 1.0 0.6

!.0 0.9 1.1 1.6 3.4

2.2 1.9 1.8 4.8 6.3 7.3 14.8 10.4 5.6 0.1

+

+

+

+

w+

+

Paraquat

Response

H20 2

PFU/plate

0.02 0.03 0.07 0.13 0.26

28 28 28 28 28 28

0 13 15 39 83 162 +

0.7 !.5 1.5 2.4 4.0 6.8

29 24 24 20 42

21 30 18 18 19 33

36 36 36 36 36

19 19 19 19 19 19

0 0 0 0 6

_

2 II O 0 0 14 Toxic

PFU/plate

Expt. 2 +$9

0.8 0.7 0.7 0.6 1.2

I.I 1.6 !.0 1.0 1.0 !.7 35 44 94 100 197 326

26 26 26 26 26 26

9 18 68 74 171 300 +

PFU/plate

-$9

!.3 !.7 3.7 3.8 7.6 12.5

18 22 19 19

24 27 28 75

19 19 19 19

19 19 19 19

0 3 0 0



5 8 9 56 Toxic

0.9 0.1 1.0 i.0

1.3 1.4 1.5 3.9

increase

Induced Fold inCompd. Conti. PFU crease plate

Induced Fold

inCompd. Contl. P F U / crease plate

Induced Fold

inCompd. Cemtl. P F U / crease plate

induced Fold

PFU/plate

Compd. Coutl. P F U / plate

-$9

Expt. i-+$9

0.0007 0.0014 0.0029 0.0057 0.011 0.023 0.046 0.28 20 0.55 41 I. 10 43 2.20 67 4.41 III 8.82 190

Compound Dose (~tM)

+

?

+$9 - $ 9

Summary response

24.(13 48.(16 96.12 192.~ 384.50 768.99 1537.99

44.01 88.03 176.06 352.1 ! 704.23 ! 408.45

26 30 52 59 266

22 46 41 47 39

41 79 95 145 91 201

28 28 28 28 28

39 39 39 39 39

33 33 33 33 33 33

+

0 2 24 31 238 Toxic

.

0 7 2 8 0

8 46 62 ! 12 58 168 Toxic +

0.9 1.1 1.8 2.1 9.5

0.6 1.2 1.0 t.2 ! .0

1.2 2A 2,9 4.4 2.8 6,1

29 16 ~ 20

45 32 39 49 38 . 19 19 19 19

39 39 39 39 3q

-

10 0 3 I Toxic

6 0 0 9 0 .

Toxic

!.5 0.8 1.2 1.0

!.2 0.8 1.0 1.2 1.0

41 61 108 161

.

51 6.~ 68 73 76

44 48 72 III i 12 240

26 26 26 26

58 58 58 58 58

28 28 28 28 28 28

15 35 82 134 Toxic +

.

0 6 10 15 18

16 20 44 83 94 212 Toxic +

1.6 2.3 4.2 6.2

0.9 I. 1 1.2 1.3 1.3

1.6 1.7 2.6 4.0 4.3 8.6

27 27 32 47

.

46 5! 62 48

19 19 19 19

46 46 46 46

8 8 13 28 Toxic -

0 5 16 2 Toxic

-

Toxic

1.4 1.4 1.7 2.4

1.0 I. I 1.4 1.0

+

+

-

-

" The positive control results for 2-AA (6.48 p M , + $ 9 ) were 568 induced P F U / p l a t e , 13.1-fold increase (mean of 7 Expts.). The positive control results for 2-NF (710.9{) /tM, - $ 9 ) were 60~ induced P F U / p l a t e , 16.2-fold increase (mean o f 7 Expts.). The P F U / p l a t e for the medium controls was 36 ( + $ 9 ) and 27 ( - $ 9 ) (mean of 8 Expts.).

Response

NaN 3

Response

KO 2

Respon~

0.52 1.05 2.09 4.19 8.39 16.78 33.55 67.! !

"-d

oxo .,e. ~w, +S9 "*"~7 ~° . ~ ~ / ~ /f~ 4~/ " ~"~ "~/ 10o so 3o

2° ~- 100.1 .......................................... 1 10 100 1,000 10,000 -9 . "~ ~l -S9 ~'"P,~ __~ 2SO ~1 ox~, / 1so so 30 20 10 , ooi

/

~"/~ /

/

/

~

...... . . . . . . . . . . . . . . ol i lo

10o

DoseOJM) Dose-responsecurves of prophage inductionby seleered agents. Data are from representativeexperimentsin Table 1. Fig. 1.

NaN 3 was examined as a possible positive control ( - $ 9 ) in the prophage-induction assay based on its use, as such, in the Salmonella assay, However, it did not induce prophage in the absence of $9, although it was genotoxic in the presence of $9 (Table 1). This is in contrast to a recent report in which NaN 3 induced prophage both in the absence and presence of $9 in the microscreen assay (Rossman et al., 1991). In the umu.test for SOS induction, NaN 3 was negative in the presence and absence of $9 (Oda et al., 1985). NaN 3 was also negative in the SOS Chromotest, $9 unspecified (Quillardet et al., 1985). The reasons for these disparate results are unknown. The metabolism of NaN3 to a genotoxin has been revic~ved by Owais and Kleinhofs (1988); however, the requirement for $9 in the present assay (compared to the absence of such a requirement in the Salmonella assay) is not clear, DNA gyrase poisons Nalidixic acid and its structural analog, oxolinic acid, induced prophage within a similar concentration range, and the addition of $9 enhanced their genotoxicity (Table 1). In this regard, nalidixic acid has been shown to be metabo-

lized to hydroxynalidixic acid, which may be a more potent poison of DNA gyrase than the parent compound (Gleckman et al., 1979). Nalidixic acid and oxolinic acid have also been shown to induce bacteriophage T4 topoisomerase ll-mediated DNA cleavage (Rowe et al., 1984; Kreuzer and Alberts, 1984), and they are mutagenie in bacteriophage T4 (DeMarini and Lawrence, 1988). The basis for the induction of prophage by nalidixic acid and oxolinic acid likely derives from the ability of these agents to poison E. cold DNA gyrase subunit A (Snyder and Drlica, 1979; Shen and Pernet, 1985). The inhibition by these agents of the ligation function of subunit A, which also provides the nicking function of DNA gyrase, likely results in DNA breaks that act as an SOSinducing signal, leading to prophage induction. Nalidixic acid may also exert its genotoxicity by additional mechanisms, such as by the production of reactive-oxygen species (discussed later), and, collectively, these mechanisms may accoun,~ for the extraordinary potency and toxicity of nalidixic acid and oxolinic acid (Liu, 1989). Nalidixic acid and oxolinic acid operate by trapping a reaction intermediate in which the DNA is broken, leaving DNA gyrase bound covalently to the DNA (Drlica, 1990). In particular, oxolinic acid has been shown to induce approximately 50 DNA breaks per E. coli chromosome, and this number is similar io the number of domains of supercoiling found in isolated E. coli chromosomes, suggesting one gyrase site per domain (Snyder and Drlica, 1979). Thus, the sites of DNA cleavage promoted by oxolinic acid (and, possibily, nalidixic acid) may correspond to chromosomal DNA gyrase cleavage sites (Snyder and Drlica, 1979). Analysis by pulsed-field gel electrophoresis has provided additional evidence that some of the 50 pieces produced by treatment of E. coli cells by these agents may arise from cleavage at specific sites on the DNA (Smith et el., 1990). In addition, transcription appears to regulate oxolinic acid-induced DNA gyrase cleavage at specific sites on the E. coil chromosome (Condemine and Smith, 1990). At low concentrations, oxolinic acid induces primarily single-strand DNA breaks; double-strand breaks might then arise from the accu-

mulation of single-strand breaks at sites that contain two independent drug targets, one on each DNA strand (Snyder and Drlica, 1979). It is likely that these DNA breaks provide the SOS-inducing signal that results in prophage induction. Although the DNA gyrase-mediated DNA damage induced by nalidixic acid and oxolinic acid elicits a strong SOS response (Fig. 1; Elespuru and White, 1983; Walker et al., 1985; Salles et al., 1987; Rossman et al., 1991), this damage does not revert the standard tester strains of Salmonella, which are DNA excision-repair deftcient (McCoy et al., 1980; Zeiger et al., 1988). Instead, nalidixic acid and oxolinic acid cause DNA damage that inhibits the growth of DNArepair-deficient bacteria (McCoy et al., 1980). However, nalidixic acid and oxolinic acid revert Salmonella strain TA102, which can revert by small (3- or 6-base pair) deletions at A-T sites (Levin et al., 1984). At the doses tested in strain TA102, approximately 50% of the revertants were deletion revertants, which was similar to the proportion induced by X-rays (Levin et al., 1984). However, it is unlikely that the ability of TA102 to revert by small deletions or at A - T sites accounts for the inability of the standard tester strains to be reverted by oxolinic acid and nalidixic acid because strain TA98 can also revert by a variety of small and large deletions, some of which involve A - T sites (Bell et al., 1991); but it is not reverted by these two agents (McCoy et al., 1980; Zeiger et al,, 1988). Perhaps more important is the excision-repair status of the strains. Strain TAI02 is repair-proficient, which may permit the conversion of the oxolinic acid- and nalidixic acid-induced DNA damage into mutations (mostly deletions). Aithough the lysogen used in the present study is DNA excision-repair-deficient, the singlestranded DNA that results from the poisoning of DNA gyrase subunit A by the two agents is an adequate SOS-inducing signal. However, such DNA damage is only lethal (not muta~enic) to the standard tester strains of SalmoneUa and must be processed (by the DNA excision-repair system) to a mutation. Thus, the excision-repair deficiency of the standard tester strains of Salmonella may account for the inability of these strains to be reverted by nalidixic acid and ox-

olinic acid. If true, t~en one prediction of this hypothesis is that the two agents should be mutagenic in excision-repair proficient strains of Salmonella, such as TA1978. Novobiocin inhibits E. coli DNA gyrase subunit B, which is an ATPase necessary for DNA gyrase to relax supertwisting (Gellert et al., 1976; Snyder and Drlica, 1979). However, novobiocin may have the more general effect of inhibiting all ATP-requiring enzymes (Downes et al., 1985). Although novobiocin causes preferential inhibition of the growth of DNA-repair-deficient bacteria (McCoy et al., 1980), it does not produce DNA damage that either elicits the SOS response (Table 1; Mamber et ai., 1986) or reverts various strains of Salmonella or E. coil WP2 (McCoy et al., 1980). Furthermore, novobiocin is not mutagenic in bacteriophage T4 or mammalian cells, and it inhibits the mutagenicity of m-AMSA a DNA topoisomerase ll-speciftc drug, in mammalian cells and bacteriophage T4 (DeMarini et al., 1987c; DeMarini and Lawrence, 1988). Thus, inhibition by novobiocin of DNA gyrase's ATPase (or other ATP-requiring enzymes) does not, in general, produce genotoxic effects, including prophage induction. This result indicates that the inability of DNA gyrase to relax supertwisting, which requires a functional subunit B ATPase, does not perturb DNA metabolism in a way that would lead to either mutation (in the other systems cited) or prophage induction. Considering the results with oxolinic ecid, nalidixic acid, and novobiocin, it appears that inhibition only of DNA gyrase subunit A (and not subunit B) is sufficient to produce an SOS-inducing signal (most likely single-stranded DNA) that can result in prophage induction. MammalianDNAtopoisomeraseHpoisons None of the 5 mammalian DNA topoisomerase II poisons examined here have been reported to affect DNA gyrase. However, all have been shown to poison bacteriophage 'I"4 and/or mammalian cell DNA topoisomerase ll (reviewed by Vosberg, 1985; Wang, 1985; Liu, 1989). Nonetheless, 3 of the agents (m-AMSA, adriamycin, and ellipticine) induced prophage, whereas 2 (actinomycin D and teniposide) did not (Table 1).

10 The inability of actinomycin D to induce prophage or the SOS response is consistent with previous studies (Rojanapo et al., 1981; Elespuru and White, 1983; Oda et al., 1985; Mamber et al., 1986; Nakamura et al., 1987; Rossman et al., 1991). Perhaps due to its lack of specificity for DNA gyrase or bacteriophage T4 topoisomerase II, actinomycin D is also not mutagenic in bacteria (Benedict et al., 1977; Xu et al., 1984) or bacteriophage T4 (DeMarini and Lawrence, 1988). Actinomycin D is known to intercalate into DNA; however, this property does not appear to be sufficient to elicit either the SOS response or gene mutation in prokaryotic systems. Although actinomycin D does not induce high frequencies of gene mutation at hemizygous loci in mammalian cells, its specificity for mammalian DNA topoisomernse I1 makes it highly clastogenic to mammalian cells and, thus, mutagenic at a heterozygous locus (reviewed by DeMarini et al., 1987a). Consequently, actinomycin D represents an agent whose potent clastogenicity in mammalian cells (presumably due to its poisoning of DNA topoisomerase !I) is in sharp contrast to its nongenotoxicity in prokaryotic systems, Similar to actinomycin D, teniposide did not induce prophage (Table 1), and is also not mutagenie in bacteriophage T4 (DeMarini and Lawrence, 1988). However, unlike actinomycin D, teniposide is weakly mutagenic in bacteria, ¢specially in excision-repair proficient strains (Matney et al., 1985). As with the other DNA topoisomerase II poisons studied here, teniposide is highly clastogenic to mammalian cells (DeMarini et al., 1987b). However, unlike the other DNA topoisomerase il poisons, teniposide does not intercalate into DNA (Chen et al., 1984). The fact that teniposide can induce DNA damage that is processed by the bacterial DNA exicision-repair system to mutations (Matney et al., 1985) suggests that the teniposide-induced DNA damage itself may not be sufficient to elicit the SOS response. A recent study by Sommer et al. (1991) has shown that DNA damage (UV-induced lesions) alone is not sufficient to produce an SOS-inducing signal in E. coll. Instead, the damage must be processed by DNA replication to form single-stranded DNA that acts as the SOSinducing signal, resulting in the activation of the

RecA protein. Thus, teniposide-induced DNA damage may simply no~-be processed to form a suitable SOS-inducing 'signal in the excision-repair-deficient strain of E. coli used in the present study. One consequence of this supposition is that teniposide might induce prophage in a DNA excision-repair proficient lysogen. Of the 3 mammalian DNA topoisomerase II poisons that induced prophage, ellipticine and adriamycin were nearly as potent as the DNA gyrase poisons, whereas m-AMSA required a concentration 3 orders of magnitude greater than that of the other DNA topoisomerase I1 poisons (Fig. 1). Adriamycin and ellipticine were genotoxic in the absence of $9, and their genotoxicities were enhanced upon the addition of S9, whereas m-AMSA required $9 in order to induce prophage (Table 1, Fig. 1). To our knowledge, this is the first report on the ability of m-AMSA to induce prophage. In addition to being a weak inducer of prophage, m-AMSA is also a weak mutagen in Salmonella strain TA1537 (Ferguson and Denny, 1979, 1991). However "E. coil DNA ~rase is refractory to m-AMSA treatment in vitro (unpublished results)" (Nelson et al., 1984). Thus, mAMSA's genotoxicity in bacteria is unlikely to be due to the poisoning of DNA gyrase; instead, it may result from m-AMSA's combined ability to intercalate into DNA (Liu, 1989; Gupta, 1990; Ferguson and Denny, 1991) and, as discussed later, to mediate the production of reactiveoxygen species that may cause SOS-inducing DNA damage. The weak genotoxicity of m-AMSA in bacteria is in sharp contrast to the finding that m-AMSA is among the most potent chromosomal mutagens identified in mammalian cells (DeMarini et al., 1987c) and one of the most potent frameshift mutagens in bacteriophage T4 (DeMarini and Lawrence, 1988), which contains a mammaliantype DNA topoisomerase Ii (Rowe ¢t al., 1984). The necessity of T4 DNA topoisomerase II for mediating the mutagenicity of m-AMSA in T4 has been demonstrated most convincingly by RipIcy et al. (1988) who showed that (a) the mutational hotspot induced by m-AMSA in T4 occurs precisely at the site of m-AMSA-induced, T4 DNA topoisomerase II-mediated DNA cleavage

!1

and (b) the mutagenicity of m-AMSA is greatly reduced in a topoisomerase mutant of T4. Thus m-AMSA represents an agent that is mutagenic in bacteria and mammalian cells but, apparently, by different mechanisms (intercalation and production of reactive-oxygen species in bacteria vs. DNA topoisomerase II poisoning in mammalian cells), Ellipticine is a potent inducer of prophage (Fig. 1), consistent with the results of Elespuru and White (1983). Eilipticine is also a moderately potent frameshifl mutagen in bacteriophage T4 (DeMarini and Lawrence, 1988) and in repairproficient strains of Salmonella (DeMarini et al., 1983). Similar to m-AMSA, eilipticine is a potent chromosomal mutagen in mammalian cells (Moore et al., 1987c) and a potent poison of DNA topoisomerase II (Tewey et al., 1984a). In addition, biophysical studies have shown that ellipticine can intercalate into DNA (Schwaller et al., 1990). As with m-AMSA, there is no evidence that eilipticine poisons DNA gyrase, Based on studies in repair-proficient and-deftcient cells, ellipticine's genotoxicity in bacteria appears to be due to its ability to intercalate and/or form covalent adducts (DeMarini et al., 1983). However, as suggested by inability of actinomycin D to induce prophage, intercalation alone does not appear to be sufficient to produce an SOS-inducing signal. Thus, intercalation alone is unlikely to account for ellipticine's ability to induce prophage. As with m-AMSA however, ellipticine may also mediate the production of reactive-oxygen species that may produce SOS-inducing DNA damage. This combination of intercalation and the production of reactive-oxygen species (as opposed to poisoning of DNA gyrase) may account for the considerable ability of ellipticine to induce prophage (discussed later), Adriamycin was another potent inducer of prophage (Fig. 1), a finding that is consistent with previous studies (Elespuru and White, 1 9 8 3 ; Mamber et al., 1986; Nakamura et al., 1987; Salles et al., 1987). The addition of S9 enhanced the genotoxicity of adriamycin, and adriamycin is also mutagenic in bacteria (reviewed by Cebula, 1986). Adriamycin exhibits a variety of direct as well as indirect interactions with DNA, including intercalation and covalent binding (Waring, 1970;

Israel et al., 1982), poisoning of mammalian DNA topoisomerase II (Tewey et al., 1984b), and the production of free radicals that may cause DNA damage (Bachur et al., 1979). Adriamycin is a potent clastogen in mammalian cells (Moore et al., 1987"o),and its ability to poison DNA topoisomerase II may account for some of adriamycin's genotoxicity in mammalian cells. However, a series of detailed studies in bacteria and a review of the literature by Cebula (1986) suggest that the likely basis for adriamycin's genotoxicity in bacteria is due to DNA binding and/or intercalation that results in the production of free radicals that cause DNA damage. Using a series of agents that inhibit the generation of free radicals by adriamycin, Cebula (1986) showed that those agents that scavanged only the hydroxyl radical ( H O - ) did not inhibit adriamycin-induced mutant yields in Salmonella. AIthough these studies implicated the involvement of free radicals, they indicated that the mutagenic species generated by adriamycin may be something other than H O . . Based on his results as well as those of others, Cebula (1986) has suggested that adriamycin may bind to specific sites on DNA, including B - Z DNA junctions, and that SOS functions might then be induced by either a stalling of the repair enzymes at the adducted site or by the production of single-stranded DNA gaps at B-Z junctions, which are known to contain regions of single-stranded DNA. This mechanism, rather than any poisoning of DNA gyrase, may account for adriamycin's potent ability to induce prophage. These mechanisms may account for the enhanced potency of adriamycin relative to mAMSA. In addition, adriamycin might generate single-stranded DNA, which would elicit the SOS response and prophage induction, by inhibiting DNA and/or RNA polymerases (Dano et al., 1972; Goodman et al., 1974). Stalled transcription complexes might release single-stranded DNA, which could be a signal for SOS induction. AIthough less likely, inhibition of polymerases by adriamycin might also affect phage lysogeny by altering levels of lambda repressor.

Reactive-oxygenspecies In the present study, H202 and paraquat in-

12 duced prophage, confirming previous SOS studies with these agents (Oda et al., 1985; lmlay and Linn, 1987; Nakamura et al., 1987; yon der Hude et al., 1988; Goerlich et al., 1989; Nunoshiba and Nishioka, !991). An interesting exception was observed by Eder et al. (1989) who found that sf/A SOS functions were induced by H 202 but not by paraquat. In the present study, KO 2 did not induce prophage; we are unaware of any previous prophage- or SOS-induction studies with this agent. The mutagenicity of these three agents has been examined extensively in bacteria (Shirasu et al., 1981; Levin et al., 1982; De Flora et al., 1989; Glatt, 1989; Abu-Shakra and Zeiger, 1990). In the course of inducing SOS repair and mutation, reactive-oxygen species also induce the expression of a number of genes that defend cells against oxidative damage. H202, for example, generates hydroxyl radicals ( H O . ) and induces the synthesis of ~ 30 proteins, 9 of which are regulated by the oxyR locus; similarly, paraquot generates superoxide :~nions ( 0 2 ' - ) and induces the synthesis of ~ 40 proteins, 9 of which are regulated by the s e x R locus (reviewed by Storz et al., 1990). Perhaps the lesions produced by KO2, as opposed to those produced by H20 2 and paraquat, were repaired effectively by this inducible system, preventing an SOS-inducing signal (such as single-stranded DNA) from being formed. Another possible reason for the inability of KO 2 tO induce prophage may be due to the fact that the mierotiter plates were sealed in plastic wrap, which may have affected aeration such that generation of reactive-oxygen species was prevented, As Imlay and Linn (1988) have demonstrated, H202 can cause DNA damage through Fentontype reactions that produce the highly reactive H O . . The generation of this radical outside of the cell likely produces toxicity; whereas generation of this radical intracellularly on the surface of DNA may cause mutagenicity, possibly by breakage of the deoryribose ring after abstraction of a hydrogen atom from the ring by H O . . In the present study, H202 and paraquat were highly toxic to the cells in the absence of $9; however, the addition of $9 reduced the toxicity of these agents and enabled the induction of prophage to occur (Table 1). It is likely that the $9 provided

an additional target for much of the HO- produced extraceUularly, reducing the toxicity and permitting the genotoxic effects of this radical to be expressed and detected. DNA-strand breakage by reactive-oxygen species has been demonstrated in a variety of mutagenesis systems in bacteria and mammalian cells (Anderson and Phillips, 1989). In bacteria, Levin et al. (1984) have suggested DNA-strand breakage as the likely lesion responsible for reversion of Salmonella strain TA102 by H 2 0 2. Consistent with this suggestion is the recent finding by Abu-Shakra and Zeiger (1990) that H 2 0 2 induced a 10-fold increase (relative to the control) of intragenic deletion revertants of SalmoneUa strain TA104. In addition to DNA-strand breakage, HO. can hydroxylate DNA bases, which may also serve as SOS inducers. This has been confirmed by Shirname-More et al. (1987) who found that 5-hydroxymethyl-2'-deoxyuridine (HMUdR), which is also a product of ionizing radiation, is a potent inducer of prophageoin the microscreen assay. Consistent with this is the finding that HMUdR induces base-pair substitutions in Salmonella strains TA1O0 (Bilimoria and Gupta, 1986) and TAI04 (Abu-Shakra, unpublished results), In addition to oxyradicals, a variety of redoxactivating d~'~,~ including the DNA topoisomerase 11 poisons (adriamycin m-AMSA and elliptieine), can mediate toxicity and, possibly, mutagenicity by producing DNA damage through redox reactions with transition metals (reviewed by lmlay and Linn, 1988). In prokaryotes, nalidixic acid, for example, has been shown to induce oxidative stress proteins (VanBogelen et al., 1987), suggesting that in addition to poisoning DNA gyrase, it may also generate reactive-oxygen species. This may be an especially important mode of action by adriamycin, m-AMSA, and ellipticine in mammalian cells because, in addition to serving as electron donors for Fenton reactions, they also can bind tightly within the DNA-drug-DNA topoisomerase II complex, ensuring that oxidants are generated on or near the DNA surface. The production of reactive-oxygen species by these agents may also be an important mechanism by which they induce prophage because even though

13 these drugs do not show any apparent specificity for DNA gyrase, they can intercalate, which would place them in intimate contact with DNA while they generate DNA-damaging, reactive-oxygen species, Conclusions

The induction of prophage by DNA gyrase subunit A poisons (nalidixic acid and oxolinic acid), DNA intercalating agents that also produce reactive-oxygen species (adriamycin, m-AMSA and ellipticine), and agents that generate reactire-oxygen species (H202 and paraquat) illustrates the variety and (relative effectiveness) of mechanisms by which the SOS response can be induced. In addition, the inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicates that inhibition of the gyrase's ATPase is insufficient to elicit the SOS response, Likewise, the ability of an agent to intercalate but not generate reactive-oxygen species (actinomycin D) is insufficient to induce prophage. An agent that neither intercalates nor produces reactiveoxygen species (teniposide) is also unable to induee prophage, even though it is a potent poison of mammalian DNA topoisomerase II. Although the genotoxic agents described here may operate by diverse mechanisms and produce various types of DNA damage, the production of DNA strand breaks, which are a strong inducing signal for SOS repair and prophage induction, may be a type of DNA damage common to all of these agents. Studies of prophage-induction by these and related agents in the presence of inhibitors of reactive-oxygen species, such as those used in similar studies of bacterial mutagenicity (Cebula, 1986; De Flora et al., 1989), might help to demonstrate whether the generation of reactive-oxygen species by various agents examined here play an essential role in the ability of these agents to induce prophage. In addition, an examination of the abilities of the DNA topoisomerase poisons to induce the SOS response in special strains of E. coli, such as those containing a deletion of the oxyR gene, would help to determine whether these agents produce reactiveoxygen species that, in turn, induce SOS repair (Goerlich et al., 1989; Quillardet et al., !989).

An interesting aspect of the results presented here is that, with the exception of oxolinic acid, all of the agents that induced prophage lambda either required $9, or their genotoxic potencies were enhanced by the addition of $9 (Fig. 1). This remarkable dependency on $9 is unexpected for many of the agents, especially for H202, which are direct-acting in other systems, especially mammalian cells. Some of the requirement for $9 may have been due to the long exposure time (overnight, ~ 20 h), which resulted in any free virus particles to be "bathed" in the agent for an extensive period of time. This may have inactirated free virus particles, preventing them from being infective in the plaque assay. Thus, the requirement for infective particles, and the long incubation period, may be two aspects of the microscreen assay that (1) prevented the detection of genotoxic activity by many of the agents in the absence of $9 and (2) made such detection possible only in the presence of $9. This may have been because the $9 served a protective effect, i.e., reactive species bound to the $9, permitring free virus particles to remain undamaged and infective. Finally, it is interesting to note that in mammalian cells, the pattern of mutational damage induced by DNA topoisomerase poisons is frequently similar to that induced by ionizing radiation and reactive-oxygen species. For example, the induction of high frequencies of chromosomal aberrations (CAs) but low frequencies of sisterchromatid exchanges (SCEs) in mammals in vivo by m-AMSA and camptothecin (a DNA topoiosmerase I poison) resembles that of ionizing radiation (Backer et al., 1990). Likewise, the induction of high frequencies of small-colony TK - / - murants and CAs but low frequencies of large-colony TK - / - mutants in mouse lymphoma cells by mAMSA is similar to the pattern produced by ionizing radiation, H 202 , and KO 2 (Moore et al., 1987a;Evans et al., 1986, 1987, 1989; DeMarini et al., 1989). These results illustrate a unifying feature of the genotoxicity of agents that induce DNA-strand breaks. With some exceptions, such agents tend to be highly clastogenic in mammalian cells and strong inducers of prophage, but they are weak gene mutagens in both eukaryotic and prokary-

14 otie systems. T h u s , it m a y b e that for agents that p r o d u c e D N A b r e a k s via a certain subset o f all

possible mechanisms, an SOS or prophage-induction assay m i g h t d e t e c t the genotoxicity o f such agents as well as a cytogenetic assay. Finally, the e x t r e m e potency of DNA topoisomerase poisons in t e r m s o f their ability to induce c h r o m o s o m a l a b e r r a t i o n s in m a m m a l i a n cells a n d p r o p h a g e

induction in E. coil demonstrates the considerable genotoxic consequences of perturbing important DNA-protein complexes, especially those formed by D N A a n d D N A topoisomerase. Acknowledgements

This paper was reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use,

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Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: role of DNA breaks.

Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay. The inability of ...
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