Frameshift mutations induced by four isomeric

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Environmental and Molecular Mutagenesis 47:82^94 (2006)

Frameshift Mutations Induced by Four Isomeric Nitroacridines and Their des-Nitro Counterpart in the lacZ Reversion Assay in Escherichia coli George R. Hoffmann,1* Catherine C.Yin,1 Caitlin E. Terry,1 Lynnette R. Ferguson,2 and William A. Denny2 1

Department of Biology, College of the Holy Cross, Worcester, Massachusetts, USA 2 Cancer Research Laboratory, University of Auckland Medical School, Private Bag 92019, Auckland 1000, New Zealand Acridines are well-known as compounds that intercalate noncovalently between DNA base pairs and induce 61 frameshift mutations at sites of monotonous repeats of a single base. Reactive derivatives of acridines, including acridine mustards and nitroacridines, form covalent adducts in DNA and exhibit mutagenic properties different from the simple intercalators. We compared the frameshift mutagenicity of the cancer chemotherapy drug nitracrine (1-nitro-9-(30 -dimethylaminopropylamino)-acridine), its des-nitro counterpart 9-(30 -dimethylaminopropylamino)-acridine (DAPA), and its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA) in the lacZ reversion assay in Escherichia coli. DAPA is a simple intercalator, much like the widely studied 9-aminoacridine. It most strongly induced 61 frameshift mu-

tations in runs of guanine residues and more weakly induced –1 frameshifts in a run of adenine residues. A nitro group in the 1, 3, or 4 position of DAPA reduced the yield of 61 frameshift mutations. DAPA weakly induced –2 frameshifts in an alternating CG sequence. In contrast, nitracrine and its 3-nitro isomer resembled the 3-nitroacridine Entozon in effectively inducing –2 frameshift mutations. The 2- and 4-nitro isomers were less effective than the 1- and 3-nitro compounds in –2 frameshift mutagenesis. The results are interpreted with respect to intercalation, steric interactions, effects of base strength on DNA binding, enzymatic processing, and a slipped mispairing model of frameshift mutagenesis. Environ. Mol. Mutagen. 47:82–94, 2006. VC 2005 Wiley-Liss, Inc.

Key words: acridine; frameshift; lacZ; slipped mispairing; nitroacridine; nitracrine; ledakrin; Entozon

INTRODUCTION Acridines are model frameshift mutagens whose genetic effects have been reported for phage, bacteria, fungi, insects, plants, cultured mammalian cells, and mammals [Ferguson and Denny, 1990, 1991]. Acridines can be classified into those whose mutagenicity stems from noncovalent interactions with DNA, most notably intercalation, and those that have reactive substituents that permit covalent reaction with DNA. Intercalation entails insertion of the planar ring system into DNA parallel to the base pairs [Lerman, 1961; Neidle and Abraham, 1984]. In contrast, reactive acridines, including acridine mustards and nitroacridines, form covalent addition products in DNA, and they differ from simple intercalating agents with respect to the potency of their mutagenicity and the spectrum of mutations induced [Brown et al., 1980; McCoy et al., 1981; Ferguson and Denny, 1991; Hoffmann et al., 1996, 2003]. The boundary between the two classes is not sharp, in that reactive acridines can both intercalate and form adducts. The predominant mode of action of nitroacridines is influenced by the position of the nitro group and C V

2005 Wiley-Liss, Inc.

its susceptibility to nitroreductase activity. The reduction potential of a nitro group at the 1, 3, or 4 position of acridine permits efficient nitroreduction, which is required for covalent reaction with DNA [Ferguson and Denny, 1991]. Nitracrine, also called ledakrin, is a cancer chemotherapy drug that has received extensive study related to antitumor activity, toxicity, and interactions with DNA [Stezowski et al., 1985; Ferguson and Denny, 1991; Gniazdowski and Szmigiero, 1995]. It has been used in Poland to treat ovarian, colon, lung, and mammary carcinomas, but its toxicity and clinical side effects have limited

*Correspondence to: George R. Hoffmann, Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395, USA. E-mail: [email protected] Received 5 April 2005; provisionally accepted 11 May 2005; and in final form 25 June 2005 DOI 10.1002/em.20171 Published online 22 September 2005 in Wiley InterScience (www.interscience. wiley.com).

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Fig. 1. Structures of nitracrine (1-nitro-9-(30 -dimethylaminopropylamino)-acridine; ledakrin; 1-nitro-DAPA); DAPA (9-(30 -dimethylaminopropylamino)-acridine); the 2-, 3-, and 4-nitro isomers of nitracrine (2-nitroDAPA, 3-nitro-DAPA, and 4-nitro-DAPA); 9-aminoacridine; ICR-191; and Entozon.

its use [Gniazdowski and Szmigiero, 1995]. Despite the limitations, nitracrine and related compounds remain of interest, in part, because of evidence that they are selectively toxic under hypoxic conditions and can function as radiosensitizers [Wilson et al., 1984; Roberts et al., 1990; Ferguson and Denny, 1991; Gniazdowski and Szmigiero, 1995]. Nitracrine (1-nitro-DAPA), its 2-, 3-, and 4-nitro isomers, and their des-nitro counterpart 9-(30 -dimethylaminopropylamino)-acridine (DAPA) can serve as model compounds in studies of frameshift mutagenesis. In the Ames assay in Salmonella enterica (serovar Typhimurium), DAPA is mutagenic only in strain TA1537, whose hisC3076 allele has a monotonous run of guanine residues at the target site. Nitracrine and its isomers also revert the hisD3052

allele, which has an alternating CG target sequence, in strains TA1538 and TA98 [Kalinowska and Chorazy, 1980; Ferguson et al., 1987]. Another nitroacridine, Entozon, which reverts hisD3052 more effectively than hisC3076 in the Ames assay [McCoy et al., 1981], is a potent inducer of 2 frameshift mutations in Escherichia coli, both in the lacZ reversion assay [Hoffmann et al., 2003] and in a reversion assay based on the tetracyclineresistance gene (tet) of pBR322 [Hoffmann et al., 1996]. The lacZ reversion assay in E. coli permits the measurement of mutation frequencies on the basis of a positive selection for reversion from the Lac to Lacþ phenotype. The chromosomal lac operon of the tester strains is deleted, and the target alleles are on F0 episomes [Cupples

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and Miller, 1989; Cupples et al., 1990; Josephy, 2000]. The episomal allele in each strain reverts by a single mutational mechanism, and selection for Lacþ thereby allows the specific identification of each of the six base-pair substitutions and of frameshift mutations in different sequence contexts. In this study, we measured the frameshift mutagenicity of the 1-nitroacridine compound nitracrine, its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA), and DAPA in the lacZ reversion assay, and we compared their activity to that of the 3-nitroacridine Entozon. MATERIALS AND METHODS Chemicals Nitracrine dihydrochloride (ledakrin; 1-nitro-9-(30 -dimethylaminopropylamino)-acridine diHCl; CAS 6514-85-8), its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA), and 9-(30 -dimethylaminopropylamino)-acridine (DAPA) were synthesized in the Auckland Cancer Society Research Centre, University of Auckland, New Zealand, using published methods [Ledo´chowski et al., 1964; Ledo´chowski and Stefanska, 1966; Ferguson et al., 1987; Denny et al., 1990]. Entozon dihydrochloride (CAS 7397250-6), originally from Hoechst AG, Frankfurt/Main, Germany, was a gift of Dr. Herbert S. Rosenkranz, Florida Atlantic University. The structures of DAPA, its nitro analogs, and related compounds are shown in Figure 1. Stock solutions at 2048 lM were frozen at 208C with no diminution of biological activity over several months.

Bacterial Strains Escherichia coli strains CC107, CC108, CC109, and CC111 (ara D(lac pro)/F0 lacI lacZ proBþ) were obtained from Dr. Claire G. Cupples, University of Victoria, Victoria, BC, Canada. These strains detect þG (G6 ? G7), G (G6 ? G5), CG (CG5 ? CG4), and A (A7 ? A6) frameshift mutations, respectively [Cupples et al., 1990].

Media Vogel–Bonner citrate medium [Vogel and Bonner, 1956], also called Medium E, containing lactose as the sole carbon source (VBCL), was used to select for Lacþ revertants. Its composition was 0.2% a-lactose, 15 lM thiamine, and 1.8% Difco Bacto Agar. Numbers of viable cells were determined on the same medium containing 0.5% D-(þ)-glucose instead of lactose (VBCG). Acridine treatments were conducted in liquid LB medium [Sambrook et al., 1989].

Mutation Assay Single colonies were picked from streaks on LB or VBCG plates and cultured to stationary phase in liquid VBCG without proline to select for the presence of the episome. Bacteria were spread on VBCL to determine the spontaneous revertant frequency while the original cultures were held at 48C. Spontaneous revertant frequencies did not vary widely, and all experiments were conducted using cultures whose spontaneous frequencies were consistent with our historical controls and published values [Cupples et al., 1990]. Bacteria were subcultured in liquid LB in a shaker for 16 hr, and 10 ll aliquots were inoculated into 1 ml LB containing acridine compounds. LB was used for the treatments because nitroreductase activity may be greater in rich medium than in minimal medium [Kranendonk et al., 1997]. After 16 hr at 378C, the treatments were quenched by adding 10 ml cold 0.9% NaCl. The bacteria were centrifuged and resuspended in 1 ml cold 0.9% NaCl for plating.

Approximately 2 3 108 cells were spread on each plate of VBCL to select for revertants, and dilutions giving about 150 colonies per plate were spread on VBCG to quantify viable cells. Plating was in triplicate, and colonies were counted after 48–54 hr at 378C. The significance of differences in mean numbers of revertants per plate and in revertant frequencies was determined by analysis of variance, and slopes of doseresponse curves were determined by linear regression analysis.

RESULTS The induction of þG frameshift mutations by nitracrine (1-nitro-DAPA), its 2-, 3-, and 4-nitro analogs, and DAPA in E. coli strain CC107 is shown in Table I. The data show that the nitro compounds all induced þG frameshifts in a run of guanine residues (G6 ? G7), but their activity tended to be less than that of DAPA. The potency of the responses was quantified in two ways: slopes calculated from the upward portion of the dose response curves, and the yield of revertants (maximum number of revertants per 108 cells plated minus that of the untreated concurrent control). An array of slopes is presented in Figure 2. In calculating slopes, we used absolute numbers of revertants, rather than revertant frequencies per survivor, so that the slopes are not strongly influenced by variation in the quantification of viable cells at toxic doses. The yield of revertants induced is shown in Figure 3. The calculation of mutagenic potency as a yield of induced revertants deemphasizes the absolute concentration at which a chemical is effective, considering only the maximum mutagenic effect independently of the concentration at which it occurs. An alternative calculation of potency as induced revertants at a constant level of toxicity gives a pattern of responses that is highly correlated with our calculation of yield (r > 0.98) and supports the same interpretations. The nitro compounds produced a smaller absolute yield of revertants than DAPA in strain CC107 (Table I; Fig. 3). The reduction in þG mutagenicity caused by the nitro group was greatest for nitracrine and 3-nitro-DAPA and least for 2-nitro-DAPA. The same trend is evident in the slopes of the dose-response curves (Fig. 2), except that the slope for nitracrine is large owing to its activity at very low concentration. Table II shows that the nitro analogs all induced G frameshift mutations in the repetitive guanine residues of the target sequence in strain CC108 (G6 ? G5). The yield of revertants (Fig. 3) was much reduced for nitracrine and 3-nitro-DAPA relative to DAPA, even though nitracrine acted at low concentrations. The 2-nitro and 4-nitro isomers retained substantial activity but at higher concentrations than DAPA. The induction of –CG frameshift mutations by DAPA and its nitro derivatives in strain CC109 is shown in Table III. The target sequence in this strain is a run of alternating cytosine and guanine residues, and revertants arise by the loss of a single unit of the repeat

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TABLE I. Induction of þG Frameshift Mutations by DAPA, Nitracrine, and the 2-, 3-, and 4-Nitro Isomers of Nitracrine in Strain CC107 of the E. coli lacZ Reversion Assay

Chemical DAPA

Nitracrine (1-nitro-DAPA)

2-nitro-DAPA

3-nitro-DAPA

4-nitro-DAPA

Dosage (lM)

Relative cell density

0 2 4 8 16 32 0 0.03125 0.0625 0.125 0.25 0.5 0 4 8 16 32 64 128 0 2 4 8 16 32 0 8 16 32 64 128

1.00 0.71 0.77 0.63 0.50 0.32 1.00 0.55 0.37 0.37 0.30 0.06 1.00 0.92 0.97 0.80 0.45 0.25 0.05 1.00 0.80 0.75 0.80 0.48 0.38 1.00 1.10 1.00 0.48 0.47 0.19

Lacþ revertants per plate (6SEM)a 40.3 59.5 111.0 358.0 3306.3 4097.3 49.0 61.3 75.7 76.7 68.3 27.0 32.7 67.7 95.3 204.3 266.0 1391.3 1029.3 38.7 38.0 43.0 60.7 81.0 89.7 37.7 43.0 42.3 64.0 106.7 156.7

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

4.3 10.5 10.1 17.6 279.9 217.2 2.1 4.3 1.2 2.0 9.4 1.5 0.88 2.2 7.7 43.7 18.2 172.8 110.1 5.2 5.3 2.1 2.2 8.6 7.5 6.3 7.6 6.4 4.6 5.6 5.0

Revertant frequency 6 SEM (Lacþ colonies/ 108 viable cells) 18.9 39.2 67.3 266.6 3090.0 5964.1 22.8 52.3 94.9 97.7 107.6 195.6 20.9 46.9 62.8 162.6 381.6 3504.6 12866.7 24.9 30.6 36.8 48.6 109.0 152.5 29.4 30.5 33.7 105.4 178.4 650.1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.00 6.9 1.3 12.0 261.6 316.2 1.0 3.6 1.5 2.6 14.7 11.1 0.56 1.5 5.1 34.8 26.0 435.4 1376.3 3.4 4.3 1.8 1.8 11.6 12.8 5.0 5.4 5.0 7.6 9.4 20.6

a The slopes of all the dose-response curves (revertants/108 cells plated/lM) are significantly greater than zero. Revertant frequencies and revertants per plate are significantly increased for all compounds (ANOVA; P < 0.01).

(CG5 ? CG4). Compared with DAPA, nitracrine and 3nitro-DAPA were stronger inducers of 2 frameshift mutations, both on the basis of slopes of dose-response curves and of absolute yields of revertants. The 4-nitro isomer showed little mutagenic activity, detectable only as a modest increase in revertant frequency at the highest concentration. Data on the induction of A frameshift mutations in a run of adenine residues (A7 ? A6) in strain CC111 are shown in Table IV. Only DAPA and its 2-nitro derivative effectively induced –A frameshifts, as indicated both by slopes of dose-response curves and by statistically significant increases in yields of revertants and revertant frequencies. Revertant frequencies were also significantly increased at the highest doses of nitracrine and 4-nitroDAPA, suggesting a weak mutagenic effect at toxic doses. The slopes in Figure 2 suggest that nitracrine is the most potent mutagen of the series. This apparent potency rests on its being mutagenic at a much lower concentration than the other compounds. Compared with DAPA, its 3-

nitro derivative shows more potent induction of 2 frameshifts. The 2-, 3-, and 4-nitro compounds all show reduced potency in the induction of 61 frameshifts, compared with DAPA. The yields of revertants in Figure 3 show the 1nitro and 3-nitro acridines to have heightened potency of induction of 2 frameshifts, compared with DAPA. These analogs also show the greatest reduction in the induction of 61 frameshifts. The 2-nitro and 4-nitro compounds more closely resemble the simple intercalator DAPA. Comparative data for Entozon, which is a 3-nitroacridine compound, are shown in Table V. The spectrum of revertants induced by Entozon differed sharply from that of DAPA, in that Entozon was a relatively weak inducer of 61 frameshift mutations but a potent inducer of 2 frameshifts. This pattern resembles that of 3-nitro-DAPA, but Entozon is a stronger mutagen. Figure 4 shows the ratio of 2 (–CG) to 1 (G) frameshifts induced by DAPA, its nitro derivatives, and Entozon. Results for the simple intercalators 9-aminoacridine (9AA) and quinacrine

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Fig. 2. Potency of mutagenic responses to nitracrine, its structural isomers (2-, 3-, and 4-nitro-DAPA), and DAPA measured as slopes of dose-response curves in the lacZ reversion assay in E. coli. Mutagenic potency is presented as a mean of the slopes of the ascending portion of dose-response curves from replicate experiments. In all cases, except

where noted NS, linear regression analysis showed the slopes to differ significantly from zero (P < 0.05), and there was a significant increase in absolute numbers of revertants. Slopes are given as numbers of revertants/ 108 cells plated/lM.

and for the acridine mustards ICR-191 and quinacrine mustard are included for comparison [Hoffmann et al., 2003]. The most striking difference from the simple intercalators (DAPA, 9AA, quinacrine) is a shift in the revertant spectrum to more 2 frameshifts induced by the acridine mustards and especially the 1- and 3-nitroacridines.

pounds and DAPA were inactive in assays for gene mutations in V79 cells, whereas nitracrine and 4-nitro-DAPA induced mutations conferring thioguanine-resistance [Ferguson and van Zijl, 1988]. The 1- and 4-nitro isomers were potent inducers of micronuclei [Ferguson and van Zijl, 1988], whereas the 2- and 3-nitro isomers gave inconclusive results in tests for chromosomal damage in V79 cells [Ferguson and Denny, 1991]. These results are consistent with the formation of covalent adducts in DNA by the 1 and 4 isomers [Ferguson and Denny, 1991]. The induction of 2 frameshifts by nitracrine in the lacZ assay (Table III) is consistent with its acting as a reactive acridine rather than a simple intercalator, but a comparable pattern was not observed for the 4-nitro isomer. In the Ames assay, DAPA was mutagenic only in strain TA1537, which is highly responsive to simple intercalators, whereas nitracrine and its isomers were more mutagenic in hisD3052 strains TA1538 and TA98 [Kalinowska and Chorazy, 1980; Ferguson et al., 1987]. The tendency to revert the hisD3052 allele is ascribable to the nitro group. The activity in TA98 was greater for nitracrine and its 3-nitro isomer than for the 2- and 4-nitro isomers [Ferguson et al., 1987]. Consistent with their acting as

DISCUSSION Nitracrine and its analogs have been tested for genotoxicity in bacteria, cultured mammalian cells, and yeast [Ferguson and Denny, 1991]. In E. coli, nitracrine strongly reverted the frameshift allele lacZ19124 [MacPhee and Liaskou, 1989]. The mutagenicity was RecA-dependent and enhanced by defective excision repair. It was also enhanced by mucAB in pKM101, but was independent of the umuC gene product [MacPhee and Liaskou, 1989], suggesting differential effects of PolRI and PolV, as have been reported in other studies [O’Grady et al., 2000; Lambert et al., 2001]. The 1- and 4-nitro isomers were potent recombinagens in yeast, whereas the 2- and 3-nitro isomers were weakly active, and DAPA was inactive or nearly so [Ferguson and Turner, 1987b]. The 2- and 3-nitro com-

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Fig. 3. Potency of mutagenic responses to nitracrine, its structural isomers (2-, 3-, and 4-nitro-DAPA), and DAPA measured as the absolute yield of revertants at the most mutagenic concentration in the lacZ reversion assay in E. coli. Revertant yield is given as a mean of maximum

numbers of induced revertants per 108 cells treated in replicate experiments (i.e., total minus spontaneous revertants). Significant increases in absolute numbers of revertants and in the slopes of the dose-response curves were detected in all cases, except where noted NS.

reactive acridines, the 1-, 3-, and 4-nitro compounds were more potent mutagens in uvrB bacteria than in repairproficient bacteria [Ferguson and Turner, 1987a]. Like nitracrine, Entozon reverted the hisD3052 allele more effectively than it reverted hisC3076 [McCoy et al., 1981]. Both 3-nitro-9AA and 2-nitro-9AA were mutagenic in TA1538, the 3-nitro compound being more potently so, and they were less mutagenic in TA1537 than 9AA, which gave no mutagenic response in TA1538 [Brown et al., 1980]. TA98 was more responsive than TA1537 to 3nitroacridine with no substituent on the 9-position, while TA78, an E. coli strain with a run of six adenine residues at the mutant site, responded only weakly [Ferguson et al., 1985]. The mutagenicity of nitracrine and 2-nitro-DAPA was strongly reduced in the nitroreductase-deficient strain TA98NR, whereas that of the 3- and 4-nitro isomers was only modestly reduced [Ferguson et al., 1987]. Our results for DAPA and its nitro derivatives in the lacZ assay are compatible with those in the Ames assay. The two assays are not equivalent, however, because the his alleles in the Ames strains revert by more than one mechanism. The reversion of hisC3076 corresponds

roughly to the induction of G frameshifts in lacZ strain CC108, in that G is the principal class of hisC3076 revertants and accounts for about 90% of those induced by 9AA [Cebula and Koch, 1990]. The correspondence for hisD3052 is less clear. The hisD3052 allele resembles CC107 in containing a 1 frameshift, but spontaneous revertants of hisD3052 include þ1 frameshifts, 2 frameshifts, other small deletions, and complex mutations [DeMarini et al., 1998]. The spectrum differs with mutagens, in that hisD3052 revertants induced by N-2-acetylaminofluorene (AAF) contain 2 frameshifts but not þ1 frameshifts [Shelton and DeMarini, 1995]. In the Ames assay, 1-nitroacridines and 3-nitroacridines [Brown et al., 1980; Ferguson et al., 1987], including Entozon [McCoy et al., 1981], revert the hisD3052 allele more effectively than hisC3076. A nitro group at the 1 or 3 position enhances the toxicity of 9AA [Brown et al., 1980], decreases its mutagenicity in TA1537, and leads to mutagenicity in TA1538 [Brown et al., 1980]. The pattern of mutations induced by DAPA in the lacZ assay (Figs. 2 and 3) resembles that induced by 9AA and quinacrine, which are also nonreactive intercalators

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Hoffmann et al. TABLE II. Induction of G Frameshift Mutations by DAPA, Nitracrine, and the 2-, 3-, and 4-Nitro Isomers of Nitracrine in Strain CC108 of the E. coli lacZ Reversion Assay

Chemical DAPA

Nitracrine (1-nitro-DAPA)

2-nitro-DAPA

3-nitro-DAPA

4-nitro-DAPA

Dosage (lM)

Relative cell density

0 2 4 8 16 32 0 0.03125 0.0625 0.125 0.25 0.5 0 4 8 16 32 64 128 0 2 4 8 16 32 0 8 16 32 64 128

1.00 0.83 0.90 0.67 0.74 0.50 1.00 0.96 0.94 0.67 0.49 0.19 1.00 0.85 0.81 0.94 0.62 0.41 0.10 1.00 1.13 0.89 1.10 0.85 0.55 1.00 0.94 0.77 0.93 0.65 0.38

Lacþ revertants per plate (6SEM)a 99.0 140.0 221.7 858.3 4761.0 5058.7 62.7 95.0 120.3 147.0 170.7 121.7 58.0 76.0 100.7 222.3 966.3 5297.0 7526.3 17.7 38.0 62.3 106.3 209.7 511.7 33.7 49.3 81.3 153.0 706.7 2698.7

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

14.5 10.7 14.2 159.4 280.9 585.4 12.2 6.8 8.3 3.6 17.3 14.3 7.1 3.8 13.4 6.9 80.0 254.8 93.9 1.4 3.2 2.2 16.2 9.5 38.9 5.6 6.6 13.8 11.6 53.1 153.6

Revertant frequency 6 SEM (Lacþ colonies/ 108 viable cells) 39.9 67.7 99.4 514.9 2573.5 4024.4 28.3 44.4 58.0 99.3 156.1 293.2 26.5 40.9 57.0 108.6 710.5 5866.0 34683.6 9.3 17.8 37.0 51.2 130.2 493.4 16.9 26.5 53.1 82.6 549.1 1530.7

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

5.8 5.2 6.4 95.6 151.8 465.7 5.5 3.2 4.0 2.4 15.8 34.6 3.2 2.0 7.6 3.4 58.8 282.2 432.7 1.0 1.5 1.3 7.8 5.9 37.5 2.8 3.5 9.0 6.3 41.3 87.1

a The slopes of all the dose-response curves (revertants/108 cells plated/lM) are significantly greater than zero. Revertant frequencies and revertants per plate are significantly increased for all compounds (ANOVA; P < 0.01).

[Hoffmann et al., 2003]. There is a predominance of 1 and þ1 frameshifts in a run of guanine residues. In previous studies, we found that the nitroacridine Entozon differs from simple intercalating acridines and acridine mustards in being an unusually potent inducer of 2 frameshifts in strain CC109 of the lacZ assay [Hoffmann et al., 2003] and in strain BR871 of a specific reversion assay based on the tet gene of plasmid pBR322 [Hoffmann et al., 1996]. It also differed by being less active in inducing 61 (G) frameshifts [Hoffmann et al., 2003]. Like Entozon, the 1-, 3-, and 4-nitro derivatives of DAPA tend to be weaker inducers of 61 frameshifts than are simple intercalators (Figs. 2 and 3). Properties of nitroacridines that are important in their biological effects are the reduction potential of the nitro group [Ferguson and Denny, 1991; Gniazdowski and Szmigiero, 1995], altered basicity [Ferguson and Denny, 1991], and steric interactions between position 1 and the side chain, resulting in changes in the amino-imino tautomeric equilibrium [Gniazdowski and Szmigiero, 1995;

Stezowski et al., 1995]. We will attempt to relate the patterns of mutagenicity that we observed (Figs. 2 and 3) to these factors. The first step in the activation of nitroaromatic and nitroheterocyclic compounds into mutagens is nitroreduction. The major oxygen-insensitive nitroreductase is encoded by the nfsA gene in E. coli and by snrA in Salmonella enterica (serovar Typhimurium) [Carroll et al., 2002; Nokhbeh et al., 2002]. The SnrA enzyme is present in some Ames tester strains (e.g., TA1535), but it is lacking in strains TA1538 and TA98 [Yamada et al., 1997; Nokhbeh et al., 2002]. The flavin-dependent classical nitroreductase of Salmonella, which is present in TA1538 and TA98, is encoded by the cnr gene and is homologous with the E. coli minor oxygen-insensitive nitroreductase encoded by nfsB [Yamada et al., 1997; Carroll et al., 2002; Nokhbeh et al., 2002]. Decreased mutagenicity of nitro compounds in strains TA1538NR and TA98NR is ascribable to a lack of Cnr, the classical nitroreductase [Ferguson et al., 1987; Josephy et al., 1997; Watanabe et al., 1998].

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TABLE III. Induction of CG Frameshift Mutations by DAPA, Nitracrine, and the 2-, 3-, and 4-Nitro Isomers of Nitracrine in Strain CC109 of the E. coli lacZ Reversion Assay

Chemical DAPA

Nitracrine (1-nitro-DAPA)

2-nitro-DAPA

3-nitro-DAPA

4-nitro-DAPA

Dosage (lM)

Relative cell density

0 4 8 16 32 0 0.03125 0.0625 0.125 0.25 0 16 32 64 128 0 2 4 8 16 0 16 32 64 128

1.00 1.02 1.03 0.80 0.54 1.00 0.86 0.77 0.56 0.30 1.00 0.82 0.94 0.55 0.24 1.00 0.96 1.23 0.86 0.64 1.00 1.11 0.77 0.87 0.35

Lacþ revertants per plate (6SEM)a 131.7 129.7 133.0 215.0 228.3 127.3 342.0 531.7 635.3 439.3 138.0 150.0 203.0 345.3 324.0 110.0 165.3 296.3 330.7 482.3 126.3 121.3 116.0 126.0 100.0

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.9 4.9 5.2 49.0 18.6 8.2 8.1 12.6 15.8 6.7 2.0 4.6 6.7 13.2 18.6 8.7 6.0 12.9 8.9 14.4 10.2 4.2 9.7 11.9 9.0

Revertant frequency 6 SEM (Lacþ colonies/ 108 viable cells) 59.0 56.9 57.6 119.9 188.7 57.7 179.3 314.0 512.4 665.7 69.4 92.2 108.7 316.8 693.8 66.5 109.5 145.5 231.7 454.2 71.8 62.1 85.5 82.2 161.3

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.4 2.2 2.2 27.3 15.3 3.7 4.2 7.4 12.7 10.1 1.0 2.8 3.6 12.1 39.8 5.3 3.3 6.3 6.2 13.6 5.8 2.1 7.2 7.8 14.5

a The slopes of the dose-response curves (revertants/108 cells plated/lM) are significantly greater than zero for all compounds, except 4-nitro-DAPA. Revertant frequencies corrected for toxicity (revertants/108 viable cells) show highly significant increases for all five compounds (ANOVA, P < 0.01). The differences in absolute numbers of revertants per plate are nonsignificant for 4-nitro-DAPA, significant for DAPA (P < 0.05), and highly significant (P < 0.01) for the other compounds.

The toxicity and mutagenicity of nitroaromatic compounds depend on efficient reduction, ultimately to nitroso [Siim et al., 1997] or arylhydroxylamine [Josephy et al., 1997] derivatives, which are interconvertible, mutagenic, and susceptible to further metabolism into other mutagenic derivatives [Josephy, 1989]. N-acetoxyesters derived from arylhydroxylamines also generate highly reactive nitrenium ions [Josephy, 1989; Gorlewska et al., 2001]. The 1-, 3-, and 4-nitro isomers have higher reduction potentials than the 2-nitro isomer, permitting more rapid enzymatic reduction [Ferguson and Denny, 1991]. Nitroreduction precedes the covalent binding of nitracrine to DNA and proteins [Gniazdowski and Szmigiero, 1995]. The induction of 2 frameshifts by nitracrine and 3-nitroDAPA (Table III) and by Entozon (Table V) can be ascribed to reactive intermediates that bind to DNA covalently, but the specific nitroreductases involved are uncertain. Entozon exhibits full mutagenic activity in TA98NR [McCoy et al., 1981], which lacks both the major (SnrA) and minor (Cnr) oxygen-insensitive nitroreductases [Carroll et al., 2002; Nokhbeh et al., 2002]. Results in Salmonella strains TA98NR and TA98 suggest that the classical nitroreductase (Cnr) is effective in activating nitracrine but less

so in the case of 3-nitro-DAPA [Ferguson et al., 1987]. The contribution of the major and minor E. coli oxygeninsensitive nitroreductases to the mutagenicity of nitroacridines in the lacZ assay cannot be resolved on the basis of results in Salmonella strains bearing or lacking homologs of these enzymes. Moreover, the possibility that an oxygensensitive nitroreductase plays a role in the activation of nitroacridines is suggested by the fact that activation of nitracrine is inhibited by oxygen, thus making it a hypoxiaselective toxicant [Siim et al., 2000]. The comparative mutagenicity data from E. coli and Salmonella suggest that the nitroacridines would be useful model compounds for further studies of metabolism by different oxygen-sensitive and oxygen-insensitive nitroreductases. In mammalian cells, nitracrine differed from its 2- and 4-nitro analogs in being cytotoxic and producing adducts recognizable by postlabeling [Bartoszek and Konopa, 1989]. The unique biological properties of nitracrine appear to stem more from the reactivity of reduced intermediates than from susceptibility to nitroreduction [Gorlewska et al., 2001]. Nitracrine binds covalently only to purine nucleotides, and binding to poly(dG) was about 40-fold greater than binding to poly(dA) [Bartoszek et al., 1997a]. It forms

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Hoffmann et al. TABLE IV. Induction of A Frameshift Mutations by DAPA, Nitracrine, and the 2-, 3-, and 4-Nitro Isomers of Nitracrine in Strain CC111 of the E. coli lacZ Reversion Assay

Chemical DAPA

Nitracrine (1-nitro-DAPA)

2-nitro-DAPA

3-nitro-DAPA

4-nitro-DAPA

Dosage (lM)

Relative cell density

0 2 4 8 16 32 0 0.03125 0.0625 0.125 0.25 0.5 0 4 8 16 32 64 128 0 2 4 8 16 32 0 8 16 32 64 128

1.00 1.35 0.87 1.09 0.89 0.64 1.00 0.81 0.64 0.55 0.46 0.10 1.00 0.96 1.11 0.84 0.66 0.32 0.21 1.00 0.97 0.83 0.85 0.57 0.36 1.00 1.02 0.99 0.92 0.45 0.33

Lacþ revertants per plate (6SEM)a 11.3 8.0 18.3 12.3 24.7 71.0 10.0 15.0 18.3 20.7 20.3 12.0 16.3 12.3 14.3 14.7 22.7 57.0 178.3 19.3 9.0 16.0 12.3 18.3 11.0 11.3 8.7 18.0 17.0 16.7 16.0

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.67 0.58 0.67 3.5 3.3 6.2 2.1 1.7 2.2 2.2 3.3 3.1 1.8 1.9 2.0 0.33 2.6 6.1 4.5 2.7 2.5 0.0 1.2 4.1 2.6 2.2 0.33 2.1 2.5 2.6 3.6

Revertant frequency 6 SEM (Lacþ colonies/ 108 viable cells) 7.8 4.1 14.6 7.8 19.1 76.8 5.1 9.4 14.6 22.8 26.6 72.4 8.8 6.9 6.9 9.4 18.5 95.0 457.3 10.1 4.9 10.1 7.6 16.8 15.8 7.6 5.7 12.2 12.4 24.8 32.5

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.46 0.30 0.53 2.2 2.5 6.8 1.1 1.1 1.7 2.4 4.4 18.4 0.94 1.0 0.98 0.21 2.1 10.2 11.5 1.4 1.4 0.00 0.74 3.7 3.8 1.5 0.2 1.4 1.8 3.9 7.3

a Only DAPA and 2-nitro-DAPA have slopes of dose-response curves (revertants/108 cells plated/lM) significantly greater than zero (P < 0.01). The increases in numbers of revertants per plate for these compounds are highly significant (ANOVA; P < 0.01). Revertant frequencies are also significantly increased at the highest doses of nitracrine and 4-nitro-DAPA.

at least four different adducts with poly (dG), the major adduct comprising about 40% of the total [Bartoszek et al., 1997a]. The cytotoxicity of nitracrine is clearly ascribable to covalent binding to DNA [Gniazdowski and Szmigiero, 1995; Bartoszek et al., 1997a]. Whether crosslinking of DNA is a major contributor to toxicity remains debatable [Pawlak et al., 1984; Stezowski et al., 1985; Wilson et al., 1989; Ferguson and Denny, 1991; Gniazdowski and Szmigiero, 1995; Bartoszek et al., 1997b; Gorlewska et al., 2001]. The structure of the adducts formed in DNA by nitracrine [Gniazdowski and Szmigiero, 1995] and other nitroacridines is unknown. Acridine mustards form adducts on the N7 position of guanine [Kohn et al., 1987; Sahasrabudhe et al., 1991], but there is little basis for extrapolating this finding to the nitroacridines. Gorlewska et al. [2001] proposed that the N-hydroxy-1-amino derivative of nitracrine may give rise to a nitrenium ion that binds to the C8 of guanine. Alternatively, carbons 2 and 4,

activated during metabolic reduction, might create a C C bond with the C8 of guanine [Gorlewska et al., 2001]. The similarities of Entozon, nitracrine, and 3-nitroDAPA to aromatic amines in their induction of 2 frameshifts [Shelton and DeMarini, 1995; Hoffmann and Fuchs, 1997] leads us to speculate that a reactive derivative of these acridines may form adducts at the C8 position that undergo similar mutagenic processing. Being in cationic form favors the electrostatic attraction of 9-aminoacridines to DNA and intercalation. The lesser 6G frameshift mutagenicity of nitroacridines than of simple intercalators can be ascribed largely to the electronwithdrawing nitro group conferring a lower pKa [O’Connor et al., 1990; Ferguson and Denny, 1990, 1991; Siim et al., 2000]. Being less basic, nitroacridines are less ionized at physiological pH and are therefore less effective in intercalation mutagenesis. In the lacZ assay, the reduction in 61 frameshift mutagenesis relative to DAPA is least for 2-nitro-DAPA, which has the highest pKa of the nitro

Frameshift Mutagenicity of Nitroacridines

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TABLE V. Induction of Frameshift Mutations by Entozon in the E. coli lacZ Reversion Assay

Strain

Reversion mechanism

Entozon (lM)

Relative cell density

CC107

þ1 (þG)

CC108

–1 (–G)

CC109

–2 (–CG)

CC111

–1 (–A)

0 1 2 4 8 16 0 1 2 4 8 16 32 64 0 1 2 4 8 16 0 1 2 4 8 16 32

1.00 1.11 1.11 0.92 0.78 0.49 1.00 1.22 0.96 0.92 1.15 0.72 0.22 0.019 1.00 0.93 0.97 0.98 0.70 0.58 1.00 1.26 1.11 0.89 0.81 0.75 0.26

Revertants per plate (6SEM)a 30.3 42.3 37.7 45.0 56.0 77.0 47.7 38.3 61.7 89.7 125.7 274.3 374.0 63.7 113.7 202.3 359.0 766.3 1391.0 1604.0 4.00 8.33 3.33 8.33 9.00 10.00 8.00

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.7 7.5 0.9 2.5 5.5 7.2 1.8 2.3 3.0 6.2 9.3 13.0 6.0 10.5 10.8 5.4 7.6 67.9 169.5 47.3 2.0 1.8 1.2 1.3 1.0 1.7 1.5

Revertant frequency (revertants/ 108 viable cells) 33.1 41.5 37.0 51.4 77.9 170.7 13.5 8.9 18.2 27.8 31.0 108.7 487.6 941.6 130.29 249.79 422.95 898.61 2287.83 3157.48 5.1 8.4 3.8 11.8 14.1 17.0 38.8

6 3.0 6 7.4 6 0.9 6 2.9 6 7.7 6 16.0 6 0.5 6 0.5 6 0.9 6 1.9 6 2.3 6 5.2 6 7.8 6 155.6 612.4 6 6.6 6 8.9 6 79.7 6 278.8 6 93.1 6 2.5 6 1.8 6 1.4 6 1.9 6 1.6 6 2.9 6 7.4

Analysis of variance showed highly significant (P < 0.01) increases in revertants per plate and revertant frequencies in strains CC107, CC108, and CC109. In strain CC111, the difference in numbers of revertants per plate was nonsignificant at all doses, and the increase in revertant frequency was significant (P < 0.05) only at the highest dose (Dunnett multiple comparisons test). a

isomers [O’Connor et al., 1990]. The yield of 6G frameshifts roughly correlates with pKa [O’Connor et al., 1990; Siim et al., 2000], in that DAPA > 2-nitro-DAPA > 4nitro-DAPA > 3-nitro-DAPA > nitracrine (Fig. 3). Besides altered basicity, steric factors can impede intercalation by nitracrine [Ferguson and Denny, 1991], which undergoes tautomeric shifts between a secondary amine and an imine at the 9 position [Gorlewska et al., 2001]. The imino form is favored in solution because of steric interactions between the nitro group and the side chain [Stezowski et al., 1985]. Consequently, the binding constant for intercalation of nitracrine is lower than those of its isomers [Ferguson and Denny, 1991]. The induction of 61 and 2 frameshift mutations by acridines can be explained by a slippage model. Slippage, first proposed by George Streisinger and colleagues, explains frameshift mutagenesis in repetitive sequences as a consequence of misaligned pairing [Streisinger et al., 1966; Ferguson and Denny, 1990; Hoffmann and Fuchs, 1997; Fujii et al., 1999; Kirchner et al., 2000; Becherel and Fuchs, 2001; Hoffmann et al., 2003]. An unpaired bulge is relatively stable in a repetitive sequence owing to correct pairing in the adjacent regions. Extension of a

growing polynucleotide chain from a slipped configuration leads to a 1 frameshift mutation if the bulge is in the template strand and a þ1 frameshift mutation if it is in the new strand. Although slippage was initially envisioned as an event in replication, it might also occur in recombination or repair. The induction of frameshift mutations by acridines is most readily explained by their binding to DNA and stabilizing bulges that arise spontaneously [Ferguson and Denny, 1990] or facilitating the formation of bulges. A variation of the classical slippage model, called ‘‘incorporation slippage’’ [Schaaper et al., 1990; Lambert et al., 1992, 1998; Garcia et al., 1993], may explain the tendency of reactive nitroacridines to induce minus frameshifts more than plus frameshifts. In this model, the formation of a –G frameshift in a run of G:C base pairs begins with the correct incorporation of cytosine opposite a guanine that bears a polymerase-hindering adduct. Slippage creates a bulge containing the adduct-bearing base, and a correct base pair is formed between a guanine residue 50 to the adducted base and the cytosine that had been inserted opposite the adduct. Slippage explains 2 frameshift mutagenesis in an alternating repetitive sequence (e.g., GCGCGC) much as

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Fig. 4. Ratio of CG to G frameshift mutations calculated from maximum yields of induced revertants per 108 cells treated in replicate experiments. Calculations for the simple intercalators 9AA and quinacrine and the acridine mustards ICR-191 and quinacrine mustard are based on Hoffmann et al. [2003].

it explains 1 mutagenesis in a monotonous run (e.g., GGGGG), except that there is a dinucleotide bulge rather than a single bulged base [Koffel-Schwartz and Fuchs, 1995; Shelton and DeMarini, 1995; Hoffmann and Fuchs, 1997; Hilario et al., 2002; Hoffmann et al., 2003]. Studies of AAF show that adducts on the C8 position of guanine at sites of alternating base pairs stabilize a slipped intermediate [Garcia et al., 1993; Milhe´ et al., 1994, 1996; Hoffmann and Fuchs, 1997]. Translesion synthesis at sites of stabilized slipped mispairing leads to frameshifts

[Becherel and Fuchs, 2001]. The lack of þ2 frameshifts induced by acridine mustards and Entozon in the tet assay in pBR322 [Hoffmann et al., 1996] suggests that the dinucleotide bulges are restricted to the template strand. The weak response with all the nitroacridines and DAPA in CC111 relative to CC108 indicates that A:T runs are less susceptible to acridine mutagenesis than G:C runs. The greater proclivity of G:C runs to frameshift mutagenesis may be ascribable to there being 3, rather than 2, hydrogen bonds stabilizing paired bases adjacent

Frameshift Mutagenicity of Nitroacridines

to the bulge. Alternatively, G:C regions may have more efficient proofreading and mismatch repair [Sagher et al., 1999]. In conclusion, the nonreactive intercalating agent DAPA resembles 9AA and quinacrine [Hoffmann et al., 1996, 2003] in inducing primarily –G and þG frameshifts in a monotonous run of G:C base pairs. The 1-, 3-, and 4nitroacridines tend to be weaker inducers of 61 frameshifts than the simple intercalators, probably owing to the effect of the nitro group on base strength and charge. The greater toxicity of nitracrine than of its isomers or of simple intercalators complicates comparisons of mutagenic potency. Nevertheless, slopes and revertant yields indicate that the 1- and 3-nitro compounds are more potent inducers of 2 frameshifts than DAPA. Thus, the present results for nitracrine, its isomers, and DAPA form a consistent pattern with previous studies of mutations induced by Entozon, 9AA, and quinacrine [Hoffmann et al., 1996, 2003]. Slipped mispairing models can explain the induction of þG, –G, and –CG frameshifts by acridines. ACKNOWLEDGMENTS The authors thank Drs. Ronald Jarret and Robert Bellin for valuable discussions and Darlene Colonna for excellent secretarial assistance. Entozon was generously given to us by Dr. Herbert S. Rosenkranz. REFERENCES Bartoszek A, Konopa J. 1989. 32P-post-labeling analysis of DNA adduct formation by antitumor drug nitracrine (Ledakrin) and other nitroacridines in different biological systems. Biochem Pharmacol 38:1301–1312. Bartoszek A, Dackiewicz P, Konopa J. 1997a. 32P-Post-labelling analysis of nucleobases involved in the formation of DNA adducts by antitumor 1-nitroacridines. Chem Biol Interact 103:131–139. Bartoszek A, Dackiewicz P, Skladanowski A, Konopa J. 1997b. In vitro DNA crosslinking by Ledakrin, an antitumor derivative of 1nitro-9-aminoacridine. Chem-Biol Interact 103:141–151. Becherel OJ, Fuchs RPP. 2001. Mechanism of DNA polymerase II-mediated frameshift mutagenesis. Proc Natl Acad Sci USA 98:8566– 8571. Brown BR, Firth WJ, III, Yielding LW. 1980. Acridine structure correlated with mutagenic activity in Salmonella. Mutat Res 72:373– 388. Carroll CC, Warnakulasuriyarachchi D, Nokhbeh MR, Lambert IB. 2002. Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB. Mutat Res 501:79–98. Cebula TA, Koch WH. 1990. Sequence analysis of Salmonella typhimurium revertants. In: Mendelsohn ML, Albertini RJ, editors. Mutation and the Environment, Part D. New York: Wiley. pp 367–377. Cupples CG, Miller JH. 1989. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci USA 86:5345–5349. Cupples CG, Cabrera M, Cruz C, Miller JH. 1990. A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations. Genetics 125:275–280.

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