Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues

Fundamentaland Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 329 (1995) 19-27

Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues Lynnette R. Ferguson

*,

William A. Denny

Cancer Research Laboratory, University of Auckland School of Medicine, Private Bag, Auckland, New Zealand

Received 9 November 1994; revised 9 February 1995; accepted 10 February 1995

Abstract A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D.5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TAlOO but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor ‘petite’ mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TAlOO) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study. 1. Introduction While DNA alkylating agents (particularly nitrogen mustard derivatives) are widely used in the combination chemotherapy of cancer, there are continuing concerns about their mutagenic potential, which provides a mechanism for accelerated

* Corresponding

author.

conversion to drug resistant phenotypes (Henderson, 19841, and their known carcinogenic properties (Pederson-Bjergaard et al., 1988). These compounds alkylate DNA predominantly at the N-7 of guanine in runs of guanines. Although relationships between patterns of DNA alkylation and mutagenic potential are complex (being mediated, among other things, by DNA repair systems) (Horsfall et al., 1990), there is a good deal of current interest in the development of alkylat-

0027-5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0027-5107(95)00013-5

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L.R. Ferguson, WA. Denny /Mutation Research 329 (1995) 19-27

ing agents with altered spectra of DNA primary lesions. One approach to such compounds is ‘DNAtargeted alkylators’, in which sequence-specific DNA-binding carriers are used to direct the pattern of alkylation (DNA-bonding) sites on DNA (Warpehoski and Hurley, 1988; Prakash et al., 1991). It has been shown that even relatively non-sequence-specific carriers such as DNA intercalating agents can result in compounds with significantly different alkylation patterns compared to those of simple nitrogen mustards (Prakash et al., 19901, and with much higher cytotoxic potency (Gourdie et al., 1990). However, while these compounds show different patterns of events compared to simple nitrogen mustards, they retain significant mutagenic potential, as shown by their ability to induce reversions in Salmonella strains (Ferguson et al., 1989b) and mitochondrial ‘petite’ mutations in yeast (Ferguson et al., 1989~). Another class of DNA-targeted nitrogen mustards are those possessing a minor groove binding carrier, and include polypyrrole (Arcamone et al., 1989) and polybenzamide (Prakash et al., 1991) structures. Compounds of this type are effective anticancer drugs, and alkylate adenines in runs of AT base-pairs. Table 1 Physicochemical and biological properties of bisbenzimidazole

MeN,)

1

R=OH

2

R = N(Et)CH,CH,CI

3:n=o

5:n=2

4:n=1

6:n=3

Fig. 1. Structural formulae of the studied compounds.

However, no mutagenicity studies have been reported to date on these compounds. In this paper, we report studies on the mutagenic potential (in Salmonella and yeast) and DNA-alkylating properties of a new series of DNA minor groove targeted aniline mustards (2-7) (Gravatt et al., 1994) (Fig. 1) based on the bisbenzimidazole carrier pibenzimol (11, better known as Hoechst 33258. This is a well-characterized DNA minor groove binder (Pjura et al., 1987; Carrondo et al., 1989).

mustards X-linking g

Compound

1O-6 K a (M-l)

n’ b

104 k,]k = (s-l)

In vitro IC,, d (PM)

In vivo OD=

ILS f

1 2 3 4 5 6 7

1.82 _ 1.52 6.73 6.30 4.55 3.89

2.4

_ 1.78 3.01 4.62 6.52 9.64

1.2 0.5 0.8 0.85 0.01 0.02 0.06

20 30 30 13.3 3.9 5.9 8.9

NAh 86 106 (2) i 71 52 NA NA

2.6 3.0 2.8 3.0 2.4

7:n=6

_ -

_ + ++

a Intrinsic association constant for binding of mustards to calf thymus DNA at 20” C in 0.01 M HEPES buffer at pH 7.00. b Site size expressed in nucleotide residues. ’ Rate constant for alkylation of calf thymus DNA measured by spectrofluorophotometry. d The concentration of drug (PM) required to reduce P388 cell numbers to 50% of controls after 70 h exposure. Initial cell numbers were 3 X lo4 cells/ml. Data from Gravatt et al., 1994. ’ Optimal dose of drug (mg/kg) administered as a single intraperitoneal injection 24 h after similar injection of 10” P388 leukemia cells. Data from Gravatt et al., 1992. f Percentage increase in lifespan of drug-treated, tumor-bearing animals when treated at the optimal dose; values above 25% are statistically significant. Data from Gravatt et al., 1994. ’ Ability to crosslink linearized pSV2gpt DNA (Tindall and Stankowski, 19891,measured by agarose gel electrophoresis (Valu et al., 1990). -, no activity; + ,weak activity; + + ,strong activity. h No activity seen at all dose levels up to the maximum tolerated dose. ’ Number in parentheses is the average number of long-term survivors (from a group of six animals).

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L.R. Ferguson, WA. Denny /Mutation Research 329 (1995) 19-27

2. Materials and methods Chemicals

The compounds of Table 1 were prepared as previously described (Gravatt et al., 1992). Compounds were stored as solids at - 10” C and solutions made up for testing were used immediately. Ail compounds were pure as judged from time to time by thin-layer chromatography in butanol/acetic acid/water solvent systems. DNA cross-linking assay

This was carried out by agarose gel electrophoresis, using linearized pSV2gpt DNA (Tindall and Stanowski, 1989) and the method reported by Valu et al. (1990). Briefly, linearized pSV2gpt DNA (1 ~1 of a 1 mg/ml solution) was incubated with drug in TE-80 buffer (10 mM Tris * HCl, 1 mM EDTA at pH 8) (shielded from ambient light) for 2 h at 30” C, then denatured by the addition of 2 ,ul of 1% sodium dodecyl sulfate and 10 ,ul of 50 mM methylmercury hydroxide and incubated for a further 30 min at 20” C in the dark. Renaturation of drug-treated DNA samples was carried out by incubation with 2.5 ~1 of 2-mercaptoethanol for 1 h. Samples were prepared for electrophoresis by the addition of 0.1 mg/ml bromophenol blue, 0.5 fig/ml ethidium bromide and 5 ~1 of 40% sucrose. Electrophoresis was carried out at 120 V in 89 mM Tris/borate buffer at pH 8 containing 2 mM EDTA and 0.5 pg/ml ethidium bromide. DNA was visualized using 302 nm trans-illumination, and was photographed using Polaroid type 55 film and a Wratten 3A filter. Chlorambucil and its halfmustard were used as controls in these experiments, as an example of a cross-linking drug and a non cross-linking analogue. Bacterial strains

Salmonella strains TA1537, TA1978 pKM101, TA98, TAlOO and TA102 were kindly supplied by B.N. Ames (Biochemistry Dept, University of California, Berkeley, CA, USA). All are deep rough derivatives of the LT2 subline of S. typhimurium. Characteristics of these strains and the rationale for their selection have been previously described (Ferguson et al., 1988). Since we

have found that the use of aliquots of frozen stock is necessary for reproducibility of experiments, the bacteria were initially grown to stationary phase in nutrient media and frozen (with 10% DMSO) in 1 ml aliquots at - 80” C. Strains were routinely characterized for spontaneous reversion characteristics and reversion rates in response to the following diagnostic mutagens. TA98: daunomycin (Sigma) and cl-nitro-o-phenylene diamine (Sigma); TAlOO: sodium azide (BDH); TA102: bleomycin; TA1978 pKM101: mitomycin C; TA1537: 9-aminoacridine (Sigma). Saccharomyces cerevisiae

The diploid strain D5 (Zimmermann, 1973) was kindly provided by Dr. B.S. Cox (Botany School, University of Oxford). A single colony isolate was inoculated into liquid yeast complete medium (YC; Cox and Bevan, 1962) and grown to stationary phase for 24 h. Dimethyl sulphoxide was added to 10%. Aliquots (1 ml> were frozen to -70” C, and stored at this temperature before use. For all experiments, the 1 ml sample was thawed, added to 10 ml of fresh medium and grown for exactly 2 h before use. Bacterial method)

mutagenicity

assay

(standard

plating

For each experiment, a 1 ml vial of bacteria was removed from the - 80” C freezer, inoculated into 20 ml of fresh bacterial complete medium and grown for 4 h. Optical density was checked at that time and at intervals thereafter until a one-in-ten dilution into fresh bacterial complete medium gave a reading of between 0.10 and 0.12 at 654 nm. (This was to ensure that all cultures were at the same stage of growth when used.) The S. typhimurium plate incorporation assay was carried out as described (Maron and Ames, 1983). Each drug was tested over a range of concentrations by adding varying amounts to 2 ml of soft agar containing 5 mM histidine-biotin maintained at 42” C in a temperature block. Bacterial suspension (100 ~1) was added, the tube was mixed and quickly poured over the surface of agar plates containing 20 ml of minimal medium (Vogel and Bonner, 1956). Plates were allowed to harden and then incubated at 37” C for 3 days

22

L.R. Ferguson, WA. Denny /Mutation

Research 329 (1995) 19-27

before scoring colonies for reversion to histidine independence. All assays were performed in triplicate, and repeated at least once. Data presented are an average. Reversion characteristics of each strain were routinely tested in each experiment. Care was taken to exclude light from both the chemical and the assay plates. Colony counts were determined on an Artek Model 880 automatic counter, calibrated with plates counted manually (Maron and Ames, 1983). Microtiter assay for mitotic crossing-over and petite mutagenesis

This assay has been described in detail (Ferguson, 1984). Briefly, a log-phase culture was washed, then diluted into 0.87% saline. A 96-well microtiter tray (A/S Nunc, Denmark) was inoculated with (usually) 100 ~1 aliquots of the diluted yeast culture, and drugs added at various dilutions to the wells, to a maximum of 1000 pgg/ml. Drugs were dissolved in dimethyl sulfoxide, and dilutions made so that there was no more than 1% DMSO in each well. Trays were incubated for either 2 h or 20 h at 3O”C, an appropriate dilution made from each well into saline, and 100 ~1 plated onto each of 10 YC plates. Cell numbers were calculated so that the dilutions at this point were at least 1/104, thereby effectively washing drugs from cells by dilution (Ferguson, 1984). Plates were incubated at 30” C for 5 days, and scored for ordinary, colored or sectored colonies by visual inspection (Zimmermann, 1973). They were then overlaid with tetrazolium in order to score petite colonies (Nagai, 1959). All experiments were performed at least twice, and the data compared for reproducibility. The presented data have been pooled.

3. Results DNA interaction The structures

of the compounds studied, and their relevant physicochemical properties, are given in Table 1. The mustards (2-7) all bind tightly to calf thymus DNA at 0.01 ionic strength (3-4-fold more tightly than does pibenzimol (1) itself). Although the binding constants decrease

Fig. 2. Agarose gel electrophoresis of pSV2gpt DNA treated with alkylating agents and visualized with ethidium bromide to determine drug crosslinking ability. After drug treatment, DNA is denatured to the single-stranded form, which runs as a lower band in the gels (see channels 1 and 2). Crosslinking by the drugs is evidenced by increasing amounts of renatured, double-stranded DNA appearing (the upper band in the gels; corresponding to the band for untreated DNA seen in channel 1). Channels are as follows: (1) Untreated DNA, no drug. (2) Denatured DNA, no drug. (4-5) Denatured DNA in the presence of chlorambucil half-mustard, 20, 50 PM. (6-7) Denatured DNA in the presence of chlorambucil, 20, 50 PM. (9-11) Denatured DNA in the presence of compound 4, 0.5, 1, 2 FM (higher concentrations are not considered to have biological relevance for these drugs). (13-15) Denatured DNA in the presence of compound 6, 0.5, 1, 2 PM. (17-19) Denatured DNA in the presence of compound 7, 0.5, 1, 2 PM.

by a factor of nearly 2 as the linker chain is increased, the binding site size 12’remains essentially constant (at 2.6 k 0.4 nucleotides per binding site), suggesting that the mode of binding and the drug/DNA contacts which comprise it are similar, and dictated by the bisbenzimidazole chromophore. The rate constants for alkylation of calf thymus DNA (determined by measuring the rate of increase in the DNA-bound fluorescence) are also given in Table 1, and show the compounds alkylate DNA more rapidly as the chain length increases. The relative DNA cross-linking ability of the compounds was estimated using agarose gel electrophoresis of plasmid DNA. An illustration of the gels is provided in Fig. 2, and a summary of the data are given in Table 1. The conditions used were similar to those employed in the yeast assays, and only the compound with the longest

23

L.R. Ferguson, W.A. Denny /Mutation Research 329 (1995) 19-27

Bacterial mutagenicity The compounds were tested in a number of strains, and were found to be completely negative in TA98, TA1978pKMlOl and TAB37 (data not presented). Some compounds showed activity in strains TAlOO and TA102, and dose-response curves for these are summarized in Table 2. The parent compound (1) and the less reactive mustard derivatives 2-4 showed no statistically significant (Mahon et al., 1989) mutagenic response in TAlOO. However, if the more toxic dose levels were not included in the analysis, compounds 5 and 6 could be considered to show a mutagenic effect in TAlOO. In TA102, the parent compound showed a weak but significant mutagenic effect. The more reactive mustard analogues 4-6 were

alkyl chain (7) cross-linked DNA effectively under these conditions, although compound 6 also shared some activity. Antitumor activity The mammalian cytotoxicities and antitumor activities of these compounds has been reported (Gravatt et al., 19921, and these data are also given in Table 1. The parent compound pibenzimol (1) is inactive in the P388 leukemia system (although it has undergone phase I clinical evaluation as an anticancer agent; Kraut et al., 1988). The shorter-chain compounds 2 and 3 both show significant in vivo antitumor activity, while the (more reactive) longer-chain compounds (4-7) are much more cytotoxic but Iess active in vivo.

Table 2 Bacterial mutagenicity data for compounds l-7 Compound

1

Concentration

Bacterial strains a

Cughlate)

TA98

0 4 10 20 40 80 0

4 10 20 40 80 120

60& 59+ 64-1 55+ 61+ 62+

60+ 9 70+ 3 62k 7 67+ 9 52* 7 65+ 3 [53 * 391

0 4 10 20 40

60+ 61k 68+ 58+ 52k

80 120

55+ 148 f

0 4 10 20 40 80 120

TAlOO 9b 3 8 6 5 9

187 f 188 + 191* 179+ 168 + 178 +

TA102 4 11 7 6 13 11

187 f 4 196 + 23 162 f 21 172 f 13 226 _+23 207 f 12 1155 f 391

9 3 2 1 7

187k 173* 175 i 167 + 210 f

2 21

192k 1 185 f 14

72k 5 71+ 2 83 f 10 87+ 6 86+ 5 77 f 10 86k 8

150 f 1545 130 + 137 f 167 + 167k 157 +

4 7 17 28 20

17 8 10 43 24 8 12

246+ 275 f 277+ 301+ 377 + 397 +

6 12 1 3 49 10

296 406 417 442 442 [194 [82

k 41 + 20 f 26 _+62 + 43 + 601 f 481

296 408 371 280 [274

f * + f +

41 31 14 14 121

[51 k 71 [lo? 11 296 297 237 304 268 [377 505

+ f + + f + *

41 38 64 16 59 351 121

24

L.R. Ferguson, WA. Denny/Mutation

Research 329 (1995) 19-27

Table 2 (continued) Compound

Concentration t~g/elate)

TA98

5

0 4 10 20 40 80 120

72+ 77+ 74 + 75f 65f t61 * [55 +

5 2 10 1 6 91

0

6OIt 63* 62+ 71 f 62f 63+ 71+

9 2 1 13 3 9 1

4 10 20 40 80 120 0

4 10 20 40 80 120

Bacterial strains a

61

72rt 5 65 + 10 69f 7 68+ 8 69f 9 96z!z 5 75f 3

TAlOO

TA102

150 + 172 + 231k 264 f 237 + 207 + 232 +

296 f 340 + 366 + 429 rt 465 + 609 + [564 +

17 14 7 12 29 10 19

187k 4 188 f 15 180+ 5 205 & 14 240 + 7 258 + 21 1204+ 281 150 + 160 f 136+ 156 + 144 + 142 + 157 +

17 13 0 29 10 10 14

41 26 49 17 41 74 191

296 + 41 342 + 32 392 f 31 368 + 32 327 f 17 480 + 17 607+28 296 + 41 271 + 31 386 &-10 342 f 14 372 + 19 336 + 19 353 + 34

a Strains of S. typhimurium (Maron and Ames, 1983). b Results are mean values (two experiments, plated in triplicate) f standard deviation. ’ Square brackets denote toxicity was becoming apparent on visual inspection of the plates.

also active in TA102 (the last two more so than the parent compound). Mitotic crossing-over and petite mutagenesis in yeast Dose-response data for each of the compounds are summarized in Table 3. Compounds l-4 caused no significant activity in either of these assays. However, the more reactive mustards 5-7 caused mitotic crossing-over, with the effect increasing with chain length; 7 is a moderately effective inducer of mitotic crossing-over. This was the only compound which also showed activity (albeit weak) in the petite mutagenesis assays.

4. Discussion As shown in Table 1, the compounds all bind strongly to DNA. Gel electrophoresis studies (un-

published data, this laboratory) show that the analogues 3-7 do not unwind closed circular supercoiled DNA, suggesting strongly that these compounds bind (as does 1) in the minor groove. The kinetic data (unpublished data, this laboratory), indicate different rates of alkylation of DNA. While the slower rates for compounds 3 and 4 are largely explained by the lower reactivities of these mustards (unpublished data, this laboratory), the increase from compounds 5-7 is probably related to their decreasing DNA binding allowing more conformational freedom for the drug; a phenomenon observed previously with DNA-targeted alkylators. However, cross-linking was only detected for the compound (7) of longest chain length. It is of considerable interest that this compound was not the most effective antitumor drug and it would appear that cross-linking is not important for antitumor activity in these drugs, as shown previously for other minor

L.R. Ferguson, W.A. Denny/Mutation

groove-targeted monoalkylating agents (Warpehoski et al., 1988). None of the compounds showed any evidence

25

Research 329 (1995) 19-27

of frameshift mutagenesis in GCGC regions, as shown by inactivity in the strains TA98 and TA1978 pKM101. They were also inactive against

Table 3 Petite mutagenesis, mitotic crossing over and total aberrant colonies following 2 h incubation of drugs with Saccharomyces strain Ds, under non-growing conditions Compound

Concentration

(mg/ml) 1

Survival a

Petites b

MRC

TAd

0 0.15 0.313 0.625 1.25

100 (4965) (4499) (3560) (2033) (1220)

0.4 0.5 0.4 0.7 1.8

0.04 0.04 0.05 < 0.05 < 0.08

0.28 0.22 0.22 0.18 0.33

0

100 (5100) 98.1 (4997) 87.8 (4478) 69.8 (3562) 12.5 (638)

0.4 0.5 0.4 0.3 0.3

0.04 < 0.04 0.05 0.06 < 0.1

0.31 0.25 0.25 0.29 0.31

100 (5620) 88.6 (5566) 70.9 (4457) 65.4 (4106) 33.6 (2101)

0.5 0.4 0.4 0.6 0.6

< 0.02 0.04 0.02 0.05 < 0.05

0.19 0.23 0.22 0.45 0.20

100 (6081) 83.5 (5246) 75.0 (4714) 48.3 (3034) 14.3 (896)

0.5 0.4 0.4 0.5 1.8

< 0.02 < 0.02 < 0.02 0.06 < 0.1

0.26 0.23 0.25 0.20 0.23

100 (5374) 82.3 (4421) 81.9 (4402) 66.2 (3558) 36.6 (1892)

0.5 0.4 0.6 0.4 0.4

0.04 < 0.04 < 0.04 0.06 0.21

0.26 0.18 0.36 0.45 0.62

100 (4672) 85.8 (4008) 76.3 (3565) 45.6 (2130) 17.7 (827)

0.5 0.7 0.7 0.4 0.5

0.04 0.05 0.06 0.09 0.11

0.26 0.26 0.45 0.47 0.94

100 (4672) 76.2 (3566) 64.3 (3008) 61.3 (2863) 24.7 (1151)

0.5 0.6 0.8 1.1 4.5

0.04 0.11 0.26 0.28 0.36

0.26 0.33 0.75 0.85 0.93

1.25 2.5 5 10 0

0.04 0.08 0.15 0.33 0

0.08 0.15 0.31 0.63 0

1.25 2.5 5 10 0

1.25 2.5 5 10 7

Effects in yeast

0

0.04 0.08 0.15 0.3

90.5 71.9 40.9 24.6

a Survival is expressed as a percentage. The actual numbers of colonies examined are given in parentheses. b The induction of petite colonies at the given dose, expressed as a percentage of total surviving cells. fi Mitotic crossing over, calculated as the frequency of red/pink or red/pink/white colonies as a percentage of total surviving cells. The total frequency of aberrant colonies, calculated as the number of colored colonies as a percentage of total surviving cells.

26

L.R. Ferguson, WA. Denny/Mutation

TA1978+, which was included as an example of a uvrB + strain (a genotype previously found necessary to detect frameshift mutagenesis by cross-linking drugs such as mitomycin C) (Ferguson et al., 1988). The compounds were also non-mutagenic in strain TA1537 (McCoy et al., 1981; Ferguson and von Borstel, 19921, and thus appeared quite different to the series of acridine-targeted aniline mustards studied earlier (Ferguson et al., 1989b,c) and also to the parent aniline mustards (Ferguson et al., 1989a). The mutagenic ability of 5 and 6 in TAlOO is consistent with DNA monoalkylation by these compounds, as is their activity in causing weak mitotic crossing-over and some aberrant colonies in yeast. The lower bacterial mutagenicity and increased mitotic crossing-over activity of 7 are consistent with its DNA cross-linking capability. Both the parent compound (1) and several mustard analogues did show activity in strain TA102. This strain carries the hisG428 ochre (TAA) mutation (Levin et al., 19821, and detects as mutagens a variety of oxidants and other compounds not detected by the other tester strains. The hisG428 mutation has AT base pairs at the mutated site, while the other standard tester strains have G:C base pairs at the critical site. Both the parent (non-alkylating) and the mustard analogues show similar effects, which may be due more to their high binding selectivity for AT sequences than to any alkylation event. Overall, these results support the view that the pibenzimol chromophore may be redirecting mustard alkylation to unusual sites (presumably in the minor groove), as has been shown for other targeted mustards (Prakash et al., 1991). Previous studies have shown that many minor groove binding drugs are very effective ‘petite’ or mitochondrial mutagens in yeast (Ferguson and Baguley, 19831, an activity which is also seen for both simple nitrogen mustards and those targeted to DNA by an intercalating (acridine) carrier (Ferguson et al., 1988, 1989~; Ferguson and von Borstel, 1992). It is therefore of considerable interest that both the parent compound 1 and most of the analogues are not active in this system. Although the present results do not rule out some mitochondrial effects for these compounds

Research 329 (1995) 19-27

(for example, some types of mitochondrial enzyme inhibition will not be detected in the petite mutagenesis assay), none of the present series appears to act similarly to the bisquaternary ammonium heterocycle class of minor groove binders (Ferguson and B aguley, 1983). Thus the most effective antitumor agent, the monoalkylator 3, was inactive as a bacterial mutagen and as a mitochondrial mutagen in yeast. The (at best weak) activity of the bisbenzimidazole-derived mustards studied here suggests that, unlike many alkylating agents, they may be less likely to be potential carcinogens (Quint0 and Radman, 1987; Schmahl, 1986; PedersonBjergaard et al., 1988).

Acknowledgements

This work was funded by the Auckland Division of the Cancer Society of New Zealand, the Medical Research Council of New Zealand, and Pharmol Pacific Ltd. We thank Hamish Pogai for technical assistance.

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