Mutation Research, 240 (1990) 47-58 47 Elsevier

MUTGEN 01504

Tests for the genotoxicity of m-AMSA, etoposide, teniposide and ellipticine in Neurospora crassa

Ranjan Gupta Genetics Group, Department of Biological Sciences, Illinois State University, Normal, 1L 61761 (U.S.A.)

(Received 9 May 1989) (Revision received 21 August 1989)

(Accepted 28 August 1989)

Keywords: m-AMSA; Etoposide; Teniposide; Ellipticine; ICR-170; Mutagenicity; ad-3; Neurospora crassa

Summary

The antitumor agents m-AMSA, etoposide, teniposide and ellipticine have been reported to be potent clastogens in mammalian cells but non- or weakly mutagenic in bacteria; these observations have been correlated to the interference of these chemicals with DNA topoisomerase II activity in the former, but not in the latter, organisms. The genotoxicity of these 4 agents was evaluated using ad-3 reverse- and forward-mutation tests in Neurospora crassa. These agents (up to 0.8/~mole/plate) did not cause reversion in conidia of the ad-3A frameshift strains N24 and 12-9-26 using the overlay plate test, as contrasted to the positive control frameshift mutagen ICRo170. Heterokaryon 12 (H-12) of N. crassa permits the recovery of all classes of forward mutation at the ad-3 + region, including multilocus deletions. Using resting conidia of H-12 in a suspension assay, ellipticine was moderately mutagenic but no increase in ad-3 mutants was noted with the other 3 agents at a dose of 100 #g/ml. In vegetative cultures of H-12 grown in the presence of these agents, all 4 agents were nonmutagenic at a dose of 100 #g/ml. The positive control mutagen ICR-170 was mutagenic in both resting conidia and growing cultures of H-12. A similarity between the topoisomerase II of N. erassa and DNA gyrase of bacteria is suggested.

4'-(9-Acridinylamino)methanesulfon-m-anisi- dide (m-AMSA or amsacrine), ellipticine, etopo- side (VP16-213) and teniposide (VM26) are a group of antitumor agents presently under clinical use. They are structurally different (Fig. 1) but functionally similar in mammalian cells, with novel mechanisms of antineoplastic activities. All 4 agents -- m-AMSA (Deaven et al., 1978; de la

Correspondence: Ranjan Gupta, Department of Biological Sciences, Illinois State University, Felmley Hall 206, Normal, IL 61761-6901 (U.S.A.).

Iglesia et al., 1984; Ferguson and Baguley, 1984; Kao-Shan et al., 1984; Larripa et al., 1984; Wilson et al., 1984; Pommier et al., 1985; Ferguson et al., 1986; DeMarini et al., 1987b; Andersson and Kihlman, 1989; Doerr et al., 1989), ellipticine (Bhuyan et al., 1972; Moore et al., 1987a), etopo- side (Huang et al., 1973) and teniposide (De- Marini et al., 1987a) - - are potent clastogens in mammalian cells. SCEs have been induced in Chinese hamster V79 cells (West et al., 1981; Pommier et al., 1985) and in human lymphocytes in vitro (Crossen, 1979; Kao-Shan et al., 1984) by m-AMSA, as well as in Chinese hamster ovary

0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

48

C H 3 0 / ~ NHSO2CH3

NH

OH rn-AMSA OH

CH 30 CH 3 CH 3 ° OCH 3

J° 7 jo O

OH OH Etoposide (VP 16-213) Teniposide (VM-26)

NH(CH2) ~ N ~CH2CH3 CH3 [ ~ ~CH2 CH2Cl

~ N ~ OCH 3

C1 ~ CH 3 ICR-170

Ellipticine

Fig. 1. Chemical structures of mutagens.

(CHO) cells by m-AMSA (Deaven et al., 1978; de la Iglesia et al., 1984), ellipticine, etoposide and teniposide (Singh and Gupta, 1983a). m-AMSA also induces morphological transformation in mouse C3H/10T1/2 cells (Ferguson et al., 1986).

The clastogenicity of these compounds is fur- ther indicated by the fact that they induce almost exclusively (and high mutant frequencies of) small-colony TK - / - mutants at the heterozygous tk locus in L5178Y/TK+/--3.7.2C mouse lymphoma cells (DeMarini et al., 1987a, b; Moore et al., 1987a, b; Doerr et al., 1989; Evans et al., 1989). Such mutants are a consequence of prim- arily chromosomal, rather than gene, mutation (Clive, 1987; DeMarini et al., 1989). Not surpris- ingly, these agents induce low mutant frequencies at the hemizygous hprt locus (DeMarini et al., 1983; Singh and Gupta, 1983a, b; de la Iglesia et al., 1984; Wilson et al., 1984; Pommier et al., 1985; Moore et al., 1987b) and are nonmutagenic at the ouabain-resistance locus (Wilson et al., 1984; Ferguson et al., 1986), both of which detect only gene mutations in mammalian cells (DeMarini et al., 1989).

The above results have been related to the observations that m-AMSA and ellipticine (Min- ford et al., 1984; Nelson et al., 1984; Pommier et al., 1984a, b, c, d, 1985; Tewey et al., 1984a; Yang et al., 1985), as well as etoposide and teniposide (Chen et al., 1984; Minocha and Long, 1984; Ross et al., 1984; Tewey et ai., 1984b; Long et al., 1985), cause DNA topoisomerase II-associated single- and double-strand breaks in mammalian cell DNA. Also, induction of SCEs by m-AMSA, etoposide and 2-methyl-9-hydroxyellipticinium in Chinese hamster cells correlates well with cyto- toxicity, further suggesting the inhibition of mam- malian topoisomerase II by these drugs (Pommier et al., 1988).

Opposed to their topoisomerase II-mediated, potent clastogenic/mutagenic activity in mam- malian cells, the mutagenicity of these agents in prokaryotes is weak or negative. In the case of m-AMSA (Ferguson et al., 1983, 1985; de la Iglesia et al., 1984; Ferguson and MacPhee, 1984) and ellipticine (DeMarini et al., 1983), where some mutagenicity has been demonstrated in Ames' Salmonella typhimurium frameshift tester strains, the mutagenicity has been explained by intercala- tion into, and/or covalent adduct formation with, DNA (Baguley and Nash, 1981; DeMarini et al., 1983). Although teniposide and etoposide have been reported to be more mutagenic in the S. typhimurium uvrB + frameshift strains UTH8413 and TA1978 than in their uvrB (nucleotide exci- sion repair-deficient) counterparts TA98 and TA1538, respectively (Matney et al., 1985), others have reported these compounds as being non- mutagenic in TA98 with or without a rat liver activation system and in the Escherichia eoli nucleotide excision repair-deficient strains WP2s (uvrA) and WP44sNF ( t i f ) (Gupta et al., 1987). The latter authors also found etoposide to be nonmutagenic in the Gal- to Gal + forward-muta- tion assay in E. coli strain 343/113 with or without permeabilization treatment by EDTA.

In contrast to m-AMSA and ellipticine, the nonmutagenicity of etoposide and teniposide in bacteria may perhaps be explained by the observa- tion that neither of them intercalates into DNA (Chen et al., 1984) and that etoposide does not bind covalently to DNA (Ross et al., 1984). None of these agents is mutagenic in the base-substitu-

tion tester strain TA100 of S. typhimurium (Fergu- son and Baguley, 1981).

It is interesting that none of the 4 agents has been reported to interact with bacterial DNA gyrase (the prokaryotic equivalent to eukaryotic DNA topoisomerase II), and according to Nelson et al. (1984), "E. coli DNA gyrase is refractory to m-AMSA treatment in vitro (unpublished re- suits)". The contrasting situations discussed here regarding both the mutagenicity and mechanism of action of these chemicals in mammalian cells and prokaryotes make it interesting to know how these agents operate in organisms between the two extremes: i.e., in lower eukaryotes. This paper reports the results of tests for the mutagenicity of m-AMSA, ellipticine, etoposide and teniposide in forward- and reverse-mutation tests in the lower eukaryote Neurospora crassa.

The two-component heterokaryon H-12 of N. crassa (de Serres and Mailing, 1971) mimics the genetic constitution of diploid organisms. The ad-3

forward-mutation test in H-12 allows the recovery and characterization of a wide variety of mutants ranging from intracistronic mutations in the ad-

3A ÷ or ad-3B ÷ cistrons to multilocus deletions (MLD) involving either or both ad-3 ÷ cistrons together with one or more adjacent cistrons that determine essential functions (de Serres and Mai- ling, 1971; de Serres, 1981, 1983). Therefore, this test in a lower eukaryote is analogous to the L5178Y/TK ÷/- mouse lymphoma assay (De- Marini et al., 1989). In the ad-3 forward-mutation test, mutagen treatment can be done with non-

49

growing (resting) conidia or growing (vegetative) cultures.

The ad-3A presumptive frameshift tester strains N24 (Ong, 1978) and 12-9-26 (Brockman and Goben, 1965) of N. crassa, each of which was derived from component II of H-12, allow rapid screening of frameshift mutagens by the overlay plate test in the ad-3 reverse-mutation test (Ong, 1978). Strains 12-9-26 and N24 are presumptive frameshift tester strains whose reversion character- istics differ. 12-9-26 is revertible by X-ray, UV, ethyl methanesulfonate, fl-propiolactone and N- methyl-N'-nitro-N-nitrosoguanidine, as well as being moderately revertible by ICR-170, whereas N24 is strongly revertible by ICR-170 but is not revertible by the other agents (Brockman, 1974; Ong, 1978). Thus, like most of the ICR-170-in- duced mutants in N. crassa (Bums et al., 1986), 12-9-26 presumably contains a single base-pair addition that can be reverted by a single base-pair deletion induced by the other agents (Brockman, 1974), whereas N24, which was induced by aflatoxin B1, presumably contains a single base- pair deletion that can be reverted only by ICR-170. These 2 frameshift testers (presumptive + 1 and -1 ) should permit detection of a variety of frameshift mutations.

In this paper, I report the results of tests for the mutagenicity of m-AMSA, ellipticine, etoposide and teniposide in the ad-3 forward-mutation test, using both resting conidia and growing cultures, and in resting conidia in the ad-3 reverse-mu- tation test, in N. crassa. No mutagenicity was

TABLE 1

GENOTYPES OF Neurospora crassa STRAINS

Strain Nuclear Linkage group

component 1 II! IV V VI

H-12 I (74-OR60-29A) II (74-OR31-16A)

12-9-26 II b

N24 II b

A, his-2 a, ad-3A, ad-3B, nic-2, +; ad-2; + ; inl; + A, + , + , + , + , al-2; + ; cot-l; + ; pan-2

A, +, ad-3A, +, +, al-2; + ; cot-l; + ; pan-2

A, +, ad-3A, +, +, al-2; + ; cot-l; + ; pan-2

a Abbreviations are: A, mating type A; his, histidine; ad, adenine; nic, nicotinamide; al, albino; cot, colonial temperature-sensitive; inl, inositol; pan, pantothenic acid.

b Identical to component II except for the ad-3A mutation, which is a different allele in the 2 strains.

50

observed, except for ellipticine, which was mod- erately mutagenic in resting conidia in the ad-3 forward-mutation test.

Materials and methods

Strains The genetic constitution of heterokaryon 12

(H-12) of N. crassa (de Serres and Malting, 1971), which was used in the ad-3 forward-mutation experiments, is shown in Table 1. It is DNA repair proficient, and the 2 nuclear components are es- sentially isogenic except for the allele differences shown. Because H-12 is heterokaryotic at the ad-3 region, both intracistronic and MLD types of for- ward mutations at the ad-3A + and/or ad-3B + cistrons of nuclear component II are easily de- tected (de Serres and Malting, 1971). H-12 was designed by and obtained from Frederick J. de Serres, Center for Life Sciences and Toxicology, Research Triangle Institute, Research Triangle Park, NC.

Strains N24 and 12-9-26 were used in the ad-3 reverse-mutation experiments. Each is homo- karyotic for component II of H-12 but carries a presumptive frameshift ad-3A mutation. Strains N24 and 12-9-26 were obtained from Tong-man Ong, Division of Respiratory Disease Studies, Na- tional Institute for Occupational Safety and Health, Morgantown, WV and Herman E. Brock- man, Department of Biological Sciences, Illinois State University, Normal, IL, respectively. Stock cultures of all 3 strains were maintained as conidia on silica gel crystals at 4°C (Brockman and de Serres, 1962).

Mutagens The 4 antitumor agents (Fig. 1) - - m-AMSA

(amsacrine, CAS No. 51264-14-3), etoposide (VP16-213, CAS No. 33419-42-0), teniposide (VM26, CAS No. 29767-20-2) and ellipticine (CAS No. 519-23-3) - - were gifts from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Be- thesda, MD. ICR-170 (CAS No. 13441-95-7), which was used as a positive control mutagen, was purchased from Raylo Chemicals Ltd., Edmonton, Canada. All solutions were prepared in dimethyl sulfoxide (DMSO) purchased from Sigma Chem-

ical Co., St. Louis, MO. Handling of all mutagens was done in yellow light to minimize photo-activa- tion or -deactivation.

Vegetative cultures and conidial suspensions Vegetative cultures and conidial suspensions of

H-12, and of N24 and 12-9-26, were prepared according to the methods described by de Serres and Mailing (1971) and Ong (1978), respectively. Conidia generally were harvested from 10-day-old cultures. Conidial suspensions were prepared in 0.067 M, pH 7 KH2PO4.Na2HPO 4 buffer, ad- justed to a given concentration appropriate for each test and stored overnight at 4°C.

Reverse-mutation test The overlay plate test of Ong (1978) was used.

1 ml of 2 x 107 conidia of N24 or 12-9-26 and 0.1 ml of mutagen solution (in 20% DMSO) were added to 2 ml of molten top agar, which was overlaid onto plates of reversion medium, giving doubling doses of 0.025-0.8 /~mole/plate. The solvent control was identical except that 0.1 ml of 20% DMSO was used in place of the mutagen. The plates were incubated at 34°C for 4 days, and the revertant colonies were counted.

Forward-mutation test The ad-3 forward-mutation test was performed

using both resting conidia and growing cultures of H-12. In the former case, conidial suspensions (4 x 107/ml) in scintillation vials were treated with an equal volume of each mutagen solution in 20% DMSO at the desired concentrations and in- cubated in a rotary shaking waterbath at 25°C. Thus, the final conidial concentration was 2 × 107/ml and the final solvent concentration was 10% DMSO. The solvent control contained 10% DMSO in place of the mutagen. Conidia were plated at 1.5-, 3-, 6-, 12-, 24- and 48-h incubation in appropriate medium to determine percent survival. Based on these survival experiments (data not shown), the 24-h treatment time and a final treatment concentration of 100 /xg mutagen/ml were chosen. In subsequent experiments, after 24 h of incubation conidia from each vial were washed twice with buffer (0.067 M, pH 7 KH2PO 4 • Na2HPO4) by centrifugation, resuspended to a final concentration of 2 × 107/ml and inoculated

into 12-1 Florence flasks, each containing 10 1 of medium, at an estimated 106 viable heterokaryotic conidia per flask.

Due to limited availability of mutagens, treat- ment of growing cultures was done in slants in 13 mm x 100 mm test tubes. 0.1 ml of mutagen solu- tion in 20% DMSO was added to a tube contain- ing 2 ml of molten (45°C) vegetative growth medium (Fries' basal medium supplemented with 1% sucrose and 1.5% agar), and the contents were mixed and allowed to solidify as a slant. The final concentration of 1% DMSO in the slant did not inhibit growth or conidiation. As with resting co- nidia, the final treatment concentration in growing cultures was 100 #g mutagen/ml. The slants were inoculated by adding 106 conidia in 1 drop of suspension to the bottom of each slant. These cultures were grown at room temperature (about 23°C) for about 10 days, after which conidia from each treatment were harvested in buffer, adjusted to 2 x 107 conidia/ml and inoculated into 12-1 Florence flasks at an estimated 106 viable hetero- karyotic conidia per flask. As before, a solvent control was included.

The inoculated Florence flasks, both from the resting-conidia and the growing-culture experi- ments, were incubated for 7 days at 30°C in the dark with aeration, after which percent survival and presumptive ad-3 mutants (purple colonies)/ 10 6 survivors were determined for each flask fol-

51

lowing the procedures of de Serres and Mailing (1971).

Results

Table 2 shows the data from the reverse-mu- tation test. The 4 antitumor agents (m-AMSA, etoposide, teniposide and ellipticine) caused no increase in reversion over the large concentration range (0.025-0.8 #mole/plate). Based on visual comparison of the background growth on the treatment and control plates, the agents caused no killing. The positive control mutagen ICR-170 in- duced reversion in a dose-dependent fashion, especially in N24.

Using resting conidia of H-12 in the ad-3 for- ward-mutation test (Table 3), no mutagenicity or decrease in survival was found for m-AMSA, etoposide or teniposide. At the same concentra- tion (100 #g/ml), ellipticine reduced percent survival to 45% and increased the frequency of ad-3 mutants about 30-fold over the spontaneous frequency. Based on this ad-3 mutant frequency of 40/106 survivors, ellipticine is classified as a mutagen of moderate potency (de Serres and Mailing, 1983). At the concentration of 100/~g/ml, the positive-control mutagen ICR-170 decreased survival to 6% and increased the ad-3 mutant frequency to 18/106 survivors. However, at 10 /~g/ml, ICR-170 gave an ad-3 mutant frequency of 512/106 survivors at 34% survival.

TABLE 2

INDUCTION OF ad-3 REVERTANTS OF N. crassa IN THE OVERLAY PLATE TEST

Strain Agent # moles/plate

0.025 0.05 0.1 0.2 0.4 0.8

N24

12-9-26

ICR-170 0.75 a 3.75 14.75 45.75 106.00 264.50 m-AMSA 0.00 0.00 0.00 0.00 0.00 0.00 Etoposide 0.25 0.25 0.00 0.00 0.00 0.25 Tertiposide 0.00 0.25 0.25 0.00 0.00 0.00 Ellipticine 0.00 0.25 0.00 0.00 0.00 0.00

ICR-170 0.00 0.00 0.50 0.00 3.75 20.25 m-AMSA 0.00 0.00 0.00 0.00 0.00 0.00 Etoposide 0.00 0.00 0.00 0.00 0.00 0.00 Teniposide 0.00 0.00 0.00 0.00 0.00 0.00 Ellipticine 0.00 0.25 0.00 0.25 0.00 0.25

a Revertants/plate are averages of duplicate plates from duplicate experiments. The solvent controls (0.1 ml of 20~ DMSO) showed no revertants.

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TABLE 3

INDUCTION OF ad-3 MUTANTS IN RESTING CONIDIA OF H-12 OF N. crassa

Agent Number Number Percent Number ad-3 of of survival of ad-3 mutants/10 6 flasks colonies mutants survivors

( X 106)

Control 4 a 3.06 100 4 1.32

Treatment b

ICR-170 4 0.39 6 7 18.1 2 c 0.69 34 356 511.7

m-AMSA 4 2.88 98 2 0.69 Etoposide 4 3.05 100 0 0.0 Teniposide 4 3.73 100 1 0.27 Ellipticine 4 2.22 45 90 40.46

a Results from duplicate flasks from duplicate experiments. b All treatments were at a final concentration of 100/~g of mutagen/ml. c Results from duplicate flasks from 1 Expt. at a final concentration of 10 /~g of ICR-170/ml. This concentration gave reduced

killing and an increased frequency of ad-3 mutants.

The frequencies of a d - 3 mutan ts in conidia from cultures of H-12 grown in the presence of 100 #g of each of the 4 agents were no t grea ter

than that of the control (Table 4). There was no or little decrease in survival, except for m-AMSA,

which decreased survival to 53%. Al though el- l ipticine was moderately mutagenic in resting co-

nidia (Table 3), it was negative in growing cultures

(Table 4). The posit ive-control mutagen ICR-170,

however, induced a high frequency of a d - 3 mutan t s (219/106 survivors) at 44% survival, which classi-

fies ICR-170 as a strong mutagen (de Serres and

Mailing, 1983). To my knowledge, this is the first report on the mutagenic i ty of ICR-170 in growing cultures of N. c r a s s a .

The relative percent survival values in Table 4 deserve further comment . Owing to the na tu re of

the experiment, as described in the Methods, de- t e rmina t ion of relative percent survivals of conidia

f rom growing cultures of H-12 may not be very

meaningful . Each 12-1 flask (control or treated) was inoculated with conidia from different sets of

growing cultures. As the heterokaryotic conidial fraction - - the sole k ind of conidia that grows

TABLE 4

INDUCTION OF ad-3 MUTANTS IN GROWING CULTURES OF H-12 OF N. crassa

Agent Number Number Percent Number ad-3 of of survival c of ad-3 mutants/10 6 flasks colonies mutants survivors

( x 10 6 )

Control 4 a 2.82 100 0 0.00

Treatment b

ICR-170 4 3.09 44 679 219.12 m-AMSA 4 2.33 53 1 0.43 Etoposide 4 1.83 81 0 0.00 Teniposide 4 3.11 100 0 0.00 Ellipticine 4 2.80 87 1 0.36

a Results from duplicate flasks from duplicate experiments. b All treatments were at a final concentration of 100 ~tg of mutagen/ml. c See text for discussion of these values.

into colonies in the flask medium - - generally varies from 10 to 30% from culture to culture, an observed decrease in relative percent survival may be due to chance differences in the fraction of conidia that are heterokaryotic.

Discussion

In this study in N. crassa, the lack of mutagen- icity of 3 of the antitumor agents (m-AMSA, etoposide and teniposide) and the weak mutagen- icity of the fourth agent, ellipticine, are in sharp contrast to the mutagenicity of the positive control frameshift mutagen ICR-170. In the ad-3 reverse- mutation test (Table 2), all 4 antitumor agents were nonmutagenic over a concentration range of 0.025-0.8 g mole/ plate whereas ICR-170 clearly reverted both ad-3 mutants; in N24, only 0.05 gmole of ICR-170/plate induced reversion. When resting conidia of H-12 were used in the ad-3

forward-mutation test, 10 gg of ICR-170/ml in- creased the frequency of ad-3 mutants almost 400-fold over the spontaneous frequency (Table 3). In contrast, when each of the 4 antitumor agents was tested at 100 #g/ml, only ellipticine induced ad-3 mutants, causing only an about 30- fold increase over the spontaneous frequency (Ta- ble 3). Finally, when growing cultures of H-12 were exposed to 100 gg of each agent/ml only ICR-170 was mutagenic, inducing a high frequency of ad-3 mutants (Table 4).

Furthermore, the mutagenic potencies of the 4 antitumor agents in an appropriate test (hetero- zygous locus) in mammalian cells (see Introduc- tion) are remarkably greater than in N. crassa

(this paper). Forward mutation at the hetero- zygous tk locus in mouse lymphoma cells has been detected at 0.05 gg of ellipticine/ml (Moore et al., 1987a) and 0.5 ng of teniposide/ml (De- Marini et al., 1987a). Higher doses of each of these agents induced very large increases over the spontaneous frequency. On the other hand, el- lipticine, the only one of the 4 antitumor agents mutagenic in N. crassa, was mutagenic only in resting conidia of H-12 at the comparatively high dose of 100/~g/ml (Table 3).

A number of possible explanations for the ab- sence of mutagenicity of m-AMSA, etoposide and teniposide in N. crassa may be suggested. Per-

53

haps, the absence of mutagenicity is due to lack of uptake of these agents. However, the chemical structures of the 3 negative compounds are differ- ent (Fig. 1). Although they share similar biological properties in that they are all mammalian DNA topoisomerase II-interactive agents, it seems rather unlikely that all 3 chemicals are not taken up by iV. crassa. Furthermore, ICR-170, which contains the acridine ring structure, as does m-AMSA, is taken up and is mutagenic in N. crassa.

Another possibility is that the cells were not in the appropriate cell cycle during exposure to the chemicals. It has been shown that DNA-topo- isomerase II enzyme content is proliferation de- pendent in CHO cells, making them more sensi- tive to the cytotoxic and DNA-cleavage effects of etoposide during log phase than the quiescent phase (Sullivan et al., 1987). However, the absence of mutagenicity of m-AMSA, etoposide and teniposide even in growing vegetative cultures, which should encompass the entire cell cycle, sug- gests that this does not account for the lack of mutagenicity of these chemicals.

Another explanation for the negative results in N. crassa may be that the DNA topoisomerase II of N. crassa is similar to bacterial DNA gyrase rather than to mammalian DNA topoisomerase II. As reviewed in the Introduction, the 4 agents tested are highly mutagenic in mammalian cells but non- or weakly mutagenic in bacterial cells, which correlates well with their interaction with mammalian DNA topoisomerase II but not with bacterial DNA gyrase. Based on studies in bacteria, the 4 agents may be broadly divided into 2 classes: the intercalators m-AMSA and ellipticine, which are mutagenic in frameshift testers via DNA inter- calation and/or covalent adduct formation (De- Marini et al., 1983; Ferguson et al., 1985), and the non-intercalators etoposide and teniposide, which do not intercalate into the DNA (Chen et al., 1984) nor form covalent adducts with DNA (Ross et al., 1984) and have been reported to be non- or weakly mutagenic in bacterial frameshift testers (Matney et al., 1985; Gupta et al., 1987). Also, with regard to their activity as mammalian DNA topoisomerase II poisons, these 4 agents have been classified as either intercalative (m-AMSA and eUipticine) or nonintercalative (etoposide and teniposide) antitumor agents (Liu, 1989). In the

54

case of the AMSA group of compounds in gen- eral, detailed studies on quantitative structure-ac- tivity relationships have shown that the mutagenic potency of a chemical is dependent upon a num- ber of factors such as lipophilic/hydrophilic bal- ance (Ferguson and Denny, 1979, 1980), substitu- tion on the anilino and/or acridine rings (Fergu- son and Denny, 1979, 1980; Ferguson and Mac- Phee, 1983; Ferguson et al., 1985) and DNA-bind- ing affinities (Ferguson and Baguley, 1981). Re- cently, 9-aminoacridine (from which m-AMSA is derived) and isopropyl-OPC, a derivative of el- lipticine (conjugate of 2-N-methyl-9-hydroxy el- lipticinium), have been shown to induce sequence-specific + 1 and - 1 frameshifts in E. coli by reversible DNA intercalation (Rene et al., 1988). However, there is no evidence for the medi- ation of DNA gyrase in the mutagenicity of any of these chemicals in bacteria. In fact, E. coli DNA gyrase has been found to be resistant to m-AMSA in vitro (Nelson et al., 1984).

A very similar set of characteristics has been found for the intercalating acridine proflavin. Proflavin has been found to be a frameshift mutagen in bacteria and bacteriophages (Kohno and Roth, 1974; Speck and Rosenkranz, 1980) and a potent mutagen/clastogen in the L5178Y/ TK +/- mouse lymphoma assay (DeMarini et al., 1988) but only a weak mutagen at the hemizygous hprt locus in L5178Y and CHO cells (Rogers and Back, 1982). Similar to m-AMSA, proflavin in- duces SCEs in mammalian cells (Ostertag and Kersten, 1965; Kato, 1974; Popescu et al., 1977; Carrano et al., 1978; Speit and Vogel, 1979; Car- rano and Thompson, 1982; Morgan and Crossen, 1982) and morphological transformation in mouse 3T3 cells (Neubort et al., 1982). In the r l IB gene of bacteriophage T4, the mutagenicity of proflavin and m-AMSA has been correlated with T4 DNA topoisomerase II-mediated cleavage (Ripley and Clarke, 1986; Ripley et al., 1988). T4 DNA topo- isomerase II is a mammalian-type DNA topo- isomerase (Rowe et al., 1984). In a frameshift mutant of T4 (rFC11), m-AMSA has been found to be an extremely potent mutagen, whereas el- lipticine is weakly mutagenic and teniposide is nonmutagenic (DeMarini and Lawrence, 1988). Interestingly, proflavin also does not induce point mutations in Neurospora (de Serres, 1964) or Drosophila (Ostertag and Haake, 1966).

All these observations lead to the supposition that the nature of the enzyme DNA topoisomerase II causes a distinctive demarcation between multi- drug-susceptible mammalian cells on the one hand and non-susceptible prokaryotes on the other hand. In prokaryotes, when mutagenicity is dem- onstrated - - as in the case of m-AMSA, ellipticine or proflavin - - it is due to physicochemical inter- actions with DNA (such as intercalation or cova- lent adduct formation) without any involvement of bacterial DNA gyrase. Apart from mammalian ceils, results from the other eukaryotic models studied remain controversial. The finding of a CHO cell line (VpmR-5) that is simultaneously cross-resistant to the epipodophyllotoxins etopo- side and teniposide, m-AMSA, mitoxantrone and adriamycin has led the authors to suggest that "multidrug resistance may be acquired by a qualitative change in type II topoisomerase that alters interaction of drug with the enzyme or enzyme-DNA complex" (Glisson et al., 1986). Similar qualitative changes could account for the insensitivity to these topoisomerase II-interacting agents in certain eukaryotes. Nelson et al. (1984) have observed that Drosophila DNA topo- isomerase II has a very weak sensitivity to m- AMSA. However, Udvardy et al. (1986) have shown that Drosophila topoisomerase II gives characteristic cleavage reactions with teniposide, just like mammalian topoisomerase II. Compari- son between the nucleotide sequence of a bacterial DNA gyrase gene and the TOP2 gene encoding DNA topoisomerase II in Saccharornyces cere- visiae, and also the deduced amino acid sequences of the two gene products, has shown a consider- able degree of homology between the two genes and between the two deduced amino acid se- quences, indicating mechanistic and structural similarities and a possible evolutionary relation- ship (Lynn et al., 1986). However, Nitiss and Wang (1988) have found that m-AMSA induces homologous recombination and is mutagenic in S. cerevisiae. Also, Worland and Wang (1989) have reported that m-AMSA, etoposide and teniposide can interact with purified DNA topoisomerase II from S. cereoisiae.

Based on the above facts, the results with m- AMSA, ellipticine, etoposide and teniposide in N. crassa reported in this paper are difficult to inter- pret at this point. However, it may be hypothe-

sized that D N A topoisomerase II in N. crassa is more similar to bacterial D N A gyrase than to mammal i an D N A topoisomerase II and that the mutagenici ty of ellipticine in Neurospora may oc-

cur independent ly of D N A topoisomerase II. Bio- chemical assays of the types done on m a m m a l i a n

cells, using crude nuclear extracts to demonst ra te e n z y m e - d r u g - D N A interactions, may help to re-

solve this quest ion in N. crassa. Also, characteri-

zat ion of the el l ipt icine-induced mutan t s to de- termine if they are solely intracistronic frameshift

mutan t s or if they include MLDs as well will help

to determine if this agent is merely acting as an intercalator (as predicted) or perhaps involves D N A topoisomerase I I -mediated clastogenesis.

Acknowledgements

I thank Dr. H e r m a n E. Brockman for his val- uable guidance throughout the course of this work and for his review of this manuscr ipt . Special

thanks also go to Dr. David DeMar in i for his encouragement and suggestions and to Megin Hayne for her technical assistance. This report is

par t of a dissertat ion to be submit ted in part ial fulf i l lment of the Ph.D. degree at Il l inois State University. The research was supported by the

Beta Lamda Chapter of Phi Sigma and Sigma Xi, The Scientific Research Society.

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