Mutation Research, 173 (1986) 193-196 193 Elsevier

MRLett 0811

Mutagenic effect of fagaronine in Drosophila melanogaster

Yvette P6rez-Chiesa and Carmen Noemi Cintr6n Department of Biology, University of Puerto Rico, R(o Piedras, PR 00931 (Puerto Rico)

(Accepted 29 October 1985)

Summary

Fagaronine, a strong anti-tumor agent, was shown to induce sex-linked recessive lethals and male sterility in Drosophila melanogaster.

The alkaloid 2-hydroxy-3,8,9-trimethoxy-5- methylbenzo[clphenanthridine (fagaronine, CAS 52259-65-1) isolated from the roots of Fagara zan-

thoxyioides (Rustaceae) represents a class of com- pounds that show potent anti-tumor activity against L1210 and P388 murine leukemias (Mesmer et al., 1972; Tin-Wa et al., 1974). Fagaronine inhibits the enzymatic activity of reverse transcriptase of RNA oncogenic viruses and of mammalian DNA and RNA polymerases (Sethi and Sethi, 1975; Sethi, 1976, 1979). It simultaneously inhibits nucleic acid synthesis and protein synthesis in KB tumor cells and it also in- hibits protein synthesis in a cell-free system derived from rabbit reticulocytes (Casiano, 1983). The in- hibition of polymerase activities is thought to oc- cur by the binding of fagaronine to regions of DNA containing both A-T and G-C pairs and to its interaction with ribonucleic acids (Pezzuto et al., 1983). Casiano (1983) suggested that it may in- hibit protein synthesis by also interacting with the ribosomal system.

Although fagaronine can bind to DNA and is bactericidal, it has not been found to be mutagenic

in Salmonella even in the presence of a microsomal subfraction from rat liver which increased its bactericidal activity 1000-fold (Pezzuto et ai., 1983). Fagaronine is under evaluation as a possible anti-leukemia agent. Because its mechanism of ac- tion appears to be at several levels, as suggested by the simultaneous inhibition of nucleic acid syn- thesis and protein synthesis (Casiano, 1983), it is important to evaluate its general ability to produce genetic damage in higher organisms in vivo, before declaring it mutagenically safe. In this study, fagaronine's mutagenicity was tested using Drosophila melanogaster as the model system. The drug was shown to induce sex-linked recessive lethals and male sterility.

Material and methods

The Base method, which detects sex-linked recessive lethals (Abrahamson and Lewis, 1971; Wfirgler et al., 1977) was used to look for the pro- duction of point mutations and small deletions. Base and wild-type Oregon R stocks were reared in standard banana-yeast-agar medium under

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laboratory conditions at 25°C. (For a complete description of the Basc flies see Lindsley and Grell, 1968.)

Oregon R males, 3-4 days old, were fed for 48 h with a 5°-/0 sucrose solution containing 1.5 mM or 3.0 mM of fagaronine (NSC 157995). The drug was dissolved, first in DMSO and then in the sucrose solution. The final concentration of DMSO was 0.0207o. Control males received only DMSO and sucrose. With the concentrations of fagaronine used, less than 40"/o of the flies died dur- ing treatment. Sterilized filter paper in clean vials was soaked with the solution and the males were introduced and handled from then on without anesthesia. After feeding, treated and control males were crossed, individually, with two virgin Basc females, 3-5 days old. Different stages of spermatogenesis were tested by transferring the males to a new set of virgin females every 3 days and collecting their progeny in separate broods. Thus, 3 successive broods were obtained: one for each group of females. On the assumption that the treatment does not result in a delay of sper- matogenesis, the first two broods represent post- meiotic stages and the last brood, meiotic and pre- meiotic ones. The number of sterile males in each brood was scored.

The F1 progeny was intercrossed and the sex- linked lethals were scored in the F2 generation. The criterion for scoring a lethal was the complete absence of wild males in a group of at least 20 flies.

All lethals were confirmed by breeding their sisters for at least two generations. To detect clusters of lethals in the F2, the progeny of each individual male was numbered. The control and experimental

groups were coded to avoid bias.

Results and discussion

The number of sterile males is significantly in- creased after treatment with fagaronine at a con- centration of 3.0 mM. Sterility (measured in terms of the number of sterile males in a given brood) is significantly different for the 3 broods when com- pared to the control broods (Table 1). The effect of the drug may include an impairment on courtship behavior, genitalia, sperm transfer and/or an in- hibition of sperm function, besides an inhibition or delay of spermatogenesis. The increase in the number of sterile males after treatment with the drug is in conformity with the toxic effects of the drug reported for other systems (Sethi, 1976; Ca- siano, 1983, Pezzuto et al., 1983).

The results of the test for detecting sex-linked recessive lethals are summarized in Table 2. The data of 5 experiments were pooled. The frequency of lethals is low in all treated groups regardless of the germ-cell stage treated. No significant increase over the control frequency of lethals was observed when individual broods were considered (Kasten- baum and Bowman, 1970). However, when the results from broods 1, 2 and 3 are pooled, there is

TABLE 1 FREQUENCY OF STERILE MALES PER BROOD AFTER FEEDING THEM WITH FAGARON1NE FOR 2 DAYS

Conc. Brood 1 Brood 2 Brood 3

(mM) Sterile/ Sterile/ Sterile/

tested b °70 tested b % tested b %

Brood 1-3

Average o7o sterile

Control a 7/39 17.9 8/39 20.5 7/39 17.9 18.8 1.5 15/46 32.6 17/46 37.0 18/46 39.1 36.2

3.0 25/49 51.0 ¢ 21/49 42.9 a 26/49 53.1 e 49.0

Sterile/tested (number of sterile males/total number of males tested).

a 0.02% DMSO in 5% sucrose. b The number of males tested was pooled from the last 3 of the 5 sex-linked recessive lethal tests performed. c x2 = 8.93, P<0.01 .

d X2 = 3.94, P<0.05.

e x2 = 9.97, P<0.01

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TABLE 2

INDUCTION OF SEX-LINKED

FAGARON1NE FOR 2 DAYS

RECESSIVE LETHALS IN DROSOPHILA MALE GERM CELLS EXPOSED TO

Conc. Brood 1 Brood 2 Brood 3 Brood 1-3

(mM) Leth/ % 1 Leth/ % 1 Leth/ % 1 Leth/ % 1

chrom, b chrom, b chrom.b chrom.b

Control a 1/1376 0.07 0/1224 0.00 1/1324 0.08 2/3924 0.05

1.5 4/1334 0.30 5/1217 0.41 4/1206 0.33 13/3757 0.35 c

3.0 4/1204 0.33 1/1129 0.09 1/ 849 0.12 6/3182 0.19

Leth/chrom., number of lethals/number of chromosomes tested; % 1, percentage lethals.

a 0.02% DMSO in 5% sucrose.

b The number of chromosomes tested was pooled from 5 Expts.

c Statistically significant 0.05 > P > 0 . 0 1 (Kastenbaum and Bowman, 1970).

a significant difference between the number of lethals produced with the 1.5 mM concentration as compared to the number produced in the control (P<0.05). All lethals came from different treated males, excluding the possibility that they arose from a single pre-meiotic mutational event.

The estimated induced mutation rate of 0.29% represents at least a 5-fold increase above the estimated spontaneous rate. (We used the method of Wiirgler et al. (1977) to calculate the induced

mutation rate from our data.) No dose effect was observed; at the 3.0 mM concentration the number of lethals produced was not significantly different from control. Nevertheless, this concentration significantly increased the percentage of sterile males (Table 1). Perhaps it is too toxic, reducing the survival of many germ cells which in turn may account for a lower production of recessive lethals.

A wide variety of intercalating agents generate protein-associated DNA breaks. Among them are adriamycin, actinomycin D, ellipticine, ethidium bromide and lucanthone (Ross et al., 1979). DNA breaks may lead to cell death and mutations. Ethidium bromide, for example, was recently found to be highly mutagenic in Drosophila as in other organisms (Sep61veda et al., 1981). Thus, fagaronine, which intercalates into DNA, could cause DNA breaks which in turn, could lead to cell death, deletions, chromosome loss and other kinds of genetic damage. Even mature sperm that are metabolically inactive at the time of treatment

would be affected since pre-mutational lesions in- duced in mature sperm are processed after in- semination by the maternal enzyme system present in the oocytes (Graf et al., 1978).

Through the sex-linked recessive lethal test in Drosophila one can detect all kinds of point muta- tions including small deletions arising from chromosome breaks (WiJrgler et al., 1977). Unrepaired DNA breaks would be lethal in bacterial systems and may account for the high bactericidal effect of fagaronine in Salmonella (Pezzuto et al., 1983), besides other toxic effects of the drug. The present results reveal a weak mutagenic activity of fagaronine in Drosophila. No peak mutagenic activity was found in any of the germ-cell stages treated. This suggests that the drug does not require metabolic activation in order to cause genetic damage (Vogel, 1977). However, the number of lethals is too small to discard this possibility.

The capacity of fagaronine to produce dominant lethals, chromosome breakage, loss and non- disjunction in Drosophila is now being in- vestigated.

Acknowledgements

This work was supported with funds from the University of Puerto Rico. The authors are in- debted to Jack Rodriguez-D~ivila and Nanette Van Loon for technical assistance. The Oregon R and

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Basc stocks were kindly given to us by the Califor- nia Institute of Technology, U.S.A. The fagaronine samples were a courtesy of the National Cancer Institute of the National Institute of Health, U.S.A. We thank Dr. David Bruck for reading the manuscript and helpful criticism.

References

Abrahamson, S., and E.B. Lewis (1971) The detection of muta- tions in Drosophila melanogaster, in: A. Hollaender (Ed.), Chemical Mutagens, Vol. 2, Plenum, New York, pp. 461-487.

Casiano-Torres, C.A. (1983) Studies on the mechanisms of ac- tion of the antitumor agents 3-nitrobenzothiazolo[3,2a]- quinolium and fagaronine: effects on nucleic acid and pro- tein synthesis, Thesis, University of Puerto Rico.

Graf, U., M.M. Green and F.E. Wiirgler (1978) Mutagen sen- sitive mutants in Drosophila melanogaster, effects of pre- mutational damage, Mutation Res., 63, 101-112.

Kastenbaum, M.A., and K.O. Bowman (1970) Tables for deter- mining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549.

Lindsley, D.L., and E.H. Grell (1968) Genetic variations of Drosophila melanogaster, Biology Division, Oak Ridge Na- tional Laboratory, Carnegie Institution of Washington,

Publ. 627, 472 pp. L6pez de Sep6lveda, J.R.M., M. Xamena and A. Creus (1981)

Mutagenicity of ethidium bromide in the sex-linked recessive lethal assay in Drosophila melanogaster, Mutation Res., 91,

337-340.

Messmer, W.M., M. Tin-Wa, H. Fong, C. Bevelle, N. Farns- worth, D. Abraham and J. Trojanek (1972) Fagaronine, a new tumor inhibitor isolated from Fagara zanthoxyloides, J. Pharm. Sci., 61, 1858-1859.

Pezzuto, J.M., S.K. Antosiak, W.M. Messmer, M.B. Slaytor and G.R. Honig (1983) Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[c]phe- nanthridine (fagaronine), with nucleic acids, Chem.-Biol. In- teract., 43, 323-339.

Ross, W.E., D. Glaubiger and K.W. Kohn (1979) Qualitative and quantitative aspects of intercalator-induced DNA strand breaks, Biochim. Biophys. Acta, 562, 41-50.

Sethi, M.L. (1979) Inhibition of reverse transcriptase activity by benzophenanthridine alkaloids, J. Natl. Prod., 42, 187-196.

Sethi, V.S. (1976) Inhibition of mammalian and oncornavirus nucleic acid polymerase activities by alkoxybenzophenan- thridine alkaloids, Cancer Res., 36, 2390-2395.

Sethi, V.S., and M.L. Sethi (1975) Inhibition of reverse transcriptase activity of RNA-tumor viruses by fagaronine, Biochem. Biophys. Res. Commun., 63, 1070-1076.

Tin-Wa, M., C.L. Bell, C. Bevelle, H. Fong and N.R. Farns- worth (1974) Potential anticancer agents, I. Confirming evidence for the structure of fagaronine, J. Pharm. Sci., 63, 1476-1477.

Vogel, E. (1977) Identification of carcinogens by mutagen testing in Drosophila, The relative reliability for the kinds of genetic damage measured, in H.H. Hiatt, J.D. Watson and J.A. Winstein (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, New York, pp. 1483-1497.

Wiirgler, F.E., F.H~ Sobels and E. Vogel (1977) Drosophila as assay system for detecting genetic changes, in: Kilbey et al. (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 335-373.

Communicated by R.J. Preston