Mutation Research, 188 (1987) 307-309 307 Elsevier

MTR01197

Effect of fagaronine on chromosome breaks and loss in Drosophila melanogaster

Yvette Prrez-Chiesa, Carmen Noemi Cintr6n and Alice Rodriguez Department of Biology, University of Puerto Rico, Rio Piedras, PR 00931 (U.S.A.)

(Received 19 November 1986) (Revision received 26 February 1987)

(Accepted 11 March 1987)

Keywords: Fagaronine; Chromosome breaks and loss; Antileukemic agent; (Drosophila melanogaster).

Summary

Fagaronine, a potent antileukemic agent, was shown to induce chromosome breaks and loss in sperm of Drosophila melanogaster.

Fagaronine (CAS Reg. No. 52259-65-1) is a potent antileukemic agent (Messmer et al., 1972; Tin-Wa et al., 1974). Its antitumor activity is attributed to its capacity to intercalate into the DNA double helix and double-stranded RNA (Pezzuto et al., 1983). Many intercalating agents are capable of inducing DNA beaks (Ross et al., 1979) and some have been found to be mutagenic a n d / o r clastogenic (Marcos et al., 1981; Alderson and Khan, 1968; Ostertag and Hacke, 1966). Pez- zuto et al. (1983) found fagaronine to be bacteri- cidal but not mutagen ic in Sa lmone l la typhimurium, even in the presence of a rat micro- somal subfraction. When fagaronine was tested in vivo using Drosophila melanogaster it was found to be mutagenic and increased by 5-fold the spontaneous frequency of sex-linked recessive lethals (Prrez-Chiesa and Cintrbn, 1986). It also produced a high frequency of dominant lethals at concentrations that have no effect on toxicity (Cintrrn, 1985). However, most of the dominant

Correspondence: Dr. Yvette P6rez-Chiesa, Department of Bi- ology, University of Puerto Rico, Rio Piedras, PR 00931 (U.S.A.)

lethals detected could be due to eggs fertilized by nonfunctional sperm or to the death of male germ cells and not to true dominant lethals. Dominant lethals are mainly chromosomal aberrations such as chromosome loss, breaks and rearrangements (Brink, 1969; WiJrgler et al., 1977). In this study we have tested the capacity of fagaronine to pro- duce chromosome breaks and loss in D. melanogaster and found it to be weakly clasto- genic.

Materials and methods

R(1)2, y f / B S y y + adult males, 3-4 days old, were exposed to a 5% sucrose solution containing 0.0 mM (control), 1.5 mM or 3.0 mM of fagaronine chloride (NSC 157995) dissolved in dimethyl sulfoxide (DMSO) as described by Prrez-Chiesa and Cintr6n (1986). After exposure for 48 h to the fagaronine-sucrose solution each male was crossed to two y2 sc w a mei-9 a virgin females, 3-5 days old. The males were discarded after 3 days. The females were subcultured 3 times, once every 3 days, to increase the progeny yield. The progeny

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collected from these females developed from eggs that were fertilized by postmeiotic sperm present in the males at the time of treatment. The flies were cultured in banana-yeas t -agar medium un- der standard laboratory conditions at 25 ° C.

The ring-X chromosome was used because of its sensitivity; it tends to be lost with a higher frequency than does the rod-X. The spontaneous frequency of loss for the Y or rod-X is less than 0.02-0.06% (Vogel and Natarajan, 1979). The m e i -

9 a excision-repair-deficient females were used be- cause they lay eggs which tend to lose the ring-X with a higher frequency than most normal, repair-proficient strains (Cooper and Zimmering, 1981; Wiargler and Graf, 1982). The progeny from these flies were scored for XO males (caused by complete loss of the X or Y, but mainly the ring-X), for partial loss of the Y chromosome (loss of the B ~ or y+ markers) and for spontaneous nondisjunction (XXY progeny, mainly maternal nondisjunction which is frequent in m e i - 9 a

females). The y2 s c w ~ markers in the X chro- mosome of the female and the B s and y+ in the Y of the male are used to distinguish maternal from paternal chromosome loss and nondisjunction. (See Lindsley and Grell (1968) for a complete description of the chromosomes and genetic markers used.)

Control and experimental groups were coded to avoid bias. Statistical analysis was done according to Kastenbaum and Bowman (1970) when fewer than 100 events had occurred. Otherwise, con- tingency tests were done.

Results and discussion

A summary of the data obtained from the m e i - 9 a test for detecting chromosome breaks and loss is shown in Table 1. In this experiment, maternal nondisjunction is high as is expected for m e i - 9 a females (Baker et al., 1976) and does not concern us here. The frequency of paternal nondisjunction detected among the treated groups is not significantly different from control, as ex- pected, because the progeny were collected from postmeiotic sperm exposed to fagaronine. The paternal nondisjunction detected was spontaneous and occurred prior to treatment.

The frequencies of complete chromosome loss (either X or Y, but mainly the X) for the 1.5 and 3.0 mM concentrations are significantly higher than the control (P < 0.025). The estimated in- duced rate of chromosome loss with treatment is from 0.40 to 0.43%. Only with the 3.0 mM concentration was the test positive for partial chromosome loss of the Y (P < 0.05). Here the estimated induced rate of breakage is 0.11%. Therefore, we can conclude that fagaronine is weakly clastogenic and could potentiate a low but significant frequency of chromosomal rearrange- ments such as translocations (Zimmering, 1983). Moreover, because of its clastogenic and muta- genic activity (P6rez-Chiesa and Cintr6n, 1986), the drug may have carcinogenic properties.

The low frequency of chromosome breaks and loss detected in the present study cannot account for the high frequency of dominant lethals (18%)

TABLE 1

CHROMOSOME BREAKS AND LOSS IN THE PROGENY FROM SPERM a OF Drosophila melanogaster MALES TREATED WITH FAGARONINE FOR 48 h

Conc. Total Normal PL b % CL b % Maternal Paternal (mM) progeny progeny ND b % ND b %

Control c 9066 8664 3 0.03 91 1.00 299 3.29 9 0.10

1.5 9565 9126 2 0.02 134 1.40 e 292 3.05 11 0.12 3.0 9262 8784 13 0.14 d 132 1.43 f 321 3.47 12 0.13

a Postmeiotic sperm from the cross: R(1)2, y f / B s y y+ males mated to y sc w a mei-9 a females. b PL, partial loss of Y chromosome (loss of B s or y+ markers); CL, complete loss of X or Y chromosome; ND, nondisjunction. ¢ 0.02% DMSO in 5% sucrose. d Statistically significant (P < 0.05; Kastenbaum and Bowman, 1970). c X2 ~ 5.83, P < 0.025. f X 2 = 6.42, P < 0.025.

detected among the sperm exposed to fagaronine in a previous study (Cintrrn, 1985). We conclude that the greatest contribution to egg mortality in the dominant lethal study may be attributed to the toxic effects of the drug on sperm function and germ cell metabolism.

Acknowledgement

This investigation was supported with research funds from the University of Puerto Rico.

The fagaronine samples were a courtesy of the National Cancer Institute, National Institutes of Health, U.S.A. We thank Dr. S. Zimmering for providing us with the ring and m e i - 9 ~ stocks and Dr. David Bruck for reading the manuscript.

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