Ž .Mutation Research 397 1998 279–285

Induction of mosaic sex-linked recessive lethals in the differentgerm cell stages of Drosophila melanogaster by bleomycin

May Fouad Sadiq ), Omar Falah KhabourDepartment of Biological Sciences, Yarmouk UniÕersity, Irbid, Jordan

Received 10 July 1997; revised 30 September 1997; accepted 2 October 1997

Abstract

The mutagenicity of bleomycin was studied in the different stages of spermatogenesis in Drosophila melanogaster.Following the injection of 2 ml of 0.1 mgrml of the chemical into young wild-type males, complete and mosaic sex-linkedrecessive lethals were scored by the Muller-5 method in five successive broods, mainly representing the different stages ofspermatogenesis. The delayed mutagenic effect of the chemical was measured by the proportion of mosaic progenyproduced. The results showed that bleomycin significantly increased the proportions of both complete and mosaic lethals inthe broods representing the meiotic and pre-meiotic stages, but did not show any significant increase in these proportions inthe broods representing the sperms and spermatids. The sizes of the mutated areas in the F gonads represented by the1

proportions of lethal-bearing females in F mosaic cultures were small, indicating that the genetic instabilities induced by2

bleomycin were transformed into actual mutations in later zygotic divisions. The significant production of mosaic progeny inthe F generation of the treated males showed that the mosaic F females produced by bleomycin were able to produce4 1

further mosaic progeny and suggested that bleomycin-induced instabilities can be transmitted as such for many futuregenerations. q 1998 Elsevier Science B.V.

Keywords: Bleomycin; Drosophila melanogaster; Mosaic sex-linked recessive lethal; Mutation

1. Introduction

Bleomycin is a chemotherapeutic drug currentlyused in the treatment of neoplastic diseases, particu-larly in some testicular squamous cell carcinomas,

w xand lymphomas 1 . It was first isolated from Strep-w xtomyces Õerticillus 2 and consists of a mixture of

about 200 different basic sulfur-containing glycopep-tides which share the same basic structure but have

) Corresponding author. Tel.: q962 2 271100 ext. 2833; Fax:q962 2 274725.

w xdifferent amino terminals 3 . Bleomycin interactswith DNA and induces different types of point muta-tions and chromosomal aberrations in a wide rangeof living systems including Salmonella typhimurium

w xTA 102, E. coli WP rPKMI101 4–6 , the purple2w xsulfur bacterium Thiocapsa 7 , bacteriophages

w xlambda and M 8,9 , Saccharomyces cereÕisiae13w x w x10–12 , Aspergillus nidulans 13,14 and different

w xmammalian systems including human 15 .Drosophila melanogaster is a convenient eukary-

otic system linked to the development of genetictoxicology and was used extensively in studying

0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0027-5107 97 00226-1

( )M.F. Sadiq, O.F. KhabourrMutation Research 379 1998 279–285280

germ line mutagenesis since Muller’s pioneeringw xwork 16 . The ingestion of bleomycin by Drosophila

increased the rates of somatic crossing over, mitoticw xrecombination and aneuploidy in the oocytes 17–19 ,

but did not induce sex-linked recessive lethals whenw xsperms and spermatids were tested 20,21 . There-

fore, it is of interest to study the mutagenic effects ofbleomycin on the different germ cell stages in thisorganism.

In this study, the classical multiple-locus sex-lin-ked lethal test was used to examine the mutagenicityof bleomycin on the different stages of spermatogen-esis, because it is the best validated Drosophilamutagenicity test that detects a broad spectrum ofgenetic alterations, mainly point mutations and small

w xdeletions 22 , and shows quite good correlationbetween mutagenic response and carcinogenic activ-

w xity of the tested chemicals 23 .

2. Materials and methods

2.1. Drosophila stocks and chemical treatment

The proper concentrations of bleomycin sulfatew xCAS reg. a 9041-93-4 were freshly prepared bydissolving in 0.45% NaCl saline. Using an Ugla

Žmicrometer syringe Welcome Research laboratories,.England , young wild-type Oregon-k males obtained

from Carolina Biological Supply, USA, were in-jected with 0.2 ml of the saline or the proper concen-

Ž .trations of bleomycin sulfate. Muller-5 Basc stock( ) S1L 8 R 8 S1 aIn 1 sc sc qS, sc sc w B, was a generous

gift from Dr. I.C.I. Eeken, Leiden University, theNetherlands. All the cultures were continuouslymaintained at 258C on wheat-sucrose agar mediumw x24 . Bleomycin was obtained from Sigma, agar wasobtained from Himedia, and other media ingredientswere obtained from the local market.

Fig. 1. Survival of Drosophila melanogaster males following their treatment with different concentrations of bleomycin.

( )M.F. Sadiq, O.F. KhabourrMutation Research 379 1998 279–285 281

2.2. Mutagenicity tests

The induction of sex-linked recessive lethals wasŽ . w xstudied using the Basc Muller-5 test 25 . 24 h

following the treatment, each male was individuallymated to three Basc virgin females. Three days later,each male was individually remated with other threenew Basc virgin females. This procedure was re-peated to yield five broods of three days each.Beginning from the first, these broods correspondedapproximately to the five different germ cell stages:spermatozoa, spermatids, spermatocytes, late sper-

w xmatogonia, and early spermatogonia 26 . In eachbrood, all the F heterozygous females were individ-1

ually mated to young Basc males, then tested for thepresence of complete sex-linked recessive lethals.The absence of wild-type males and the presence ofseveral Basc males in their F cultures were indica-2

tions of the presence of complete sex-linked reces-Ž .sive lethal mutation s . All lethals were reconfirmed

by separately mating four of their heterozygous F2

females with Basc males and scoring lethality intheir F progeny.3

The induction of mosaic sex-linked recessivelethals was studied by individually mating randomlyselected F heterozygous Basc females from non-2

lethal cultures to Basc males. These F heterozygotic2

females were selected from all lethal and non-lethallines. The progeny of these F females were then3

examined for the presence of sex-linked recessivelethals. Gonadic mosaic lethal F females showed1

their lethality when their F females were individu-2

ally mated to Basc males and their lethal mutationsappeared in F . The proportion of lethal bearing F3 2

females among all daughters of an F female re-1

flected the size of the mutated tissue in their gonads.

3. Results

Fig. 1 shows that bleomycin had highly toxiceffects and induced sterility in Drosophila males.Both toxicity and sterility were increased and ex-tended with time following the treatment. The sur-vival of the treated males one day following theinjection with 0.2 mgrml was 98%, then droppedafter thirteen days to 71%, and few of the survivingmales were fertile. Due to the toxicity and sterilityeffects, 0.1 mgrml bleomycin was used in the muta-genicity studies.

As shown in Table 1, the frequency of sex-linkedcomplete lethals in the five different broods, variedbetween 0.09% and 0.26% in the control group, andvaried between 0.17% and 1.07% in the group treatedwith bleomycin. The difference between the frequen-cies of spontaneous and bleomycin-induced completesex-linked recessive lethals was significant at 5%

w xlevel 27 in both the meiotic and the pre-meiotic butnot in the post-meiotic stages. Table 2 shows that thefrequencies of mosaic lethals induced by bleomycinin the different broods appeared to be higher thanthose of the control, but the increase was significant

Table 1Frequencies of sex-linked recessive lethals induced in Drosophila melanogaster by 0.1 mgrml bleomycin treatment

Ž .Treatment Brood 3 days No. of tested X-chromosomes No. of complete lethals % of complete lethals

Bleomycin A 1191 2 0.17B 1181 2 0.17

aC 1171 12 1.03aD 1123 12 1.07aE 1075 9 0.84

Control A 1165 3 0.26B 1168 2 0.17

bC 1134 2 0.18bD 1130 1 0.09bE 1096 2 0.18

Controls were treated with 0.45% NaCl.a,b Ž . a bStatistical analysis Kastenbaum–Bowman test showed a significant increase at 5% level in the induced over the spontaneousfrequencies.

( )M.F. Sadiq, O.F. KhabourrMutation Research 379 1998 279–285282

Tab

le2

Freq

uenc

ies

ofm

osai

cse

x-lin

ked

rece

ssiv

ele

thal

sin

duce

din

Dro

soph

ila

mel

anog

aste

rm

ales

trea

ted

with

0.1

mgr

ml

bleo

myc

in

Tre

atm

ent

Bro

odN

o.of

non-

leth

alN

o.of

Fm

osai

c%

ofF

mos

aic

No.

ofF

No.

ofle

thal

-bea

ring

%of

tota

l2

22

Ž.

3da

yscu

lture

ste

sted

leth

alcu

lture

scu

lture

spr

oduc

edfe

mal

este

sted

Ffe

mal

esle

thal

sin

F2

3

Ble

omyc

inA

205

31.

4613

153

0.29

B20

13

1.49

1282

30.

23a

C18

37

3.83

1213

100.

82D

173

52.

8911

647

0.60

E18

34

2.19

1135

60.

53

Con

trol

A19

92

1.01

1439

20.

14B

202

20.

9914

222

0.14

bC

188

31.

5613

363

0.22

D18

62

1.08

1365

20.

15E

185

10.

5413

412

0.15

Con

trol

sw

ere

trea

ted

with

0.45

%N

aCl.

a,b

Ž.

Ž.

ab

Stat

istic

alan

alys

isK

aste

nbau

m–

Bow

man

test

show

edsi

gnif

ican

tin

crea

se5%

leve

lin

the

indu

ced

over

the

spon

tane

ous

freq

uenc

ies.

Ž .5% level only in brood C. Both induced completeand mosaic sex-linked lethals were detected only insingles in the post meiotic stages, and were producedin both singles and clusters in the pre-meiotic and

Ž .meiotic stages Table 3 .The frequencies of the induced mosaic lethals

were generally less than those of the induced com-plete lethals in all broods. The ratios of mosaic- tocomplete-sex-linked recessive lethals are shown inTable 4, they varied between 2.6 and 8.6 in thetreated group with an overall average of 5.3, andvaried between 3.0 and 12 in the control group withan overall average of 6.7.

All of the F mosaic females were heterozygous2

for a lethal derived from the F mosaic mother. The1

sizes of the mutated tissue sectors in the F mosaic1

gonads, represented by the proportion of their F2

lethal-bearing daughters, varied in the pre-meioticand the post-meiotic stages of the treated groupbetween 11% and 17% respectively, with an overallaverage of 14.4% and varied between 6.3% and

Table 3Distribution of complete and mosaic lethals, as singles and clus-

Ž .ters two or more lethals per cluster in Drosophila melanogastermales treated with 0.1 mgrml bleomycin

Treatment Test Brood No. of No. ofsingle lethals clusters

Bleomycin Complete lethals A 2 0B 2 0C 8 2D 7 2E 7 1

Mosaic lethals A 3 0B 3 0C 4 3D 4 1E 2 2

Control Complete lethals A 1 1B 2 0C 2 0D 1 0E 2 0

Mosaic lethals A 2 0B 2 0C 3 0D 2 0E 0 0

Controls were treated with 0.45% NaCl.

( )M.F. Sadiq, O.F. KhabourrMutation Research 379 1998 279–285 283

Table 4Ratio of mosaic to complete lethals produced in successive broodsof Drosophila melanogaster males treated with 0.1 mgrmlbleomycin

aŽ .Treatment Brood 3 days Ratio of mosaic to complete lethals

Bleomycin A 8.6B 8.8C 3.7D 2.7E 2.6

Control A 3.9B 5.8C 8.7D 12.0E 3.0

Controls were treated with 0.45% NaCl.a This ratio was calculated by dividing the percentage of F2

mosaic cultures by the percentage of complete lethals.

8.3% in the control group with an average of 6.9%.Most of the broods in the treated group had mosaicF females that again produced mosaic F daughters1 2

in both singles and clusters, while the production ofmosaic F females was limited to brood C in the2

control group.

4. Discussion

The observed toxicity of bleomycin to Drosophilawas proportional to the concentration of the drug,and was in agreement with earlier studies on mam-

w xmalian and non-mammalian systems 4–15 , wherethe bases for this toxicity were related to the induc-tion of chromosomal aberrations, RNA sequencespecific hydrolysis and inhibition of DNA synthesisw x15,28–32 . Our results also confirmed the earlierobservation where non-toxic concentrations ofbleomycin proved to be not mutagenic to Drosophila

w xsperms and spermatids 20 , but also showed thatŽ .bleomycin is mutagenic at the 5% significance level

to the dividing spermatocytes, late spermatogonia,and early spermatogonia represented by broods C, Dand E respectively. These mutagenic effects ofbleomycin to Drosophila were in agreement with theinduction of reciprocal translocations in bone mar-row cells and stem-cell spermatogonia in micew x33,34 . According to earlier reports, the mutagenic-

ity of bleomycin in different systems was due to thew xinduction of abasic sites 35 , release of thymine

w x36–39 , and increase in the rate of induced DNAw xstrand breaks 40 . Our observed selective mutagenic

effects of bleomycin on the dividing stages inDrosophila could be explained by either the prefer-ential action of bleomycin on the DNA replicationfork, or by its dependence on the actual relativelyhigher amounts of bleomycin reaching the DNA inmitotic cells compared to those amounts reaching the

w xintact nuclei of non-mitotic cells 41,42 .The ability of bleomycin to produce both DNA

w x w xsingle-strand breaks 17 and abasic sites 43 couldbe involved in the production of the genetic instabili-ties that produce mosaic lethals. The repeated occur-rences of mosaic lethals in Drosophila male linestreated with bleomycin, suggested that bleomycin iscapable of inducing delayed mutations for severalgenerations, and supported the view that chemicallyinduced instabilities can be transmitted as such over

w xseveral generations 44 . Similarly, the small sizes ofthe mutated areas in the F gonads, represented by1

the proportions of lethal-bearing females in the F2

mosaic cultures, indicated that the instabilities in-duced by bleomycin were transformed into actualmutations in later zygotic divisions, during whichthey were replicated as such for many cellular gener-ations before they actually became fixed mutations.As a result of the genetic risks of bleomycin on thegerm cell lines, and since the factors influencinggenetic aberrations in germ cells are not different

w xfrom those of somatic cells 45 , it is important toconsider the results of this study in future evaluationof the genetic risks for patients treated withbleomycin.

Acknowledgements

The authors thank Dr. I.C.I. Eeken, Leiden Uni-versity, The Netherlands, for his precious gift of theDrosophila Basc stock. This investigation was par-tially supported by grant 53r90 from the Deanshipof Research and Graduate Studies at Yarmouk Uni-versity, Jordan.

( )M.F. Sadiq, O.F. KhabourrMutation Research 379 1998 279–285284

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