Mutation Research, 92 (I 982) 69-87 69

Elsevier Biomedical Press

A search for some common characteristics of the

effects of chemical mutagens in Drosophila

E . W . Vogel, W . G . H . Blijleven, M . J . H . K o r t s e l i u s
a n d J.A. Z i j l s t r a
Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratoria, Wassenaarseweg 72,
State University of Leiden, 2333 A L Leiden (The Netherlands)
(Received 7 September 1981)
(Accepted 14 September 1981)

1. Introduction

An observation of utmost importance for estimates of genetic hazards posed by

chemical mutagens or radiation is the difference in response that a given dose of
reagent elicits in different types of germ cell. Auerbach (1954) was among the first to
demonstrate in experiments with X-rays that in Drosophila, X-linked and autosomal
recessive lethals show a peak incidence in spermatids; whereas spermatozoa, meiotic
stages and spermatogonia were less sensitive to the mutagenic action of ionizing
radiation. Since then, much work has been directed at a clarification of the
underlying causes of these radiosensitivity differences. Among the many factors
involved, are: initial radiosensitivity to the induction of premutational damage,
efficiency of repair processes, degree of oxygenation of the cells, amounts of
radiosensitizing and radioprotective material in the cytoplasm (Sobels, 1954, 1955,
1963; Sankaranarayanan and Sobels, 1976).
The situation is far more complex in the case of chemical mutagenesis, when we
consider all the possible physiological effects and chemical interactions that can
occur after exposure to a mutagen (uptake, transport across cell membranes,
metabolic activation and deactivation pathways). In view of the complexicity of
these problems, a central question in chemical mutagenesis is whether general
characteristics and relationships can be established for the genetic effects of chemical

Abbreviations: AA, alkylating agents; A 139, 2,5-(methoxy-ethoxy)-3,6-bisethyleneimino-l,4-

benzoquinone; BM, bleomycin; DEN, N-nitrosodiethylamine; DMN, N-nitrosodimethylamine; DEB,
1,2:3,4-diepoxybutane; DMBA, 7,12-dimethylbenzanthracene; EMS, ethyl methanesulfonate; ENNG,
N-ethyl-N'-nitro-N-nitrosoguanidine; ENU, N-nitroso-N-ethylurea; HMPA, hexamethylphosphoramide;
iPMS, isopropyl methanesulfonate; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-
nitrosoguanidine; MNU, N-nitroso-N-methylurea; TEB, 2,3,5,6-tetra(ethyleneimino)-l,4-benzoquinone;
TEM, triethylenemelamine; Trenimon, 2,3,5-tris(ethyleneimino)-1,4-benzoquinone.

0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press

70

reagents that are valid beyond the particular species, strain, cell stage, and set of
environmental conditions under which the correlations were obtained. The purpose
of the present paper is (1) to seek for such general patterns and common elements of
the effects of chemical mutagens in Drosophila, and (2) to trace some of the more
recent developments that have taken place in this respect during the past years.
A judgement of general characteristics of mutagenic effects in Drosophila germ
cells requires special definition of how sensitivity, cell-stage mutability and changes
in mutational response are to be measured. Because it is known that the extent of
changes in sensitivity depends very much on the kinds of damage under observation,
it seems most desirable to look at several kinds (Auerbach, 1976; Parker, 1963). But
this is not always possible, owing to the paucity of the available data. The bulk of
information on stage response in Drosophila has been collected by tests for recessive
lethal mutations in males, by using a wide array of direct-acting agents and
promutagens. Thus, in the present survey we are going to depend rather heavily on
data obtained from experiments on the induction of X-chromosomal and, though to
a minor extent, autosomal recessive lethals in male germ cells. Cell-stage compari-
sons for other types of genetic damage will also be included where feasible.

2. Recessive iethals in males

2.1. Direct-acting mutagens

General pattern. After treatment of the male germ-cell cycle with direct-acting
reagents, for each mutagen a specific pattern is characteristic in terms of the stage
response observed and the type as well as the magnitude of damage produced. This
seems to be a logical consequence of the large number of factors affecting chemical
mutagenesis. In spite of this diversity in response, there are some characteristic
features direct-acting mutagens have in common. Firstly, as a general rule, the
highest percentages of X-chromosomal recessive lethals are mostly obtained from
cells that have passed the meiotic divisions. This observation has been amply
confirmed, e.g. for MMS (Fahmy and Fahmy, 1961) (Fig. 1), EMS (Browning, 1970;
Fahmy and Fahmy, 1961) (Table 1), ethyleneimine (Alexander and Glanges, 1965),
epichlorohydrin (reviewed in Vogel et al., 1981), DEB (Shukla and Auerbach, 1980),
TEPA (Sram, 1972), TEM and Trenimon (reviewed in Vogel et al., 1981). The
differences in mutational response found between postmeiotic and premeiotic stages
are generally large for those agents. Browning (1970) reported for EMS a 4-5-fold
higher yield in postmeiotic cells of recessive lethals compared with the yield in
meiotic and premeiotic cells, whereas in experiments by Fahmy and Fahmy (1961)
the difference between postmeiotic and premeiotic cells was even more than one
order of ma~titude. Analysis of the trifunctional alkylating agent TEM (reviewed in
Vogel et al., 1981) revealed a reaction pattern that is typical of alkylating agents with
more than one reactive site: at high concentrations (> 2.0 X 10-1 mM), postmeiotic
stages are the only ones that can survive the treatment, showing a high rate of
recessive lethals, whereas meiotic and premeiotic stages are destroyed by TEM as
indicated by complete sterility of the later broods. In the concentration range from
 71
x

20" ~x
18- '~mM

16-
14- x
\\ MMS
"~1.0rnM , \
12" ~e ~ \ ~x
10" \~xsterile
:=
i 8" \\
x 0.5 mM \x~x~
6- ~ 4 \\x x

o0.2 mM \ = ~

2 4 6 8 10 12 DAYS(brooding}
sp st sc sc sg sg
Fig. 1. Percentages of X-linked recessive lethals produced by MMS in consecutive germ-cell stages.
Treatment of adult males for 48 h.

1.0 × 10 - ' to 3.0 × 10 -2 mM TEM, younger cells survive, but recessive lethal yield
was 5-10 times below that determined for postmeiotic cells similarly treated.
Mutagens that do not follow the general pattern described here are ICR 170
(Carlson and Southin, 1963) and mitomycin C (Mukherjee, 1955). For both reagents,
no major differences in response of premeiotic and postmeiotic stages are seen when
X-linked recessive lethal induction is determined.
Germinal selection. The reason that has been advanced to mainly account for
these large changes in sensitivity within the male germ-cell cycle is known under the
term "germinal selection" introduced by Muller (1954). It refers to the fact that
X-linked deletions that include a gene required for development of a spermatogonial
cell into a spermatozoon are removed by germinal selection. Thus, to obtain a
realistic picture of the intrinsic stage mutability throughout the cell cycle, only
autosomal lethals can be used to compare the sensitivity to a given mutagen, and the
ratio of autosomal to X-linked lethals in postroeiotic cells as compared with that in
spermatogonia, is a measure of germinal selection (Shukla and Auerbach, 1980). If
this concept is valid, it follows that the relative difference in yield between X-
chromosomal and autosomal lethals should be greater in spermatogonia as opposed
to postmeiotic cells, if the mutagen in question is capable of inducing multi-locus
deletions and gross structural aberrations. Mutagens for which this approach has
been followed are DEB, DEN, EMS and 2-chloroethyl methanesulfonate (Purdom,
1957; Shukla and Auerbach, 1980). The work of Shukla and Auerbach (1980) on
DEB and DEN has shown that germinal selection takes place in the case of DEB,
known to produce chromosome breaks in Drosophila. With DEN, the ratio of
autosomal to X-linked lethals also tended to be somewhat higher in premeiotic cells
as opposed to that in spermatozoa; but the differences observed were only small.
DEN is a potent mutagen in terms of point mutations, but seems fairly ineffective in
72

TABLE I
COMPARISON OF 2nd-CHROMOSOME TO X-CHROMOSOME RECESSIVE LETHAL FRE-
QUENCIES IN CELLS TREATED WITH EMS OR DEN
Spermatozoa Spermatids Spermatogonia
l./tests % l./tests % l./tests %
EMS
II 167/724 23.1 116/691 16.8 79/1 604 4.9
X 106/807 13.1 78/795 9.8 10/I 880 0.53
II:X 1.8 1.7 9.3

DEN
II 24/495 4.9 60/423 14.2 89/ 789 11.3
X 15/821 1.8 53/797 6.7 41/1 617 2.5
II:X 2.7 2.1 4.5
Pooled data from 2 Expts.: X, X-chromosome; II, 2nd chromosome.

breaking the chromosomes of Drosophila (Vogel and Leigh, 1975; Vogel and
Natarajan, 1979a).
The results of experiments in our institute with EMS and DEN are summarized in
Table 1. After treatment with EMS and, somewhat unexpectedly, also with DEN, a
substantial portion of spermatogonia carrying X-linked lethals does not pass through
meiosis. When the yields of autosomal lethals produced by EMS and DEN are
compared, it is evident that EMS extends maximal activity in spermatozoa and
spermatids, while DEN preferentially acts in spermatids and spermatogonial stages.
With DEB, there appears no stage specificity for autosomal recessive lethals (Shukla
and Auerbach, 1980); this picture is different from that obtained with other
multifunctional agents in the X-linked recessive lethal test, indicating the signifi-
cance of strong germinal selection for this type of mutagen.

2.2. Stage specificity of promutagens

In the preceding section, recent data on induced recessive lethals were sum-
marized and examined from the point of view of relationship to cell stage of
induction. It is now instructive to review the results of those experiments that have
provided evidence for sensitivity differences within the male germ-cell cycle after
treatment with mutagens that require metabolic activation.
A striking feature of promutagen mutagenesis in Drosophila is the significance of
intragonadal metabolism for their genetic effectiveness. Insects do not have a
specific organ for detoxication, but several tissues appear to be involved. These are
the fat bodies, various parts of the digestive tract (Casida, 1969) and, crucial for the
explanation of the pattern induced by promutagens, certain parts of the gonadal
tissue. There is considerable evidence, derived from experiments with alkaryltria-
zenes and other promutagens, that release of mutagenic metabolites from the parent
promutagen takes place directly in gonadal tissue, the ceils that are tested in the
recessive lethal assay. In the majority of cases, triazenes show a persistent peak
mutagenic activity in metabolically active stages, i.e., in various spermatid stages,
 73

whereas spermatocytes are highly susceptible to killing (Vogel and Sobels, 1976;
Vogel et al., 1981; Vogel, 1981). Tates (1971) has reported that these stages have a
highly developed endoplasmic reticulum.
A pattern of sensitivity similar to that described for alkaryltriazenes has also been
recorded for other classes of promutagen: nitrosamines, oxazaphosphorines, aflato-
xins, azoxyalkanes, halo-olefins, haloalkanes, pyrrolizidine alkaloids (reviewed in
Vogel, 1981). The observation that maximal mutation frequencies were observed in
spermatids and spermatocytes also accounts for DEN (Table 1) and aflatoxin B 1
(Lamb and Lilly, 1971), the only promutagens extensively studied in the autosomal
recessive lethal assay. It should be realized, however, that the occurrence of peak
mutagenic activity in spermatids or earlier stages alone is no proof that the chemical
under study is a promutagen.
The picture described above does not apply for aromatic hydrocarbons, which are
difficult to detect in Drosophila. With aromatic hydrocarbons, e.g. with DMBA, we
were unable to identify a clear-cut brood-specific pattern in mutagenic response
(Table 2). The mutagenic metabolite(s) in this particular case might have to migrate
from other tissue to the germ cells, and this would mean that germinal tissue of
Drosophila is not very effective in activating aromatic hydrocarbons. This interpre-
tation is supported by a recent experiment in which males and females were
pretreated with phenobarbital before benzo[a]pyrene hydroxylation was measured
(J.A. Zijlstra, unpublished observations). The result was a 7-fold increase in the
specific benzo[a]pyrene hydroxylating capacity in both sexes. However, the relative
inducibility of the enzymes was not the same in different parts of the body. Enzyme
induction was about equal in various parts of thebody, except the testis where it was
lowest.
The second piece of evidence supporting the significance of intragonadal activa-
tion for most classes of promutagens comes from concentration-effect studies with
procarbazine (Blijleven and Vogel, 1977), DMN (Fahmy and Fahmy, 1975), and
DEN (Vogel and Leigh, 1975). With those mutagens, concentration-effect relations
are different for metabolically inert sperm versus metabolically active spermatids. In

TABLE 2
I N D U C T I O N OF RECESSIVE LETHALS IN MALES INJECTED WITH DMBA

Strain Treatment In spermatozoa In spermatids

(mM)
Tests Effect a Tests Effect a
Berlin-K I 0.0 1 631 12 × 1 535 5×
Haag-6 I 0.0 27 (43 × ) 197 27 ×
Oregon-K 10.0 382 63 X 269 98 ×
91-R 10.0 281 36x 106 17X
Canton S (V) 10.0 102 0 118 4x
Canton S (F) 10.0 107 34 × 20 59 X
Canton S (F) 5.0 800 21 x 680 31 ×
a n-fold increase in mutation frequency above spontaneous rate.
74

15-

sperrnatids

u, 10-
-6
procarbazine

~ 5
sper rnalozoo

10 15 2'0 25 30
mM
Fig. 2. Mutagenic activity of procarbazine in spermatozoa and spermatids. (Data from Blijleven and
Vogel, 1977.)

spermatids, a steep increase is found for recessive lethals between 0.5 and 10.0 mM
procarbazine (Fig. 2) (with a plateau above 20.0 mM) and between 1.0 and 10.0 mM
D M N (Fig. 3). This stands in marked contrast to the flat curves found after
treatment of spermatozoa. In spermatozoa, there is no single proportionality be-
tween the concentration applied and the response obtained in the recessive lethal
test.
The metabolically inert spermatozoa, then, must have been attacked by more
stable material from the surrounding tissue, and the extent to which this happens
may determine the extent of sensitivity differences seen between sperm and sperma-
tids. Whether this interpretation is correct is not known, but the consequences for

25'
DMN
20'
jo
-~ 15" °~~ st

10-
8
5-

(Fohmy and Rlhmy,1978) injected dose ( n-fold]

Fig. 3. Mutagenic activity of dimethylnitrosamine in spermatozoa and spermatids. (Data from Fahm y and
Fahmy, 1975.)
 75

EMS

t~
25

20'

15"

10- /
.f MN
..

:t
injected dose {n-fold}

Fig. 4. Exposure-effect relationship for the induction of recessive lethals by MNU, ENU, EMS and
iPMS. (Data from Vogel and Natarajan, 1979a.)

mutagenicity testing of an intragonadal activation are obvious - - particular atten-

tion must be paid to metabolically active germ-cell stages. A further implication
from these results is that there is a strongly marked concentration dependence of
stage sensitivity and of stage-sensitivity differences, which are presumably linked to
differences in metabolic activation. This is a good example of the care that has to be
taken when interpreting on the basis of only one dose, differences between the
responses of germ cells to mutagens.
On the other hand, with direct-acting alkylating agents, this pattern in mutagenic
response is no longer present (Fig. 4). This type of mutagen, over a wide range of
doses, generally shows the expected linear concentration-effect relationship, as can
be seen from Fig. 4 for MNU, EMS, iPMS and ENU. The shapes of the dose-effect
curves are similar for EMS and ENU, and also for iPMS and MNU, in spite of the
fact that the types and the distribution of DNA adducts formed by these reagents
are quite different.
Dose-effect relationships of the type reported for postmeiotic cells have yet to be
established for spermatogonia.

2.3. Role of repair

In recent years, a number of recombination-defective and repair-defective mutants
has been isolated in Drosophila to study the genetic control of mutagenesis in this
eukaryote (Baker et al., 1976). One large class of mutagen-sensitive mutants has been
isolated on the basis of hypersensitivity to killing by MMS (Smith, 1973; 1976; Boyd
et al., 1976a). C0mplementation tests suggest that there are about 60 loci in the
Drosophila genome which control sensitivity to MMS (Mason, 1980; Smith et al.,
1981). Some of these mutants have been reported to be defective in meiotic
recombination and female fertility (Baker and Carpenter, 1972; Boyd et al., 1976a,b;
Smith, 1976), excision repair (Boyd et al., 1976b) and post-replication repair (Boyd
and Setlow, 1976).
76

In attempts to explore, in vivo, the function of repair mutants on chemically

induced mutagenesis, and the possibility that these mutants could enhance the
sensitivity of Drosophila for detecting environmental mutagens, several of these
mutants were analyzed for their sensitivity to AAF, BP, aflatoxin B~, nitrogen
mustard and a series of monofunctional alkylating agents. Most work has been
carried out with the single mutants mei-9 a, mei-9 Ll, mus(1)101, mei-4 t)5 and the
double mutagen-sensitive mutant mei-9 a mei-45 D5 (Graf and Wiirgler, 1981; Graf et
al., 1979; Nix et al., 1980; Nguyen et al., 1979; Vogel, 1981). The picture obtained is
far from clear, some observations even being controversial. One experimental
approach has been to treat either wild-type or mutagen-sensitive males and compare
mutation induction in postmeiotic and meiotic cells. Because mature sperm do not
have the biochemical machinery to repair premutational lesions or to convert these
into mutations (Maddern and Leigh, 1976; Sankaranarayanan and Sobels, 1976;
WiJrgler and Ulrich, 1976), differences between repair-proficient and repair-defective
strains should appear only in broods corresponding to the treatment of spermatids
or spermatocyte stages. Nix et al. (1980) observed a significant increase in the
frequency of X-linked recessive lethals when mei-9 a males were treated with several
alkylating agents. Treatment of males from strains Oregon R (wild type), mei-9 a,
mei-9 L1, mei-41 a and mei-41 D5 with EMS and DEN gave no significant differences
between the 5 genotypes. DMN and MMS treatments produced even fewer lethals in
mei-9 a and mei-9 L1 than in mei-41 a, mei-41DS, or wild-type males.
The results with m e i - 9 El and Oregon R for EMS and MMS, obtained by Nix et
al. (1980), differ from the picture obtained in similar experiments in our laboratory
with mei-9 El and Berlin K, the strain from which mei-9 El was isolated. Fig. 5 shows
summarized data from results to be published elsewhere. Treatment of m e i - 9 El males
with MMS or EMS, at various concentrations, led to a consistent and significant
enhancement in the lethal frequency, but only in progeny corresponding to treated
spermatid or spermatocyte stages. By contrast, the effects of ENU and DEN
treatments are virtually the same in the 2 types of male.

1~] SPERM SPERMATIDS ~ 9.I SPERMATOCYTES IM

584 R I lii?l
~7~ HIH
FIITil I lii!l

MMS EMS MNU ENU DEN MMS EMS MNU ENII DEN MMS EMS MNU ENU DEN
(raM) 20 50 10 1.0 100 (raM} 20 50 10 10 100 {rnM) 20 50 10 10 100

Fig. 5. Influence of the genotype on m u t a t o n induction (recessive lethals) by alkylating agents. Treatment
of either Berlin K males (white columns) or mei-9 m males (dotted columns).
 77

20" 20"
18" 18-
16" sperm 16- spermatids
1/*" 14"
~ 12"
~ 10" 10-
8' g-
8
-- 4"
2' ::1

MMS EMS MNU ENU MMS - EMS MNU ENU

[mMI 20 150 10 10 (mM] 20 150 1.0 1.0

Fig. 6. The role of maternal repair on the induction of recessivelethals after mutagen treatment of Basc
males for 2 days. Crosses performed with either Berlin K females (white columns) or mei-9LI females
(dotted columns).

We also performed the reciprocal cross, i.e., repair-proficient males were treated
and mated to either mutagen-sensitive or wild-type females, to explore the effects on
mutation induction of repair-deficient females. Graf and Wiirgler (1981) and Graf et
al. (1979) reported that D N A alkylation by MMS, EMS, M N U or ENU in
spermatozoa always led to increased percentages of X-linked recessive lethals in
oocytes with an excision-repair deficiency (mei-9) compared with wild-type oocytes.
This pattern is consistent with the picture observed in similar experiments in our
laboratory with MMS, EMS and M N U (Fig. 6). There is, however, no agreement
concerning the effects produced by ENU. Although a whole series of ENU experi-
ments was performed, utilization of mei-9 L~ females did not alter the yield of lethals
determined at several ENU concentrations which ranged from 5 X 10 -3 to 2.5 X 10 -
mM. At high concentrations of 0.5 and 1.0 mM ENU, a marked decline in male and
female fertility and mutation induction was noted for the cross mei-9 L~ ~ £ X Basc
d d , as compared with the combination Berlin K ££ × Basc d ~ . Therefore, one of
the questions that can be asked is whether selective killing of certain cell stages can
explain part of the rather puzzling inter-laboratory discrepancies. To clarify the
issue, we feel that there is a stringent need for extensive concentration-effect studies.

3. Other types of genetic damage in males

In the preceding sections, data on induced recessive lethal mutations were

presented and considered from the point of view of the relationship to cell stage of
induction, metabolic activation and repair. In the present section, an attempt will be
made to summarize information on chromosome aberrations (chromosome loss,
translocations, deficiencies) for an overall view of relative sensitivities of the various
germ-cell stages of Drosophila to the induction of chromosome aberrations. How-
TABLE 3
I N D U C T I O N OF 2 - 3 T R A N S L O C A T I O N S IN T H E G E R M - C E L L C Y C L E OF MALES T R E A T E D W I T H M M S F O R 48 h

Concentration Storage Broods (days after treatment)

(mM) condition
I(0-2) II(2-4) III(4-6) IV(6-8) V(8-1 O) VI( I O- 12)
0.1 U N 367 395 403 405 384 398
%T 0 0 0 0 0 0

S N 1 219 1 222 843 644 735 1066

%T 0 0 0 0 0 0

0.2 U N 778 757 791 755 436 772

%T 0 0 0 0 0 0

S N 2072 2 103 2013 1 215 690 1 829

%T 0.19 0.05 0.15 0.25 0 0

0.5 U N 399 396 397 92 271 390

%T 0 0 0.3 (1.0) 0 0

S N 1 140 981 648 68 207 561

%T 1.3 0.7 1.9 0 0 0

1.0 U N 336 303 228 28 341 400

%T 0.6 2.6 2.2 (7.1) 0 0

S N 446 178 98 190 778

%T 4.9 5.6 8.2 sterile 0 0

2.0 U N 52 91 34 sterile 43 373

%T 0 5.5 14.7 (0) 0

S N 108 42 7 sterile 15 532

%T 4.6 4.8 (14.3) (0) 0
U, unstored; S, stored for .3-9 days; %T, % 2 - 3 translocations; N, gametes tested.
 79

ever, as already pointed out, we shall be concerned again entirely with male
germ-cell stages.
Comparisons of spermatozoa, spermatids, spermatocytes and spermatogonia give
generally consistent results in various studies. Thus, on the basis of induced
sex-chromosome aberrations and translocations, postmeiotic cells yield higher fre-
quencies of aberrations than do spermatogonia (Alexander and Glanges, 1965;
Browning, 1969; Sram, 1972). Spermatocytes are readily eliminated by cell killing at
moderate and higher concentrations. To illustrate this aspect with an example, in a
detailed brood fractionation analysis with 10 3-day broods after treatment with
MNNG, 4.3% translocations (14/412 gametes) were detected in brood 1, 2.5% (5 in
201) in brood 2, 0.5% (1 in 211) in brood 3, but none thereafter in about 1400 tests
(Browning, 1969). Determination of MMS-induced translocations in stored and
unstored germ cells shows a similar distribution in translocation yield (Table 3).
There are, however, basic difficulties in comparing spermatocytes and spermato-
gonia with postmeiotic stages. The possibility of strong segregational elimination
during meiosis makes it impossible to draw any conclusion concerning initially
induced breakage frequencies from the final yield observed.
This leaves us with the question whether there are differences in relative sensitiv-
ity between the postmeiotic spermatozoa and spermatid stages. In an analysis with a
series of monofunctional AA, 2 parameters were applied for comparing, in these 2
stages, the chromosome-breaking efficiency of some AA: the lowest exposure
concentration which produced both chromosome aberrations (translocations and
ring-X loss) and recessive lethal mutations, and the proportion of translocations to
mutations (T:M) at equal or at least similar mutation frequencies. In some experi-
ments with MMS, the T : M ratios tended to be somewhat higher in spermatids, but
the differences observed were only small (Vogel and Natarajan, 1979a). Experiment~
with ring-X chromosomes (Vogel and Natarajan, 1979b) provided firmer evidence of
an essential difference in sensitivity to chromosome aberration induction between
spermatozoa and spermatids. Again, the comparisons were made on the basis of
similar percentages of recessive mutations. These comparisons revealed a lower
response of spermatozoa relative to spermatids for DMN, MNU, EMS and MMS.
The new data for MMS on recessive lethals (Fig. 1) and on translocations (Table 3)
point in the same direction.

4. Genetic damage in females

The bulk of mutation work on Drosophila has been carried out in males, whereas
there is only sparse information about the effects of chemical mutagens in females.
The reason for this perhaps lies in the relatively high sensitivity of oocytes to
induced cell death. Another complication is that females may contain pre-existing
lethals which must be crossed out before an experiment is begun.
The experimental evidence available shows that in Drosophila the sensitivity of
germ cells to damage induced by chemical mutagens varies strikingly during oogene-
sis. The development of the Drosophila egg has been subdivided into a series of 14
80

consecutive stages, ending with stage 14, the mature primary oocyte (Cummings and
King, 1969; King et al., 1956; Koch et al., 1970). The 2 stages that have received
most attention are stages 7 and 14. These are both meiotic prophases, stage 7 being
the oldest and most posterior oocyte in each ovariole in the newly emerged female.
By the time the adult female is 2 days old, stage 7 has advanced to stage 14 and
remains in this stage until the egg is laid (Parker, 1963).
In general, comparisons of relative sensitivities of stage 7 and stage 14 to
mutagens yield close parallelism. Thus, with mitomycin C treatment of females,
Schewe et al. (1971) noted that the frequency of X-chromosome recessive lethals was
twice as high in the first 2-day brood (4.2%) as in the next one (presumably
immature oocytes), followed by a decline in mutation rate down to 0.4-0.6% in
gonial cells. With the alkaryltriazenes 1-phenyl-3,3-dimethyltriazene and 1-(pyridyl-
3-N-oxide)-3,3-dimethyltriazene, stage-14 oocytes were about 3-4 times as sensitive
as stage-7 oocytes for the induction of sex-linked recessive lethals (Vogel, 1971).
Data on mutation induction by EMS at the dumpy locus showed similar differences
in response between mature and immature oocytes. In a study with DES (Pelecanos
and Alderson, 1964) mutation induction tended to be highest in the first brood. The
authors concluded that no significant difference in mutational sensitivity was
detectable between the 2 oocyte stages; however, the number of gametes tested was
only very small in those experiments.
With the aziridine analogs A 139, Trenimon and TEB, about 100-fold higher
concentrations of these 3 strong mutagens are required to produce the same rates of
cell death in immature oocytes as in mature ones (Vogel, 1972). Thus, immature
oocytes appear to be relatively immune to both cell killing and mutation induction
by chemical mutagens.
There are only few mutagens (mitomycin C, EMS, bleomycin, DES, PDMT, and
PNDMT) that have been extensively tested in both Drosophila sexes. Among those,
BM (bleomycin) has been reported to be far more active in females. Thus, after
feeding BM to Drosophila males, Traut (1980) observed only a low (0.63%) induc-
tion of recessive mutations in treated spermatozoa and spermatids. However, under
conditions similar or identical to those of the experiments with males, a considerable
sensitivity was observed of Drosophila oocytes to BM-induced recessive lethals
(1.7-4.5%) and X-chromosomal aneuploidy (non-disjunction and chromosome loss).

5. Genetic pattern in relation to interaction with D N A

5.1. Germ cells

In the foregoing paragraphs, the discussion of experimental findings was con-
cerned mainly with the description of phenomenological observations, primarily
owing to inadequate understanding of the mechanism of mutation at the molecular
level. In assessing the mutagenic potential of a chemical, consideration should be
given to the mechanism by which the chemical induces the genetic effect. This, to
some extent, seems possible for monofunctional alkylating agents, because their
reaction principles are relatively well understood. This makes AA particularly
 81

valuable as model compounds for comparisons between different systems or species.

The ethylating agents EMS, DES, ENNG, ENU and DEN are particularly suited for
approaches aimed at identifying links between the mode of interaction with DNA
and the kind of genetic damage observed, because their effects of relative extents of
alkylation of various DNA sites have been investigated in some detail (Lawley et al.,
1975; Lawley and Shah, 1972; Pegg and Nicoll, 1976; Singer, 1976; Sun and Singer,
1975). The critical point with multicellular organisms, such as Drosophila, is
measurement of the actual dose at the genetic material of the target cell under
analysis, usually the germ line. The exposure concentration is an entirely inap-
propriate parameter for comparing the genetic effects of chemicals, because of the
large variation in the rates of absorption, distribution in cells and tissues, and
kinetics of inactivation. To overcome this problem, the percentages of recessive
lethal mutations can be used to serve as a biological dosimeter for the extent of
interaction of alkylating agents with target DNA in the germ line (Vogel and
Natarajan, 1979a,b). On this basis, the induction of recessive lethal mutations was
used as a unit to compare in the same population of treated cells the ability of the
reagents to induce 2-3 translocations. One of the comparisons made was the
calculation of the proportions T: M of translocations to mutations at different
mutation rates (doses). Thus, for MMS, the starting point for the production of
translocations and chromosome losses was relatively low; translocations already
occurred at MMS doses producing a 5-fold increase in the percentage of lethals
(Vogel and Natarajan, 1979a). However, the results also showed that with an
ENU-type mutagen, the recovery of chromosome aberrations can be expected only
at an extremely high degree of DNA ethylation, i.e., at a dose leading to about a
100-300-fold increase in the percentage of recessive lethals. ENNG and DEN
showed the same behavior as ENU, while DES and EMS took an intermediate
position between these 3 and MMS. The similarities in the mutational spectra
observed for ENU, ENNG and DEN are consistent with their very similar ethyla-
tion pattern. Over 80% of DNA modifications by ENU, ENNG and DEN are on
oxygen atoms (Singer, 1976; Sun and Singer, 1975). The corresponding values for
the DES-EMS pair are 8-20%. The major conclusion drawn from the marked
specificity of DEN, ENU and ENNG for point mutations was that the molecular
events that characterize the action on DNA of these agents, O-ethylation, in
particular the formation of ethylphosphotriesters which are the predominant prod-
ucts (Singer, 1976, 1979; Sun and Singer, 1975), are not critical for the processes
leading to chromosome breakage in Drosophila.
Another striking feature of these AA which is instructive to examine, is the lower
yield of delayed mutations in passing from MMS, EMS, DES, through DEN and
ENU. Recessive lethals that arise with a delay give rise to mosaic gonads in F~ and
cannot be detected with the conventional F2 recessive lethal test. An F3 generation
must be set up to determine these effects, and the ratio of F2:F3 lethals can serve to
express the relative effectiveness of a reagent to cause delayed mutations.
It is striking that the agents relatively inefficient in inducing chromosome
breakage, ENU and DEN, gave high F2-lethal to F3-lethal ratios. MMS, EMS, DES
and DMS at various levels of exposure tended to induce more delayed mutations,
82

this being reflected by lower F2-1ethal:F3-1ethal ratios (Vogel and Natarajan,

1979a,b). Thus, for the AA considered here, ability to induce delayed mutations and
efficiency to cause chromosome breakage, seem to be positively related. In other
words, ability to produce delayed mutations seems to correlate with preference for
alkylation of base nitrogens, especially the guanine N-7, the major product of DNA
reacted with MMS, EMS, DES or DMS (Lawley and Brookes, 1963; Lawley and
Shah, 1972; Sun and Singer, 1975; Swenson and Lawley, 1978). Alkylation at the
7-position and the 3-position of purine bases in nuclei acids is known to cause
depurination from DNA (Lawley, 1974; Lawley and Brookes, 1963). Depurination
has been equated with strand breakage, a potentially lethal lesion (Brookes and
Lawley, 1961, t963). More recently, Shearman and Loeb (1977) showed a possible
relationship between depurination and mutagenesis through incorporation of non-
complementing nucleotides during DNA synthesis in vitro. It has been suggested
that, besides its involvement in lethal effect, depurination may contribute to chro-
mosome breakage and occasional base-pair deletions, which may occur as delayed
effects (Auerbach, 1976; Freese, 1971; Oeschger and Hartman, 1970).
The hypothesis on the link between alkylation at base nitrogen and the occur-
rence of delayed mutations in Drosophila has been advanced before (Vogel and
Natarajan, 1979a) and most recently has received further experimental support from
work with hexamethylphosphoramide (HMPA). The mutational pattern of HMPA
is, in many respects, entirely different from that seen for mono- and poly-functional
alkylating agents. When HMPA-treated males are mated to untreated females, the
percentage of 2-3 translocations is the same whether the females are allowed to lay
eggs at once (unstored) or whether they are prevented from doing so for several days
or weeks (Vogel, unpublished observations). This indicates no delayed formation of
exchanges from HMPA-induced breaks. When the same type of experiment is
carried out with MMS, for example, a gradual increase in the percentage of
translocations is observed in the later broods. This phenomenon shows that MMS-
induced breakage is a slow process. Auerbach (1976) has suggested that "depurina-
tion leading to strand breakage is a conceivable candidate for the potential lesions
that, in the chromosomes of Drosophila and plants, mature gradually into chro-
mosome breaks". HMPA, and again this stands in contrast to the picture seen with
MMS-type mutagens, is also not very efficient in the production of delayed
mutations, measured as F3 recessive lethals. The frequencies of recessive lethal
mutations in the F2 were 4-7-fold higher than the percentages of mutations that
could be detected by an analysis of the F3 test (Vogel, unpublished observations).
Preliminary experiments were carried out to see whether specific interactions with
DNA could explain the peculiar genetic pattern observed for HMPA. An experiment
was designed in which Drosophila DNA was reacted in vivo with [14C]HMPA
(0.23%) for 2 h (Van Zeeland, unpublished observations). By far the major part of
radioactive label was at a position of unidentified products, possibly the pyrimi-
dines, whereas HMPA did not bind to a measurable extent with the O 6 or N-7
position of guanine. If HMPA reacted with guanine or purine sites, then this activity
must have been very small. Experiments are now in progress to analyze further and
possibly identify the reaction products of DNA reacted with HMPA.
 83

5.2. Somatic tissue

The last part of this paper concerns the consideration of genetic effects of some
alkylating mutagens in somatic cells of Drosophila in relation to their interaction
with DNA. We felt that a brief consideration of the effect produced by these agents
in somatic cells would be of interest in the present context, the possible relation
between initial alkylation and the kind of genetic effects observed. There exist a
number of assays that measure the induction of somatic recombination, mutation
and non-disjunction in Drosophila larvae. A system devised by Becker (1966) is
based on the determination of somatic mutation (single spots) and recombination
(twin spots) in the eyes of WC°sn2//w; se h / s e h females, and of single spots in w c°
sn 2, se h / s e h males following treatment of larvae. We used this system to compare
the ability of MMS, EMS, DES, DEN, ENNG and ENU for producing twin spots
and single spots. All 6 compounds were efficient in producing twin spots (TS) and
single spots (MS) in females treated as larvae. However, the proportion of TS and
MS varied considerably, depending on the type of alkylating agent investigated
(Table 4). MMS produced the highest TS:MS ratio, followed by EMS and DES
which gave similar ratios. DEN, ENNG and ENU were the least effective com-
pounds in the induction of twin spots. This is obvious from the low TS:MS indices
determined for these 3 agents. It is interesting to note that for the reagents which
alkylate DNA in a similar fashion, the indices are also similar: for the EMS-DES
pair (0.76 versus 0.78) and for DEN (0.23), ENNG (0.29) and ENU (0.36). In other
words, reagents such as MMS, which in germ cells are very efficient in the
production of chromosome aberrations, give relatively high TS:MS indices in
somatic tissue. The reverse is true for DEN, ENNG and ENU. Experiments are now
in progress to investigate for other mutagens to what extent this quick somatic
mutation assay has predictive value for the relative ability of a reagent to produce
chromosome aberrations in the germ line.

TABLE 4
I N D U C T I O N OF S O M A T I C M U T A T I O N A N D R E C O M B I N A T I O N BY A L K Y L A T I N G A G E N T S
IN F E M A L E w e ° / w L A R V A E

Compound MMS EMS DES DEN ENNG ENU

N u m b e r of experiments 9 5 5 5 4 9

Eyes tested 5 558 4 062 3 694 4118 3 948 5 370

% TS 4.84 1.92 0.68 0.56 0.91 2. l0

MD 2.0 1.62 0.60 1.75 1.98 3.37

ME 2.16 0.91 0.27 0.68 1.11 2.46

TS : (M o + M E) 1.16 0.76 0.78 0.23 0.29 0.36

M o :M E 0.93 1.78 2.22 2.57 1.78 1.37

S u m m a r y table from data to be published elsewhere.
TS, twin spots; M D, dark spots; M L, light spots.
84

6. Concluding remarks

It is felt that the analysis of shared general features is the best way to compare the
actions of genetically active agents in a complex multicellular organism such as
Drosophila.
(1) From the relationship of cell-stage sensitivity to action of promutagens, it is
clear that with this type of mutagen, germ-cell metabolism is the most significant
factor determining their effectiveness in various cell stages.
(2) To obtain a realistic picture of the intrinsic stage mutability throughout the
Drosophila germ-cell cycle, only autosomal recessive lethal tests can be used to
compare stage sensitivity to a given mutagen.
(3) Response of Drosophila to some, but not all, alkylating mutagens can be
modified considerably by introducing into the assay system repair-deficient mutants
(e.g. mei-9). There is a tendency to add repair-deficient strains to the battery of
tester strains in common use and thereby assume that the Drosophila assay has been
improved. This can only be determined if each new strain is validated before general
use with a wide selection of mutagens. From our experience with the mutants
mei-9 El and mei-9 a mei-41DS, these strains offer no apparent advantage over
repair-proficient tester strains.
(4) Attention has been drawn to the relation of molecular mode of interaction
with DNA and the genetic effects produced in germ cells by some methylating and
ethylating reagents. The results reviewed in this paper indicate that several genetic
parameters, i.e., the proportion of chromosome aberrations in relation to mutations
and the ability of a reagent to cause delayed mutations, are valuable tools to get
more insight into basic mechanisms of chemical mutagenesis. A new parameter
which seems useful in this context is the proportion in somatic cells of mosaic twin
spots in relation to mosaic single spots. It is concluded that, for MMS, EMS, DES,
ENU, DEN and ENNG, there is a positive relationship between their ability to
produce chromosome aberrations in germ cells and mosaic twin spots in somatic
tissue.

Acknowledgement

This work was supported by the National Institute of Environmental Health

Sciences (U.S.A.), Contract No. ESO 1027-04/06, and the "Stichting Koningin
Wilhelmina Fonds" (The Netherlands), Contract No. 81.90. Part of this investigation
also received support from the Association Contract No. 139-77-1 ENV N between
the European Communities (Environmental Research Programme) and the State
University of Leiden.

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