[CANCER RESEARCH 32, 1273-1277,
June 1972]
Genetic Characterization of Diethylnitrosamine-induced
Adenine (ad-3) Mutants in Neurospora crassa1
Purple
H. V. Mailing and F. J. de Serres
Carcinogenesis Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
SUMMARY
closely related carcinogen
The mutagenic effect of diethylnitrosamine was studied in
Neurospora crassa. The metabolic activation required to make
diethylnitrosamine carcinogenic in vivo was mimicked in vitro
with one of Udenfriend's hydroxylation mixtures (a mixture
consisting of ascorbic acid, Few, and EOT A); only under these
tested have been nonmutagenic in Escherichia coli (11, 29),
Serratici (11), and yeast (27) and require metabolic activation
in the host-mediated assay to become mutagenic in bacteria
(10). DMN and diethylnitrosamine
have induced reverse
mutations in Neurospora crassa (20), but only when treatment
has been performed in Udenfriend's hydroxylation mixture
conditions was the compound found to be mutagenic. Mutants
were induced
in a gentically
marked
2-component
heterokaryon and recovered as purple colonies among the
white, nonmutant background colonies. In this system, purple
colonies result from two types of genetic damage in the
adenine-3 region: point mutations at ad-3A and ad-3B loci and
multilocus deletions covering one or both loci simultaneously.
Genetic
analysis
showed
that
99%
of
the
diethylnitrosamine-induced
ad-3
mutants
were
point
mutations.
Mutants in the ad-3B locus show allelic
complementation, whereas those at the ad-3A locus do not.
Among the diethylnitrosamine-induced
ad-3B mutants, 65.5%
had nonpolarized
complementation
patterns, 4.5% had
polarized complementation
patterns,
and 29.9% were
noncomplementing.
Our previous studies have shown that
mutants with nonpolarized patterns result from base-pair
substitutions and probably produce a complete polypeptide
chain with only a single erroneous amino acid substitution.
Thus, our data on diethylnitrosamine support our hypothesis
(Ann. N. Y. Acad. Sci., 163: 788-800, 1969) that potent
carcinogenic activity is associated with gene products that have
altered functions rather than with gene products that have no
function.
INTRODUCTION
Diethylnitrosamine is known to be a potent carcinogen in
many animal species (1, 12, 30, 33, 34), including primates
(14). Diethylnitrosamine is metabolized by enzymes in many
different organs to a strong alkylating agent (19). All
suggestions (7, 8, 16, 18) about the active metabolic product
involve an initial enzymatic oxidative dealkylation
of
nitrosamines.
In addition, diethylnitrosamine is mutagenic in Drosophila
(9, 28). It is not mutagenic in Arabidopsis thaliana, but the
' Research jointly sponsored by the National Cancer Institute, NIH,
and by the United States Atomic Energy Commission under contract
with the Union Carbide Corporation.
Received June 17, 1971; accepted March 7, 1972.
JUNE
DNM2 is (39). Most nitrosamines
under 02 aeration (38).
The spectra of genetic alterations induced by 2 methylating
compounds, MMS and MNNG, have been studied in TV.crassa
(25, 26). MMS is a weak carcinogen as compared to DMN (37)
or MNNG (35). Comparison of the spectra of genetic
alterations induced by MMS and MNNG has led to a working
hypothesis (25) that carcinogenic activity of nitrosamines and
nitrosamides is associated with the induction of a particular
type of genetic alteration, those that give rise to gene products
with altered function.
The mutagenic activity of diethylnitrosamine
has been
studied previously (20) in a reverse-mutation system, which
did not give any quantitative indication of the types of genetic
alteration induced. The present studies utilized the ad-3 test
system in a 2-component heterokaryon of TV.crassa developed
by de Serres and coworkers (6, 40). With this system, the
induction of recessive-lethal mutations can be studied at 2
closely linked specific loci, ad-3A and ad-3B. These mutations
can be due either to a point mutation in 1 of the loci or a
multilocus (or chromosome) deletion covering 1 or both of
these loci in the ad-3 region. The complementation patterns of
the ad-3B mutants can be determined and, by comparing the
complementation pattern of diethylnitrosamine-induced ad-3B
mutants with those of ad-3B mutants in which genetic
alterations have been analyzed, tentative conclusions can be
reached
about
the genetic
alterations
induced
by
diethylnitrosamine in Neurospora. This study has shown that
99% of the diethylnitrosamine-induced
mutations are point
mutations and that a high percentage of these point mutations
specify proteins with altered function.
MATERIALS AND METHODS
Strains. All purple (ad-3) mutants were isolated by the
direct method (5) after diethylnitrosamine treatment of a
genetically marked 2-component heterokaryon of TV. crassa
2The abbreviations used are: DMN, dimethylnitrosamine; MMS,
methyl methanesulfonate; MNNG, Ar-methyl-jV'-mtro-A'-nitrosoguanidine.
1972
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1273
H. V. Mailing and F. J. de Serres
(40) (strain 12) with the following genetic markers in each
component: 1 (strain 74-OR60-29A), A, hist-2, ad-3A,ad-3B,
nic-2, ad-2, inos; and 2 (strain 74-OR31-16A), A, al-2, cot,
pan-2. Mutant types are designated as A, mating type; hist-2,
histidine-requiring; ad-2, ad-3A, and ad-3B, adenine-requiring;
nic-2, niacin-requiring; cot, colonial, temperature-sensitive;
inos, inositol-requiring; pan-2, pantothenic acid-requiring. The
techniques involved in such forward-mutation
experiments
have recently been summarized (5).
Preparation of the Culture and Conidial Suspension. The
procedure for preparing the conidia has been described in
detail by Webber and de Serres (40).
Mutagenic Treatment. The conidia were suspended in
Udenfriend's hydroxylation mixture (38), which is composed
of ascorbic acid (20 mM), sodium salt of EDTA (2 mM), and
Fe4* as FeS04 (1 mM) dissolved in 1 liter phosphate buffer
(0.06 M) at pH 7.4. The final conidial concentration was 2 X
107/ml. The diethylnitrosamine
treatment was started by
adding 110 /il diethylnitrosamine to 10 ml of the conidial
suspension in the mixture. The final concentration
of
diethylnitrosamine was 100 mM. The control was treated in
the same mixture without diethylnitrosamine.
During the
treatment, N2 or 02 was bubbled continually through the
mixture. The gases were prewashed in Udenfriend's mixture
before being bubbled through the reaction mixture in order to
avoid evaporation.
The reaction was quenched by centrifugation and washing 3
times with Fries' minimal salt solution (13) adjusted to pH 8.
After being washed, the conidia were resuspended in the same
solution.
Survival. The conidia from the heterokaryotic culture are of
3 different
types: homokaryotic
for Component
1,
homokaryotic
for Component 2, and heterokaryotic.
The
survival of each type was determined by plating diluted
suspensions of conidia on 4 different media (40).
Dikaryon Test. This test is designed to determine whether
the induced ad-3 mutations are homokaryotic viable or lethal.
The ad-3 mutants are induced in Component 2 of the
heterokaryon
that carried
the cot mutation.
Those
heterokaryons that at 35°produced any of the tiny dense
colonies made by conidia homokaryotic for cot in a total of
1000 to 2000 colonies were classified as homokaryotic viable.
The test has been described in detail previously by de Serres
(3).
Trikaryon Test. The mutants that were homokaryotic lethal
in the dikaryon test were further classified to determine
whether they were chromosome deletions covering the ad-3
region (ad-3IR ) or point mutations (ad-3R ) with a separate site
of recessive-lethal damage. This recessive-lethal mutation can
either be closely linked with the ad-3 regions (closely linked
recessive lethal, RLCL) or elsewhere in the genome (RL). The
trikaryon test has been described in detail previously by de
Serres (4).
Homology Tests. Since a number of mutants in the control
samples resulted from ad-3R + RL mutations, the induced
frequency
of this type
of mutation
among
the
diethylnitrosamine-induced
mutants
was determined
by
performing trikaryon tests to test for homology of the RL
damage. In general, the spontaneous ad-3R + RL mutations
1274
were used as testers to determine which of the mutants
recovered after diethylnitrosamine treatment were preexisting
mutants of spontaneous origin.
Genotype and Complementation Test. The genotype of the
ad-3 mutants and the type of complementation pattern among
point mutations
at the ad-3B locus were determined
simultaneously by heterokaryon tests (for procedures, see Ref.
2).
RESULTS
Biological Parameters Measured with Heterokaryon Strain
12. In TV.crossa, mutants in either of the 2 genes involved in
adenine biosynthesis, ad-3A or ad-3B, accumulate a purple
pigment in the vacuoles of the hyphae when placed on
medium supplemented with low levels of adenine. Such
mutants can be recovered by a direct method (5). Neurospora
is a haploid organism, but by use of a forced heterokaryon
between 2 biochemically marked strains heterozygous for
ad-3A and ad-3B, both point mutations and chromosome
deletions can be isolated in the ad-3 region (6). Thus, de
Serres' ad-3 specific-locus system in Neurospora effectively
corresponds to Russell's specific-locus system in mice (31, 32)
and can be used to study the genetic effects that produce
specific locus mutations in higher diploid organisms. For the
study of the mutagenicity of diethylnitrosamine,
we used
Neurospora heterokaryon strain 12. Mutants were isolated in
the ad-3 region of Component 2 of this heterokaryon, and the
following biological parameters were studied: (a) nuclear and
cytoplasmic inactivation, |(e)| frequency of chromosomal
deletions in the ad-3 region, (c) frequency of point mutations
in the ad-3A and ad-3B loci, and (d) the spectrum of
complementation patterns within the ad-3B locus.
Inactivation
of Conidia from a Heterokaryon
by
Diethylnitrosamine.
Conidia
from
the
2-component
heterokaryon contain an average of 2 nuclei. The conidia can
either be homokaryotic for 1 or the other type of nucleus or
they can contain 1 nucleus of each type. The fraction of each
of these 3 types of conidia produced by a heterokaryon can be
determined by plating on differentially supplemented media.
If 1 nucleus in a heterokaryotic conidium is inactivated, the
conidium
is functionally
homokaryotic.
If mutagenic
treatment causes inactivation of nuclei, the heterokaryotic
fraction of the conidia is the most sensitive and is inactivated
at a faster rate than the 2 homokaryotic fractions. In contrast,
if mutagenic treatment causes cytoplasmic inactivation, all
fractions of conidia are inactivated to the same degree.
The inactivation of the different fractions of conida was
measured for various lengths of time in Udenfriend's
hydroxylation mixture with and without diethylnitrosamine,
with N2 or O2 bubbling through the reaction mixture. The
inactivation curves are given in Chart 1. They show no
difference between the inactivation of the heterokaryotic
fraction and of the total population of conidia. Thus, none of
the
treatments
resulted
in
nuclear
inactivation.
Diethylnitrosamine is much more toxic under N2 bubbling
than under O2 bubbling, which is in contrast to the earlier
results by Mailing (20) for an ad-3 mutant of N. crossa, where
CANCER RESEARCH VOL. 32
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Diethylnitrosamine
the toxicity of diethylnitrosamine was the same with both N2
and 02. At present we can give no rational explanation for this
discrepancy. Diethylnitrosamine
is mutagenic only in the
mixture with 02. These data show, then, that the mechanism
of inactivation with diethylnitrosamine and the mechanism of
mutation induction are apparently unrelated.
1
23456
TIME OF TREATMENT (hr)
Chart 1. Inactivation effect of diethylnitrosamine dissolved in
Udenfriend's hydroxylation mixture on the different fractions of
conidia from a 2-component heterokaryon of N. crossa. The conidia
were treated with 0.1 M diethylnitrosamine with O2 or N2 aeration, o,
heterokaryotic fraction in diethylnitrosamine + N2 ; •¿,
total population
of conidia in diethylnitrosamine + N2; A, heterokaryotic fraction in
diethylnitrosamine
+ O2 ; *, total population of conidia in
diethylnitrosamine + O2.
Mutagenicity
Diethylnitrosamine.
diethylnitrosamine
as a Mittagen
and
Mutagenic
Specificity
of
The treatment of Neurospora conidia with
dissolved in Udenfriend's hydroxylation
mixture and under 02 aeration was the only treatment that
induced any significant increase in the forward-mutation
frequency (Table 1). This agrees with the earlier observation
(20) that diethylnitrosamine
must be activated through
hydroxylation to become mutagenic and that it is mutagenic
only under those conditions that mimic the metabolic
activation required for carcinogenic activity. Thus we have
demonstrated
a correlation
between
mutagenic
and
carcinogenic activity for diethylnitrosamine.
For determining whether there is a correlation between
carcinogenic activity and the production of a particular type
of genetic alteration, the spectrum of genetic alterations was
determined by complementation tests and by dikaryon and
trikaryon tests. These data show that only 1 mutant was
classified as a multilocus deletion (ad-3IR ) in the ad-3 region
(Table 2); 99% of the diethylnitrosamine-induced
ad-3
mutations are point mutations. Of the point mutations, 26.8%
of the mutants were of genotype ad-3A and 73.2% were of
genotype ad-3B. ad-3A mutants do not show interallelic
complementation,
but ad-3B mutants
do, and their
complementation responses can be grouped in 3 main classes:
complementing
mutants with nonpolarized,
those with
polarized complementation patterns, and noncomplementing
mutants (3). Of the ad-3B mutants induced by hydroxylated
diethylnitrosamine, 65.6% had nonpolarized complementation
patterns, 4.5% had polarized complementation patterns, and
29.9% were noncomplementing.
Table 1
Induction of ad-3 mutations in heterokaryotic conidia of N. crossa strain 12
Conidia were suspended in Udenfriend's hydroxylation mixture with and without
diethylnitrosamine, with either N2 or O2 bubbling through the reaction mixture.
ad-3mutations/106survivors01.015.30.1
oÃad-3mutations2051041Frequency
of
TreatmentControlControl
O2Control
+
N2Diethylnitrosamine
+
O2Diethylnitrosamine+
+ N2Time(hr)02222Survival(%)1001001078986No.
Table 2
Percentages of different types of ad-3 mutations induced by diethylnitrosamine + 02 in heterokaryotic conidia of N. crossa
ad-3R
purple
coloniesTotal
TreatmentControl6
Diethylnitrosamine + O2No.Recovered7
104of
analyzed7
93Too
leaky
for analysis2
NC0
3.2(3)
0ad-3AR(3)c26.8 (25)NP°047.4 (44)ad-3BRP
(1)
21.5(20)ad-3IR(1)
1.1 (1)
0 NP, nonpolarized complementation pattern; P, polarized complementation pattern; NC, noncomplementing mutant.
b Includes the untreated control and the 2 treated controls.
e The number in parentheses indicates the total number of mutants in that class.
JUNE
1972
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1275
H. V. Mailing and F. J. de Serres
DISCUSSION
The present experiments with diethylnitrosamine
show a
correlation between carcinogenic and mutagenic activity.
Diethylnitrosamine
is not mutagenic per se but is only
mutagenic with an in vitro treatment that mimics the in vivo
metabolic activation that results in carcinogenic activity.
There is also a correlation between carcinogenic activity and
the production of a particular type of genetic alteration.
Genetic analysis of the diethylnitrosamine-induced
ad-3
mutants shows that 99% are due to point mutations (alteration
of the genes) and only 1% to multilocus deletions (physical
removal of the gene from the chromosome by chromosome
breakage). Genetic analysis of the point mutations to obtain a
presumptive identification of the genetic alterations at the
molecular level was made by tests for allelic complementation
among the ad-3B mutants. Our previous studies (22-24) have
shown a correlation between complementation
pattern and
genetic alteration at the molecular level. These data have led to
the
conclusion
that
mutants
with
nonpolarized
complementation
patterns are due mainly to base-pair
substitutions,
whereas
mutants
with
polarized
complementation
patterns and noncomplementing
mutants
can arise from many types of genetic alteration. We also
concluded that mutants with nonpolarized complementation
patterns produce a complete protein with only a single
erroneous amino acid substitution, whereas mutants with
polarized complementation
patterns and noncomplementing
mutants produce nonfunctional
proteins or polypeptide
fragments. Mutants with nonpolarized patterns, which specify
a complete polypeptide chain with altered function, are the
only mutants that can be leaky (can grow on minimal medium
without
adenine); mutants with polarized patterns or
noncomplemented mutants are never leaky (3).
The complementation spectrum among mutants induced by
different mutagens varies considerably (3, 25), as illustrated in
Table 3. The frequency of nonpolarized complementation
patterns among MNNG- and diethylnitrosamine-induced ad-3B
mutants is significantly higher than the frequency among
MMS-induced ad-3B mutants (25), showing a correlation
between carcinogenic activity and the production of a
particular type of genetic alteration (those base-pair transitions
that produce missense mutations).
MNNG, diethylnitrosamine,
and MMS all result in
7-alkylated
guanine
(15,
17, 36, 37). MNNG and
diethylnitrosamine
result in a higher proportion
of
0-6-alkylated guanine than MMS. It seems likely that the
differences in the complementation
patterns could be
Table 3
Percentages of different types of complementation patterns among
ad-3B mutants induced by 4 different mutagens
Complementation pattern
Mutagen
NP°
NC
MMNGDiethylnitrosamine
O2MMSHydroxylamine8166374614131118305043
+
0 NP, nonpolarized
complementation
pattern; P,
complementation pattern; NC, noncomplementing mutant.
1276
polarized
explained on the basis of the alkylated minor component of
the DNA and that this minor component could result in
mutants with nonpolarized complementation patterns. If we
assume that 0-6-methyl-guanine induces base-pair transitions
from GC to AT and plays the major role in the induction of
mutations,
then
the
complementation
spectrum
of
MNNG-induced ad-3B mutants should be similar to the
complementation spectrum of hydroxylamine-induced ad-3B
mutants, which mainly have AT at the mutation site (21);
however, this is not the case (Table 3). For an explanation of
our data, we must assume that minor alkylation products of
DNA, not yet identified, play a major role in the induction of
mutation.
In summary,
the data from the genetic tests on
diethylnitrosamine-induced
ad-3 mutants show a correlation
between potent carcinogenic activity and the production of a
particular type of genetic alteration. Essentially all of the
diethylnitrosamine-induced
mutants result from alterations of
DNA that produce point mutations, many of which specify
proteins with altered function. Thus the data from the present
experiments support our working hypothesis (25) that potent
carcinogenic activity is associated with the production of gene
products with altered function rather than with gene products
with no function.
ACKNOWLEDGMENTS
We acknowledge the capable technical assistance of Mr. David Carrol.
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1277
Genetic Characterization of Diethylnitrosamine-induced Purple
Adenine ( ad-3) Mutants in Neurospora crassa
H. V. Malling and F. J. de Serres
Cancer Res 1972;32:1273-1277.
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