Enhanced somatic mutation rates induced in stem cells by low chronic exposures

Abstract

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
92,
pp.
11470-11474,
December
1995
Genetics
Enhanced
somatic
mutation
rates
induced
in
stem
cells
of
mice
by
low
chronic
exposure
to
ethylnitrosourea
(dose
rate/intestine/D1b-1/lacI)
P.
M.
SHAVER-WALKER,
C.
URLANDO,
K.
S.
TAO*,
X.
B.
ZHANG,
AND
JOHN
A.
HEDDLEt
Department
of
Biology,
York
University,
Toronto,
ON,
Canada
M3J
1P3
Communicated
by
Richard
B.
Setlow,
Brookhaven
National
Laboratory,
Upton,
NY,
August
21,
1995
(received
for
review
April
28,
1995)
ABSTRACT
We
have
found
that
the
somatic
mutation
rate
at
the
Dlb-1
locus
increases
exponentially
during
low
daily
exposure
to
ethylnitrosourea
over
4
months.
This
effect,
enhanced
mutagenesis,
was
not
observed
at
a
lacI
transgene
in
the
same
tissue,
although
the
two
loci
respond
very
similarly
to
acute
doses.
Since
both
mutations
are
neutral,
the
mutant
frequency
was
expected
to
increase
linearly
with
time
in
response
to
a
constant
mutagenic
exposure,
as
it
did
for
lacI.
Enhanced
mutagenesis
does
not
result
from
an
overall
sen-
sitization
of
the
animals,
since
mice
that
had
first
been
treated
with
a
low
daily
dose
for
90
days
and
then
challenged
with
a
large
acute
dose
were
not
sensitized
to
the
acute
dose.
Nor
was
the
increased
mutant
frequency
due
to
selection,
since
animals
that
were
treated
for
90
days
and
then
left
untreated
for
up
to
60
days
showed
little
change
from
the
90-day
frequency.
The
effect
is
substantial:
about
8
times
as
many
Dlb-1
mutants
were
induced
between
90
and
120
days
as
in
the
first
30
days.
This
resulted
in
a
reverse
dose
rate
effect
such
that
90
mg/kg
induced
more
mutants
when
delivered
at
1
mg/kg
per
day
than
at
3
mg/kg
per
day.
We
postulate
that
enhanced
mu-
tagenesis
arises
from
increased
stem
cell
proliferation
and
the
preferential
repair
of
transcribed
genes.
Enhanced
mutagen-
esis
may
be
important
for
risk
evaluation,
as
the
results
show
that
chronic
exposures
can
be
more
mutagenic
than
acute
ones
and
raise
the
possibility
of
synergism
between
chemicals
at
low
doses.
Somatic
mutations
are
important
in
carcinogenesis,
yet
little
is
known
about
their
origins.
The
large
number
of
mutations
found
in
tumors
seems
to
be
at
variance
with
the
estimated
spontaneous
mutation
rates
(1).
There
are
several
possible
explanations
for
this.
Loeb
(1)
suggests
that
one
of
the
early
events
may
be
a
mutation
that
inactivates
a
DNA
repair
gene
and
creates
a
mutator
phenotype.
This
is
supported
by
the
finding
of
high
mutant
frequencies
in
murine
tumors
at
a
locus
not
involved
in
carcinogenesis
(a
lacI
transgene)
and
by
the
frequent
occurrence
of
mutations
at
a
gene
involved
in
mis-
match
repair
in
human
colonic
tumors
(2,
3).
Others
have
suggested
that
sequential
expansion
of
selected
clones
pro-
vides
a
large
enough
cell
population
that
the
mutation
rate
is
not
limiting
(4).
Environmental
mutagens
may
also
influence
the
mutation
rate,
as
epidemiological
studies
show
that
envi-
ronmental
factors
are
important
for
human
cancer
rates
(5).
Clearly
it
is
the
mutation
rate
in
the
stem
cells
that
is
important
for
carcinogenesis,
as
differentiated
cells
are
often
unable
to
divide
and
are
lost
from
the
epithelial
cell
populations
where
many
cancers
arise.
Until
recently,
it
has
been
difficult
to
study
somatic
muta-
tions
in
stem
cells,
but
the
Dlb-1
mutation
assay
makes
this
easy
for
the
small
intestine
(6).
Dlb-1
determines
the
presence
or
absence
of
Dolichos
biflorus
lectin
binding
sites
on
the
surface
of
cells
of
the
small
intestine
and
elsewhere.
The
presence
of
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
these
sites
can
be
detected
by
means
of
peroxidase
staining
of
the
small
intestine
after
reaction
with
peroxidase-bound
lectin.
In
the
small
intestine,
the
Dlb-1b
allele
(presence
of
the
binding
site)
is
dominant
over
the
Dlb-la
allele
(absence
of
the
binding
site).
In
heterozygous
mice
(Dlb-lb/Dlb-la),
mutation
of
the
dominant
Dlb-1b
allele
in
an
intestinal
stem
cell
results
in
a
mutant
clone
that
lacks
the
lectin
binding
site
and
is
unstained
by
the
peroxidase.
Since
the
stem
cells
of
the
small
intestine
are
located
in
the
crypts
(invaginations
of
the
cell
sheet)
and
feed
cells
inward
to
the
villi
(projections
of
the
cell
sheet
into
the
lumen
of
the
intestine),
a
mutant
stem
cell
produces
a
ribbon
of
nonstaining
cells
extending
from
the
crypt
to
the
tip
of
the
villus.
There
are
about
10
crypts,
each
with
a
single
stem
cell,
maintaining
each
villus.
The
time
required
for
the
appearance
of
these
mutant
ribbons
corresponds
roughly
to
the
5-7
days
required
for
the
progeny
of
a
stem
cell
to
reach
the
tip
of
the
villus
(7).
Similarly,
mutations
arising
at
the
transgenic
lacI
locus
in
the
stem
cell
can
be
measured
by
allowing
at
least
1
week
after
the
end
of
treatment
for
the
epithelium
to
turn
over.
Hemizygous
transgenic
mice
carry
a
A
vector
that
contains
the
lacI
target
gene
and
an
a-complementing
lacZ
reporter
gene
(8,
9).
This
vector
can
be
recovered
from
the
DNA
of
the
mice
in
the
form
of
a
viable
A
phage.
When
these
phage
are
plated
on
Esche-
richia
coli
on
plates
containing
5-bromo-4-chloro-3-indolyl
,3-D-galactopyranoside
(X-Gal),
those
that
carry
a
functional
lacI
will
form
clear
plaques,
whereas
those
in
which
the
repressor
has
been
mutated
will
produce
03-galactosidase,
which
cleaves
the
X-Gal
to
produce
blue
plaques
(8, 9).
Comparisons
of
the
behavior
of
the
lacI
transgene
and
the
Dlb-l
host
locus
in
the
small
intestine
showed
that
they
behaved
very
similarly
in
most
respects
(10).
Indeed,
both
behaved
as
if
mutations
at
these
loci
are
neutral:
the
frequency
of
mutations
was
constant
from
1
to
8
weeks
after
mutagenesis
(10),
and
the
effect
of
weekly
treatments
was
additive
(11,
12).
The
concept
of
neutrality
led
to
the
suggestion
that
chronic
protocols
would
enhance
the
sensitivity
of
transgenic
mutation
assays,
provided
time
is
allowed
for
tissue
turnover
(12-14).
It
also
inspired
these
experiments,
which
began
as
an
attempt
to
quantify
the
mutant
frequency
induced
at
much
lower
doses
by
treating
the
animals
many
times
with
such
a
dose
and
assuming
additivity.
This
approach
has
been
used
for
thioguanine-
resistant
mutants
in
cultured
TK6
cells
(15).
The
doses
used
correspond
to
about
1%
and
0.3%
of
the
acute
LD50,
whereas
most
previous
studies
have used
doses
about
20
times
higher
or
more.
MATERIALS
AND
METHODS
Animals.
An
independent
animal
care
committee
approved
all
experimental
protocols
in
advance.
All
mice
were
housed
in
Abbreviations:
ENU,
ethylnitrosourea;
X-Gal,
5-bromo-4-chloro-3-
indolyl
,B-D-galactopyranoside.
*Present
address:
Hospital
for
Sick
Children,
Toronto,
ON,
Canada
M5G
1X8.
tTo
whom
reprint
requests
should
be
addressed.
11470

Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
11471
plastic
cages
with
wood
chip
bedding
at
70%
humidity
and
22
±
2°C
and
a
12-h
light/12-h
dark
cycle.
Water
and
food
were
supplied
ad
libitum.
The
hemizygous
lacI
C57BL/6
(Dlb-1b/
Dlb-1b)
transgenic
mice
were
obtained
from
Stratagene
and
bred
with
SWR
(Dlb-la/Dlb-la)
mice,
obtained
from
The
Jackson
Laboratory.
Those
F1
that
carry
the
transgene
are
suitable
for
mutation
detection
at
both
loci.
Their
nontrans-
genic
siblings
were
also
used
for
some
of
these
experiments.
For
the
initial
measurements,
only
the
high-dose
group
con-
tained
transgenic
animals.
Since
the
animals
differed
some-
what
in
age,
they
were
stratified
into
four
age
groups,
41,
43-44,
47-52,
and
53-56
weeks
old.
There
is
very
little
difference
in
the
spontaneous
mutation
frequency
in
adult
mice
in
this
age
range
(16,
17).
The
test
groups
were
structured
to
contain
similar
contributions
from
each
age
group,
and
the
treatments
were
assigned
to
each
group
randomly
after
their
composition
had
been
determined.
Initially,
each
group
con-
tained
five
animals,
two
males
and
three
females
or
three
males
and
two
females.
Subsequently,
somewhat
younger
animals
were
used,
but,
again,
it
was
difficult
to
obtain
enough
animals
of
the
same
age,
and
they
were
stratified
into
three
groups
of
17-19,
23,
and
28-29
weeks
of
age.
Treatment
groups
were
constructed
and
assigned
as
before.
There
were
two
males
and
two
females
in
each
group.
Mice
were
sacrificed
by
cervical
dislocation
immediately
after
treatment
in
the
first
study
and
1
week
after
treatment
in
all
others.
The
animals
were
treated
daily
between
9:00
and
11:00
a.m.
Controls
were
not
treated.
Mutagens.
Ethylnitrosourea
(ENU)
was
obtained
from
Sigma.
Test
solutions
were
made
by
dissolving
ENU
in
di-
methyl
sulfoxide
at
20
times
the
highest
final
concentration
and
then
frozen
at
-70°C.
Each
day
an
individual
vial
of
frozen
concentrate
was
diluted
to
the
proper
concentration
of
1
or
3
mg/kg
with
phosphate-buffered
saline
(PBS)
at
pH
7.2
and
injected
intraperitoneally
within
20
min.
lacI-
Mutations.
After
sacrifice,
the
entire
small
intestine
was
removed
and
two-thirds
of
it
was
used
for
DNA
extraction.
There
is
no
difference
in
the
response
at
either
locus
in
different
regions
of
the
small
intestine
to
acute
doses
of
ENU
(10).
These
sections
were
flushed
with
PBS
(pH
8.0)
and
inverted.
The
inverted
small
intestine
was
placed
in
3
ml
of
Douncing
buffer
containing
RNase
A
at
a
concentration
of
100
jig/ml
and
pushed
in
and
out
of
a
5-ml
syringe
in
order
to
loosen
the
cells.
The
cell
suspension
was
then
digested
with
a
proteinase
K
solution
(2
mg/ml),
and
the
DNA
was
extracted
with
phenol/chloroform
and
precipitated
with
ethanol
(9).
The
A
phage
shuttle
vector,
which
contains
the
entire
lacI
mutational
target
gene
and
the
odacZ
reporter
gene,
was
to
0
x
>1
L)
cs
IL.
C4-
a
200
150
100
50
recovered
by
in
vitro
packaging
with
Transpack
packaging
extract
(Stratagene).
The
extracts
were
incubated
with
SCS-8
E.
coli
(Stratagene),
which
produce
the
complementing
car-
boxyl-terminal
end
of
the
lacZ
gene.
The
infected
bacteria
were
grown
on
NZY
agar
containing
70
mg
of
X-Gal
per
25-cm2
agar
plate.
When
a
mutation
inactivates
the
lacI
repressor,
the
alacZ
reporter
gene
is
produced
and
complementation
results
in
a
functional
,3-galactosidase,
which
cleaves
the
X-Gal,
and
blue
plaques
result.
The
number
of
plaques
recovered
from
each
animal
differed
greatly:
the
mean
was
16,400
plaques;
the
range
was
6100-50,000
plaques.
Dlb-)
Assay.
Whole
mounts
were
prepared
as
described
in
Winton
et
al.
(6)
with
a
few
modifications
(10).
The
middle
one-third
of
the
small
intestine
was
flushed
with
PBS,
inflated,
and
fixed
with
10%
formal
saline
[0.85%
(wt/vol)
NaCl
in
10%
buffered
formalin].
It
was
then
cut
open
and
clipped,
villi
up,
to
a
microscope
slide
with
plastic-coated
paper
clips.
The
slides
were
fixed
for
1
h
in
10%
formal
saline,
rinsed
with
PBS,
and
then
incubated
in
20
mM
dithiothreitol
for
45
min
to
remove
the
mucus.
They
were
stored
in
10%
formal
saline
until
staining.
The
slides
were
incubated
in
0.1%
phenylhydrazine
hydrochloride
for
30
min
to
block
endogenous
peroxidases,
incubated
20
min
in
0.5%
albumin
in
PBS,
and
then
stained
with
5
,ug
of
D.
biflorus
agglutinin-peroxidase
conjugate
(Sigma)
per
ml
in
the
PBS/albumin.
The
peroxidase
was
developed
by
using
3,3'-diaminobenzidine
(Sigma)
at
0.5
mg/ml
for
20
min.
The
slides
were
rinsed
twice
with
PBS
and
stored
in
PBS
until
analyzed.
The
slides
were
coded
and
then
scored
with
a
dissecting
microscope
at
x50.
The
Dlb-lb/Dlb-la
epithelial
cells
stained
brown,
whereas
mutant
cells
(Dlb-1
-/Dlb-la)
were
not
stained
and
appeared
as
vertical
white
ribbons
on
the
villi.
Fifty
fields,
defined
by
a
rectangle
in
an
eyepiece
graticule,
were
scored.
The
first
and
last
fields
were
each
counted
twice
and
averaged
to
estimate
the
total
number
of
villi,
which
was
always
about
104
per
slide.
Statistics.
The
statistical
analyses
were
conducted
with
the
MINITAB
software.
All
means
and
standard
errors
reported
are
based
on
the
mutant
frequency
observed
in
each
animal,
regardless
of
the
number
of
plaques
or
villi
analyzed.
RESULTS
The
mutant
frequency
as
a
function
of
time
is
shown
in
Fig.
1
for
both
lacI
and
Dlb-1b.
The
time
shown
is
the
actual
treatment
time
less
the
expression
time
of
4
days,
since
in
this
case
there
was
only
1
day
between
the
last
treatment
and
the
30
60
90
0
30
60
90
0
1
2
3
Days
of
Treatment
(ENU)
Days
of
Treatment
Dose
Rate
(mg/kg/day)
FIG.
1.
Mutant
frequency
(±
SEM)
observed
after
daily
treatments
with
ENU.
In
several
cases
the
error
bars
are
smaller
than
the
symbols.
(A)
ENU
at
3
mg/kg
per
day.
v,
Individual
animals;
0,
means.
(B)
o,
3
mg/kg
per
day;
v,
1
mg/kg
per
day;
*,
control.
(C)
Open
symbols
represent
individual
animals;
closed
symbols
represent
the
means.
Genetics:
Shaver-Walker
et
al.

11472
Genetics:
Shaver-Walker
et
al.
sacrifice.
The
accumulation
of
mutations
at
lacI
appears
to
be
linear
(F
=
0.054;
df
=
1,14;
P
=
0.82),
as
expected,
but
the
accumulation
of
mutations
at
Dlb-]
is
not
linear
at
either
1
mg/kg
per
day
(F
=
14.6;
df
=
1,17;
P
=
0.001)
or
3
mg/kg
per
day
(F
=
17.1;
df
=
1,17;
P
=
0.0007)
and
appears
to
be
exponential.
In
acute
experiments,
the
frequencies
of
muta-
tions
at
the
two
loci
are
about
equal
(10),
but
this
was
not
observed
at
the
early
samples
where
there
are
fewer
mutations
at
Dlb-1
than
at
lac.
The
two
curves
do
not
have
the
same
shape
(F
=
4.79;
df
=
2,33;
P
=
0.02).
In
spite
of
the
nonlinear
accumulation
of
Dlb-l
mutations
with
time,
the
dose-response
curve
at
any
one
time
is
linear
(Fig.
1C).
The
nonlinear
accumulation
of
mutants
at
the
Dlb-]
locus
with
continued
treatment
is
reproducible
and
continues
to
accelerate
past
90
days,
as
shown
in
Fig.
2.
In
this
experiment,
a
1-week
expres-
sion
time
was
allowed
after
the
last
treatment
before
mea-
surements
were
made.
A
reverse
dose
rate
effect
is
evident
when
the
mutant
frequency
is
plotted
as
a
function
of
cumulative
dose
(Fig.
3).
The
mutant
frequencies
induced
by
the
same
total
dose
at
a
dose
rate
of
1
mg/kg
per
day
are
greater
than
those
accumulated
at
3
mg/kg
per
day.
This
can
also
be
seen
in
Fig.
2,
where
the
mutant
frequency
observed
after
1
mg/kg
for
90
days
is
higher
than
that
after
3
mg/kg
for
30
days,
although
the
total
doses
are
the
same.
The
same
effect
was
observed
in
the
initial
experiment.
One
explanation
for
the
nonlinear
accumulation
of
Dlb-]
mutants
with time
is
that
the
mutations
are
not
neutral,
so
that
the
increasing
mutant
frequency
does
not
reflect
an
increasing
mutation
rate
but
rather
a
selective
advantage,
in
spite
of
previous
results.
To
test
this,
some
animals
were
exposed
for
90
days
afild
then
left
untreated
for
30
or
60
days
in
addition
to
the
1-week
expression
time.
As
shown
in
Fig.
4,
the
mutant
frequency
remained
essentially
constant
in
animals
whose
treatment
had
ceased
at
90
days,
although
it
continued
to
increase
at
an
accelerating
rate
in
those
animals
whose
treat-
ment
continued.
This
is
consistent
with
the
conclusions
of
Tao
and
Heddle
(12)
and
shows
that
the
increased
frequency
is
not
the
result
of
selection.
It
should
be
noted
that
even
if
a
mutant
stem
cell
had
a
selective
advantage,
this
would
be
expected
to
produce
a
larger
mutant
ribbon
rather
than
more
ribbons.
Thus
the
higher
mutant
frequency
at
the
longer
times
results
from
a
higher
mutation
rate
rather
than
selection
of
mutant
cells.
The
higher
mutation
rate
observed
at
later
times
might
result
from
some
sort
of
sensitization
of
the
animals,
although
the
lacI
data
are
not
consistent
with
this.
As
a
further
test
of
LO
0
X
c)
a)
LI
0)
CZ
LI
10
360
320
280
240
200
160
120
80
40
0
30
60
90
120
150
Days
of
Treatment
(ENU)
FIG.
2.
Mutant
frequency
(+
SEM)
observed
after
daily
treat-
ments
with
ENU.
In
several
cases,
the
error
bars
are
smaller
than
the
symbols.
oi,
3
mg/kg
per
day;
,
1
mg/kg
per
day;
0,
control.
LI)
0~
a)
U-
0
.0
Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
360
320
280
240
200
160
120
80
40
o
I
I/
Ie
IaI
y
1
mg/kg
per
day/
r . .
.
.
...
.~~~~~~~~~~~~I
0
60
120
180
240
300
360
ENU
Dose
(mg/kg)
FIG.
3.
Mutant
frequency
(+
SEM)
observed
after daily
treat-
ments
with
ENU.
In
several
cases
the
error
bars
are
smaller
than
the
symbols.
this
hypothesis,
animals
that
had
been
treated
for
90
days
with
daily
low
doses
were
challenged
with
a
series
of
high
doses,
together
with
untreated
controls.
The
challenge
doses
induced
approximately
the
same
number
of
mutations
in
animals
that
had
previously
been
treated
for
90
days
with
either
1
or
3
mg/kg
per
day
as
in
controls
(Fig.
5).
No
evidence
of
increased
sensitivity
to
high
doses
could
be
found.
Thus
the
higher
mutation
rate
observed
is
not
the
result
of
some
overall
sensitization
of
the
animals
and
is
a
low-dose
effect.
The
mutations
being
detected
arise
in
the
stem
cells
of
the
small
intestine,
which
may
divide
infrequently.
It
is
thought
that
many
mutations
arise
during
DNA
synthesis
and
that
dividing
cells
may
be
more
sensitive
to
mutation
as
a
result
(18).
We
wondered
if
increased
proliferation
of
the
stem
cells,
induced
by
prior
treatment,
might
increase
the
sensitivity
of
the
small
intestine
to
mutation.
As
a
test
of
this
hypothesis,
we
have
given
animals
two
doses
of
50
mg
of
ENU
per
kg
separated
by
different
intervals
from
0
to
7
days.
The
results
(Fig.
6)
show
that
the
treatments
were
additive
at
7
days,
as
reported
earlier
for
ENU
and
other
mutagens
(12).
With
fractionation
intervals
of
1-3
days,
however,
the
mutant
fre-
quency
was
greater
than
that
produced
by
the
unfractionated
dose.
This
shows
that
the
tissue
can
be
transiently
sensitized
by
a
previous
exposure.
Compared
to
the
effect
of
the
initial
dose
of
50
mg/kg,
the
second
dose
of
50
mg/kg
2
days
later
produced
about
twice
as
many
mutations.
400
U.)
o
360
X
320
T
o
280
U)
Continued
v
240
-
treatment
200
200
Stopped
treatment
T
C
160
-
D
120
Continued
80
treatment
Q
40
Stopped
treatment
0
30
60
90
120
150
180
Day
of
Experiment
FIG.
4.
Mutant
frequency
(±
SEM)
observed
after
daily
treat-
ments
with
ENU.
In
several
cases,
the
error
bars
are
smaller
than
the
symbols.
m,
3
mg/kg
per
day;
o,
1
mg/kg
per
day.

Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
11473
U-)
0
x
c
a)
03
U1)
-D
C
400
360
320
260
240
200
160
120
60
40
0
a
D
30
60
90
120
150
180
ENU
Challenge
(mg/kg)
FIG.
5.
Mutant
frequency
(±
SEM)
observed
in
mice
that
had
been
chronically
exposed
to
the
daily
doses
shown
for
90
days
and
then
treated
with
a
high
(challenge)
dose
of
ENU.
In
several
cases
the
error
bars
are
smaller
than
the
symbols.
DISCUSSION
The
results
clearly
demonstrate
that
the
mutation
rate
at
Dlb-1
is
not
constant
during
chronic
exposure
to
ENU
but
increases
exponentially
with
time
over
a
4-month
period.
It
is
apparent
that
neither
a
selective
advantage
of
preexisting
mutations
nor
a
generally
increased
sensitivity
of
the
animals
to
the
mutagen
is
responsible
for
the
increasing
rate
of
accumulation
of
mutants
as
a
function
of
exposure
time.
The
selective
advan-
tage
is
ruled
out
by
the
constant
mutant
frequency
observed
after
the
cessation
of
chronic
exposure,
as
had
been
observed
after
both
single
and
weekly
exposures.
These
control
exper-
iments
do
not
rule
out
the
possibility
that
Dlb-1
-
mutants
are
at
a
selective
advantage
during
exposure,
but
this
seems
unlikely
when
a
higher
rate
of
cell
division
would
lead
to
a
larger
ribbon
(mutant
clone)
rather
than
more
ribbons.
Equally,
a
general
sensitization
of
the
tissue
by
any
mechanism
that
changes
the
effective
dose
to
the
DNA
is
inconsistent
with
both
the
linear
accumulation
of
mutants
at
the
lacI
locus
and
the
normal
response
to
a
large
acute
dose
after
90
days
of
exposure.
The
phenomenon
is
a
low-dose
effect
and
is
appar-
ent
only
after
an
extended
exposure.
Any
proposed
mechanism
must
explain
these
facts
and
the
difference
between
lacI
and
to)
0
0~
c
0-
L-n
S4
180
160
140
120
100
80
60
4
40
20
0
0
1
2
3
4
5
6
7
Days
Between
Treatments
of
50
mg/kg
8
FIG.
6.
Mutant
frequency
(±
SEM)
observed
when
animals
were
treated
with
a
single
dose
of
50
mg
of
ENU
per
kg
(0)
or
two
doses
(0)
separated
by
the
intervals
shown.
The
horizontal
line
is
that
expected
if
the
two
doses
were
additive
(=
twice
that
observed
for
50
mg/kg
minus
the
control
frequency).
Dlb-1.
At
this
time,
we
can
only
speculate
as to
the
mechanism
involved
and
the
generality
and
importance
of
the
effect.
The
two
loci
differ
in
several
ways
that
may
affect
mutagen-
esis.
Obviously
their
sequences
and
their
locations
in
the
genome
differ.
In
addition,
the
lacI
transgenes
are
embedded
in
2
megabases
of
prokaryotic
DNA.
Since
it
was
constructed
without
mammalian
promoter
sequences
and
is
heavily
meth-
ylated
(19),
the
transgene
is
probably
unexpressed
in
any
tissue.
The
Dlb-1
locus
is
a
single
endogenous
locus,
which
is
expressed
in
the
small
intestine
and
many
other
tissues.
Given
the
coupling
of
some
forms
of
DNA
repair
and
transcription
(20,
21),
some
differences
between
the
two
loci
would
not
be
surprising.
Nevertheless,
the
two
loci
respond
very
similarly
to
acute,
single-dose
mutagenesis
by
ENU,
even
quantitatively
(10),
as
do
lacI
and
hprt
in
splenic
lymphocytes
(22).
X-rays,
however,
are
more
mutagenic
at
Dlb-1
than
at
lacI,
showing
that
the
mutation
spectrum
differs
(10).
Obviously
deletions
having
one
end
in
the
vector
will
not
be
recovered
as
viable
phage,
so
the
large
deletions
characteristic
of
x-rays
will
be
underrepresented.
Unfortunately,
no
information
about
the
molecular
nature
of
the
mutations
detectable
at
Dlb-1
is
available,
except
by
inference
from
the
relative
responsiveness
to
different
agents.
If
the
nonlinear
component
of
the
Dlb-1
mutant
accumulation
were
all
deletion
mutants,
then
a
difference
between
lacI
and
Dlb-1
would
be
expected,
but
it
would
not
be
the
observed
difference.
What
would
be
expected
would
be
a
consistent
excess
of
Dlb-1
mutations
at
all
times,
whereas
a
deficiency
exists
at
early
times.
Furthermore,
ENU
produces
primarily
base
substitutions.
It
is
also
unclear
what
mechanism
could
lead
to
an
increasing
frequency
of
deletion
mutations
but
not
to
sensitization
of
the
cells
to
a
large
acute
dose.
It
cannot
be
the
accumulation
of
DNA
adducts,
as
the
tissue
turns
over
weekly.
The
other
difference
between
the
loci,
transcription
of
Dlb-1
and
nontranscription
of
lad,
is
more
suggestive.
Preferential
repair
of
transcribed
genes
does
occur
in
mammalian
cells
(19).
It
has
also
been
shown
that
quiescent
normal
cells
are
more
resistant
to
mutation
by
some
agents,
such
as
UV,
whereas
repair-deficient
cells
are
not
(18).
Since
alkylating
agents
are
S-phase-dependent
mutagens,
enhanced
mutagenesis
can
be
explained
by
the
biology
of
the
stem
cell
in
the small
intestine.
In
the
small
intestine,
cells
proliferate
in
the
crypts
and
then
migrate
from
the
crypts
up
the
villus
to
be
sloughed
off
at
the
tip
(7).
Stem
cells
typically
have
a
much
longer
cell
cycle
time
than
the
proliferative
population,
although
this
is
not
certain
for
the
small
intestine,
where
the
stem
cells
have
not
been
identified
definitively
(7).
There
is
evidence
for
a
slowly
dividing
population
of
cells
in
the
small
intestine
from
the
dilution
of
[14C]thymidine
with
time
(G.
Dawod,
I.
Kogan,
J.
Moody,
P.
B.
Moens,
R.
R.
Swiger,
J.
D.
Tucker,
K.
W.
Tur-
teltaub,
and
J.A.H.,
unpublished
data)
and
for
the
existence
of
a
brief
sensitive
period
in
the
life
of
these
stem
cells
in
the
small
intestine
(23).
When
an
animal
is
initially
treated
with
a
very
low
dose
of
ENU,
few
of
the
stem
cells
are
in
S
phase,
and
thus
few
are
susceptible
to
mutation
and
killing.
Most
of
the
stem
cells
are
nonproliferating
and
resistant.
If
a
stem
cell
is
killed,
one
of
the
cells
from
the
proliferating
pool
must
replace
it
in
order
to
maintain
the
flow
of
epithelial
cells
toward
the
villus.
This
possibility
is
well
established
from
radiation
experiments
that
show
there
are
many
more
potential
than
actual
stem
cells
in
the
small
intestine
(7).
The
death
of
cells
in
the
proliferating
pool
may
also
stimulate
the
stem
cells
to
proliferate
more
frequently.
When
these
concepts
are
applied
to
the
chronic
low-dose
exposure,
the
stem
cells
will
be
stimulated
to
prolif-
erate
slightly
more
often
after
the
initial
exposure
to
compen-
sate
for
a
small
amount
of
cell
death,
so
slightly
more
cells
will
be
in
the
sensitive
phase
at
the
time
of
the
next
dose.
This
second
dose
will
thus
induce
a
few
more
mutations
than
the
first
dose,
slightly
more
cell
death,
and
still
more
proliferation.
Genetics:
Shaver-Walker
et
al.

11474
Genetics:
Shaver-Walker
et
al.
With
continuing
exposures,
this
process
will
continue
so
that
the
mutant
frequency
will
rise
exponentially.
The
split-dose
experiments
are
consistent
with
a
transient
sensitization
induced
by
an
acute
exposure
to
a
high
dose
of
ENU,
as
would
be
expected
under
the
mechanism
proposed
above.
However,
the
results
of
the
challenge
experiment,
in
which
animals
treated
daily
for
90
days
responded
normally
to
high
doses
of
ENU,
do
not
seem
consistent
with
this
hypoth-
esis.
They
can
be
made
compatible
by
assuming
(i)
that
the
preferential
repair
of
transcribed
genes
is
readily
saturable
and
(ii)
that
high
doses
induce
more
proliferation.
It
is
reasonable,
although
untested,
that
the
preferential
repair
of
transcribed
genes
is
readily
saturated;
why
else
would
unexpressed
genes
not
be
repaired
as
well?
Such
a
saturable
repair
would
lead
to
a
large
difference
in
sensitivity
to
low
doses
between
S-phase
cells,
where
the
time
available
for
repair
before
a
replication
fork
reaches
the
lesion
is
short,
and
non-S
cells,
where
the
time
available
for
repair
is
much
longer.
At
high
doses,
the
repair
system
is
saturated
in
all
cells,
so
that
many
lesions
survive
until
a
replication
fork
arrives,
even
in
cells
exposed
while
not
in
S
phase,
and
the
difference
in
sensitivity
between
S
and
non-S
cells
is
small.
At
high
doses
the
sensitivity
is
observable
in
a
split-dose
experiment,
even
though
small,
as
many
cells
are
stimulated
to
enter
S
phase.
In
the
chronic
experiments,
relatively
few
cells
are
involved,
and
the
overall
increased
sensitivity
to
the
high
challenge
doses
is
negligible.
But
in
the
chronic
experiments,
the
relative
sensitivity
difference
is
large
and
so
detectable
when
summed
over
many
daily
repetitions.
The
magnitude
of
the
sensitization
would
be
in
proportion
to
the
increase
in
the
rate
of
proliferation
of
the
stem
cells;
this
prediction
is
testable.
The
generality
of
enhanced
mutagenesis
is
not
known.
If
the
effect
is
the
result
of
differential
sensitivity
of
cycling
and
noncycling
stem
cells,
then
only
agents
with
S-phase
depen-
dence
would
show
enhanced
mutagenesis.
A
single
experiment
with
x-rays,
which
mutate
cells
at
all
stages
of
the
cell
cycle,
showed
no
indication
of
enhanced
mutagenesis
(data
not
shown).
Similarly
chronic
exposures
to
2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine
(PhIP;
X.B.Z.,
J.
D.
Felton,
C.
U.
Tucker,
and
J.A.H.,
unpublished
data)
show
a
linear
accumulation
of
mutants
with
time
at
both
lacI
and
at
Dlb-1
over
a
90-day
period,
but
the
mechanism
by
which
PhIP
is
mutagenic
is
not
known.
Ames
and
Gold
(24)
have
suggested
that
the
usual
method
of
running
the
cancer
bioassay
produces
artifactual
positive
results
because
very
high
doses
are
used.
If
these
doses
induce
cellular
toxicity
and
increased
cellular
proliferation
as
a
re-
sponse,
then
the
cells
might
be
more
sensitive
to
mutation
and,
as
a
consequence,
more
cancers
may
result.
Implicit
in
this
suggestion
is
the
assumption
that
the
mutable
cells
are
not
proliferating
at
their
maximum
rate
and
that
the
cells
are
more
sensitive
when
proliferating.
Our
assumptions
are
identical,
but
we
suggest
that
a
similar
effect
can
occur
at
low
doses
if
the
exposure
is
chronic.
This
would
mean
that
extrapolations
from
high
to
low
dose
would
require
consideration
of
the
pattern
of
exposure.
Possibly
intermittent
exposure
is
less
hazardous
than
chronic
exposure
at
low
doses.
It
is
noteworthy
that
the
chronic
dose-response
curves
observed
at
any
one
time
were
linear
in
all
of
our
experiments.
A
sequential
series
of
measurements
is
required
to
detect
the
enhanced
mutagenesis.
It
has
often
been
thought
that
combinations
of
mutagens
would
be
additive
at
low
environmental
doses.
These
data
suggest
that
combinations
of
mutagens
could
be
synergistic,
with
each
one
sensitizing
the
cells
to
the
other
by
inducing
enhanced
mutagenesis.
If
increased
proliferation
is
involved,
any
agent,
mutagenic
or
not,
that
increased
cellular
prolifer-
ation
would
increase
the
sensitivity
to
mutation
by
other
agents
and
to
further
cell
proliferation,
thus
initiating
the
enhanced
mutagenesis.
Further
experiments
are
required
to
test
this
possibility.
It
may
also
be
that
more
or
less
continuous
exposures
to
low
doses
are
more
hazardous
than
intermittent
ones,
even
if
the
total
dose
is
the
same.
Possibly
enhanced
mutagenesis
is
one
of
the
factors
involved
in
the
origin
of
large
numbers
of
mutations,
both
point
mutations
and
chromosomal
deletions
and
rearrangements,
found
in
human
cancer.
In
any
case,
enhanced
mutagenesis
indicates
that
the
existing
trans-
genic
loci
in
widespread
use
for
assessing
somatic
mutation
may
not
reflect
all
of
the
biological
phenomena
of
interest
in
vivo.
We
are
grateful
to
Stratagene
for
determining
which
of
the
F1
carried
the
transgene.
We
thank
Gemma
Vomiero-Highton
and
Jason
Halberstadt
for their
help
with
these
experiments.
This
work
was
supported
by
grants
from
the
National
Cancer
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of
Canada
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