Download induced Mutagenesis in Cultured Chinese

[CANCER RESEARCH 38, 4527-4533, December 1978]
0008-5472/78/0038-0000$02.00
Effects of Harman and Norharman on Spontaneous
and Ultraviolet Light
induced Mutagenesis in Cultured Chinese Hamster Cells
Chia-cheng Chang,' Marc Castsblazzl,2Thomas W. Glover, and James E. Trosko3
Department of Human Development, College of Human Medicine, Michigan State University, East Lansing. Michigan 48824
were identified. These compounds are known to exist in
cigarette tar (18), charred meat, and naturally occurring
Nontoxic concentrations of harman and norharman foods (21). It was found that, when these 2 compounds are
were tested in cultured Chinese hamster cells for their added to a reaction mixture containing rat liver enzymes
effects on DNA repair and mutagenesis. The following and other chemical mutagens, a significant increase in
effects of harman were observed: (a) the survival of mutagenicity was observed with the Ames assay (11, 15).
uitravloletbight-or X-ray-damagedcells was reduced;(b) Moreover,aniline ando-tobuidine,which are not mutagenic,
the ultravioletlight-inducedunscheduledDNA synthesis were capable of inducing mutations in the presence of
was slightly inhibited; and (C) the frequency of sponta harmanor norharman (14). Nagaoet a!. (15) introduced the
neousor ultravioletbight-induced
ouabain-resIstant(oua―)term, “comutagenesis,―
to describe this phenomenon,
or 6-thioguanine-resistant(6-TG')mutationswas reduced. which in principle seems to parallel similar observations
Furthermore, the effect of harman on survival and muta
made by Van Duuren and Goldschmidt (27) related to the
genesis was greater than that of norharman and was phenomenon of “cocarcinogenesis.―
detected primarily In treatments in which cells were cx
Experimental evidence exists that links the role of muta
posed to harman immediately following uftraviolet light genesis to carcinogenesis (9, 24). From a wide variety of in
irradiation. Our data clearly indicate that harman de vitro studies with either tumor promoters (23) or antitumor
creases the capacity to repair DNA damage and fix muta promoters (2), it now appears that carcinogenic initiation
tions in Chinese hamstercells, possiblybecause of the might be due to a mutagenic process, whereas carcino
intercalation properties of this compound.
genic promotion might be due to an epigenetic effect (22).
Accordingly, the experiments reported here were designed
to demonstrate whether such a comutagenic phenomenon
INTRODUCTION
exists in eukaryotic cells and whether this would be related
A wide variety of studies has implicated tryptophan (and
to a cocarcinogenic effect in vivo. As a first step in a
some of its precursors and metabolites) in the carcinogenic comparative series of molecular and biological in vitro and
process in mice, rats, hamsters, and dogs. A cocarcino in vivo experiments, we report here the effect of harman
genic effect of DL-tryptophanhasbeendemonstrated in rats and norharman on the recovery of spontaneous and UV
(3) and dogs (19). Indole, a precursor of tryptophan,
has
induced mutations in cultured Chinese hamster V79 cells.
been shown to enhance the carcinogenicity of 2-acetylami
Using2 genetic markers, resistanceto 6-thioguanine and to
nofluorene in the bladder of rats (17) and hamsters(16) and ouabain, we have shown that harman and norharman in
yet suppress the carcinogenicity of 2-acetylaminofluorene hibit rather than enhance mutagenesis in our assay sys
in the liver of rats (17) and hamsters (16). Matsumoto et a!. tems.
ABSTRACT
(10) have shown that indole appears to act as an antitumor
promoter of dibutylnitrosammne-mnduced
bladder cancers in
hamsters.
In search of possible chemical factors in the typical
Japanese diet that might be correlated with a high fre
quency of stomach cancer in the Japanese population,
charred material of fish and meat has been tested for its
mutagenic potential with the use of the Ames assay(13). It
was found that mutagenic principles were formed by pyrob
ysis of protein but not of other compounds (12, 13). Subse
quently, some mutagenic principles in pyrolytic products of
MATERIALSAND METHODS
Cell Culture. A Chinese hamster cell line (V79), derived
originally from lung tissue (6), was used for the experi
ments. Cells were grown in modified Eagle's medium (5)
(Earle's balanced salt solution with a 50% increase of
essential amino acids and vitamins) supplemented with
“nonessential―
amino acids (100%increase), 1 mM sodium
pyruvate, and 5% fetal calf serum. Under the incubation
condition with 5% CO2 in humidified air at 37°,the cells
DL-tryptophan
were identified
(20). In addition,
2 nonmuta
havea generation time of about 12 hr.
genic f3-carboline derivatives, harman and norharman,
UnscheduledDNASynthesis.To determinewhetherhar
which are the main products of DL-tryptOphanpyrolysate, man affects DNA repair synthesis (“unscheduled―
DNA
synthesis), we used the method described by Trosko and
Yager(25)in a slightly modified form. Cells were inoculated
I
Recipient
of
National
Institute
of
Environmental
Health
Sciences
Young
Environmental Scientist Award E501809-01. To whom requests for reprInts
should be addressed.
a On
postdoctoral
leave
from
Unite
do Genétique
Cellulaire,
Institut
do
Rochorches en Biologie Moléculaire,2. p1. Jussieu, F-75221 Paris, Cedex
05. France.
3 Recipient
of
National
Cancer
Instftute
Grant
CA
21104-01.
Received May 15, 1978; accepted September 7. 1978.
into6-cm Falcon plastic @tri
plates(3 x 10°/plate)
and
grown overnight to confluency. The medium was then
replaced with an arginine-deficient medium for 3 days. One
hr prior to UV treatment, hydroxyurea (Sigma Chemical
Co., St. Louis, Mo.) was added (5 mM final concentration).
DECEMBER 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
4527
C-c. Chang et a!.
The medium was decanted, and the cells were then exposed
to various UV doses. The decanted medium, now contain
ing [3H]thymidine (5 MCi/mI; 50 Ci/mmol; New England
Nuclear, Boston, Mass.), was added, and the cells were
incubated for various times before they were collected and
frozen. Cells were later analyzed for DNA content and
rad ioactivity incorporated into DNA.
Table 1
Effect of harman inChinese
and norharman on colony-forming
cellsCells
periodof were exposed to harman or norharman
colony development.No.of
Harman
Platingml)
(lLg/
Mutagenesisand Cell Survival.Quantitativemutagene
sis, using ouabain resistance as a genetic marker, has been
systematically characterized (1). Cells were trypsinized with
0.01% crystalline trypsin in phosphate-buffered saline with
out calcium and magnesium ions and plated for attachment
for 3.5 hr before UV irradiation. With the medium removed
from the plates, the attached cells were exposed to UV from
a (General Electric G25T8-25W) germicidal lamp positioned
to deliver a dose rate of 10 ergs/sq mm/sec (1 J/sq m/sec).
Growth medium with or without harman (Aldrich Chemical
Company, Milwaukee, Wis.) was added at various times for
various durations described in the protocols accompanying
the tables. After sufficient expression times (when colonies
contain 8 to 16 cells, usually 2 to 4 days depending on the
severity of UV dose), the cells were exposed to selective
medium with 1 mM ouabain. The resistant colonies that
developed in selective medium were scored 1 week later.
The number of cells seeded and plates (9 cm) used for each
experiment are indicated in the tables. The cells plated for
survival studies were treated the same way as the cells for
mutation. experiments, except they contained fewer cells
and were not exposed to the selective medium. The per
centage of survivors was determined by dividing the total
number of colonies developed in 3 to 4 plates by the total
number of cells plated, multiplied by 100.
For mutation experiments in which 6-thioguanine resist
ance was used as a genetic marker, the replating technique
was used.The cellswere replated
at2 x 10°/plate
(9cm)
after sufficient expression time and exposed to 6-thiogua
nine (10 @tg/mI)after they had attached (3.5 hr after cell
plating). The 6-thioguanine-resistant mutations require 6 to
8 days for maximum mutation expression. The mutation
frequencies are, however, very stable after the maximum
expression time (1, 28).
Norharman
(@.tg/ml)
efficiency—
cells
plated
1001
1025(5.5)°
(28)
515b20(110)
10 (55)
—
—
—
—
01
985 (5.9)
(30)
9320(120)
10 (60)
a Numbers
in parentheses,
b Colony
size slightly
C
size
Colony
ability
hamster(V79)
greatly
for the entire
No.ofcol
onies
formed
600
603
600
600
600
600
613
605
600
600
600
593
583
563k
97
600
391@'
65
100
0
@LM.
reduced.
reduced.
reduced to 10 @M.
The sensitization effect of harman was
found in a narrow range (10 to 50 SM). Higher concentra
tions of harman were toxic to cells. Results presented in
Table 2 indicate that the reducing effect of harman on
survival was found only when the chemical is present
immediately
following
UV irradiation.
Itwas alsofoundthat,
at same molar concentration, norharman was not as effec
tive as harman for this sensitization.
The effect of harman on reduction of the survival of
radiation-damaged cells was not limited to UV. A similar
effect was also found in X-ray-irradiated cells (Chart 1b).
Effect of Harmanon UnscheduledDNA Synthesis.The
unscheduled DNA synthesis induced by UV was slightly, but
consistently, inhibited by the presence of nontoxic concen
trations of harman (Chart 2). The inhibition appears to be
specific for unscheduled DNA synthesis, since normal DNA
synthesis was found not to be decreased by this treatment
for 3 hr in exponentially growing cells (data not shown).
Effect of Harman on UV Mutagenesis.In a preliminary
experiment with the use of only 1 UV dose (18 J/sq m)
(Table 2), harman and norharman were found to reduce the
RESULTS
frequency of UV-induced ouabain-resistant mutations. The
Cytotoxicity of Harman and Norharman. The effects of reducing effect was greater when the cells were exposed to
different concentrations of harman or norharman on the harman or norharman immediately following UV irradiation.
At a similar
concentration,
harman showed a greatereffect
colony-forming ability of Chinese hamster cells were tested.
As shown in Table 1 the colony-forming ability of Chinese than did norharman. For this reason harman was chosen
hamster cells was slightly reduced by 10 @g
(55 @M)
harman for a more detailed study.
Two additional experiments testing the effect of harman
per ml and completely eliminated by 20 @g
(110 @M)
harman
on mutation induction by different doses of UV at the
treatment per ml. Furthermore, at the same molar concen
trations, harman was more toxic than was norharman to ouabain-resistant locus were carried out. The results of one
experiment showing that harman reduces both the survival
Chinese hamster cells.
of UV-damaged cells and the frequencies of UV-induced
Effects of Harman on Survival of UV and X-ray-irradiated
Cells. The survivalof UV-damagedChinesehamstercells mutations are presented in Chart 3. The results were essen
when exposed to nontoxic concentratrons of harman after tially repeated in another experiment (Table 3) in which
irradiation was significantly reduced compared to cells not more UV doses were used.
To confirm the observation that harman treatment re
treated with harman. The effect was concentration depend
duces the frequency of UV-induced mutations, we repeated
ent (Chart la). The cells were more sensitive to UV killing
when exposed to higher concentrations of harman. The experiments in a different mutation system, namely, for
effect was not detectable when the concentration was ward mutation from 6-thioguanine sensitivity to 6-thiogua
4528
CANCERRESEARCHVOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Inhibition
of Mutagenesis by Harman
100
‘@‘
lb
Ia
\‘
10
l(
‘V
\
—
- Harmon
———
+ Harmon (40MM)
\
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01
>
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.1
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(l0@M)
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\
\
\
@Hormon
(30@J4
‘.Korma@
(50MM)
0
5
10
15
20
2@
uv Dose(J/sqm)
30
X-Ray Dose(R)
Chart 1 a, survival of UV-irradiated Chinese hamster cells posttreated with various nontoxic concentrations of harman. Cells were exposed to harman
immediately after UV irradiation for the entire period. b, survival of X-ray-irradiated Chinese hamster cells posttreated with harman. Cells were exposed to
harman immediately after X-ray-irradiation for the entire period. X-rays were generated by a General Electric Maxitron 300 X-ray machine. The exposure rate
was180R/min (250kV,20 ma,with 3 mm aluminumfiltration).
nine resistance. Since the ouabain mutation system appar
ouar mutants does not increase as the cell number in
ently can detect only “
point―mutations (excluding deletion
creases; therefore the frequencies are bower than the con
type mutations) (1), the 6-thioguanine system was chosen trol not treated with harman. Once the cells were exposed
to pick up any possible effect of harman on the recovery of to the selective agent, the presence of harman did not
mutations,includingdeletion-type
mutations.The results affect the mutant frequency.
from 2 experiments involving a replating technique after an
The harman effect on frequencies of spontaneous muta
8-day expression time (Table 4) consistently showed that tions was similarly observed in the two 6-TG―mutation
harman treatment reduces UV-induced 6@TGrmutations. To experiments (Table 4). Therefore the observation was re
test whether the reduction is due to a delay in the mutation
peatable in 2 different mutation systems.
expression because of the harman treatment, a 12-day
Effectof Harmanon Survivalof Preexistingouar and 6expression time was included in one of the experiments.
TGr Mutants. Treatment by nontoxic concentrations of
The results clearly indicate that the mutation reduction by harman has been shown todecreasebothspontaneousand
harman treatment is not due to a delay effect on mutation
UV-induced oua'@mutants. The results could be due to its
expression.
effect on mutagenesis or to its specific inhibitory effect on
Effect
of Harman
on the Spontaneous
Mutation
Fre
the survival ofouar mutants in growth or selective medium.
quency. Experimentspresentedin Table 3 and Chart 3 As a test of the latter possibility, 2 reconstruction experi
show that harman reduces both the spontaneous and UV
ments involving wild-type and ouar mutant cells were done,
induced oua'@mutations. Since spontaneous frequencies
although results presented in Table 5 (Protocol A) have
are very low, the difference could be due to a random shown that the frequencies of spontaneous ouar mutants
variation. Three more experiments were carried out to verify were not reduced by harman treatment in selective medium.
whether the difference is real. The results from these 5 The results from these reconstruction experiments clearly
experiments (Table 5) consistently show that, when cells show that harman treatment, comparable to that used in
replicate in the presence of harman before exposure to the mutation experiments, did not reduce the recovery of
selective agent (ouabain), the spontaneous frequency of preexisting ouar mutants derived from a single clone or
DECEMBER1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
4529
C-c. Chang et a!.
Table2
Effect of harman and norharman
treatment
on the frequency
of
UV-induced ouar mutations
For Experiment1, the number of cells per plate (9 cm) usedwas
reduced; the UV-induced unscheduled DNA synthesis was
slightly inhibited; and the frequencies of spontaneous or
UV-induced mutations were reduced. Furthermore the ef
2.1 x 10°/21
. For Experiments 2 to 6, the number of cells per plate
(9 cm) used was 4.2 x 10°/21.
Protocol:
3.5hr
1.5days
Cell plating
2days
UV
Score for
mutants
Ouabain
I//I/I/I,
Control
[@JTdR Incorporation
—lweek
______________
f//f//I',
za
o—o———oI.5hr
Harmon (50MM)
0—a——a 3 hr
Horm@
(25MM)
0'
E
0.
±Harman, norharman
>@
UV
(18J/
(28
survivors1———9912
sq m)Harman@M)Norharman(30
j.tM)Sur
fre
quency/10°
vival
(%)Mutation
(25)°2
(160)
+
3
+
4
(73)6+—+
5+ +—
a Numbers
+ ([email protected])b
+ (1 .5—3.5)
—
——
300― (39)
521d
+ (0-1 .5)b6.1
47625 37CC
(1.5-3.5)5.9609
in parentheses,
b Numbers
3.1
5.8
in parentheses,
number
of mutants.
time
of treatment
(days)
d
Mutation
frequency
of
harman-
or
U
Control
Harmon(5O@M)
Hormon([email protected])
U
U
4,
0.
U)
(127)
(151)
after
UV
irradiation.
C'
>
norharman-treated
cells
compared to control cells at equivalent dose of UV was significantly
reduced (Footnote c , p < 1%; Footnote d, p < 5%).
5
UVDose (J/sq m)
Chart 2. Effect of harman on uv-induced unscheduled DNA synthesis in
Chinese hamster cells. Various concentrations of harman were added
immediately after uv irradiation for 1.5 or 3.0 hr. Statistical tests (t test on
paired observations) indicate that harman treatments significantly reduced
the unscheduled DNA synthesis with the exception of 25 @M
harman (1.5 hr
measurement).
from a mixture of many clones (Table 6). A reconstruction
experiment with 6@TGrmutants and a protocol similar to
that of the UV mutagenesis experiments also indicates that
harman did not reduce the recovery of preexisting sponta
neous or UV-induced 6-TGT mutants (Table 7).
0
S
DISCUSSION
Harman and norharman have been shown to exhibit a
variety of effects on biological systems. Both these /3carboline derivatives have been shown to intercalate into
DNA in vitro, causing the unwinding
of the double helix (7).
The binding was more efficient with harman than norhar
man. Both compounds also inhibit the binding of
benzo(a)pyrene metabobites to DNA in vitro (8). In bacteria,
both compounds have been shown either to enhance (11,
15, 26) or to inhibit (8) the mutagenicity of mutagens, as
well as to develop the mutagenicity of nonmutagens (14) in
the presence or absence of microsomal enzymes. In the
mouse both harman and norharman inhibited benzo(a)
pyrene metabolism as measured by aryl hydrocarbon hy
droxylase activity (8). On the basis of these observations, 3
possible comutagenic mechanisms have been considered
(26): alteration
of the extracellular
metabolic
activation
.@
C/)
C
of
mutagens; alteration of the bacterial metabolism involved
in mutagenesis; and a more direct action such as interac
tion of comutagens with DNA. The first mechanism affects
the initiation of DNA damage. The latter 2 mechanisms
could be combined as one mechanism, i.e., alteration in
the process of DNA repair and mutagenesis.
From present studies in Chinese hamster cells, the fob
lowing effects of nontoxic concentrations of harman were
observed: the survival of UV or X-ray damaged cells was
4530
I
U)
UVDose (J/sqm)
Chart 3. The effect of a nontoxic concentration of harman on the survival
(left ordinate)and ouar mutations(right ordinate)of UV-irradlatedChinese
hamster cells. The results were obtained from the same experiment with the
same population of cells. The protocol used was identical with that presented
in Table 3. The mutation frequencies of harman-treated cells compared to
control cells at an equivalent dose of UV were significantly reduced (p <
1%).
CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Inhibition
Table 4
Effect of harman
6@TGrmutants
treatment on frequenciesof UV-induced
Table 3
Effect of harman treatment on frequencies of UV-induced ouar
mutants in Chinesehamster(V79)cells
For Experiments
1 to 3 and 6 to 8, the number
cellsProtocol:3.5hr
in Chinese hamster (V79)
of cells per plate
3days
(9 cm) used was 2.1 x 10'/21 ; for Experiments 4 and 9, the number
of cells per plate (9 cm) used was 4.2 x 10°/21; for Experiments
5
and 10, the number of cells per plate (9 cm) used was 21.0 x 10°/
Cell plating
4days
Replating for mu
tation analysis
,,,,,,,,,,,
3.5hr
48hr
Cell plat
ing
8days
UV
21.
Protocol:
@
of Mutagenesis by Harman
l6hr
2Ohr
—lweek
Ouabain
UV
± Harman
Scorefor
mutants
Mutation frequency―Il
0'
‘‘‘‘I'''''
survivors8-day
±Harman
12-dayUV
(40
expression
(J/sq
time01
m)ExperimentHarman
timeb
@M)expression
Expres
UV (JI
time
quencyIl0°
(40@tM)sion
vors10—4810317
sqm)Harman
(hr)Survival
(%)Mutation
fre
suM
+
(37)a25—489553
22―
—
2—
88
+44
52―51
79
56―
(106)310—6455109
(126)420—844.4351
+
(65)530—840.61525
(67)6
2—
284@'101
159―
—
318
+287
207―
+
322―
316
(9)
70
(66)8
5+
+48
48104
954―
8444
2.492
38b
(85)
+64
910 20+
(20)1030+840.28404
2—
199@'
(24)
a Numbers
in parentheses,
b Mutation
frequency
number
of mutants.
a Number
of cells
used
—
569
567
+447
349―
394C
for mutation
assay:
4.2
x 10'
(cells
not
trol cells at equivalent dose of UV was significantly reduced (p <
irradiated with UV) and 1 .5 x 10' (5 or 10 J/sq m UV-irradiated).
b Time interval between removal of harman and exposure of cells
1%).
to selectivemedium.
of
harman-treated
cells
compared
to
con
C
Mutation
frequency
trol cells at equivalent
of
harman-treated
cells
compared
dose of UV was significantly
to
reduced
con
(p <
1%).
fect of harman on survival and mutation frequencies was
greater than that of norharman and was detected primarily
in treatments in which cells were exposed to harman
immediately following UV irradiation.
By using UV light as a mutagen, the possible effect of
harman on metabolic activation and initiation of DNA dam
age in our system can be eliminated.The timing and the
specific effect of harman on survival of radiation-damaged
cells appear to indicate that harman inhibits the DNA repair
of radiation-damaged cells. This notion is supported by the
observed inhibiting effect of harman on unscheduled DNA
synthesis. However, since unscheduled DNA synthesis pri
manly measures the error-free excision repair process [as
suming that excision repair in Chinese hamsters cells is
error free as it seems to be in human cells (9)] and if harman
specifically inhibits this repair process, an increase in
mutation frequency would be expected. The results ob
tamed apparently contradict this prediction. An alternative
hypothesis that can explain all these results is that unsched
uled DNA synthesis could include an error-prone postrepli
cation repair component and that harman specifically in
hibits this process. Experiments to test this hypothesis are
now inprogress.
Harman was also found to reduce the survival of X-ray
damaged cells. There is a report that X-ray-induced DNA
single strand breaks seem to be repaired except those that
are sufficiently close to each other on complementary
strands, which would constitute double strand breaks,
which are not repairable (4). The harman treatment could
possibly affect the final yield of these double strand breaks
as a consequence of its intercalation into DNA. Alterna
tiveby, there might be a common step in the repair of UV
and X-ray-induced DNA damage that is inhibitable by har
man.
Reduction of mutagenesis by harman has been demon
strated in 2 different mutation systems in Chinese hamster
cells. Experiments have shown that it is not due to its effect
on mutation expression or its specific effect on survival of
oua'@or 6-TG'@mutants. The effect is more likely due to a
harman effect on DNA repair and mutagenesis similar to
that observed for benzo(a)pyrene in bacteria (8). From the
resultspresented here and resultsreportedinthe literature,
an interesting correlation is observed. Norharman is more
effective than harman for inducing a comutagenic effect
with carcinogens tested in the Ames assay (6, 25). On the
contrary, harman is more effective than norharman in its
ability to intercalate into the DNA (7), to reduce the surival
of radiation-damaged cells (our data), and to reduce the
mutagenic effect of the damage caused by another mutagen
(Ref. 8; present results). This correlation not only seems to
separate the 2 major effects of /3-carboline derivatives, i.e.,
the effect on mutagen activation and mutation fixation, but
also points to the possible causal relationship between DNA
DECEMBER 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
4531
C-c. Chang et a!.
Table 6
Effect of harman on the recovery of wild-type and ouabain
resistant Chinese hamster cells
Protocol:
3.5 hr
1 day
—1
week
Table 5
Effect of harman on recovery of spontaneous ouar mutants in
Chinese hamster (V79) cells
Protocol:
A.
3.5 hr
—1week
Score for
Ouabain
Cell plating
Score for
Cell plat
mutants
ing
± Ouabain
colonies
‘‘‘I''''''
HarmanNo.
± Harman
B.
3.5hr
Cell plat
ing
1_1.5daysa
—1week
±
ofcells/plate
(9cm)Har
Score for
Ouabain
‘‘“‘‘‘/‘“/
mutants
%ouar@Wild
Oua
No. of col
(1
@M)bain
mM)oniesformed―Recov
(ouar@141)mixture@man
ery°200——167841200
(40
typeouar@135
± Harman
@
(200)
(40
@.tM)survivorsProtocol
B1—
A
—
+
—
200(200)
—
—
+
+
——
+165
200(200)+
74(85)1xlO'++0.51x
Protocol
165 (168)d
(84)d
170(168)
85(84)
153(173)
77(87)
—
200(200)
mutants/lO'Experiment'@Harman Frequency ofouar
xlO'200
147(169)
083
17.1―
4.1c2—
+—
—
3.6e3—
+—
—
2.2°4—
+0.13@
ozi@
<O.9@5—
+025h
0.49―
10'200(200)—+148(170)74(85)1x
10'200
(200)++146
(84)200——18894200200
8.8'
<0.69i
94
—
—
188
—
+
200
176
—
+
192
200
+—
+182 18291
911xlO'—+01xlO'++01xlO'200—+192961xlO°200++18894
200+
6.7'
+<0.69'
(167)73
9.0°
0.7'
88
96
a One day for Experiment 1 , 1 .5 days for Experiments 2 to 5.
b Experiments
1 and
3 are
experiments
presented
in Table
3 and
Chart 3, respectively.
C
Numberofcellstested:2.1
d Number
x
of cells tested:
10g.
a Determined by dividing the total number of colonies developed
1 .65 x 10'.
by the total number of cells plated that were expected to grow in
the medium provided, multiplied by 100.
e Number of cells tested: 1 .8 x 10g.
I Numberofcellstested:
4.2
U Numberofcellstested:
1.05
h Numberofcellstested:
8.4
x 10'.
1
1.5
x
Numberofcellstested:
x 10'.
b Mixture
x 10'.
of an equal
C
10g.
Average
of
(I Numbers
intercalation, cell survival, and the process of mutagenesis.
Harman was also found to reduce the frequency of
spontaneous mutations. If the reduction is due to the
intercalation of harman into DNA, we suspect that sponta
neous mutagenesis may share a common step with an
error-prone postreplication repair process. If the harman
effecton spontaneousmutagenesiscan be found to be a
general phenomenon, harman can be an important chemi
cal to study the mechanism of spontaneous mutations.
If mutagenesis does play a role in carcinogenesis, and
because of the potential role of harman and norharman in
nutritionally
related
human
carcinogenesis,
additional
from
17
indepe―-'
it
4
plates.
in parentheses,
results
for ouar@141.
Table 7
Effect of harman on the recovery of preexisting 6@TGrmutants in
Chinese hamster cells
Protocol:
3.5hr
9days@'
3days
Replating for
recoveryof
/////‘‘‘‘‘‘‘‘‘/////
Cell plating
6-TGT mu
tants
± Harman
Proportion
of initial cell mixtureHarman
of
6@TGrmutant
type(xlO')6@TGr
recoveredWild
(12O)'@1
(44)
1
1
125―
(spontaneous)
l25@(spontaneous)
1
125d (UV-induced)
—
10
125d
+0.9
a Cells
were
b Numbers
The authors gratefully acknowledgethe technical assistanceof Judy
(40Frequency
(origin)@M)(x
10')10—3.3
ACKNOWLEDGMENTS
4532
of cells
stud
ies must be made to examine the implications of our results.
Comparative studies, with the use of mutagenesis assays in
both bacteria and cultured human fibroblasts as well as the
initiation-promotion mouse skin assay, are now in progress.
Funston and the typing assistance of Sharon Austin.
number
spontaneous ouar clones.
C
A
mixture
d A mixture
+
(UV-induced)+
subcultured
every
in parentheses,
of
—
18
63 (368)
67 (368)
123 (940)
139 (1062)
2 days.
number
of mutants.
independent6@TGr
clones.
of 116 independent6@TGr
clones.
CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
4533
Effects of Harman and Norharman on Spontaneous and
Ultraviolet Light-induced Mutagenesis in Cultured Chinese
Hamster Cells
Chia-cheng Chang, Marc Casteliazzi, Thomas W. Glover, et al.
Cancer Res 1978;38:4527-4533.
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