Genotoxicity assessment of new synthesized acridine derivative - 3,6-diamino-10-methyl-9,10-dihydroacridine

Article abstract of DOI:10.1016/S1383-5718(99)00112-6

A new synthesized acridine derivative, 3,6-diamino-10-methyl-9,10-dihydroacridine (AcrH), was tested for in vitro reverse mutations with Salmonella TA strains, chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes, and for in vivo chromosome aberrations in bone marrow of mice. Using the classic plate incorporation method, mutagenicity of AcrH in bacterial cells (TA97a, TA98, TA100 and TA102) was observed in the experiments performed with, and without, rat liver S9 metabolic activation. The reverse mutation assay showed no difference in mutagenic activity between AcrH and acriflavine (Acr⁺) in the test with TA97. The results of in vitro chromosome aberrations assay revealed potential clastogenicity. The test using macroculture of human lymphocytes induced mainly chromatid gaps. The experiments with human lymphocytes revealed SCE-inducing effect of AcrH and Acr⁺. In an in vivo study, AcrH given intraperitoneally to Balb/c mice did not cause any significant increase in the percentage of cells with aberrations compared to the negative control. Copyright (C) 1999 Elsevier Science B.V.

Full text of DOI:10.1016/S1383-5718(99)00112-6

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Mutation Research 444 1999 463–470
www.elsevier.comrlocatergentox
Community address: www.elsevier.comrlocatermutres
Genotoxicity assessment of new synthesized acridine derivative
— 3,6-diamino-10-methyl-9,10-dihydroacridine
a
a
Tomasz Ferenc , Ewa Janik-Spiechowicz b,), Wanda Bratkowska ,
a
c
d
a
Dobrosława Łopaczynska , Henryk Strozynski , Andrzej Denys , Anna Mordalska
´
´˙ ´
a
Department of Biology and Medical Genetics, Military Medical UniÕersity, 90-647 Łodz, Pl. Hallera 1, Poland
Department of Toxicology and Carcinogenesis, The Nofer Institute of Occupational Medicine, PO Box 199, 90-950 Łodz, Poland
´ ´
b
´ ´
c
Department of Infections Diseases and Gastroenterology, Military Medical UniÕersity, Łodz, ul. Kniaziewicza, Poland
´ ´
´ ´
d
Department of Microbiology, Military Medical UniÕersity, 90-647 Łodz, Poland
Received 16 February 1999; received in revised form 17 June 1999; accepted 18 June 1999
Abstract
Ž
.
A new synthesized acridine derivative, 3,6-diamino-10-methyl-9,10-dihydroacridine AcrH , was tested for in vitro
Ž
.
reverse mutations with Salmonella TA strains, chromosome aberrations and sister chromatid exchanges SCE in human
lymphocytes, and for in vivo chromosome aberrations in bone marrow of mice. Using the classic plate incorporation method,
Ž
.
mutagenicity of AcrH in bacterial cells TA97a, TA98, TA100 and TA102 was observed in the experiments performed
with, and without, rat liver S9 metabolic activation. The reverse mutation assay showed no difference in mutagenic activity
q
Ž
.
between AcrH and acriflavine Acr in the test with TA97. The results of in vitro chromosome aberrations assay revealed
potential clastogenicity. The test using macroculture of human lymphocytes induced mainly chromatid gaps. The experi-
ments with human lymphocytes revealed SCE-inducing effect of AcrH and Acrq. In an in vivo study, AcrH given
intraperitoneally to Balbrc mice did not cause any significant increase in the percentage of cells with aberrations compared
to the negative control. q 1999 Elsevier Science B.V. All rights reserved.
Keywords: 3,6-Diamino-10-methyl-9,10-dihydroacridine; Salmonella typhimurium; Chromosome aberrations; Sister chromatid exchange;
Human lymphocytes; Mouse bone marrow
1. Introduction
Commercial mixture of proflavine 3,6-di-
DNA intercalators plays an important role due to its
outstanding antibacterial properties. It is assumed
that Acrq binds rapidly to saturate the surface of the
membrane cell, which is negatively charged due to
presence of phosphate anions in the outer leaf.
Therefore, only very small fractions of Acrq
molecules can penetrate into the bacterial cells to
interact with the genetic material. To overcome
transportation barriers through the plasma mem-
Ž
.
aminoacridine and its N-methyl quaternary salt, eu-
Ž
q. Ž
Ferguson
flavine, is known as acriflavine Acr
and Denny 1 . Acrq representing a group of cationic
w x.
)
Corresponding author. Fax: q48-42-6314-610; E-mail:
ksitarek@porta.imp.lodz.pl
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
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PII: S1383-5718 99 00112-6

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)
464
T. Ferenc et al.rMutation Research 444 1999 463–470
branes, a strategy of ‘Trojan horse’ can be envis-
aged. This strategy may involve application of an
uncharged, hydrophobic Acrq precursor, which can
release Acrq inside bacterial cells. The preparation
of a stable Acrq precursor of unique properties,
namely 3,6-diamino-10-methyl-9,10-dihydroacridine
2. Materials and methods
2.1. Chemicals
Acrq neutral CAS No. 8048-52-0, product of
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.
Aldrich Chemical was used to synthesize AcrH by
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.
AcrH and its antibacterial, antiviral and antifungal
NaBH4 reduction of 3,6-diamino-10-methyl-acri-
dinium chloride. The synthesis was performed ac-
cording to the method described by Gilliot 5 ;
w x
activity was described earlier by Adamus et al. 2 . It
was shown that, upon dissolving AcrH in aerated
water or other polar solvent, spontaneous reoxidation
takes place and Acrq is reformed quantitatively.
Reoxidation takes place during several hours in H2O,
and it can be much longer in less polar solvents or in
solvents characterized by poor oxygen solubility.
w x
w x
Adamus et al. 2 describe the characteristics and
properties of AcrH. The chemical structure is shown
in Fig. 1. In all tests, AcrH was assayed after it had
Ž
been solved in 50% ethanol or 1,2-propanediol pro-
pylene glycol; POCH, Gliwice on the day of the
assay.
.
Ž
.
The time-dependent experiments 0.5–24 h showed
that Acrq and OP2y generated from AcrH inactivated
bacteria of Escherichia coli, Proteus Õulgaris, and
Pseudomonas aeruginosa about five times faster and
Staphylococcus aureus over 15 times faster than
Acrq in reference solution of similar concentration.
The mechanism of AcrH reoxidation to Acrq in-
volves a sequential electron–proton–electron transfer
2.2. ReÕerse mutations assay with Salmonella TA
strains
The Salmonellarmicrosome plate-incorporation
assay was employed according to Ames et al. 6 as
revised by Maron and Ames 7 . The S. typhimurium
tester strains TA97a, TA98, TA100 and TA102 were
supplied by B.N. Ames, University of California,
Berkeley, CA. The cultures were grown in ‘Oxoid’
nutrient broth No. 2 for 10 h at 378C with shaking.
The test was performed both with, and without, S9
w x
w x
w x
w x Ž
after Marcinek et al. 3 , and Ge˛bicki et al. 4 see
.
Fig. 1 .
The aim of this study was to evaluate the geno-
toxic activity of AcrH using in vitro and in vivo
screening tests and additionally, to compare the
genotoxic activity of AcrH and Acrq under the
experimental conditions.
Ž
.
fraction 50 mlrplate prepared from liver of male
outbred Imp:WIST rats. The inducing agent for S9
Fig. 1. The mechanism of AcrH reoxidation to Acrq involves a sequential electron–proton–electron transfer.

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T. Ferenc et al.rMutation Research 444 1999 463–470
465
(
)
preparation was Aroclor 1254. The protein concen-
tration of S9 fraction, determined by the Lowry
2.4. Sister chromatid exchanges SCE assay without
S9-mix in human lymphocytes
w x
method 8 , was 38.3 mgrml. The test was con-
Peripheral blood was obtained from five male
donors. The lymphocyte cultures were incubated for
ducted up to the soluble concentration limit of AcrH
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.
100 mgr100 ml, ca. 50% ethanol . Each dose level
Ž
72 h in the presence of bromodeoxyuridine BrdU,
was tested in duplicate on three separate experi-
ments. Revertant colonies were counted 48 h after
incubation with automated bacterial colony counter
Biotran II from New Brunswick Scientific, NJ. A
mutagenic test was positive if the compound induced
a concentration-dependent increase in the number of
revertants per plate as compared to the number of
revertants per control plates, and the increase had to
be at least twice the controls rate.
q
.
Ž
Sigma, 10 mgrml . AcrH and Acr
0.1 and 0.5
.
mgrml were added to the cultures at the beginning
of the incubation or 24 h afterwards. Colcemid was
added to the culture 2 h prior to harvesting. The
slides were coded and stained for sister chromatid
w
x
differentiation according to Perry and Wolff 11 .
Analysis of SCE was performed in M2 metaphases
with a minimum of 25 mitoses per point.
2.3. Chromosome aberrations assay without S9-mix
in human lymphocytes
2.5. In ÕiÕo chromosome aberrations assay in bone
marrow of mice
Venous blood from 10 healthy adult male non-
smoker donors without a known drug or medical
history was used in the experiment. The necessary
metaphases were obtained in the conventional man-
ner from the series of 48 h macroculture of lympho-
cytes. The culture medium was Eagle’s fluid 1959
The assays were performed on bone marrow cells
Ž
from male Balbrc mice weighing about 25 g, 8
.
weeks old according to the standard procedures as
w
x
described by Brusick 12 . Based on preliminary
experiments, AcrH was given by intraperitoneal in-
Ž
.
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.
jection i.p. at 28 mgrkg 50% of LD50 . The
Ž
.
Ž
.
MEM . Colcemid Serva, 0.15 mgrml was added 2
genotoxicity of Acrq in this test was not examined.
h before termination to arrest cells in the metaphase.
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.
Colchicine Sigma, 3.3 mgrkg i.p. was given 2 h
Ž
Chlormethine hydrochloride Nitrogranulogen, Polfa;
before the mice were sacrificed to inhibit cell mito-
.
0.1 mgrml was used as the positive control. Two
Ž
sis. 1,2-Propanediol and cyclophosphamide Endo-
doses 0.1 and 0.5 mgrml of AcrH and Acrq
Ž
.
xan, Asta Medica, the negative and positive control,
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.
prepared and diluted in MEM negative control
.
respectively were injected i.p. to four mice at 48 h
Ž
were added to the culture of lymphocytes protected
before the end of the experiment. Bone marrow
samples were collected in the 30th, 48th and 72nd h
after the injection of AcrH. The chromosome prepa-
rations from hipotonized and fixed bone marrow
.
from exposure to visible light . The basic experiment
in this study was when a compound was added to 24
Ž
.
h culture of lymphocytes S phase . Experiment when
a compound was added at the beginning of incuba-
Ž
.
cells both femurs of each mouse were air-dried and
stained by Giemsa. The analysis of chromosome
aberrations was performed in average of 50
metaphases per mouse.
Ž
.
tion stage G0 of the cell cycle was used for cytoge-
netic testing of S-independent compounds which can
produce aberrations at all stages of the cell cycle. In
a pilot experiment, 0.5 mgrml was the highest dose
which made it possible to obtain metaphases for
analysis. Preparations were made by the air-drying
method and were stained by Giemsa. Slides were
coded for data entry and observed according to the
3. Statistical analysis
The recorded results were statistically analyzed
w x
Ž
standard procedure after Preston et al. 9 . Analysis
with the help of: Fisher’s exact test chromosome
.
for chromatid and chromosome type aberrations was
performed in all satisfactorily spreading and stained
metaphases. The classification of gaps and breaks for
non-banded preparations was in accordance with the
aberrations in vitro and in vivo and Mann–
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.
Whitney’s test SCE , with the level of significance
set at pF0.05 or higher. The numerical data in
Table 2 compared with the historical control were
analyzed by Chi-square test with Yates’ correction.
w
x
ISCN 10 .

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)
466
T. Ferenc et al.rMutation Research 444 1999 463–470
4. Results
toxic effect of AcrH as evidenced by reduced growth
of the hisy background lawn.
4.1. Induction of reÕerse mutation in bacterial cells
4.2. Analysis of chromosomal aberrations: in Õitro
tests
The results of mutation tests, without and with the
supplementation of S9 rat liver supernatant are sum-
marized in Table 1. AcrH was detectably mutagenic
at 50 mgrplate in the absence of S9 activation
The frequency of aberrations for the tested com-
pounds are presented in Table 2. No aberrations were
found in the cells analyzed in the control cultures.
However, for both compounds at 0.1 mgrml, the
percentages of cells with aberrations were similar to
the control frequency of aberrations found in other
Ž
.
TA97a, TA98 and TA102 . At this dose applied to
Ž
.
reports about 2.0% . For AcrH at 0.5 mgrml and
Acrq at 0.1 and 0.5 mgrml, a significant increase of
cells with aberrations as compared to the negative
control was observed for both lymphocyte treatment
times. In another experiment, the mean number of
Ž
.
chromosome aberrations per cell including gaps of
lymphocyte culture containing only culture fluid was
3.0%. When the percentages of the aberrated cells
were compared for both chemicals given at two time
intervals vs. the historical control, a significant in-
crease in the percentage of the aberrated cells was
Table 1
Induction of reverse hisy mutation in S. typhimurium strains
Compoundrdose per plate,
mg or ml
Mean number of revertants per plate and standard deviation
TA97a
TA98
TA100
TA102
yS9
qS9
yS9
qS9
yS9
qS9
yS9
qS9
Spontaneous reversion
Negative control
114"8
119"8
124"7
130"7
24"2
24"3
29"3
32"5
116"7
119"7
104"9
244"14 344"23
114"10 249"19 345"28
Ž
.
50% ethanol
1
10
123"13
159"21
30"5
44"7
98"9
35"7
109"17 127"14 130"14 279"32 382"32
425"48 157"21 242"15 442"58 522"67
643"48 189"19 361"18 576"80 700"58
618"79 126"22 254"29 536"63 686"73
524"32
a
Ž
Ž
Ž
Ž
.
Ž
Ž
Ž
Ž
.
.
.
.
120"7
180"23
289"25
310"27
427"47
454"40
599"51
638"60
214"22
161"22
325"24
279"28
187"31
383"19
.
.
.
50
100
Positive controls: 4-
nitroquinoline-N-oxide
4-nitro-o-
phenylenediamine
Sodium azide
0.5
3.0
271"15
123"10
357"26
27"2
1.5
5.0
5.0
787"53
2-aminofluorene
575"30
116"11 663"35 235"32 516"40
328"27
Ž .
Benzo a pyrene
a The values in parentheses represent the number of revertants in terms of mean"S.D. for Acrq.

(
)
T. Ferenc et al.rMutation Research 444 1999 463–470
467
Table 2
Chromosome aberrations frequency — in vitro tests in culture of human lymphocytes
Compoundsrdose
Number
of cells
scored
Number of aberrations
GX GY BX
Percent of
cells with
aberrations
BY
DIC
EF
AF
Ž
.
mgrml culture
a
Ž
.
.
.
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž
.
Negative control
3,6-diamino-10-methyl-
9,10-dihydroacridine
0.1
0.5
Acriflavine
0.1
0.5
450 240
0.00 0.00
349 140
0.29f 1.43
.
Ž
Ž .
0 2
Ž .
5 2
Ž .
0 0
Ž .
0 0
Ž .
1 0
Ž .
1 0
Ž .
0 0
Ž .
1 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž
230 70
3.04d 2.86
Ž
b
b
Ž
.
.
300 140
2.33c 2.86
Ž
Ž
Ž .
5 3
Ž .
6 3
Ž .
1 1
Ž .
0 0
Ž .
0 0
Ž .
1 5
Ž .
0 0
Ž .
1 1
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
0 0
Ž .
1 0
Ž .
1 0
.
d,f
160 80
5.63d 11.25
Ž
.
Ž
.
Positive control
Ž
chloromethine
.
hydrochloride
0.1
c
30.91d,f 4.29
3.00
Ž
.
Ž .
9 3
55
Ž .
5 0
1
Ž .
9 1
1
Ž .
4 0
0
Ž .
0 2
0
Ž .
2 0
0
Ž .
5 0
0
Ž
.
110 140
1900
Historical controle
a
Ž
.
The table gives the results of the effects observed for the test compounds, when added at the beginning of incubation data in parentheses
X
Y
X
Y
Ž
.
and 24 h before harvesting of cells data without parentheses ; G — chromatid gap, G — chromosome gap, B — chromatid break, B —
chromosome break, DIC — dicentric, EF — exchange figures, AF — acentric fragment.
b
Ž
.
.
Significant differences vs. negative control at pF0.05 .
c
d
e
Ž
Significant differences vs. negative control at pF0.01 .
Ž
.
Significant differences vs. negative control at pF0.001 .
w
x
Relevant literature: Ferenc et al. 18 .
fSignificant differences vs. historical control.
noted only for Acrq added at 0.5 mgrml at the
beginning of the incubation period. However, the
percentage of the aberrated cells for AcrH at 0.1
mgrml was significantly lower compared to the
historical control. The recorded percentages of cells
with aberrations were compared between Acrq and
AcrH for the doses 0.1 mgrml and 0.5 mgrml. In
experiments when the compounds were added 24 h
before harvesting, the percentage increase of cells
with aberrations was observed, but the difference
between the compared numerical values was statisti-
cally insignificant. In experiments when the com-
pounds were added at the beginning of incubation, a
significant percentage increase of cells with aberra-
Table 3
SCE frequency — in vitro tests in culture of human lymphocytes
Compounds
Dose
Number of cells
scored
SCErcell
Ž
.
Ž
.
mean"S.D.
mgrml
Ž
.
.
Ž
.
Negative control
3,6-diamino-10-methyl-
9,10-dihydroacridine
Acriflavine
0.0
0.1
0.5
0.1
0.5
162 138
8.56"2.71 7.93"2.60
a
c
c
126 118
10.77"3.85b 10.98"4.46
Ž
Ž
.
.
Ž .d
d
–
–
149 126
15.56"4.89c 12.63"4.74
Ž
Ž
.
Ž .d
d
–
–
a
Ž
.
The table gives the results of the effects observed for the test compounds added to culture of lymphocytes 48 h data without parentheses
Ž
.
and 72 h data in parentheses before harvesting.
b
Ž
.
Significant at pF0.05 vs. negative control .
c
Ž
.
Significant at pF0.001 vs. negative control .
d No M2 cells or a few mitoses.

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468
T. Ferenc et al.rMutation Research 444 1999 463–470
Table 4
Chromosome aberrations frequency — in vivo tests in bone marrow of mice
Compoundsrdose
Duration
of action
Ž .
h
Number of
cells scored
Number of aberrations
Percent of
of cells with
aberrations
GX
GY
BX
BY
0
DIC
0
AF
0
POL
0
Ž
.
mgrkg body weight
Negative control
72
200
1
0
0
0.5
Ž
.
1,2-propanediol
vrv 0.1 ml
3,6-diamino-10-methyl-
9,10-dihydroacridine
28.0
30
48
72
48
200
100
140
80
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0.5
1.0
0.0
Positive control
12
16.2a
Ž
.
cyclophosphamide 40.0
GX — chromatid gap, GY — chromosome gap, BX — chromatid break, BY — chromosome break, DIC — dicentric, AF — acentric fragment,
POL — polyploid.
a
Ž
.
Significant at pF0.001 as compared to the negative control .
tions was observed for Acrq at 0.5 mgrml pF
Ž
Ž
.
AcrH and negative control pF0.001 and dis-
.
0.05 .
played clastogenicity.
4.3. Analysis of SCE: in Õitro tests
5. Discussion
Mean SCE frequency per cell for the tested com-
pounds are presented in Table 3. For both com-
pounds at 0.5 mgrml, insufficient numbers of
metaphases with differentially stained chromatids
were observed. Therefore, the SCE values presented
above were recorded for one dose only of the com-
pounds in the two time intervals. For AcrH and
Acrq at 0.1 mgrml, in both intervals, a significant
increase of number SCErcell as compared to the
negative control was observed. The comparison of
mean SCErcell count between Acrq and AcrH for
the dose of 0.1 mgrml showed a significant increase
in SCE frequencies for Acrq added to culture at 72 h
The genetic toxicity of proflavine and related
Ž
compounds was studied by many authors Ferguson
w x.
and Denny 1 . In a bacterial model with S. ty-
phimurium TA series, mutagenic activity of
proflavine was observed in the presence and absence
of metabolic activation mainly in the strains which
detect frameshift mutagens, but also in basepair sub-
Ž
w x.
stitution mutagens Speck and Rosenkranz 13 . In
our studies, indication that AcrH is a mutagenic
compound was observed in both series of experi-
Ž
.
ments with and without the metabolic system . The
mutagenic effect was found in all four tester strains.
However, AcrH was somewhat more active in induc-
ing frameshift mutations, especially in TA98. An
increase of the mutagenic activity was observed in
the presence of S9-mix. The mutagenic potential of
AcrH was the same as that of Acrq in TA97a at
Ž
.
before the termination of the experiment pF0.05
and a higher effect for Acrq added at 48 h before the
Ž
.
termination pF0.001 .
Ž
4.4. Analysis of chromosome aberrations: in ÕiÕo
tests
comparable doses 50 mgrplate, without S9 and 10
.
mgrplate, with S9 .
w
x
DeMarini et al. 14 showed that proflavine had
strong clastogenic activity in mouse lymphoma cells.
Nishi et al. 15 observed that proflavine induced
SCE in Chinese hamster V79 cells. Speit and Vogel
16 tested intercalating dyes acridine orange,
proflavine and methylene blue and drugs chlor-
In the cells from bone marrow of mice exposed to
AcrH, only two cells with chromosome aberrations
w
x
Ž
.
were observed Table 4 . The recorded percentage of
cells with the aberrations did not differ significantly
as compared to the negative control. The observed
percentage of cyclophosphamide-exposed cells with
aberrations was significantly higher as compared to
w
x
Ž
.
Ž
.
promazine, promazine and chlorprothixene for their
ability to induce SCEs with and without photoactiva-

(
)
T. Ferenc et al.rMutation Research 444 1999 463–470
469
tion by visible light. They found that while in the
dark, all these substances increased the frequency of
SCEs; they also observed a superimposed effect of
visible light on SCE formation for the proflavine and
acridine orange, but not for the phenothiazine deriva-
tives, methylene blue and chlorpromazine. In our
study, the chromatid type aberrations were mainly
observed in the cells exposed in vitro to AcrH and
Acrq. However, a very low number of mitoses with
chromosome gaps, chromosome breaks and acentric
fragments was observed. A significant increase in the
aberration frequencies vs. the negative control was
genic effect of AcrH is quantitatively similar to
that of Acrq;
-the results of in vitro chromosome aberrations
assay compared vs. the negative control point to a
potential clastogenicity of AcrH.
-AcrH induced a significantly higher number of
SCEs in human lymphocytes, which indicates that
AcrH is genotoxic.
Acknowledgements
observed at 0.5 mgrml of AcrH and at both doses
Reverse mutagenicity assays with Salmonella has
been performed at the Nofer Institute of Occupa-
q
Ž
.
0.1 and 0.5 mgrml of Acr for both lymphocyte
exposure times. The frequency of aberrations was
Ž
.
tional Medicine NIOM , Łodz, Poland under Con-
q
´ ´
w x
higher with Acr . When using the Preston et al. 9
tract No. ZU 10r98 from the Military Medical Uni-
criteria, the results point to the clastogenic activity of
both chemicals. However, clastogenic activity was
not observed when the aberration frequency was
compared vs. the historical control. A significant
increase in the aberration frequency as compared to
the historical control was observed at 0.5 mgrml of
Acrq when added at the beginning of the incubation.
In our study, the mean SCE frequency per cell for
AcrH and Acrq was significantly higher as com-
pared to the negative control at 0.1 mgrml for 48-
and 72-h exposures. However, it is not clear why
Acrq induced a higher number of SCEs than AcrH.
Data from in vivo experiments for proflavine and
related compounds in mammalian cells are rare.
versity.
References
w x
1
L.R. Ferguson, W.A. Denny, The genetic toxicology of
Ž
.
acridines, Mutat. Res. 258 1991 123–160.
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