Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

Article abstract of DOI:10.1016/j.mrgentox.2007.11.003

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.

Full text of DOI:10.1016/j.mrgentox.2007.11.003

Available online at www.sciencedirect.com
Mutation Research 650 (2008) 140–149
Genotoxicity of aminohydroxynaphthoquinones in bacteria,
yeast, and Chinese hamster lung fibroblast cells
Luis Fernando da Costa Medinaa,1, Cassiana Macagnan Viaua,1, Dinara Jaqueline Mouraa,
c
d
a,b,∗
Jenifer Saffia,b, Valter Stefani , Adriano Brandelli , Joao Antonio Pegas Henriques
˜
ˆ
a Laborato´rio de Genotoxicidade-Instituto Royal-Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
b Laborato´rio de Gene´tica Toxicolo´gica, Universidade Luterana do Brasil, Canoas, RS, Brazil
c Laborato´rio de Novos Materiais Orgaˆnicos, Departamento de Qu´ımica Orgaˆnica, Universidade Federal
do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
d Laborato´rio de Bioqu´ımica e Microbiologia Aplicada, Departamento de Cieˆncia de Alimentos,
Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
Received 9 March 2007; received in revised form 9 November 2007; accepted 14 November 2007
Available online 22 November 2007
Abstract
There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells.
We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-
OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity
were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic,
whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT),
mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any
recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak
mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared
to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone
compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the
bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced
DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic
activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a
significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones
were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced
oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of
DNA intercalation, have an important role in mutagenic and genotoxic activities.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Salmonella microsome/Ames; Saccharomyces cerevisiae; Mutagenesis; Comet assay; Quinones
1. Introduction
respiratory chain-dependent energy production, allelopathy, and
defense [1–3]. During the last decades, a large number of natural
and synthetic NQs and its derivates have been extensively stud-
ied as to their antitumoral [4], antiparasitic [5], and antimicrobial
[6–9] activities.
Naphthoquinones (NQs) and their derivates play an impor-
tant role in several biological processes, such as photosynthesis,
The classic mechanism of NQs toxicity is to produce reactive
oxygen species (ROS). NQs can be reduced to semiquinones by
NADPH–cytochrome P-450 reductase or to hydroquinones by
DT-diaphorase NAD(P)H: quinoneoxireductase (EC 1.6.99.2).
∗
Corresponding author at: Centro de Biotecnologia – UFRGS, Avenue Bento
Gonc¸alves 9500, 91501-970 Porto Alegre, Brazil. Fax: +55 51 3308 6069.
E-mail address: pegas@cbiot.ufrgs.br (J.A.P. Henriques).
These authors contributed equally to this work.
1
1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2007.11.003

L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
141
In the presence of oxygen, reduced NQs auto-oxidize, produc-
ing superoxide anion radical. This redox cycling of quinones
promotes oxidative stress conditions, resulting in destabiliza-
tion of lysosomes, release of cathepsin D, and mitochondrial
membrane potential decrease [10,11]. Furthermore, the capac-
ity to produce free radicals is influenced by the nature and the
position of substituents in the quinone molecule [12].
Cell membranes are often permeable to NQs, which, once
present in the cell, may form adducts with DNA, leading to
abasic sites and single or double strand breaks [12–14]. NQs
also react with proteins and lipids, destroying their functionality,
thereby causing cell death [15].
Tikkanen et al. [16] investigated the influence of substituents,
such as hydroxy, methyl or both moieties in different positions
of NQs molecules. Upon metabolic activation, naphthaz-
arin(5,8-dihydroxy-1,4-naphthoquinone)(NTZ)andplumbagin
(2-methyl-5-hydroxy-1,4-naphthoquinone) induced frameshift
mutations in Salmonella typhimurium strain TA98. Hakura et al.
[17] demonstrated that 4-amino-1,2-naphthoquinone was muta-
genic in TA97, TA100, and TA102 strains. However, few studies
were conducted to determine the mutagenic activity of amino-
hydroxy substituents in this class of compounds.
Considering the use of some quinones as antineoplas-
tic agents [4] and their ability to form adducts with
DNA [12,13], we conducted a study on the toxic and
genotoxic potential of two aminohydroxynaphthoquinones –
5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-
2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) – by employing
the three test organisms: Salmonella/microsome assay, Sac-
charomyces cerevisiae, and permanent lung fibroblast cell line
derived from Chinese hamsters (V79 cells). The genotoxicity of
these compounds was determined using standard Comet assay
(single-cell gel—SCG). Standard Comet assay sensitivity and
specificitycanbeimprovedbyincubatinglysedcellswithlesion-
specific endonucleases, which recognize particular damage
bases and create breaks [18]. The most commonly used endonu-
cleases in the modified Comet assay are formamidopyrimidine
DNA-glycosylase (FPG, also known as MutM), and endonu-
clease III (ENDOIII, also known as NTH). FPG is specific
for oxidized purines, specially for 8-oxo-7,8-dihydroguanine
(8-oxoGua), and ENDOIII recognizes oxidized pyrimidines,
including thymine glycol and uracil glycol [18,19]. Using this
technique, we were able to determine the specific oxidative
lesion induced by these naphthoquinones in mammalian culture
cells, and thereby obtain insight as to their toxicity mechanisms.
Fig. 1. Chemical structures of 1,4-naphthoquinones tested. (1,4-NQ) 1,4-
naphthoquinone, (ANQ) 5-amino-8-hydroxy-1,4-naphthoquinone and (ANQ-
OH) 2,8-dihydroxy-5-amino-1,4-naphthoquinone.
ice. The solution is filtered and extracted with chloroform in a liquid/liquid
extractor. Crude quinine is purified by short-column chromatography in sil-
ica gel (chloroform) [20]. 1,4-Naphthoquinone (1,4-NQ), CAS [130-15-4],
was purchased from ACROS (Geel, Belgium). d-Biotin, aflatoxin B1, amino
acids (l-histidine, l-threonine, l-methionine, l-tryptophan, l-leucine and l-
lysine), nitrogen bases (adenine and uracil), cycloheximide, methyl methane
sulfonate (MMS), hydrogen peroxide (H2O2), 4-nitroquinoline-oxide (4-NQO),
cyclophosphamide (CP), and sodium azide were purchased from Sigma (St.
Louis, MO, USA). FPGandENDOIIIwereobtainedfromNewEnglandBioLabs
(USA). Nutrient broth no. 2 was obtained from Oxoid (MD, USA), and Yeast
extract, Bacto-peptone and Bacto-agar were obtained from Difco Laboratories
(Detroit, USA). Dulbecco’s modified Eagle Medium (DMEM), fetal bovine
serum (FBS), trypsin–EDTA, l-glutamine, antibiotics, and trypan blue (TB)
were purchased from Gibco BRL (Grand Island, NY, USA). The S9 fraction,
prepared from livers of Sprague–Dawley rats, pre-treated with the polychlori-
nated biphenyl mixture Araclor 1254, was purchased from Moltox (Annapolis,
MD, USA). For all treatments, 5 mg/mL dimethylsulfoxide (DMSO, Merck,
Darmstadt) stock solutions of NQ were prepared immediately prior to use. All
others reagents were of an analytical grade.
2.2. Strains
S. typhimurium TA97, TA98, TA100, and TA102, described in [21], were
kindly provided by Ames and Maron (University of California, Berkeley, CA,
USA). S. cerevisiae strain XV185-14c (MAT␣ ade2-2 arg4-17 his1-7 lys1-1
trp5-48 hom3-10) [22] was used in mutagenicity assay. Induced mitotic recom-
bination was measured in diploid yeast strain XS2316 (MATa/␣ his1-1/his1-1
leu1-1/leu1-12 +/cyh2 trp5-48 +/met13) [23]. Chinese hamster lung fibroblasts
(V79 cells) were cultivated under standard conditions in Dulbecco’s modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 2 mM
l-glutamine [24].
2. Materials and methods
2.3. Yeast culture media
2.1. Chemicals
Media, solutions, and buffers were prepared as previously described [25].
Complete YPD medium, containing 0.5% yeast extract, 2% bacto-peptone, and
2% glucose was used for routine growth of yeast cells. For plates, the medium
was solidified with 2% bacto-agar. Minimal medium (MM), containing 0.67%
yeast nitrogen base without amino acids, 2% glucose, and 2% bacto-agar, was
supplemented with the appropriate amino acids. Synthetic complete medium
(SC) consisted of MM supplemented with 2 mg adenine, 2 mg arginine, 5 mg
lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg uracil, 2 mg tryp-
tophan, and 24 mg threonine per 100 mL MM. For mutagenesis of the strain
The compounds 5-amino-8-hydroxy-1,4-naphthoquinone, CAS [68217-36-
7] (ANQ), and 5-amino-2,8-dihydroxy-1,4-naphthoquinone, CAS [500733-87-
9] (ANQ-OH) (Fig. 1) were synthesized as described. A mixture of sulfur (7.5 g)
and fuming sulfuric acid (60% SO3, 85 mL) is added dropwise, with stirring to
an ice-cooled slurry of 1,5-dinitropnaphthalene (20.0 g) in concentrated sulfu-
ric acid (45 mL), as well as crushed ice. The solution is continuously stirred
for 1 h, and then it is warmed at 50 ◦C for 10 min. The mixture is allowed to
stand at room temperature for 18 h for cooling, and finally poured on crushed

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L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
XV-185-14c, omission media, lacking lysine (SC-lys), histidine (SC-his), or
homoserine (SC-hom), were used. For recombinogenesis, leucine was omitted
from the synthetic complete medium (SC-leu) or supplemented with 0.2% (w/v)
cycloheximide (SC + Cyh). Cells were harvested and washed with phosphate
buffer saline solution pH 7.4. Cell titer and percentage of budding cells were
determined by microscopic counting/observation, using a Neubauer chamber.
as from monosomy of chromosome VII. Therefore, cycloheximide-resistant
colonies were replica-plated on a series of plates with SC-lys, SC-met, and
SC-ade media to confirm the recombination origin of cyh2 homozygocity. Irra-
diation at UV 254 nm (30 J/m2 and 60 J/m2) was used as positive control.
Assays were repeated three times, and plates were performed in triplicate for
each dose.
2.8. Treatment of V79 cells and trypan blue exclusion
2.4. Growth and survival assay in S. cerevisiae
V79 cells were maintained in tissue-culture flasks at 37 ◦C in a humidi-
fied atmosphere containing 5% CO2, and were harvested by treatment with
0.15% trypsin and 0.08% of EDTA PBS. Cells (2 × 105 cells) were seeded
into each flask and grown/incubated 1 day prior to treatment. The assays
performed in the presence of metabolic activation used 3% final S9-mix
concentration.
The concentrations used for each sample were based on trypan blue exclu-
sion, which was determined as described by Robichova and Slamenova [31].
Treated and washed, as well as control V79 cells were trypsinized, stained with
trypan blue (0.4%), and the number of viable (uncolored) and dead (colored)
cells was counted. The ratio of number of viable cells/all cells results in the
percentage of viable cells. Dose was considered cytotoxic when cell survival
was <70%.
Stationary phase (STAT) cultures were obtained by inoculation of liquid
YPD bearing a single colony. After 48 h incubation at 30 ◦C with aeration
by shaking, cultures contained 1–2 × 108 cells/mL. Cells were harvested by
centrifugation, and washed with PBS. Cytotoxicity of the compounds was
assayed by suspending 2 × 108 STAT cells/mL in PBS containing different
compound concentrations, and incubation with aeration by rotary shaking at
30 ◦C for 4 h. Cell number was determined by microscopic count and colony
formation on solid SC. Plates were incubated at 30 ◦C for 3–5 days before
counting.
2.5. Salmonella/microsome mutagenic assay
Mutagenicity was assayed by the pre-incubation procedure [26]. S9
metabolic activation mixture (S9 mix) was prepared according to Maron and
Ames [21]. The appropriate concentrations of compounds were obtained by
dilution in DMSO stock solution. One hundred microliters of test bacterial cul-
tures (1–2 × 109 cells/mL) were incubated in the dark at 37 ◦C with different
amounts of naphthoquinones (0.01–20 ␮g per plate) in the presence or absence
of the S9 mix for 20 min, without shaking. Then, 2 mL of soft agar (0.6% agar,
0.5% NaCl, 50 ␮M histidine, 50 ␮M biotin, pH 7.4, 42 ◦C) were added, and the
contents of the test tube were immediately poured onto a plate of minimal agar
(1.5% agar, Vogel-Bonner E medium, containing 2% glucose). Aflatoxin B1
(0.5 ␮g/plate) was used as positive control for all strains in the metabolic assay
with S9 mix. In the absence of the S9 mix, the positive control was 4-NQO
(0.5 ␮g/plate), except in TA 100, where sodium azide (5 ␮g/plate) was used
as positive control. Plates were incubated in the dark at 37 ◦C for 48 h before
revertant colony counting.
2.9. Comet assay
Alkaline Comet assay was performed as described by Singh et al. [32], with
minor modifications [33]. At the end of the treatment, cells were washed with
ice-cold PBS, and trypsinized with 100 ␮L trypsin (0.15%). After 30 s, 100 ␮L
complete medium was added and the cells were gently re-suspended. Imme-
diately thereafter, cells (106 cells/mL) were suspended in 0.75% low melting
agarose (LMA), and spread on agarose-precoated microscope slides. Cells were
lysed (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH 10.0, with freshly added
1% Triton X-100 and 10% DMSO) at 4 ◦C for 24 h. In the modified Comet assay,
slides were removed from the lysing solution, and washed three times in enzyme
buffer (40 mM hepes, 100 mM KCl, 0.5 mM Na2EDTA, 0.2 mg/mL BSA, pH
8.0), drained, and incubated with 70 ␮L FPG (30 min 37 ◦C) or ENDOIII (45 min
37 ◦C). Slides were placed in a horizontal electrophoresis chamber, containing
freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13.0) at 4 ◦C
for 20 min. An electric current of 300 mA and 25 V (0.90 V/cm) was applied for
20 min to allow DNA migration. Slides were neutralized (0.4 M Tris, pH 7.5),
and stained with silver [34]. Images of 100 randomly selected cells (50 cells
from each of two replicate slides) were analyzed per group. Cells were also
visually scored into five classes, according to tail size: (1) class 0: undamaged,
without a tail; (2) class 1: tail shorter than the diameter of the head (nucleus); (3)
class 2: tail length 1 − 2x the diameter of the head; (4) class 3: tail longer than 2x
the diameter of the head; (5) class 4: comets with no heads. Damage frequency
(%) was calculated based on number of cells with tails as a percentage of total
number of scored cells.
International guidelines and recommendations for the Comet assay consider
that visual scoring of comets is a well-validated evaluation method as it is highly
correlated with computer-based image analysis [35,36]. Damage index is based
on migration length and on the amount of DNA in the tail, and it is considered
a sensitive DNA measure. Therefore, a damage index (DI) was assigned to
each comet according to its class, and ranged from 0 (completely undamaged:
100 cells × 0) to 400 (with maximum damage: 100 cells × 4) [37]. Results are
expressed as mean and standard deviation of three independent experiments.
DMSO was used as negative control, whereas MMS and CP were used as positive
controls, without or with metabolic activation, respectively.
2.6. Detection of aminohydroxynaphthoquinone induced reverse
mutation in S. cerevisiae
Mutagenesis was measured in haploid cells of STAT strain XV185-14c, as
described in Section 2.4. A suspension of 2 × 108 STAT cells/mL was incubated
at 30 ◦C for 4 h with different concentrations of naphthoquinones. Cell sur-
vival was determined in SC (3–5 days, 30 ◦C), and mutation induction (LYS,
HIS or HOM revertants) in appropriate omission media (5–7 days, 30 ◦C).
Whereas his 1–7 is a non-suppressible missense allele and reversions result
from mutations at the locus itself [27], lys1-1 is a suppressible ochre non-
sense mutant allele [28], which can be reverted either by locus-specific or by
forward mutation in a suppressor gene [22,29]. True reversions and forward
(suppressor) mutations at lys1-1 locus were differentiated according to Schuller
and von Borstel [30]. It is believed that hom3–10 contains a frameshift muta-
tion due to its response to a range of diagnostic mutagens [22]. Assays were
repeated at last three times, and plating was performed in triplicate for each
dose.
2.7. Detection of induced mitotic recombination in S. cerevisiae
Suspension of STAT diploid XS2316 (2 × 107 cells/mL) cells was incu-
bated at 30 ◦C for 4 h in PBS, which contained different NQ concentrations.
Treated cells were diluted in PBS, plated on three different media (SC, SC-leu
and SC + cyh), and incubated at 30 ◦C for 5–7 days. Colonies grown on SC
medium indicated cell survival and colonies grown on SC-leu and SC + cyh
were scored for intragenic mitotic recombination (mitotic gene conversion) and
intergenic recombination (crossing-over), respectively. The exact frequency of
reciprocal crossing-over was determined by subtracting possible revertants to
cycloheximide-resistance, resulting from reversion at the CYH2 locus, as well
2.10. Statistical analysis
Mutagenicity data were analyzed using Salmonel software [38]. A com-
pound was considered positive for mutagenicity only when: (a) the number
of revertants was at least double the spontaneous yield (MI ≥ 2; mutagenic
index (MI): number of induced colonies in the sample/number of sponta-
neous in negative control), (b) analysis of variance yielded significant response
(P ≤ 0.05) and (c) a reproducible positive dose response (P ≤ 0.01) was present,

L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
143
Table 1
Induction of his+ revertants in Salmonella typhimurium TA100 and TA97 by naphthoquinones, with and without metabolic activation
Substance
Dose (␮g/plate)
TA100
TA100 + S9
Rev/plate
TA97
TA97 + S9
Rev/plate
Rev/platea
MIb
3.38
MI
Rev/plate
MI
MI
NCc
PCd
208
592 62**
6
175 10
362 21**
287 31
751 33**
203 68
509 24**
0.5
2.12
2.62
2.51
1,4-NQ
0.1
0.5
1
240 28
232 16
124 19
97 41
1.15
1.12
0.58
0.47
199 11
193 10
187 11
1.06
1.11
1.07
1.17
362 27
360 30
361 10
267 16
1.26
1.27
1.26
0.93
301 51
358 59
340 41
345 27
1.48
1.76
1.68
1.70
2
204
185 12
175
8
ANQ
1
5
10
20
216
2
1.04
0.74
0.75
0.30
1.06
1.00
1.22
1.55
324 10
388 12
347 26
324 30
1.13
1.35
1.21
1.09
315 63
320 43
314 53
366 104
1.55
1.58
1.54
1.80
154 44
155 34
61 28
9
213 23
271 29
ANQOH
1
5
10
20
263
2
1.27
1.58
1.55
1.77
161 14
218 25
222 22
330 27
0.92
1.25
1.27
1.89
361 33
375 24
335 55
338 63
1.26
1.31
1.17
1.18
280 43
284 51
275 53
284 38
1.38
1.40
1.35
1.40
328 44
323 35
367 28
Data significant in relation to negative control (solvent) at *P < 0.05; **P < 0.01; ***P < 0.001.
a
Number of revertants/plate: mean of three independent experiments S.D.
b
MI mutagenic index: number of his+ induced in the sample/number of spontaneous his+ in the negative control.
Negative control: dimethylsulfoxide.
c
d
Positive control (4NQO—S9mix to TA98 and TA102 and aflatoxin B1 + S9).
as evaluated by Salmonel software. Cytotoxic effect was considered when
MI ≤ 0.6.
Descriptive statistical analysis for mutagenesis assays in S. cerevisiae and
Comet assay was expressed as means and standard deviation from three inde-
pendent experiment data submitted to one-way ANOVA and Tukey’s multiple
comparison tests, using GraphPad Prism version 4.00, GraphPad Software (San
Diego, USA).
3. Results
3.1. Salmonella/microsome assay
When the NQs dose range was evaluated with the TA
100 strain, with and without metabolization, cytotoxicity was
observed in concentrations higher than 3 ␮g/mL for 1,4-NQ
and 30 ␮g/mL for ANQ and ANQOH (data not shown).
No mutagenicity of NQ compounds was observed in TA97a
Table 2
Induction of his+ revertants in S. typhimurium TA102 and TA98 by naphthoquinones, with and without metabolic activation
Substance
Dose (␮g/plate)
TA102
TA102 + S9
Rev/plate
TA98
TA98 + S9
Rev/plate
Rev/platea
MIb
MI
Rev/plate
MI
MI
NCc
PCd
310 31
2910 300***
380
1173 15***
5
32
1
46
4
0.5
9.38.38
3.08
301 35***
9.40
562 31***
12.2
1,4-NQ
0.1
0.5
1
400
421
462 10
429
3
5
1.29
1.35
1.49
1.38
719 70
681 21
516 42
509 53
1.89
1.79
1.36
1.33
27
29
32
25
2
3
8
6
0.85
0.90
1.00
0.78
49
42
44
49
6
1
5
7
1.06
0.91
0.95
1.07
2
5
ANQ
1
5
10
20
287 30
255 20
0.93
0.82
0.94
0.86
838 72*
781 54*
1106 64**
1155 140**
2.21
2.06
2.91
3.04
42
60
59
1
7
4
1.31
1.88
1.84
3.71
47
53
93 14*
2
4
1.02
1.15
2.02
3.11
290
4
268 15
119 20**
144 6**
ANQOH
1
5
10
20
309 27
302 26
281 21
308 39
0.99
0.97
0.91
0.99
891 128**
790 151**
844 88**
886 122**
2.35
2.08
2.22
2.33
34
48
76 3**
3
5
1.06
1.50
2.37
4.96
47
39
48
60
9
3
8
7
1.02
0.84
1.04
1.30
159 4**
Data significant in relation to negative control (solvent) at *P < 0.05; **P < 0.01; ***P < 0.001.
a
Number of revertants/plate: mean of three independent experiments S.D.
b
MI mutagenic index: number of his+ induced in the sample/number of spontaneous his+ in the negative control.
Negative control: dimethylsulfoxide.
c
d
Positive control (4NQO—S9mix to TA98 and TA102 and aflatoxin B1 + S9).

144
L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
Table 3
Naphthoquinone-induced reversion of point mutation for his1–7, frameshift mutation (hom3–10) and ochre allele (lys1–1) in haploid strain XV185-14c of Saccha-
romyces cerevisiae in stationary phase (STAT)
Agent
Treatment (␮g/mL)
Survival (%)
His1/107 survivorsa
Hom3/107 survivorsa
Lys1/107 survivorsb
STAT
DMSOb
4NQOc
0
1
100
86.8
7.73 0.87
275.5 1.36***
1.19 0.06
4.91 2.60**
4.36 0.62
51.42 2.61**
1,4-NQ
1
10
15
30
85.7
80.2
86.5
4.5
7.51 1.76
6.23 2.74
5.85 2.51
8.86 2.04
1.05 0.67
3.05 0.95
1.44 1.01
8.86 2.00**
5.11 1.05
4.82 0.86
3.45 0.45
7.74 0.84
ANQ
1
10
30
60
103.0
92.7
97.9
97.0
4.14 0.92
6.82 2.29
7.03 1.53
8.31 1.93
1.63 0.55
1.45 0.27
2.01 1.17
1.70 0.79
5.03 2.02
4.23 1.55
5.12 0.48
4.12 0.41
ANQ-OH
1
10
30
60
96.4
101.0
103.1
99.1
5.01 0.85
4.38 0.83
4.38 1.35
5.66 2.21
1.45 0.29
1.48 0.65
0.71 0.02
1.16 2.60
5.18 0.61
6.30 0.41
3.66 1.16
5.85 0.54
Data significant in relation to negative control group (solvent) at *P < 0.05; **P < 0.01; ***P < 0.001/one-way ANOVA with Tukey’s post-test.
a
Locus-specific revertants.
Locus non-specific revertants (suppression by forward mutation).
Mean and S.D. per three independent experiments.
b
c
(detects frameshift mutations in C–C–C–C–C–C; +1 cyto-
sine), or in TA100 (base-pair substitution mutation results from
the substitution of a leucine [GAG] by a proline [GGG]),
in absence or presence of metabolic activation. However, at
their highest concentration, 1,4-NQ (1–2 ␮g/plate) and ANQ
(20 ␮g/plate) were toxic to TA100, without metabolic activa-
tion (Table 1). Table 2 shows the absence of mutagenicity
effects of all NQS compounds on TA102, which detects
oxidative and alkylating mutagens [21], in the absence of
metabolic activation. ANQ and ANQ-OH were able to induce
mutagenicity in TA102, in the presence of metabolic activa-
tion. However, ANQ induced mutagenic response in TA98
(detects frameshift in DNA target –C–G–C–G–C–G–C–G), in
the presence and absence of metabolic activation, whereas
for ANQ-OH this effect was only detected in absence of
S9-mix.
3.2. Cytotoxic and mutagenic effects in S. cerevisiae
The results of the cytotoxicity and mutagenicity tests in
the yeast S. cerevisiae are shown in Tables 3 and 4. Cyto-
toxicity of naphthoquinones in yeast STAT cells was more
significant in diploid cells as compared to that observed in hap-
loid cells. This cytotoxic effect was critical in diploid cells,
when 1,4-NQ concentration was higher than 2 ␮g/mL. However,
ANQ and ANQ-OH showed mild cytotoxicity in all treatments
(Tables 3 and 4). None of the naphthoquinones was muta-
genic in strain XV185-14c during treatment in PBS (STAT)
Table 4
Induction of crossing-over (+/cyh2) and gene conversion (leu-1/leu1–12) by naphthoquinones in diploid yeast XS2316 in stationary phase
Substance
Treatment
Survival (%)
Crossing-over/105 survivorsa
Gene conversion/105 survivorsa
NC
UVC (J/m2)c
UVC (J/m2)c
100
77.9
56.3
56 1.00
708 44.0**
1150 32.0***
22 1.07b
691 85.3**
1210 87.1***
30 J
60 J
1,4-
NQ
0.5 ␮g/mL
1 ␮g/mL
2 ␮g/mL
79.1
71.0
2.03
20.2 13.0
42.0 3.43
1180 21***
20.8 8.3
17.1 1.53
50.0 10.0
ANQ
1 ␮g/mL
5 ␮g/mL
10 ␮g/mL
30 ␮g/mL
79.7
85.1
81.2
81.0
30.2 12.5
35.9 10.7
42.8 18.2
71.5 18.4
33.1 1.59
14.4 7.00
10.0 1.00
24.4 12.0
ANQ-
OH
1 ␮g/mL
10 ␮g/mL
30 ␮g/mL
84.3
72.0
68.0
48.4 0.45
33.6 0.19
47.9 1.29
26.8 6.10
27.6 5.40
23.9 2.60
Data significant in relation to negative control group (solvent) at *P < 0.05; **P < 0.01; ***P < 0.001/one-way ANOVA with Tukey’s post-test.
a
Mean and S.D. per three independent experiments.
Negative control.
Positive control.
b
c

L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
145
Table 5
Effect of naphthoquinones in V79 cells exposed for 3 h and evaluated by standard
Comet assay without metabolic activation
Substance Treatment
NCb
Damage indexa
Damage frequency (%)a
66.67 3.51a
4.0 × 10−5 M 318.33 92.1***
51.33 7.37a
100.00 0.0***
MMSc
1,4-NQ
0.01 ␮g/mL
0.1 ␮g/mL
1 ␮g/mL
63.66 26.57
48.33 14.01
76.66 5.03
97.33 4.61***
99.33 1.15***
147.33 40.20**
316.66 75.08***
341.00 70.15***
5 ␮g/mL
ANQ
0.1 ␮g/mL
1 ␮g/mL
5 ␮g/mL
10 ␮g/mL
27.00 4.24
34.00 19.79
145.50 44.54**
325.50 4.95***
21.00 1.41
28.00 16.97
75.50 23.30
100.00 0.0***
ANQ-OH
0.1 ␮g/mL
1 ␮g/mL
5 ␮g/mL
10 ␮g/mL
41.66 25.85
66.67 15.00
80.00 40.28
166.00 36.37**
32.66 18.58
48.00 7.55
49.33 18.14
81.00 9.6
Fig. 2. Cell survival of V79 after exposure to naphthoquinones for 3 h in the
absence of metabolization.
(Table 3), except for locus hom3+ at the highest NQ concen-
tration (P < 0.01).
Data significant in relation to negative control (DMSO: solvent) group at
*P < 0.05; **P < 0.01; ***P < 0.001/one-way Tukey’s multiple comparison test.
a
Mean values and S.D. obtained from average of 100 cells per
experimental—total of three experiments for each substance.
3.3. Recombinogenic effects in S. cerevisiae
b
Negative control.
c
Positive control.
The recombinogenic effect of naphthoquinones was investi-
gated in diploid STAT cells. Table 4 shows that 1,4-NQ induced
crossing-overathighestdose(P < 0.001);however, 1,4-NQcyto-
toxicity was high (2% survival). Aminohydroxynaphthoquinone
did not induce any recombinogenic events in STAT yeast cells.
DNA-repair enzymes ENDOIII and FPG, without metabolic
activation. It can be seen in Fig. 3 that the post-incubation results
with the enzymes clearly shows increased DNA migration of
the positive control H2O2. It shows that the enzyme activity was
appropriated. The results indicate that the extent of oxidative
DNA damage caused by NQs, as recognized by ENDOIII and
FPG in V79 cells, was significantly higher (Fig. 3A). More-
over, a high level of DNA damage was recognized by FPG was
observed at intermediate ANQ doses (Fig. 3B). However, both
3.4. Trypan blue exclusion (TB)
Cytotoxicity of naphthoquinones for the 3-h treatment with-
out the S9 mix in V79 cells is shown in Fig. 2. Significant
cytotoxicity was induced in cells treated with 1,4-NQ at con-
centration of 5 ␮g/mL or higher. ANQ and ANQ-OH did not
show any cytotoxic effect in any concentration.
Table 6
Effect of naphthoquinones in V79 cells exposed for 3 h and evaluated by standard
Comet assay with metabolic activation
3.5. NQ induced DNA damage in V79 cells
Substance
Treatment
Damage indexc
Damage frequency (%)a
NCb
CPc
33.3 8.28
272.5 9.19***
23.0 5.79
87.0 1.10***
Table 5 shows DNA damage induced by compounds tested in
V79 cells, without metabolic activation. Both 1,4-NQ and ANQ
caused pronounced DNA damage in the standard Comet assay. A
concentration-dependent increase in damage index and damage
frequency was observed for both compounds. As compared to
the negative control, concentrations >0.1 ␮g/mL of 1,4-NQ, and
5 ␮g/mL of ANQ induced significant DNA damage. V79 cells
treated with 0.1–5 ␮g/mL ANQ-OH did not show any significant
DNA damage. Increased DNA damage was observed only at the
highest dose (Table 5). In presence of metabolic activation, all
substances caused genotoxic effects in V79 cells (Table 6). The
extent of DNA damage in cells exposed to these compounds was
concentration dependent.
4.5 × 10−5 M
0.01 ␮g/mL
0.1 ␮g/mL
1 ␮g/mL
1,4-NQ
ANQ
63.3 7.45
50.6 1.41
142.6 4.24**
244.3 12.16***
282.0 1.41***
75.5 2.12**
88.3 0.70***
93.7 2.0***
5 ␮g/mL
0.1 ␮g/mL
1 ␮g/mL
5 ␮g/mL
10 ␮g/mL
45.0 0.08
36.3 3.24
93.5 12.02*
223.0 12.72***
268.5 0.70***
62.0 4.55*
92.3 4.95***
90.6 4.24***
ANQ-OH
0.1 ␮g/mL
1 ␮g/mL
5 ␮g/mL
10 ␮g/mL
27.8 5.65
21.3 2.12
150.6 3.53**
189.6 2.82***
212.0 15.86***
77.5 0.70**
84.3 1.41***
88.5 0.7***
Data significant in relation to negative control (DMSO: solvent) group at
*P < 0.05; **P < 0.01; ***P < 0.001/one-way Tukey’s multiple comparison test.
3.6. DNA repair enzymes recognize oxidative DNA damage
a
Mean values and S.D. obtained from average of 100 cells per
experimental—total of three experiments for each substance.
Fig. 3A–C shows mean DNA damage caused by NQs,
expressed as DNA damage index after treatment with
b
Negative control.
c
Positive control.

146
L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
We observed that all NQS at higher concentrations cause oxida-
tiveDNAdamage, asrecognizedbyFPGenzyme(Fig. 4A–C). A
significant level of DNA damage, recognized by ENDOIII, was
only observed for ANQ and ANQ-OH at highest concentrations
(Fig. 4B and C). 1,4-NQ-incubated cells treated with ENDOIII,
in the presence of S9-mix, did not show significant increase in
strandbreaksatalltestedconcentrations(Fig. 4B). Itisimportant
to note that H2O2 was only used as a control for enzyme activity,
and therefore was not included in figure. This positive control
demonstrated significant increase in ENDOIII and FPG sites
as compared to incubation in reaction buffer (data not shown).
Cyclophosphamide (CP) is an alkylating agent widely used as
positive control in tests using metabolic activation [39,40]. The
damage generated by CP is expected to be recognized only by
FPG, as few authors reported that FPG is able to recognize and
to remove alkylating damage as well [18]. We found a signif-
icant difference between buffer level and incubation with FPG
after CP treatment (Fig. 4A–C).
4. Discussion
Quinone cellular toxicity has been extensively studied, and
it is generally accepted to be a function of (a) the capacity
of quinones to produce ROS, and (b) the electrophilicity of
quinones, which enables them to form adducts with cellular
macromolecules [41]. However, there are few studies on amino-
hydroxynaphthoquinone mutagenesis [17]. In the present study,
we investigated the influence of hydroxy and amino substituents
on 1,4-naphthoquinone genotoxicity in prokaryotic and eukary-
otic cell systems.
InSalmonella/microsomeassay, ANQandANQ-OHinduced
mutagenesis, in the presence of the S9-mix, in the strain TA102,
which is sensitive to oxidative mutagens [21]. The reduction of
these compounds by cytochrome P-450 produces a semiquinone
radical, and, in the presence of oxygen, most semiquinone
rapidly auto-oxidize, producing a hydroxyl and a superoxide
anion radical [42]. Both ANQ and ANQ-OH were mutagenic in
TA98 (sensitive to frameshift mutagens), without metabolic acti-
vation, whereas in the presence of S9-mix, only ANQ exhibited
mutagenicity. This confirms results of Tikkanen et al. [16], who
showed that naphthazarin and plumbagin induced frameshift
mutations in strain TA98, with metabolic activation. The only
tested naphthoquinone with an amino substituent – 4-amino-1,2-
naphthoquinone – was mutagenic in strains TA97, TA100, and
TA104 [17].
Chemical compounds bearing planar topologies and elec-
trophilicity are often capable of intercalating between DNA
bases [43]. As aminohydroxynaphthoquinone is an electrophilic
molecule with an aromatic planar structure, inducing frameshift
mutation only in S. typhimurium (where access to DNA is facili-
tated), we suggest that ANQ and ANQ-OH are weak intercalator
mutagens.
Fig. 3. (A) Effect of ENDOIII and FPG post-treatment on 1,4-naphthoquinone
(1,4-NQ), with metabolic activation induced DNA migration in modified Comet
assay. Columns from left to right: solvent; positive control (H2O2 10 ␮M);
NQ: 0.01 ␮g/mL, 0.1 ␮g/mL, 1 ␮g/mL and 5 ␮g/mL. (B) Effect of ENDOIII
and FPG post-treatment on 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ)
with metabolic activation induced DNA migration in modified Comet assay.
Columns from left to right: solvent; positive control (H2O2 10 ␮M); ANQ:
0.1 ␮g/mL, 1 ␮g/mL, 5 ␮g/mL and 10 ␮g/mL. (C) Effect of ENDOIII and
FPGpost-treatmenton5-amino-2,8-dihydroxy-1,4-naphthoquinone(ANQ-OH)
with metabolic activation induced DNA migration in modified Comet assay.
Columns from left to right: solvent; positive control (H2O2 10 ␮M); ANQ-OH:
0.1 ␮g/mL, 1 ␮g/mL, 5 ␮g/mL and 10 ␮g/mL. Mean S.D. of three indepen-
dent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001/one-way ANOVA
Tukey’s multiple comparison test. All asterisks indicate significant data after
enzyme treatment when compared the same concentration in buffer conditions.
enzymes were not able to detect ANQ-OH-induced oxidative
DNA damage in the absence of metabolic activation (Fig. 3C).
Fig. 4A–C shows mean DNA damage caused by NQS
expressed as DNA damage index after treatment with DNA-
repair enzymes ENDOIII and FPG, with metabolic activation.
1,4-NQ did not induce mutagenesis in any of the tested
Salmonella strains, in the presence or absence of metabolic
activation, confirming the results of Sakai et al. [44]. How-
ever, Hakura et al. [17], using a pre-incubation variant of the
Ames-test, observed 1,4-NQ mutagenic activity in TA100, both

L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
147
in the presence and absence of metabolic activation, and in
TA97 and TA102 with metabolic activation. These controver-
sial results may be explained by the fact that 1,4-NQ mutagenic
activity is relatively weak, as compared to its cytotoxicity.
In the yeast S. cerevisiae, 1,4-NQ induced mutagenesis and
recombinogenesis(crossing-over)werealsocoupledwithhigher
cytotoxicity (4.5–2% survival). This higher 1,4-NQ cytotoxic-
ity, observed mainly in yeast diploid cells, could be associated
to a more efficient one-electron reduction to semiquinone by
NADPH–cytochrome P-450 reductase, leading to a better capac-
ity to produce free radicals in the presence of oxygen (10–12).
In addition, it is known that the diploid cells of the yeast S.
cerevisiae have a higher level of cytochrome P-450 than to the
haploid cells [45].
All S. cerevisiae strains were less sensitive to aminohy-
droxynaphthoquinones than to 1,4-NQ, indicating that the
introduction of amino and hydroxy groups decreases toxicity in
bacteria, yeasts, and mammalian cell lines. Lower 1,4-NQ toxi-
city after mono or dimethyl substitution (at positions 2 or 2 and
3, respectively) has been reported [12], and this was attributed to
a lower rate of redox cycling caused by the lower redox potential
imposedbymethylsubstitution, ortoalowerrateofelectrophilic
addition. Consistent with this last assertion, Rodriguez et al. [46]
suggested that 1,4-NQ toxicity depends not only on the rate of
superoxide production, but also on the electrophilicity of the
quinone molecule.
The alkaline SCG assay is a sensitive procedure to quantify
DNA damage in cells, which includes alkali-labile sites (ALS)
and DNA single- and double-strand breaks [35]. The standard
Comet assay showed a significant increase in DNA damage after
1,4-NQ and ANQ treatment, without metabolization (Table 5).
By employing ENDOIII and FPG proteins, usually applied in
the modified Comet assay, we found that 1,4-NQ and ANQ pro-
moted an important additional increase in strand breaks, without
S9-mix, suggesting that these breaks are caused by ROS (Fig. 3A
and B). This observation, along with the fact that this increase
was not observed in all tested doses, suggests that, in addition to
naphthoquinone-induced oxidative DNA damage, other types of
lesions are involved in the genotoxic effects of these compounds.
Indeed, it was shown that NQs are able to produce adducts with
DNA, which may lead to a basic sites and single or double
strand breaks [13,14]. 1,4-NQ-generated oxygen-free radicals
can attack DNA at both pyrimidine and purine bases, and lead
to strand breaks (Fig. 3A). At intermediate concentrations, ANQ
predominantly induced damage in purines (Fig. 3B). Based on
these results, we suggest that amino and hydroxy groups (at posi-
tions C8 and C5, respectively) facilitate DNA oxidative damage
by free radicals, such as superoxide radical (O2•−) and hydro-
gen peroxide (H2O2). H2O2 is not toxic by itself, but its high
in vivo reactivity, through the Fenton reaction, where it reacts
with partially reduced metal ions, generates hydroxyl radical
(•OH), the most important radical in ROS-related DNA damage
[47]. These radicals cause a variety of base lesions in DNA. In
the case of ANQ, the lesion produced is probably 8-oxo-Gua,
the preferred substrate for FPG [48]. Interestingly, ANQ-OH
showed a relevant genotoxic effect in the standard Comet assay
at the highest dose, but it had no significant genotoxic effects
Fig. 4. (A) Effect of ENDOIII and FPG post-treatment on 1,4-naphthoquinone
(1,4-NQ), with metabolic activation induced DNA migration in modified
Comet assay. Columns from left to right: solvent; positive control (cyclophos-
phamide 6.0 × 10−5 M); NQ: 0.01 ␮g/mL, 0.1 ␮g/mL, 1 ␮g/mL and 5 ␮g/mL.
(B) Effect of ENDOIII and FPG post-treatment on 5-amino-8-hydroxy-1,4-
naphthoquinone (ANQ), with metabolic activation induced DNA migration in
modified Comet assay. Columns from left to right: solvent; positive control
(cyclophosphamide 6.0 × 10−5 M); ANQ: 0.1 ␮g/mL, 1 ␮g/mL, 5 ␮g/mL and
10 ␮g/mL. (C) Effect of ENDOIII and FPG post-treatment on 5-amino-2,8-
dihydroxy-1,4-naphthoquinone (ANQ-OH) with metabolic activation induced
DNA migration in modified Comet assay. Columns from left to right: sol-
vent; positive control (cyclophosphamide 6.0 × 10−5 M); ANQ-OH: 0.1 ␮g/mL,
1 ␮g/mL, 5 ␮g/mL and 10 ␮g/mL. Mean S.D. of three independent exper-
iments. *P < 0.05, **P < 0.01, and ***P < 0.001/one-way ANOVA Tukey’s
multiple comparison test. All asterisks indicate significant data after enzyme
treatment when compared the same concentration in buffer conditions.

148
L.F. da Costa Medina et al. / Mutation Research 650 (2008) 140–149
in the modified Comet assay, without S9 metabolic activation
(Table 5 and Fig. 3C), indicating that the introduction of one
additional hydroxy group at the C2 position does not produce a
genotoxic molecule.
[4] R.M. Phillips, M. Jaffar, D.J. Maitland, P.M. Loadman, S.D. Shny-
der, G. Steans, P.A. Copper, A. Race, A.V. Patterson, I.J. Stratford,
Pharmacological and biological evaluation of a series of substituted
1,4-naphthoquinone bioreductive drugs, Biochem. Pharmacol. 68 (2004)
2107–2116.
However, all NQs with metabolic activation presented impor-
tant genotoxic potential in V79 cells (Table 6), and, as indicated
by the presence of FPG sensitive sites, all tested compounds
at higher doses can predominantly damage purines, which is
suggestive of their ability to produce ROS (Fig. 4A–C).
Therefore, naphthoquinone capacity to produce free radicals
is strongly influenced by the substituents present in the quinoid
molecule and by its reduction. The compound 1,4-NQ is able
to generate free radicals, reduced or not to semiquinone. Nev-
ertheless, ANQ, and particularly ANQ-OH, must be reduced to
semiquinone intermediates and enter in a process called redox
cycling, generating superoxide anion, hydrogen peroxide, and
hydroxyl radical, in order to cause oxidative stress [12]. We
suggest that the presence of the hydroxy group in position C2
can stabilize the quinoid form, and modify the one-electron
reduction of ANQ-OH, which can be associated a lesser effi-
cient one-electron reduction. A similar effect was described
for 2-hydroxy-1,4-naphthoquinone [12]. Furthermore, Roberg
et al. [49] showed that NTZ induced oxidative stress in human
foreskin fibroblasts, which caused lysosomal membrane desta-
bilization, releasing proteases and causing loss of membrane
potential in mitochondria, resulting in cell death. Considering
their structural similarities with NTZ, we suggest similar effects
in 1,4-NQ and ANQ-treated V79 cells.
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gens in prokaryotic cells, whereas in mammalian cells, all NQs
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The authorsthank Dr. MartinBrendel, Dr. Diego Bonatto, and
MSc Renato Moreira Rosa for critically reading the manuscript.
This work was supported by grants from the Brazilian Agencies
´
Conselho Nacional de Desenvolvimento Cientıfico e Tec-
´
˜
nologico (CNPq), Fundac¸ao de Amparo a Pesquisa do Estado
do Rio Grande do Sul (FAPERGS) and GENOTOX—Instituto
Royal - Centro de Biotecnologia - UFRGS.
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