Mutation Research 665 (2009) 51–60
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis
journal homepage: www.elsevier.com/locate/molmut
Community address: www.elsevier.com/locate/mutres
Differences in DNA damage and repair produced by systemic, hepatocarcinogenic
and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor
Zuzana Valoviˇ cováa, Soˇ na Marvanováb, Monika Mészárosováa, Annamária Sranˇ cíkováa,
Lenka Trilecováb, Alena Milcovác, Helena Líbalovác, Jan Vondráˇ cekb,d, Miroslav Machalab,
Jan Topinkac, Alena Gábelováa,∗
aLaboratory of Mutagenesis and Carcinogenesis, Cancer Reserach Institute, SAS, Vlárska 7, 833 91 Bratislava, Slovakia
bDepartment of Chemistry and Toxicology, Veterinary Research Institute, 621 00 Brno, Czech Republic
cLaboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, v.v.i. 142 20 Prague, Czech Republic
dLaboratory of Cytokinetics, Institute of Biophysics, AS CR, 612 65 Brno, Czech Republic
a r t i c l ei n f o
Received 29 December 2008
Received in revised form 17 February 2009
Accepted 28 February 2009
Available online 13 March 2009
DNA strand breaks
Kinetics of DNA repair
a b s t r a c t
Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9-
dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole
(N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, an
in vitro model of liver oval cells [J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z.
Andrysik, K. Chramostova, S. Marvanova, Z. Valovicova, A. Kozubik, A. Gabelova, M. Machala, 7H-
Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing
to tumor promotion in rat liver epithelial ‘stem-like’ cells, Mutat. Res. Fundam. Mol. Mech. Mutagen.
596 (2006) 43–56]. In this study, we focused on the genotoxic effects generated by these dibenzo-
carbazoles in WB-F344 cells to better understand the cellular and molecular mechanisms involved
in hepatocarcinogenesis. Lower IC50 values determined for DBC and DiMeDBC, as compared with
N-MeDBC, indicated a higher sensitivity of WB-F344 cells towards hepatocarcinogens. Accordingly,
DBC produced a dose-dependent DNA-adduct formation resulting in substantial inhibition of DNA
replication and transcription. In contrast, DNA-adduct number detected in DiMeDBC-exposed cells
was almost negligible, whereas N-MeDBC produced a low level of DNA adducts. Although all diben-
zocarbazoles significantly increased the level of strand breaks (p<0.05) and micronuclei (p<0.001)
after 2-h treatment, differences in the kinetics of strand break rejoining were found. The strand break
level in DiMeDBC- and N-MeDBC-exposed cells returned to near the background level within 24h after
treatment, whereas a relatively high DNA damage level was detected in DBC-treated cells up to 48h
after exposure. Additional breaks detected after incubation of DiMeDBC-exposed WB-F344 cells with a
repair-specific endonuclease, along with a nearly 3-fold higher level of reactive oxygen species found in
these cells as compared with control, suggest a possible role of oxidative stress in DiMeDBC genotoxicity.
We demonstrated qualitative differences in the DNA damage profiles produced by hepatocarcinogens
DBC and DiMeDBC in WB-F344 cells. Different lesions may trigger distinct cellular pathways involved in
hepatocarcinogenesis. The low amount of DNA damage, together with an efficient repair, may explain
the lack of hepatocarcinogenicity of N-MeDBC.
© 2009 Elsevier B.V. All rights reserved.
formamidopyrimidine-DNA glycosylase/AP endonuclease; ?H2AX, histone H2AX
phosphorylated on Ser139; MI, mitotic index; MNi, micronuclei; MTT, methyl
thiazolyl blue tetrazolium bromide; N-MeDBC, N-methyldibenzo[c,g]carbazole;
ROS, reactive oxygen species; SCGE, single-cell gel electrophoresis.
E-mail address: firstname.lastname@example.org (A. Gábelová).
7H-Dibenzo[c,g]carbazole (DBC) (Fig. 1), a potential human
carcinogen (Group 2B) [1,2], is a ubiquitous environmental pol-
lutant. This agent has been found in various complex mixtures
of organic compounds resulting from incomplete combustion of
synthetic fuel material , coal and oil processing , and tobacco
smoke . DBC has been shown to be a potent multi-species and
0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
Fig. 1. Chemical structure of 7H-dibenzo[c,g]carbazole.
multi-site carcinogen, with local and systemic effects (reviewed
in ). DBC carcinogenicity was demonstrated in different species
(mouse, rat, hamster, and dog) and tissues (skin, lung, forestomach,
and urinary bladder), with the liver being the prime target organ
after DBC administration via different routes. In addition to hepa-
tocellular adenoma and carcinoma, severe hepatocellular toxicity,
DBC-treated mice [9–11].
dimethyldibenzo[c,g]carbazole (DiMeDBC), is one of the most
efficient strictly organ-specific carcinogens. This derivative is
devoid of activity in the skin, but produces multiple malignant
liver tumors with lung metastases in 100% of animals after sub-
cutaneous injection or skin application [10,12,13]. In contrast to
DBC, sex dimorphism in response to DiMeDBC treatment was
observed in mice; males were much less sensitive than females
to this derivative . The strict hepatocarcinogen DiMeDBC is
remarkably less hepatotoxic than DBC; no histologically detectable
toxicity has been found in the liver at lower (10mg/kg) concentra-
tion . At high concentration (90mg/kg), an early hepatocellular
degeneration/necrosis was observed, mostly in centrilobular areas
In the liver, DBC and DiMeDBC produce tumors, DNA adducts
induces fewer mutations and DNA adducts than the parent com-
pound , which is probably due to a reduced access to the
NH group for drug metabolizing enzymes, caused by a steric hin-
drance of two methyl groups at the C5 and C9 positions .
The heterocyclic nitrogen is supposed to have an important role
in liver carcinogenicity and strongly affects the biological activity
of DBC. Substitution of methyl groups at the C6 and C8 posi-
tions or directly at the heterocyclic nitrogen resulted in the loss
of activity in the liver [16,18]. The synthetic methyl derivative N-
methyldibenzo[c,g]carbazole (N-MeDBC) induces sarcomas, respi-
tial [19,20]. Analogs of DBC bearing sulphur, oxygen or carbon in
Cell lines established from a particular tissue are a valuable tool
for better understanding of cellular and molecular mechanisms
which may underlie the tissue specificity of chemical carcinogens.
The diploid rat epithelial cells WB-F344 are considered to be an in
vitro model of liver oval cells . The latter can proliferate when
regenerative hepatocyte proliferation is compromised, and can dif-
ferentiate into hepatocytes  and biliary epithelial cells .
There is increased evidence that the small epithelial oval-shaped
(hepatic progenitor) cells are, in addition to hepatocytes, a possible
target cell population for hepatocarcinogenesis [25,26]. These cells
have been observed in various models of rodent experimental car-
cinogenesis and human liver diseases associated with an increased
incidence of hepatocellular carcinoma or cholangiocarcinoma .
Experiments with polycyclic aromatic and heterocyclic aromatic
hydrocarbons have shown that the WB-F344 cell line is a useful
tool for analysis of cellular and molecular mechanisms involved in
toxic effects of environmental carcinogens [28–31].
In our recent study, the liver carcinogens DBC and DiMeDBC
significantly affected cellular pathways associated with tumor pro-
motion in WB-F344 cells . DBC was an efficient inhibitor of
gap-junctional intercellular communication (GJIC), and DiMeDBC
manifested the strongest aryl hydrocarbon receptor (AhR)-
mediated activity. The tissue-specific sarcomagen N-MeDBC failed
to substantially affect the cellular events associated with tumor
promotion. DBC and DiMeDBC, in contrast to N-MeDBC, induced
The differences in cell response to DBC and DiMeDBC treatment
suggested that these liver carcinogens may produce qualitatively
(or at least quantitatively) different DNA lesions in WB-F344 cells.
Because the genotoxic potential of DBC, DiMeDBC and N-MeDBC
has not been studied in liver progenitor cells or in their mod-
els such as WB-F344 cells, the primary objective of this work
was to assess the DNA damage profile generated by individual
dibenzocarbazoles in this liver “stem-like” cell line. In addition
to clastogenic effects (DNA adducts, DNA breakage, micronuclei),
cellular responses (DNA repair kinetics, induction of oxidative
stress, histone H2AX phosphorylation) were investigated to obtain
a more comprehensive picture of the genotoxic effects generated
by the tissue-specific dibenzocarbazole derivatives in the WB-F344
2. Materials and methods
DBC (CAS No. 194-59-2), DiMeDBC and MeDBC were kindly provided by Dr.
Francois Périn (Institute Curie, France). Benzo[a]pyrene (B[a]P, CAS No. 50-32-8),
methyl thiazolyl blue tetrazolium bromide (MTT, CAS No. 298-93-1), and 4?,6-
diamidino-2-phenylindole (DAPI, CAS No. 28718-90-30), anti-?-actin, Clone AC-15,
anti-Mouse IgG, spleen phosphodiesterase, RNases A and T1, proteinase K, micro-
coccal nuclease and nuclease P1 were purchased from Sigma–Aldrich (Deisenhofen,
Germany). T4 polynucleotide kinase was from USB (Cleveland, OH, USA); ?-32P-
ATP (3000Ci/mmol, 10?Ci/?L) from GE Healthcare (Little Chalfont, UK); and
0.1mm polyethylene-imine cellulose thin-layer chromatography (TLC) plates from
NY, USA), peroxidase-conjugated swine antirabbit immunoglobulin antisera was
from Sevapharma (Prague, Czech Republic), ECL Plus reagent was from GE Health-
care (Little Chalfont, UK), and aflatoxin B1 (AFB1, CAS No. 1162-65-8) from Serva
(BioTech, Slovakia). Stock solutions of DBC, MeDBC, DiMeDBC, B[a]P and AFB1 in
dimethyl sulfoxide (DMSO; 2mM) were kept at −20◦C and diluted immediately
before use in DMSO. Media, fetal calf serum (FCS) and other chemicals used for cell
cultivation were purchased from Gibco (KRD Limited, Slovakia). All other chemicals
and solvents were of high-performance liquid chromatography (HPLC) or analytical
2.2. Cell culture
WB-F344 cells were kindly provided by Dr. J. E. Trosko (MSU, East Lansing, MI,
USA). They were cultivated in modified Eagle’s minimum essential medium (MEM)
with 50% increased concentrations of essential and non-essential amino acids, and
supplemented with sodium pyruvate (110mg/L), 10mM HEPES, 10% FCS and antibi-
otics (penicillin 200U/mL, streptomycin and kanamycin 100?g/mL). Cells were
cultured in a humidified atmosphere of 5% CO2at 37◦C.
2.3. Treatment of cells
Exponentially growing WB-F344 cells were exposed to carcinogens for 2h and
MeDBC, DiMeDBC, B[a]P: 0.1–100?M, and AFB1 0.001–1?M). The final concentra-
tion of vehicle (DMSO) never exceeded 0.5% (v/v) in any of the samples; control
cells (negative control) were therefore exposed to 0.5% DMSO. At treatment end,
cells were washed twice with culture medium and then processed immediately, or
incubated in fresh medium for different time intervals, and then processed.
2.4. MTT assay
Viability determination was carried out in plastic 96-well cell culture cluster
plates at 5×103cells/well and photometric evaluation (at 540nm excitation and
690nm emission wavelengths) using the Multiskan Multisoft plate reader (Labsys-
tems, Finland) and Genesis software provided by the producer. IC50 values were
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
calculated from the dose–response curves using CalcuSyn software (Biosoft, Cam-
2.5. DNA isolation and32P-postlabeling
DNA was isolated using RNases A and T1 and proteinase K treatment fol-
lowed by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation
. DNA concentrations were estimated spectrophotometrically by measuring UV
absorbance at 260nm. DNA samples were stored at −80◦C until analysis.
32P-Postlabeling analyses were done as previously described . Briefly, DNA
samples (exact amount of DNA was 6?g) were digested by a mixture of micrococcal
nuclease and spleen phosphodiesterase for 4h at 37◦C. The nuclease P1 proce-
dure was used for adduct enrichment. Adducted nucleotides were enzymatically
labelled using ?-32P-ATP and T4 polynucleotide kinase, and separated by multidi-
used were: D1, 1M sodium phosphate, pH 6.8; D2, 3.54M lithium formate, 8.5M
urea, pH 3.5; D3, 0.8M lithium chloride, 0.5M Tris, 8.5M urea, pH 8.0; D4=D1,
same direction as D3. After screen-enhanced autoradiography at −80◦C, the distinct
DNA adduct spots were cut out and evaluated by measuring32P-radioactivity using
ple, aliquots of the enzymatic DNA digest were analyzed for nucleotide content by
reverse-phase HPLC with UV detection, which simultaneously allowed for control-
ling DNA purity. DNA adduct levels were expressed as adducts per 108nucleotides.
A BPDE-derived DNA adduct standard derived from the liver of rats treated with
benzo[a]pyrene (100mg, i.p.) was run in triplicate to control inter-assay variability
and to normalize calculated DNA adduct levels. Data are mean values of total DNA
adducts derived from at least three biological replicates.
2.6. Cumulative synthesis of macromolecules
Exponentially growing WB-F344 cells were exposed to dibenzocarbazoles
(1–10?M) or positive controls for 2h. After treatment, fresh medium con-
taining [14C]thymidine (1?Ci/mL) or [14C]uridine (1?Ci/mL) or [14C]–L-leucine
(0.2?M/mL) was added to all dishes. At different time intervals (30min, 60min,
90min, 120min, 180min, and 300min), cells were rinsed with SSC buffer (0.15M
sodium chloride, 0.015M sodium citrate) and ice-cold 5% trichloroacetic acid (TCA)
was added. The next day, cells were harvested and filtered through a membrane
(0.45?m pore), washed and dried. Radioactivity was measured on a liquid scintil-
lation counter Beckman LS 1801.
2.7. Single-cell gel electrophoresis (SCGE)
The procedure of Singh et al. , modified by Collins et al.  and Gabelova
et al.  was followed. In brief, liver cells embedded in 0.75% LMP agarose and
spread on a base layer of 1% NMP agarose in PBS buffer (Ca2+and Mg2+free)
were placed in a lysis solution (2.5M NaCl, 100mM Na2EDTA, 10mM Tris–HCl,
pH 10 and 1% Triton X-100) at 4◦C for 1h. In experiments focused on detection
of oxidative DNA damage, slides were washed three times for 5min in endonuclease
buffer (40mM HEPES–KOH, 0.1M KCl, 0.5mM EDTA, pH 8.0) and incubated with
formamidopyrimidine–DNA glycosylase/AP nuclease (Fpg; 30min) at 37◦C. Slides
were transferred to an electrophoretic box and immersed in an alkaline solution
(300mM NaOH, 1mM Na2EDTA, pH>13). After 40min unwinding time, a voltage of
25V (300mA) was applied for 30min at 4◦C. Slides were neutralized with 3× 5min
washes with Tris–HCl (0.4M, pH 7.4), and stained with 20?L of ethidium bromide
(EtBr, 10?g/mL). EtBr-stained nucleoides were examined with an Olympus BX51
fluorescence microscope by image analysis using Komet 5.0 (Kinetic Imaging, Ltd.,
Liverpool, UK) software. The percentage of DNA in the tail (% tail DNA) was used as
a parameter for measurement of DNA damage (DNA strand breaks). One hundred
comets were scored per each sample in one electrophoretic run.
2.8. Detection of reactive oxygen species (ROS)
Confluent WB-F344 cells were exposed to test compounds for 24h. Hydro-
gen peroxide (exposure, 5min) was the positive control. After exposure, cells
were washed twice with PBS, trypsinized, centrifuged, and resuspended with
Hank’s balanced salt solution with 5% heat-inactivated fetal bovine serum
(FBS). The cell suspension was incubated for 15min with the fluorescent probe
2?,7?-dichlorofluorescein diacetate (DCFH-DA, 20?M). Cells were washed again,
centrifuged, and cooled on ice (except hydrogen peroxide-exposed cells). The flu-
orescence of dichlorofluorescein (DCF) was analyzed on FACSCalibur (at 505nm
(Becton Dickinson, San Jose, CA, USA).
2.9. Micronucleus assay
Two sampling time intervals, 24h and 48h after treatment, were used to deter-
mine the number of micronuclei (MNi) induced by individual dibenzocarbazole
derivatives after cell exposure for 2h. WB-F344 cells were washed with 0.9% NaCl,
incubated in mild hypotonic solution (0.075M KCl/0.9% NaCl, 1:19) for 10min at
37◦C, fixed with methanol–glacial acetic acid (3:1) for 15min at 37◦C, rinsed with
The IC50values of DBC, DiMeDBC, N-MeDBC, B[a]P and AFB1 after 2h and 24h cell
treatment. IC50values were calculated using the CalcuSyn software.
Cell exposure (h) 2h [?M]24h [?M]
in the dark at room temperature, rinsed with McIlvaine’s buffer and distilled water,
and dried and mounted with glycerol. MNi were identified based on the criteria
specified by Miller et al. . Proliferation status (mitotic index, MI) of WB-F344
cells was measured according to Eckl and Raffelsberger ; cell death (apoptosis
after Oberhammer et al. . Two thousand cells per dish were analyzed using the
fluorescence microscope Olympus BX51. Data are mean±S.D. of at least two parallel
dishes per one experiment from three independent experiments.
2.10. Western blotting
Confluent WB-F344 cells were exposed for 24h to test compounds or to 0.1%
DMSO as vehicle control. The effects of test compounds on histone H2AX phos-
phorylation were determined in whole-cell lysates prepared by harvesting cells in
cocktail). Total protein concentrations were determined with DC Protein Assay (Bio-
Rad, Hercules, CA, USA). For Western blot analyses, equal amounts of total protein
were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS PAGE), electrotransferred onto polyvinylidene fluoride membrane Hybond-P
(GE Healthcare), immunodetected using appropriate primary and secondary anti-
bodies, and visualized by ECL Plus reagent according to manufacturer’s instructions.
form. The fold of ?H2AX expression was estimated semi-quantitatively as the ratio
of induced to control level of band density.
Data are given as means values ±S.D. The differences between treated sam-
ples and untreated control were evaluated by the Student’s t-test. The threshold of
statistical significance was set at p<0.05.
3.1. Cytotoxicity of DBC and its derivatives in the rat liver
progenitor WB-F344 cells
Cell viability after exposure to dibenzocarbazoles and the pos-
itive controls B[a]P and AFB1 was measured after short-term (2h)
ing from 5?M to 100?M were used for dibenzocarbazoles and
B[a]P, whereas 0.05–10?M were chosen for AFB1. The param-
eter IC50was applied to compare the cytotoxicity of chemicals
DiMeDBC and B[a]P for 24h.
CompoundsConc. [?M]DNA adducts/108nucleotides
DBC129.6 ± 10.6
56.3 ± 10.4
0.2 ± 0.4
0.5 ± 0.3
0.8 ± 0.4
3.0 ± 1.8
27.6 ± 1.5
The mean±S.D. from at least two independent experiments on each triplicate of
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
Fig. 2. Representative autoradiograms of32P-labeled DNA isolated from rat liver epithelial WB-F344 cells incubated with vehicle (0.5% DMSO) (A), 1?M B[a]P (B), 1?M and
10?M DBC (C and D), 1?M and 10?M DiMeDBC (E and F), and 1?M and 10?M N-MeDBC (G and H). Chromatographic conditions (see Section 2) were the same for all
compounds. Film exposure was as followed: control, B[a]P and DBC 24h, DiMeDBC and N-MeDBC 72h.
under study. Substantial differences in IC50values between hep-
atocarcinogenic and sarcomagenic carcinogens have been assessed
(Table 1). Considerable lower IC50 values were determined for
the liver carcinogens DBC and DiMeDBC, as well as the posi-
tive control AFB1 (IC50: 48?M, 30?M, and 2.2?M, respectively)
compared with B[a]P and tissue-specific sarcomagen N-MeDBC
(IC50>100?M for both agents) after 2h exposure. Although long-
term treatment (24h) increased the cytotoxicity of all carcinogens
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
Fig. 3. The course of synthesis of DNA (A), RNA (B) and proteins (C) in WB-F344 cells after treatment with DBC. Cells were exposed to DBC at 1–10?M for 2h. The intensity
of synthesis of macromolecules was analyzed at several time intervals after treatment. Each point represents the mean from at least two independent experiments.
under study, N-MeDBC was less cytotoxic (IC50>50?M) even after
24h treatment, as compared with DBC and DiMeDBC (IC50: 11?M
and 9?M, respectively). These preliminary experiments suggested
a higher sensitivity of the liver progenitor cells towards both hepa-
tocarcinogens, DBC and DiMeDBC.
3.2. Formation of DNA adducts by DBC and its derivatives
DNA adducts resulting from covalent binding of chemicals to
DNA are a critical event in the initiation of cancer . Therefore
Based on our previous study , long-term (24h) cell treatment
was applied to assess the level of stable DNA adducts in exposed
cells. Representative autoradiograms of DNA adducts formed in
WB-F344 cells treated with individual compounds under study are
determined among individual dibenzocarbazoles. The DBC “finger-
print” was difficult to analyze due to close marked spots (Fig. 2C
and D), while only two weak (but distinct) spots were found in cells
exposed to the organ-specific hepatocarcinogen DiMeDBC (Fig. 2E
matograms (Fig. 2G and H). B[a]P, the positive control, produced
one dominant spot representing a well-known BPDE-DNA adduct
(Fig. 2B) and no DNA adducts were found in control cells (Fig. 2A).
At equimolar (1?M) concentrations, DBC induced a comparable
levels of DNA adducts as the reference compound B[a]P, whereas
the DNA-adduct level generated by DiMeDBC at 1?M and 10?M
was very low, close to the detection limit (Table 2). The DNA adduct
than that of DBC. To elucidate and verify the inability of DiMeDBC
to produce DNA adducts in WB-F344 cells, a time-course study
was undertaken. DiMeDBC-treated cells were harvested at 2h time
intervals during exposure for 24h, and DNA adducts measured by
32P-postlabeling. No DiMeDBC-related adducts were found at early
time points, suggesting that the lack of DNA adducts in DiMeDBC-
during 24-h treatment (data not shown). Because the cytotoxic
effects of DiMeDBC were comparable with those of DBC, further
experiments were undertaken to clarify the mechanism(s) which
may underlie the toxic activity of DiMeDBC in WB-F344 cells.
3.3. The effect of DBC and its derivatives on the replication and
transcription of DNA and proteosynthesis
the short-term (2h) treatment interval was chosen to evaluate the
genotoxic effects of dibenzocarbazoles induced in WB-F344 cells.
The effect of dibenzocarbazoles on replication (DNA synthesis),
transcription (RNA synthesis) and proteosynthesis was assessed
at concentrations 1–10?M after 2h cell exposure. DBC caused a
substantial dose-dependent delay of DNA and mainly RNA synthe-
ses during 5h post-cultivation of WB-F344 cells in fresh medium
(Fig. 3A and B), while proteosynthesis was less affected (Fig. 3C).
In contrast, neither DiMeDBC nor N-MeDBC, as well as the posi-
tive controls B[a]P and AFB1, significantly influenced the course of
synthesis of macromolecules under identical treatment conditions
(data not shown).
3.4. DNA strand breaks and the kinetics of DNA repair
DNA strand breaks are readily detected as the cellular response
to exposure; therefore DNA strand break formation has been pro-
posed to be a standard biomarker of DNA damage. The capacity of
dibenzocarbazoles to generate DNA breaks was measured within
the concentration range 0.1–20?M (Fig. 4). All dibenzocarbazoles,
regardless of their tissue specificities, induced significant levels of
strand breaks in exposed cells (p<0.05 to p<0.001). Although DBC
appeared to be the most efficient producer of DNA strand breaks,
in the kinetics of DNA strand break rejoining were determined in
cells exposed to dibenzocarbazoles (Fig. 5). Relatively fast removal
of strand breaks was observed in DiMeDBC-treated cells; the level
of strand breaks reached the steady-state level within 16h after
treatment. A significant delay in DNA strand break rejoining was
was observed even 48h after treatment. The level of breaks pro-
duced by N-MeDBC reached the background level within 24h after
Fig. 4. Detection of DNA damage in WB-F344 cells exposed to DBC, DiMeDBC and
N-MeDBC for 2h by the alkaline SCGE. Cells were treated with equimolar concentra-
tions 0.1–20?M and DNA breakage was analyzed immediately after treatment. The
bars represent the mean±S.D. from three independent experiments on each tripli-
cate of slides. The data were analyzed statistically by Student’s t-test. Significantly
different from the control, *p<0.05; **p<0.01; ***p<0.001.
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
The frequency of micronuclei induced by DBC, N-MeDBC, DiMeDBC, AFB1 and B[a]P in WB-F344 cells after 2h treatment and the mitotic index of exposed cells determined
24h and 48h after cell exposure.
Compound Conc. [?M] Micronuclei (%)Mitotic index (%)
Control0 2.7 ± 0.3
6.3 ± 1.8***
8.0 ± 1.5***
8.4 ± 1.1***
6.6 ± 1.4***
6.2 ± 1.1***
6.9 ± 1.7***
6.1 ± 1.2***
7.7 ± 1.1***
6.5 ± 1.3***
10 ± 0.9***
10.7 ± 1.3***
13.1 ± 0.9***
4.4 ± 0.7***
5.0 ± 1.0***
5.3 ± 0.5***
3.8 ± 0.7
8.2 ± 1.1***
8.8 ± 0.9***
11.0 ± 1.1***
8.3 ± 1.4***
8.8 ± 1.0***
8.5 ± 1.5***
7.3 ± 2.0**
9.3 ± 1.7***
8.2 ± 1.4***
9.3 ± 1.1***
9.2 ± 1.3***
11.3 ± 1.9***
6.0 ± 0.8***
6.0 ± 0.6***
6.6 ± 0.5***
4.1 ± 1.4
4.1 ± 1.4
5.8 ± 1.5
5.4 ± 1.8
6.4 ± 1.6
5.6 ± 0.2
6.2 ± 2.2
6.0 ± 2.0
6.9 ± 1.5
5.6 ± 1.4
7.0 ± 0.8
6.4 ± 1.4
6.0 ± 2.0
5.1 ± 1.3
4.8 ± 1.1
5.3 ± 1.7
4.4 ± 1.0
6.1 ± 2.0
7.0 ± 2.0
7.7 ± 1.3
5.9 ± 1.1
8.0 ± 1.0
7.0 ± 2.0
5.7 ± 1.8
7.9 ± 2.3
6.6 ± 1.2
4.2 ± 1.9
5.1 ± 1.7
5.4 ± 1.3
4.5 ± 1.16
4.9 ± 2.2
5.9 ± 1.6
Data represent the mean±S.D.
**Significantly different from control group at p<0.01.
***Significantly different from control group at p<0.001.
3.5. Clastogenic activity of DBC and its derivatives
Micronucleus formation has become an important endpoint in
genotoxicity studies because a positive correlation exists between
carcinogenicity and clastogenicity of chemical agents . The
clastogenic activities of dibenzocarbazoles and the reference com-
pound B[a]P were assessed in concentration range 0.5–2.5?M,
whereas concentrations 0.1–1?M were used for AFB1. WB-F344
cells were exposed to dibenzocarbazoles and positive controls for
2h. The frequency of MNi and MI were assessed in WB-F344 cells
at 24h and 48h after treatment (Table 3). In agreement with
experiments focusing on the strand break formation, all diben-
zocarbazoles induced a significant level of MNi compared with
control cells. A dose-dependent increase of MNi was detected in
DBC-treated cells at both sampling times (r=0.802 and 0.880,
respectively), but the rise in MNi generated by the strict hepato-
carcinogen DiMeDBC and the tissue-specific sarcomagenN-MeDBC
were less significant (DiMeDBC: r=0.687 and 0.624; N-MeDBC:
r=0.576 and 0.629, respectively). B[a]P, the positive control, was
the most potent inducer of MNi in WB-F344 cells. Under these
treatment conditions, no substantial variation in the frequency of
Fig. 5. The kinetics of DNA strand break rejoining in WB-F344 cells exposed to
equimolar (1?M) DBC, DiMeDBC and N-MeDBC for 2h. DNA strand break level
was measured at several time intervals after treatment. The columns represent the
were analyzed by Student’s t-test. Significantly different from the control, *p<0.05;
apoptotic and necrotic cells was determined in WB-F344 cells due
to exposure to chemicals under study (data not shown). A trend
towards increased MI that did not reach a statistical significance
was found in exposed cells (Table 3).
3.6. Oxidative DNA damage and oxidative stress
It is generally accepted that accumulation of oxidative dam-
age to DNA is implicated in various human diseases, including
cancer and aging . In chemical carcinogenesis, highly redox-
active molecules produced during biotransformation of organic
compounds can contribute to oxidative stress generation result-
ing in oxidative damage to DNA. To evaluate the mechanism by
which the strict hepatocarcinogen DiMeDBC could mediate geno-
toxic effects (strand breaks, MNi) in WB-F344 cells, the modified
SCGE assay was utilized . This modification involves incuba-
(FAPY-adducts) and oxidized (8-oxodG) purines. A slight but sta-
tistically significant level of Fpg-sensitive sites was determined in
DiMeDBC-treated cells (Fig. 6A). Neither DBC nor N-MeDBC pro-
duced a significant level of base modifications in exposed cells. The
kinetics of Fpg-sensitive sites removal produced by DiMeDBC was
relatively slow; a detectable level of DNA breaks was determined
up to 14h after treatment (Fig. 6B). To verify the capacity of diben-
zocarbazoles to induce oxidative stress and generate ROS which
may cause oxidative DNA lesions, flow cytometric analysis of ROS
production was carried out (see Section 2). WB-F344 cells were
exposed to dibenzocarbazoles for 24h; hydrogen peroxide (5min,
on ice) was the positive control in these experiments. At equimolar
concentration (1?M), DiMeDBC was the most potent producer of
ROS compared with DBC and N-MeDBC (Table 4). Although the ROS
level generated by DiMeDBC was lower than that produced by the
positive control, it might still be sufficient to induce oxidative DNA
damage in exposed cells.
3.7. Phosphorylation of histone H2AX by DBC and its derivatives
Phosphorylation of histone H2AX (termed ?H2AX) represents
one of early events occurring in cells due to genotoxic stress .
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
Fig. 6. The level of oxidative DNA damage detected in WB-F344 cells exposed to equimolar (1?M) DBC, DiMeDBC and N-MeDBC concentration for 2h (A) and the kinetics
of oxidative DNA damage repair (B). Opened portions of the bars represent DNA strand breaks (strand breaks and alkali-labile sites) detected immediately after treatment in
the absence of Fpg endonuclease, the filled portion of the bar represent additional DNA strand breaks detected in the presence of Fpg endonuclease (Fpg-sensitive sites). The
columns represent the mean±S.D. from at least two independent experiments on each triplicate of slides. The kinetics of Fpg-sensitive site removal induced by DiMeDBC
in WB-F344 cells was analyzed at several time intervals after treatment. Points represent the mean±S.D. from at least two independent experiments on each triplicate of
slides. Data were analyzed statistically by Student’s t-test. Significantly different from the control, *p<0.05; ***p<0.001; significantly different from DiMeDBC-treated cells
in the absence of Fpg endonuclease;ap<0.01.
The relative level of reactive oxygen species (ROS) generated by equimolar (1?M)
DBC, DiMeDBC and N-MeDBC concentration in WB-F344 cells after 24h treatment.
Hydrogen peroxide (250?M, 5min treatment) was used as the positive control.
Data represent the mean±S.D. of three independent experiments.
aSignificantly different from DiMeDBC at p<0.05.
*Significantly different from control (DMSO) at p<0.05.
carbazoles at 0.1–10?M (Fig. 7). Dibenzo[a,l]pyrene, the strongest
known genotoxin among polycyclic aromatic hydrocarbons (PAHs)
was the positive control in these experiments. Exposure of WB-
Fig. 7. Histone H2AX phosphorylation (?H2AX) detected in WB-F344 cells exposed
to equimolar (0.1?M, 1?M and 10?M) DBC, DiMeDBC, N-MeDBC, 0.1?M DB[a,l]P
(positive control), and 0.1% DMSO (negative control) for 24h. For Western blot anal-
yses, equal amounts of total protein were subjected to 10% SDS PAGE mini gel. Actin
was used as loading control. For immunodetection, appropriate primary and sec-
to control level of band density. All RI values were checked and corrected relative to
F344 cells to the liver carcinogens DBC and DiMeDBC resulted in
a significant induction of histone H2AX phosphorylation; up to
fivefold increase of ?H2AX was determined at the highest concen-
tration (Fig. 7). Only a weak increase in ?H2AX level was detected
in cells exposed to the tissue-specific sarcomagen N-MeDBC.
This study is a follow-up of a previous investigation which
suggested that the “stem-like” rat liver epithelial cells may be
a potential target cell population for the liver carcinogens DBC
and DiMeDBC . Variability in cellular processes triggered by
these agents in WB-F344 cells suggested differences in DNA lesions
produced by DBC and DiMeDBC. Because the genotoxic effects of
dibenzocarbazole derivatives have not been studied in this type
of liver cells, this work was initiated as part of ongoing research
devoted to understanding the cellular and molecular mechanisms
involved in chemical hepatocarcinogenesis.
A higher susceptibility of WB-F344 cells towards the liver car-
cinogens, as compared with sarcomagens, was observed after 2h
treatment (Table 1). The 24-h exposure to the systemic carcino-
gen DBC resulted in a significant, dose-dependent increase of DNA
adduct level was comparable with that of B[a]P (Table 2). The DNA
adduct pattern produced by DBC in WB-F344 cells was similar to
that found in human hepatoma HepG2 cells , but distinct from
the one detected in mouse liver cells in vivo [8,16,18]. These obser-
vations seem to imply that additional drug-metabolizing enzymes
the differences in tissue distribution patterns of DBC-DNA-adducts
detected in vivo [16,18] and in vitro [18,47,48], two metabolic path-
ways have been proposed for DBC: (i) activation involving the
ring-carbon atoms, as in the case of PAHs, and (ii) metabolism
at the pyrrolic NH group [8,19,49]. Although stable DNA adducts
are supposed to be major DNA lesions responsible for the carcino-
genicity of this agent in vivo , DBC, which has a relatively low
ionization potential , can also form unstable, depurinating DNA
adducts through one-electron oxidation mediated by radical cation
pathway . Aldo–keto reductases (AKR), which compete with
cytochrome P450 (CYP) enzymes, can play a significant part in DBC
metabolism [53,54]. Phenols are the major DBC metabolites found
in vitro using mouse and rat liver microsomes [55–57] as well as
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
human CYP1 enzymes . The proximate carcinogens 3-OH-/4-
OH-DBC  may be further activated to yield DBC-o-quinones
via the AKR pathway, which can form stable and unstable DNA
adducts. o-Quinones were also identified as a minor component
of DBC metabolites in vitro . DBC-3,4-dione is supposed to be
the ultimate DBC metabolite . DNA lesions produced by DBC
syntheses (Fig. 3), caused DNA breakage (Fig. 4) and micronucleus
formation (Table 3). DBC-bulky adducts generate serious distortion
in DNA, resulting in a considerable delay in DNA damage removal
(Fig. 5); even 48h after exposure, a significant level of DNA strand
breaks was detected in DBC-treated cells. Significant phosphoryla-
tion of histone H2AX (Fig. 7) and p53 protein  were determined
in DBC-treated cells.
Surprisingly, the strict hepatocarcinogen DiMeDBC produced
an almost negligible level of stable DNA adducts under identical
treatment conditions (Fig. 2E and F; Table 2). A time-course study
aimed at the kinetics of DNA-adduct formation in cells exposed
to DiMeDBC excluded a loss of bulky adducts already during cell
treatment owing to rapid DNA repair. No specific DNA adduct spots
were detected after the exposure of WB-F344 cells by DiMeDBC
for 2h, 4h, 6h, and 12h. DBC and DiMeDBC are potent liver
carcinogens, but quantitative and qualitative differences in their
biological effects were detected in vivo. At equimolar concentra-
tions, DiMeDBC induced fewer mutations and DNA adducts even
with different chromatographic mobility than the parent com-
pound DBC [10,13,16,18]. DBC hepatocarcinogenicity is associated
ditions, no histologically detectable toxicity has been found in the
liver of DiMeDBC-treated mice .
Despite the lack of stable DNA adducts, a significant level of
(Fig. 4, Table 3). In contrast with DBC, the increase in strand breaks
was not concentration dependent. One of the reasons of such phe-
nomenon might be a rapid repair of DNA damage at these low
concentrations as suggested, e.g., by Doak et al. . Indeed the
results depicted in Fig. 5 seem to suggest that the repair of breaks
induced by DiMeDBC is more rapid, when compared with DBC; no
DNA strand breaks were determined in cells 16h after exposure.
An explanation might be induction of oxidative stress. It is sup-
posed that ROS generated by various agents are eliminated to some
degree by inherent cellular defence mechanisms as presented, e.g.,
by Jenkins and colleagues . DiMeDBC (Table 4) induced a sig-
nificant level of ROS, as compared with both control and DBC.
The modified comet assay was used to analyze the DNA-damage
profiles induced by individual dibenzocarbazoles to evaluate the
mechanism of DiMeDBC genotoxicity in WB-F344 cells. A signifi-
cant rise in strand breaks due to incubation of DiMeDBC-exposed
cells with Fpg protein, a repair-specific endonuclease, suggested
that base modifications such as oxidized damage (e.g., 8-oxodG) or
ring-opened DNA adducts (Fapy-DNA adducts) may be responsi-
ble for DiMeDBC genotoxicity in these cells (Fig. 6A). In addition,
similarity in the kinetics of DNA damage removal in the presence
or absence of Fpg protein was found in DiMeDBC-exposed cells
(Figs. 5 and 6B). No DiMeDBC-DNA adducts were detected also in
V79MZh1A2 cells stably expressing human CYP1A2 despite a sig-
nificant level of mutations and micronuclei detected in exposed
revealed possible role of oxidative DNA damage or unstable DNA
adducts in DiMeDBC genotoxicity in V79MZh1A2 cells.
DiMeDBC is a potent aryl hydrocarbon receptor (AhR)-agonist
and accordingly increased significantly the expression of AhR-
mediated genes in WB-F344 cells, mainly CYP1A1 , although
it is poorly metabolized by this cytochrome P450. No mutations,
DNA strand breaks, micronuclei or DNA adducts were detected in
V79MZh1A1 cells stably expressing CYP1A1 [48,62–64]. Based on
these data, we hypothesized that DiMeDBC might cause a release
of ROS due to uncoupling of the catalytic cycle of CYP1A1 similarly
as the planar halogenated hydrocarbons. Both events, oxidative
damage and CYP1A1 induction were detected in cells exposed to
dioxin (TCDD) or polychlorinated biphenyl (PCB) congeners, which
are AhR-agonists but are, at the same time, poorly metabolized by
CYP1A1 [65–67]. On the other hand, AhR activation alone may also
tive stress [68,69]. Oxidative DNA damage produced by DiMeDBC
may lead to a replication fork collision resulting in histone H2AX
phosphorylation (Fig. 7). H2AX, a variant of histone H2A, is a crit-
ical factor for cellular protection . It is rapidly phosphorylated
at serine 139 in response to DNA double strand breaks , repli-
cation arrest , apoptosis , transcription inhibition , and
oxidative stress .
Although the level of stable adducts determined in N-MeDBC-
treated WB-F344 cells was very low (Fig. 2G and H, Table 2), it
was probably sufficient to produce DNA strand breaks and MNi
(Fig. 4, Table 3). DNA strand break formation has been proposed
to be a standard biomarker of DNA damage; however, this parame-
ter is probably not specific enough to identify differences in tissue
specificity of chemical compounds. DNA strand breaks are formed
as a consequence of various events; they are induced directly by
the agent, produced due to DNA damage removal in the process of
DNA repair or can result spontaneously by release of unstable DNA
adducts leading in alkali labile sites. DNA lesions generated by N-
ination of N-MeDBC metabolites via conjugation reactions cannot
be excluded, as, e.g., Perin et al.  showed that high N-MeDBC
mutagenicity in vitro due to activation by pure microsomes was
significantly reduced in the presence of cytosolic fraction. Despite
the lack of biological activity of this tissue-specific sarcomagen in
the liver ; a low level of DNA adducts (a level ∼300-fold lower
than that produced by DBC) was detected in mouse liver exposed
to N-MeDBC . Contrary to DBC and DiMeDBC, N-MeDBC did
not exhibit cytotoxicity (Table 1), cell-cycle arrest or apoptosis in
WB-F344 cells . No significant histone H2AX (Fig. 7) and p53
phosphorylation was found in N-MeDBC-treated WB-F344 cells
In conclusion, the present study clearly demonstrated that
different mechanisms may underlie genotoxic effects of liver car-
specific sarcomagen N-MeDBC may result in DNA strand breaks
and MNi. The extent of these genotoxic effects was insufficient to
produce additional cellular events related to hepatocarcinogene-
sis. Stable DNA adducts may mediate DBC genotoxicity in WB-F344
cells, but oxidative stress resulting in oxidative damage to DNA
is likely to be the causal factor in DiMeDBC genotoxicity. Differ-
ent DNA lesions may trigger distinct cellular pathways, resulting
in diverse cell responses detected in WB-F344 cells. The previous
study also showed that the strict hepatocarcinogen DiMeDBC is a
relatively potent agonist of AhR, which is an important player in
carcinogenesis. High levels of apparently active AhR characterize
various tumors, even in the absence of exogenous ligands [77–79].
AhR can interact via molecular cross-talk with multiple signalling
pathways involved in cellular growth, differentiation, and regula-
tion of cell adhesion and migration [80,81]. Future studies should
under in vivo conditions, as well as its relevance to hepatocarcino-
Conflict of interest
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
The authors wish to thank Professor F. Périn, Department of
Genotoxicity and Carcinogenicity, Institute Curie, France, who pro-
vided the dibenzocarbazole derivatives; and Professor J. E. Trosko,
MSU, East Lansing, MI, USA, who kindly offered the rat liver oval
WB-F344 cells. The authors express their appreciation to Mrs. A.
Vokáliková for excellent technical assistance.
This study was supported by the grants awarded by the Sci-
entific Grant Agency of SAS (No. 2/6063/26), Czech Ministry
of Education (No.2B08005), and Czech Ministry of Agriculture
(MZE0002716201). Zuzana Valoviˇ cová, M.Sc. was a fellow of the
European Social Fund Project (13120200038).
 IARC, Certain polycyclic aromatic hydrocarbons and heterocyclic compounds,
Monogr. Eval. Carcinog. Risk Chem. Man IARC, 1973.
 IARC, Polynuclear aromatic compounds, Monogr. Eval. Carcinog. Risk Chem.
Man IARC, 1983.
 L. Tomatis, The IARC program on the evaluation of the carcinogenic risk of
chemicals to man, Ann. N. Y. Acad. Sci. 271 (1976) 396–409.
 M.L. Yu, R.A. Hites, Identification of organic compounds in diesel engine soot,
Anal. Chem. 53 (1981) 951–954.
 C.H. Ho, B.R. Clark, M.R. Guerin, B.D. Barkenbus, T.K. Rao, J.L. Epler, Analytical
and biological analysis of test materials from the synthetic fuel technologies,
Mutat. Res. 85 (1981) 335–345.
 R.W. Serth, T.W. Hughes, Polycyclic organic matter (POM) and trace element
contents of carbon black vent gas, Environ. Sci. Technol. 14 (1980) 298–301.
 S.S. Hecht, Tobacco smoke carcinogens and lung cancer, J. Natl. Cancer Inst. 91
 D. Warshawsky, G. Talaska, W. Xue, J. Schneider, Comparative carcinogenicity,
metabolism, mutagenicity, and DNA binding of 7H-dibenzo[c,g]carbazole and
dibenz[a,j]acridine, Crit. Rev. Toxicol. 26 (1996) 213–249.
 M.E. Schurdak, D.B. Stong, D. Warshawsky, K. Randerath, 32P-postlabeling
analysis of DNA adduction in mice by synthetic metabolites of the environ-
mental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for
3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver
but not skin, Carcinogenesis 8 (1987) 591–597.
 O. Perin-Roussel, F. Perin, N. Barat, M.J. Plessis, F. Zajdela, Interaction of
7H-dibenzo[c,g]carbazole and its organspecific derivatives with hepatic mito-
chondrial and nuclear DNA in the mouse, Environ. Mol. Mutagen. 25 (1995)
 D. Warshawsky, G. Talaska, M. Jaeger, T. Collins, A. Galati, L. You, G.
Stoner, Carcinogenicity, DNA adduct formation and K-ras activation by 7H-
dibenzo[c,g]carbazole in strain A/J mouse lung, Carcinogenesis 17 (1996)
 F. Tombolan, D. Renault, D. Brault, M. Guffroy, O. Perin-Roussel, F. Perin, V. Thy-
baud, Kinetics of induction of DNA adducts, cell proliferation and gene muta-
tions in the liver of MutaMice treated with 5,9-dimethyldibenzo[c,g]carbazole,
Carcinogenesis 20 (1999) 125–132.
ity of 7H-dibenzo[c,g]carbazole and two derivatives in MutaMouse liver and
skin, Mutat. Res. 417 (1998) 129–140.
 D. Valero, F. Perin, M.J. Plessis, F. Zajdela, Sexual differences in the expression
induced hepatocarcinogenesis in mice, Cancer Lett. 27 (1985) 181–197.
genic or regenerative cell proliferation on lacz mutant frequency in the liver of
MutaTMMice treated with 5,9-dimethyldibenzo[c,g]carbazole, Carcinogenesis
20 (1999) 1357–1362.
 D. Taras-Valero, O. Perin-Roussel, M.J. Plessis, F. Zajdela, F. Perin, Tissue-specific
activities of methylated dibenzo[c,g]carbazoles in mice: carcinogenicity, DNA
adduct formation, and CYP1A induction in liver and skin, Environ. Mol. Muta-
gen. 35 (2000) 139–149.
 D. Renault, D. Brault, Y. Lossouarn, O. Perin-Roussel, D. Taras-Valero, F. Perin, V.
Thybaud, Kinetics of DNA adduct formation and removal in mouse hepatocytes
following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole, Carcinogen-
esis 21 (2000) 289–294.
 O. Perin-Roussel, N. Barat, F. Zajdela, F. Perin, Tissue-specific differences in
dibenzo[c,g]carbazole in mouse parenchymal and nonparenchymal liver cells,
Environ. Mol. Mutagen. 29 (1997) 346–356.
 F. Perin, D. Valero, J. Mispelter, F. Zajdela, In vitro metabolism of N-methyl-
dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and
hepatocarcinogenic properties, Chem. Biol. Interact. 48 (1984) 281–295.
the DNA-binding activity of 7H-dibenzo[c,g]carbazole approximately 300-fold
in mouse liver but only approximately 2-fold in skin: possible correlation with
carcinogenic activity, Carcinogenesis 8 (1987) 1405–1410.
 J.C. Arcos, M.F. Argus, Chemical iduction of cancer, Struct. Basis Biol. Mech. IIB
 M.S. Tsao, J.D. Smith, K.G. Nelson, J.W. Grisham, A diploid epithelial cell line
from normal adult rat liver with phenotypic properties of ‘oval’ cells, Exp. Cell
Res. 154 (1984) 38–52.
 W.B. Coleman, A.E. Wennerberg, G.J. Smith, J.W. Grisham, Regulation of the
by the hepatic microenvironment, Am. J. Pathol. 142 (1993) 1373–1382.
 D. Couchie, N. Holic, M.N. Chobert, A. Corlu, Y. Laperche, In vitro differentiation
of WB-F344 rat liver epithelial cells into the biliary lineage, Differentiation 69
 M.R. Alison, M.J. Lovell, Liver cancer: the role of stem cells, Cell Prolif. 38 (2005)
 T. Roskams, Liver stem cells and their implication in hepatocellular and cholan-
giocarcinoma, Oncogene 25 (2006) 3818–3822.
 M.L. Dumble, E.J. Croager, G.C. Yeoh, E.A. Quail, Generation and characteriza-
tion of p53 null transformed hepatic progenitor cells: oval cells give rise to
hepatocellular carcinoma, Carcinogenesis 23 (2002) 435–445.
Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic
epithelial stem-like WB-F344 cells, Toxicol. Appl. Pharmacol. 196 (2004)
 L. Svihalkova-Sindlerova, M. Machala, K. Pencikova, S. Marvanova, J. Neca, J.
Topinka, O. Sevastyanova, A. Kozubik, J. Vondracek, Dibenzanthracenes and
benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver
‘stem-like’ cells, Toxicology 232 (2007) 147–159.
 J. Topinka, S. Marvanova, J. Vondracek, O. Sevastyanova, Z. Novakova, P. Krcmar,
K. Pencikova, M. Machala, DNA adducts formation and induction of apoptosis
in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aro-
 S. Marvanova, J. Vondracek, K. Pencikova, L. Trilecova, P. Krcmar, J. Top-
inka, Z. Novakova, A. Milcova, M. Machala, Toxic effects of methylated
benz[a]anthracenes in liver cells, Chem. Res. Toxicol. 21 (2008) 503–512.
 J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z. Andrysik, K.
7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert mul-
tiple toxic events contributing to tumor promotion in rat liver epithelial
‘stem-like’ cells, Mutat. Res. Fundam. Mol. Mech. Mutagen. 638 (2006) 43–56.
 R.C. Gupta, Enhanced sensitivity of 32P-postlabeling analysis of aromatic car-
cinogen:DNA adducts, Cancer Res. 45 (1985) 5656–5662.
 B. Binkova, M. Cerna, A. Pastorkova, R. Jelinek, I. Benes, J. Novak, R.J. Sram, Bio-
logical activities of organic compounds adsorbed onto ambient air particles:
comparison between the cities of Teplice and Prague during the summer and
winter seasons 2000–2001, Mutat. Res. 525 (2003) 43–59.
 N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quanti-
tation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988)
 A.R. Collins, A.G. Ma, S.J. Duthie, The kinetics of repair of oxidative DNA damage
 A. Gabelova, D. Slamenova, L. Ruzekova, T. Farkasova, E. Horvathova, Measure-
ment of DNA strand breakage and DNA repair induced with hydrogen peroxide
using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elu-
tion of DNA, Neoplasma 44 (1997) 380–388.
 B.M. Miller, E. Pujadas, E. Gocke, Evaluation of the micronucleus test in vitro
using Chinese hamster cells: results of four chemicals weakly positive in the in
vivo micronucleus test, Environ. Mol. Mutagen. 26 (1995) 240–247.
 P.M. Eckl, I. Raffelsberger, The primary rat hepatocyte micronucleus assay: gen-
eral features, Mutat. Res. 392 (1997) 117–124.
 F.A. Oberhammer, M. Pavelka, S. Sharma, R. Tiefenbacher, A.F. Purchio, W.
Bursch, R. Schulte-Hermann, Induction of apoptosis in cultured hepatocytes
and in regressing liver by transforming growth factor beta 1, Proc. Natl. Acad.
Sci. U.S.A. 89 (1992) 5408–5412.
carcinogenic potency of DNA adducts, Mutat. Res. 424 (1999) 237–247.
 A. Gabelova, Z. Valovicova, G. Bacova, J. Labaj, B. Binkova, J. Topinka, O. Sev-
astyanova, R.J. Sram, I. Kalina, V. Habalova, T.A. Popov, T. Panev, P.B. Farmer,
Sensitivity of different endpoints for in vitro measurement of genotoxicity of
extractable organic matter associated with ambient airborne particles (PM10),
Mutat. Res. Fundam. Mol. Mech. Mutagen. 620 (2007) 103–113.
 S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W.P. Chang, N. Holland, M.
Wasilewska, E. Fabianova, A. Fucic, L. Hagmar, G. Joksic, A. Martelli, L. Migliore,
E. Mirkova, M.R. Scarfi, A. Zijno, H. Norppa, M. Fenech, An increased micronu-
cleus frequency in peripheral blood lymphocytes predicts the risk of cancer in
humans, Carcinogenesis 28 (2007) 625–631.
 M. Dizdaroglu, Substrate specificities and excision kinetics of DNA glycosylases
involved in base-excision repair of oxidative DNA damage, Mutat. Res. Fundam.
Mol. Mech. Mutagen. 531 (2003) 109–126.
 C. Zhou, Z. Li, H. Diao, Y. Yu, W. Zhu, Y. Dai, F.F. Chen, J. Yang, DNA damage eval-
uated by gammaH2AX foci formation by a selective group of chemical/physical
stressors, Mutat. Res. 604 (2006) 8–18.
 T. O’Brien, G. Babcock, J. Cornelius, M. Dingeldein, G. Talaska, D. Warshawsky,
K. Mitchell, A comparison of apoptosis and necrosis induced by hepatotoxins
in HepG2 cells, Toxicol. Appl. Pharmacol. 164 (2000) 280–290.
60 Download full-text
Z. Valoviˇ cová et al. / Mutation Research 665 (2009) 51–60
 A. Gabelova, O. PerinRoussel, Y. Jounaidi, F. Perin, DNA adduct formation
in primary mouse embryo cells induced by 7H-dibenzo[c,g]carbazole and
its organ-specific carcinogenic derivatives, Environ. Mol. Mutagen. 30 (1997)
 A. Gabelova, B. Binkova, Z. Valovicova, R.J. Sram, DNA adduct formation by 7H-
dibenzo[c,g]carbazole and its tissue- and organ-specific derivatives in Chinese
hamster V79 cell lines stably expressing cytochrome P450 enzymes, Environ.
Mol. Mutagen. 44 (2004) 448–458.
 F. Perin, D. Valero, V. Thybaud-Lambay, M.J. Plessis, F. Zajdela, Organ-specific,
carcinogenic dibenzo[c,g]carbazole derivatives: discriminative response in S.
typhimurium TA100 mutagenesis modulated by subcellular fractions of mouse
liver, Mutat. Res. 198 (1988) 15–26.
 H.V. Dowty, W. Xue, K. LaDow, G. Talaska, D. Warshawsky, One-electron oxi-
dation is not a major route of metabolic activation and DNA binding for the
carcinogen 7H-dibenzo[c,g]carbazole in vitro and in mouse liver and lung, Car-
cinogenesis 21 (2000) 991–998.
 W. Xue, D. Zapien, D. Warshawsky, Ionization potentials and metabolic acti-
vations of carbazole and acridine derivatives, Chem. Res. Toxicol. 12 (1999)
Synthesis of depurinating DNA adducts formed by one-electron oxidation of
7H-dibenzo[c,g]carbazole and identification of these adducts after activation
with rat liver microsomes, Chem. Res. Toxicol. 10 (1997) 225–233.
 W. Xue, A. Siner, M. Rance, K. Jayasimhulu, G. Talaska, D. Warshawsky, A
metabolic activation mechanism of 7H-dibenzo[c,g]carbazole via o-quinone.
Part 2. Covalent adducts of 7H-dibenzo[c,g]carbazole-3,4-dione with nucleic
acid bases and nucleosides, Chem. Res. Toxicol. 15 (2002) 915–921.
matic hydrocarbons and DNA damage: a review, Toxicol. Appl. Pharmacol. 206
 F. Perin, M. Dufour, J. Mispelter, B. Ekert, C. Kunneke, F. Oesch, F.
Zajdela, Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-
dibenzo[c,g]carbazole metabolism by microsomal enzymes from mouse and
rat liver, Chem. Biol. Interact. 35 (1981) 267–284.
 L.P. Wan, W.L. Xue, J. Schneider, R. Reilman, M. Radike, D. Warshawsky, Com-
parative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by
mouse and rat liver microsomes, Chem. Biol. Interact. 81 (1992) 131–147.
 D.B. Stong, R.T. Christian, K. Jayasimhulu, R.M. Wilson, D. Warshawsky, The
chemistry and biology of 7H-dibenzo[c,g]carbazole: synthesis and charac-
terization of selected derivatives, metabolism in rat liver preparations and
mutagenesis mediated by cultured rat hepatocytes, Carcinogenesis 10 (1989)
 H.G. Shertzer, M.B. Genter, G. Talaska, C.P. Curran, D.W. Nebert, T.P. Dalton, 7H-
Dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of
enzymes, Carcinogenesis 28 (2007) 1371–1378.
 G. Talaska, R. Reilman, M. Schamer, J.H. Roh, W. Xue, S.L. Fremont, D. War-
shawsky, Tissue distribution of DNA adducts of 7H-dibenzo[c,g]carbazole and
 S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mecha-
nistic influences for mutation induction curves after exposure to DNA-reactive
carcinogens, Cancer Res. 67 (2007) 3904–3911.
 G.J. Jenkins, J. Cronin, A. Alhamdani, N. Rawat, F. D’Souza, T. Thomas, Z. Eltahir,
A.P. Griffiths, J.N. Baxter, The bile acid deoxycholic acid has a non-linear dose
response for DNA damage and possibly NF-kappaB activation in oesophageal
cells, with a mechanism of action involving ROS, Mutagenesis 23 (2008)
 A. Gabelova, G. Bacova, L. Ruzekova, T. Farkasova, Role of cytochrome
P4501A1 inbiotransformationofa tissuespecific sarcomagen
methyldibenzo[c,g]carbazole, Mutat. Res. Genet. Toxicol. Environ. Mutagen.
469 (2000) 259–269.
 A. Gabelova, T. Farakasova, G. Bacova, S. Robichova, Mutagenicity of
7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engi-
neered Chinese hamster V79 cell lines stably expressing cytochrome P450,
Mutat. Res. Genet. Toxicol. Environ. Mutagen. 517 (2002) 135–145.
 T. Farkasova, A. Gabelova, D. Slamenova, Induction of micronuclei by 7H-
dibenzo[c,g]carbazole and its tissue specific derivatives in Chinese hamster
V79MZh1A1 cells, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 491 (2001)
suppression of hepatic cytochrome P450 1A1 by 3,3?,4,4?-tetrachlorobiphenyl,
Biochem. Pharmacol. 53 (1997) 1029–1040.
 J.J. Schlezinger, R.D. White, J.J. Stegeman, Oxidative inactivation of cytochrome
P-450 1A (CYP1A) stimulated by 3,3?,4,4?-tetrachlorobiphenyl: production of
reactive oxygen by vertebrate CYP1As, Mol. Pharmacol. 56 (1999) 588–597.
 J.J. Schlezinger, W.D. Struntz, J.V. Goldstone, J.J. Stegeman, Uncoupling of
cytochrome P450 1A and stimulation of reactive oxygen species production
by co-planar polychlorinated biphenyl congeners, Aquat. Toxicol. 77 (2006)
 D.W. Nebert, A.L. Roe, M.Z. Dieter, W.A. Solis, Y. Yang, T.P. Dalton, Role of the
aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress
response, cell cycle control, and apoptosis, Biochem. Pharmacol. 59 (2000)
 T.P. Dalton, A. Puga, H.G. Shertzer, Induction of cellular oxidative stress by aryl
hydrocarbon receptor activation, Chem. Biol. Interact. 141 (2002) 77–95.
 J.A. Meador, M. Zhao, Y. Su, G. Narayan, C.R. Geard, A.S. Balajee, Histone H2AX is
a critical factor for cellular protection against DNA alkylating agents, Oncogene
27 (2008) 5662–5671.
 J. Fillingham, M.C. Keogh, N.J. Krogan, GammaH2AX and its role in DNA double-
strand break repair, Biochem. Cell Biol. 84 (2006) 568–577.
 I.M. Ward, J. Chen, Histone H2AX is phosphorylated in an ATR-dependent man-
ner in response to replicational stress, J. Biol. Chem. 276 (2001) 47759–47762.
 E.P. Rogakou, W. Nieves-Neira, C. Boon, Y. Pommier, W.M. Bonner, Initiation of
at serine 139, J. Biol. Chem. 275 (2000) 9390–9395.
 H.E. Mischo, P. Hemmerich, F. Grosse, S. Zhang, Actinomycin D induces his-
tone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70
and nuclear DNA helicase II, J. Biol. Chem. 280 (2005) 9586–9594.
 T. Tanaka, X. Huang, H.D. Halicka, H. Zhao, F. Traganos, A.P. Albino, W. Dai, Z.
Darzynkiewicz, Cytometry of ATM activation and histone H2AX phosphoryla-
A 71 (2007) 648–661.
 D. Szafarz, F. Perin, D. Valero, F. Zajdela, Structure and carcinogenicity of
dibenzo(c,g)carbazole derivatives, Biosci. Rep. 8 (1988) 633–643.
 P. Andersson, J. McGuire, C. Rubio, K. Gradin, M.L. Whitelaw, S. Pettersson, A.
Hanberg, L. Poellinger, A constitutively active dioxin/aryl hydrocarbon receptor
induces stomach tumors, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 9990–9995.
 O. Moennikes, S. Loeppen, A. Buchmann, P. Andersson, C. Ittrich, L. Poellinger,
M. Schwarz, A constitutively active dioxin/aryl hydrocarbon receptor promotes
hepatocarcinogenesis in mice, Cancer Res. 64 (2004) 4707–4710.
 M.H. Yang, M.J. Kong, Y. Choi, C.S. Kim, S.M. Lee, C.W. Park, H.S. Lee, K. Toe,
Associations between XPC expression, genotype, and the risk of head and neck
cancer, Environ. Mol. Mutagen. 45 (2005) 374–379.
 A. Puga, C.R. Tomlinson, Y. Xia, Ah receptor signals cross-talk with multiple
developmental pathways, Biochem. Pharmacol. 69 (2005) 199–207.
 R. Barouki, X. Coumoul, P.M. Fernandez-Salguero, The aryl hydrocarbon
receptor, more than a xenobiotic-interacting protein, FEBS Lett. 581 (2007)