Mutagenesis, 2015, 30, 593–602
doi:10.1093/mutage/gev021
Original Article
Advance Access publication 17 June 2015
Original Manuscript
Micronucleus formation by single and mixed
heavy metals/loids and PAH compounds in
HepG2 cells
Cheng Peng1,2, Sasikumar Muthusamy1,2, Qing Xia1,2, Vincent Lal1,2,
Michael S. Denison3 and Jack C. Ng1,2,*
National Research Centre for Environmental Toxicology-Entox, The University of Queensland, 39 Kessels Road,
Coopers Plains, Brisbane 4108, Australia, 2Cooperative Research Centre for Contamination Assessment and
Remediation of the Environment, Adelaide 5095, Australia and 3Department of Environmental Toxicology, University
of California, Davis, CA 95616, USA
1
*To whom correspondence should be addressed. Tel: +61 7 3274 9009/+61 4 1474 7147; Fax: +61 7 3000 9687;
Email: [email protected]
Received 9 December 2014; Revised 3 February 2015; Accepted 10 February 2015.
Abstract
Humans and other organisms are exposed to multi-chemical mixtures including commonly found
carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heavy metal/loids. The joint
effects of these chemicals as beyond the binary mixtures have not been well characterised. In
this study, we evaluated the combined genotoxicity of mixtures of PAHs and heavy metal/loids
containing benzo(a)pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe), pyrene (Pyr), arsenic
(As), cadmium (Cd) and chromium (Cr) using in vitro micronucleus (MN) test in HepG2 cells. The
induction of aryl hydrocarbon receptor (AhR) by single and mixed PAHs was also measured.
The results indicated that individual and mixed Nap, Phe and Pyr did not induce significant MN
frequencies. PAHs mixture containing B[a]P and B[a]P alone caused significant but similar level of
MN frequencies. The same pattern was found in their AhR induction. Individual metal/loids induced
significant cytostasis and MN formation of which Cd was found the most potent inducer. Mixture
of metal/loids caused higher frequency of MN suggesting a possible additive effect among metal/
loids. In addition, binary mixture of metal/loids and B[a]P, namely As/B[a]P, Cd/B[a]P and Cr/B[a]
P, increased MN formation. Mixture of Cd and B[a]P induced the highest level of MN. Exposure
of cells to the mixture containing B[a]P and Cd/Cr/As at lower concentration (0.25 µM) resulted in
significant MN frequency, the level of which was equal to that by Cd/B[a]P at 1.0 µM. The results
of the study suggested that an additive effect may exist between PAHs and heavy metal/loids in
a compound- and concentration-dependent manner. The compounds with highest potencies of
genotoxicity in the mixture seem dominant as driving sources in the final combined genotoxicity
of PAHs and heavy metal/loids.
Introduction
The toxicological studies to support human health risk assessment have mainly focused on effects of single compound. In most
cases, humans are exposed simultaneously to multiple pollutants
among which polycyclic aromatic hydrocarbons (PAHs) and heavy
metal/loids are the common and important chemicals co-existing
in ambient air (1), soil (2,3) and water (4–6). PAHs are generated
during combustion process of fossil fuels and organic matters (7).
Exposure to PAHs through inhalation and ingestion has been casually associated with higher risk of cancer incidence (8,9). Benzo[a]
pyrene (B[a]P) is a representative member of the PAHs and has
been classified as Group 1 carcinogen by the International Agency
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for Research on Cancer (IARC) (7). It has been known that B[a]P
exerts its carcinogenic effects through its metabolites transformed
by Phase I enzymes cytochrome P450 (CYP) family, mainly CYP1A1
and CYP1B1 (10,11). In addition to DNA-adduct formation, B[a]P
elicits its genotoxic effects through the formation of reactive oxygen
species (ROS) and resultant DNA damages through the aldo–keto
reductase pathway (12,13). Heavy metal/loids are another category
of environmental contaminants, which can accumulate in soils and
plants. Chronic low exposure of heavy metal/loids is known to cause
genomic instability, endocrine disruption, neurotoxicity, carcinogenicity and immunotoxicity (14). Arsenic (As), chromium (Cr) and
cadmium (Cd) are among the most toxic heavy metal/loids and have
been classified as human carcinogens by IARC (15). Their genotoxicities have been tested positive in different in vitro systems/assays.
Extensive studies have been conducted for health effects of individual compounds of PAHs and heavy metal/loids. Although the
effects of these chemicals as individual compounds have been documented, their combined effects in particular the genotoxicity have
not been well addressed. The combined effects of PAHs-heavy metal/
loids mixture may not be simply described using the model of concentration addition or independent action due to the prerequisite of
the metabolisms of PAHs for their biological effects, different action
pathways and possible interactions within/between PAHs and/or
heavy metal/loids at different stages. For PAHs only, it was proposed
that there may be a competition of P450 among the compounds in
PAH mixtures resulting in the decreased level of reactive metabolites and consequently less genotoxicity of the PAHs mixtures (16).
However, the interaction among PAHs and resultant genotoxicity
are compound-dependent (17).
Studies on combined effects of heavy metal/loids and PAHs have
found that heavy metal/loids inhibit the PAH-induced CYP1A1 and
1A2 at the transcriptional and post-translational levels (18–20).
Because CYP1A1-mediated metabolism is a prerequisite for B[a]
P-exerted genotoxicity, the reduced CYP1A1 activity by heavy metal/
loids may conceivably result in a decreased genotoxicity of B[a]P.
However, the overall consequences of the joint effects of mixtures may
depend on not only the metabolic process but also other factors such
as DNA repair. Heavy metal/loids have been shown to affect the base
excision repair (BER) through inhibiting the repair activity of apurinic/
apyrimidinic (AP) endonuclease 1 (21,22). BER pathway is responsible for repairing the B[a]P-induced oxidative DNA damages (23,24).
Furthermore, heavy metal/loids can induce oxidative damages through
H2O2 production (25). Hence, the overall level of ROS from co-exposure of PAHs and heavy metal/loids may increase as PAHs can also
cause ROS stress and DNA damage (25,26). The co-mutagenic effects
of PAHs and heavy metal/loids have been extensively studied not only
for B[a]P and arsenite (27) but also for B[a]P and Cd (28), B[a]P and
nickel (29) and B[a]P and Cr (30). However, these studies concentrated
on only binary mixtures, whereas this study investigated the effects
of multi-compound mixtures. The co-mutagenic/genotoxic effects of
this kind of multi-compound mixtures are much more complicated
as there may be interactions not only among PAHs but also between
heavy metal/loids and PAHs and vice versa in terms of bioavailability,
metabolisms and other cellular responses. To the best of our knowledge, studies on multi-compound mixtures of PAHs and metal/loids
have not been reported. Therefore, this study provides more insightful
information about the co-mutagenic/genotoxic effects of multi-compound mixtures of PAHs and heavy metal/loids, which will be useful
for better health risk assessment as multi-contaminants are more commonly found in the real environment. Recently, it has been reported
that the PAHs mixture from real samples such as PAHs-contaminated
sediment and soil caused an increased genotoxicity (31,32). However,
these studies did not report the contents of the PAH mixtures and
therefore the mode of combined effects could not be addressed. In this
study, we evaluated the combined genotoxic effects of single and mixed
PAHs containing B[a]P, naphthalene (Nap), phenanthrene (Phe) and
pyrene (Pyr) with their structures shown in Figure 1 and heavy metal/
loids including As, Cd and Cr. These compounds can be co-existed in
the soil of contaminated sites. People, especially kids, may be exposed
to these soil contaminants through the ingestion route by which these
compounds can move to the liver where they undergo metabolic transformation resulting in potential harm. Therefore, human liver cells
(HepG2) were used for this study. HepG2 cells have metabolic competency (33,34) and DNA repair capacity with p53 proficiency (35)
and have been demonstrated to be a suitable model for studying the
effects of B[a]P (36). We evaluated the micronucleus (MN) formation
using the cytokinesis-block micronucleus (CBMN) assay based on the
Organization for Economic Cooperation and Development (OECD)
Guideline for Testing of Chemicals 487 (37) and other references
(38,39). MN formation can be used to assess the DNA damages in the
cells because MN is originated from unrepaired DNA damages (40).
CBMN assay can not only assess MN formation and other nuclear
anomalies but also provide information of cell toxicity/cytostasis (41).
We also measured the induction of aryl hydrocarbon receptor (AhR),
which is known to up-regulate the CYP1A1 expression (42) and associate with DNA damage (43,44).
Materials and Methods
Chemicals
B[a]P (B10102-100MG), Nap (147141-25G), Phe (P11409-25G),
Pyr (82648-10G), sodium arsenite (NaAsO2; S7400-100G), cadmium chloride (CdCl2; 202908-50G), Cr (316512-100G), mitomycin C (MMC; M4827-2 MG), acridine orange (A6014-10G) and
cytochalasin B (Cyto B; C6732-1MG) were obtained from Sigma–
Aldrich Pvt Ltd (Castle Hill, NSW, Australia). Dulbecco’s Modified
Eagle’s Medium (DMEM), minimum essential media, foetal bovine
serum (FBS), l-glutamine, penicillin/streptomycin and geneticin
(G418; 11811031) were from Life Technologies Australian Pvt Ltd
(Mulgrave, VIC, Australia). All other reagents were of analytical
grade. The testing compounds were dissolved in DMSO (V900090500ML, Sigma–Aldrich Pvt Ltd) as stock solutions, which were kept
in the dark and stored at −20°C.
Cell and culture conditions
Human hepatocellular carcinoma cell line, Hep G2 (ATCC®
77400™), cells were cultured in DMEM, containing 10% foetal
Figure 1. The chemical structures of (A) Nap, (B) Phe, (C) Pyr and (D) B[a]P.
Combined genotoxicity of heavy metals/loids and PAHs, 2015, Vol. 30, No. 5
calf serum, 2 mM of l-glutamine, 100 U/ml penicillin (GIBCO BRL),
100 U/ml streptomycin and maintained in a humidified incubator at
37°C under 5% CO2. Because HepG2 cells are adherent and tend to
clump up, we used a little longer time for the trypsinisation (3–5 min
compared with other cell types such as A549) and more rounds of
pipetting of the cell culture up and down in order to break up clumps
during subculture.
Chemical treatment
Stock solutions of B[a]P, Nap, Phe and Pyr were dissolved in DMSO
and the mixture was made by mixing equal volume of each solution.
Stock solutions of NaAsO2, CdCl2 and potassium dichromate were
made in H2O, and the mixture was made by mixing equal volume of
each solution. The treatments included single and mixed compounds
of PAHs and heavy metal/loids. For the treatment, compound solutions were added in 1:100 to the culture medium without FBS. Cells
were seeded into 12-well plates at 2–5 × 104/well 24 h prior to the
chemical treatment. MMC (2 µM) was used for the positive control
for metal/loids. Solvent vehicle, the mixture of DMSO and H2O, was
used as the negative control. The final DMSO concentration in the
medium did not exceed 1%.
Cytotoxicity testing using MTS assay
To get primary cytotoxicity data of these compounds, we conducted
the MTS assay using the Promega CellTiter 96 AQueous NonRadioactive Cell Proliferation (MTS) assay (Promega, Alexandria,
Australia). The assay was performed according to the manufacturer’s instructions (Promega) with slight modifications. Briefly, cells
in 100 µl (1 × 105 cells/ml) were seeded into each well of a 96-well
plate and cultured for 20 h before treatment. The cells were treated
with testing compounds at different concentrations for 24 h. Control
groups were treated with the reagent vehicle DMSO only. At the
end of each incubation time, the cells were washed three times
with the medium without FBS followed by the addition of 120 μl
medium containing 20 μl of MTS and further incubated for 2 h. The
absorbance of the formazan product was then recorded at 490 nm
using a microplate reader (FLUOstar OPTIMA by BMG Labtech,
Offenburg, Germany). Each experiment was performed in triplicate and repeated independently three times. Data were taken as
the mean ± SD from three independent experiments and analysed
using the GraphPad Prism version 5 (GraphPad, La Jolla, CA, USA).
The results were expressed as the percentage growth inhibition with
respect to the untreated cells.
CBMN assay
Based on our cytotoxicity results and previous studies (45,46),
we selected lower concentrations between 0.25 and 2 µM for the
metal/loids, and 20 µM for B[a]P and <1000 µM for Phe, Pyr and
Nap. Obvious precipitation was found for PAHs when their concentrations are higher than 1000 µM. Chemical treatment was performed according to the published method (38,47) and OECD Test
Guideline 487 for the In Vitro Mammalian Cell MN Test (37) in
the presence of cytokinesis block by the addition of Cyto B. Briefly,
cells were seeded into 12-well plates at 5 × 104/well 24 h prior to the
chemical treatment. After 24-h incubation, the medium was replaced
with a fresh medium (without FBS) containing the testing compounds. Each treatment had duplicate for each concentration. After
being treated with chemicals for 24 h, the cells were washed and
cultured with fresh normal medium containing Cyto B (6 µg/ml) for
another 48 h to inhibit the cell division. Then, the cells were washed
twice with PBS and fixed with a mixture of methanol and acetic acid
595
(3:1 v/v), stained with 2 µM of acridine orange and scored under a
fluorescent microscope (Nikon, ECLIPSE TS100, Japan). A total of
1000 binucleated cells (BNC) were scored blindly and analysed for
each treatment. MN number was counted, and the mononucleate,
binucleate and multinucleate cells were categorised for the evaluation of cytokinesis-block proliferation index (CBPI) and cytostasis.
MN frequency was expressed as MN/1000 BNC. The number was
expressed as mean ± standard error of the mean (SEM) from three
independent experiments. Cytotoxicity was evaluated by % cytostasis using the formula (37):
% Cytostasis = 100 − 100[(CBPI T _ 1) ÷ (CBPIC _ 1)]. Where,
(No. mononucleate cells) + (2 × No. binucleate cells) +



(3 × No. multinucleate cells)


CBPI =
Total number of cells
T = test chemical treatment culture and C = vehicle control culture.
Stable transfections and green fluorescent protein
reporter assay
HepG2 cells were transfected with reporter plasmid, pGreen1.1,
using Tfx™-20 reagent (Promega; E2391). The plasmid contains the
480-bp dioxin-responsive domain from the mouse CYP1A1 gene
inserted upstream of the mouse mammary tumour virus (MMTV)
promoter and it confers dioxin responsiveness upon the MMTV
promoter and adjacent enhanced green fluorescent protein (EGFP)
reporter gene (48). After transfection, the cells were selected with
G418 (1 mg/ml) and cell clones isolated. The derived stable cell
lines were maintained in the medium containing G418 (200 µg/ml)
and screened with 2,3,7,8-tetrachlorodibenzo-p-dioxin to get the
cell lines (pGreen1.1/HepG2) with strongest inducibility (data not
shown). For induction analysis, pGreen1.1/HepG2 cells were cultured in black 96-well flat/clear-bottom plates at 2.5 × 104 cells/ml
at a volume of 100 μl per well for 14–16 h. After treatment with
PAHs compound(s), the cells were incubated in a 5% CO2 atmosphere for 72 h at 33°C to maximise EGFP expression and inducible
fluorescence (49). EGFP activity (fluorescent intensity) was observed
under fluorescent microscopy and quantitated after 72 h of incubation in a FLUOstar plate reader (excitation 485 nm, emission 520 nm
and gain 1500). Each experiment was performed in triplicate and
repeated independently three times. After subtraction, the background fluorescence in cells incubated with the carrier solvent, EGFP
activity was expressed as relative fluorescence units (RFU).
Statistical analysis
The number was expressed as mean ± SEM from three independent
experiments. The data were analysed by one-way analysis of variance followed by the Student–Newman–Keuls test using GraphPad
Prism 6 (GraphPad) and the differences were considered significant
for P < 0.05.
Results
Cytotoxicity of single PAHs and heavy metal/loids
Cytotoxicity of HepG2 cells induced by single PAHs and heavy
metal/loids are presented in Figure 2. The results showed that the
C. Peng et al., 2015, Vol. 30, No. 5
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Figure 2. Evaluation of the viability of HepG2 cells exposed to single heavy metal/loid and PAH compound using the MTS assay. (A) Effects of heavy metal/
loids, As, Cd and Cr at different concentrations on the growth of HepG2 cells. (B) Effects of PAH compounds including B[a]P, Phe, Pyr and Nap at different
concentrations on HepG2 cell. Each data point represents the mean and standard deviation of three replicates and three independent experiments.
metal/loids elicited different degrees of cytotoxicities to HepG2 cells
with IC50 of 2.5, 9.1 and >100 µM for Cd, Cr and As, respectively
(Figure 2A). B[a]P showed the most toxicity in HepG2 cells followed
by Nap, Phe and Nap as shown in Figure 2B, but the values of IC50
were not available from the experiment due to the formation of precipitation at higher doses of these compounds.
MN formation and cytostasis by individual PAH
compounds
The MN formation (left Y axis) and cytostasis (right Y axis) in
cells treated with PAH compounds, Phe, Pyr and Nap are shown in
Figure 3. Single PAH compound appeared to increase cytostasis at
higher concentrations, but there was no significant difference comparing with that in the control. Nap (Figure 3C) induced more cytostasis
than Phe (Figure 3A) and Pyr (Figure 3B). No obvious formation of
MN was found even at higher concentrations where the precipitations were found. Cells treated with B[a]P (20 µM) and MMC (2 µM)
as the positive control caused 60.5 and 68.6 MN/1000 BNC, respectively, compared with the negative control of 3.4 MN/1000 BNC.
MN formation and cytostasis by PAH mixtures
Figure 4 shows the MN formation and cytostasis induced by two
PAHs mixtures. One mixture contained equal molar of Phe, Pyr and
Nap at different concentrations and the second mixture also contained equal molar of Phe, Pyr and Nap at different concentrations
with the addition of 20 µM of B[a]P. Figure 4A shows MN formation and cytostasis induced by the Phe/Pyr/Nap mixture. Increased
level of cytostasis by the Phe/Pyr/Nap mixture was observed but
not much difference in MN formation was found when compared
with that of the control. Cytostasis and MN frequencies induced by
the mixture of Phe/Pyr/Nap/B[a]P were compared with single B[a]P
exposure as shown in Figure 4B. Cytostasis and MN frequencies by
this PAHs mixture were significantly increased with no significant
differences between the concentrations.
MN formation and cytostasis by individual and
mixed heavy metal/loids
Results of MN formation and cytostasis in HepG2 cells treated with
single heavy metal/loids As, Cd, Cr and their mixture are presented
in Figure 6. Arsenic induced slight cell cytostasis but significant MN
frequencies at 1.0 µM as shown in Figure 6A. Exposure to Cd at
0.5 µM and above resulted in significant MN formation (Figure 6B).
Cr caused MN frequencies at 2.0 µM as shown in Figure 6C. The
cytostasis and MN frequencies induced by heavy metal/loids was
in the order of Cd > Cr > As and Cd > As > Cr, respectively. Much
higher cytostasis and MN formation were found when cells were
treated with the mixture of As, Cd and Cr compared with that by
individual compounds as shown in Figure 6D.
MN formation induced by mixtures of PAHs and
heavy metal/loid(s)
MN formation and cytostasis in HepG2 cells treated with the mixtures of heavy metal/loids and B[a]P comparing to that by B[a]P only
are shown in Figure 7. When combined with B[a]P (20 µM), As caused
increased cytostasis and significant MN frequencies at all concentrations as shown in Figure 7A, but MN formation was not significantly
different between the mixtures and B[a]P alone. Exposure of HepG2
cells to the mixture of Cd and B[a]P led to significant increased formation of MN (Figure 7B). Furthermore, when Cd at 1.0 µM, the
Cd/B[a]P mixture caused significantly higher MN frequencies than
that by B[a]P. Similar to the effect of the As/B[a]P mixture, the mixture of Cr and B[a]P induced significant MN at all concentrations of
Cr with no difference between the MN induction by Cr/B[a]P mixture and B[a]P as shown in Figure 7C. The highest level of MN and
cytostasis was induced by the combination of metal/loid(s) mixture
(Cd/Cr/As) and B[a]P as shown in Figure 7D. In addition, the metal/
loid(s) mixture (Cd/Cr/As) at 0.5 µM when mixed with B[a]P induced
110 MN/1000 BNC, significantly higher than that by B[a]P.
Discussion
AhR induction by single and mixed PAHs
compounds
AhR inductions by single and mixed PAHs are shown in Figure 5.
Exposure of pGreen1.1/HepG2 cells to individual Phe, Pyr and Nap
and their mixture for 72 h caused no induction of AhR-dependent
EGFP activity. Treatment of B[a]P at concentrations between 0.01
and 20 µM resulted in a concentration-dependent increase in AhR
induction. PAHs mixture of Phe, Pyr and Nap with B[a]P showed
almost the same induction pattern of AhR as B[a]P did alone.
PAHs and heavy metal/loids are contaminants commonly found
co-existing in the environment such as sites near gas-works plants.
Individual toxicity of PAHs and heavy metals has been studied extensively, but their joint effects have been rarely addressed.
Therefore, in this study, we evaluated the MN formation in HepG2
cells exposed to single and mixed compounds of PAHs and heavy
metal/lids for 24 h using the CBMN assay. In order to get proper
treatment concentrations for the interaction study, we initially tested
the cytotoxicity of these compounds using MTS assay for which the
Combined genotoxicity of heavy metals/loids and PAHs, 2015, Vol. 30, No. 5
597
Figure 4. MN formation and cytostasis in the HepG2 cells exposed to PAH
mixtures at various concentrations and comparison with that induced by B[a]P
(20 µM). No obvious MN formation was found in the HepG2 cells exposed to PAH
mixture, Phe/Pyr/Nap (A). PAH mixture of Phe/Pyr/Nap/B[a]P caused significant
induction of MN (B). The MN formation was analysed from at least 1000 BNC
of each treatment. Cell viability (%) was calculated from the CBPI. The number
of MN in 1000 BNC (MN/1000 BNC) was used to express the MN frequency.
The MN frequency was expressed as mean ± SEM from three independent
experiments. *P < 0.05 was considered to be statistically significant.
Figure 3. Assessment of MN formation and cytostasis in the HepG2 cells
exposed to individual PAHs compounds Phe, Pyr and Nap at various
concentrations using CBMN assay. No obvious MN formation was found
in the HepG2 cells exposed to Phe (A) and Pyr (B). Nap (C) induced higher
MN frequencies. The MN formation were analysed from at least 1000 BNC of
each treatment. Cell viability (%) was calculated from the CBPI. The number
of MN in 1000 BNC (MN/1000 BNC) was used to express the MN frequency.
The MN frequency was expressed as mean ± SEM from three independent
experiments. *P < 0.05 was considered to be statistically significant.
cells were treated with the testing compounds at various concentrations for 24 h. Compared with PAHs, heavy metal/loids elicited
higher toxicities in the order of Cd > Cr > As (Figure 2A). The results
are in agreement with that from previous studies (49,50).
Evaluation of MN formation was performed using the CBMN
assay for which HepG2 cells were exposed to the compounds for
24 h followed by further culture with Cyto B for 48 h. The timing of
treatment and harvest for MN test may be different depending on
cell proliferation rate of the cells (or doubling time), which varies
with the cell type and culture conditions. HepG2 cells have been
used for MN test in many studies most of which used 24 h as the
treatment time as we did in this study. It has been reported that the
doubling time of HepG2 cells varies from 19 to 48 h depending on
the culture condition including the concentrations of glucose in the
medium (51,52). For the recovery time of 48 h used in this study, we
referred to the OECD MN test guideline 487 (37), which suggests
to harvest cells 1.5–2 cell cycle later. The same time for harvest was
used by another study using HepG2 cells (53).
598
B[a]P is a well-known carcinogen to animals and humans. It has
been proved that DNA-adduct formation and resultant gene mutations is one of the main pathways for its genotoxicity (54). However,
B[a]P-induced ROS stress and DNA breaks also play an important
role in its genotoxicity (55,56). Unrepaired DNA breaks may result
in MN during the anaphase (41). MN formation by B[a]P has been
shown in vivo and in vitro models (57,58). Both Phe and Pyr are noncarcinogenic to humans according IARC classification with controversial results from in vitro genotoxicity studies (59–62). We did not find
increased MN level in the HepG2 cells treated with Phe and Pyr even
at a higher concentration of 1000 µM (Figure 3A and B). Nap is a
possible carcinogen to humans according to IARC classification, but
animal and in vitro data gave mixed results in vitro for its genotoxic
effects (63,64). Recent studies showed that Nap can cause significant
DNA damages in human lymphocytes (65) and human TK6 cells (66).
A recent study reported significant MN in human TK6 cells induced
by Nap at lower concentrations between 2.5 and 10 µM (66). We
observed insignificantly increased level of MN formation in HepG2
cells treated with Nap (Figure 3C). Induction of MN in HepG2 cells by
Nap at higher concentration observed in this study was possibly due to
higher DNA repair capacity of HepG2 cells than that of TK6 cells (67).
We then examined the MN frequencies in cells exposed to PAHs
mixtures at various concentrations with and without B[a]P (20 µM).
Mixture of Phe/Pyr/Nap treatment did not cause increased MN frequencies when compared with the negative control (Figure 4A),
but the mixture containing B[a]P induced significant level of MN,
which was similar to that by B[a]P itself (Figure 4B). The result suggested that the additions of Nap, Phe and Pyr have no effects on B[a]
P-induced MN.
C. Peng et al., 2015, Vol. 30, No. 5
We also evaluated the induction of AhR by single and mixtures of
PAHs using pGreen1.1/HepG2 cells. The AhR is a ligand-dependent
basic helix-loop-helix transcription factor that mediates the toxic and
biological effects of a variety of environmental chemicals by regulating
the induction of the gene expression through ligand binding, translocation of the liganded:AhR into the nucleus and its interaction with specific
regulatory DNA elements (dioxin-responsive elements) that stimulate/
repress promoter activity and gene transcription (48). AhR has been
shown to be mediated with the CYP1A1 induction and toxic effects
Figure 5. Induction of AhR-dependent EGFP activity in the HepG2 cell by
single and mixed PAHs compounds. pGreen1.1/HepG2 cells were incubated
with single and mixed PAH compounds for 72 h and EGFP activity determined
in a microplate fluorometer. Fluorescence intensity was measured by
FLUOstar plate reader as described in the Materials and methods. Results
shown represent the mean ± SD of the EGFP activity of three replicates
(expressed as RFUs) after subtraction of the background EGFP activity
present in the control solvent (1% DMSO) exposed cells.
Figure 6. Assessment of MN formation and cytostasis in the HepG2 cells exposed to individual and mixed heavy metal/loids using CBMN assay. Heavy metal/
loids of As (A), Cd (B) and Cr (C) and mixture of As/Cd/Cr (D) induced significant cytostasis and MN formation. The MN formation was analysed from at least 1000
BNC of each treatment. Cell viability (%) was calculated from the CBPI. The number of MN in 1000 BNC (MN/1000 BNC) was used to express the MN frequency.
The MN frequency was expressed as mean ± SEM from three independent experiments. Significant differences were set at *P < 0.05 and **P < 0.01.
Combined genotoxicity of heavy metals/loids and PAHs, 2015, Vol. 30, No. 5
599
Figure 7. MN formation and cytostasis in the HepG2 cells exposed to mixtures of heavy metal/loids and B[a]P and comparison with that induced by B[a]P (20 µM).
Significant induction of MN was caused by the treatment of As/B[a]P (A), Cd/B[a]P (B), Cr/B[a]P (C) and As/Cd/Cr/B[a]P (D). The MN formation were analysed from
at least 1000 BNC of each treatment. Cell viability (%) was calculated from the CBPI. The number of MN in 1000 BNC (MN/1000 BNC) was used to express the MN
frequency. The MN frequency was expressed as mean ± SEM from three independent experiments. Significant differences were set at *P < 0.05 and **P < 0.01.
of high molecular PAHs and used as a measurement tool for monitoring PAHs compounds in environmental matrices including food (68).
The results showed the degrees of AhR induction by PAHs mixtures are
mainly dependent on the most potent compounds, i.e. B[a]P in the mixture (Figure 5). The similar induction pattern of AhR by PAHs mixtures
partially confirmed the results of MN by PAHs mixture as expression
of AhR has been shown to up-regulate the CYP1A1 expression (42)
and associate with DNA damage (43,44). Our results indicated that the
genotoxicity of PAHs in terms of MN formation was predominated by
the compound, which has the most genotoxic potency such as B[a]P.
However, the joint effects may be dependent on the compounds in the
mixtures because previous studies showed an increased DNA damage
by PAH mixtures containing other compounds with genotoxic potency
such as benzo[b]fluoranthene and dibenz[a,h]anthracene (69,70).
Cd, Cr and As are among the most harmful heavy metal/loids
commonly found in the environment according to the Environmental
Protection Agency and the Agency for Toxic Substances and Disease
Registry (71–73) and have been classified as human carcinogens by
IARC (74). Results from this study indicated that Cd induced prominent inhibition of cell division, followed by Cr and As (Figure 6B).
The degree and order of the cytostasis by these compounds correlated
well with the results from toxicity testing using the MTS assay, which
may suggest the inhibition of cell division is one of the main pathways
leading to their cytotoxicities. The mixture of As, Cd and Cr induced
high levels of cytostasis in a dose-response manner when compared
with that of a single compound (Figure 6D). The elevated cytostasis
by the mixture of the compounds indicates a potential additive effect
of cell division inhibition. The detailed combined effects and mode of
action of these metal/loids are being studied in our group. All of these
compounds caused significant MN formation as shown in Figure 6,
which was expected because they have been shown to induce DNA
damages in various in vitro systems (75–78).
We then examined the combined effects of single metal/loids mixed
with B[a]P and found the highest frequency of MN in the cells exposed
to the mixture of Cd/B[a]P (Figure 6). Increased MN frequencies by
single meal/loids with B[a]P suggest possible additive effects. It was
proposed that heavy metal/loids including As, mercury, lead, Cd, Cr,
copper and vanadium may decrease the carcinogenic potency of PAHs
because they have been shown to inhibit the PAH-induced CYP1A1
(16,19,79,80). CYP1A1/2 is the main P450 enzyme for transformation of B[a]P to reactive metabolites that can bind to DNA to form
DNA adducts. A very recent study reported that Cd decreased DNA
adducts by B[a]P (81). However, the same study also found that higher
levels of gene expression in Nrf2 antioxidant pathway and total
glutathione when cells co-exposed to Cd and B[a]P, which indicates
additive or synergistic effects of heavy metal/loids and B[a]P on ROS
formation. Heavy metal/loids have been known to induce DNA damages and carcinogenesis mainly mediated by ROS formation and stress
(82–84), which is also one of the main pathways for B[a]P (85,86).
In this respect, heavy metal/loids may interact with B[a]P resulting in
an enhanced oxidative stress and consequent DNA damages and MN
formation as observed in this study. When cells were exposed to the
mixture containing Cd/Cr/As and B[a]P, we found unexpectedly significant MN levels occurred at lower concentration (0.25 µM) and was
similar to that of Cd/B[a]P. Reduced MN levels when the concentrations of metal/loids increased in the mixture, which indicates the combined effects of the metal/loid and B[a]P mixture, is dose-dependent
(Figure 7D). Several factors may contribute to this result. This study
600
(data not shown) showed there are interactions among these metal/
loids compounds, which may affect their bioavailability and speciation
of these compounds in the cells. These interactions may regulate their
combined effects (87). Besides, we observed the exposure of mixture of
metal/loids and B[a]P caused higher level of cytostasis of HepG2 cells,
which may also contribute the results observed. The data obtained in
this study were not enough to set up a model for characterising the
combined genotoxic effects of heavy metal/loids and PAHs. Further
work needs to be conducted to understand the mechanisms behind
the combined effects of heavy metal/loids and PAHs. In addition, it
is noted that ortho/para-quinones and other PAH metabolites are
important for PAHs-induced toxicity and genotoxicity. It can be speculated that the amount of these metabolites may change when PAH
compounds co-exist with heavy metal/loids and exposed to the same
cells. This study is being carried out by our group using mass spectroscopic methods including Gas Chromatography with triple quadrupole
Mass Spectrometer and Orbitrap Liquid Chromatography with Mass
Spectrometer (Orbitrap LC-MS) and will be reported in due course.
In conclusion, we compared single and mixed heavy metal/loids and
PAHs for their genotoxicity in HepG2 cells. Based on the results, we
tentatively propose that the combined potency of DNA damage of the
PAHs mixture of, in our case, B[a]P, Nap, Phe and Pyr is dependent on
the compound with the most potency in the mixture which is the B[a]P.
For the mixture of heavy metal/loids and PAHs (Cd/Cr/As/B[a]P), the
combined effects may be more genotoxic in a compound- and concentration-dependent manner. The compounds with highest potencies of
genotoxicity in the mixture seem dominant as driving sources in the
final combined genotoxic effect of PAHs and heavy metal/loids.
Funding
The project was funded by Cooperative Research Centre for
Contamination Assessment and Remediation of the Environment
(CRC CARE, 3.1.01.11-12).
Acknowledgement
Entox is a partnership between Queensland Health and The University of
Queensland.
Conflict of interest statement: None declared.
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