Radiol Oncol 2002; 36(2): 156-8. 

Cadmium induced DNA damage in human hepatoma (Hep G2) 
and Chinese hamster ovary (CHO) cells 

Tanja Fatur and Metka Filipič 

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, 
Večna pot 111, 1000, Ljubljana, Slovenia 


Introduction 

Cadmuim (Cd) is one of the most important 
heavy metal environmental toxicants. It accumulates 
in human tissues, particularly in kidney 
and liver. Cd is classified as probable human 
carcinogen by IARC.1 The genotoxic 
potential of Cd is rather weak and restricted to 
high cytotoxic concentrations. However, at 
low concentrations Cd enhances genotoxicity 
of other DNA damaging agents.2 It was shown 
that Cd interferes with nucleotide excision repair 
(NER) by inhibiting DNA damage recognition 
and incision step of NER.3 The aim of the 
study was explore the DNA damaging potential 
of Cd and its interference with the repair of 
UV induced DNA damage in vitro, using Comet 
assay. We studied the induction of DNA 
single strand breaks (ssb) by low nanomolar 
concentrations of CdCl2 after different duration 
of exposure on human hepatoma cell line 
(Hep G2). The effect of CdCl2 on the repair of 
UV induced DNA damage was studied in Chinese 
hamster ovary cells (CHO). The results of 
the formation and disappearance of DNA ssb 
after different periods of recovery after the UV 
irradiation, reflecting the nucleotide excision 
repair (NER) kinetics, are presented. 

Materials and methods 

Cell lines 

Human hepatoma cells (HepG2) cells were 
cultured in Williams Medium containing 10 % 

FBS at 37 °C in humidified atmosphere with 
5% CO2. Chinese hamster ovarian (CHO) 
cells were cultured in F-12 medium containing 
10% FBS at 37°C in humidified atmosphere 
with 5 % CO2. 

Treatment of cells 

HepG2 cells were incubated with low, non-toxic 
concentrations of CdCl2 (10 nM, 100 nM 
and 1000 nM) in complete growth medium 
for 3, 6, 9, 12, 24 and 72 h. After the incubation 
period the cells were harvested and subjected 
to alkaline single cell electrophoresis 
(Comet assay). CHO cells were seeded in 25 
cm2 tissue culture flasks one day prior to treatment 
with Cd. Five hours prior to the UV irradiation 
the cells were treated with 1 µM and 
10 µM CdCl2 in complete growth medium. After 
the treatment the cells were washed with 
PBS, harvested, suspended in PBS and UV irradiated 
(20’’, 50-cm distance). The irradiated 
cells were subjected to Comet assay after the 
recovery period of 0, 10, 20, 30 and 60 minutes 
at 37 °C. 

Comet assay 

The cells (HepG2 or CHO) were embedded in 
1 % LMP agarose on pre-coated microscope 
slide and lysed at 4 °C for 1 h (2.5M NaCl, 
100mM EDTA, 10 mM Tris, 1 % Triton X-100, 
pH10). The slides were then placed in the 
electrophoresis solution (300 mM NaOH, 
1 mM EDTANa2, pH13) for 20 min at 4 °C to 


Fatur T et al. / Cadmium induced damage on cells 

allow DNA unwinding and electrophoresed 
for 20 min at 25V (300 mA). After electrophoresis 
slides were neutralized (0,4 M Tris, pH 
7,5) and stained with EtBr. 50-100 cell nuclei 
per each experimental point were examined 
at 400 magnification using a fluorescence microscope 
(Olympus) and analyzed with the 
software VisCOMET. The DNA damage is expressed 
as comet tail moment, which is defined 
as product of the comet length and percentage 
of DNA in tail. 

Results and discussion 

Figure 1 shows the effects of low non-toxic 
concentrations of CdCl2 on DNA damage in 
HepG2 cells. Level of DNA damage was assessed 
after different periods of incubation 
with CdCl2 by measuring the increase of comet 
tail moments. Incubation with 10 nM, 
100 nM and 1000 nM CdCl2 caused a dose-
dependent increase of DNA damage. The 
DNA damage increased also with the increasing 
time of exposure to CdCl2 up to 12 h. 
However, when the cells were incubated for 
24 h the DNA damage was lower than after 
12 h incubation. After 72 h incubation DNA 
damage was detected only in cells treated 
with 1000 nM CdCl2. This time dependent 
decrease of DNA damage can be due to the 
Cd mediated induction of the synthesis of 
metallothioneins, which are known to play 

Figure 1. Comet tail moment of HepG2 cells incuba-
ted with CdCl2 for 3, 6, 12, 24, and 72 h. 100 comets 
per experimental point were analysed by image analy-
sis system. 


role in cellular defence mechanism against 
Cd toxicity.4 

UV irradiation induces pyrimidine dimers 
and 6-4 photoproducts that are repaired predominantly 
by NER. With the Comet assay 
ssb are detected, which reflect the incision 
step of the NER. In CdCl2 pretreated cells the 
tail moment was lower than in control cells 
(Figure 2) indicating that CdCl2 prevented the 
incision step of NER. After 60 minutes of recovery 
the residual ssb were higher in CdCl2 
treated cells compared to the control, which 
might reflect either slower NER or inhibition 
of ligation step of NER. This result confirms 
the interference of Cd with NER. 


Figure 2. DNA repair kinetic of UV induced DNA damage 
in CHO cells. Non-treated or CdCl2 pre-treated 
cells were exposed to UV irradiation and than incubated 
at 37oC. At different intervals samples were taken 
for comet assay. 100 comets per experimental point 
were analysed by image analysis system. 

Conclusion 

In conclusion, our results in Hep2G cells showed 
that ssb were induced at 10 nM of CdCl2, 
which is the concentration that corresponds 
to the concentrations of Cd found in blood of 
environmentally exposed population.5 This 
damage was detected only after short time of 
exposure. In CHO cells, we demonstrated, 
that when cells are exposed to CdCl2 for a 
short time, the repair of UV induced DNA damage 
was inhibited. Further experiments are 
in progress to explore the role of metallothioneins 
in protection against genotoxic and cogenotoxic 
effects of Cd. 

Radiol Oncol 2002; 36(2): 156-8. 


Fatur T et al. / Cadmium induced damage on cells 

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Radiol Oncol 2002; 36(2): 156-8.