Previous studies (Burns et al. 2004; Rossman et al. 2001) showed
that arsenite markedly increased the cancer incidence of solar-simulation
ultraviolet radiation (UVR) in hairless mice. The relationship
between arsenite concentration in drinking water and the yield
of squamous cell carcinomas in UVR-exposed mouse skin was linear
up to 5 mg/L. UVR is a complete carcinogen that readily induces
skin cancer (Zhuang et al. 2000). Although UVR induces a wide range
of DNA damage, such as protein-DNA crosslinks, oxidative base damage,
single-stand breaks, and double-strand breaks (de Gruijl et al.
2001), the major DNA damage is cyclobutane pyrimidine dimers (CPDs)
and 6-4 photoproducts (6-4PPs). Both of the latter lesions are
repaired by nucleotide excision repair (NER) (Mitchell et al. 1985).
Failure to repair CPDs and 6-4PPs leads to the signature mutations
containing CC to TT and C to T transitions (Miller 1985; Ziegler
et al. 1993) that are common in UVR-induced human skin cancers
but not in other epithelial cancers (Brash et al. 1996). UVR can
stimulate many signal transduction pathways (Bender et al. 1997)
that may contribute to skin cancer progression.
Exposure to arsenite is associated with skin, bladder, lung,
and probably kidney and liver cancers in humans (Rossman 2003).
Studies of human cell lines have demonstrated that arsenite can
reduce NER capacity by inhibiting the DNA incision step (Hartwig
et al. 1997). Inhibition of the ligation step in base excision
repair (Li and Rossman 1989) by arsenite has also been reported.
Arsenite is known to alter the methylation status of the cell,
which affects expression of a variety of genes (Zhao et al. 1997).
Arsenite also shows high affinity toward vicinyl sulfhydryl groups,
including zinc finger proteins identified as transcription factors
and several DNA repair enzymes (Hartwig 2001).
The aim of the present study was to examine how arsenite affected
the solar-simulation UVR-induced apoptosis and photodamage repair
in mouse keratinocyte line 291.03C in vitro. The results
showed that arsenite reduced the repair rate of 6-4PPs by about
50% at 5.0 µM but not at 2.5 µM and inhibited apoptosis
in mouse keratinocyte cell line 291.03C.
Cell culture. Mouse keratinocyte cell line 291.03C,
generously provided by M. Kulesz-Martin, was grown in Eagle’s
minimum essential medium (EMEM; Mediatech Cellgro, Herndon, VA)
with nonessential amino acids (Cellgro) containing 5% fetal bovine
serum, 1% antibiotic-antimycotic, and 10 ng/mL epidermal growth
factor at 37°C in 5% CO2. The 291.03C line is a
7,12-dimethylbenz[a]anthracene-initiated clone derived from
nontransformed 291 cells (Kulesz-Martin et al. 1985). It is a precursor
of squamous cell carcinoma, which was selected on the basis of
its ability to resist extracellular Ca2+-induced terminal
differentiation, and has distinctive keratinocyte morphology. The
gene expression patterns are substantially similar between the
nontransformed cell line 291 and 291.03C. The p53 protein in 291.03C
is wild type, even if its function is somewhat compromised (Wang
et al. 2002).
Ultraviolet irradiation. The UVR source was a bank
of four FS20 sunlamps (Westinghouse, Bloomfield, NJ) mounted in
parallel 15 cm apart. The UVR intensity was measured (30 cm below
the source)
by a calibrated radiometer/photometer
(model IL1400A; International Light Inc., Newburyport, MA). According to manufacturer
specifications, 85% of the lamp output was in the UVB (290-320 nm) range, < 1%
was in the UVC (200-290 nm) range, 4% was in the UVA (320-400 nm) range, and
the remainder was in the visible (> 400 nm) range.
For assays of photodamage repair, apoptosis, and caspase activity,
we seeded 2 106 291.03C
keratinocytes in 100 mm culture dishes and incubated them for 24
hr with different concentrations (0.0, 2.5, and 5.0 µM) of
sodium arsenite (Sigma, St. Louis, MO) beginning at about 70% of
confluence. At 24 hr the cells were washed with Dulbecco’s
phosphate-buffered saline (DPBS; Sigma) twice and exposed to UVR
in the presence of 5 mL DPBS.
Colony survival assay. Mouse 291.03C keratinocytes
were seeded at a density of 300 cells/60 mm dish in EMEM. After
24 hr, sodium arsenite was added to the medium from a freshly prepared
stock solution to final concentrations of 0.0, 0.05, 0.1, 0.5,
1.0, and 5.0 µM, and the cells were incubated for 7 days,
followed by fixation in methanol and then staining with 0.5% crystal
violet in 50% methanol. We counted colonies and determined the
percentage of survival as the ratio of treated to control 100.
The survival after exposure to solar-simulation UVR was determined
similarly at 7 days after single doses of 0.0, 0.05, 0.10, 0.20,
and 0.30 kJ/m2.
Measurement of CPDs and 6-4PPs in genomic DNA by ELISA. We
isolated genomic DNA using the QIAamp Blood Kit (QIAGEN Inc., Valencia,
CA). DNA concentrations were calculated from the absorbance at
260 nm measured by a Beckman DU 650 spectrophotometer (Beckman
Instruments, Fullerton, CA). We determined the quantities of CPDs
and 6-4PPs by enzyme-linked immunosorbent assay (ELISA) as described
by Mori et al. (1991). In brief, Falcon polyvinylchloride flat-bottom
96-well assay plates (Becton Dickinson Labware, Franklin Lakes,
NJ) precoated with 1% protamine sulfate (Sigma) were incubated
with purified genomic DNA (15 ng for CPD detection and 150 ng for
6-4PP detection) in PBS at 37°C for 20 hr. For CPD detection,
we used the TDM-2 antibody, and for 6-4PP detection we used the
64M-2 antibody (both antibodies were generously provided by T.
Mori, Nara, Japan). After adding biotinylated F(ab´)2 goat
anti-mouse IgG fragments and streptavidin-peroxidase (Zymed, San
Francisco, CA), we measured the optical density from o-phenylene
diamine at 492 nm using a Bio Assay Reader HTS7000 (Perkin-Elmer
Corp., Norwalk, CT). The percentage of the initial number of photoproducts
was calculated at various times after UVR exposure by using standard
curves obtained from DNA samples irradiated with UVR doses of 0.0,
0.06, 0.12, 0.24, and 0.36 kJ/m2.
Measurement of apoptosis by flow cytometry. Apoptosis
was detected using the APO-BRDU kit (Phoenix Flow Systems, Inc.,
San Diego, CA) following the protocol provided by the manufacturer.
Briefly, attached cells were harvested by trypsinization and combined
with free-floating cells that were harvested by centrifugation.
After washing with PBS, the cells were fixed with 1% paraformaldehyde
in PBS, followed by fixation with 70% ice-cold ethanol overnight.
The fixed cells were washed twice with wash buffer included in
the kit, and freshly prepared DNA labeling solution [containing
terminal deoxynucleotidyl transferase and 5-bromo-2´-deoxyuridine
(BrdU) 5´-triphosphate] was added to the cell pellet and
incubated overnight at room temperature. Cells were labeled by
fluorescein isothiocyanate-conjugated monoclonal antibody to BrdU
(FITC~PBR-1 mAb), washed again, resuspended in staining solution
containing propidium iodide and RNase, and incubated for 30 min
at room temperature. After this, cells were immediately analyzed
using a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter, Miami,
FL). We calculated the percentage of R1 (normal), R2 (apoptotic
without loss of DNA), and R3 (hypodiploid) cells using EXPO32 Multifile
software (Beckman Coulter).
Measurement of caspase-3/7 activity. We measured
Caspase-3/7 activity using the Apo-one Homogenous Caspase-3/7 Assay
(Promega, Madison, WI) following the protocol provided by the manufacturer.
In brief, cells were trypsinized, and 20,000 cells/sample were
mixed with the same volume of the Apo-one Homogenous Caspase-3/7
reagent. After incubation at room temperature for 2 hr, caspase-3/7
activities were estimated from the fluorescence of each sample
at the excitation wavelength of 485 nm and the emission wavelength
of 535 nm using the Bio Assay Reader HTS7000. EMEM mixed with the
same volume of Apo-one Homogenous Caspase-3/7 reagent served as
a negative control.
Toxicity of arsenite and UVR to 291.03C mouse keratinocytes. Figure
1 shows the effects of arsenite (Figure 1A) and solar-simulation
UVR (Figure 1B) on clonal survival of 291.03C mouse keratinocytes.
The median lethal dose (LD
50) of UVR was 0.05 kJ/m
2.
There was no measurable colony survival at UV doses > 0.30
kJ/m
2.
The median lethal concentration (LC
50) of sodium arsenite
was 0.9 µM. Arsenite did not show significant lethality < 0.5 µM
and showed nearly 100% lethality > 5.0 µM.
In a previous study (Burns et al. 2004), hairless mice were fed
sodium arsenite in drinking water at concentrations ranging from
1.25 mg/L (9.6 µM) to approximately 10 mg/L (77.0 µM),
and solar spectrum UVR exposure was applied to the dorsal skin
at 1.0 kJ/m2 three times weekly. The arsenite concentrations
and solar UVR dose used in the present in vitro study were
2.5 and 5 µM and 0.3 kJ/m2. These two arsenite
concentrations were estimated to be equivalent to 26 and 52% of
the lowest arsenite concentration (1.25 mg/L) used in the in
vivo carcinogenesis study (Burns et al. 2004).
Arsenite effects on DNA photodamage repair. The
two photolesions, CPDs and 6-4PPs, were detected by ELISA. The
6-4PPs were 80% removed by 12 hr, whereas CPDs were not removed > 10%
by 24 hr (Figure 2). According to the regression analysis of the
data, arsenite showed no significant effect on CPDs repair. The
6-4PP repair rate after UVR was 11.95%/hr; when combined with 2.5 µM
or 5.0 µM arsenite, the 6-4PP repair rates were 11.3%/hr
and 6.19%/hr, respectively. Arsenite slowed the 6-4PP repair rate
by 48% at 5.0 µM, but no difference was detected at 2.5 µM.
Arsenite inhibits UVR-induced apoptosis. Figure
3 shows that at 24 hr after UVR alone (0.30 kJ/m2) the
percentage of apoptotic cells was 27.6% (Figure 3D). When UVR-treated
cells were incubated in 2.5 µM or 5.0 µM arsenite,
the percentage of apoptotic cells decreased to 21.4% (77.36% of
UVR only; Figure 3E) and 10.5% (38.1% of UVR only; Figure 3F),
respectively. Untreated control cells showed few apoptotic cells
(Figure 3A), whereas 5.0 µM arsenite only showed 4.9% apoptotic
cells at 48 hr after treatment (Figure 3B) and 8.1% at 60 hr (Figure
3C) after treatment. Apoptosis was not detected at 0 hr and 14
hr after UVR and was 51.56, 39.42, and 36.47% at 36 hr after UVR,
UVR + 2.5 µM arsenite, and UVR + 5 µM arsenite, respectively
(data not shown), indicating that apoptosis is progressing with
the time. As shown in Figure 3, the R3 population was 5.28% (UVR
alone), 3.71% (UVR + 2.5 µM arsenite), and 1.32% (UVR + 5.0 µM
arsenite), indicating that apoptosis is more extensive after treatment
with UVR alone compared with UVR plus arsenite.
The caspase-3/7 activities 24 hr after UVR are shown in Figure
4. Arsenite decreased the UVR-induced caspase-3/7 activity to 88.48%
at 2.5 µM and to 58.83% at 5 µM. Arsenite alone did
not affect the caspase level significantly. These results are consistent
with the results in Figure 3 indicating arsenite inhibited UVR-induced
apoptosis (Figure 4).
The photoproducts (CPDs and 6-4PPs) produced by UVR may lead
to mutations and cancer development if the damage is not removed
from the DNA. There are two mechanisms for a cell to remove DNA
damage: repairing the DNA damage or inducing apoptosis. Arsenite
indeed increased the mutagenicity of UVB in Chinese hamster V79
cells (Li and Rossman 1991). As reported here, mouse keratinocytes
did not repair UVR-induced CPDs efficiently, and arsenite did not
affect the DNA photodamage repair rates significantly. The apoptosis
inhibiting activity of arsenite may have converted a greater amount
of DNA damage to mutations without substantially affecting DNA
repair. If so, these findings might help to explain why skin cancer
in mice is markedly increased by prolonged exposure to the combination
of UVR and dietary arsenite.
Although it has been reported that arsenite inhibits DNA repair
in a variety of cell types (Hartwig et al. 1997; Li and Rossman
1989; Yager and Wiencke 1997), the present study in a mouse keratinocyte
cell line (Figure 2) shows little effect of arsenite on the removal
of UVR-induced photoproducts from the genomic DNA except the reduced
6-4PP repair rate at 5 µM. In normal human epidermal keratinocytes
(NHEK, Cambrex BIO Science, Walkersville, MA), 6-4PPs were removed
at a rate of 30%/hr, whereas CPDs were removed at a rate of 2%/hr
after 0.3 kJ/m2 solar-simulation UVR (data not shown).
The mouse 291.03C keratinocyte line exhibited a 6-4PP removal rate
of 13%/hr and a CPD removal rate of < 0.4%/hr (Figure 2). The
repair rates of mouse keratinocytes were not > 20% for CPDs
and > 40% for 6-4PPs compared with those of human keratinocytes.
There are two subpathways of NER: transcription-coupled repair
(TCR) and global genomic repair (GGR). TCR refers to the preferential
repair of transcribed strands of active genes, and GGR refers to
repair anywhere else in the DNA. Many rodent cells have normal
TCR, which is very important for clonal survival, but are deficient
in GGR of CPDs, which is more important for suppressing mutagenesis
(Hanawalt 2001). Because the ELISA method used here detects GGR,
these results confirm that mouse keratinocyte line 291.03C performs
GGR of DNA photodamage less efficiently than do human keratinocytes.
UVR can trigger apoptosis by damaging the DNA and activating
the death receptors on the cell surface (Kulms and Schwarz 2000).
Arsenite can induce Fas/FasL-dependent apoptosis at higher concentrations
(≥ 5.0 µM) in primary
human keratinocytes (Liao et al. 2004). The results reported here
show that 5.0 µM arsenite produced a small increase (8%)
in apoptosis after 60 hr of treatment, whereas a single 0.30 kJ/m2 dose
of solar-simulation UVR produced 27% apoptosis by 24 hr and 51%
by 36 hr, indicating that apoptosis increased gradually after exposure
to UVR. Paradoxically, arsenite at the 5.0 µM concentration
produced a small incidence of apoptosis by itself, but it was inhibitory
when combined with a strongly apoptotic dose of UVR (Figure 3).
The caspase results generally confirmed the apoptosis results obtained
by flow cytometry. At 36 hr after UVR + 5.0 µM arsenite,
apoptosis increased to 37% (30% less than UVR alone), indicating
that arsenite delayed the onset of the apoptosis but did not prevent
it completely.
In a previous study of Chinese hamster V79 cells (Li and Rossman
1991), the combination of UVB (0.2 kJ/m2) and sodium
arsenite (10 µM and 15 µM) increased the mutation rates
by 1.65-fold and 2.06-fold respectively, whereas survival was decreased
to 43 and 11.8%, respectively. The inhibition of apoptosis may
help to explain the higher mutation rates in the presence of arsenite.
In conclusion, arsenite lessened the rate of DNA repair and inhibited
apoptosis at 24 hr after a single exposure of mouse keratinocyte
line 291.03C to 0.3 kJ/m2 of solar-simulation UVR.
The consequences of this delay on mutation rates will be investigated
in future studies.