1670 Articles | JNCI Vol. 101, Issue 24 | December 16, 2009
Inorganic arsenic is a common contaminant of human drinking
water, and chronic arsenicosis affects tens of millions of people world-
wide ( 1 , 2 ). Arsenic is clearly carcinogenic in humans and has multiple
in vivo targets that include the skin, lung, bladder, prostate, and liver,
although the carcinogenic mechanisms remain incompletely defined
( 1 , 2 ). Chronic low-level exposure to arsenic compounds induces in
vitro malignant transformation of human and rodent cells derived
from similar organs ( 3 – 7 ). Inorganic arsenic also undergoes multistep
biomethylation in some, but not in all cells, through specific methyl-
transferases, notably arsenic – (+3 oxidation state) – methyltransferase
(AS3MT), using S -adenosyl- l -methionine as the methyl donor ( 8 –
10 ). Inorganic arsenic was once believed to be detoxified by biom-
ethylation ( 11 , 12 ), but more recent work has convincingly shown that
it is not ( 13 – 18 ). In fact, trivalent methylated arsenic compounds, such
as methylarsonous acid, are much more reactive and toxic than their
unmethylated forms and consequently have been hypothesized to be
the ultimate carcinogenic metabolites of inorganic arsenic ( 15 – 19 ).
Because of their reactivity, if the methylated forms of arsenic were to
Affiliations of authors: Inorganic Carcinogenesis Section, Laboratory of
Comparative Carcinogenesis, Center for Cancer Research, National Cancer
Institute at National Institute of Environmental Health Sciences, Research
Triangle Park, NC (CK, EJT, MPW); Laboratory of Pharmacology, Division of
Intramural Research, National Institute of Environmental Health Sciences,
Research Triangle Park, NC (DCR, RPM); Laboratory of Molecular Nutrition
and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima, Japan (SH); Department of Nutrition,
School of Public Health and of Medicine, University of North Carolina at
Chapel Hill, Chapel Hill, NC (ZD, MS) .
Correspondence to: Michael P. Waalkes, PhD, Inorganic Carcinogenesis
Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute
at National Institute of Environmental Health Sciences, PO Box 12233, Mail
Drop F0-09, 111 Alexander Dr, Research Triangle Park, NC 27709 (e-mail:
See “Funding” and “Notes” following “References.”
Published by Oxford University Press 2009.
Advance Access publication on November 23, 2009.
Requirement of Arsenic Biomethylation for Oxidative
Chikara Kojima , Dario C. Ramirez , Erik J. Tokar , Seiichiro Himeno , Zuzana Drobná , Miroslav Stýblo ,
Ronald P. Mason , Michael P. Waalkes
Background Inorganic arsenic is an environmental carcinogen that may act through multiple mechanisms including
formation of methylated derivatives in vivo. Sodium arsenite (up to 5.0 µ M) renders arsenic methylation –
competent TRL1215 rat liver epithelial cells tumorigenic in nude mice at 18 weeks of exposure and arsenic
methylation-deficient RWPE-1 human prostate epithelial cells tumorigenic at 30 weeks of exposure. We
assessed the role of arsenic biomethylation in oxidative DNA damage (ODD) using a recently developed
immuno-spin trapping method.
Methods Immuno-spin trapping was used to measure ODD after chronic exposure of cultured TRL1215 vs RWPE-1
cells, or of methylation-competent UROtsa/F35 vs methylation-deficient UROtsa human urothelial cells, to
sodium arsenite. Secreted matrix metalloproteinase (MMP)-2 and -9 activity, as analyzed by zymography,
cellular invasiveness by using a transwell assay, and colony formation by using soft agar assay were
compared in cells exposed to arsenite with and without selenite, an arsenic biomethylation inhibitor, to
assess the role of ODD in the transition to an in vitro cancer phenotype.
Results Exposure of methylation-competent TRL1215 cells to up to 1.0 µ M sodium arsenite was followed by a sub-
stantial increase in ODD at 5 – 18 weeks (eg, at 16 weeks with 1.0 µ M arsenite, 1138% of control, 95% confi-
dence interval [CI] = 797% to 1481%), whereas exposure of methylation-deficient RWPE-1 cells to up to 5.0 µ M
arsenite did not increase ODD for a 30-week period. Inhibition of arsenic biomethylation with sodium
selenite abolished arsenic-induced ODD and invasiveness, colony formation, and MMP-2 and -9 hyperse-
cretion in TRL1215 cells. Arsenic induced ODD in methylation-competent UROtsa/F35 cells (eg, at 16 weeks,
with 1.0 µ M arsenite 225% of control, 95% CI = 188% to 262%) but not in arsenic methylation-deficient
UROtsa cells, and ODD levels corresponded to the levels of increased invasiveness, colony formation, and
hypersecretion of active MMP-2 and -9 seen after transformation to an in vitro cancer phenotype.
Conclusion Arsenic biomethylation appears to be obligatory for arsenic-induced ODD and appears linked in some
cells with the accelerated transition to an in vitro cancer phenotype.
J Natl Cancer Inst 2009;101:1670–1681
be the ultimate carcinogens, methylation of arsenic would need to
occur at, or near, the target cells of carcinogenic initiation in vivo.
JNCI | Articles 1671
Arsenic has genotoxic potential and is associated with DNA
damage, such as chromosomal aberrations and sister chromatid
exchange ( 13 , 20 – 22 ). Several in vivo and in vitro studies have
shown that inorganic arsenic or methylarsonous acid induced the
formation of reactive oxygen species and increased 8-oxo-7,
8-dihydro-2 ′ -deoxyguanosine (8-oxo-dG) generation in vitro or in
vivo ( 13 , 22 – 24 ). Widely used as a biomarker of oxidative DNA
damage (ODD), 8-oxo-dG has been used as an indicator of geno-
toxicity by many chemicals, including arsenic ( 22 – 24 ). However, it
is diffi cult to reliably measure 8-oxo-dG levels by standard
methods, such as mass spectrometry, chromatography, or immu-
nohistochemistry, because artifactual adventitious oxidation gen-
erated during DNA extraction and sample preparation results in
high background readings ( 25 , 26 ) that often exceed any chemically
induced increases in 8-oxo-dG, creating major questions about the
relevance of relatively small chemically induced increases.
Furthermore, 8-oxo-dG – based methods do not directly measure
Recently, a new method was developed for detection of oxidative
DNA radicals using immuno-spin trapping (IST; 27,28). IST di-
rectly traps DNA radicals by formation of 5,5-dimethyl 1-pyrroline
N -oxide (DMPO) – DNA radical adducts, which are then converted
to stable DNA – nitrone adducts before DNA isolation and immuno-
chemical quantifi cation for a simple, sensitive, and reliable means of
ODD measurement. DNA radicals induced by reactive oxygen spe-
cies are short-lived species, with half-lives in the nanoseconds to
milliseconds because of their high reactivity, but when DMPO –
DNA radical adducts are formed and converted to DNA – nitrone
adducts, the half-lives are extended to months or years ( 27 , 28 ). Thus,
the measurement of ODD by the IST method circumvents many of
the potential errors of other methods heretofore in common use.
To address recent suspicions that arsenic biomethylation could
be important to its mechanism of carcinogenesis, we used the IST
method to assess the ability of chronic arsenite exposure to produce
ODD in a rat liver epithelial cell line, TRL1215, which methylates
arsenic ( 7 ), and in a human prostate cell line, RWPE-1, which
methylates arsenic only very poorly ( 3 , 29 ). We used levels of
chronic arsenite exposure (0.5 – 5.0 µ M) that are known to induce
malignant transformation in these cells by the appearance of a can-
cer phenotype in vitro (as defi ned by increased invasiveness,
anchorage-independent growth in soft agar, and hypersecretion of
matrix metalloproteinase [MMP]-2 and -9) and the ability to form
tumors in mice ( 3 , 7 ). We also directly tested the hypothesis that
arsenic-induced ODD and arsenic-induced acquisition of an in
vitro cancer cells phenotype are mediated by arsenic methylation by
comparing these features in human urinary bladder epithelial cells
(UROtsa) that do not methylate arsenic vs UROtsa-derived cells
expressing AS3MT (UROtsa/F35) that do methylate arsenic ( 30 ).
Cell Lines and Culture Conditions
The TRL1215 cell line is a rat liver epithelial cell line and was cul-
tured in William E medium (Gibco/Invitrogen, Carlsbad, CA)
containing 10% fetal bovine serum (Gibco/Invitrogen), 31 µ g/mL
penicillin, and 50 µ g/mL streptomycin. The RWPE-1 cell line
(passage 14) is a human prostate epithelial cell line kindly provided
by Dr Mukta M. Webber (Michigan State University) and was cul-
tured in keratinocyte serum-free medium (Gibco/Invitrogen) con-
taining 50 µ g/mL bovine pituitary extract (Gibco/Invitrogen) and 5
ng/mL epidermal growth factor (Gibco/Invitrogen) supplemented
with 1% antibiotic/antimycotic mixture (100 U/mL penicillin and
100 µ g/mL streptomycin). The UROtsa cell line is a human urothe-
lial cell line and the UROtsa/F35 cell line is the UROtsa cell line
transduced with the rat As3mt gene (30; both cell lines obtained
from laboratory of Dr M. Stýblo); both were cultured in Eagle
minimum essential medium (Gibco/Invitrogen) containing 10%
fetal bovine serum supplemented with 1% antibiotic/antimycotic
mixture. All cells were maintained in a humidified atmosphere of
5% CO 2 and 95% air at 37°C. Untreated TRL1215, RWPE-1, and
UROtsa cells are normally nontumorigenic ( 3 , 7 , 31 ). Phenotypically,
TRL1215, RWPE-1, UROtsa, and UROtsa/F35 all have typical
epithelial cell morphology in culture as previously reported ( 3 , 7 , 31 ).
Genotypically, TRL1215 cells are periodically verified as liver epi-
thelial cells with hepatocyte markers (P450 enzymes, metallothion-
ein), RWPE-1 cells are verified as prostate epithelial cells via
prostate-specific antigen, and UROtsa and UROtsa/F35 cells were
recently extensively verified via genomics ( 32 ).
CONTEXT AND CAVEATS
Metabolic conversion of inorganic arsenic to methylated arsenic
compounds was suspected to increase its carcinogenicity, but the
mechanism was unclear.
Two arsenic biomethylation – competent cell lines (TRL1215,
UROtsa/F35) and two arsenic biomethylation – deficient cell lines
(RWPE-1, UROtsa) were continuously exposed to up to 5.0 µ M in-
organic sodium arsenite for up to 55 weeks. Oxidative DNA
damage was measured by a new immuno-spin trapping proce-
dure, and arsenic-treated cells were subjected to invasion, colony
formation, and matrix metalloproteinase secretion assays as in
vitro measures of transformation.
Exposure of methylation-competent cells, but not methylation-
deficient cells, was followed by a sharp rise in oxidative DNA
damage. Subsequent to the peak of oxidative DNA damage, meth-
ylation-competent cells, more than methylation-deficient cells,
acquired the in vitro characteristics of transformed cells, including
growth in soft agar, increased invasiveness, and increased secre-
tion of matrix metalloproteinases. This coincided with the time at
which the cells became tumorigenic in nude mice.
Biomethylation of arsenic compounds appears to cause oxidative
DNA damage and to increase their carcinogenicity. Alternate mech-
anisms appear to work less efficiently in methylation-deficient
Methylated arsenicals were not directly measured. All work was
done in vitro in four cell lines, and in vivo correlates rested on pre-
vious experiments. In vivo testing in arsenic methylation-deficient
mice has not yet been performed.
From the Editors
1672 Articles | JNCI Vol. 101, Issue 24 | December 16, 2009
Chronic Arsenic Exposure and Cancer Phenotype
TRL1215, UROtsa, and UROtsa/F35 cells were continuously
exposed to 0.25, 0.5, or 1.0 µ M sodium arsenite (Sigma Chemical
Co, St Louis, MO), and RWPE-1 cells to 1.0, 2.5, or 5.0 µ M
sodium arsenite for various times in their respective media in
75-cm 2 tissue culture flasks. The medium was changed at half-
weekly intervals, and cells were passaged weekly with new cultures
seeded at 1 × 10 6 cells/flask. The concentrations of arsenite used
had no effect on cell growth. Acquisition of a transformed pheno-
type in vitro was defined by increased invasiveness, anchorage-
independent growth in soft agar, and hypersecretion of MMP-2
and -9, whereas malignant transformation was defined as all of
these changes in addition to the ability to produce malignant
tumors upon injection into nude mice.
DNA Isolation and ODD Measurement
ODD was measured in all cell lines by the previously established
IST method ( 26 , 27 ), which directly measures the formation of
DNA-centered radicals by adduction with DMPO, conversion to
stable nitrone adducts, and immunochemical quantification. After
growth in medium with or without sodium arsenite (0 – 5 µ M) for up
to 55 weeks, cells were incubated with 20 mM DMPO (Alexis
Biochemicals, San Diego, CA) for 30 minutes at 37°C, harvested by
incubation in trypsin – EDTA, and washed three times with calcium-
and magnesium-free phosphate-buffered saline (CMF-PBS). Cells
(approximately 1 × 10 6 ) were then incubated in 500 µ L of digestion
buffer (1% sodium dodecyl sulfate, 100 mM NaCl, 25 mM diethyl-
enetriaminepentaacetic acid, and 10 mM Tris – HCl; pH 8.0) con-
taining 25 µ L proteinase K (Sigma Chemical Co.; from a 20 mg/mL
stock solution of proteinase K in 50 mM Tris – HCl containing 1
mM CaCl 2 ) for 1 hour at 52°C. After the addition of 10 µ L RNase
A (20 mg/mL), the incubation continued for 1 hour at 37°C. DNA
was purified by a three-step procedure that used successive extrac-
tions in ultrapure buffer-saturated phenol (Invitrogen), 1 mM
diethylenetriaminepentaacetic acid, phenol:chloroform:isoamyl
alcohol (25:24:1), and chloroform:isoamyl alcohol (24:1) ( 27 , 28 ).
DNA was resuspended in 100 µ L TE buffer (10 mM Tris – HCl [pH
8.0], 1 mM EDTA), and its purity and concentration were deter-
mined by absorbance at 260 and 280 nm. This method produced
DNA preparations with an Abs 260/280 ratio between 1.8 and 2.0
( 27 , 28 ). The purified DNA containing the nitrone adducts was then
diluted to 5 µ g/mL in CMF-PBS, and 25 µ L of DNA solution were
mixed with 25 µ L of React-bind DNA coating solution (Pierce
Chemicals, Rockford, IL) in each well of flat-bottom 96-well
microtiter plates (PGC Scientifics, San Diego, CA). The reactions
were incubated for 4 hours at 37°C to allow the DNA to bind to the
wells, after which wells were washed once with 300 µ L washing
buffer (CMF-PBS containing 0.05% nonfat dry milk and 0.1%
Tween-20). To block nonspecific binding of antisera, 120 µ L of
blocking solution (CMF-PBS containing 3% nonfat dry milk) was
added to each well and incubated for 1.5 hours at 37°C. Each well
was then washed once for 5 minutes with 300 µ L washing buffer on
an orbital shaker at room temperature. Next, 100 µ L of rabbit anti-
DMPO polyclonal serum (1:10 000 dilution in washing buffer;
Cayman Chemical, Ann Arbor, MI) was added to each well and
incubated for 1 hour at 37°C. Plates were washed three times
with 300 µ L washing buffer, and 100 µ L of goat anti-rabbit IgG Fc
conjugated to horseradish peroxidase (1:10 000; Pierce) was added
and incubated for 1 hour at 37°C. After washing the plates three
times with 300 µ L washing buffer, 50 µ L of LumiGLO chemilumi-
nescent substrate (Upstate, Temecula, CA) was added to each well
and incubated for 30 seconds, and luminescence was read using
Xfluor4 Software (Tecan, Männedorf, Switzerland). ODD measure-
ments represent the mean of nine samples from three independent
Assessment of Acquired Cancer Phenotype
Several common assays were used to assess whether the cells
acquired an in vitro cancer phenotype during arsenic exposure and
were applied to all cell lines used. All assays represent the mean of
nine samples from three independent experiments. The assays in-
cluded zymographic analysis of secreted MMP-2 and -9 activity
after the method of Achanzar et al. ( 3 ), in which cells were cultured
in basal medium (without serum or supplements) for 48 hours, the
conditioned medium was collected, and secreted MMP-2 and -9
activity was measured by a standard zymographic method.
Enhanced MMP-2 and -9 activity is common for cancer cells and
frequently found in cells transformed by arsenic ( 3 , 29 , 33 – 36 ).
Cellular invasiveness was assessed as described previously ( 37 ).
Culture medium containing 10% fetal bovine serum served as a
chemoattractant and was loaded in the lower well of a blind-well
chamber (NeuroProbe, Inc, Gaithersburg, MD). A polycarbonate
8.0- µ m fi lter (Fisher Scientifi c, Hanover Park, IL) coated with
Matrigel (BD Bioscience, San Jose, CA) was layered on top of the
lower well. Cells (2 × 10 5 cells/chamber) were suspended in basal
medium and added on top of the fi lter and incubated for 48 hours.
Cell invasion was then quantifi ed as the number of cells that pen-
etrated the Matrigel and fi lter. Cells were fi xed and stained
(HEMA 3 Manual Staining System; Fisher Scientifi c), stain was
extracted, and absorbance determined at 630 nm and quantifi ed as
Colony formation in soft agar was performed as previously
described method ( 38 ) to assess the cells ’ capacity for anchorage-
independent growth. Cell line – dependent medium (see above)
containing 0.5% agar was fi rst added to 35-mm culture plates, and
1.25 × 10 4 cells were then suspended in medium containing 0.33%
agar and layered on top. Cultures were incubated at 37°C in 5%
CO 2 for 3 weeks, and then, the number of colonies (all sizes) was
counted with an automated colony counter.
Real-Time Reverse Transcription – Polymerase Chain
Expression levels for glutathione- S -transferase- ? ( GSTP1 ; accession
number: NM_000852; primers, forward: 5 ′ -AGAGCTGG AGGA-
GGAGGTG-3 ′ ; reverse: 5 ′ -AGGTCTCCGTCCTGG-AACTT-
3 ′ ) , ATP-Binding Cassette C1 ( ABCC1 ; accession number: L05628;
primers, forward: 5 ′ -GAGGAGGTGGAGG CTTTGATC-3 ′ ;
reverse: 5 ′ -AAGTAGGGCCCAAAGGT CTTG-3 ′ ), nuclear factor
(erythroid-derived 2)-like 2 ( NFE2L2 ; accession number:
NM_006164; primers, forward: 5 ′ -AACC AGTGGATCTGCCAA-
CTACTC-3 ′ ; reverse: 5 ′ -CTGCGCC AAAAGCTGCAT-3 ′ ), and
heme oxygenase-1 ( HMOX1 ; accession number: NM_002133; for-
ward: 5 ′ -GCCTGGAA GACACC CTAATGTG-3 ′ ; reverse: 5 ′ -
GGCCGTGTCAA CAAGGATACTT-3 ′ ) were quantified by
JNCI | Articles 1673
real-time reverse transcription – polymerase chain reaction
analysis by the reaction conditions described in Liu et al. ( 39 ).
The selected genes were first normalized with ? -actin levels
within the same sample and then expressed as percent control (0
µ M arsenite in each case), which was set to 100%. Results are
expressed as the mean of nine samples from three independent
Influence of Selenite on Arsenite-Induced Acquired Cancer
TRL1215 cells were continuously exposed to 0.5 µ M sodium arse-
nite in the presence or absence of 1.0 µ M sodium selenite (Sigma)
for up to 24 weeks in 75-cm 2 tissue culture flasks. The medium was
changed at half-weekly intervals, and cells were passaged weekly
with new cultures seeded at 1 × 10 6 cells/flask. The levels of arse-
nite and selenite had no effect on cell growth. ODD, MMP-2 and
-9 secretion, cellular invasiveness, and colony formation in these
cells were measured approximately biweekly as above. The activity
of glutathione peroxidase was measured using a Glutathione
Peroxidase Assay Kit (Cayman) in control and selenite-treated cells
at 0, 4, 8, 16, and 24 weeks of exposure. Control cells and cells
exposed to 1.0 µ M selenite for 20 weeks were further treated with
0.0, 1.0, 2.5, and 5.0 mM hydrogen peroxide for 24 hours, and
then, ODD in these cells was measured. Results are expressed as
the mean of nine samples from three independent experiments.
Data are expressed as the mean with 95% confidence intervals
(CIs). Statistical significance was determined by Student t test or
analysis of variance followed by Dunnett multiple comparison test,
as appropriate. In some cases, correlations were calculated by
linear regression and further assessed by Pearson correlation. A P
value of less than .05 from two-sided tests was considered to be
statistically significant in all cases.
ODD and Chronic Arsenite Exposure at Levels Known to
Induce Malignant Transformation
In our initial experiments, arsenic biomethylation – competent
TRL1215 rat liver cells and arsenic methylation – deficient RWPE-1
human prostate cells were continuously exposed to sodium arsenite
concentrations known to induce malignant transformation as defined
by the acquisition of a transformed phenotype in vitro and the ability
to produce malignant xenograft tumors in immunodeficient mice
( 3 , 7 ). Cellular ODD was measured during exposure to arsenite at
approximately biweekly intervals for up to 30 weeks ( Figure 1 ).
Exposure of TRL1215 cells to arsenite induced a remarkable, but
delayed, increase in ODD in an arsenite concentration – dependent
manner, starting at 5 weeks of exposure ( Figure 1, A ). For example,
after growth in medium with 0.5 µ M arsenite for 16 weeks, ODD
reached 836% compared with control cells without arsenite (95%
CI = 368% to 1304%, P < .01), and after growth in medium with 1.0
µ M arsenite for 16 weeks, ODD reached 1138% of control (95%
CI = 797% to 1481%, P < .01). The ODD levels then precipitously
declined to baseline after about 18 – 20 weeks of exposure. Previous
work showed that malignant transformation of TRL1215 cells, as
established by production of malignant xenograft tumors in mice,
occurs at 18 weeks of exposure to similar levels of arsenite as used in
this study and that tumor formation rate is directly related the ar-
senic concentration used for in vitro transformation ( 7 ). In sharp
contrast, after chronic exposure of RWPE-1 cells to arsenite, there
was no evidence of ODD ( Figure 1, B ). For example, for cells grown
in medium with 5.0 µ M arsenite for 30 weeks, ODD was 119% com-
pared with that in control cells grown without arsenite (95% CI =
103% to 135%, P > .05). RWPE-1 cells undergo malignant
transformation with these arsenic levels, as established by produc-
tion of malignant xenograft tumors, although it requires approxi-
mately 30 weeks of continuous arsenic exposure in vitro, they are
able to do so after inoculation into mice ( 3 ).
Both the TRL1215 and the RWPE-1 cell lines acquired an in
vitro transformed phenotype after arsenite exposure, based on inva-
siveness, colony formation, and hypersecretion of MMP-2 and -9
( Figure 1, C – F). All three parameters were increased over the course
of 18 weeks of arsenite exposure for TRL1215 cells, or over 30
weeks of arsenite exposure for RWPE-1 cells, which were time
frames similar to those reported for these cell lines to acquire the
ability to form xenograft tumors in mice ( 3 , 7 ). For example, more
than 18 weeks of arsenite exposure invasiveness of TRL1215 cells
increased to 547% of unexposed control cells (95% CI = 509% to
585%, P < .001), and colony formation increased 719% above that
in control cells (95% CI = 595% to 845%, P < .001). MMP-2 and -9
secretion after 18 weeks of arsenite exposure increased 836% above
that in control cells (95% CI = 796% to 876%, P < .001). By con-
trast, RWPE-1 cells exposed to 5 µ M arsenite for 30 weeks showed
a more modest 191% increase in invasion compared with control
cells (95% CI = 172% to 209%, P < .001). Likewise, at 30 weeks of
arsenite exposure, RWPE-1 cells showed a comparatively modest
increase in colony formation at 260% of control (95% CI = 104% to
415%, P = .023), and an increase of MMP-2 and -9 secretion of
179% of control (95% CI = 172% to 186%, P < .001). MMP-2 and
-9 are frequently secreted by cancer cells ( 33 – 35 ), including arsenic-
transformed cells ( 3 , 29 , 36 ). Levels of increased MMP-2 and -9 se-
cretion in TRL1215 cells at the approximate time of malignant
transformation (week 18) showed a robust positive correlation
(linear regression followed by Pearson correlation, r = .98, P = .013)
with the levels of maximal ODD for all of the various concentrations
of arsenite used for treatment, suggesting that ODD was positively
linked to the acquired cancer phenotype ( Figure 1, G ). In fact, when
MMP-2 and -9 secretion was measured in TRL1215 cells that had
acquired a cancer phenotype in vitro after 18 weeks of arsenite expo-
sure in present work, and correlated with either in vitro arsenite
concentration-related xenograft tumor formation rate or the rate of
metastasis seen previously ( 7 ), both showed robust positive correla-
tions (linear regression followed by Pearson correlation, P < .001;
Supplementary Figure 1 , A and B , available online). From these
results, it appeared that arsenic methylation might be obligatory for
ODD, and although cells could acquire a cancer phenotype in its
absence, the presence of ODD might hasten the process. However,
TRL1215 and RWPE-1 cells differ widely in terms of genetic back-
ground, which made direct comparisons of time to acquired cancer
phenotype in vitro somewhat problematic. Because arsenic methyl-
ation can vary widely with target site in vivo, the relative role of
ODD formation in vivo will also require direct study.
1674 Articles | JNCI Vol. 101, Issue 24 | December 16, 2009
Figure 1 . Oxidative DNA damage (ODD) and acquisition of an in vitro
transformed phenotype upon chronic sodium arsenite exposure in ar-
senic methylation – competent TRL1215 cells and arsenic methylation –
defi cient RWPE-1 cells. A ) TRL1215 cells were exposed to various
concentrations of sodium arsenite for up to 30 weeks, and ODD was
assessed by the formation of DNA – nitrone adducts using the immuno-
spin trapping method ( 27 , 28 ). B ) RWPE-1 cells were exposed to sodium
arsenite for up to 30 weeks, and ODD was assessed as in ( A ). C ) Cellular
invasiveness, as an in vitro marker of malignant transformation, was
measured in a transwell assay with a Matrigel-coated membrane for
TRL1215 cells (0.5 µ M) after 18 weeks of arsenite exposure and for
RWPE-1 cells (5.0 µ M) at 30 weeks of arsenite exposure. D ) Anchorage
independence, as an in vitro marker of malignant transformation, was
measured by colony formation in soft agar for TRL1215 cells (0.5 µ M)
after 18 weeks of arsenite exposure and for RWPE-1 cells (5.0 µ M) after
30 weeks of arsenite exposure. Colonies (all sizes) were scored. E )
Secreted matrix metalloproteinase (MMP)-2 and -9 activity during chronic
arsenite exposure in TRL1215 cells. Cells were exposed to 1 µ M sodium
arsenite for 6 – 24 weeks, and MMP-2 and -9 in conditioned media were
assayed by zymography. F ) Secreted MMP-2 and -9 activity during
chronic arsenite exposure in RWPE-1 cells. Cells were exposed to 5 µ M
and otherwise treated as in ( F ). G ) Correlation between arsenite-induced,
concentration-response ODD (from A ) and secreted MMP-2 and -9 activity
at 18 weeks at the various concentrations of chronic arsenite exposure.
Statistical analysis performed by using linear regression and further
assessed by Pearson correlation. Results in all panels ( A – G ) are expressed
as the mean of nine samples from three independent experiments. The
95% confi dence intervals have been included in panels ( C – G ) but have
been omitted for clarity in ( A) and ( B ) and can be found in Supplementary
Tables 1 and 2 , available online. In panels ( A ), ( B ), ( E ), and ( F ), an arrow
marked “Xenograft Tumor Formation” marks the time point at which the
specifi c cells in question acquired the ability to form malignant tumors
after inoculation into nude mice in our prior work with these same cells
under similar conditions ( 3 , 7 ). All P values are from two-sided tests.
JNCI | Articles 1675
ODD in TRL1215 cells rapidly diminished with time after
transformation. We initially suspected that this decline was
because of progressive arsenic adaptation. Various genes could
participate in this adaptation including those related to oxidative
stress, glutathione metabolism, and arsenic transport. Therefore,
we looked at expression of GSTP1 , ABCC1 , NFE2L2 , and HMOX1
using real-time reverse transcription – polymerase chain reaction.
ABCC1 encodes for a transport protein that is responsible for
arsenic effl ux in complex with a glutathione trimer produced by
GST- ? and is a key to arsenic tolerance ( 40 ). In TRL1215 cells,
arsenic increased expression of GSTP1 and ABCC1 ( Figure 2, A ).
Glutathione levels were also increased by arsenite (at 24 weeks of
arsenite exposure, maximal levels = 264%,of unexposed control
cells, 95% CI = 184% to 344%, P < .001).
Arsenic also enhanced the expression of oxidative stress-related
genes like NFE2L2 , HMOX1 ( Figure 2, A ), superoxide dismutase
1 (eg, at 18 weeks, 211% of control, 95% CI = 162% to 260%, P <
.001), and metallothionein-1 (eg, at 18 weeks, 1727% of control,
95% CI = 1315% to 2138%, P < .001). However, these genes were
also induced at 30 weeks of arsenic exposure in RWPE-1 cells
( Figure 2, B ), which suggests that cell-specifi c increases in ex-
pression as an adaptive phenomenon are unlikely to explain the
precipitous loss of ODD in TRL1215 cells.
Treatment of cells with sodium selenite very effectively inhibits
arsenic methylation ( 41 ). Therefore, to further test whether ar-
senic methylation might be responsible for ODD, TRL1215 cells
were exposed to a concentration of sodium arsenite, 0.5 µ M, that
induces ODD and is known to produce malignant transformation
( 7 ), together with or without a nontoxic level of sodium selenite,
1.0 µ M, for up to 24 weeks. Co-exposure to selenite abolished
arsenite-induced ODD ( Figure 3, A ). Furthermore, at 18 weeks
exposure to arsenite [the approximate time of malignant transfor-
mation for TRL1215 cells ( 7 )], arsenite-induced increases in
MMP-2 and -9 secretion, invasion, and colony formation were all
abolished by selenite ( Figure 3, B – D). Selenite could potentially
have antioxidant effects, most likely as a component of antioxidant
enzymes, the most prominent being glutathione peroxidase; sub-
optimal selenium nutritional status in humans reduces glutathione
peroxidase activity, which can increase cellular reactive oxygen
species ( 42 ). However, chronic selenite exposure had no effect on
glutathione peroxidase activity in TRL1215 cells ( Figure 3, E ).
Furthermore, if selenite treatment were blocking oxidative stress
to eliminate ODD, rather than inhibiting arsenic methylation, it
would be expected to block ODD from externally applied oxidants.
However, when cells chronically treated with selenite were
exposed to hydrogen peroxide, they showed the same level of
ODD as in control ( Figure 3, F ), indicating that selenite treatment
had no effect on ODD induced by reactive oxygen species or at
least from the species generated by hydrogen peroxide. These data
support a non-antioxidative mechanism for selenite-mediated inhi-
bition of arsenite-induced ODD. This conclusion is consistent
with the scenario that biomethylated arsenicals could be key fac-
tors in arsenite-induced ODD, and the ability of selenite to reduce
ODD appears to be related to a blockade of acquired cancer
phenotype in vitro.
Association of Arsenic-Induced ODD With Acquisition of a
Cancer Phenotype in UROtsa/F35 Cells but not UROtsa
To test the role of arsenic biomethylation in ODD in an isogenic
pair of cell lines that differed only in their ability to methylate ar-
senic, we also did experiments in UROtsa human urothelial cells
and in UROtsa/F35 cells, which are stably transduced with As3mt
and methylate arsenic ( 30 ). Chronic exposure of arsenic methyla-
tion – deficient UROtsa human urothelial cells to 0.25 – 1.0 µ M
sodium arsenite did not induce ODD ( Figure 4, A ). However, expo-
sure of UROtsa/F35 cells to the same arsenite concentrations
induced a remarkable, but delayed, increase in ODD starting after
6 weeks of exposure that rose to 192% of control (10 weeks, 95%
CI = 180% to 205%, P < .01) for cells exposed to 0.5 µ M or to 225%
Figure 2 . Expression of adaptive response genes
during chronic arsenite exposure in TRL1215 and
RWPE-1 cells. A ) Expression of the genes for gluta-
thione- S -transferase- ? ( GSTP1 , here shown as
GST- ? ), the ATP-Binding Cassette Transporter C1
( ABCC1 ), an antioxidant response transcription
factor ( NFE2L2 ), and heme oxygenase-1 ( HMOX1 ,
here shown as HO-1 ) in TRL1215 cells exposed to
arsenite. Expression was quantifi ed by reverse
transcription – polymerase chain reaction and nor-
malized to ? -actin expression levels within each
sample. An arrow points to the approximate time of
rapid decline in oxidative DNA damage (ODD) (ODD
Loss; see Figure 1, A ). Arrows with P values indi-
cate statistical signifi cance of specifi c data at spe-
cifi c time points. B ) Adaptive response gene
expression in RWPE-1 cells at their time of malig-
nant transformation (30 weeks). Assays were per-
formed as in ( A ). Results are expressed as the mean
of nine samples from three independent experi-
ments with 95% confi dence interval. All P values
are from two-sided tests.
1676 Articles | JNCI Vol. 101, Issue 24 | December 16, 2009
Figure 3 . Infl uence of exposure to sodium selenite (1.0 µ M) on arsenite-
induced (0.5 µ M) oxidative DNA damage (ODD) and acquired in vitro
cancer phenotype in TRL1215 cells. A ) ODD in cells exposed to sodium
arsenite in the presence or absence of sodium selenite for up to 24
weeks, as assessed by the immuno-spin trapping method. Control
cells and those grown in the presence of selenite alone showed no
ODD and are omitted for clarity. B ) Infl uence of selenite on secreted
matrix metalloproteinase (MMP)-2 and -9 activity induced by chronic
arsenite exposure at 18 weeks, the time of malignant transformation
as assessed by arsenic-induced xenograft tumor formation in a prior
study ( 7 ). Assays were performed as in Figure 1, E . C ) Infl uence of
selenite on invasiveness induced by 18 weeks of chronic arsenite
exposure. Assays were performed as in Figure 1, C . D ) Infl uence of
selenite on colony formation induced by 18 weeks of chronic arsenite
exposure. Assays were performed as in Figure 1, D . E ) Infl uence of
selenite on glutathione peroxidase activity for more than 24 weeks of
exposure. F ) Infl uence of 20 weeks of chronic selenite treatment on
hydrogen peroxide – induced ODD. Hydrogen peroxide was added to
cells at indicated levels for 24 hours before ODD measurement.
Untreated cells served as a control. Results in all cases are expressed
as the mean with 95% confi dence intervals of nine samples from three
independent experiments. In panel ( A ), an asterisk indicates a statisti-
cally signifi cant ( P < .05) difference from control. All P values are from
two-sided tests. P values were calculated by using analysis of variance
followed by Dunnett multiple comparison test ( B – D ) or Student t test
( A and F ).
of control (16 weeks, 95% CI = 198% to 252%, P < .01) for cells
exposed to 1.0 µ M ( Figure 4, B ). ODD, as measured by levels of
DNA – nitrone adducts, then precipitously returned to baseline after
approximately 28 weeks of arsenic exposure. This pattern of delayed
ODD onset and then precipitous loss was remarkably similar to that
seen in methylation-competent TRL1215 cells ( Figure 1, A ).
We next tested our hypothesis that arsenite methylation medi-
ated arsenite-induced ODD and accelerated the acquisition of a
JNCI | Articles 1677
Figure 4 . Oxidative DNA damage (ODD) induced by
chronic low-level sodium arsenite exposure in UROtsa
cells and UROtsa/F35 cells. UROtsa cells ( A ) and UROtsa/
F35 cells ( B ) were exposed to 0.25, 0.5, or 1.0 µ M
sodium arsenite for up to 30 weeks, and ODD was
assessed by the immuno-spin trapping method.
Results are expressed as mean of nine samples from
three independent experiments (the 95% confi dence
intervals have been omitted for clarity but can be
found in the Supplementary Tables 3 and 4 , available
online). An asterisk indicates a statistically signifi cant
( P < .05) difference from control ( Supplementary
Tables 3 and 4 , available online, for complete data).
P values were calculated by analysis of variance
followed by Dunnett multiple comparison test.
cancer cell phenotype in vitro by using arsenic biomethylation –
defi cient UROtsa vs methylation – competent UROtsa/F35 cells.
Arsenite exposure in biomethylation-competent UROtsa/F35 cells
markedly increased MMP-2 and -9 secretion, with dramatic in-
creases by 24 weeks (510% of control at 0.5 µ M arsenite, 95%
CI = 323% to 698%, P < .01; 1055% of control at 1.0 µ M arsenite,
95% CI = 634% to 1476%, P < .01; Figure 5, A ). Enhanced MMP
secretion is common in urothelial cancer and in arsenic-transformed
cells ( 3 , 29 , 33 – 36 ). Indeed, MMP-2 and -9 secretion, a marker of an
acquired cancer phenotype in vitro and in vivo ( 3 , 29 , 33 – 36 ), was
strongly correlated ( r = .998, P = .002, linear regression followed by
Pearson correlation) with ODD in arsenite-exposed UROtsa/F35
cells ( Figure 5, B ). Chronic exposure to 1 µ M sodium arsenite also
increased invasiveness and colony formation in arsenic methyla-
tion – competent UROtsa/F35 cells by 18 weeks of exposure, at
which time, invasion was 285% of control (95% CI = 263% to
307%, P < .001) and colony formation was 224% of control (95%
CI = 95% to 355%, P = .027) ( Figure 5, C and D ). By contrast,
exposure of methylation-defi cient UROtsa cells to arsenite only
showed increases in MMP-2 and -9 secretion, invasiveness, or
colony formation beginning at 55 weeks of arsenite exposure. For
example, invasiveness was increased 300%, compared with control,
in UROsta/F35 cells exposed to 1.0 µ M arsenic for 55 weeks (95%
CI = 254% to 346%, P < .001; Figure 6, C ). Because UROtsa/F35
cells are isogenic with UROtsa cells except that the former con-
tains the AS3MT transgene, which allows arsenic methylation ( 30 ),
it seems reasonable to attribute the more rapid acquisition of an in
vitro transformed phenotype in arsenite-exposed UROtsa/F35
cells to their ability to methylate arsenic and thereby acquire
In this study, we used the IST method, which measures DNA
radicals directly, to investigate the ability of inorganic arsenite to
induce ODD in arsenite methylation – deficient and – competent
cells. A major advantage of the IST method is that it converts un-
stable DNA radicals induced by reactive oxygen species into stable
nitrone adducts allowing for direct measurement of DNA radicals
and avoiding sample preparation – induced artifactual ODD, making
results more reliable than previous methods ( 27 , 28 ). Furthermore,
we used biologically relevant levels of arsenite exposure: chronic
exposure to inorganic arsenic levels in the 5.0 µ M range in drinking
water has been associated with oxidative stress in humans ( 43 ).
In our initial experiments, we found ODD to occur in TRL1215
cells, which can methylate arsenic, but not in RWPE-1 cells, which
cannot, even though both cell lines were exposed to arsenite con-
centrations and durations suffi cient to cause an acquired malignant
phenotype in vitro (in the present work) and instill the ability when
inoculated to cause formation of malignant tumors in immunode-
fi cient mice ( 3 , 7 ). An inhibitor of arsenic methylation, sodium
selenite ( 40 ), abolished arsenite-induced ODD and the acquisition
of an in vitro cancer phenotype in arsenic methylation–competent
TRL1215 cells. These observations were fortifi ed by our observa-
tion that arsenic methylation–competent ( As3mt- transduced)
UROtsa/F35 cells showed much more ODD upon arsenic expo-
sure than its parental UROtsa cell line and acquired a transformed
phenotype in vitro in much less time. Hence, it appears that ar-
senic methylation is obligatory for ODD in some cells and hastens
the acquisition of cancer phenotype. However, because cells un-
able to methylate arsenic can still acquire an arsenic-induced can-
cer phenotype in vitro over time, it is likely that arsenic is also
carcinogenic by mechanisms that neither require biomethylation
nor ODD. It is possible that multiple mechanisms may account for
carcinogenesis by arsenic even within the same cell line and that
the speed with which a cancer phenotype is acquired depends on
the number of mechanisms at play.
Chronic exposure to low levels of sodium arsenite caused a re-
markable, but delayed, increase in ODD only in arsenic methylation –
competent cells. Accumulating evidence indicates that the methylation
of arsenic is not a detoxifying event and that arsenic biomethylation
may produce highly toxic compounds with genotoxic potential
1678 Articles | JNCI Vol. 101, Issue 24 | December 16, 2009
Figure 5 . Evidence for acquired cancer phenotype
in vitro and correlation with oxidative DNA
damage (ODD) in UROtsa/F35 cells. UROtsa/F35
cells were exposed to sodium arsenite for up to 30
weeks, and secreted matrix metalloproteinase
(MMP)-2 and -9 activity by zymography, cellular
invasiveness by Boyden chamber assay, and
colony formation by agar assay were assessed.
Secreted MMP-2 and -9 activity was also correlated
with ODD as assessed by the immuno-spin trap-
ping method. A ) Secreted MMP-2 and -9 activity
during chronic arsenite exposure. B ) Correla tion
between arsenite-induced, concentration-response
ODD and secreted MMP-2 and -9 activity (24
weeks). Statistical analysis done by linear regres-
sion and further assessed by Pearson correlation.
C ) Cellular invasiveness. D ) Colony formation.
Results are expressed as the mean with 95%
confi dence intervals (nine samples from three
independent experiments). All statistical tests
were two-sided. P values were calculated by
using analysis of variance followed by Dunnett
multiple comparison test ( A ) or Student t test
( C and D ).
( 13 – 19 ). Although inorganic arsenic can stimulate reactive oxygen
species production, trivalent methylated arsenic compounds appear
to stimulate the production of reactive oxygen species more effi -
ciently than inorganic arsenic compounds ( 13 ). Indeed, ex vivo evi-
dence indicates that dimethylarsinous acid is an indirect genotoxin
that forms hydroxyl radicals through an unknown mechanism ( 44 ).
In addition to directly increasing reactive oxygen species, arsenic
compounds might also induce ODD indirectly by inhibiting impor-
tant detoxifying enzymes. Generally speaking, trivalent methylated
arsenicals are more potent inhibitors of enzymes when compared
with inorganic forms, possibly because of higher affi nities for critical
thiol groups ( 45 ). It is important to note that high concentrations of
inorganic arsenic that are lethal to the cells can acutely induce ODD
in cells without arsenic biomethylation, like RWPE-1 cells (data not
shown), but the relevance of fi nding ODD in dead or dying cells to
cancer is undoubtedly limited.
Several striking, mutually supportive similarities were evident in
the temporal pattern of ODD generation in the arsenic methyla-
tion – competent cells (TRL1215 and UROtsa/F35) in this study.
First, both the human and the rat cells showed a delay of several
weeks before the onset of ODD [ Figures 1, A and 4, B ( 7 )]. Second,
both cell lines showed a rapid drop-off in levels of ODD at about
the point in time they acquired a cancer phenotype [ Figures 1, A
and 4, B ( 7 )]. The reasons for both phenomena are unclear. The
delay may involve buildup of a key toxic metabolite, such as a
methylated arsenic compound. Initially, we suspected that the pre-
cipitous loss of ODD might be attributed to adaptation to arsenic;
adaptive increases in arsenic transport – related gene expression
JNCI | Articles 1679
Figure 6 . Evidence for acquired cancer phenotype in vitro in UROtsa cells.
UROtsa cells were exposed to sodium arsenite for up to 55 weeks, and
secreted matrix metalloproteinase (MMP)-2 and -9 activity by zymography,
cellular invasiveness by Boyden Chamber assay, and colony formation by
agar assay were assessed. A ) MMP-2 and -9 secreted activity during
chronic arsenite exposure. B ) Cellular invasiveness. C ) Colony formation.
Results are expressed as the mean with 95% confi dence intervals of nine
samples from three independent experiments. All P values are from
two-sided tests. P values were calculated using analysis of variance fol-
lowed by Dunnett multiple comparison test ( A ) or Student t test ( B and C ).
(eg, GSTP1 and ABCC1 ) and in glutathione levels are not un-
common, all of which promote arsenic effl ux from exposed cells
( 40 , 46 , 47 ). Chronic arsenic exposure also typically increases the
expression of stress-response genes such as NFE2L2 , HMOX1 , and
metallothionein ( 39 , 48 , 49 ). We had suspected that a differential
adaptive response might account for the loss of ODD in TRL1215
cells, but we found these genes to also be overexpressed in
RWPE-1 cells (which show no ODD) at about the time of arsenic-
induced malignant transformation, so this gene expression pattern
is not likely to be an adaptation caused by ODD loss. In any event,
by the time of the precipitous drop in ODD, it appears that suffi -
cient damage had already occurred to hasten malignant transfor-
mation in methylation-competent cells.
Some limitations of this study have already been discussed. It is
unknown why arsenic biomethylation – competent cells show a delayed
onset of ODD with arsenic exposure or why there is a precipitous
drop near the point of acquired cancer phenotype. Furthermore, the
observed ODD, although consistently linked to more rapid acquisi-
tion of cancer phenotype, is not linked to precise precipitating events
(ie, mutations) that would drive cells more rapidly toward malig-
nancy. This is an unproven assumption. Only four cell lines were used
in this work, two of which are isogenic with the exception of the
transduction of As3mt . Methylated arsenicals were never actually
measured, and it is only assumed that they were differentially pro-
duced based on prior work. Cell lines exposed here to arsenite in vitro
were not directly used in in vivo xenograft studies but were assumed
to be transformed based on in vitro phenotype and timing relative to
earlier experiments. In vivo testing in arsenic methylase knockout
mice would be critical to confi rm our hypothesis. In vitro cell models
of cancer may not be strictly concordant in timing, and so on, with
much more complicated formation of tumors in vivo.
In summary, chronic exposure to inorganic arsenic consistently
caused a delayed increase in ODD, but only in cells able to methylate
arsenic, which was then precipitously lost at about the same time that
these methylation-competent cells acquired a cancer phenotype in
vitro. Arsenite-induced ODD appeared to accelerate acquisition of
cancer phenotype in vitro and only required the transduction of a
single gene, As3mt . Human data are emerging that clearly show that
polymorphisms in AS3MT exist that affect arsenical methylation
patterns ( 50 ) and thereby may alter susceptibility to carcinogenesis.
AS3MT -null humans are not known, but As3mt knockout mice have
very recently been introduced ( 51 ). Although inorganic arsenicals
have not yet been tested for carcinogenic effects in these genetically
altered mice, this clearly should be a high priority. AS3MT polymor-
phism analysis may one day provide a metric of human susceptibility
to arsenical carcinogenesis at critical target sites.
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Intramural Research Program of the National Cancer Institute, Center for
Cancer Research (C.K., E.J.T., M.P.W.); National Institute of Environmental
Health Sciences (D.C.R., R.P.M.).
The authors are solely responsible for the design of the study, the collec-
tion and analysis of data and the interpretation of the results, the prepa-
ration of the manuscript, and the decision to submit the manuscript for
Present address: Free Radical Biology and Aging Research Program,
Oklahoma Medical Research Foundation, Oklahoma City, OK (D. C.
We thank Drs J. Liu and L. Keefer for careful review of this material and
Mr M. Bell for his help with the graphics.
Manuscript received February 19 , 2009 ; revised September 25 , 2009 ;
accepted October 13 , 2009 .