Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats

Abstract

Background: Potassium bromate (KBrO3), used in both the food and cosmetics industry, and a drinking water disinfection by-product, is a nephrotoxic compound and rodent carcinogen. To gain insight into the carcinogenic mechanism of action and provide possible biomarkers of KBrO3 exposure, the gene expression in kidneys from chronically exposed male F344 rats was investigated.Methods: Male F344 rats were exposed to KBrO3 in drinking water for 52 and 100 wk. Kidneys were removed, frozen, and stored at –80ºC, then used for Affymetrix microarray analysis. Gene expression patterns were examined using a non-carcinogenic (20 ppm) and carcinogenic dose (400 ppm) at 52 wk, and compared to 100 wk high dose (400 ppm) and adenoma gene expression.Results: Statistical analysis revealed 144, 224, 43, and 994 genes out of 15866 from the 52 wk low, 52 wk high, 100 wk high, and adenomas respectively, were differentially expressed when compared to control kidneys. Gene ontology classification of the 52 wk high dose showed alterations of gene transcripts involved in oxidative stress, lipid metabolism, kidney function/ion transport, and cellular function. In a comparison of kidney development gene expression, alterations were seen in the adenomas but not in the 52 wk bromate-treated kidneys. However, the normal kidney from the high dose group resembled the adenoma expression pattern with early kidney development genes being up-regulated and adult phase genes being down-regulated. Moreover, eight genes were identified which could serve as biomarkers of carcinogenic exposure to bromate. The most promising of these was Pendrin, or Slc26a4, a solute carrier of chloride and iodide active in the kidney, thyroid, and inner ear. All these tissues are targets of KBrO3 toxicity. Expression array results were verified with quantitative real-time rtPCR.Conclusions: These data demonstrate that the 400 ppm carcinogenic dose of KBrO3 showed marked gene expression differences from the 20 ppm non-carcinogenic dose. Comparison of kidney development gene expression showed that the adenoma patterns were more characteristic of embryonic than adult kidneys, and that the normal kidney from the high dose group resembled the adenoma-like gene expression pattern. Taken together, the analysis from this study identifies potential biomarkers of exposure and illuminates a possible carcinogenic mode of action for KBrO3.

Full text

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Translational Oncogenomics 2006: 2 33–52
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Correspondence: Dr. Don Delker, U.S. Environmental Protection Agency, Environmental Carcinogenesis
Division,109 TW Alexander Drive (B143-06), Research Triangle Park, NC 27711. Tel: (919) 541-7639; Fax: (919)
541-0694; Email: delker.don@epa.gov
ORIGINAL RESEARCH
Kidney Toxicogenomics of Chronic Potassium Bromate
Exposure in F344 Male Rats
David R. Geter1,3, William O. Ward1, Geremy W. Knapp1, Anthony B. DeAngelo1,
Jessica A. Rubis2, Russell D. Owen1, James W. Allen1 and Don A. Delker1
1National Health and Environmental Effects Research Laboratory, Office of Research and
Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
2CIIT Centers for Health Research, Research Triangle Park, NC 27711.
3National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711.
Abstract
Background: Potassium bromate (KBrO3), used in both the food and cosmetics industry, and a drinking water disinfection
by-product, is a nephrotoxic compound and rodent carcinogen. To gain insight into the carcinogenic mechanism of action
and provide possible biomarkers of KBrO3 exposure, the gene expression in kidneys from chronically exposed male F344
rats was investigated.
Methods: Male F344 rats were exposed to KBrO3 in drinking water for 52 and 100 wk. Kidneys were removed, frozen,
and stored at –80ºC, then used for Affymetrix microarray analysis. Gene expression patterns were examined using a
non-carcinogenic (20 ppm) and carcinogenic dose (400 ppm) at 52 wk, and compared to 100 wk high dose (400 ppm) and
adenoma gene expression.
Results: Statistical analysis revealed 144, 224, 43, and 994 genes out of 15866 from the 52 wk low, 52 wk high, 100 wk
high, and adenomas respectively, were differentially expressed when compared to control kidneys. Gene ontology classifi -
cation of the 52 wk high dose showed alterations of gene transcripts involved in oxidative stress, lipid metabolism, kidney
function/ion transport, and cellular function. In a comparison of kidney development gene expression, alterations were seen
in the adenomas but not in the 52 wk bromate-treated kidneys. However, the normal kidney from the high dose group re-
sembled the adenoma expression pattern with early kidney development genes being up-regulated and adult phase genes
being down-regulated. Moreover, eight genes were identifi ed which could serve as biomarkers of carcinogenic exposure to
bromate. The most promising of these was Pendrin, or Slc26a4, a solute carrier of chloride and iodide active in the kidney,
thyroid, and inner ear. All these tissues are targets of KBrO3 toxicity. Expression array results were verifi ed with quantitative
real-time rtPCR.
Conclusions: These data demonstrate that the 400 ppm carcinogenic dose of KBrO3 showed marked gene expression dif-
ferences from the 20 ppm non-carcinogenic dose. Comparison of kidney development gene expression showed that the
adenoma patterns were more characteristic of embryonic than adult kidneys, and that the normal kidney from the high dose
group resembled the adenoma-like gene expression pattern. Taken together, the analysis from this study identifi es potential
biomarkers of exposure and illuminates a possible carcinogenic mode of action for KBrO3.
Abbreviations: CD: collecting duct, DBPs: disinfection by-products, DCT: distal convoluted tubules, DEG: differently
expressed genes, K: potassium, KBrO3: potassium bromate, LH: loop of Henle, ppm: part per million, PCT: proximal
convoluted tubules, QRT-rtPCR: Quantitative Real Time- rtPCR, THMs: trihalomethanes.
Keywords: drinking water, bromate, disinfection by-product, gene expression, biomarker.
Background
The disinfection of public drinking water over the past century has dramatically decreased infectious
waterborne diseases and is a hallmark of American public health policy. The benefi ts of drinking water
disinfection are well recognized, however, an undesirable side effect is the production of disinfection
by-products (DBPs). These DBPs are formed when disinfectants such as chlorine, chloramine, and
ozone react with organic and inorganic matter in water. In the mid 1970’s, it was discovered that

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Translational Oncogenomics 2006: 2
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Geter et al
trihalomethanes (THMs) were produced in chlo-
rination of drinking water and that they are carci-
nogenic in laboratory animals (Rook, 1974; Bellar
et al. 1974; NCI, 1976). Because of the potential
health risks associated with THMs, in 1979 the
U.S. Environmental Protection Agency (U.S. EPA)
began the regulation of DBPs in the water supply.
Initially, concern focused on the trihalomethanes
(THMs), but it is now recognized that a wide
variety of DBPs are produced during chlorination.
In an effort to reduce exposure to halogenated
DBPs, many utilities have switched to ozonation
as an alternative treatment method to chlorination.
Ozonation is also preferred because it reduces the
turbidity of the water and is effective in treating
chlorine resistant organisms. However, if ozonation
is performed using surface water high in bromide
content, brominated by-products, such as bromate
ion (BrO3
1985). Potassium bromate (KBrO3), a salt of the
bromate ion, is nephro- and neurotoxic in humans
and carcinogenic in rodents (IARC, 1986; Kuro-
kawa et al. 1990). Under the current guidelines for
cancer risk assessment (U.S. EPA, 1986), bromate
is classifi ed as a probable human carcinogen due
to its kidney carcinogenicity in male and female
rats following exposure in drinking water (Kuro-
kawa et al. 1983, 1986a, 1986b; DeAngelo et al.
1998; Wolf et al. 1998). A dose-response relation-
ship in rat kidneys was observed in progressive
severity from renal dysplastic foci, preneoplastic
lesions, through renal adenomas, and fi nally renal
carcinoma (Kurokawa et al. 1986a; DeAngelo
et al. 1998; Wolf et al. 1998).
Mechanistic studies by Umemura et al. (2004,
2006) demonstrated dose-dependent changes in
oxidative stress and cell proliferation parameters
at carcinogenic doses of potassium bromate in male
and female rat kidneys. After four weeks of
continuous drinking water exposure, 8-oxodeoxy-
guanosine levels (8-oxodG), an indicator of pro-
mutagenic oxidative DNA damage (Wood et al.
1990; Shibutani et al. 1991), were signifi cantly
elevated in male and female rats administered
potassium bromate at concentrations of 250 ppm
and higher. In addition, BrdU labeling, an indicator
of cell proliferation, was also increased in the
proximal tubule of female rats at similar concentra-
tions and in male rats at concentrations as low as
30 ppm. It was suggested, however, that the suscep-
tibility of the male rat to increased cell proliferation
at lower concentrations was probably attributable
-), can be generated (Fiessinger et al.
to increased α2u-globulin in the male rat proximal
tubule. These studies provide important informa-
tion regarding the potential mechanism of action
of potassium bromate carcinogenicity.
The primary objective in this study was to
examine renal gene expression differences in male
F344 rats exposed to a non-carcinogenic and
carcinogenic dose (20 and 400 ppm, respectively)
of KBrO3 in drinking water for 52 wk. Further-
more, renal gene expression from the high dose
(400 ppm) and adenomas from 100 wk exposed
animals were examined and compared to the 52 wk
exposure groups. This was accomplished by
extracting kidney and adenoma RNA from rats
exposed in the previous 1998 DeAngelo et al.
study. Comparisons of gene expression profi les
between these groups were used to identify func-
tional pathways and individual genes that might
contribute to the carcinogenic mechanism of
action for KBrO3 and provide insight into poten-
tial biomarkers of exposure.
Methods
Animal maintenance
Complete study details were published previously
(DeAngelo et al. 1998). Briefl y, KBrO3 (99%;
CAS 7758–01–2) dissolved in deionized water at
concentrations of 0, 20, 100, 200, and 400 ppm
was administered to male F344 rats as the sole
water source for 12, 26, 52, 78, or 100 wk. Rats,
28 to 30-days-old, were allowed to acclimate for
1 wk and then randomly assigned to treatment
groups. Treatment rooms were maintained at
20–22ºC and 40–60% humidity with a 12-hr light:
dark cycle. Rats were housed 3 per cage on wood
chips and provided Purina Rodent Laboratory
Chow (St. Louis, MO) and water ad libitum.
Animals were observed daily and moribund
animals were euthanized and necropsied. Six
animals from each group were euthanized by CO2
asphyxiation and necropsied after 52 wk of treat-
ment. At necropsy, kidneys were removed,
washed, fl ash frozen in liquid nitrogen, and stored
at –80ºC.
Microarray experiment
For the microarray experiment, rats exposed to 0,
20, and 400 ppm KBrO3 for 52 wk, were selected
because they represent a control, non-carcinogenic,

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
and carcinogenic dose, respectively (DeAngelo
et al. 1998; Wolf et al. 1998). Kidneys from
animals exposed to 20 and 400 ppm KBrO3 for
52 wk were compared to kidneys from 52 wk
control animals. In addition, kidneys from rats
exposed to 0 and 400 ppm KBrO3 and two
adenomas from animals exposed for 100 wk were
also examined. For this comparison, kidneys from
the 100 wk high dose and adenomas were
compared to kidneys from 100 wk control
animals. Kidney RNA was extracted from three
animals per dose group in the 52 wk exposure,
and two animals per dose group in the 100 wk
samples. RNA from two 100 wk adenomas were
also extracted. RNA extraction was performed by
acid guanidinium isothiocynate-phenol-bromoch-
loropropane treatment (Tri reagent; Molecular
Research Center, Inc., Cincinnati, OH, U.S.A.)
and purifi ed on an affi nity resin (RNeasy; Qiagen,
Valencia, CA) according to manufacturer instruc-
tions. Extractions were tested on an Agilent
Bioanalyzer to determine RNA quality. Clearly
defi ned 28S and 18S bands were observed on all
samples used for microarray analysis. Samples
showing degradation were not used for microarray
analysis. Microarray procedures were performed
as recommended by the manufacturer of the
GeneChip system (Affymetrix, Inc, Santa Clara,
CA, U.S.A.). The gene expression probe array
used was the Rat Expression Array 230A gene
chip containing 15,866 probe sets. For each
animal, one kidney sample was used for gene
expression analysis. Chips were examined by M
versus A plots, chip clustering, and principal
component analysis to determine outlying chips.
Arrays demonstrating poor hybridization were
not used for analysis.
Data analysis
The resulting image fi les (.cel) were normalized
using the procedure of Li and Wong [D-chip]
(2001a; 2001b) and the determination of signifi cant
differences between groups were performed by
using the web interface of Cyber-T [http://visitor.
ics.uci.edu/genex/cybert/](Baldi and Long, 2001).
A tutorial for using both D-chip and Cyber-T, found
on the Cyber-T website, was used as the procedure
for this analysis. Multiple testing correction and
false discovery rate test were conducted by using
the method of Benjamini and Hochberg (1995) on
P-values: P = < (i/m)q, where i = # of genes
accepted at selected p, m = total # genes, and q =
desired false discovery rate. Determination of false
positives was performed by: fp = (Pbh * total #
genes). This resulted in a unique P-value for each
comparison. Genes expressed at levels different
than control such that a corrected P-value less than
0.05 were classifi ed as differentially expressed
genes (DEG) and annotated using NetAffx (http://
www.affymetrix.com). Individual literature review
on each DEG was conducted and used to classify
genes into functional groups.
Kidney developmental gene response
in KBrO3 induced adenoma
Gene expression analysis has become suffi ciently
standardized that comparisons between experi-
mental datasets can yield insights into biological
processes such as the differentiation of renal cells.
In this vein, we compared our dataset of gene
expression in whole adult rat kidney to that
collected and analyzed by Stuart et al. (2001). This
study performed the fi rst high density oligonucle-
otide microarray investigation of kidney develop-
ment using the 8,740 gene Affymetrix rat U34A
microarray. Multiple developmental stages were
examined, including embryonic day 13 (E13), E15,
E17, E19, newborn, 1 wk, and adult. Cluster
analysis defi ned fi ve temporal expression groups.
Early group consisted of genes with very high
expression in the early embryonic kidney, many
with roles in protein translation and DNA replica-
tion. Prenatal group consisted of genes that peaked
in mid-embryogenesis and contained many tran-
scripts specifying proteins of the extracellular
matrix. Neonatal group consisted of transcripts that
peaked in the neonatal period and contained a
number of retrotransposon RNAs. Steady group
contained genes that steadily increased in relative
expression levels throughout development,
including many genes involved in energy metabo-
lism and ion and water transport. Adult group
consisted of genes with relatively low levels of
expression throughout embryogenesis but with
markedly higher levels in the adult kidney; this
group included a heterogeneous mix of trans-
porters, detoxifi cation enzymes, and oxidative
stress genes.
Lists of the fi ve groups of genes identifi ed
in Stuart et al. (2001), were obtained from the
supplemental data website (http://organogenesis.
ucsd.edu/). These gene-lists used U34A probe set

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Translational Oncogenomics 2006: 2
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Geter et al
identifi ers. We determined the Rat Expression
Array 230A probe set identifi ers that corresponded
to the U34A probe sets using the best match
comparison fi le from Affymetrix. For the fi ve
kidney development groups mentioned above,
there were 838, 168, 61, 510, and 193 genes located
on the U34 array and 676, 126, 42, 428, and 155
genes located on the Rat Expression Array 230A
array. The minimum sequence identity for a given
probe set between the U34A and Rat Expression
Array 230A array was 93% and the average iden-
tity was greater than 99%. For each group, the
genes that were identifi ed in the Affymetrix best
match comparison file constituted the gene
members for that group in subsequent analysis.
Real time PCR determination
of gene expression
Quantitative real-time rtPCR (QRT-rtPCR) data
was collected from fresh aliquots of the total RNA
samples used to obtain array data. On average,
85 ng of total RNA was loaded in a one-step QRT-
rtPCR reaction. Multiscribe reverse transcriptase
was used to generate the cDNA template followed
by amplifi cation with AmpliTaq Gold DNA poly-
merase (Applied Biosystems). Target specific
assays were either custom designed or purchased
from Applied Biosystems’ TaqMan Assays on
Demand. All reactions were performed according
to manufacturer’s procedures. The QRT-rtPCR
cycling parameters were: 48ºC, 45 min cDNA
synthesis; 95ºC, 10 min AmpliTaq Gold (Applied
Biosystems) activation; and 40 cycles of amplifi ca-
tion at 94ºC, 15 sec melting followed by 60ºC,
1 min annealing/extension. Reaction volumes
totaled 20 μl and were run in triplicate in 384 well
plates on an ABI prism 7900HT. Based on the array
data, Rpl27 (ribosomal protein L27) was chosen
as a reference gene since it exhibited no differential
gene expression across treatment groups and
showed similar expression levels to the genes of
interest. Confi rmation that Rpl27 was not differ-
entially expressed across treatment groups was
confirmed by QRT-rtPCR analysis (data not
shown).
Results
Microarray analysis
The results of the statistical comparison among
tissues from the 52 wk control, low, and high dose
groups, and the 100 wk control, high, and adenoma
groups are given in Table 1. This study has been
archived in ArrayExpress under accession number
E-TOXM-21.
Analysis of gene expression
Gene ontology (GO) analysis of DEG expression
in the kidney from male F344 rats exposed to low
(20 ppm) and high (400 ppm) KBrO3 for 52 wk
are shown in Table 2. The individual functional
group information composed of gene symbol and
name, Affymetrix and accession numbers, and fold
change are given for oxidative stress (Table 3), and
kidney function / ion transport (Table 4) genes. In
addition to the above mentioned groups, a large
amount of lipid metabolism, oxidoreductase, and
cellular function genes were observed and are
shown as additional fi les: “lipid metabolism table.
pdf, oxidoreductase table.pdf and cellular function
table.pdf. ” A total of 99 and 139 genes were used
to provide an interpretive basis for differences in
Table 1. Results of the statistical comparison between 52 wk low, 52 wk high, 100 wk high, and 100 wk adeno-
ma and their corresponding control. Shown are the numbers of genes less than 0.01 that contribute to the
p-value calculation as given in the methods. The resultant p-value and the number of false positives are shown,
as well as the number of differentially expressed and annotated genes. The gene expression probe array used
was the Affymetrix Rat Expression Array 230A gene chip containing 15,866 probe sets.

Treatment
Genes
< 0.01
False
Positives
Differentially
Expressed P-value Annotated
52 wk low (3)*
52 wk high (3)
100 wk high (2)
100 wk adenomas (2)
470
603
244
1431
0.00128
0.00189
0.000766
0.0045
21
30
13
72
144
224
43
994
99
139
27
671
* The sample size for the comparison is shown with the treatment group.

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
expression that were observed in the kidneys of
rats treated with non-carcinogenic and carcino-
genic doses, respectively.
Analysis of the low dose showed a general
suppression of gene expression with 71% of genes
down-regulated relative to control tissues
compared to 56% in the high dose. The most
notable down-regulated groups in the low dose
were oxidative stress and kidney function with
100% and 77% down-regulated, respectively. In
the high dose, 87% of genes in kidney function and
90% of genes in lipid metabolism groups were
down-regulated. Overall these changes suggest a
general suppression of gene expression in the low
dose group, especially those genes involved
in oxidative stress and kidney function. This is in
contrast to the high dose, where kidney function
and lipid metabolism genes are suppressed
in concert with an increased amount of oxidative
stress related genes.
A comparison of genes whose expression was
signifi cantly altered in the 52 wk high dose (400
ppm) and the 100 wk adenomas is given as an
additional fi le: “similar 52,100, adenomas.pdf .”
In this comparison, 35 genes were down-regulated
relative to the kidney of control animals, 3 were
Table 3. List of differentially expressed oxidative stress genes in kidney from male rats exposed to 20 ppm (low)
and 400 ppm (high) potassium bromate in drinking water for 52 wk. All comparisons were made between the
specifi c treatment group and their corresponding control.
Gene
Symbola
Gene
Namea
Affymetrix
No.a
Accession
No.a
Fold
Change
Low dose
Dscr1
Hspb1
Txnrd1
Xdh
Down syndrome critical region homolog 1
Heat shock protein
Thioredoxin reductase 1
Xanthine dehydrogenase
1388686_at
1367577_at
1386958_at
1369973_at
NM_153724
NM_031970
NM_031614
NM_017154
–1.4
–1.5
–1.4
–1.4
High dose
Dscr1
Gnmt
Gsta2
Hspbap1
Pex11a

Down syndrome critical region homolog 1
Glycine N-methyltransferase
Glutathione S-transferase A2
Heat shock associated protein
Peroxisomal biogenesis factor 11a

1388686_at
1387672_at
1368180_s_at
1368195_at
1379361_at

NM_153724
NM_017084
NM_017013
NM_134419
NM_053487

–1.3
–1.3
–1.2
–1.8
–1.3





Ccng1
Cp
Gclm
Gstm1
Gstp1
Cyclin G1
Ceruloplasmin (ferroxidase)
Glutamate-cysteine ligase, modifi er subunit
Glutathione S-transferase M 1
Glutathione S-transferase Pi 1
1367764_at
1368418_a_at
1370030_at
1386985_at
1388122_at
NM_012923
NM_012532
NM_017305
NM_017014
NM_012577
1.3
1.3
1.3
1.4
1.3
a Gene symbols and accession numbers from Affymetrix Netaffx (http://www.affymetrix.com/analysis/index.affx).
Table 2. Gene ontology (GO) analysis of differentially expressed genes (DEG) in kidneys from male rats exposed
to 20 ppm (low) and 400 ppm (high) potassium bromate in drinking water for 52 wk. Results are the number of
differentially expressed genes within a functional group that were either up or down regulated, with group total.
Functional analyses for all groups were compiled by individual gene review.

Functional Group*
Control versus Low
Up
Control versus High
Up Down Total Down Total
Oxidative Stress
Lipid Metabolism
Kidney Function
Oxidoreductase
Cell Function
4
6
10
10
30
0
4
3
1
10
4
10
13
11
40
5
9
20
13
20
5
1
3
0
22
10
10
23
13
42
*A total of 99 and 139 genes were used for gene ontology analyses from low and high exposure concentrations respectively.

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Translational Oncogenomics 2006: 2
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Geter et al
Table 4. List of differentially expressed kidney function/ion transport genes in kidney from male rats exposed to
20 ppm (low) and 400 ppm (high) potassium bromate in drinking water for 52 wk. All comparisons were made
between the specifi c treatment group and their corresponding control.
Gene
Symbola
Gene
Namea
Affymetrix
No.a
Accession
No.a
Fold
Change
Low dose
Aqp1
Atp1a1
Dscr1
G6pc
Ramp3
Sgk
Slc15a1
Slc16a1
Slc22a1
Xdh
Aquaporin 1
ATPase, Na+/K+ transporting, alpha 1
Down syndrome critical region homolog 1
Glucose-6-phosphatase,
Receptor (calcitonin) activity modifying protein 3
Serum/glucocorticoid regulated kinase
Solute carrier family 15, member 1
Solute carrier family 16, member 1
Solute carrier family 22, member 1
Xanthine dehydrogenase
1387651_at
1371108_a_at
1388686_at
1386944_a_at NM_013098
1387389_at
1367802_at
1369381_a_at NM_057121
1386981_at
1368191_a_at NM_012697
1369973_at
NM_012778
NM_012504
NM_153724
–1.3
–1.2
–1.4
–1.7
–1.9
–1.6
–1.5
–2.8
–1.5
–1.4
NM_020100
NM_019232
NM_012716
NM_017154



Calca
Cldn16
Slc21a4
Calcitonin/calcitonin-related polypeptide, alpha
Claudin 16
Kidney specifi c organic anion, scf 21, member 4
1370775_a_at NM_017338
1369184_at
1368498_a_at NM_030837
1.4
1.4
1.4
NM_131905
High dose
Aqp2
Aqp3
Calb1
Clcnk11
Dscr1
Edn1
Kcnj16
Kcnq1
Ngfg
Prkwnk4
Scnn1a
Scnn1g
Slc5a2
Slc9a3
Slc12a3
Slc13a3
Slc22a5
Slc22a8
Slc26a4
Slc37a4
Aquaporin 2
Aquaporin 3
Calbindin 1
Chloride channel K1-like
Down syndrome critical region homolog 1
Endothelin 1
Potassium inwardly-rectifying channel, J16
Potassium voltage-gated channel, KQT-like 1
Nerve growth factor, gamma subunit
Protein kinase, lysine defi cient 4
Sodium channel nonvoltage-gated 1A
Sodium channel nonvoltage-gated 1G
Solute carrier family 5, member 2
Solute carrier family 9 (Na/H exchanger), isoform 3 1387542_at
Solute carrier family 12 (Na/Cl transporters), 3
Solute carrier family 13 Na+ transport, member 3
Solute carrier family 22, member 5
Solute carrier family 22, member 8
Pendrin, solute carrier family 26, member 4
Solute carrier family 37, member 4
1368568_at
1387100_at
1370201_at
1388175_at
1388686_at
1369519_at
1373991_at
1368371_at
1367961_at
1389662_at
1387104_at
1370481_at
1368414_at
NM_012909
NM_031703
NM_031984
NM_173103
NM_153724
NM_012548
AI411366
NM_032073
NM_031523
NM_175579
NM_031548
NM_017046
NM_022590
NM_012654
NM_019345
NM_022866
NM_019269
NM_031332
NM_019214
NM_031589
–1.4
–1.4
–1.3
–1.4
–1.4
–1.4
–1.2
–1.4
–1.9
–1.6
–1.3
–1.5
–1.4
–1.3
–1.4
–1.3
–1.3
–1.3
– 4.6
–1.3
1387230_at
1368047_at
1367950_at
1368461_at
1368193_at
1386960_at




Agtr2
Cldn16
Cp
Slc12a1
Angiotensin II receptor, type 2
Claudin 16
Ceruloplasmin (ferroxidase)
Solute carrier family 12 (Na/K/Cl), member 1
1369711_at
1369184_at
1368418_a_at NM_012532
1368548_at
NM_012494
NM_131905
1.5
1.6
1.3
1.5NM_019134
a Gene symbols and accession numbers from Affymetrix Netaffx (http://www.affymetrix.com/analysis/index.affx).
up-regulated, and 9 genes did not show the same
direction of change. The majority of these down-
regulated genes were associated with kidney
function.
A common list of genes whose expression was
signifi cantly altered relative to control tissue in the
52 wk high (400 ppm), the 100 wk high (400 ppm),
and the 100 wk adenoma groups is given in
Table 5. In most cases, the magnitude of fold
change increases with duration of exposure and/or
in tumor tissue. These genes may represent poten-
tial biomarkers of bromate exposure and effect

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
because the magnitude of their alteration is time-
dependent and/or a larger number of renal cells
incorporate these transcript changes with continued
exposure. Since tumor tissue is theoretically the
product of clonal expansion of target cells, these
eight genes might also aid in the development of
useful tumor markers of KBrO3 carcinogenicity.
These genes were: Calb1 (Calbindin 1), Gp2
(Glycoprotein 2), Klk7 (Kallikrein 7), an EST
(LOC362802), Ngfg (Nerve growth factor,
gamma), Prps2 (Phosphoribosyl pyrophosphate
synthetase 2), Slc12a3 (Solute carrier family 12,
member 3), and Slc26a4 (Solute carrier family 26,
member 4).
Kidney developmental gene response
in KBrO3 induced adenoma
The processes involved in organ development,
including cell proliferation, apoptosis, cell
adhesion, and differentiation, are processes that
are disregulated during carcinogenesis. Therefore,
a comparison was made between a previous gene
expression profi le observed in rat kidney develop-
ment to that of the kidney adenoma profi le from
this study. Stuart et al. (2001) identifi ed fi ve groups
of genes whose expression characterized stages of
rat kidney development. As described in the
Methods, genes were identifi ed in the current study
that matched specific development groups.
Figure 1A shows the average fold change of each
group compared to control for low bromate, high
bromate and adenoma. Early and prenatal genes,
associated with cell proliferation and laying down
the extracellular matrix, are up-regulated in the
Table 5. List of similar genes between 52 wk high dose potassium bromate (400 ppm), 100 wk high dose, and
adenomas (n = 2) that occurred at 100 wk. This list of genes may be usable as tumor marker genes. All com-
parisons were made between the specifi c treatment group and their corresponding control.

Gene
Symbola

Affymetrix
52 wk
High Dose
Fold Change Fold Change Fold Change
100 wk
High Dose Accession
No.a
Adenoma
No.a
Calb1
Gp2
Klk7
LOC362802
Ngfg
Prps2
Slc12a3
Slc26a4








1370201_at
1386933_at
1387820_at
1376239_at
1367961_at
1375932_at
1387230_at
1368193_at
NM_031984
NM_134418
NM_012593
NM_001014199
NM_031523
NM_012634
NM_019345
NM_019214
–1.3
–2.2
–1.4
–1.9
–1.9
–1.4
–1.4
–4.6
–11.4
–11.9
–12.1
–3.6
–20.5
–2.9
–4.8
–6.0
–153.4
–18.6
–57.3
–2.6
–77.3
–2.6
–6.6
–6.3
a Gene symbols and accession numbers from Affymetrix Netaffx (http://www.affymetrix.com/analysis/index.affx).
adenoma. Steady and adult genes, associated with
energy metabolism, transport, detoxifi cation, and
oxidative stress response, are down-regulated
in the adenoma. This adenoma expression profi le
shows the up-regulation of genes prevalent in early
kidney development and down-regulation of adult
stage genes. This observation is in agreement with
the proliferation and de-differentiation profi les
seen in classical tumor development.
Adenoma expression profi le resemble
high dose bromate kidney
The adenoma demonstrated an expression profi le
for kidney development gene groups early,
prenatal, steady, and adult that was distinct from
control and bromate treated kidneys. In order to
determine if the adenoma profi le, as determined
above, was present in part, in either the low or high
KBrO3 exposed animals, the top ten DEGs were
selected from each of the four groups mentioned
above. Figure 1B shows the average fold change
of each group compared to control for low and high
dose bromate. This fi gure illustrates that the high
dose kidney, but not the low, did resemble the
adenoma expression pattern with the early and
prenatal phase genes being up-regulated and the
steady and adult phase genes being down-regulated.
Real time PCR determination
of gene expression
Quantitative Real Time rtPCR assays were
conducted to verify gene expression of genes
deemed biologically relevant based on pathway

Page 8

Translational Oncogenomics 2006: 2
40
Geter et al
Figure 1A. Kidney development gene response in F344 male rats exposed to KBrO3 in drinking water. Kidney development gene groups
were defi ned in Stuart et al. (2003) and applied to our data as described in the methods section. For each group of genes, the expression
was normalized to control and an average response was calculated for: 52 wk low (20 ppm) KBrO3, 52 wk high (400 ppm) KBrO3, and
100 wk KBrO3-induced adenomas. For each group, the average fold change normalized to control was plotted on the y-axis.
Figure 1B. Gene expression pattern in F344 male rats exposed to high KBrO3 mimics adenoma expression pattern. Kidney development
gene groups were defi ned in Stuart et al. (2003) and applied to our data as described in the methods section. The differentially expressed
genes in the 100 wk KBrO3-induced adenomas were categorized into kidney development groups. The top ten genes from each develop-
mental group were then used in the 52 wk low (20 ppm) and high (400 ppm) KBrO3 exposed animals. For these 50 genes, the high exposure
group showed a similar expression pattern to the adenomas across kidney development groups.
Low Bromate
High Bromate
Adenoma
Adenoma
Average Normalized Expression
2 .0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
Early
Prenatal
Kidney Development Genes
Neonatal
Steady
Adult
Early Prenatal Neonatal Steady Adult
Kidney Development Genes
1.25
1.20
1.15
1.10
1.05
1.00
0.95
0.90
0.85
Average Normalized Expression
A
B
Low Bromate
High Bromate
Low Bromate
High Bromate

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
analysis and genes that could possibly be used as
biomarkers of exposure. Figure 2 shows that
QRT-rtPCR results confirmed the expression
patterns of the genes tested. However, differences
in magnitude of change were observed, for example
Aqp2 shows a –1.4 microarray fold change, but an
–18.5 QRT-rtPCR fold change. This discrepancy
in the magnitude of fold change between micro-
array data and rtPCR data has been noted in other
genomics studies and may be attributable to the
superior utility of the rtPCR procedure in
quantifying transcript abundance (Crosby et al.
2000). The specifi c genes selected, including target
sequence are given in additional fi le: “QRT-rtPCR_
table.pdf .”
Discussion
In the DeAngelo et al. (1998) and Wolf et al. (1998)
studies, male F344 rats exposed to 400 ppm KBrO3
for 52 wk developed kidney cancer, while those
exposed to 20 ppm did not. This study used the
same animal kidneys from that study to determine
if a difference in gene expression could be
discerned between the two doses. Examining
global gene expression for each dose allowed
categorizing of genes into the following groups;
oxidative stress, lipid metabolism, kidney
function/ion transport, cellular function, and oxido-
reductase function.
Oxidative stress response
Reactive oxygen species (ROS) are natural by-
products produced by the metabolism of O2 during
aerobic respiration. Oxidative stress occurs when
there are increases in ROS production, and/or
depressed antioxidant defense. Glutathione (GSH)
is an important component of the antioxidant and
detoxifi cation systems in most tissues. However,
under normal cellular conditions, KBrO3 induces
oxidative DNA damage in the form of 8-oxode-
oxyguanosine (8-oxodG) that is dependent on GSH
and other sulfhydryls (Ballmaier and Epe, 1995;
Murata et al. 2001). Bromate-induced oxidative
DNA damage is caused by the reduction of bromate
to bromine oxides and bromine radicals generated
by sulfhydryls like GSH. In vivo, it has been
suggested that sulfhydryls, present at the brush
borders of the proximal convoluted tubule (Zager
and Burkhart, 1998), are important in the generation
of bromine oxides and bromine radicals (Murata
et al. 2001). The relative reduction of extracellular
and intracellular bromate and its metabolites also
Figure 2. Affymetrix array and QRT-rtPCR fold-change results of select kidney genes of interest from male F344 rats exposed to 400 ppm
KBrO3 for 52 wk.
Fold Change
5
0
5
-10
-15
-20
Array
rt-PCR
Slc12a4
Fabp5
Ccng1
Slc26a4
Ngfg
Klk7
Calb1
Aqp2
Aldh1a1

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Translational Oncogenomics 2006: 2
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Geter et al
affect the delivery of bromate ions to target macro-
molecules. Under high dose conditions, more
bromate may be delivered to target cells for intra-
cellular activation due to reduced systemic metab-
olism and/or antioxidant defenses (Delker et al.
2006).
Altered gene expression associated with oxida-
tive stress in the low dose gave only four down-
regulated genes Dscr1, Hspb1, Txnrd1, and Xdh.
It has been reported that in oxidative stress condi-
tions, Dscr1, Hspb1, and Txnrd1 are elevated
(Ermak et al. 2004; Dunlop and Muggli, 2000;
Yamawaki and Berk, 2005), and increased levels
of Xdh are associated with free radical generation
(Sanhueza et al. 1992). Dscr1 is regulated by both
oxidative stress and Ca+ levels (Lin et al. 2003),
thus it may be influenced more by Ca+ ion
concentration than oxidative stress. Although
these genes are associated with oxidative stress,
they are all down-regulated and appear to refl ect
inhibition, chronic adaptation, or simply no induc-
tion of oxidative stress in animals exposed to 20
ppm KBrO3.
A larger involvement of proposed oxidative
stress genes are seen in the high dose group with
10 DEG; 5 down- and 5 up-regulated. In the high
dose response, the Dscr1 and a heat-shock gene
Hspbap1, which are up-regulated in response to
ROS, are down-regulated in this study. This is an
unexpected response following exposure to a
known producer of ROS. However, the expression
observed in this experiment is that of a kidney
chronically exposed to a carcinogenic dose of
KBrO3, and may represent an adaptive gene
response. Two additional down-regulated genes,
Gnmt and Gsta2, are involved with GSH homeo-
stasis and GSH conjugation, respectively.
The up-regulated oxidative stress genes include
Ccng1, Cp, Gclm, and two glutathione transferases,
Gstm1 and Gstp1. Studies have demonstrated that
cyclin G1 (Ccng1) plays roles in G2/M arrest,
damage recovery, and growth promotion after
cellular stress (Kimura et al. 2001). Cyclin G1
accomplishes this by regulating the activity of p53
(Jensen et al. 2003). This may indicate possible
oxidative stress induced DNA damage followed
by increased cellular proliferation. Cp is a plasma
protein that is up-regulated in response to oxidative
stress and functions as a copper transporter and
antioxidant. Gclm is involved in glutathione
biosynthesis and is up-regulated as a defensive
response to oxidative stress (Mathers et al. 2004).
Two glutathione S-transferases are up-regulated,
Gstm1 and Gstp1. Both are involved in the conju-
gation of reduced GSH to a wide number of exog-
enous and endogenous hydrophobic electrophiles.
Taken together, these data support the fi nding that
KBrO3 does produce an oxidative stress response
in the kidney at high doses, however the magnitude
of expression was not large, resulting in ten genes
with relatively small fold-change. This may be
characteristic of a kidney that has developed a
resistant morphological phenotype to KBrO3-
induced oxidative stress due to long-term chronic
exposure.
Lipid metabolism
Altered lipid metabolism and lipid peroxidation
have been associated with KBrO3 exposure
(Chipman et al. 1998). Cellular fatty acids are
readily oxidized by reactive oxygen species to
produce lipid peroxyl radicals and lipid hydroper-
oxides (Rice-Evans and Burdon, 1993). It is gener-
ally accepted that oxidative stress can lead to the
oxidative degradation of lipids (Moller and Wallin,
1998), which can disrupt normal lipid metabolism.
Examination of genes associated with lipid metab-
olism in the low dose exposure showed 6 down-
regulated (Apoe, Cebpb, Chk, Cyp2e1, Cyp4a10,
and Fads1) and 4 up-regulated genes (Abcg1,
Calca, Gpd2, and Nr1d1). The down-regulated
genes dealt primarily with fatty acid metabolism
and lipid biosynthesis, while the up-regulated
genes were associated with lipid homeostasis and
metabolism. In the high dose, nine genes were
down-regulated (Acaa2, Acsl3, Apoc3, Apom,
Cyp2e1, Cyp4a10, Edn1, Ehhadh, Hadhsc),
and one up-regulated (Fabp5). These genes
were primarily involved in fatty acid and lipid
metabolism. Overall, gene expression for lipid
metabolism is suppressed, with only one up-regu-
lated gene observed in the high dose, contrary to
expectations of KBrO3 induced lipid peroxidation.
This is consistent with the work of Umemura et al.
(2004) which contained no evidence of lipid
peroxidation by chronic treatment of rats with
carcinogenic doses of KBrO3.
Kidney function and ion transport
The location of DEG genes involved with kidney
function and ion transport within the nephron of
the kidney is shown in Figure 3. For the low dose
(Fig. 3A), of the 9 genes where specifi c locations
could be identifi ed, 6 were in the proximal convo-

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
luted tubules (PCT), 2 in the ascending loop of
Henle (LH), and 1 in the distal convoluted tubules
(DCT). Within the PCT, only the kidney specifi c
organic anion transporter was up-regulated. For
the high dose (Fig. 3B), 7 genes were found in the
PCT, 4 in the ascending LH, 5 in the DCT, and 6
genes in the collecting duct (CD). All genes in the
PCT and CD were down-regulated, whereas
Slc12a1 and Cldn16 were up-regulated in the
ascending loop and Agtr2 was up-regulated in the
DCT. When comparing the low and high fi gures,
the high dose has more altered gene expression in
the PCT and signifi cantly more gene involvement
down-stream of the PCT with 14 altered genes
compared to only 3 in the low. The localized gene
expression changes depicted in this fi gure reinforce
the hypothesis that as the KBrO3 dose increases,
additional cellular alterations occur in the PCT. As
these changes occur, altered gene expression within
and down-stream of the PCT is initiated as an
attempt to maintain kidney function.
Kidney function and ion transport gene expres-
sion in the kidney from the 20 ppm exposure group
showed a total of 10 genes down- and 3 up-regu-
lated. Within the down-regulated group were genes
mainly dealing with transport of water (Aqp1),
sodium (Atp1a1 and Skg), potassium (Atp1a1),
glucose (G6pc), and organic cations (Slc22a1).
Although Aqp1 was down-regulated, no increase
in water consumption was observed. In the up-
regulated group were genes involved in calcium
homeostasis (Calca), magnesium and calcium
transport (Cldn16), and organic anion transport
(Slc21a4). These gene expression changes suggest
a physiological response that helps maintain elec-
trolyte balance in light of the chronic, low dose
KBrO3 exposure. Although the low concentration
of KBrO3 used in this study (20 ppm) was not
carcinogenic, results from a similar study using a
dose of 60 ppm in the drinking water found
increased proliferation and mild degeneration of
the proximal convoluted tubules (Umemura et al.
2004). However, data collected in this paper indi-
cate that physiological adaptation to KBrO3
exposure is beginning to occur even at a non-
carcinogenic dose.
Kidney function and ion transport gene expres-
sion in the kidney from the 400 ppm exposure
group showed 20 down- and 4 up-regulated genes.
Within the down-regulated group were genes
involved with transport of water (Aqp2, and Aqp3),
calcium (Calb1), chloride (Clcnk11 and Prkwnk4,
Slc12a3, and Slc26a4), potassium (Kcnj16, Kcnq1,
and Prkwnk4), sodium (Ngfg, Prkwnk4, Scnn1a,
Scnn1g, Slc5a2, Slc9a3, Slc12a3, and Slc13a3),
organic ions (Slc22a5 and Slc22a8), glucose
(Slc5a2 and Slc37a4), and regulation of blood
pressure (End1). In the up-regulated group were
Agtr2, an angiotensin II receptor; Cldn16, a magne-
sium and calcium transporter (also up-regulated in
the low dose); Cp, an iron transporter, and Slc12a1,
a sodium, potassium, chloride co-transporter.
These results imply signifi cant alterations in the
expression of genes involved in kidney function,
and possibly decreased organ function.
In this light, when examining the DeAngelo et al.
(1998) and Wolf et al. (1998) study, a dose-depen-
dent increase in water consumption was observed
starting at wk 4 and continuing through the dura-
tion of the study. Overall, it was determined that
rats from the high dose (400 ppm) group drank
over 30% more than controls. Taken together with
the large number of down-regulated kidney func-
tion and ion transport genes from the high dose
KBrO3 exposure, these data possibly indicate a
kidney in chronic renal insuffi ciency/failure. This
condition occurs when the kidneys are unable to
conserve water as they perform their blood fi ltering
function. The amount of water conserved is
controlled by antidiuretic hormone (ADH), also
known as vasopressin. ADH controls kidney
osmosis by inserting water pores into the collecting
ducts. The water pores, Aqp2 and 3, are responsible
for the fi nal adjustment of urine concentration. In
the high dose animals, Aqp2 and 3 transcripts are
down-regulated. This would result in fewer water
pores in the collecting duct leading to increased
amounts of dilute urine. With the increase in urine
production, the kidney may respond to maintain
ionic balance and blood volume by down-regu-
lating the genes contributing to loss of nutrients
and electrolytes and up-regulating transporter
genes necessary for reabsorption, such as Slc12a1
and Cldn16.
Within the high dose PCT and CD is a down-
regulated gene called pendrin, or Slc26a4. Pendrin
functions as a sodium-independent transporter of
chloride and iodide where it is expressed in kidney,
thyroid, and inner ear (Scott et al. 1999; Soleimani
et al. 2001). It should be noted that KBrO3, in addi-
tion to being a kidney carcinogen, is also a thyroid
carcinogen, and can cause deafness (Yoshino et al.
2004). Moreover, pendrin expression was lower in
thyroid carcinomas than in normal thyroid tissue

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Translational Oncogenomics 2006: 2
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Geter et al
Figure 3A and 3B. Figure 3A (20 ppm) and 3B (400 ppm) show the location within the kidney nephron of specifi c genes associated with
kidney function from male rats exposed to KBrO3 for 52 wk. Genes in green and red font are down-regulated and up-regulated respectively.
G: glomerulus, PCT: proximal convoluted tubules, LH: loop of Henle, DCT: distal convoluted tubules, and CD: collecting duct.

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Translational Oncogenomics 2006: 2
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Kidney Toxicogenomics of Chronic Potassium Bromate Exposure in F344 Male Rats
(Skubis-Zegadlo et al. 2005; Kondo et al. 2003).
Pendrin transcripts examined by QT-rtPCR in this
study were not altered in the low dose (20 ppm),
suppressed in high dose (400 ppm), and virtually
absent in kidney adenomas, mirroring the results
seen in the thyroid carcinomas. Due to pendrin
already being identifi ed as a biomarker in thyroid
carcinogenesis, and its involvement in kidney
function following KBrO3 exposure, we propose
this gene as a possible biomarker of carcinogenic
KBrO3 exposure.
Cellular function
The primary objective of this study was to deter-
mine if a discernable difference in kidney gene
expression could be observed between a non-
carcinogenic and carcinogenic dose of KBrO3.
Gene ontology analysis of altered gene expression
revealed an accumulation of changes in the
following functionally-related categories: cancer,
cell cycle, cell death, cell-to-cell signaling and
interaction, cellular development, and cellular
growth and proliferation. Thus, the genes that fell
into these categories were placed into a general
cellular functional group for analysis. Within this
group were 30 down- and 11 up-regulated genes
in the low KBrO3 exposure group, and 20 down-
and 22 up-regulated genes in the high exposure
group.
In the low KBrO3 concentration, the majority
of genes associated with cellular function were
down-regulated (30 versus 11 up-regulated).
Several low dose genes when down-regulated are
associated with decreased cell proliferation and
apoptosis (Akap12, Arhb, Cebpb, Csf 2rb, Dusp6,
Epim, Gadd45a, Id2, Id3, Ig f bp3, Ig f bp6, Jun,
Pim1, Rgc32, Tieg, and Veg f b). This is countered
by only one up-regulated gene Ccnd1 (Cyclin d1),
which is associated with increased cell prolifera-
tion and apoptosis. From this observation, it
appears that the gene expression from the low dose
kidney does not support increased levels of cell
proliferation similar to the BrdU findings of
Umemura et al. (2004) whose no-effect level was
15 mg/L.
The gene expression in the high KBrO3 concen-
tration was more equally distributed with 22 down-
and 20 up-regulated genes. There were 10 genes
shared in the cellular function category between
the low and high KBrO3 concentration. These were
Cyp2e1, Dscr1, Dsipi, F3, Id2, Ig f bp1, Ms4a2,
Rgc32, Ptgds, and Vipr1. All were directionally
concordant except for Ig f bp1.
In contrast to the low dose, fewer genes were
associated with cell proliferation and apoptosis
(Ccng1, Eno1, Hrasls3, Id2, Igfbp1, Madh7, and
Nupr1). Eno1 and Hrasls3, when down-regulated
(Subramanian and Miller, 2000; Feo et al. 2000;
Sers et al. 2002), and the up-regulated genes,
Nupr1, and Ccng1 promote cellular growth and
proliferation. However, Id2 and Madh7 when
down-regulated support decreased proliferation
(Lasorella et al. 1996; Lallemand et al. 2001).
Although there are only a few genes in this group,
the majority suggest increased cell proliferation
and apoptosis.
Adenoma comparison
Stuart et al. (2001) established a benchmark
expression profi le for normal kidney development.
By comparing the gene expression changes in the
present study to this profi le, expression changes
that deviated from normal kidney were detected.
In four defi ned kidney development groups, the
52 wk bromate-treated kidneys did not deviate
from normal developmental patterns whereas the
adenoma samples did. In these samples, the
adenoma expression patterns were more charac-
teristic of embryonic than adult kidneys. Although
the bromate-treated kidneys did not show a strong
expression pattern that matched the adenoma, the
high dose kidney, but not the low, did resemble the
adenoma expression pattern with genes prevalent
in early kidney development being up-regulated
and adult phase genes being down-regulated. This
observation is in agreement with the proliferation
and de-differentiation profi les seen in classical
tumor development. Furthermore, the method of
comparing developmental gene expression patterns
between tumor and exposed animals could serve
as a mechanism to identify biomarkers of tumor
initiation.
Conclusion
These data suggest the 400 ppm carcinogenic dose
of KBrO3 showed marked gene expression differ-
ences from the non-carcinogenic dose. These
include gene expression changes in oxidative stress
and kidney function/ ion transport genes. Compar-
ison of kidney development gene expression
showed that the adenoma patterns were more char-
acteristic of embryonic than adult kidneys, and that

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Translational Oncogenomics 2006: 2
46
Geter et al
the high dose kidney gene expression resembled
an adenoma-like expression pattern. Taken
together, these analyses from this study identify
potential biomarkers of exposure and illuminate a
possible carcinogenic mode of action for KBrO3.
Competing Interests
The authors declare that they have no competing
interests.
Authors’ Contributions
DG aided in study design, carried out Affymetrix
experiments, analyzed data, and wrote the manu-
script. WW analyzed adenoma comparison with
kidney development genes and aided in manuscript
and fi gure preparation. GK performed QRT-rtPCR
experiments and data analysis, and aided in manu-
script and fi gure preparation. JR aided in manu-
script and fi gure preparation. AD, RO, and JA were
involved in study design with RO assisting in data
analysis. DD was involved in study design, carried
out Affymetrix experiments, analyzed data, and
aided in manuscript preparation.
Acknowledgements
The authors would like to thank Drs. Chris
Corton and Kevin Morgan for their review of this
manuscript.
The research described in this article has been
reviewed by the Health and Environmental
Effects Research Laboratory, United States
Environmental Protection Agency, and approved
for publication. Approval does not signify that
the contents necessarily refl ect the views of the
Agency, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use.
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Additional Files
File name: Oxidoreductase_table.pdf
File format: .pdf
Title of data: Oxidoreductase table
Description of data: List of differentially expressed
oxidoreductase gene expression in kidney from
male rats exposed to 20 ppm (low) and 400 ppm
(high) potassium bromate in drinking water for
52 wk.
File name: Lipid metabolism_table.pdf
File format: .pdf
Title of data: Lipid metabolism table
Description of data: List of differentially expressed
lipid metabolism gene expression in kidney from
male rats exposed to 20 ppm (low) and 400 ppm
(high) potassium bromate in drinking water for
52 wk.
File name: Cellular function_table.pdf
File format: .pdf
Title of data: Cellular function table