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Nongenotoxic mechanisms involved in bromateinduced cancer in rats
R I CHARD J . BULL A ND JO SE PH A . C O TRU VO
http://dx.doi.org/10.5942/jawwa.2013.105.0155
Risk assessments for bromate (BrO3–) in drinking water
are based on linear extrapolation from the total incidence
of tumors in male rats. The only genotoxic effects that
might result from carcinogenic doses of BrO3– in vivo are
the formation of 8-oxodeoxyguanosine (8-oxoG) in
deoxyribonucleic acid (DNA) and the production of
micronuclei. The mutations in tumors are consistent with
the 8-oxoG adduct, and both effects are nonlinear with
respect to dose. Treatment of rats with BrO3– resulted in
bromination of protein tyrosines. The accumulation of
these proteins in the kidney appeared to contribute to
kidney cancer in male, but not female, rats. BrO3– increased
the rate of apoptosis (programmed cell death) in the
kidneys of rats of both sexes, an effect associated with
increasing expression of antiapoptotic genes and proteins.
Consequently, suppression of apoptosis is a likely
mechanism for BrO3–-induced kidney cancer. More limited
data suggest nongenotoxic modes of action for thyroid
tumors and mesotheliomas. If these data are confirmed,
linear extrapolation of risk to low doses is inappropriate.
Bromate (BrO 3 – ) is an established carcinogen in
rodents, producing kidney, testicular, and thyroid tumors
in rats and kidney cancer in mice and hamsters (Gold et
al, 2012). The current maximum contaminant level
(MCL) for BrO3– is 10 µg/L. The US Environmental Protection Agency (USEPA) estimated that the additional
cancer risk posed by BrO3– at this concentration was two
in 10,000 per lifetime (USEPA, 2001). The California
Environmental Protection Agency corrected this estimate
to one in 10,000 (OEHHA, 2009) by including data that
were not available to USEPA. BrO3– in drinking water is
most closely associated with the use of ozone to treat
water sources containing high concentrations of bromide.
This is complicated by the fact that some hypochlorite
solutions used in secondary disinfection can contain significant amounts of BrO3– (Weinberg et al, 2003).
rodents must be questioned. Studies in rats have shown
increases in the amount of 8-oxoG in the genomic DNA
of the kidney at the highest doses studied. This adduct
also results from normal energy metabolism. The mutations that have been detected in the kidney of rats treated
with BrO3– have a mutation spectrum consistent with
8-oxoG being their cause. However, unlike the predictions
of linear risk models, the increased mutation frequencies
were not linear with dose (Yamaguchi et al, 2008).
The key question is: Are there other mechanisms by
which BrO3– might act at doses that are just sufficient to
induce cancer in rodents? Studies have now shown that
BrO3– doses in this range increase necrosis and subsequent
replication of cells in the kidney of male rats that develop
cancer but not in the kidney of females (Umemura et al,
2004). Such effects contribute to the development of cancer. Subsequent studies have shown that this difference
between the sexes can be distinguished at the molecular
level (Kolisetty et al, 2013a, b). A more important finding
was that both sexes displayed an increased rate of apoptosis (Kolisetty et al, 2013a). As BrO 3– doses were
increased, evidence of suppressed apoptosis was observed
in the kidneys of both sexes. Suppression of apoptosis
allows damaged cells, including cells with damaged DNA,
to survive and is recognized as a mechanism by which
cancer can be induced. Effects on apoptosis extend to doses
as low as 1 mg/kg body weight—lower than doses that
result in measurable increases in kidney cancer. Suppression of apoptosis is more likely to account for the increases
in 8-oxoG than the mechanisms identified from in vitro
studies. Suppressed apoptosis is an indirectly genotoxic
mechanism that is initiated by signaling processes in
response to cellular injury. This mechanism probably
THE ROLE OF GENOTOXIC EFFECTS
The risk from genotoxic carcinogens is assumed to be
linear with dose, implying no threshold in the dose–
response. The basis of this assumption lies in the notion that
the probability of genotoxic events (e.g., mutation) leading
to cancer is a linear function of dose (USEPA, 2005). However, it has become increasingly apparent that many carcinogens do not interact directly with DNA to produce a
mutation. An MCL goal (MCLG) of zero is not assigned to
nongenotoxic carcinogens.
Very high concentrations of BrO3– clearly are capable
of inducing genotoxic damage in vitro (USEPA, 2001;
OEHHA, 2009). These concentrations exceed those
observed in the blood plasma of rats treated with carcinogenic doses of BrO3– by a factor of 6,000. Thus the
role of genotoxic effects in the induction of cancer in
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accounts for the nonlinear dose–response observed for
8-oxoG formation and the frequency of mutations in rats.
higher MCL that would allow the wider use of ozone as a
disinfectant and oxidant at significantly lower costs.
EFFECTS OF BrO3 – REDUCTION TO BROMIDE
REFERENCES
At least 90% of the BrO3– administered to rats is reduced
to bromide (Bull et al, 2012). In short-term studies, bromide
has been shown to alter thyroid function in rats treated
with the same range of BrO3– doses that induce thyroid
cancer (Velický et al, 1997). Although no studies of bromide-induced thyroid cancer have been conducted, the type
of alteration produced (i.e., inhibition of the sodium iodide
symporter, or NIS) is known to induce thyroid cancer in
rats (NRC, 2005) by a nongenotoxic mechanism.
When BrO3– was ingested by rats, a side reaction
occurred along with its chemical reduction—bromination
of endogenous biochemicals in a dose-dependent manner
(Bull et al, 2012). Proteins with brominated tyrosines were
detected immunohistochemically in individual cells of the
testes and kidney. Phosphorylation and dephosphorylation
of protein tyrosines are involved in the control of a variety
of biochemical systems in vivo but also control the degradation of these proteins and account for their accumulation in the kidney and testes of rats treated with BrO3–.
The accumulation of the protein a-2u-globulin resulted in
necrosis of the renal tubules of male rats and in the development of kidney cancer, but this effect did not occur in
female rats. The brominated proteins that accumulated in
the testes have not been identified.
The cells in which the bromine-modified proteins accumulated in the testes are responsible for the synthesis of
sex hormones. In the study of gene expression in the kidney
of rats treated with BrO3–, expression of the gene for the
kidney androgen-activated protein was down-regulated in
the male kidney (Cotruvo et al, 2012), indicating that
androgen (e.g., testosterone) synthesis was inhibited by
BrO3– treatment.
Bull, R.J.; Kolisetty, N.; Zhang, X.; Muralidhara, S.; Quiñones, O.; Lim,
K.Y.; Guo, Z.; Cotruvo, J.A.; Fisher, J.W.; Yang, X.; Delker, D.; Snyder,
S.A.; & Cummings, B.S., 2012. Absorption and Disposition of Bromate in F344 Rats. Toxicology, 300: 1–2:83.
IMPLICATIONS FOR OZONATION
USEPA, 2001. Toxicological Review of Bromate (CAS No. 15541-45-4): In
Support of Summary Information on the Integrated Information
System (IRIS). EPA/635/R-01/002. USEPA, Washington.
Review of more recent data on the mode of action of
BrO3– shows that the case for genotoxic mechanisms in its
induction of cancer in the rat is poorly supported by in
vivo studies. In contrast, the new data indicate that apoptosis and its suppression are more probable causes of
cancer of the kidney. Thyroid cancer is likely induced by
the reduction of BrO3– to bromide, an established inhibitor
of the NIS. This leads to depressed thyroid hormone synthesis, a condition that is carcinogenic to the rat thyroid
gland. Finally, there is evidence of BrO3–-induced hormonal
changes that are consistent with this chemical’s induction
of mesothelial tumors in the testes. Although additional
studies will be necessary to show conclusively that BrO3–
acts solely by nongenotoxic mechanisms at sublethal doses,
confirmation of the hypotheses put forward in this review
can be evaluated by straightforward experimentation. If
these hypotheses are confirmed, a nonzero MCLG for
BrO3– would be appropriate, and this could lead to a
48
Cotruvo, J.A.; Bull, R.J.; Cummings, B.S.; Delker, D.; Guo, Z.; Fisher, J.;
Quiñones, O.; Snyder, S.A.; & Ong, C.N., 2012. Bromate Disposition
and Mechanisms of Toxicity at High and Low Doses. Water RF
(Water Research Foundation), Denver.
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edu/chempages/BROMATE,%20POTASSIUM.html (accessed Apr.
12, 2012).
Kolisetty, N.; Bull, R.J.; Muralidhara, S.; Costyn, L.J.; Guo, Z.; Cotruvo,
J.A.; Fisher, J.W.; & Cummings, B.S., 2013a. Association of Brominated Proteins and Changes in Protein Expression in the Rat Kidney With Subcarcinogenic to Carcinogenic Doses of Bromate.
Toxicology and Applied Pharmacology, 272:2:391.
Kolisetty, N.; Delker, D.A.; Muralidhara, S.; Bull, R.J.; Cotruvo, J.A.;
Fisher, J.W.; & Cummings, B.S., 2013b. Changes in mRNA and Protein Expression in the Renal Cortex of Male and Female F344 Rats
Treated With Bromate. Archives of Toxicology.
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2004. Dose-Related Changes of Oxidative Stress and Cell Proliferation in Kidneys of Male and Female F344 Rats Exposed to Potassium Bromate. Cancer Science, 95:5:393. http://dx.doi.
org/10.1111/j.1349-7006.2004.tb03221.x.
USEPA (US Environmental Protection Agency), 2005. Guidelines for Carcinogen Risk Assessment. EPA/630/P-03/001F. USEPA, Washington.
Velický, J.; Titlbach, M.; Dušková, J.; Vobecký, M.; Štrbák, V.; & Raška, I.,
1997. Potassium Bromide and the Thyroid Gland of the Rat: Morphology and Immunohistochemistry, RIA, and INAA Analysis.
Annals of Anatomy, 179:5:421. http://dx.doi.org/10.1016/S09409602(97)80041-6.
Weinberg, H.S.; Delcomyn, C.A.; & Unnam, V., 2003. Bromate in Chlorinated Drinking Waters: Occurrence and Implications for Future
Regulation. Environmental Science & Technology, 37:14:3104.
http://dx.doi.org/10.1021/es026400z.
Yamaguchi, T.; Wei, M.; Hagihara, N.; Omori, M.; Wanibuchi, H.; & Fukushima, S., 2008. Lack of Mutagenic and Toxic Effects of Low Dose
Potassium Bromate on Kidneys in the Big Blue Rat. Mutation
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Corresponding author: Richard J. Bull is a professor
emeritus at Washington State University, 1928
Meadows Dr. N., Richland, Wash. 99352 USA;
[email protected].
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