United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1-87/007 Nov. 1987
4>EPA Project Summary
Carcinogenic Effects of Arsenic
Compounds in Drinking Water
Donald Y. Shirachi, Shing-Hui Tu, and John T. McGowan
The primary objective of this research
project was to determine whether there
was a dose-response effect of arsenic
(Aslll) as a promoter of DENA-initiated
tumors in the kidneys of partially
hepatectomized rats. Secondary objec-
tives were to complete the study ex-
tending from the previous grant which
was to determine whether arsenite
(Aslll). arsenate (AsV) and dimethylar-
sinic acid (DMA) were initator car-
cinogens and/or promoters of DENA-
initiated tumors in the rat kidney. In
addition an analytical method for mea-
suring the above three arsenic species
simultaneously in the same biological
sample was to be developed.
A dose-response was observed for
Aslll promotion in partially hepatecto-
mized DENA-initiated rats at concen-
trations of 40.80 and 160 mg/L in the
drinking water but it was not statistically
significant.
Aslll and AsV did not have any initia-
tor activity in intact male Wistar rats
but significantly promoted DENA-initi-
ated renal tumors in these animals. DMA
had neither initiator nor promoter
activity.
A continuous flameless HPIC-electro-
thermal atomic absorption spectre-
photometric method was developed to
detect and to quantitate simultaneously
nanograms quantities of Aslll, AsV and
DMA in biological fluids.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park, NC
to announce key findings of the research
protect that Is fully documented In a
separate report of the same title (see
Project Report ordering Information at
back).
Introduction
Arsenic has been associated with
cancer in humans for a number of years
based mainly on epidemiological studies
(IARC, 1973; Yeh, 1973; Zaldivar, 1974;
Bencko, 1976; NAS Committee, 1977;
and Wildenberg, 1978). Experimental
evidence in animal studies have basically
been negative (Wildenberg, 1978) or the
epidemiological evidence has been com-
plicated by the presence of other chemi-
cals or conditions which complicated the
interpretation of the data (Pershagen,
1981). Thus, arsenic has not been un-
equivocally demonstrated to be a car-
cinogen; at best it may be a very weak
carcinogen.
More recently, the contractor's labora-
tory has been involved in determining
whether arsenic might be a promoter of
carcinogenesis rather than acting as an
initiator carcinogen. Thus, the project
used an initiator-promotion experimental
design in partially hepatectomized rats
utilizing diethylnitrosamine as the initi-
ator. Results showed that arsenite (Aslll)
was a promoter of DENA-initiated renal
tumors which were cortical in origin and
associated with the renal tubular cells
(Shirachi etal., 1983).
Associated with this effect was also a
decrease in thymus weight, implicating a
possible immunosuppressive effect as a
contributory factor in the carcinogenesis
process. In addition, these effects were
observed in partially hepatectomized rats,
a surgical procedure used to study hepatic
carcinogenesis. Thus, it was possible that
the partial hepatectomy might have been
also a contributory factor in the promotion
effect of Aslll, complicating the interpreta-
tion of the data obtained.
-------
Therefore, the first objective of this
study was to determine whether partial
hepatectomy was a contributory factor by
repeating the previous study in intact
rats, using the same experimental design.
If the results showed that partial hepatec-
tomy was not contributory, then the
second objective was to determine
whether this promotion effect was a
dose-response related phenomenon. This
latter effect would be important in
determining safe drinking water stan-
dards Since we have determined in pre-
liminary studies that the various arsenic
species are interconvertible in the liver
(unpublished results), a final objective
was to develop an analytical methodology
for determining simultaneously the levels
of the different arsenic species in bio-
logical samples. This would allow for
blood and tissue level measurements to
determine the effects of endogenous bio-
transformation of the different arsenics
administered.
Materials, Methods and Kidneys
THE PROMOTION EFFECTS OF AR-
SENIC COMPOUNDS ON LIVERS FROM
CONTROL AND DENA-INITIATED WISTAR
RATS Cronically Treated with Aslll, AsV
and DMA in the Drinking Water for 10,
15 and 24 Months.
A two stage initiation-promotion proto-
col was utilized for these experiments.
Male Wistar rats 5 to 6 weeks old were
injected with either 0.9% saline or DENA
(Eastman Kodak) at a single i.p dose of
30 mg/kg and placed on regular Purina
rat chow and deionized water ad lib. On
day 7 the animals were placed in treat-
ment groups and the promotion was in-
stituted The control groups were con-
tinued on deionized water, the Aslll, AsV
and DMA treatment groups pretreated
with 0 9% saline were put on drinking
water ad lib containing elemental arsenic
m the form of sodium arsenite (Aslll),
sodium arsenate (AsV) or dimethylarsmic
acid (DMA) in deionized water equivalent
to elemental arsenic at concentration of
160, 160 and mg/L respectively, and the
Aslll, AsV and DMA treatment groups
pretreated with DENA were put on drink-
ing water ad lib containing sodium
arsenite and dimethylarsmic acid in
deionized water also equivalent to 160,
160 and 80 mg/mL respectively These
animals were sacrificed at 10, 15 and 24
months
Livers were taken at necropsy pro-
cessed and examined microscopically for
basophilic foci, neoplastic modules and
hepatocellular carcinomas Kidneys were
also taken and examined microscopically
for atypical hyperplasia, adenoma and
adenocarcinomas
THE DOSE-RESPONSE EFFECT IN
TUMORIGENESIS IN DENA-INITIATED
MALE WISTAR RATS PROMOTED WITH
Aslll OR AsV FOR 6, 9 AND 12 MONTHS
IN THE DRINKING WATER
Materials and Methods
A two-stage initiation-promotion
protocol utilizing partially hepatectomized
rats was used for these experiments. Male
Wistar rats weighing 70-1 OOg were par-
tially hepatectomized (PH) under ether
anesthesia and allowed to recover. The
animals were injected 18-24 hours later
with a single i.p. dose of 0.9% saline or
DENA (Eastman Kodak) 25 mg/kg and
placed on regular Purina rat chow and
deionized water ad lib. On day 7 the
animals were randomly placed in treat-
ment groups and the promotion protocol
was instituted. The saline control and
DENA control treatment groups were
continued on deionized water; the Aslll
treatment group pretreated with 0.9%
saline was put on drinking water ad lib
containing elemental arsenic in the form
of sodium arsenite (Aslll) in deionized
water equivalent to 160 mg/L; and the
Aslll treatment group pretreated with
DENA were put on drinking water ad lib
containing sodium arsenite in deionized
water equivalent to 40, 80 and 160 mg/L.
In the nonhepatectomized rats the animals
were injected with a single i.p dose of
0.9% saline or DENA 40 mg/kg. On day 7
the animals were randomly placed in
treatment groups and the promotion
protocol was instituted.
The saline control and DENA control
treatment groups were continued on
deionized water; the Aslll and AsV treat-
ment groups pretreated with 0.9% saline
were put on drinking water ad lib con-
taining elemental arsenic in the form of
sodium arsenite and sodium arsenate
(AsV) in deionized water equivalent to
160 mg/L; and the Aslll and AsV treat-
ment groups pretreated with DENA were
put on drinking water ad lib containing
sodium arsenite or sodium arsenate in
deionized water equivalent to 80 and 160
mg/L. These animals were sacrificed at
6, 9, and 12 months
At necropsy, all animals were weighed
and the kidneys removed, weighted and
placed in 10% buffered formalin The
tissues were then processed for paraffin
embedding and prepared by routine his-
tological techniques for H and E staining.
The slides were examined microscopically
for atypical hyperplasia, adenoma and
adenocarcmoma in renal tissues.
SIMULTANEOUS DETERMINATION OF
ARSENIC COMPOUNDS SPECIES BY
HPLC-ELECTROTHERMAL ATOMIC
ABSORPTION SPECTROSCOPY IN BIO-
LOGICAL FLUIDS
The analytical method consisted of a
HPLC connected to a low capacity anion
exchange column which separated the
different arsenic species and converted
by post-column modification by means of
a hydride generator to their respective
arsine gases. The arsine gas was sepa-
rated from the liquid by an on-line gas-
liquid separator through which a carrier
gas passed to deliver the arsine gas into
a quartz cell mounted on an atomic ab-
sorption spectrophotometer. The hyride
generator system was driven by a propor-
tioning pump and manifold.
MONITORING OF FOOD CONSUMP-
TION, BODY WEIGHT AND WATER CON-
SUMPTION DURING CHRONIC EXPO-
SURE TO ARSENIC IN THE DRINKING
WATER
Body weight. Because of the number of
animals involved, they were weighed as
a group from each cage rather than in-
dividually. Except for a few exceptions the
number of animals in each cage did not
exceed five. Because the animals from
each cage were either sacrificed or may
have died, the changes were noted but
the remaining animals were not moved
to other cages. Therefore, all animals
remained in the same cage during the
entire study. Animals in each cage were
weighed as a group once a week and the
weight recorded.
Food consumption. As with the body
weight monitoring, food consumption of
animals in each cage was group moni-
tored Usually, the food was weighed on
the same day, depending upon the num-
ber of cages and animals. The total
amount of food provided for each cage
was weighed and recorded periodically.
Conclusions and
Recommendations
Promotion Effects In Liver
The study concluded that (1) basophilic
foci are probably precursor cells tc
neoplastic nodules m the liver; however
it is equivocal whether these cells are
preneoplastic lesions, (2) it is probable
that neoplastic nodules are precursoi
lesions to hepatocellular carcinomas
however, the presence of these lesions
does not indicate necessarily a patho
genesis to hepatocellular carcinoma; (3
2
-------
formation of basophilic foci and neoplastic
nodules are delayed in nonhepatecto-
mized animals; (4) Aslll and DMA may be
weak promoter carcinogens for DENA-
initiated hepatocellular carcinomas in the
rat liver; (5) one of the mechanisms by
which Aslll may be acting as a promoter
is suppression of the immune system;
and (6) hepatocellular carcinoma appears
to take a longer time to occur when com-
pared with the appearance of renal
adenoma or adenocarcinoma.
It is recommended that further study of
the Aslll and DMA potential promoter
effects for hepatocellular carcinoma be
made in partially hepatectomized rats.
However, since diethylnitrosamine
(DENA) appears to be a good initiator for
the inorganic arsenics, Aslll and AsV, in
renal tumorigenesis, we recommend that
several different initiators other than
DENA be tested because the animals will
more than likely die from the kidney
lesions before the liver effects can be
observed with DENA as the initiator.
Promotion Effects In Kidney
The data from this study suggest that
(1) there is no direct correlation between
formation of atypical hyperplasia and the
appearance of renal tubular adenomas
and/or adenocarcinomas; (2) atypical
hyperplasia is a general response to toxic
substances and occurs to the same degree
in response to all three arsenic treatments
studied; (3) Aslll and AsV are promoters
for DENA-initiated renal tumors; (4) partial
hepatectomy is not a prerequisite for
DENA-induced renal tumorigenesis pro-
moted by the inorganic arsenic com-
pounds; (5) inorganic arsenics are not
weak carcinogens under the experimental
conditions of this study; and (6) a possible
mechanism for the promotion effects of
Aslll and AsV might be at least partially
due to immunosuppression. Thus, the
results from this study support earlier
findings in partially hepatectomized rats
that Aslll is a promoter of DENA-initiated
renal tumors. Now AsV can also be said
to have the same effect.
Since the doses used for both inorganic
arsenics were the same, 160 mg/L in the
drinking water, and they both promoted
renal tumorigenesis initiated by DENA, it
is important to determine whether these
arsenic species were exerting their pro-
motion effects via a common mechanism
due to biotransformation or via different
mechanisms. Since the chemistry of the
two inorganic arsenics are very different
but can be interconverted by biotrans-
formation; and since the promotion effect
occurred at the same dose, a common
mechanism may have been involved. A
systematic study of the metabolism of
the various arsenic species should be
done both in the liver and in the kidney to
determine the metabolic fate of each
specie after it is absorbed from the G.I.
tract. This should indicate to which arsenic
specie the kidney would be exposed, ir-
respective of which arsenic specie was
ingested via the drinking water. This effort
should provide some insight into the
arsenic species responsible for the pro-
motion effect. In addition, a followup study
should be made to determine whether
the immune response is implicated in
some way and whether this might explain
at least partially the occurrence of the
tumorigenesis process in the renal
tubules.
THE DOSE-RESPONSE TUMORIGENIC
PROMOTION EFFECTS OF Aslll AND AsV
Some experimental evidence in this
study indicates that Aslll and AsV might
increase kidney growth to some extent,
and also that the dose-response curve for
renal tumorigenesis promoted by Aslll is
very steep and not easily determined.
Further studies conducted on dose-
response effects of Aslll and AsV in terms
of promotion of renal tumorigenesis,
should include' (1) use of the partial
hepatectomy model; (2) an increased initi-
ation dose to 30 mg/kg; and (3) observa-
tion of tumorigenesis at an earlier time
period (between 3-6 months).
SIMULTANEOUS DETERMINATION OF
ARSENIC SPECIES BY HPLC-ELECTRO-
THERMAL ATOMIC ABSORPTION SPEC-
TROSCOPY IN BIOLOGICAL FLUIDS
This method will be able to detect
some of the arsenic species down to 5-
10 ng and others down to 20-25 ng.
Thus, the method will be useful in mea-
suring nanogram quantities from blood,
urine and tissue samples. It has the
advantage over most of the other anlaytical
methods in that it is sensitive and is a
continuous flow system. This analytical
method should be further developed for
studying arsenic metabolism and mea-
suring the various arsenic species in
biological fluids.
MONITORING OF CUMULATIVE BODY
WEIGHT, WATER AND FOOD CONSUMP-
TION
When Aslll and AsV are put into drink-
ing water, they have an inhibitory effect
on the cumulative body weight gained at
the doses used in this study. This was a
primary effect of Aslll and AsV because it
occurred in the presence or absence of
DENA-initiation. This decrease in cumula-
tive body weight gain probably was due
to the inhibitory effect these compounds
has on water consumption which was
significantly decreased at these doses.
Aslll and AsV also inhibited food con-
sumption during the initial rapid growth
stage, and probably decreased weight
gain. With respect to the tumorigenesis,
it is not clear whether any of these in-
hibitory effects played a role. The decrease
in food consumption could have had an
adverse effect on the nutritional require-
ments of these animals, thereby contri-
buting to the promotion of DENA-initiated
renal tumors.
References
Bencko, V, Benes, B., and M. Cikrt,
Biotransformation of As(lll) to As(V) and
Arsenic Tolerance. Arch. Toxicol., 36:159
(1976).
Buchet, J.P. Lauwerys, R. and Roels, H.
Comparison of the Urinary Excretion of
Arsenic Metabolites After a Single Oral
Dose of Dosium Arsenite, Monomethylar-
sonate, or Dimethylarsinate in Man. Int.
Arch. Occup. Environ. Health. 48:71
(1981)
Committee on Medical and Biologic
Effects of Environmental Pollutants.
Arsenic. National Academy of Sciences.
Washington, D.C. pp. 332 (1977).
Crecelius, E.A., Changes in the Chemi-
cal Speciation of Arsenic Following Injec-
tion by Man. Environmental Health
Perspectives, 19:147(1977).
Emmelot, P. and E. Scherer, The First
Relevant Cell Stage in Rat Liver Carcino-
genesis: A Quantitative Approach. Bio-
chim. Biophys. Acta., 605:247 (1980).
IARC Monographs on the Evaluation of
the Carcinogenic Risk of Chemicals to
Man. Vol. 2. Some Inorganic and Or-
ganometallic Compounds. Lyon pp. 181
(1973).
Ivankovic, S., G. Eisenbrand, and R.
Preussmann, Lung Carcinoma Induction
in BD Rats After Single Intratracheal In-
stillation of An Arsenic-Containing Pesti-
cide Mixture Formerly Used in Vineyards.
Int. J. Cancer, 24:786 (1979).
Johansen, M.G., J.P. McGowan, S-H.
Tu, and D.Y. Shirachi, Tumorigenic Effect
of Dimethylarsinic Acid in the Rat. Proc.
West. Pharmacol. Soc., 27:289 (1984).
Lakso, J.U., L.J. Rose, S.A. Peoples,
and D.Y. Shirachi, A Colorimetric Method
for the Determination of Arsenite, Ar-
senate, Monomethylarsonic Acid and
Dimethylarsinic Acid in Biological and
Environmental Samples. J. Agric. Food
Chem., 27(6):1229(1979).
Manneh, V.A., J.P. McGowan, and D.Y.
Shirachi, The Determination of Arsenic
-------
by HPLC-Electrothermal Atomic Absorp-
tion Spectrophotometry. Proc. West.
Pharmacol. Soc., 29:137 (1986).
Pershagen, G., The Carcinogenicity of
Arsenic. Environ. Health Perspect. 40:93
(1981).
Ricci, G., S. Shepard, N. Hester and G.
Colovos, Particulate Arsenicals, in NIOSH
Manual of Analytical Methods 6:320-1
(1980).
Shirachi, D.Y., M.G. Johansen, J.P.
McGowan and S-H Tu, Tumorigenic Effect
of Sodium Arsenite in Rat Kidney. Proc.
West. Pharmacol. Soc., 26:413 (1983).
Shirachi, D.Y., S-H Tu, and J.P.
McGowan, Carcinogenic Potential of
Arsenic Compounds in Drinking Water,
EPA Technical Report (EPA/600/S1-
86/0031(1986).
Tam, G.K.H., Charbonneau, S.M., Bryce,
F., Pomroy, C. and Sandi, E. Metabolism
of inorganic Arsenic in Humans Follow-
ing Oral Injection, Toxicol. Appl. Pharm.
50:319(1979).
Wildenberg, J., An Assessment of
Experimental Carcinogenic-Detecting
System with Special Reference to In-
organic Arsenicals. Environ. Res. 16:139
(1978).
Yeh, S., Skin Cancer in Chronic Ar-
senicalism. Human Pathol. 4:4469(1973).
Zaldivar, R., Arsenic Contamination of
Drinking Water and Food Stuffs Causing
Endemic Chronic Poisoning. Beitr. Path.
Bd., 151:384(1974).
Donald Y. Shirachi, Shing-Hui Tu. and John T. McGowan are with the University
of the Pacific, Stockton, CA 95211.
Merrel Robinson is the EPA Project Officer (see below).
The complete report, entitled "Carcinogenic Effects of Arsenic Compounds in
Drinking Watei," (Order No. PB 87-232 542/AS; Cost: $13.95, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
US.OFFICIALM
J^-^
Official Business
Penalty for Private Use S300
EPA/600/S1-87/007
0000329 PS
u s M
s!8i!"o
CHICAGO
*GE"eT
------- |