AEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600/1-78 066
November 1978
Research and Development
Effect of
Insecticides on
Benzo(a)pyrene
Carcinogenesis
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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3. Ecological Research
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This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
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ances of man for unhealthful substances or conditions. This work is generally
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clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-78-066
November 1978
EFFECT OF INSECTICIDES ON BENZO(A)PYRENE
CARCINOGENESIS
by
Anthony J. Triolo
Jefferson Medical College of
Thomas Jefferson University
Philadelphia, PA 19107
Gr-.nt No. R-803486
Project Officer
Thomas M. Scotti
Toxic Effects Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or recom-
mendation for use.
11
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FOREWORD
The many benefits of our modern, developing, industrial
society are accompanied by certain hazards. Careful assessment
of the relative risk of existing and new man-made environmental
hazards is necessary for the establishment of sound regulatory
policy. These regulations serve to enhance the quality of our
environment in order to promote the public health and welfare and
the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle
Park, conducts a coordinated environmental health research
program in toxicology, epidemiology, and clinical studies using
human volunteer subjects. These studies address problems in air
pollution, non-ionizing radiation, environmental carcinogenesis
and the toxicology, of pesticides as well as other chemical
pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for
which national ambient air quality standards exist or are proposed,
provides the data for registration of new pesticides or proposed
suspension of those already in use, conducts research on hazardous
and toxic i aterials, and is preparing the health basis for non-
ionizing radiation stand rds. Direct support to the regulatory
function of the Agency is provided in the form of expert testimony
and preparation of affidavits as well as expert advice to the
Administrator to assure the adequacy of health care and surveillance
of persons having suffered imminent and substantial -endangerment
of their health.
The human population is constantly exposed to low levels of
many different types of chemicals in the environment. Experimen-
tal investigations have emphasized the testing of individual
chemical compounds for their oncogenic potential, but relatively
little is known about the oncogenic potential of the various
chemicals in combination exposures in the environment. The
present investigation, therefore, is important since it is
designed to learn more about the oncogenic potential associated
with combined environmental exposure to different chemicals.
Specifically, it is concerned with the interaction between
commonly used pesticides and .benzo(a)pyrene, a known oncogenic
agent.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
111
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ABSTRACT
The insecticides parathion, toxaphene and carbaryl were
tested for their ability to induce tumors in forestomach and
lung of female Ha/ICR and A/J mice, respectively. None of these
prototype insecticides, when fed alone in the diet of mice,
showed significant tumorigenic activity.
Results of our research show that increased benzo(a)pyrene
(BP) hydroxylase activity generally enhances tumor formation
while a decrease in BP hydroxylase activity has a protective
effect against tumors. For example, when BP was administered
chronically or acutely to Ha/ICR mice, forestomach showed the
greatest inducibility of BP hydroxylase while it was also the
site of BP-induced tumors. Papillomatous tumors developed in
the forestomach of mice fed BP for 12 weeks, but no tumors
developed in the glandular portion of stomach, or in the lung
or liver.
Various dosage levels of insecticides were investigated as
to their ability to alter tissue BP hydroxylase activity. In
the same Ha/ICR strain of mice, the organochlorine insecticide
toxaphene enhanced BP-induced forestomach tumors and increased
enzyme activity in forestomach. Also, the carbamate insecticide
carbaryl, when fed in the diet for' 20 weeks, was found to in-
crease BP hydroxylase activity in lung while enhancing BP-
induced tumors in that organ of female A/J mice. These results
are discussed in connection with the hypothesis that toxaphene
and carbaryl have a cooncogenic effect in enhancing BP-
induced tumors. Conversely, when toxaphene inhibited the BP
hydroxylase activity in lung there was an associated decrease
in BP-induced tumors in A/J mice. Feeding mice the organophos-
phate insecticide parathion, did not affect tissue BP hydroxy-
lase activity or the incidence of tumors induced by the adminis-
tration of BP.
The relationship between enzyme inducibility and tumor
formation may be due to the level of oncogenic epoxides formed
at target organs. As the enzyme levels increase, more epoxide
may be formed. Conversely, as enzyme activity decreases, less
epoxide is formed. Enzyme inducibility does not always produce
tumors, as evidenced by the finding that liver BP hydroxylase
was induced after toxaphene feeding but no tumors were observed
at this site after treatment with insecticide and/or BP. Fur-
ther studies of the formation of specific oncogenic epoxides
of BP in tissues after treatment with these insecticides would
more clearly define the relationships between BP hydroxylase
inducibility and BP oncogenesis.
iv
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CONTENTS
Disclaimer ii
Foreword iii
Abstract iv
Abbreviations and Symbols vi
1* Introduction 1
2. Material and Methods 3
Chemicals ' 3
Tissue BP Kydroxylase Activity 3
Treatment of Animals 4
Statistical Analysis 4
3. Results and Discussion 5
4. Tables 13
References 28
v
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
NADPH
NADH
min
po
ppm
wk
SD
'SYMBOLS
BP
mi-
rag
g
y
n
P
Km
M *
3-Nicotinamide adenine dinucleotide phosphate,.
reduced form
3-Nicotinamide adenine.dinucleotide , reduced
form
minute
orally
parts per million
wk
standard deviation
ben zo(a}pyrene
milliliter
milligram
gram
micro
nano
pico
Michaelis constant
Molar
percent
VI
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SECTION I
INTRODUCTION
The human population is exposed chronically to low levels
of insecticides and benzo(a)pyrene (BP). The latter agent is
generally acknowledged to be a carcinogenic polycyclic hydro-
carbon present throughout our environment. BP is present in
tobacco smoke, charcoal grilled foods and as an atmospheric
pollutant emitted into air from the general industrial combus-
tion of fuels and organic matter (1,2,3). In addition, studies
have shown that in certain strains of mice (4,5) the organo-
chlorine insecticides,DDT, aldrin and dieldrin are hepatic
jarcinogens. Traces of insecticides are also commonly found in
food, and like BP, human exposure could occur from inhalation of
tobacco smoke (6). Since man is exposed chronically to the well-
known carcinogen BP as well as to insecticides, it is important
to know mere about the oncogenic potentials associated with
combined e.-vironmental exposure to these two types of chemicals.
The metabolism of BP is mediated by the microsomal mixed
function oxidase enzyme system. In this system, BP hydroxylase
has been shown to be the enzyme which activates BP to form strong
electrophilic metabolites (7,8). These metabolites possess the
ability to bind with nucleophilic groups of nucleic acids and
other macromolecules of target organs (9,10). The mutagenic
activity of a large number of BP derivatives suggest that highly
reactive epoxide metabolites (e.g. Diol-epoxide I and Diol-
epoxide II) might be the ultimate carcinogens of BP (11,12,13,
14). Recently it has been reported that the administration of
BP7,8-dihydrodiol, a metabolite of BP, to newborn mice caused
more malignant lymphomas and pulmonary adenomas than that induced
by BP (15).
Various insecticides have been reported to alter the activi-
ty of enzymes of the microsomal mixed function oxidase system in
different tissues. Parathion, an organophosphate insecticide,
inhibits the hepatic microsomal metabolism of aniline and ethyl-
morphine in mice (16). Studies from our laboratory show that
parathion inhibits the metabolism of BP in liver, lung and small
intestine in rats (17). Furthermore, experiments with the
organochlorine insecticide toxaphene, show that feeding various
levels of this insecticide to rats increased the activity of
hepatic enzymes that metabolize EPN, p-nitroanisole and amino-
pyrine (18). Similar effects on hepatic microsomal enzyme induc-
tion were observed with the carbamate insecticide carbaryl.
Subacute administration of carbaryl produces an increase in the
rate of metabolism of hexobarbital, aniline and benzphetamine in
mice (19,16).
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Few reports have appeared in the literature regarding the
possible carcinogenic and/or cocarcinogenic action of these
widely used and chemically different insecticides such as para-
thion, toxaphene and carbaryl. A recommendation by the National
Cancer Advisory Board Subcommittee on Environmental Carcinogene-
sis suggests that one criterion which may be used for assessing
whether chemicals are potentially carcinogenic could include
"Bioassays in which, in addition to the test agent, animals are
treated with a known carcinogen, or some other foreign material
which itself may be carcinogenic or cocarcinogenic" (20) . Since
insecticides alter microsomal enzyme systems which may be respon-
sible for the metabolism of BP to an active carcinogen, we inves-
tigated the effects of 3 different chemical prototype insecti-
cides (parathion, toxaphene and carbaryl) on the oncogenic
action of BP. The influence of these insecticides on BP hydroxy-
lase activity in various tissues was also determined in order to
help elucidate possible mechanisms that may be involved with
changes observed in BP-induced oncogenesis. The results of
this study could be of value for the determination of potential
human health hazards associated with exposure to commonly used
insecticides alone and, perhaps more importantly, in the pre-
sence of the carcinogenic polycyclic aromatic hydrocarbons such
as BP, which is present in the environment.
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SECTION 2
MATERIALS AND METHODS
Chemicals - BP (practical grade, minimum purity, 95%) Tris
buffer and the cofactors NADH and NADPH were obtained from Sioma
Chemical Co. (St. Louis, Mo.). Parathion (95% .purity), 0,0-
diethyl-0-(p-nitrophenyl) phosphorothioate was purchased from K and
K Laboratories (Jamaica, N.Y.). Carbaryl (technical grade, 100%
purity) was donated by the Union Carbide Corp. (Salinas, Ca.).
Toxaphene (technical grade) was donated by Hercules Incorporated
(Wilmington, De.). All control and experimental diets containing
EP arid/or insecticides were prepared by Bio-Serv, Inc. (French-
town , N. J.) .
Tissue BP hydroxylase activity - Mice were sacrificed after
chronic- f< iding- ad libi -am of the various levels of parathion,
toxctphene, carbaryl and BP. All homogenates- were ma-de-in- ice—
cold 1.15% potassium chloride solution for assay of BP hydroxy-
lase activity by the method of Nehert and Gelboin (21). The
final" incubation mixture had a volume- of" 1.05 nrl which was- com-
posed ofr 0.3 ml of NADPH (0.5 umoles) , 0-3 ml of NADJL (Q..5.
Tnrnrles-):,,
Md_). rmiis-intj -FT nrnnpsc?grtr;c' PtjTTT-yal t»n-»- to the total hydrOXylated
metabolites produced per milligram protein per min of incubation.
flliijimlisr afrtire SQQi xr gxeoiityj snpetma.tan.t- from, eacit tissue b-oaao—
genate were- used for protein determination by the method of Lowry
et al. (22) . When assaying for BP hydroxylase activity in fore-
stomach, and glandular stomach, stomach tissue was dissected into
2 parts. The pooled forestomach of 3 animals were used to pre-
pare homogenates. Under the conditions of the assay, the rate
of hydroxylation of BP was linear for 60 min for both forestomach
and glandular stomach from untreated control mice. The Michaelis
constant (Km) determined at a 30 min period of incubation was
1.20x10-5^ for forestomach and 1.09xlO~5M for glandular stomach.
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Treatment of animals - All experiments were conducted with female
Ha/ICR mice purchased at 8 weeks of age from ARS/Sprague Dawley
(Madison, Wisconsin) or female A/J mice purchased at 8 weeks of
age from the Jackson Laboratory (Bar Harbor, Maine). After one
week of acclimation to the quarters, the animals were randomized
and placed on either diets of Purina Rat Chow to which had been
added 5% corn oil (control group), or experimental diet contain-
ing 5% corn oil as the vehicle and various levels of insecticides
and/or BP. In studies involving Ha/ICR mice, BP was incorporated
into the diet. The A/J mice, however, were administered 3 mg
BP (po) on the 7th and 21st day of the experiment. Animals were
housed in suspended wire mesh cages, five to a cage, and weighed
weekly. After various time periods (12-20 weeks) of feeding, the
mice were sacrificed for tumor analysis and/or enzyme activity.
The presence of stomach tumors in all experiments was determined
by expanding the stomachs with an intragastric injection of a
10% buffered formalin solution. Within 2-3 days the stomachs were
opened, and observed grossly for tumor counts under a dissecting
microscope as described by Wattenberg (23). Lungs were placed
in Tellyesniczky's acetic bichromate solution for 2 days and then
examined grossly. In addition, liver and other internal organs
were observed grossly for possible tumor formation. Tumors
measuring 1.0 mm or larger were counted, recorded, fixed in
buffered formalin and embedded in paraffin. They were sectioned
and stained with hematoxylin-eosin by the standard procedures,
and assessed microscopically for tumor type. The relative sus-
ceptibility to BP-induced tumors is expressed by the "tumori-
genic index", derived by multiplying the percentage of animals
in which tumors develop by the average number of tumors per
tumor-bearing animal as proposed by Shimkin (24).
Statistical Analysis. All data shown for weight gain and BP
hydroxylase activity in this study were analyzed statistically
by Student's t-test. For the analysis of tumor incidence between
the control and experimental groups the "non-parametric" rank
t-test of Bross (25) or Fisher's exact test were used. Only
probability values of 0.05 or less were considered significant.
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SECTION 3
RESULTS AND DISCUSSION
• The effect of feeding two dosage levels of BP in the diet
of Ha/ICR mice for 12 or 20 weeks on the development of fore-
stomach tumors is shown in Table 1. The Ha/ICR mouse was used
in this study because of its susceptibility to forestomach
tumors after exposure to polycyclic hydrocarbons such as BP.
The incidence of tumor formation expressed as the carcinogenic
index is shown to be dose-dependent for the 12 week feeding
period. More than one tumor was found in each tumor-bearing
animal, and microscopic examination of forestomach revealed
that these tumors were of the squamous papilloma type. After
20 weeks of BP feeding, all of the mice in the 200 ppm dietary
group were shown to have developed tumors, and in animals fed
higher dietary levels of BP (300 ppm), the tumors could not be
accurately counted because the entire lumen of the forestomach
was occluded by the formation of we11-developed squamous cell
papillomas. No tumors were found in the forestomach of the
respective control mice. In addition, gross examination of
glandular stomach, lung and liver from control mice and those
fed BP were also found to be negative for the presence of tumors.
This report shows that the forestomach is susceptible to the
development of tumors after a 12-week feeding period of BP in the
diet of mice. Glandular stomach did not form tumors, thus
appearing to be resistant to the carcinogenic action of BP. The
mechanism by which squamous cells of the forestomach gi-ve rise
to papillomatous tumors by feeding BP is not known. Selectivity
for tumor formation in forestomach has been previously reported
for BP in different strains of mice (23,26). Previous studies
(27,28,29,30) using different strains of mice have shown a
correlation between induction of BP hydroxylase activity and the
formation of tumors in various target organs by the polycyclic
hydrocarbon, 3-methylcholanthrene. Studies on DNA binding (10)
and mutagenic activity (11,12,13,14) in vitro of a large number
of BP derivatives suggest that the most active carcinogenic meta-
bolite of BP is the 7B, 8a-dihydroxy-9a, 10a-epoxy-7,8,9,10-
tetrahydrobenzo(a)pyrene. Since the BP hydroxylase enzyme system
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is thought to be essential for the metabolic activation of BP to
form this reactive epoxide , it is important to know whether the
basal level and/or the inducible level of BP hydroxylase activity
in tissues is associated with tumor formation by the administra-
tion of BP.
The induction of BP hydroxylase activity at the site of
tumor formation in forestomach, and in other tissues, is presen-
ted in Table 2. The basal level of BP hydroxylase in control
forestomach (0.212+0.03) was significantly higher than in the
glandular stomach (0.060±0 . 01) ; p<0.01. After the mice were
fed for 12 weeks with BP, the BP hydroxylase activity was
increased by 4.2- to 5. 2- fold in forestomach, whereas induction
of BP hydroxylase was lower (2.2-3.9-fold) in glandular stomach.
In lung, the BP hydroxylase activity was significantly increased
by 1.6-fold only at the higher dietary level of BP. However,
in liver where the basal level of BP hydroxylase activity was
found to be highest of all tissues studied, chronic feeding of
BP did not cause induction of BP hydroxylase activity. BP
hydroxylase activity was also determined in these same tissues
and in small intestine and kidney, 24 hr after a single p.o. dose
(20 mg/kg) of BP (Table 3). Under these experimental conditions,
BP was found to increase significantly this enzyme system in
forestomach, glandular stomach, small intestine, and lung, but
kidney and* liver BP hydrdxylase was not significantly changed.
Similar to our previous results with the chronic feeding of BP
presented in Table 2, after a single acute dose of BP, BP
hydroxylase activity is induced to the greatest extent in fore-
stomach (4.3-fold); this is followed by glandular stomach (2.4-
fold), small intestine (2.3-fold), and lung (1.9-fold). The
basal level in control mice of BP hydroxylase activity was again
significantly higher in forestomach than in glandular stomach
The results presented in Tables 2 and 3 of this report show
that the basal level of BP hydroxylase was higher in forestomach,
a site sensitive to tumor .formation, than in glandular stomach.
Since no tumors were formed in tissues with higher basal enzyme
activity than forestomach, such as in lung, small intestine,
or liver, it seems unlikely that the basal level of BP hydroxy-
lase is the major factor in the tumorigenic effects of BP.
It was further shown in this report that, in all tissues
examined, the induction of BP hydroxylase activity was highest
in forestomach after either acute p.o. administration or chronic
dietary feeding of BP. Data similar to that reported in this
paper on induction of BP hydroxylase activity in various areas
of rat stomach were reported by Wattenberg et al. (31) . They
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found that rat forestomach had moderate basal BP hydroxylase,
whereas, in the glandular portion, it was barely detectable.
After p.o. administration of 1,2-benzanthracene, forestomach was
approximately 5 times more active in enzyme activity than glandu-
lar stomach. In our experiments with mice, the BP hydroxylase
activity was also approximately 3 to 5 times higher in fore-
stomach than glandular stomach after the administration of BP.
It is generally believed that BP must be metabolically
activated by the BP hydroxylase enzyme to form reactive carcino-
genic epoxides. Our findings in Ha/ICR mice of tumor formation
only in the forestomach associated with the highest level of BP
hydroxylase inducibility at this site following the administra-
tion of BP supports the hypothesis previously proposed by other
investigators (27,28,29,30,32) that induction of BP hydroxylase
is related to the formation of tumors following the administra-
tion of various carcinogenic polycyclic hydrocarbons. An expla-
nation as to why BP hydroxylase activity was also inducible in
glandular stomach, small intestine and lung, sites that had been
found to be resistant to tumor formation by the administration of
BP, is not available at the present time. Further studies com-
paring forestomach with other tissues having inducible AHH
activity to form and/or inactivate the various tumorigenic
epoxides formed from BP may resolve the question as to why the
forestomach develops tumors and why other tissues with inducible
AHH activity do not develop tumors.
Studies on the effects of various levels of insecticides in
the diet on weight gain and forestomach tumor formation in Ha/
ICR mice can be seen in Table 4. In the case of the organophos-
phate insecticide parathion two experiments were carried out.
In the first experiment, levels of 100 and 150 ppm of parathion
were found to inhibit significantly weight gain after 12 weeks
of feeding. In the tumorigenic testing of chemicals, factors
such as body weight changes are extremely important, for it 'is
well known that all other factors being equal, a decreased gain
in body weight reduces the occurrence of tumors (33,34). Since
the mice could not tolerate these high levels of parathion in the
diet, the experiment was repeated using a lower dose of 75 ppm.
Animals fed the lower dietary level of parathion (75 ppm), the
organochlorine insecticide toxaphene (100 to 400 ppm), or the
carbamate insecticide carbaryl (500 to 2000 ppm) showed no
significant difference in weight gain from their respective
controls after 12 weeks. Examination of forestomachs from the
control, parathion, toxaphene and carbaryl groups of mice re -
vealed no tumors grossly or microscopically. In addition, the
glandular stomach, liver, and lung from the controls and all
experimental groups showed no evidence of tumor formation when
examined grossly.
The data on tissue BP hydroxylase activity from mice ex-
posed to various levels of the three insecticides are presented
in Table 5. Parathion at a dosage level of 75 ppm showed no
change in BP hydroxylase activity in the liver and stomach; how-
ever, enzyme activity was significantly decreased in the lung.
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Toxaphene, fed at a level of 100 to 400 ppm, produced increases
in liver BP hydroxylase activity, and was found to decrease lung
BP hydroxylase activity. No significant change in enzyme activity
in the stomach was observed for any of the dietary levels of
toxaphene tested. Furthermore, the data in Table 5 show that
carbaryl feeding did not affect BP hydroxylase activity in liver
and lung, but significantly decreased activity in stomach at the
highest dietary level (2000 ppm) tested. These results on the
inducibility of tissue BP hydroxylase activity after insecticides
are in agreement with the work previously reported by others
(35,36) which showed that enzyme induction after the adminis-
tration of various polycyclic hydrocarbons varies from tissue to
tissue in a given species or strain of animal.
Studies carried out on the effects of these insecticides on
BP-induced tumors in the forestomach of Ha/ICR mice are pre-
sented in Table 6. A feeding level of 200 ppm BP was chosen for
these experiments based on our previous data (Table 1) showing
that this level of BP resulted in a tumor incidence of 67% of
the test animals, and did not affect weight gain during a 12-
week feeding period. The data show that neither parathion, nor
carbaryl affects the tumorigenic action of BP in forestomach.
When toxaphene was tested for its effects on BP-induced tumori-
genesis, 60% of the mice fed 200 ppm BP had squamous papillomas
of the forestomach. In mice fed a combination of 400 ppm toxa-
phene and 200 ppm BP, 87% had similar gastric tumors. Toxaphene
was found to increase the mean number of tumors per mouse, and
the Bross (25) rank t-test comparison revealed a statistically
significant difference between groups. It should also be noted
from the data presented in Table 4, that the weight gain in the
experimental group fed toxaphene and BP was significantly less
than in the control group fed BP only. Thouqh nutritional factors
tional factors are known to affect tumor incidences and might
account for the observed increase of tumors, other investiga-
tors have reported that decreased body weight gain due to a
restricted caloric intake results in a decreased incidence of
tumors induced by carcinogenic hydrocarbons in various target
organs of mice (33,34). Therefore, the increased incidence of
tumors found after feeding both toxaphene and BP does not appear
to be related to the observed decreased growth rate in this
group.
A possible explanation for the increased incidence of BP-
induced forestomach tumors in Ha/ICR mice after exposure to the
organochlorine insecticide toxaphene, is that toxaphene itself
may be oncogenic. A study by the National Cancer Institute
reported that mice fed toxaphene for 22 months developed an
increased incidence of tumors in liver (unpublished data, 37).
Under the conditions of the present experiments, the shorter
time period (12 weeks) of feeding toxaphene may be inadequate to
demonstrate an oncogenic effect. However, a suboncogenic
effect by toxaphene may be synergistic for the development of
forestomach tumors in mice fed the carcinogen BP in combination
with toxaphene. Some early experiments with male rats (38) show
that simultaneous feeding of the carcinogens 2-acetylamino-
fluorene and 3'-methyl-4-dimethylaminoazobenzene resulted in a
8
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synergistic effect for the development of liver tumors.
Another more likely explanation for the increase in BP-
induced tumors found in forestomach is that toxaphene may act
as a cocarcinogen as defined by Berenblum (39). The type of
cocarcinogenic influence that toxaphene may be exerting is "a
permissive influence" affecting the metabolism of the oncogen
prior to its action. Detailed studies have shown that the
microsomal mixed function oxidase system has the capacity both
to activate chemicals such as BP to ultimate carcinogenic forms
and to detoxify them to inactive products (9,10,11,12,13,14).
BP hydroxylase is thought to be essential for the metabolism of
BP to form reactive carcinogenic epoxides. Before we can explain
any possible interactions between insecticides and BP, it is
important to know whether these insecticides alter BP hydroxy-
lase activity, especially in target organs.
Since toxaphene was found to enhance BP-induced tumors in
forestomach, but not affect BP hydroxylase activity in whole
stomach, a short term (2 weeks) feeding experiment was conducted
to determine the effects of the various chemical prototype insec-
ticides on BP hydroxylase activity in forestomach, a site speci-
fic for induction of tumors by BP. For comparative purposes
enzyme activity was also measured in the glandular portion of
stomach. The results of this study are shown in Table 7. After
feeding mice for only 2 weeks with these insecticides, BP hydroxy-
lase activity was significantly increased in the forestomach by
parathion and toxaphene. No change was observed in forestomach
BP hydroxylase activity after the feeding of carbaryl. In the
glandular portion of the stomach, which is resistant to BP
carcinogenesis, there was no effect by the insecticides on BP
hydroxylase activity. From this data, we see that toxaphene
acts as an inducer of BP hydroxylase activity in that portion
of the stomach (forestomach) which is susceptible to the car-
cinogenic action of BP.
Similar studies on the interaction of insecticides and BP
were conducted on A/J mice, a strain with a high spontaneous
rate of lung tumors. A pilot study utilizing a small number of
A/J mice revealed that their weight gain was inhibited signi-
ficantly when the three prototype insecticides (parathion,
toxaphene and carbaryl) were fed at the maximum tolerated doses
previously established for the Ha/ICR strain of mice. For this
reason, it was necessary to reduce the dosage level of each
insecticide in the diet for further studies with the A/J strain
of mice. Data concerning the effects of feeding parathion in
the diet for 20 weeks on lung tumors are found in Table 8.
The data show that although mice treated with parathion had a
higher percentage of tumors than controls (43% vs. 13%), this
difference was not statistically significant. Because these
results were inconclusive, a second experiment was conducted
using larger groups of mice. The results with regard to the
gross examination of lung lesions found after parathion adminis-
tration appeared at first to be similar to those of the first
experiment and are presented at the bottom of Table 8. In this
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second experiment, however, careful microscopic examination re-
vealed that most of the lung lesions in the parathion group
were probably inflammatory rather than neoplastic in nature,
while those in the control group were considered to be neoplas-
tic. Thus the lesions seen grossly in the parathion group were
more likely due to infection rather than oncogenesis.
Data pertaining to the effect of parathion on BP-induced
tumors may be found in Table 9. Since BP treatment in the first
experiment caused all of the animals to develop lung tumors
after 20 weeks, a possible enhancing effect by parathion feeding
on BP-induced tumors could not be demonstrated. Therefore, we
repeated the experiment for a shorter time period (16 weeks) in
an attempt to cause a BP-induced tumor incidence of approximately
.50%. Table 9 shows no difference in the incidence of BP-
induced tumors after parathion feeding for 16 weeks when compared
to the control group. Microscopic examination of tumors induced
by BP alone for the data presented in Table 9 indicated that 70%
of the lung tumors induced were alveolar, while 30% were of the
bronchiolar type. In the group administered parathion and BP,
approximately 95% of the lung tumors were alveolar and the re-
maining 5% were bronchiolar.
Data relating to BP hydroxylase are shown in Table 10. The
results indicate that parathion fed in the diet for 20 weeks
and/or administration of BP orally did not affect BP hydroxylase
activity in liver, lung or forestomach.
Studies to determine whether the organochlorine insecti-
cide toxaphene affects the incidence of lung tumors, or affects
BP-induced tumors were carried out, and the data are presented
in Table 11. While toxaphene alone failed to affect the tumor
incidence in the lung of the A/J mouse, it was fcund to pro-
foundly decrease the formation of BP-induced lung tumors in
this strain of mice. Toxaphene decreased the percentage and
the mean number of tumors per mouse induced by BP, and a rank
t-test comparison of the lung tumor incidence revealed that
there was a statistically significant difference between groups.
Microscopic examination of the lung from animals treated with
toxaphene and/or BP revealed that in mice given BP alone, 76%
of the lung tumors were the alveolar type and 23% were bronch-
iolar. In mice administered toxaphene and BP, 33% were alveolar
and 67% were bronchiolar. No inflammatory lesions were observed
in either of these groups. The data pertaining to BP hydroxy-
lase activity after toxaphene alone and in combination '•'ith BP
are presented in Table 12. By feeding 200 ppm of toxaphene
enzyme activity was induced in liver but not affected in fore-
stomach. More importantly, in lung where toxaphene feeding
was found to markedly decrease BP-induced tumors, there was
a significant decrease in BP hydroxylase. Thus, in the A/J
strain of mice toxaphene exerts a protective effect against
BP-induced tumors. This effect of toxaphene is associated
with a corresponding decrease in BP hydroxylase activity in
lung as the target organ. Other studies showing that inhibi-
tion of BP hydroxylase activity protects against tumorigenesis
have been reported by Gelboin et al. (40). These investigators
showed that 7,8-benzoflavone, when added to homogenates of skin
epidermis in vitro, inhibits BP hydroxylase activity. When
10
-------
applied topically, 7,8-benzoflavone inhibits the formation of
mouse skin tumors after repeated treatment with 9,10-dimethyl-
benzanthracene.
The results presented in Table 12 on enzyme activity for
toxaphene are similar to those observed for 7,8-benzoflavone
on BP hydroxylase activity in different tissues of rats as
reported by Weibel et al. (41). These investigators found a
decrease in the lung, but an increase in liver BP hydroxylase
activity when these tissues were incubated with 7,8-benzo-
flavone in vitro. They concluded that at least two forms of
the BP hydroxylase enzyme complex exist. One form predominates
in hepatic tissue and the other predominates in extra-hepatic
tissues such as the lung. This nay partly explain why toxa-
phene does not cause tumor formation in liver, although BP
hydroxylase is induced by this insecticide in this tissue.
It may be that liver has a different cytochrome P-450, which
forms metabolites of BP that do not covalently bind DNA, RNA
or other macromolecules which in turn initiate the carcino-
genic process in this organ.
As shown in Table 13, the data with regard to carbaryl
feeding were similar to that observed for parathion. Mice that
were fed carbaryl alone had more tumors than the control mice
but the difference was not statistically significant. As with
the parathion study, we. repeated the original carbaryl study
using larger numbers of animals in order to obtain more con-
clusive results. Data for the second experiment are presented
at the bottom of Table 13 and show no significant difference
in gross lung tumor incidence after carbaryl feeding. This
indicates that the previous results, which cast suspicion on
carbaryl as being tumorigenic, were probably due to chance.
In contrast to the parathion interaction study with BP,
carbaryl feeding for 20 weeks was found to enhance BP-induced
tumors in lung (Table 14). Since the tumor incidence for mice
given BP alone was close to 100% after 20 weeks, the experiment
was repeated for 16 weeks, a lesser time period, in an effort
to decrease BP-induced tumor incidence to approximately 50%.
The data from this second experiment in Table 14 show» that
carbaryl did not affect the incidence of BP-induced lung tumors
after 16 weeks. The difference in the results of the two
experiments could be due to the different time periods.
Perhaps a longer time period is necessary for carbaryl to have
an effect. Further studies could be done utilizing a longer
time period (i.e. 24 weeks) and a lower dose of BP than we
used which would yield approximately a 50% tumor incidence.
After microscopic examination of lung tissue, 84% of the lung
tumors induced by BP alone were found to be alveolar and 16%
were bronchiolar. In mice given BP and carbaryl, 70% of the
lung tumors were alveolar, while 30% were bronchiolar. No in-
flammatory lesions were found in the lungs of these animals.
The data found in Table 15 show that lung BP hydroxylase was
significantly higher in mice fed carbaryl for 20 weeks and
given BP orally than in those administered BP only. As in
previous data of this report, this indicates an association
exists between induction of BP hydroxylase and enhancement
11
-------
of BP-induced tumors. Data in Table 15 show carbaryl slightly
Inhibited enzyme activity in forestomach, but this insecticide
did not affect liver BP hydroxylase activity.
Evidence from our research shows that increased BP hydroxy-
lase generally enhances tumor formation while a decrease in BP
hydroxylase has a protective effect against tumors. The rela-
tionship between enzyme inducibility and tumor formation may
be due to the level of tumorigenic epoxides formed at target
organs. As the enzyme levels increase, more epoxide may be
formed, and conversely, as enzyme activity decreases, less
epoxide is formed. Enzyme inducibility, however, does not
always produce tumors. Additional research on these insecti-
cides is needed to study the binding of the epoxides to the
DNA, RNA and macromolecules which initiate the tumorigenic
process so that the whole mechanism for tumor enhancement may
be understood.
12
-------
U)
TABLE 1. EFFECT OF VARIOUS LEVELS OF BP IN THE DIET ON BODY WEIGHT GAIN AND TUMOR FORMA-
TION IN FORESTOMACH OF Ha/ICR MICE
Mice were fed a diet to which had been added 5% corn oil (control) or 5% corn oil with 2
dosage levels of BP in the diet. Mice were 9 weeks old at the start of the experiment
and, after 12 or 20 weeks on the diet, were sacrificed for tumor count.
I
Experimental
Group
Control
BP
BP
Control
BP
BP
Amount of
Carcinogen
added to Diet
(ppm)
0
200
300
0
200
300
Duration
of
Experiment
(wk)
12
12
12
20
20
20
Number
of
Mice
14
15
14
5
6
5
Weight
Gain*
(g)
8.3
7.2
6.7
11.5
11.2
8.5
Mice
with
Tumors
(%)
0
67
100
0
100
100
Number
of Tumors/
Mouse T
0.0
1.810.7
4.012.1
0.0+0.0
4.812.2
*•
Tumor i-
genic
Index -f
0
121
400
0
480
'"
*Mean weight gain per group during interval between start of experimental diets and time
mice were killed.
'''Mean 1 SD calculated for only tumor-bearing mice.
* Percentage of mice with tumors times the mean number of tumors per tumor-bearing mouse.
•^Tumor count not available due to the tumors forming a large-mass in the forestomach.
-------
TABLE
MICE
2. EFFECT OF VARIOUS LEVELS OF BP IN THE DIET ON TISSUE BP HYDROXYLASE ACTIVITY OF Ha/ICR
Mice were fed a diet to which had been added 5% corn oil (control) or 5% corn oil with 2 dosaae
levels of BP in the diet. Mice were 9 weeks old ab the start of the experiment and, after 12
weeks, were sacrificed for enzyme assay. The results are expressed as prooles of 3-hydroxybenzo(a)
pyrene per mg of protein per min. Each value represents the mean ± S.D. of 4 determinations.
.. BP Hydroxylase
Experimental
Group
Control
BP
BP
Amount of
Carcinogen
Added to Diet
(ppm)
0
200
300
Fore stomach
0
0
1
.21210
.833±0
.10010
.03
.28*
.43 +
Ratio* .Glandular
stomach
0
•4.2 0
5.2 0
.06010.01
.13110. 01*
.22810. 09+
Ratio
2.2
3.9
2
3
4
Activity
Luna
.95±0.74
.6910.49
.5710.88*
Ratio Liver
60.971 9.91
1.3 50.971 7.00
1.6 55.62+17.49
Ratio
0.8
0.9
*-Ratio/ BP hydroxylase activity of mice receiving BP divided by the activity of the control group
'*"Significantly different from control group (P< 0.01)
* Significantly different from control group (P* 0.05)
-------
TABLE 3. ACUTE EFFECTS OF BP ON TISSUE BP HYDROXYLASE ACTIVITY OF Ha/ICR MICE
Mice were given BP (20 mg/kg) p.o. in a 10% solution of ethanol in corn oil of 1% of body
weight 24 hr prior to sacrifice for measurement of enzyme activity. The corresponding controls
were given an equivalent amount of vehicle. The results are expressed as pmoles of 3-hydroxy-
benzo(a)pyrene per mg of protein per min. Each value represents the mean 1 S.D. of 3 to 4
determinations.
Experimental
Group
Control
BP
Amount of Forestomach
Carcinogen
Given Orally
(mg/kg)
0 0.24110.06
20 1.037±0.18*
Ratio* 4.3
Glandular
stomach
0.103±0.02
0. 251 ±0.09 •*•
2. 4
BP Hydroxylase Activity
Small
Intestine Lung Kidney
8.49+4.02 3.81±1.36 0.297±0.08
19. 48 + 4. 371" 7.3110.77* 0.46310.17
2.3 1.9 1.6
Liver
45.2714. 38
53.4516.35
1.2
*Significantly different from control group (P< 0.001)
'''Significantly different from control group (P< 0.05)
"* Significantly different from control group (P< 0.01)
*Ratio BP hydroxylase activity of mice receiving BP divided by the activity of the control group
-------
TABLE 4. EFFECT OF VARIOUS LEVELS OF PARATHION, TOXAPHENE AND
CARBARYL IN THE DIET ON BODY WEIGHT GAIN AND TUMOR FORMATION IN
THE FORESTOMACH OF Ha/ICR MICF
Mice were fed a diet to which had been added 5% corn oil (control)
or 5% corn oil with various dosage levels of insecticide. Mice
were 9 weeks old at the start of the experiment and, after 12
weeks on the diet, were sacrificed for tumor count.
Experimental
Group
Amount of
Insecticide
Added to Diet
(ppm)
Number of
of
Mice
Weight
Gain*
(g)
Mice
with
Tumors
Control
Parathion
Pa rath ion
Control
Parathion
Control
Toxaphene
Toxaphene
Toxaphene
Control
Carbaryl
Carbaryl
Carbaryl
0
100
150
0
75
0
100
200
400
0
500
1000
2000
15
15
15
15
16
15
14
15
15
15
15
14
15
10.1
6.8"^
6.9-r
9.5
8.3
9.0
6.8
6.8
7.2
7.2
8.4
6.1
5.4
—
•
0
0
0
0
0
0
0
0
0
0
*Mean weight gain per group during interval between start of
experimental diets and time mice were killed
"^Significantly different from control group (P<.0.01)
16
-------
TABLE 5. EFFECT OF VARIOUS LEVELS OF PARATHION, TOXAPHENE AND CARBARYL IN THE DIET ON
TISSUE BP HYDROXYLASE ACTIVITY OF Ha/ICR MICE
Mice were fed a diet to which had been added 5% corn oil (control) or 5% corn oil with
various dosage levels of insecticide. Mice were 9 weeks old at the start of the experi-
ment and, after 12 weeks, were sacrificed for enzyme assay. The results are expressed
as pmoles of 3-hydroxybenzo(a)pyrene per mg of protein per min. Each value represents
the mean ± S.D. of 4 or 5 determinations.
Experimental
Group
Amount of
Insecticide
BP Hydroxylase Activity
Control
Parathion
Control
Toxaphene
Toxaphene
Toxaphene
Control
Carbary 1
Carbaryl
Carbaryl
Added to Diet
(ppm)
0
75
0
100
200
400
0
500
1000
2000
Liver
50.7±10.8
46. 4± 5.4
66.8111.4
103. 1± 8.7*
102.2+10.4*
99. 3114. 9-*-
54. 9± 6.7
56. 9± 3.8
61.51 6.9
61.5111.5
Lung
5.4210.65
3.40+0.44*
2.43+0.30
0.5410.18*
0.3310.10*
0.3610.18*
4.3211.54
3.76+1.18
3.4710.81
3.3910.38
Stomach
0.1581.092
0.1951.074
0.1661.018
0.1951.069
0. 3021. 211
0. 2321. 103
0.1691.035
0.1441.024
0.2061.140
0.1101.042*
"Significantly different from control group (P<0.05)
+ Significantly different from control group (P< 0.005)
^Significantly different from control group (P< 0.001)
-------
TABLE 6. EFFECT OF PARATHION, TOXAPHENE AND CARBARYL IN THE DIET ON BODY WEIGHT GAIN AND BP-
INDUCED TUMOR FORMATION IN THE FORESTOMACH OF Ha/ICR MICE
Mice were fed a diet to which had been added 5% corn oil (control) and BP, or 5% corn oil,
insecticide and BP. Mice were 9 weeks old at the start of the experiment and, after 12 weeks
on the diet, were sacrificed for tumor count.
00
Experimental group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Added to Diet
(ppm) (ppm)
Control
Parathion 75
Control
Toxaphene 400
Control
Carbaryl 2000
BP
BP
BP
BP
BP
BP
200
200
200
200
200
200
Duration
of
Experiment
(wk)
12
12
12
12
12
12
Number
of
Mice
13
13
15
15
14
14
Weight
Gain *
(g)
4.3
5.2
I: Is
7.9
7.5
Mice
with
Tumors
53
53
S°7«
43
43
Numbe r
of Tumors/
Mouse r
2
2
1
2
1
2
.5±0.
.1 + 1.
.9±1.
.5 + 1.
.7 + 0.
.3±1.
5
3
2
2
8
9
Tumorigenic
Index *
133
111
114
217
73
99
*Mean weight gain per group during interval between start of experimental diets and time mice
were killed
* Mean ± SD calculated from tumor-bearing mice
^Percentage of mice with tumors times the mean number of tumor per tumor-bearing mouse
*Signif icantly different from control group (P<0.05)
SSignificantly different from control group (P< 0.01)
-------
TABLE 7. EFFECT OF PARATHION, TOXAPHENE AND CARBARYL IN THE
DIET ON TISSUE BP HYDROXYLASE ACTIVITY OF Ha/ICR MICE
Mice were fed a diet to which had been added 5% corn oil (con-
trol) or 5% corn oil with an insecticide. Mice were 9 weeks
old at the start of the experiment and, after 2 weeks, were
sacrificed for enzyme assay. The results are expressed as-
pmoles of 3-hydroxybenzo(a)pyrene per mg of protein per mi.n.
Each value represents the mean ± S.D. of 5 or 6 determinations
in each of which the pooled tissue of three mice were -used.
Experimental
Group
Amount of
Insecticide
Added to Diet
(ppm)
BP Hydroxylase Activity
Forestomach
Glandular Stomach
Control
Parathion
Toxaphene
Carbaryl
0
75
400
2000
0.116+0.008
0.136±0.011*
0. 145+0. 017f
0.11410. .011
0.061+0.031
0.054+0.022
0.064+0.025
0.047+0.026
"Significantly different from control group (P-^ 0.02)
"*" Significantly different from control group (P< 0.01)
19
-------
TABLE 8. EFFECT OF THE ORGANOPHOSPHATE INSECTICIDE PARATHION IN THE DIET ON WEIGHT GAIN AND
TUMORS OF THE LUNG OF A/J MICE
Mice were fed diets containing either: 5% corn oil (control) or 5% corn oil and 50 ppm para-
thion. Mice were 9 weeks old at start of experiment and after 20 weeks on the diet were
sacrificed for gross tumor count.
to
o
Experimental Group
Experiment 1
Control
Parathion
Exp e r i men t 2
Control
Parathion
Amount of
Insecticide
Added to Diet
(ppm)
0
50
0
50
Number
of
Mice
16
14
32
32
Weight Mice with
Gain* Tumors
(g) (%)
6.8 13
7.8 43 *
7.0 19.
7.5 37*
Number of
Tumors/
Mouse +
1.010.0
l.Oifr.O
1.2 ±0.4
—
Tumor i genie
Index *"
13
43
23
—
*Mean weight gain per group during interval between start of experimental diets and time
mice were sacrificed
"''Mean ± SD calculated from tumor bearing mice
£ Percentage of mice with tumors times the mean number of tumors per tumor bearing mouse
significant difference between control group
Upon microscopic examination most of the lesions were found to be inflammatory rather
than neoplastic
-------
TABLE 9. INTERACTION BETWEEN THE ORGANOPHOSPHATE INSECTICIDE PARATHION IN THE DIET AND THE
CARCINOGEN BP (P.O.) ON GROWTH RATE AND NEOPLASIA OF THE LUNG OF A/J MICE
Mice were fed diets containing either 5% corn oil (control) and 3 mq BP given P.O. on the 7th
and 21st day of the experiment or 5% corn oil and 50 ppm parathion and 3 mg BP qiven p.o. on
the 7th and 21st (day of the experiment. Mice were 9 weeks old at start of experiment and
after 16 or 20 weeks on the diet were sacrificed for gross tumor count.
Experimental Group
Amount of
Insecticide
Added to Diet
(ppm)
Amount of
Carcinogen
Given Orally
Duration Number
of of
Experi- Mice
ment
(wkF
Weight
Gain* Mice with
Tumors
(.g) (%)
Number Tumoriaenic
of
Tumors/
Mouse "*"
Index +
Experiment 1
Control I
Parathion
50
Experiment
BP
3 mg p.o. (2X)
BP
3 mg p.o. (2X)
Control II BP
3 mg p.o. (2X)
Parathion
50
BP
3 mg p.o. (2X)
20
20
16
16
15
15
15
25
6.8
7. 3
7.1
6.4
100
100
4.312.4
3.912.1
67 2.111.0
68 1.810.6
430
390
141
122
*Mean weight gain per group during interval between start of experimental diets and time mice
were sacrificed
* Mean ± SD calculated from tumor bearing mice
* Percent of mice with tumors times the mean number of tumors per tumor bearing mouse
-------
TABLE 10. INTERACTION BETWEEN THE ORGANOPHOSPHATE INSECTICIDE PARATHION IN THE DIET
AND THE CARCINOGEN BP ON BP HYDROXYLASE ACTIVITY OF THE LIVER, LUNG AND FORESTOMACH OF
A/J MICK
Mice were fed diets containing one of the following: 5% corn oil (control I), 5% corn oil
and 50 ppm ppm parathion, 5% corn oil (control II) and 3 mg BP given p.o. on the 7th and
2lst day of the experiment, or 5% corn oil and 50 ppm parathion and 3 mg BP given p.o. on
the 7th and 21st day of the experiment. Mice were 9 weeks old at start of experiment and
after 20 weeks on the diet were sacrificed for enzyme assay. The results are expressed
as pmole of 3 hydroxybenzo(a)pyrene per mg of protein per min. Each value represents the
mean ± S.D. of 4 determinations.
NJ
Experimental Group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Given Orally
(ppm)
Liver
BP Hydroxylase Activity
Lung Forestomach
Control I
Parathion
50
Control II
Parathion
50
None . 42.90+10.74 4.51+0.54
None 44.45+ 7.68 4.4610.21
BP 47.55114.09 4.6510.50
3 mg p.o. (2X) .
BP 45.08+ 5.48 5.2210.68
3 mg p.o. (2X)
0. 1391. 136
0.12410.0
0. 1771. 064
0.1521.042
Three forestomachs were pooled per determination
-------
TABLE 11. INTERACTION BETWEEN THE ORGANOCHLORINE INSECTICIDE TOXAPHENE IN THE DIET AND THE
CARCINOGEN BP ON WEIGHT GAIN AND TUMORS OF THE LUNG OF A/J MICE
Mice were fed diets containing one of the following: 5% corn oil (control I), 5% corn oil
and 200 ppm toxaphene, 5% corn oil (control II) and 3 mg BP given p.o. on the 7th and 21st
day of the experiment, or 5% corn oil and 200 ppm toxaphene and 3 mg BP given p.o. on the
7th and 21st day of the experiment. Mice were 9 weeks old at start of experiment and after
20 weeks on the diet were sacrificed for gross tumor count
U)
Experimental Group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Given Orally
(ppm)
Number Weight Number Tumorigenic
of Gain* Mice with of Tumors/ Index *
Mice (g) Tumors Moused
Control I
Toxaphene
200
Control II
Toxaphene
200
None
None
BP
3 mg p.o. (2X)
BP
3 mg p.o. (2X)
17
15
18
15
7.
6.
6.
6.
3
7
2
9
23
7*
100
67*
1.
1.
7.
1.
0±0
0±0
2±3
6±0
,0
.0
.5
.8
23
7
720
107
*Mean weight gain per group during interval between start of experiment and time mice xvere
sacrificed
"t" Mean ± SD calculated from tumor bearing mice
"* Percentage of mice with tumors times the mean number of tumors per tumor-bearing mouse
*NS: no significant difference between control I group
$ Significantly different from control II given BP (P-^- 0.001)
-------
TABLE 12. INTERACTION BETWEEN THE ORGANOCHLORINE INSECTICIDE TOXAPHENE IN THE DIET AND
THE CARCINOGEN BP ON BP HYDROXYLASE ACTIVITY OF THE LIVER, LUNG AND FORESTOMACH OF A/J
MICE
Mice were fed diets containing one of the following: 5% corn oil (control I), 5% corn oil
and 200 ppm toxaphene, 5% corn oil (control II) and 3 mg BP given p.o. on the 7th and 21st
day of the experiment, or 5% corn oil and 20*0 ppm toxaphene and 3 mg BP given p.o. on the
7th and 21st day of the experiment. Mice were 9 weeks.old at start of experiment and.after
20 weeks on the diet were sacrificed for enzyme assay. The results are expressed as pmole
of 3-hydroxybenzo(a)pyrene per mg protein per min. Each value represents the mean ± S.D.
of 4 determinations
to
Experimental Group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Given Orally
(ppm)
Liver
BP Hydroxylase Activity
Lung
200
Control .II
Toxaphene
200
BP 31.17± 9.98
3 mg p.o. (2x)
BP
3 mg p.o. (2x)
64.23117.61
2.51±0.57
0.8510.06"
Fore s tomach
Control I
Toxaphene
None
None
26.74+ 5.57
49. 43113. 78f
. 2.58±0.60
0.6110.16*
0.082±0.012
0.10110.018
0.07510.020
0.07510.010
* Three forestomachs were pooled per determination
"^"Significantly different from control I group (P^~ 0.025)
* Significantly different .from control I group (P< 0.001)
^Significantly different from control II group•(P^ 0.020)
s Significantly different from control II group (P^. 0.005)
-------
TABLE 13. EFFECT OF THE CARBAMATE INSECTICIDE CARBARYL IN THE DIET ON WEIGHT GAIN AND
TUMORS OF THE LUNG OF A/J MICE
Mice were fed diets containing either 5% corn oil (control) or 5% corn oil and 1000 ppm
carbaryl. Mice were 9 weeks old at start of experiment and after 20 weeks on the diet
were sacrificed for gross tumor count.
to
Experimental
Group
Experiment 1
Control
Carbaryl
Experiment 2
Control
Carbaryl
Amount of
Insecticide
Added to Diet
(ppm)
0
1000
0
1000
Number
of
Mice
11
16
31
31
Weight
Gain*
5.4
6.0
6.3
5.9
Mice with
Tumors
9
31 #
23
10*
Number
of Tumors/
Mouse "*"
110.0
1.210.4
1.1±0.4
1.310.6
Tumorigenic
Index *
9
37
25
13
*Mean weight gain per group during interval between start of experiment and time mice
were sacrificed
TMean ± SD calculated from tumor bearing mice
* Percentage of mice with tumors times the mean number of tumors per tumor bearing mouse
**"NS: no significant difference between control group
-------
TABLE 14. INTERACTION BETWEEN THE CARBAMATE INSECTICIDE CARBARYL IN THE DIET AND THE CARCINO-
GEN BP (P.O.) ON GROWTH RATE AND NEOPLASIA OF THE LUNG OF A/J MICE
Mice were fed diets containing eo.th.er 5% corn, oil (control) and 3 mg BP given p.o. on the 7th
and 2ist day of the experiment or 5% corn oil and 100Q ppm carbaryl and 3 mg BP given p.o. on
the 7th and 21st day of the experiment. Mice were 9 weeks old at start of experiment and after
16 or 20 weeks on the diet were sacrificed for gross tumor count.
Experimental Group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Given Orally
(ppm)
Duration
of
Experiment
(wk)
Number Weight of Number Tumori-
of Gain* ''lice with of Tumors/ genie
Mice (g) Tumors Mouse * index*
Experiment 1
Control I
Carbaryl
1000
Experiment 2
Control II
Carbaryl
1000
BP
3 mg p.o.
BP
3 mg p.o.
BP
3 mg p .0 .
BP
3 mg p.o.
20
(2X)
20
(2X)
16
(2X)
16
(2X)
17 5.6 88 3.7±2.3 326
18 5.7 100 ** 5.7±5.9 570
31 6.0 55 1.3±0.8 71
34 5.7 47 1.410.6 66
*Mean weight gain per group during interval between start of experiment and time mice were
sacrificed
^ Mean ± SD calculated from tumor bearing mice
*Percent of mice with tumors times the mean number of tumors per tumor bearing mouse
•^Significantly different from control I group (P-< 0.05)
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TABLE 15. INTERACTION BETWEEN THE CARBAMATE INSECTICIDE CARBARYL IN THE DIET AND THE
CARCINOGEN BP ON BP HYDROXYLASE ACTIVITY OF THE LIVER, LUNG AND FORESTOMACH OF A/J MICE
Mice were fed diets containing one of the following: 5% corn oil (control I) , 5% corn
oil and 1000 ppm carbaryl, 5% corn oil (control II) and 3 mg BP benzo(a)pyrene given p.o,
on the 7th and 21st day of the experiment, or 5% corn oil and 1000 ppm carbaryl and
3 mg BP given p.o. on the 7th and 21st day of the experiment. Mice were 9 weeks old at
start of experiment and after 20 weeks on the diet were sacrificed for enzyme assay.
The results are expressed as pmoles of 3-hydroxybenzo(a)pyrene per mg of protein per
min. Each value represents the mean ± S.D. of 4 determinations.
N)
-J
Experimental Group
Amount of Amount of
Insecticide Carcinogen
Added to Diet Given Orally
(ppm)
Liver
BP Hydroxylase Activity
Lung Fore stomach?^
Control I
Carbaryl
1000
Control II
Carbaryl
1000
None
None
BP
3 mg p.o. ( 2 x )
BP
3 mg p.o. ( 2 x )
39.
45.
42.
41.
76±7.
68 + 4.
11±6.
45±6.
45
27
14
32
3.
3.
3.
3.
87±0.
60±0.
06±0.
86±0.
57
36
28
21*
0.
0.
0.
0.
099±0
084±0
102±0
070±0
.000
.008 "*"
.015
.008 **"
*Three forestomachs were pooled per determination
"^Significantly different from control I group (P
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SECTION 4
REFERENCES
1. Duean, R.M. A Gas Chromatographic Determination of Benzo(a)
pyrene Using Electron Capture. Amer. Ind. Hyg. Ass. J. 30:
624-629, 1969.
2. Hoffman, D. and Wynder, E. Smoke of Cigarettes and Little
Cigars: An Analytical Comparison. Science 178: 1197-1199,
1972.
3 .. Kuratsune,. M. Benzo (a)pyrene Content of Certain Pyrogenic
Materials. J. Natl. Cancer Inst. 16: 1485-1496, 1969.'
4. Davis, K.J. Tumorigenic Potential of Aldrin and Dieldrin in
Mice. Toxicol. Appl. Pharmacol. 4: 187"189, 1962.
5. Tomatis, L., Turusov,*V., Day, N. and Charles, R.T. The Ef-
fect of Long Term Exposure to DDT in CF-1 Mice. Int. J.
Cancer 10: 489-506, 1972.
6. Dorough, H.W. and Atallah, Y.H. Cigarette Smoke as a Source
of Pesticide Exposure. Bull. Environ/ Contam. Toxicol. 13:
101-107, 1975.
7. Heidelberger, C. Current Trends in Chemical Carcinogenesis.
Fed. Proc. 32: 2154-2161, 1973.
8. Miller, J.A. Carcinogenesis by Chemicals: An Overview - GHA
Clowes Memorial Lecture. Cancer Res. 30: 559-576, 1970.
9. Gelboin, H.V. A Microsomal Dependent Binding of BP to DNA.
Cancer Res. 29: 1272-1276, 1969.
10. Sims, P.-, Grover, P.L. , Swaisland, H. , Pal, K. and Hewer, A.
Metabolic Activation of Benzo(a)pyrene Proceeds by a Diol-
epoxide. Nature 252: 326-328, 1974.
11. Huberman, E., Sachs, L., Yang, S.K. and Gelboin, H.V. Iden-
tification of Mutagenic Metabolites of Benzo (a)pyrene in
Mammalian Cells. Proc. Natl. Acad. Sci. (USA), 73: 607-611,
1976.
2.8
-------
12. Weinstein, I.E., Jeffrey, A.M., Jennette, K.W., Blobstein,
S.H., Harvey, R.G., Harris, C., Kasai, H. and Nakanishi, K.
Benzo (a)pyrene Diol-Epoxides as Intermediates in Nucleic Acid
Binding in In Vitro and Ir^ Vivo . Science 193: 592-594, 1976.
13. Wislocki, P.G., Wood, A.V., Chang, R.L., Levin, W., Yagi, H.,
Hernandez, O., Jerina, D.M. and Conney, A.H. High Mutageni-
city and Toxicity of a Diol-Epoxide Derived from Benzo(a)pyrene.
Biochem. Biophys. Res. Commun. 68: 1006-1012, 1976.
14. Wood, A.W., Wislocki, P.G., Chang, R.L., Levine, W., Lu,
A.Y.H., Yagi, H., Hernandez, 0., Jerina, D.M. and Conney,
A.H. Mutagenicity and Cytotoxicity of Benzo(a)pyrene Benzo-
Ring Epoxides. Cancer Res. 36: 3358-3366, 1976.
15. Kapitulnik, J., Levin, W., Conney, A.H., Yagi, H., Jerina,
D.M. Benzo(a)pyrene 7,8-dihydrodiol Is More Carcinogenic than
Benzo (a)pyrene in Newborn Mice. Nature 266: 378-380, 1977.
16. Stevens, J.T., Stitzel, R.E. and McPhillips, J.J. Effects
of Subacute Administration of Anticholinesterase Insecticides
on Hepatic Microsomal Metabolism. Life Sci. 11: 423-431,
1972.
17. Weber, R.P., Coon, J.M. and Triolo, A.J. Effect of the Organo-
phosphate Insecticide Parathion and its Active Metabolite
Paraoxon on the Metabolism of Benzo(a)pyrene in The Rat.
Cancer Res. 34: 947-952, 1974.
18. Kinoshita, F.K., Frawley, J.P. and Dubois, K.P. Quantitative
Measurement of Induction of Hepatic Microsomal Enzymes by
Various Dietary Levels of DDT and Toxaohene in Rats. Toxicol.
Appl. Pharmacol. 9: 505-513, 1966.
19. Cress, C.R. and Strother, A. Effects on Drug Metabolism of
Carbaryl and 1-Naphthol in the Mouse. Life Sci. 14: 861-
872, 1974.
20. Report of the Subcommittee on Environmental Carcinogenesis:
General Criteria for Assessing the Evidence for Carcinogenicity
of Chemical Substances. J. Natl. Cancer Inst. 58: 461-465,
1977.
21. Nebert, D.W. and Gelboin, H.V. Substrate-Inducible Microsomal
Aryl Hydrocarbon Hydroxylase in Mammalian Cell Culture. I.
Assay and Properties of Induced Enzyme. J. Biolog. Chem.
243: 6242-6249, 1968.
22. Lowry, O.H., Rosebrough, N.J. and Farr, A.L. Protein Measure-
ment with Folin Phenol Reagent. J. Biolog. Chem. 193: 265-
275, 1951.
29
-------
23. Wattenberg, L.W. Inhibition of Carcinogenic and Toxic Effects'
of Polycyclic Hydrocarbons by Phenolic Antioxidants and Ethoxy-
quin. J. Natl. Cancer Inst. 48: 1425-1430, 1972.
24. Shimkin, M.B. Induced Pulmonary Tumors in Mice. II. Reaction
of Lungs of Strain A Mice to Carcinogenic Hydrocarbons. Arch.
Path. 29: 239-255, 1940.
25. Bross, I'.D.J. Is There an Increased Risk? Fed. Proc. 13:
815-819, 1954.
26. Collins, V.J., Gardner, W.V. and Strong, L.C. Experimental
Gastric Tumors in Mice. Cancer Res. 3: 29-35, 1943.
27. Kouri, R.E., Ratrie, H. and Whitmire, C.E. Evidence of a
Genetic Relationship between Susceptibility to 3-Methyl-
cholanthrene-Induced Subcutaneous Tumors and Inducibility of
Aryl Hydrocarbon Hydroxylase. J. Natl. Cancer Inst. 51: 197-
200, 1971.
28. Kouri, R.E., Rude, T., Thomas, P.E. and Whitmire, C.E. Studies
on Pulmonary Aryl Hydrocarbon Hydroxylase Activity in Inbred
Strains of Mice. Chemical Biol. Interactions 13: 317-331,
1976.
29. Kouri, R.E., Salerno, R.A. and Whitmire, C.E. Relationship
between Aryl Hydrocarbon Hydroxylase Inducibility and Sensi-
tivity' to Chemically Induced Subcutaneous Sarcomas in
Various Strains of Mice. J. Natl. Cancer Inst. 50: 363-
368, 1973.
30. Nettesheim, B. and Hammons, A.S. Induction of Squamous Cell
Carcinoma in the Respiratory Tract of Mice. J. Natl. Cancer
Inst. 47: 697-701, 1971.
31. Wattenberg, L.W., Leong, J.L. and Strand, P.J. Benzpyrene
Hydroxylase Activity in the Gastrointestinal Tract. Cancer
Res. 22: 1120-1125, 1962.
32. Kellerman, G. , Shaw, C.R. and Kellerman, M.L. Aryl Hydrocarbon
Hydroxylase Inducibility and Bronchogenic Carcinoma. New Engl.
J. Med. 289: 934-937, 1973.
33. Tannenbaum, A. and Silverstone, H. The Genesis and Growth of
Tumors IV. Effects of Varying the Proportion of Protein
(Casein) in the Diet. Cancer Res. 9: 162-173, 1949.
34. Tannenbaum, A. and Silverstone, H. The Influence of the
Degree of Caloric Restriction on the Formation of Skin Tumors
and Hepatomas in Mice. Cancer Res. 9: 724-727, 1949.
35. Wiebel, F.J.., Leutz, J.C. and Gelboin, H.V. Aryl Hydrocarbon
(Benzo(a)pyrene) Hydroxylase: Inducible in Extrahepatic Tis-
sues of Mouse Strains not Inducible in Liver. Arch. Biochem.
Biophys. 154: 292-294, 1973.
30
-------
36. Thomas, P.E., Kouri, R.E. and Hutton, J.J. The Genetics of
Aryl Hydrocarbon Hydroxylase Induction in Mice: A Single Gene
Difference between C57BL/6J and DBA/2J. Biochem. Genet. 6:
157-168, 1972.
37. Johnson, E.L.: Pesticide Products Containing Toxaphene
(1976 Experimental Design Status Report: Carcinogenesis
Bioassay) Federal Register 42: No. 101, 26859-26920, 1977.
38. MacDonald, J.C., Miller, E.G., Miller, J.A. and Rusch, H.P.
The Synergistic Action of Mixtures of Certain Hepatic
Carcinogens. Cancer Res. 12: 50-54, 1952.
39. Berenblum, I. A Re-evaluation of the Concept of Cocarcino-
genesis. Progr. Exptl. Tumor Res. 11: 21-30, 1969.
40. Gelboin, H.V., Wiebel, F. and Diamond, L. Dimethylbenzan-
thracene Tumorigenesis and Aryl Hydrocarbon Hydroxylase in
Mouse Skin: Inhibition by 7,8-Benzoflavone. Science 170:
169, 1970.
41. Weibel, F.J., Leutz, J.C., Diamond, L. and Gelboin, H.V.
Aryl Hydrocarbon (Benzo(a)pyrene) Hydroxylase in Microsom.es
from Rat Tissue: Differential Inhibition and Stimulation by
Benzoflavones and Organic Solvents. Arch. Biochem.
Biophys. 144: 78-86, 1971.
31
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TECHNICAL REPORT DATA
(Please read instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-066
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
EFFECT OF INSECTICIDES ON BENZO(A)PYRENE
CARCINOGENESIS
5. REPORT DATE
November 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Anthony J. Triolo
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Jefferson Medical College
Thomas Jefferson University
Philadelphia, PA 19107
10. PROGRAM ELEMENT NO.
15
11. CON
7GRANT NO.
R-803486
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The pesticides parathion, toxaphene, and carbaryl were tested for their ability
to induce tumors in the forestomach and lungs of female Ha/ICR and A/J mice
respectively. None of these pesticides, when fed alone in the diet of the mice,
showed significant oncogenic activity. On the other hand, toxaphene enhanced
benzo(a)pyrene (BP)-induced tumors and increased BP hydroxylase activity in the
forestomach of the Ha/ICR mice and carbaryl enhanced BP-induced tumors and increased
BP hydroxylase activity in the lungs of the A/J mice. In each instance, it is
possible that toxaphene and carbaryl exhibited a cooncogenic effect in enhancing the
BP-induced tumors. Conversely, toxaphene decreased the incidence of BP-induced
tumors and inhibited BP hydroxylase activity in the lungs of the A/J mice. These
results suggest that increased BP hydroxylase activity in tissues tends to enhance
tumor formation and a decrease in the enzyme activity may have a protective effect
against tumors. The relationship between enzyme inducibility and tumor formation
may be due to the level of oncogenic epoxides formed in target organs. Further,
studies of the formation of specific oncogenic epoxides of BP in tissues after
treatment with these pesticides would help towards defining more clearly the
relationship between BP hydroxylase inducibility and BP oncogenesis.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
neoplasms
pesticides
carcinoid tumors
toxicology
oncogenesis
carcinogenesis
benzo(a)pyrene hydroxylas
aryl hydrocarbon hydroxyl
06 F, T
ase
18. DISTRIBUTION STATEMEN1
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report I
UNCLASSIFIED
. NO. OF PAGES
38
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE
32
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