EPA-600/1-78-044
June 1978
EFFECT OF PESTICIDE INTERACTIONS UPON THE REPRODUCTIVE SYSTEM
by
John A. Thomas
Department of Pharmacology
West Virginia University Medical Center
Morgantown, West Virginia 26506
Grant No. R-803578
Project Officer
Ronald L. Baron
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 recommendation for use.
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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 materials, and is primarily responsible for providing
the health basis for non-ionizing radiation standards. 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.
This report evaluates the effects of different pesticides upon the
mammalian reproductive system, and to what extent these agents can
induce changes in biochemical and hormonal activities.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
iii
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PREFACE
Metabolic interactions can occur between a variety of chemicals in the
environment (xenobiotics) that can produce biologic effects different from
those caused by the compounds individually. Many of these effects have been
found to be mediated by alterations in the hepatic microsomal enzyme system(s).
Changes in the activities of the microsomal enzymes can affect the biologic
activity of xenobiotics, such as pesticides, herbicides, carcinogens, drugs,
and hormones. Hormonal imbalances may be magnified by pesticide-induced
changes in certain enzyme systems.
While the volume of literature regarding the effects of a single pesti-
cide upon the male reproductive system of mammals continues to increase, few
studies have been devoted to investigating the simultaneous effects of more
than one pesticide.
The present report reveals the interaction of major classes of pesticides
on hormone metabolism, and attempts to explain molecular actions of certain
pesticides by demonstrating their affinity for hormone receptor molecules.
IV
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ABSTRACT
The metabolism of l,2-3H-testosterone in vitro was studied in prostate
glands and livers of rats and mice treated with different pesticides including
dieldrin and parathion. The metabolism of 1,2-3H-testosterone (T-^H) ^n viTro_
by mouse anterior prostate glands or hepatic microsomes has been studied after
the oral administration of dieldrin (2.5 rag/kg daily x 5 or 10) and/or para-
thion (1.3,2.6,or 5.2 mg/kg daily x 5 or 10). T-3H metabolism in the prostate
was unaffected by the various treatment regimens. Dieldrin (10 days) caused
some reduction in the microsomal production of androstenedione-^H but failed
to affect the biotransformation to androstanediol—^H or dihydrotestosterone-
^H. Only treatment regimens with dieldrin stimulated hepatic testosterone
hydroxylases; parathion alone had no effect. This study revealed that dieldrin
and parathion can interact and produce biological effects different from those
caused by either pesticide alone.
Liver microsomal steroid hydroxylacing enzyrr.fs and prostatic testosterone-
5a-reductase were studied in rat and mouse. Organochlorine and organophosphate
pesticides tended to inhibit liver steroid hydroxylations, while carbofuran
slightly stimulated them. Neither species was consistently more sensitive to
pesticide effects than the other. All the pesticides (viz, heptachlor,
carbofuran, diazinon and parathion) bound to cytochrome P-450, producing type I
spectral charges. Values of Ks ranged from 1.9 to 8. 7mM for Organochlorine and
organophosphate compounds. Affinity for carbofuran was much lower (Ks=100-200mM).
The binding of [%] dihydrotestosterone ([^K]DKT) to cellular components
from prostate, seminal vesicles, kidney, and, liver of the male mouse was studied
using a dextran-coated charcoal method to separate bound steroid from free
steroid. Optimum conditions for binding include incubating tissues from animals
3 days postcastration for 12 hr at 0°C. Separation of bound steroid from free
steroid was found to be optimal when the samples were incubated with the char-
coal suspension for 15 min. Two components of DHT binding were found in all
tissues studied, but a higher capacity was noted in androgen target tissues
such as the prostate gland. The high affinity binding was also very specific
for DHT as evidenced by competition studies employing various hromones, such as
estriol, corticosterone, estrone, progesterone, estradiol, cyproterone acetate,
testosterone, and DHT. The effects of various pesticides of ^H-DHT binding in
these tissue cytosols were also assessed. Parathion (10~8 _ 10~^M) was found
to be an effective inhibitor of total -^H-DHT binding in the prostate, seminal
vesicle, kidney and liver. This organophosphate was unable to compete with
%-DHT for cytosol binding sites in the intestine. Similar in vitro binding
studies using bieldrin, DDT, or carbaryl failed to reveal any interference
with ^H-DHT binding in any of the tissues studied. The mechanism of parathion's
interference with 3H-DHT binding in unclear.
This report was submitted in fulfillment of Grant No. R0803578 by West
Virginia University under the full sponsorship of the Environmental Protection
Agency. Work was completed as of February 14, 1978.
v
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CONTENTS PAGE
Foreword iii
Preface iv
Abstract v
List of Illustrations vii
List of Tables viii
Abbreviations and Symbols ix
Acknowledgement x
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods 4
5. Results 7
6. Discussion 26
References 31
Publications Supported by R0803578 36
Glossary 38
VI
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LIST OF ILLUSTRATIONS
Number; Page
1 Binding of hepatachlor/methoxychlor to hepatic microsomes .... 14
2 Binding of parathion/diazinon/carbofuran to hepatic microsomes. . 16
3 Optiminal conditions for dihydrotestosterone (DHT) binding to
prostate cytosol 19
4 Scatchard analysis of DHT and various organs 20
5 Ability of various hormones to compete with DHT 21
6 Binding of parathion/carbaryl/DDT/dieldrin to prostate cytosol
p rote in (s) 24
7 Binding of parathion/carbaryl/DDT/dieldrin to intestinal cytosol
protein(s).... 25
vii
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LIST OF TABLES
Number Page
1 Dleldrin/parathion effects on prostate weight and hepatic
protein 3
2 Dieldrin/parathion effects on testosterone-^H metabolism in
prostate 8
3 Dieldrin/parathion effects on testost:erone-3H metabolism in
liver 9
4. Heptachlor/methoxychlor/carbofuran/diazinon/parathion effects
on testosterone-^H metabolism in liver 12
5. Pesticide binding affinities in liver microsomes 13
6. Specific binding sites in different organs 21
7. Inhibitory actions of parathion on dihydrotestosterone (DHT)
binding 23
Vlll
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ABBREVIATIONS A-ND SYMBOLS
ABBREVIATIONS
DHT — dihydrotestosterone
^H — tritium
S.E.M. — standard error of mean
X — mean
dpm — disintegrations/minate
p.o. — S^L _2Ji> oral administration
Ka — binding affinity (liters/mol x Id")
+ — plus one and minus one S.E.M.
IX
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ACKNOWLEDGEMENT S
The Principal Investigator gratefully acknowledges the scientific interest
and support of several graduate students and post-doctoral fellows involved
in various facets of this research. It was partly through these different
investigators that the information was gathered and dissendmated to the
scientific community.
The Principal Investigator wishes to also acknowledge the scientific
assistance and administrative aid of the EPA scientific staff including Drs.
William Durham, James Stevens and Ronald Barren.
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SECTION I
INTRODUCTION
The presence of many chemical classes of pesticides in the environment
offers numerous opportunities for metabolic interactions to occur in various
species of animals including man. Investigations have demonstrated that many
environmental chemicals (xenobiotics) can interact to produce effects different
from those effects produced by the compounds individually (Richardson e t al.,
1952; Conney £t_ al_. , 1956; Conney, 1967; Schein and Thomas, 1976). Many of
these pesticide interactions have been found to be due to altered hepatic
microsomal enzyme activity (Conney and Burns, 1972; Krampl e_t_ al_. , 1973).
Such pesticide interactions may result in lowered chronic toxicity of
certain pesticide combinations (DuBois, 1969; Murphy, 1969). Endocrine
imbalances may be caused due to pesticide-induced alterations in certain
enzymes (Peakal, 1967; Street e^ al.., 1969). Methoxychlor decreases the storage
of dieldrin in the body fat of rats (Cueto and Hayes, 1965). Joint lethality
of DDT and dieldrin appeared to be additive in both Japanese quail and ring-
necked pheasants (Kreitzer and Spann, 1973) .
Although there has been an increasing interest in studying the effects of
single pesticides on the mammalian reproductive system, few investigations have
examined the simultaneous effects of more than one pesticide upon the male
reproductive system. DDT and dieldrin have been shown to interfere with male
rodent sex accessory gland metabolism of testosterone (Smith et jil_. , 1972;
Schein and Thomas, 1975). Studies by Wakeling and Visek (1973) have shown that
o,p-DDT can inhibit the binding of dihydrotestosterone to specific receptor
proteins in the cytoplasmic fraction of the rat prostate gland.
Various effects of pesticides have been demonstrated on hepatic metabolism
of androgens in rodents. Studies by Kuntzman ££ sd. (1966) have shown that DDT
treatment caused a marked increase in testosterone 16a-hydroxylase activity in
immature male rats. In the mouse DDT-inhibited hepatic microsomal testosterone
16a-hydroxylase activity while dieldrin stimulated this activity. Dieldrin
stimulated the 16a- hydroxylase to a much greated extent than either the 66- or
7a-hydroxylase (Thomas and Lloyd, 1973).
The presence of parathion has been shown to inhibit oxidation of hexobar-
bital and hydroxylation of aniline by mouse hepatic microsomes in vitro (Stevens
et_ al_. , 1971; Welch ej^ al_. , 1967). On the other hand, administration of one-
half the oral LD5Q of this pesticide to mice for 5 consecutive days reduced
hexobarbital sleeping time, indicating stimulation of oxidation by hepatic
microsomal enzymes (Stevens et_ al., 1972) . Thomas and Schein (1974) using oral
doses of one-sixteenth to one-fourth the LD5Q demonstrated no alterations in
mouse hepatic microsomal androgen hydroxylase activity. Swann et al. (1958)
reported that the acute toxicity of parathion was related to the sex of the
animal, and that sexual maturity conferred added protection against the toxic
action of this organophosphate.
1
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bECTION 2
CONCLUSIONS
1. Dieldrin and/or parathion can affect androgen metabolism in the prostate
gland and the liver, but the extent of this effect is related to the dose
and duration of the pesticide(s).
2. Both organochlorine-type and organophosphate-types pesticides tend to
inhibit androgen hydroxylatlcn reactions in hepatic microsomes.
3. Several pesticides such as heptachlor, carfaofuran, diazinon and parathion
can bind in yitr_p. to hepatic cytochrome P-450 producing type I spectral
changes.
4. Isolation of a cytosolic protein reveals that in some tissues, it not
only has an affinity for hormones and certain drugs, but also for pesti-
cides .
5. The effects of a single pesticide on hormone metabolism and the reproduc-
tive system are different than those occurring from the administration of
more than one pesticide.
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SECTION 3
RECO?C-:£NDATIONS
1. Efforts should continue to disclose the toxico!ogical effects of the
differanc chemical classes of pesticides neon the reproductive system,
particularly since so little is known about their molecular mechanism(s)
of action(s).
2. Further consideration should be given to correlating pesticide-induced
changes in different; hormone target organs with the entire endocrine sys-
tem.
3. Studies must continue to be devoted to examining the effects of new (and
even "old') pesticides on the male reproductive system with some considera-
tion devoted to spermatogenesis, fertility and possible teratogenicity.
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SECTION 4
MATERIALS AND METHODS
METHODS
Animals—Male Swiss-Webster mice and sprague-Dawley rats (Hilltop Lab-
oratory Animals; Scottsdale, PA) were acclimated for at least two weeks in the
Medical Center Animal Quarters and were maintained on a standard diet of lab-
oratory chow (Purina laboratory chow) and water _ad j.ibiturn. Castrations were
performed via abdominal incision under pentobarbitai anesthesia.
Preparation of cytosol_—All preparative procedures were performed at 0° to
4°C. Immediately upon sacrifice of the aninals by cervical dislocation, whole
anterior prostate glands, seminal vesicles, kidneys, and samples of liver were
excised, blotted, and placed temporarily in 0.05 M Tris-HCl (pH 7.2) containing
0.60 mM Na2EDTA and 2.6 mM mercaptoethanol (Tris/EDTA buffer). Tissues were
subsequently blotted, weighed, and homogenized (Kontes Teflon-glass homogenizer,
15-20 manual strokes) in 10 vol of ice-cold Tris/EDTA buffer. The prostate and
seminal vesicle were chopped (Mcllwain tissue chopper) just before homogeni-
zation. Homogenates were centrifuged(2000g for 20 min), and the supernatants
were decanted and recentrifuged (100,000g for 60 min) on a Beckmaa Model L
ultracentrifuge. The resulting supernatants were designated as the cytosols or
cytoplasmic fractions.
Steroid binding—Purity of the [JH] DHT was assessed using thin-layer
chronuitography with a chloroform: ether (7:3, v/v) solvent system and was con-
sidered acceptable only when it exceeded 95%. Stock solutions of [JH] DHT in
ethanol/benzene were dried under N2 and redLssolved in an appropriate volume of
Tris/EDTA buffer immediately before each experiment.
Quantities of [3H] DHT (sp,act, 80 Ci/mmol; New England Nuclear, Boston,
Mass.) were incubated with cytosol prepared in Tris/EDTA buffer in a total vol-
ume of 0.45 ml. The concentrations of cytosol protein, [^H] DHT, and competing
steroid were among the experimental variables studied, as were the duration of
incubation and the incubation temperature. Corticosterone, cyproterone acetate,
dihydrotestosterone, estradiol, estrone, progesterone, and testosterone were
used as competing steroids. Samples of stock solutions of these compounds in
methanol were dried under nitrogen in the incubation tubes before cytosol pro-
tein and [^H] DHT were added. Final concentrations of competing steroids were
varied from 10~8 to 10~6M.
Separation of bound and free steroid—To remove free steroid from the in-
cubation mixture and allow the counting on only the bound steroid, a dextran-
coated charcoal method was employed (Binoux and Odell, 1973). A suspension of
activated charcoal (0.25%, w/v) was prepared in 0.9% NaCl containing 0.025%
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dextrari T-70 and stirred at U-4°C for ac least 1 hr. After incubation of
cytosol and [%] DHT, 1.0 ml of the charcoal suspension was added. The dura-
tion of incubation was varied from _L tj 15 min. The charcoal and the free
steroid adsorbed from the solution were sedimented by centrifugation (lOOOg
for 10 min) , and the supernacants were decanted into scintillation vials.
Initial studies established the conditions for the optimum removal of
free steroid by adsorption to charcoal and consistently revealed that no ad-
ditional steroid was removed after 10 min of exposure to cr.arccal. Therefore,
a 15-min charcoal incubation interval was routinely employed. No so-called
"stripping" of steroid from protein binding sites occurred during this 15-min
incubation period. Suitable blank tubes assessed the efficiency of removal
of unbound steroids, and revealed that nore than 98% of the free pH] DHT was
removed under these experimental conditions.
Cytpchrome P-450—Difference spectra were recorded in a Gary 15 spectro-
photometer using hepatic microsomai suspensions at approximately 2 rag protein/
ml at room temperature. Pesticides were added to the sample cuvette in ace-
tone solutions; acetone alone was added to the control cuvette. Total acetone
added never exceeded 1%. Spectral dissociation constants (Ks) were calculated
by the method of Schenkman, £t_ a^. (1967). Cytochrome P-450 was determined
from CO difference spectra (Omura and Sato, 1964).
V.
Tissue Protein—Protein was estimated by the method of Lowry et al. (1951)
using bovine serum albumin as standard.
Incubation techniques—Twenty-four hours after the last daily pesticide
dose, animals were sacrificed by cervical dislocation and anterior prostate
glands and samples of liver we.re rapidly excised. The liver samples were
homogenized in ice-cold 0.154 M KC1-0.05 M Tris HC1 (pH 7.4). Cell debris,
nuclei, and mitochondria were removed by centrifugation of the homogenate at
10,000g for 25 minutes. Microsomes were sedimented from the postmitochondrial
supernatant at 100,000g for 60 minutes, then resuspended in cold buffer.
Whole prostates or aliquots of hepatic microsomes (about 600 _jig of protein)
were incubated with 1,2-%-testosterone (New England Nuclear, sp. act. 40 Ci/
mmole, 98.5%) in 0.1 M sodium phosphate (pH 7.4) containing 4 mM NADP, 5 mM
glucose-6-phospha'te, 5 mM MgSo4 , 4 mM nicotinamide, and 5 1.If/ml of glucose-6-
phosphate dehydrogenase. Incubations were carried out aerobically in a total
volume of 1 ml at 37°C with shaking.
Incubations of hepatic microsomes were terminated by adding cold chloro-
form-ether (7:3). Prostate incubations were stopped by immersing the tissue
in cold 0.4 N perchloric acid and simultaneously adding chloroform-ether to the
medium. The prostate tissues were homogenized and radiometabolites of 3H-T
extracted from these homogenates as well as the incubation mixtures with
chloroform-ether. The extracts were evaporated to dryness under nitrogen and
redissolved in 500 ,ul of chloroform.
Twenty-five-microliter samples of the redissolved extracts were spotted on
prepared thin-layer chromatography plates (Eastman Company) or on hand-made
Silica Gel-G plates. Chromatograms were developed with chloroform-ether (7:3)
and spots visualized by iodine vapor (nonpolar metabolites) on ultraviolet light
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(polar metabolites). Spots vere scrapped from the plates and radioactivity
determined by a Packard Tri-Carb Scintillation Counter. These methods are
described in detail elsewhere (Schein and Thorns.-, 1976),
Liquid scintillation counting—Quantitation of radioactivity was carried
out in scintillation counting solution containing 0.05 g PQPOP, 4 gm PPO, and
200 ml of Beckman Biosolv in 1 liter of toluene. Samples were counted on a
Packard Tricarb scintillation spactromatiar at an efficiency of 25 to 30%,
using [3H] toluene (New England Nuclear) as an external standard. All data
were expressed in dpm or moles of [^H] DHT.
Statistics—Data were analyzed by Dunnett's method for comparing multiple
treatment means (Dunnett, 1955). Scatchard analyses (1949) were calculated
for several tissues to determine binding affinity of both hormones and pesti-
cides.
Chemicals and Pesticides — Solutions of dieldrin [1,2,3,4,10 ,10a-
hexachloro-6,7-epoxy-l,4 4a, 5,6,7,8,8a-octahydroendoexo-l,1,4:5,8-dimethano-
naphthalene] (K & K Laboratories, Plainfiew, NY., 95-99%) and parathion [0,0-
diethyl-O-(p-nitrophenyl) ester phosphothioic acid] (City Chemical Corp., NY,
98.76%) were prepared in corn oil. Daily doses of dieldrin (2.5 mg/kg) and/
or parathion (1.3,2.6 or 5.2 mg/kg) were administered by gastric intubation in
a volume of 0.1 ml. Methoxychlor (DuPont), carbofuran (FMC), and diazinon
(CIBA-Geigy) were received from the Environmental Protection Agency. Henry K.
Suzuki, Velsicol Chemical Corporation, Chicago, kindly supplied a sample of
chlordane. DDT was obtained from Nutritional Biochemical Corporation.
Cyproterone Acetate (1,2a-methylene-6-chloro-A4,6-pregnadien-173ol 3,20-dion-
17a-acetate) was obtained from Schering Corporation. DHT (5a-androst-2-ane-
173~ol) and corticosterone (4~pregnene-ll$, 21-diol-3,20-dione) were obtained
from Sigma Corporation. Dextran was purchased from Pharmacia Fine Chemicals,
and scintillation fluors and toluene from Fisher Scientific Company. All
other reagents were obtained commercially from Sigma Chemical Corporation.
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SECTION 5
RESULTS
Regardless of the dose or dose sequence, neither parathion nor dieldrin
caused any significant changes in prostate gland weights (Table 1). Similarly
these dose regimens produced no significant alterations in hepatic microsomal
proteins. Thus, these dosing protocols represent no-effect levels insofar as
weights and protein levels are concerned.
Testosterone-^H metabolism by the prostate gland was largely unaffected
by the pesticide regimens (Table 2). There was an unexplained increase in pro-
duction of androstanediol-^H in a dieldrin-parathion group, but it is evident
that the enzymes catalyzing formation of the major nonpolar radiometabolites of
testosterone-^H are not substantially altered by the organochlorine-or the organo-
phosphate dose regimens.
Unlike the prostate gland (Table 2) , hepatic microsomal testosterone-^H
metabolism was extensively altered by certain of the pesticide regimens (Table
3A). With the exception of deiJdrin plus the highest dose of parathion, a 10-
day regimen of dieldrin significantly reduced the formation of androstenedione-
^H (P<0.05). The highest dose of parathion administered concomitantly with diel-
drin appeared to abolish or negate the inhibitory effect of the organochlorine
on formation of androstenedione-^H. Testosterone-3H levels were generally lower
in those groups receiving dieldrin for a period of 10 days. Again, the highest
dose of parathion seemed to counteract the lowering effects of the dieldrin upon
testosterone-^H levels. Neither androstanediol-^H nor dihydrotestosterone-^H
was significantly affected by the different pesticide dose regimens.
The most conspicuous alterations produced by parathion and/or dieldrin were
observed in the hepatic polar metabolites (Table 3B). Of the three testosterone
hydroxylase activities examined, two (the 63- and 7a-hydroxylases) were signifi-
cantly stimulated by several treatment regimens. These stimulatory effects most
consistently seen on the 63- and 7a-hydroxylases were not restricted to either
dieldrin or to parathion, but were recorded after all combined treatments as
well as after administration of dieldrin only. The greatest stimulatory effect
appeared to occur in the 7ct-hydrcxytestosterone levels. No significant changes
were evident in the 16a -hydroxytestosterone-^H levels. It is apparent that only
some of the androgen hydroxylases present in hepatic microsomes (viz., 63- and
7ct-hydroxytestosterone) were affected by these dose regimens of pesticides.
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TABLE 1
DlELDRIN (D) AND/OR PARATHION (P) EFFECTS ON PROSTATE
WEIGHT AND HEPATIC PROTEIN OF THE MOUSE"
Ten-day
treatment regimen
1
C
P,
P*
P,
D
P,
Pa
P3
D
D
D
P,
D
P,
D
Pa
D
2
C
P,
Pa
P,
D
P,
Pa
Pa
D
D
D
P,
D
P,
D
?a
D
" Dieldrin
3
C
P,
Pa
Pa
D
P,
Pa
Pa
D
D
D
P,
D
Pa
D
Pa
D
(D)(2
4
C
P,
P*
Pa
D
P,
Pa
Pa
D
D
D
P,
D
Pa
D
P3
D
5
C
P,
P2
Pa
D
P,
Pa
P3
D
D
D
P,
D
P2
D
Pa
D
.50 me/kg
6
C
P,
Pa
P3
D
D
D
D
P,
P*
Pa
P,
D
P2
D '
P3
D
daily x
7
C
P,
Pa
Pa
D
D
D
D
P,
P2
Pa
P,
D
ft
D
Pa
D
5 or
8
C
P,
Pa
Pa
D
D
D
D
P,
P2
Pa
P,
D
P2
D
Pa
D
9
C
P,
P2
Pa
D
D
D
D
P,
Pa
Pa
P,
D
Pa
D
Pa
D
10
C"
P,
Pa
Pa
D
D
D
D
P,
P2
Pa
P,
D
P2
D
Pa
D
10) and/or parathion
Prostate wt
(mg)
20.10 ± \.53f
22.82 ± 1.21
17.70 ± 0.62
18.92 ± 0.94
18.62 ± 0.51
16.76 ± 1.42
17.02 ± 0.76
16.02 + 1.42
17.82 ± 0.69
18.26 * 0.46
18.40 ± 1.13
18.40 ±0.13
19.04 ± 0.40
12.15 ± 1.91
(P)(P,. 1.3: P,,
Hepatic
microsomal protein
(mg/g)
4.05 ± 0.36
4.10*0.31
4.20 ± 0.24
4.37 ± 0.28
4.66 ± 0.31
4.26 ± 0.22
4.29 ± 0.41
3.85 ± 0.22
3.78 ± 0:44
3.09 ± 0.17
3.68 ±0.17
4.20 ± 0.26
3.82 ± 0.17
4.62 i 0.34
2.6; PJ, 5.2 mg/ke daily
x 5 or 10).
* Corn oil vehicle.
c x ± SEM of at least six samples.
TABLE 2
EFFECTS OF DIELDRIX (D) AND/OK PARAIHION (P) ON im 10-MiNinr. In Vitro
METABOLISM OF 'H-TESTKRONE BY THE MOUSE PROSTATE Gi AND"
Ten-day
treatment regimen
1
C
P,
Pa
PI
D
P,
P,
Pa
D
D
D
P,
D
Pa
D
f,
D
2
C
P,
P,
P,
D
P,
Pa
Pa
D
D
D
P,
D
P*
D
Pa
D
3
C
P,
P,
Pn
D
P,
Pa
Pa
D
D
D
P,
D
P.
D
Pn
D
4
C
P,
Pa
PI
D
PI
Pa
P3
D
D
D
P,
D
Pa
D
Pa
D
5
C
P,
Pa
P,
D
P,
Pa
Pa
D
D
D
P,
D
Pa
D
P3
D
6
C
P,
Pa
P,
D
D
D
D
P,
Pa
Pa
P,
D
Pa
D
P,
D
7
C
Pr
Pa
PI
D
D
D
D
P,
P.
Pa
P,
D
Pa
D
Pa
D
8
C
P,
Pa
PI
D
D
D
D
P,
Pa
P,
P,
D
Pa
D
P,
D
9
C
P,
P2
Pa
D
D
D
D
P,
Pa
Pa
P,
D
Pa
D
Pa
D
10
C"
P.
Pa
P,
D
D
D
D
P,
Pa
Pa
P,
D
P,
D
Pa
D
Radiometabolites (dpm/100 ^g protein)
•'H -Testosterone
544 ±
552 ±
729 ±
564 ±
534 ±
610 ±
513 ±
495 ±
401 ±
575 ±
556 ±
'465 ±
fill ±
614 ±
2#
116
127
96
52
62
69
59
23
72
36
57
40
105
3H-Androstancdiol 3H-Dihydrotestosterone
215 ± 62
220 ± 70
198 ± 56
179 ± 20
212 ± 23
174 ± 47
216 ± 39
295 ± 49
369 ±51"
316 ± 52
199 ± 22
219 ± 46
200 ± 19
228 ± 50
428 ± 45
460 ± 82
568 ± 94
382 ± 58
436 ± 72
394 ± 45
383 ± 14
401 ± 48
389 ± 27
391 ± 33
398 ± 48
370 ± 31
343 ± 26
395 ± 40
'H-AndroMencdione
43 ± 13
30 ± 8
40 ± 20
38 ± 13
38 ± 8
44 ± 10
30 ± 6
49 ± 11
45 ± 10
43 ± 17
29 ± 8
43 ± 8
40 ± 1
40 ± 10
« Dieldrin (D) (2.50 mg/kg daily x 5 or 10) and/or parathion (P) (P,, 1.3; P2> 2.6; P3, 5.2 mg/kg daily x 5 or 10).
* Corn oil vehicle.
<• x ± SEM of at least six samples.
" Significantly different from control (P < 0.05).
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Pesticides were tested at three concentrations for their ability to inhib-
it %-testosterone metabolism. With rat liver mi ; r.-.-. or.e s , none were particular-
ly potent (Table 4). Heptachlcr and methoxychlcr significantly inhibited
hydroxylation reactions at 10~^M but n , c at 1G~JM or less. Carbofuran did not
inhibit hvdroxiTl "ti^np, out actually seemed to stimulate formation of I6a-
hydroxytestosterone and b5-L:vdi:^:-:.'-~si:----<"c"~ viien present_ at low concentration.
Organophosphates had mixed effects (Table 4).
Diazinon inhibited 16a hydroxylation at 10~"^M to a greater extent than it
reduced formation of the other hydroxysteroids. Parathion, on the other hand,
had no effect on I6c:-hydroxylation but at 10~^M severely reduced 7a and 6 fc!
hydroxylations.
A similar array of effects was seen men .-.epari^;
were studied (Table 4). In general, the steroid hydroxylations were somewhat
less sensitive to organochlorines than were these reactions in the rat. The
exception was 16a and 63 hydroxylat L^ub anch were inhibited by low concentra-
tions of heptachlor. Carbofuran had the same tendency to slightly stimulate
metabolism that it showed in the rat. Mouse microsomes were very sensitive to
diazinon at all concentrations,but were nearly refractory to parathion even
at 1CT4M.
Spectral changes produced by direct interaction of pesticides with hepatic
microsomal cytochrome P-450 were measured because this hemoprotein is a part
of the enzymic complex responsible for steroid hydroxylation. All the pesti-
cides in this study produced typical Type I spectral changes with an absorbance
peak ac 385nm and a trough at 420nin.
Double reciprocal plots of the pesticide concentration dependence of the
peak-trough difference showed complex interactions with rat (Figs. 1A and 2A)
and mouse (Figs. IB and 2B) microsomes. In both species, heptachlor appeared
to bind at two sites, one having a high affinity and the other having low-
affinity (Fig. 1). The total capacity of the low affinity binding sites was
about twice that of the high affinity type (Table 5). Methoxvchlor also bound
to sites in both species with affinities comparable to the high affinity
heptachlor binding (Fig. 1). However, at concentrations greater than 2 x 10~^M,
methoxychlor apparently disrupts microsomal membranes with consequent loss
of the Type I cytochrome P-450 spectrum. This change was accompanied by an
increase in turbidity which was visible to the unaided eye and accounts for the
increase in 1AA at low values of 1/S.
Carbofuran produced Type I spectral changes in both rat and mouse (Fig. 2),
but only at concentrations greater than 10~4M. The affinity for this compound
was very low and the concentration of binding sites for it was also low (Table 5).
Organophosphates had complex binding properties. In both species, diazinon
bound to one type of high affinity site and caused disruption of membranes and
reversal of spectral changes when its concentration exceeded 10~^M (Fig, 2).
The only aajor differences between the species was in the binding of parathion
(Fig. 2). Both mouse and rat liver microsomes showed high affinity binding
with K* approximately 7mM (Table 6). Only mice, however, had low affinity
binding <;ites for parathion.
11
-------
The pesticides studied had very little apparent effect on reduction of
3H-testosterone to 3H-dihydrotestosterone by prostate glands from these animals.
Only mouse tissue showed reduced formation of 3H-dihydrotestosterone and that
only when 10~^M heptachlor or parathion was present.
Chlordane showed similar inhibitory properties to the other organochlorines,
that is, it inhibited microsomal hydroxylations in both species at high concen-
trations and showed weak inhibition of mouse prostate testosterone reduction.
Since chlordane is a mixture of many components, its cytochrome P-450 binding
spectra were not recorded.
TABLE 4
EFFECTS OF PESTICIDES ON FORMATION OF 3H-HYDROXYTESTOSTERONE
ISOMERS FROM 3H-TESTOSTERONE BY RODENT LIVER MTCROSOMES
660HT
1611-OUT
MOUSE
7a-OUT
Heptachlor
10 -8M
10-6M
10 -4 M
Methoxychlor
10 ~8M
10~6M
10-^M
Carbofuran
10-8M
10~6M
1 Q-4M
Diazinon
10-6M
10 -4 M
Parathiou
1 0 ~~ M
i n — 6 v
10~/tM
82 A
77
25
85
79
25
294
224
137
90
76
33
105
133
96
111
12]
38
113
120
37
93
• S3
77
78
67
108
144
22
96
104
54
93
106
53
150
149
114
104
142
60
92
96
40
56
36
104
100
63
139
141
1 7 0
31
39
20
96
112
22 80
90
103
93
93
111
85
86
Q/,
50
32
32
86
81
78
52
63
42
84
Si
10/4
117
i in
67
70
32
100
105
76
A. All values are expressed as % of control dpm/nig i'roteiu/5inin,
12
-------
TABLE 5
A.
Rat
HepLachlor
MctiioxychJ or
Carbofuran
Dinz Lnon
Parathion
Heptachlor
Methoxychlor
Car bofuran
Dinziuon
Paratrhiou
2.8
2S
o
100
3.6
7.1
35
3.8
200
2.0
6.7
14.3
30.1
15.0
10.5
28.Q
41.4
13
-------
FIGU?.H la
(R;
,iver Microsomes
020
B
c ^
o <
CNl <3
-o- ~^-
i o
LT) L-1
co
-------
T^-r.^lT-T-177 -!1
i1 JLLrl r -. ID
[Mouse Liver Micros oraes)
g
o <"
I O
U"| LO
oo -i
< CJ
rH
A Heptachlor
o Metho:rychlor
1/S [PESTICIDE] (1/M x 1CT4)
Figure 1. Double reciprocal plot of binding of organochlorine pesticides to
rodent liver microsomes. Absorbance spectra were recorded using a sample con-
taining 2 mg/ml microsomal protein plus pesticide and a reference containing
only microsomes. The peak-trough difference (385-420 nm) was divided by the
cytochrome P-450 concentration as determined from CO difference spectra and the
reciprocal of this quantity was plotted as a function of the reciprocal of the
pesticide concentration.
15
-------
FIGURE 2a
(RAT LIVER MICROSOMES)
6
C /-j
o <:
CN <3
oo •<»•
016
012
008
004
A
o Parathion
A Diazmon
° Carbofuran
A
1/S [PESTICIDE] (1/M x
Figure 2. Double reciprocal plot of binding of organophosphate and carbamate
pesticides to rodent liver microsomes. Conditions and calculations were as
described in Figure 1.
16
-------
FIGURE 2b
(MOUSE LIVER MICROSOMES)
DIG -
6
C x-x
o <:
CM <3
•o- —
I O
to m
CO
-------
Figure 3 reveals that several parameters can influence the amount of
fetmnd [^H] DHT. Increasing the duration of postcastration time expectedly led
to a loss of endogenous androgen resulting in more [^H] DHT being bound to cvto-
plasmic receptors in several tissues (Fig. 3A) . The increased availability of
binding sites following castration was particularly evident in the sex acces-
sory organs (viz . , prostate and seminal vesicles). A 3-day , postcastration
time seemed to reveal considerable binding of [-^Hj DHf,and I': was arbitrarily
chosen as the interval affording substantial binding (Fig. 3A) . Using several
in vitro incubation temperatures indicated that the total binding of [^H] DHT
to prostate cytosoi receptor increased exponentially (Fig. 3B) . However, no
specific binding, as determined according to method of Charanes and McGuire
(1975) , was detected when the incubation temperature was elevated beyond 10°C,
and all further experiments were carried out at 0°C to maximize the degree of
specific [3H] DHT binding by prostate cytosoi proteins. Although not shown,
similar temperature curves were also obtained for other organ cytosols (viz . ,
seminal vesicles, liver, and kidney).
If the duration of incubation was monitored over a 24-hr period, it
observed that the total binding of [^H] DHT increased until about 10 hr and there-
after remained on a plateau until 24 hr (Fig. 3C) . Subsequent experiments there-
fore utilized a 12-hr incubation interval in order to ensure the establishment
of an equilibrium between bount and free [^H] DHT in the tissue cytosols.
Just as an increase in incubation time led to an increase in binding of
pH] DHT, as increase in the concentration of prostate cytosolic protein in the
incubation mixture lead to increased pH] DHT binding (Fig. 3D). Based on this
dependence pattern, a protein concentration of 2 mg/ml was selected for sub-
sequent experiments. The dependence of binding on protein concentration was
similar in all tissues studied, and the same standard concentration was chosen.
Using the optimum experimental conditions of postcastration interval,
temperature, incubation time, and protein concentration, Scatchard analysis
(1949) was performed on [-*H] DHT binding data from prostate gland and other
organs (Fig. 4) . The affinity of the cytosolic protein for PH] DHT was sim-
ilar in all the tissijes, but the total binding capacity varied among them
(Table 6) . Capacity was highest in the prostate and lowest in the liver.
The specificity of the prostate cytosolic steroidophil was demonstrated
by competition studies using various agents (Fig. 5). As expected, nonradio-
active DHT and testosterone were very effective competitors of [^H] DHT binding
by prostate cytosoi. Of intermediate inhibitory activity were cyproterone
acetate and estradiol followed by progesterone. Over a wide range of in vitro
concentrations, estrone, estriol, and corticosterone failed to compete effective-
ly with [%] DHT for its binding sites (Fig., 5). The specificity of the seminal
vesicles, liver, and kidney binding profiles for the various steroids was
analogous to the prostate response (not shown) .
18
-------
Figure j. Effaces of incubation conditions on [JHj DHT binding by cytoscl.
All cytosol incubations were performed in the presence of 5 x !0~'M [~H] DHT
in Tris/EDTA buffer (pH 7.2). Vertical bars show SE of the mean, and each
point represents the mean of at least six values. (A) Relationship of [ H]
DMT bound/rag of protein to cytosols prepared from tissues ta!>en from animals
at varying times postcastration and ir.rubatsc £-:r 12 hr at 0°C. (B) Relation-
ship of total nonspecific (NSP) and specific (SP) [^H] DHT bound to cytosols
prepared from tissues taken from -Anirv.lr 3 jr/'s ~os':c:.?crcricn .:r.d incubated
for 12 hr at varying temperatures. The 5E of each point is approximately 10%.
(C) Relationship of [-^H] DHT bound/my of protein to cytosols prepared from tis-
sues taken from animals 3 days postcastration and incubated for varying times
at 0°C. (D) Relationship of [^H] DHT bound to protein concentration of cytosols
prepared from tissues taken from animals 3 days postcastration and incubated
for 12 hr at
FIGURE 3
3 >?
* L
2 4 6 8 iQ :2
PCST-CASTPATlCrt (Gays)
lOOrg
"0 ' o r0)al
300
1 250
t-~
f.
O
i ^SP
a SP
P
/
u
% 1501-
30L
E 251-
6 S .0 12
E; : N ing/mj I
19
-------
STTKCin .ilNDING IN MOLSh TISSl KS
S/F
DHT BOUND (1CT11 Moles/Liter)
Figure 4. ScaCchard analysis of [-H] DHT (radioactive) binding to specific
cytosol-binding proteins in various tissues from, mice 3 days postcastration.
Incubations of the cytosols were performed in the presence of various concen-
trations of [3H] DHT'for 12 hr at 0°C.
20
-------
TABLE 6
S.~ MCIIARU ANALYSIS OF 3H-DHT BINDING ro SPICIHC
OTOSCL RrcLPTORS most SsMss-WtBSTr.R Mio 3 D\YS
AFTER CASTRATION
k.
BindriK sues
(liters/mol x 10") (moi-'g of protein x 10"")
Prostate
Seminal vesicle
Kidney
Liver
1.7 - 0 I"
1.7 ±0.2
1.3 ±0 1
1.3 ±0.2
5.1 -i-O.l
3.7 ± 0 1
2.9 ± 1.6
1.2 ± 0.5
" Values are expressed as means ± SE of results of three experiments.
each containing triplicate values
100
a 80
s? 20
PROSTATE
COMPETITOR
Figure 5. Ability of various hormones to compete with [3H] DHT for its
binding sites in prostate cytosol from castrate mice. Incubations of pro-
state cytosol, prepared from animals 3 days postcastration with 5 x 10~9M
[3H] DHT either alone or with competitor were carried out for 12 hr at 0°C.
Percentage total [3n] DHT bound was determined by comparing the amount of
[ H] DHT bound in the presence of the competitor to the amount bound in the
absence of the competitor.
21
-------
Having extablished the specificity of the prostate cytosol binding compon-
ents for 3t[_E)HT, another series of experiments was conducted in order to examine
the ability of certain pesr Lcic-ie to parturb the ir.caraction of DHT with its
cytosolic binding components (Fig. 6) . Parathion (10~° - 10~^M) was observed to
be an effective inhibitor of -'H-TjHT binding in the proscate cytosoL (Table 7).
Comparable cone-ant rations of c-r'oaryl and the o- ^r.noehlorirvas, DBT an-.: aijldrin,
had no effect of -^H-DHT binding in the cytosol fraction of the mouse prostate.
These findings of the lack of effect of the organochlcrines on incus? prostate
cytosol binding of JH-DHT are in contrast to tnose in the rat wherein either DDT
or dieldrin inhibited 3H-DHT binding (Wakeling and Visek, 1973). Although not
shown, -^H-DKT binding in the seminal vesicle, kidney and liver was similarly
affected by the various pesticides investigated. Again, parathion was a potent
inhibitor of %-DHT binding in the cytosols of these tissues, whereas carbaryl,
DDT, and dieldrin had no effect.
Q
In order to determine whether parathion's inhibitory effects on H-DHT
binding were specific for organs known to be influenced by androgens (e.g.
sex accessory organs, liver and kidney), a series of studies were undertaken
using cytosol prepared from the srr.all intestine of the mouse (Fig. 7). Although
%-DHT can be bound to cytosol components of the intestine, such binding is
totally non-specific (Schein and Donovan, urjniVLished). None of the pesticides
studied caused inhibition of 3H-DHT binding in the cytosol of the small intestine
(Fig. 7). It is evident, therefore, that among the tissues utilized in these
investigations parathion's inhibitory effect, on -'H-DHT binding resides prin-
cipally in those that are influenced by male sex steroids, and not in a non-
target tissue such as the small intestine.
22
-------
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23
-------
MOLAR
CONCENTRATION
r5 H IO'7
• 10-8
Die-
o
oo
100
80
I
i 60
ro
O 40
20
0
DHT
PARATHION CARBARYL
DDT
DIELDRIN
Figure 6. Percent of total ^H-DHT bound in vitro to cytosol of the mouse
anterior prostate gland in the presence of various concentrations of different
pesticides. Each value represents the mean of at least 10 observations. Cyto-
sols prepared from tissues taken from animals 3 days post-castration were in-
cubated with 10~9\i 3jj_DHT and various concentrations of pesticides for 12 hours
at 0°C.
24
-------
100
Q
1 80
CD
I
? 60
o
0 40
3?
20
0
•
I
1
1
MOLAR
CONCENTRATION
v-7
D'°~5
^ IO'6
10"
ID" 8
DHT
RftRATHION CARBARYL
DDT DIELDRIN
Figure 7. Percent total H-DHT bound in vitro to cytosol of the mouse intes-
tine in the presence of various concentrations of different pesticides. Each
value represents the mean of at least 10 observations. Cytosols prepared from
tissues taken from animals 3 days post-castration were incubated with 10~'M
and various concentrations of pesticides for 12 hours at 0°C.
25
-------
SECTION 6
DISCUSSION
The effects of various pesticides on hepatic steroid metabolism have
been previously reported (Conney e_t _al_. , 1957; Welch et_ al. , 1967; DuBois,
1969). In the liver, the affects of organochlorine pesticides seem to be
mediated through stimulation of the microsomal hydroxylating enzyme systems
(Hart and Fouts, 1965; Kuntzmann _e_t al_. , 1966; Murphy, 1969). In the pro-
state gland, pesticides have been shown to alter the uptake and metabolism
of radiolabeled testosterone (Schein and Thomas, 1975), to interfere with
the binding of androgen metabolites to proteins (Wakeling and Visek, 1973)
and to change the composition of prostatic secretions (Blend and Visek, 1972).
Early studies showed that the toxicity of various pesticides could be
either increased or decreased by the previous or simultaneous exposure to
other agents (Murphy 1969; DuBois 1969). These interactions among pesticides
seemed to occur as a result of differential effects on oxidative enzyme
activities (Conney and Burns, 1972). Resultant changes occurred in residue
accumulation (Street and Blau, 1966), detoxication (Murphy, 1969) and hormonal
balance (Street e_t_ al_. , 1969). Such changes, resulting from pesticide inter-
actions might be responsible for altered sexual function (impotence) in farm
workers exposed to pesticides (Espir et_ al. , 1970) .
Recently, the administration of dieldrin and parathion was shown to pro-
duce effects on testosterone metabolism different from those produced by either
agent alone. Such effects occurred in liver and in androgen-dependent organs
(Schein and Thomas, 1976). Likewise, the present studies demonstrate some
differential effects of these two pesticides on androgen metabolism, but using
dieldrin doses twice as large as previously administered.
In the mouse prostate gland, parathion treatment followed by administra-
tion of dieldrin stimulated the production of nonpolar metabolites of -^H-tes-
tosterone in vitro (Schein and Thomas, 1976). In the present study, using
twice the dose of dieldrin previously reported (Schein and Thomas, 1976), the
effect upon nonpolar metabolites was absent and had presumably been abolished
(Table 2). Unlike the previous study using lower doses of dieldrin (Schein
and Thomas, 1976), the formation of metabolites of ^H-T was not significantly
different from that of the controls.
Similar to the response in the prostate gland (Table 2) , there were changes
in androgen metabolism in hepatic microsomes (Table 3). Previous studies showed
that parathion alone stimulated the production of ^H-androstanediol (Thomas and
Schein, 1974) while dieldrin alone reduced the formation of 3H-aridrostenedione
(Schein and Thomas, 1975). When both pesticides were administered simultane-
ously, both effects were observed; formation of ^H-androstanediol was enhanced
26
-------
and of %-androstenedione was reduced (Schein and Thomas, 1976). Table 3
shows, however, that when the dose of dieldrin was doubled, both the stimula-
tion of ^H-androstanediol and the inhibition of -^H-androstenedione were abolished.
Parathion is an inhibitor of hepatic nic/o = o^.al steroid hydroxylases
(Kuntzman et al., 1966) while dieldrin induces these activities (Welch et al.,
1971). When these two pesticides were administered simultaneously for 10 days
or when dieldrin was administered for 5 days followed by 5 days of treatment
with parathion, no significant changes were recorded in androgen hydroxylations
(Schein and Thomas, 1976). The present findings extend these earlier studies
since higher doses of dieldrin resulted in further stimulation of androgen
hydroxylases (Table 3).
The present studies reveal that different pesticides can interact in
mammalian systems to produce effects different from those produced by a single
pesticide. The mechanism of interaction may be similar with different doses
or dose regimens. This seems apparent in the response of hepatic androgen
hydroxylases to various doses of dieldrin and parathion. Increasing doses of
pesticides result in increasing stimulation of enzyme activities. It is pos-
sible, however, that pesticide interactions change eqalitatively with different
dose regimens. This was made evident by the effects of these same pesticides
on nonpolar metabolites of testosterone in liver and prostate. The effects of
lower doses of pesticides on these activities disappear when higher doses are
administered. Such pesticide interactions should be considered when evaluat-
ing environmental and toxicological impacts. The effects shown here on hepatic
androgen metabolism have the potential to alter the hormonal balance. In ad-
dition, the effects on the prostate gland androgen metabolism could represent a
direct action upon hormone-dependent organs. The importance of pesticide in-
teractions is emphasized by the widespread agricultural use of many different
types of these agents.
Organochlorines have high affinity for the cytochrome P-450 protein (Table
5) as well as high lipid solubility. This could easily lead to their accumula-
tion in cellular membranes and produce concentrations sufficient to inhibit
steroid hydroxylations (Table 4). Likewise.organophosphates bind with high
affinity to liver microsomes, and at high concentrations inhibit testosterone
hydroxylases. Mice seem especially sensitive to diazinon with a low Ks value
and significant enzyme inhibition at 10~8ji pesticide. In addition, methoxychlor
and diazinon both appear to physically disrupt membranes at higher concentrations.
Carbofuran appears to have a different sort of toxic liability. It inter-
acts only minimally with hepatic cytochrome P-450. but by some mechanism it
appears to stimulate microsomal hydroxylations reactions.
Direct effects on prostate metabolism did not appear to be very important
in this study. It is always possible that these pesticides did not adequately
permeate the tissue and were not present in sufficient amounts at potential
sites of action.
27
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Another objective in this study was to determine the optimum conditions
for [%] DHT cytoplasmic binding in various tissues of the mouse. It was
decided that the best postcastration interval was 3 days. At this time the
total binding capacity of the prostate gland is at a maximum, but tissue
regression is minimal. It is essential that both preparation and incubation
with labeled steroid be carried out at 0 to 4°C. The high-affinity, hormone-
specific component of binding is extrsmaly heat-labile and is destroyed at
10°C ; but the amount of the low-affinity, nonspecific component was increased
by higher temperatures. The heat lability of the specific binding compote. ic
in the present studies was typical of androgen binding in various species
(Mainwaring and Morgan, 1973). The optimum total concentration of protein in
the incubation mixture is about 2 mg/ml . At protein concentrations greater
than 3 mg/ml [ H] DHT binding was no longer a linear function of protein con-
centration. A 12-hr period of incubation is necessary in order to reach
equilibrium between free and bound hormone.
The slow kinetics of clearance of endogenous androgen after castration
and of approach to [%] DHT binding equilibrium in vitjro in the mouse are
unusual. In the rat (Robinette and Mawhinney, 1978) and guinea pig (Belis
e_t al_. , 1978), both of these processes are much more rapid. For example, rat
prostates obtained 24 hr after castration and incubated for just 4 hr with
steroid yielded maximum binding of androgen to cytosol fractions (Robinette
and Mawhinney, 1978). In the present studies, it is not clear whether both
high and low affinity components of [^H] DHT binding exhibit this slow reac-
tion rate nor is it clear just what structural of functional differences
in the mouse prostate account for this slow clearance rate of endogenous
androgens following castration.
Still another purpose was to examine the manner in which [-*H] DHT was
bound in the cytosols prepared from the various tissues of the mouse. Two
types of binding were found: (1) a heat-stable, high capacity type with lew
affinity, and (2) a heat-labile type with high affinity (Ka 1.5 x 10^ liter/
mol) and low capacity. This two-component androgen binding is commonly found
in cytosols of mammalian cells (Wilson and French, 1976; Aakvaag et al_. , 1972).
Table 6 shows that the affinity constants of the high affinity [^H] DHT
binding component are similar in all tissues studied. On the other hand, the
capacity of this binding component varies considerably among the tissues
studied, being greatest in andro gen-dependent organs. These findings are con-
sistent with the distribution of binding activities among tissues of other
animals (Mainwaring and Morgan, 1973).
The high-affinity type of binding present in most tissues was very specific
with respect to competitors. At 10~^M (ij_e.. , at ten times the concentration
[3H] DHT) , either DHT or testosterone significantly reduced binding of [3H] DHT
to this binding component. Cyproterone acetate, estradiol, or progesterone
competed with [%] DHT for the high affinity binding sites at concentrations
100- to 1000-fold greater than the [3H] DHT concentration. Estriol, esterone,
or corticosterone did not reduce [-^H] DHT binding at any concentration studied.
28
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The high affinity component of [%] DHT binding in mouse tissues behaves
in the manner expected of a cytoplasmic androgen receptor. Its affinity for
androgens is high. It is present in cells of tissues dependent on androgens
for normal function and is also found in tissues responsive to but not dependent
on androgens. Binding to this high affinlf.-/ c^-.pcn:nt is specific for androgens
and is inhibited by anciandrogens such as cyproterone acetate, progesterone,
and estradiol.
These investigations have demonstrated a relatively simple method for
studying the in vitro effects of various compounds on the interaction of
DHT with its specific binding proteins in cytosol preparations of various
tissues of the mouse. These methods can be useful to determine whether or not
exogenous compounds (e.g., steroids, pesticides, etc.) have the ability to
interfere with androgen-receptor dynamics at the cellular level.
Previous studies in our laboratory have shown that parathion has little
or no direct effect upon -^H-testosterone assimilation and/or metabolism by the
mouse prostate gland if administered orally for a period of 5 days (Thomas
and Schein, 1974). In the present studies, using an in vitro cytosol system,
this pesticide was particularly effective in interfering with androgen binding.
Parathion was a potent inhibitor of ^H-DHT binding in the cytosol of several
tissues including the anterior prostate gland, seminal vesicle, kidney and
liver (Table 7). All of these tissues are subject to androgenic stimulation,
but vary in their magnitude of response. On the other hand, the intestine
does not appear to be stimulated by male sex hormones. Correspondingly, para-
thion had little effect on -'ll-Dttl binding to cytosol components from this organ.
Although the organochlorine pesticides DDT and dieldrin have been shown
to interfere with the assimilation and metabolism of %-testosterone in mouse
sex accessory organs (Thomas and Lloyd, 1973; Thomas e_t_ al_., 1973), neither of
these compounds altered the binding of H-DHT to cytosol binding components
from any of the tissues studied in this series of experiments. Cytosol binding
studies with the rat prostate (Wakeling and Visek, 1973), but not with the
mouse prostate (Fig. 6), reveal that either dieldrin or DDT can inhibit binding
of ^H-DHT to cytosol binding components. The differences seen in these two
studies might be due to species differences or technique differences. Wakeling
and Visek (1973) used sedimentation gradient techniques to study the binding of
%-DHT. These studies used Dextran-coated charcoal method to isolate the bound
steroid. Also, the constituents of their incubation buffer system (N-2-
hydroxyethylpiperazine-N1-2-ethanesulfonic acid [pH 7.4], containing 1.5 mM
EDTA, 2.0 mM mercaptoethanol and 0.4 M KC1) differed from that used in these
investigations. Furthermore, their in vitro incubation procedure included
freezing the incubate overnight. These studies did not allow samples to freeze.
Finally, the DDT used in these investigations was not analysed to determine the
amount of the o,p'-DDT isomer present. This isomer has been found to be much
more potent as an estrogen that the p,p'-DDT isomer (Welch ej^ al_. , 1969).
Therefore, o,p'-DDT should be a more potent antiandrogen than the p,p'-DDT isomer.
The lack of effect in this series of experiments may be due to a relatively
large percent of p,p'-DDT present.
29
-------
In the present studies, carbaryl was found to have little effect on
-%-DHT binding to cytosol binding components of the various tissues studied..
This finding was not unexpected, since previous studies have shown that this
pesticide had little effect on -%-testosterone uptake and metabolism in the
various androgen dependent tissues (Dieringer and Thomas, 1974; Thomas et_ aL^. ,
1974). As a chemical class, the cnrbamates seer: to be unable to produce changes
in the reproductive syscem (Guthrie et_ al_. , 1971).
C;ruiey £j^ ££. (197'+) and Kunczinan et_ al_. (1966) have suggested that organo-
chlorine and organophosphate pesticides, respectively, can perturb endocrine
responses in mammals by induction of liver nicrosomal steroid hydroxylase
enzymes. On the basis of the results presented in this report, an alternative
mechanism for modifying hormonal action in ~nale reproductive organs may be
through the more direct inhibition of DHT binding to target tissue cytosol
binding proteins.
In summary, the results of this investigation reveal that parathicn is a
potent inhibitor of JH-DHT binding to cytosol binding components present in
androgen sensitive tissues. It would be of interest to examine the effects of
paraoxon (the active metabolite of parathion) on -%-DHT binding in these tissues,
30
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26. Kuntzman, R., Welch, R., and Conney, A.H. (1966). Factors influencing
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32
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27. Levin, W., Welch, R.M. and Conney, A.M. (1969). Inhibitory effect of
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29. Mainwaring, W.I.P., and Morgan, F.R. (1973). A study of the androgen
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30. Murphy, S.D. (1969). Mechanisms of pesticide interactions in vertebrates.
Residue Rev. 25, 201-221.
31. Naess, 0., Hansson, V., Djocaeland, 0., and Attramadal, A. (1975).
Characterization of the androgen receptor in the anterior pituitary of the
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32. Nelson, J.A. (1974). Effects of dichlorodiphenyltrichloroethane (DDT)
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34. Peakall, D.B. (1967). Pesticide induced enzyme breakdown of steroids in
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35. Richardson, H.L., Stier, A.R., and Borsos-Nachtnebel, E. (1952). Liver
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36. Robinette, L., and Mawhinney, M.G. (1978). Androgen receptors in rat sex
accessory organs. Arch. Biochem. Biophys. (in press).
37. Roy, A.K., Milin, B.S., and McMinn, D.M. (1974). Androgen receptor in rat
liver: Hormonal and developmental regulation of the cytoplasmic receptor
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38. Scatchard, A., (1949). Molecular interaction. Ann. N.Y. Acad. Sci. 51,
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39. Schein, L.G., and Thomas, J.A. (1975). Effects of dieldrin on the uptake
and metabolism of testosterone-1,2-^R by rodent sex accessory organs.
Environ. Res. 9, 26-31.
40. Schein, L.G., and Thomas, J.A. (1976). Dieldrin and parathion interaction
in the prostate and liver of the mouse. J. Toxicol. Environ. Health 1,
829-838.
33
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41. Schein, L.G., Donovan, M.P. and Thomas, J.A. (1973). Effects of Pesticides
on H-dihydrotestosterone Binding to Cytosol Proteins from Various Tissues
of the Xo..se. Toxl^l. Appl, Pliarnacol., i:> press.
42. Schein, 1., T>?ncva:i, M.P. :md Tr_-?s, .T.A. 'I0;7,?) . Charactarizat icn of
cytoplasmic binding of dihydrotestosterone by the prostate gland and
other tissues of the rr.ouse. Toxicol . Applied. Pharmacol. in p re 3 s.
43. Smith, M.T., Thomas, J.A., Smith, C.G., Mawhinney, M.G., and Lloyd, J.W.
(1972). Effects of "CDT on radioactive uptake from testcsterone-1,2-3H by
mouse prostate glands. Toxicol. Appl. Pharmacol. 23, 159-164.
44. Stevens, J.T., McPhillips, J.J., and Stitzel, R.E. (1971). The effect of
organophosphate pesticides on h/pa^ic drug metabolism in the mouse (abstr.)
Pharmacologist 13, 289.
45. Stevens, J.T., Stitzel, R.E., and McPhillips, J.J. (1972). The effects of
subacute administration of anticholinesterase insecticides on hepatic
microsomal metabolism. Life Sci. 11, 423-431.
46. Street, J.C., and Blau, A.I). (1966). Insecticide interaction affecting
residue accumulation in animal tissues. Toxicol. Appl. Pharmacol. 8, 497-
504.
47. Street, J.C., Mayer, F.L., and Waystaff, D.J. (1969). Ecological signifi-
cance of pesticide interactions. Ind. Med. Surg. 38, 409-414.
48. Swann, H.E., Woodson, G.S., and Ballard, T.H. (1958). The acute toxicity
of intramuscular parathion in rats and the relation of weight, sex and sex
hormones to this toxicity. Amer. Ind. Hy
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54. Wakeling, A.E., and Visek, W.J. (1973). Insecticide inhibition of 5a-
dihydrotestosterone binding the rat ventral prostate. Science 181, 659-661,
55. Welch, R.M., Levin, W., and Conney, A.H. (1967). Insecticide inhibition
and stimuls-ion of steroid hydroxylases in rat liver. J. Pharmacol.
Exp. Ther. 155, 167-173.
56. Welch, R.M., Levin, W. and Conney, A.H. (1969). Estrogenic action of DDT
and its analogs. Toxicol. Appl. Pharmacol. ,14_, 358-467.
57. Welch, R.M., Levin, W., Kuntzman, R., Jacobson, M. , and Conney, A.H. (1971),
Effects of halogenated hydrocarbon insecticides on the metabolism and
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19, 234-246.
58. Wilson, E.M., and French, F.S. (1976). Binding properties of androgen
receptors: Evidence for identical receptors in rat testis, epididymis,
and prostate. -J. Biol. Chem. 254, 5620-5629.
35
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PUBLICATION RESULTING FRCIJ THIS EPA GRANT
1. Parathion-Dieldrin Interactions on Androgen Metabolism in the Mouse. L.G.
Schein and J.A. Thomas, Proc. Society Toxicology, 14th Annual Meeting, p. 71,
March, 1975 (Abstract).
2. Effects of Dieldrin on the Uptake and Metabolism of Testosterone-1,2-^H by
Rodent Sex Accessory Organs. L.G. Schein and J.A. Thomas. Environmental
Research, 9_: 25, 1975.
3. Interaction of Carbaryl and Dieldrin on the Metabolism of -^[{-Testosterone.
L.G. Schein, J.A. Thonas, P. A. Klase and W.D. Edwards. Prcc. 15th Annual
Toxicology Society, p. 149, 1976 (Abstract).
4. Combined Effects of Parathion and Carbaryl on the Biotransformat ion of ^H-
Testosterone in the Mala Mouse. J.A. Thonas and L.G. Schein. Proc. 15th
Annual Toxicology Society, p. 150, 1976 (Abstract).
5. Dieldrin and Parathion Interaction in the Proscate and Liver of the Mouse.
L.G. Schein and J.A. Thonas, Journal Toxicology and Environmental Health
1_: 829, 1976.
6. Some Action of Parathion and/or Dieldrin on Androgen Metabolism. J.A. Thomas,
L.G. Schein, and M.P. Donovan, Environmental Research 1_3_: 441, 1977.
7. Perturbation by Organochlorine Pesticides of %-testostercne Metabolism in
Rodent Hepatic Microsomes and Prostate Gland In Vitro. M.P. Donovan, J.A.
Thomas, L.G. Schein, and P.A. Klase. The Pharmacologist 1_8: 234, 1976
(Abstract) .
8. Effects of Diazinon, Parathion and Carbaryl on the In Vitro Metabolism of
^H-testosterone by Rodent Prostate Glands and Hepatic Microsomal Enzymes.
L.G. Schein, J.A. Thomas, M.P. Donovan and P.A. Klase. The Pharmacologist
18_: 243, 1976 (Abstract).
9. Effects of Pesticides on Steroid Hormone Binding in Cytoplasm of Rodent
Tissues. L.G. Schein, J.A. Thomas and M.P. Donovan. Society of Toxicology
Proceedings, p 95, March, 1977 (Abstract).
10. A Biochemical Basis for Insecticide-induced Changes in the Male Reproductive
System. J.A. Thomas, M.P. Donovan and L.G. Schein, 17th Annual Proceedings
of Society of Toxicology, 145: 1978 (Abstract).
36
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11. Characterization of Cytoplasm Binding of Dihydrotestosterone by the Pro-
state Gland and Other Tissues of th.; Mouse. L.G. Schein, M.P. Donovan,
and J.A. Thomas, Toxicol. Appl. Pharmacol. 1978 (in press).
12. Effects of Pesticides on %-dihydrotestosterone Binding to Cytosol Protein
from Various Tissues of the MOM:". L.G. Schein, M.P. Donovan, and J.A.
Thomas. Toxicol. Appl. Pharmacol. 1978 (in pr^ss).
37
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GLOSSARY
Carbaryl: (1-Naphthyl-N-methyl carbamate)
Carbofuran: (2,3-dihydro-2,2-dimethyl-benzofuran-7-yl N-methylcarbamate)
DDT: (l,l-bis[p-chlorophenyl]-2,2,2-trichloroethane)
DHT: (dihydrotestosterone)
Diazinon: (diethyl 2-isoprophyl-6-methyl-4-pyrimidinyl phosphorothionate)
Dieldrin: (1,2 ,3,4,LO,10-hexachloro-6,7-epoxy-l,4,4a,6,7,3,8a-octahydro~
endo-exo-1,4:5,3-dimethanonaphthalene)
Heptachlor: (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro~4,7-metharioindene)
Methoxychlor : (1,1,l-trichloro-2,2,-bis-4~(methoxyphenyl) ethane)
Parathion: (0 ,0-diethyl-0-[p-nitrophenylJester phosphorothioic acid
38
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-044
2.
4. TITLE ANDSUBTITLE
EFFECT OF PESTICIDE INTERACTIONS UPON THE
REPRODUCTIVE SYSTEM
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
.limp 1Q7«
7 AUTHOR(S)
John A. Thomas
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Pharmacology
West Virginia University Medical Center
Morgantown, W. VA 26506
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R 803578
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Trianplp Park. NC. 77711
13. TYPE OF REPORT AND PERIOD COVERED
RTP,NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16, ABSTRACT
The metabolism of 1, 2--'H-testosterone in vitro was studied in prostate glands
and livers of rats and mice treated with different pesticides including dieldrin and
parathion. The metabolism of 1,2- H-testosterone (T-%) in vitro by mouse anterior
prostate glands or hepatic microsomes has been studied after the oral administration
of dieldrin (2.5 mg/kg daily x 5 or 10) and/or parathion (1.3,2.6, or 5.2 mg/kg
daily x 5 or 10). T-^H metabolism in the prostate was unaffected by the various
treatment regimens. Dieldrin (10 days) caused some reduction in the microsomal
production of androstenedione-% or dihydrotestosterone-^H. Only treatment regimes
with dieldrin stimulated hepatic testosterone hydroxylases; parathion alone had no
effect. This study revealed that dieldrin and parathion can interact and produce
biological effects different from those caused by either pesticide alone.
Liver microsomal steroid hydroxylating enzymes and prostatic testosterone-5 -
reductase were studied in rat and mouse. Organochlorine and organophosphate slightly
stimulated them. Neither species was consistently more sensitive to pesticide
effects than the other. All the pesticides (viz, heptachlor, carbofuran, diazinon
and parathion) bound to cytochrome P-450, producing type I spectral charges. Values
of Ks ranged from 1.9 to 8.7mM for organochlorine and organophosphate compounds.
Affinity for carbofuran was much lower (Ks=100-200mM).
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
pesticides
metabolism
reproductive system
dieldrin
parathion
06 T, A
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
49
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
39
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