EPA-660/2 73-027
May 1974
Environmental Protection Technology Series
A Tissue Enzyme Assay
for Chlorinated
Hydrocarbon Insecticides
•o
3D
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1
JJ
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Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
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2. Environmental Protection Technology
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This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
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-------
EPA-660/2-73-027
May 1971*
A TISSUE ENZYME ASSAY FOR
CHLORINATED HYDROCARBON INSECTICIDES
By
Laurence K. Cutkomj)
University of Minnesota
St. Paul, Minnesota 55101
Project 16030 ELZ
Program Element 1BA027
Project Officer
H. Page Nicholson
Southeast Environmental Research Laboratory
Environmental Protection Agency
Athens, Georgia 30601
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20U60
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.0.20402 - Price 85 cents
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ABSTRACT
Certain chlorinated hydrocarbon insecticides, especially DDT and
closely related chemicals, tested at low concentrations, adversely
affect the ATPase enzyme system. DDT inhibited oligomycin-sensitive
2+
Mg ATPase (mitochondrial) both in vitro and in vivo. About 1 uM
(1 x 10"6 M) gave 5C# inhibition in fish brain and 0.5 ppb of DDT in
water inhibited about 5C# of mitochondrial Mg ATPase. Na+-K+ATPase
was not inhibited in brain, but was inhibited in vivo in fish gills.
Certain discriminating effects were found among chlorinated hydrocarbons,
2+
particularly with respect to inhibition of Mg ATPase, but the ranking
of compounds by enzymic effects does not always parallel toxicity
values. Organophosphate and carbamate insecticides were ineffective.
Further research is needed both in vitro and in vivo to determine how
the adverse effects on the enzymes relate to practical interpretations
of effects. The abnormally low ATPase activity in chronically treated
fish is the first report of an adverse biochemical effect with sublethal
doese of DDT. All effects appear to be primarily within the group of
insecticides and'acaricides which are persistent in parts of the environ-
ment and in organisms.
-------
CONTENTS
SECTION PAGE
I. Conclusions 1
II. Recommendations 3
III. Introduction 5
IV. Experimental 7
V. Results 9
VI. Discussion 13
VII. References . 33
VIII. Publications 37
IX, Glossary 39
iii
-------
FIGURES
PAGE
2+
Figure 1. In vitro inhibition of Mg ATPase from cockroach muscle
homogenates treated with DDT 23
2+
Figure 2. In vitro inhibition of Mg ATPase from fish brain
homogenates treated with DDT 2*t
Figure 3« In vitro inhibition of mitochondrial Mg ATPase from
fish brain homogenates with DDT, 2 analogs of DDT, and
DDE, the principal metabolite 25
2+
Figure k. In vitro inhibition of Mg ATPase from fish brain
homogenates treated with dicofol (Kelthane) 26
2+
Figure 5. In vitro inhibition of Mg ATPase from fish brain
homogenates treated with tetradifon (Tedion) 2?
2+
Figure 6. In vitro inhibition of Mg ATPase from fish brain
homogenates treated with dichlorodiphenylethanol
(DMC or Dimite) and chlorobenzilate 28
2+
Figure 7» In vitro inhibition of mitochondrial Mg ATPase from
fish brain homogenates treated with ovex, Genite and
chlorbenside (Mitox) 2°
Figure 8. Structural formulas of chlorinated hydrocarbon
acaricides tested on ATPase system of blue gill fish 30
2+
Figure 9» In vitro inhibition of mitochondrial Mg ATPase from
brain homogenates of fish and homogenates of two-
spotted spider mites 3L
iv
-------
-TABLES
Table 1. In vitro inhibition of ,fish brain mitochondria!
Mg ATPase by DDT and some analogues
Table 2. Inhibition of ATPases by DDT using k different
tissues from blue gill fish
Table J>. Inhibition of ATPases from fish brain by DDT and
several cyclodiene insecticides
Table k. Reduction of mitochondria! Mg +ATPase of brain
from fish continuously exposed to DDT
Table 5- Stimulation (increase) in Na -K ATPase of brain
from fish continuously exposed to DDT
Table 6. Stimulation (increase) of oligomycin-insensitive'
2+
Mg ATPase of brain from fish continuously exposed
to DDT
Table 7« Per cent changes in ATPases of gill tissue from
fish continuously exposed to DDT
PAGE
16
18
19
22
22
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ACKNOWLEDGEMENTS
We wish to acknowledge the research facilities of the University of
Minnesota., Dept. of Entomology, Fisheries and Wildlife, St. Paul, Minne-
sota. In particular, we are indebted to Dr. Robert B. Koch, Honeywell
Research Center, Bloomington, Minnesota, for his valuable active consult-
ant ship work. Research Fellows were Dr. Han Heng Yap and Dr. D. Desaiah.
Graduate Research Assistants were Dr. Elymar V. Vea and Mr. E.Y. Cheng
and initially graduate students F. M. Do and Y. C. Chu. Laboratory
assistance was carried out by Misses Jennifer Mather and Jean Jahr and
Mr. Thomas Lorsbach.
vi
-------
SECTION I
CONCLUSIONS
DDT and related insecticides and acaricides cause a significant
inhibition or depression of mitochondrial Mg ATPase from fish brain
when tested in vitro or when fish are continuously exposed to % to 2
ppb of DDT. ATPases from fish gills of treated fish, in contrast,
showed some inhibition of Na -K ATPase and also Mg ATPases. Organo-
phosphate and carbamate insecticides do not produce an inhibition of
the ATPases. Therefore the enzyme sensitivity to most chlorinated hydro-
carbons, particularly DDT and PCB compounds is a discriminating charac-
teristic and may be useful in identifying the causative agents and in
giving an indication of exposure time in contaminated water. These
studies are the first to show a significant quantitative reduction of
enzyme activity in any vertebrate exposed chronically to DDT.
-------
SECTION II
RECOMMENDATIONS
It is recommended that chronic toxicity studies on fish be made with
other persistent materials, notably PCBs and mercury, to relate any
adverse effects in the ATPase system to the residue burden in selected
tissues, e.g., brain and gills. Some additional experiments should
include detectable, but less persistent, pesticides. Of great impor-
tance is the need to determine whether the abnormal ATPase enzymic con-
dition is a critical factor in survival of young fish or whether the
residue burden is the most important, or whether the combination may
be a limiting factor in reproduction.
-------
SECTION III
INTRODUCTION
The ATPase enzyme system from several animal species had been shown to
be sensitive (usually inhibited) to chlorinated hydrocarbon insecticides.
However, there were many unknown aspects. The plan of study was to
examine homogenates from numerous tissues, brain, muscle, liver and
kidney - possibly gills - and determine their relative usefulness for
determining the effect on the enzyme system. The ATPases could be
obtained from any of the tissues but selective sensitivity was not known.
Furthermore, a number of chemically related pesticides needed to be tested
for-their effectiveness, along with unrelated components to determine
discriminatory effects on ATPases. The ATPase system itself required
experimentation into the important components, Na -K ATPase, and two
2+
forms of Mg ATPase.
The above considerations required study by in vitro techniques primarily,
i.e. with tissue homogenates centrifuged at a speed (13,000 g) which
retained most of the mitochondria and nerve endings. A second phase of
the study was to relate the enzyme assay to insecticide toxicity. Both
acute and chronic toxicity needed to be considered. Some of the compari-
sons were made on cockroaches which could be used in greater numbers
than fishes. There were established insecticide doses with both species.
Many similarities were evident when comparing responses of ATPases in
fish and insects.
-------
Objectives
To determine whether an enzyme assay, using the ATPases, can be used
as a detecting system in fish or related organisms for insecticide-
polluted water.
To utilize ATPase enzymes from fish tissues as sensitive detectors of
contamination due to chlorinated hydrocarbon insecticides and ralafcfcd
compounds.
To determine the most suitable fish tissues for analyzing inhibition.
of ATPases by chlorinated hydrocarbons.
To determine the usefulness of selectivity of pesticide effects on
components of the ATPase system.
To determine whether in vitro and in vivo effects on ATPase can be
related to overall toxicity to fish.
-------
SECTION IV
EXPERIMENTAL
MATERIALS AND METHODS
The enzyme source for in vitro studies and in vivo was brain tissue
from blue gill fish, Lepomis machrochirus. For a study of chronic effects
of DDT brain and gill tissues were used from the fat head minnow,
Pimephales promelas. The tissue was dissected and homogenized under
iced conditions using 0.32 M sucrose, 1 mM EDTA and 10 mM imidazole.
The homogenate fraction (B) used was the sediment obtained by centrifuging
(n p "5 4 5)
at 13,000 x g for 20 minutes, and prepared as described ' ' ' ' .
ATPase activities were determined using a continuous enzymatic procedure
essentially described by Pullman et al. and Fritz and Hamrick ' and
/Q\
reported by Yap and Cutkomp . Protein determinations were by the
(9)
Lowry method .
Total ATPase activity was measured with Mg , Na , K in the reaction
mixture. Mg ATPase activity was measured when ImM ouabain was in the
mixture. Ouabain is a specific inhibitor of Na+-K+ATPase(l° . Na+-K+
ATPase activity is total activity minus the Mg ATPase activity.
Mg ATPase activity was further delineated by adding one ul of oligomycin
in ethanol (0.03 Mg per ml reaction mixture); the oligomycin-sensitive
portion is designated mitochondrial Mg ATPase activity in this study.
The oligomycin contained approximately 15$ oligomycin A and 85$ oligo-
mycin B as obtained from Sigma Chem. Co., St. Louis, Missouri.
A 3 ml reaction mixture contained: 4.3 mM ATP, 5 mM Mg++, 100 mM Na+,
20 mM K+ (all as chlorides), 'l20 mM imidazole buffer pH 7.5, 0.19 mM
NADH 0.5 mM PEP (phosphoenol pyruvate), 0.02$ BSA (bovine serum albumin),
approximately 9 units pyruvate kihase., and. 12 units lactic dehydrogenase,
and 100 pi homogenate fraction. Absorbance changes were measured at
3^0 nm using a Beckman DU spectrophotometer with temperature controlled
-------
for 37 in the reaction mixture. This temperature was used to insure
comparable results with earlier extensive studies with mammals (rats
and rabbits).
Analytical grades of insecticides and designated PCBs (Arodors) were
used. Each insecticide was dissolved in ethanol and 1 to 5 1*1 were
added to a rapidly stirred reaction mixture using a Hamilton micro-
syringe. Ethanol had no effect at the amounts used.
The dosage-response relationships suited to statistical treatment were
analyzed according to Finney's probit analysis ' programmed follow-
ing Daum and calculated on an electronic computer. The regression
lines and 50$ inhibitory values (Icn) are given in each figure.
y\J
The blue gill fish used in the study were collected as young in an
isolated lake believed to be free of any possible pesticide contamination.
They were maintained in deionized water for several weeks before ex-
perimental use. Fat head minnows used for pesticide treatments were
reared throughout their lifetime in uncontaminated water at the Duluth
Water Quality Laboratory, Environmental Protection Agency.
8
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SECTION V
RESULTS
The research has clarified numerous points regarding the sensitivity
of the ATPases by chlorinated hydrocarbon pesticides and polychlorinated
biphenyl compounds. Twelve research publications give this information
in great detail.
The major points determined are as follows, with the first eight points
referring to in vitro studies:
~^~ ~~™~"~~ ?4- 2-f-
(l) The oligomycin-sensitive Mg ATPase (known as mitochondrial Mg ATPase)
is the most sensitive of the ATPase systems to DDT and closely related
compounds , followed by Na -K ATPase and oligomycin- insensitive
Mg +ATPase . Fig. 1 shows DDT effects on Mg +ATPases in cockroach
muscle and Fig. 2 the effects in blue gill fish brain. Fig. 3 gives a
comparison of 2 analogs and DDE, the chief metabolite of DDT. Table 1
also shows DDT to be more effective than TDE, Perthane and methoxychlor
and from 7 to 8x as effective as DDE. Dicofol (Kelthane) is somewhat
exceptional and Fig. *f shows the effect resulting from this acaricide
which is closely related to DDT.
(2) The inhibition of ATPases occurs in all tissues studied, including
brain, muscle, liver, kidney and gill, but generally is somewhat greater
(~\ .
in muscle v ' ' ' ' . Fig. 2 shows the DDT inhibition of fish
brain Mg ATPase to be "about 8x more DDT than within muscle (Fig. l).
However, consistency of inhibition is somewhat greater in brain homo-
genates.
A tabular comparison of the inhibition of ATPases by DDT using dif-
ferent tissues is given in Table 2. It will be noted, not only that
2+ 2+
Mg ATPase from muscle is affected more prominently, but that Mg ATPase
is inhibited to a greater extent than Na+-K+ATPase . Comparisons of this
enzyme were possible in brain and kidney.
(3) The toxicity to fish by DDT and closely related chemicals show a
certain parallelism to ATPase enzyme inhibition; however, several
9
-------
acaricides (miticides) such as chlorobenzilate , tetradifon and other
chlorinated bridged biphenyl compounds also are good inhibitors of
mitochondrial Mg +ATPase and relatively ineffective on Na+-K+ATPase ''
Fig. 5 gives the results with tetradifon and Figs. 6 and 7 with 5 addi-
tional compounds having chemical structures given in Fig. 8. Tejbradifon
was highly effective, inhibiting 5C$ of mitochondrial Mg +ATPase at
o
4.9 x 10 M concentration.
The inhibition of ATPases by chlordane-type compounds also shows
( 20 21 )
some distinctive characteristics consistent with toxicity ' ; a
greater inhibition of Na -K+ATPase occurs than with DDT-type compounds
as shown in Table 2. The remainder of the Cyclodiene compounds, such
as aldrin and dieldrin, do not show a good correlation between enzyme
inhibition and toxicity (Table 2). The manner of biodegradation and
penetration of the cyclodiene compounds to the site of action within the
fish undoubtedly affects the results.
(5) The organophosphates and carbamates insecticides tested (cholines-
(21)
terase inhibitors) are not ATPase inhibitors
(6) The inhibitory effects of chlorinated hydrocarbons in insect homo-
genates are similar to fish homogenates of the same type of tissue with
minor differences in sensitivity (also see Figs. 1 and 2).
(7) At least one chemically distinct compound, Plictran, an acaricide,
2+ (22)
was found to be an outstanding inhibitor of mitochondrial Mg ATPase
The compound was also a better inhibitor of Na -K ATPase and oligomycin-
p.
insensitive Mg ATPase than DDT (see Fig. 9). In these respects its
broad inhibiting effects are readily distinguished from DDT which
2+
inhibits mitochondrial Mg ATPase to a much greater extent than either
Na -K ATPase or oligomycin-insensitive Mg ATPase.
In vivo results, involving the treatment of fish or insects, followed by
an analysis for enzyme activity, gave the following results:
(8) Fish or insects treated with sublethal doses of DDT and closely
related chemicals had the mitochondrial Mg ATPase reduced or inhibited
21)
when compared with untreated ; also see Table k.
10
-------
(9) Brain tissues taken from fat head minnows continuously exposed to
DDT showed a reduction of mitochondrial Mg ATPase amounting to 36,
46.5, 52.5 and 45$ when examined 56, 118, 225 and 266 days, respectively,
after treatment started (see Table 4). The values given above referred
to fish treated with a combination of 0.5 ppb DDT in water and 57 ppm in
food. The maximum reduction of the enzyme activity when 0.5 ppb DDT
was in the water alone was 4l.9$ after 225 days and 56.6$ after 266 days
continuous exposure. The effects on Na -K ATPase and oligomycin-insensi-
tive Mg +ATPase from brain were a modest stimulation (never any inhibi-
tion) which gradually increased through the 266th day of exposure. A
summary is given in Tables 5 and 6.
(10) Gill tissue from chronically exposed minnows gave contrasting results
with respect to Na -K ATPase. Using the same specimens as examined for
brain sensitivity, the activity of Na+-K ATPase was reduced by over 30$
(see Table ?)• The Mg +ATPases from gills were also reduced, the great-
est reduction being 49.7$. Thus, the effect on the Mg ATPases in gills
was similar, but less pronounced than in brain, but contrasted sharply
in having the additional significant reduction in Na -K ATPase activity.
(ll) Injection of several small doses of DDT in cockroaches (chronic
treatments) resulted in a maximum of 35$ inhibition of mitochondrial
Mg ATPase in nerve cords 25 days -after the initial treatment, indicating
a similar sensitivity to that of brain in fish, and also corresponding to
(21)
the^ effects obtained in vitro . However, it was found that moderately
high levels of DDT (acute U^n doses) resulted in a stimulation of mito-
2+
chondrial Mg ATPase, amounting to 50$ in muscle and 30$ in nerve cord.
(12) The polychlorinated biphenyl compounds (PCBs), like DDT, inhibited
total Mg +ATPase both jln vitro and _in vivo. They differed in that the
~~~~~~ 2+ (2"5
in vitro effect was greater oh the oligomycin-insensitive Mg ATPase '
The in vivo effects were rather similar to those of DM?' f 2^' .
Aroclor 1242 appeared to be quite effective with Aroclor 1254 giving a
somewhat smaller effect.
11
-------
SECTION VI
DISCUSSION
The consistent inhibition of the ATPase enzyme system jin vitro by
chlorinated hydrocarbon insecticides and acaricides has been demon-
strated. The DDT-group of compounds give a more predictable pattern,
2+
primarily inhibiting mitochondrial Mg ATPase. We must emphasize that
only with mitochondrial Mg ATPase (oligomycin-sensitive) can one show
inhibition values which can be illustrated as regression lines which
parallel toxicity. DDE, for example, is a much poorer inhibitor than
DDT and it was not possible to obtain ?C# inhibition of mitochondrial
Mg ATPase. The criticism of Jackson and Gardner (1973) is invalid
because they compared insecticidal effects on total ATPases instead of
discrete ATPases as we have done. Further, in their techniques of adding
the insecticide in ethanol to .the reaction mixture they used a 25 ul
aliquot while we never exceeded 5 ul» and usually used 1 ul with constant
stirring conditions. The larger volume which they used would be con-
ducive to precipitation, a feature which we avoided. We could detect
such an occurrence because we were continuously monitoring the reaction
in a spectrophotometer and only used results which showed the same rate
of reaction to be consistent over a total of 15 or 20 minutes.
Results with cyclodiene compounds are not comparable to the DDT analogs
because of greater variability in their inhibitory characteristics;
lindane and dieldrin are poor inhibitors of all the ATPases tested
under our conditions. The compound Kepone was an excellent inhibitor
of mitochondrial, yet its close chemical relative, Mirex, was almost
without effect. Thus, the usefulness of the bioassay lies primarily
with the DDT analogs.
Response differences between ATPases from different tissues are not
very great; most comparisons show muscle homogenates to be slightly
more sensitive than brain; a lesser number of comparisons were made
with kidney, liver and gills. Considerable detailed information is
13
-------
given in the research papers, and the figures and tables present the
important findings.
In general, the inhibition of mitochondrial Mg ATPase by DDT and re-
lated compounds has some parallels to acetylcholinesterase inhibition
by organophosphates and carbamates. In both cases the in vitro inhibi-
tion is readily established, but for various reasons, notably metabolic
changes in the organisms, varying degradation patterns of the pesticide
and penetration to the site of action cause some compounds to show a
poor effect in vivo. We have principally studied the jln vivo effects of
DDT. The effects obtained, however, indicate the value of a follow-up
study, along with the further development of a gill assay.
One relevant feature of the findings is the fact that only the pesticides
which are persistent in the environment and in biological systems, and
are capable of bioconcentration are effective inhibitors of the ATPase
system. We do not find the inhibitory characteristic among the organo-
phosphates and carbamates, which are relatively non-persistent, and although
pyrethrins have some inhibitory effect on Na -K ATPase, the concen-
tration required is one or 2 magnitudes higher than DDT and related com-
pounds. It is of course, this group of compounds (chlorinated hydrocarbons)
which cause the greatest concern because residues continue to be found in
soils, in certain predator birds, in certain fish, and in some bodies
of water.
Based on this survey of compounds capable of ATPase inhibition, further
research should investigate ways of utilizing this type of information
for studying prolonged effects in various key organisms.
Chronically treated fish deserve further discussion. Fat head minnows
treated with DDT showed a 50$ average depression of mitochondrial
Mg ATPase from brain after 266 days exposure. The first sampling, at
56 days, showed about a 30-^O$ depression, thus there appeared to be a
slow, but progressive adverse effect on the enzyme. There was no marked
difference attributable to the method of DDT treatment, whether in water
alone, or a combination with food. There is, of course, the possibility
of small amounts of DDT getting into the water from food. Earlier in
-------
the experiment (56 days) the greatest effects of brain ATPases seemed
to result from mixed treatments of both food and water. Gill- tissue
ATPases reacted differently in two respects. First, Na -K ATPase from
gills was 30$ less (presumably inhibited) than the control, yet this
enzyme had increased activity in brain tissue (presumably stimulated).
Second, in the earlier treatments, the greatest effect on gills occurred
in DDT-treated water, rather than in treated food. Furthermore, the
combined ATPase inhibition was greatest in gill tissue at the last deter-
mination of 266 days.
The results show that chronically treated fish do have an impressive
reduction of the energy-related mitochondrial Mg ATPase with DDT-con-
taminated water, and the ATPase affected included a depression of Na -
K ATPase in gills. Thus the exposure of the fish is very important in
this differential effect because jln vitro effects show no significant
differences in sensitivity between brain and gill enzymes.
We believe this is the first report of significant abnormal biochemical
or physiological conditions in a vertebrate exposed to controlled sub-
lethal chronic doses of DDT.
15
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Table 1. IN VITRO INHIBITION OF FISH BRAIN
MITOCHONDRIA!, Mg2+ATPase BY DDT AND SOME ANALOGUESa'b
uM for Inhibition
DDT
Dicofol
TDE
Perthane
Methoxychlor
DDE
i°
1.3
0.8
2.7
3.8
M
8.8
I i
70 90
3.5 1^.1
3.7 37.^
7.6 3^.3
13.^
27.3
30.8
fReaction mixtures given under Materials and Methods.
Specific activity of untreated mitochondria! Mg ATPase
in u moles P.mg" protein hr~ ) was 10.91 - 0.86
(mean value of 6 determinations).
16
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Table 2. INHIBITION OF ATPases BY DDT USING
If DIFFERENT TISSUES FROM BLUE GILL FISH
% Inhibition
DDT Cone, x 10"6 M Total Mg2+ ATPase Na* -K* ATPase
(A) Muscle
Specific activity = *f9-9 - 5-3 " 6.2 - 1.6
1.3 15
5.2 J>k
10.4 51
20.8 63
(B) Brain
Specific activity = 22.1 - 1.0 33.0 - l.*f
1.3 16 16
5.2 32 22
10.4 ifl 23
20.8 Mf 31
(C) Kidney
Specific activity = *f0.5 - 3.0 k6.7 - 1.0
0.8 22 6
1.5 32 9
6.1 46 26
12.2 53 31
(D) Liver
Specific activity = 28.2 - 3.7 *f.O*- 1.8
0.8 25
1.5 33
6.1 48
12.2 51
*Specific activity too low for accurate inhibition determinations.
Specific activity expressed as pinoles P. mg~ protein hr~ .
17
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Table 3- INHIBITION OF ATPases FROM FISH BRAIN BY DDT
AND SEVERAL CYGLODIENE INSECTICIDES
c
Compounds
DDT
Indenes or Non-Naphthalenic
alpha chlordane
gamma chlordane
heptachlor
heptachlor epoxide
isobenzan (Telodrin )
Naphthalenes
aldrin
dieldrin
isodrin
endrin
Miscellaneous
Kepone
toxaphene
endosulfan
pentachlorophenol
lindane
mirex
Mean specific activity
of untreated
Per
Mg +ATPase
cent inhibition
Oligomycin
Sensitive Insensitive
95
93
85
63
35
66
55
+15
+48
+37
95
77
69
+31
3
+2
12.85 15
20
69
61
40
42
36
50
20
62
15
60
61
50
+7
10
+3
.26
Na+ -K+ATPase
31b
53
61
43
55
68
48
40
18
36
62
32
37
14
0
29.69
a Mol. Wt. calculated as ^13.85 based upon Cno^O^S "
DDT inhibited 63.9$ of Na+ -K+ATPase from cockroach nerve cord.
£
All compounds compared at a concentration of 20.8 urn
18
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Table *t. REDUCTION OF MITOCHONDRIA!, Mg2+ATPase OF BRAIN
FROM FISif CONTINUOUSLY EXPOSED TO DDT
DDT
Concentration
57 ppm
in food
0.5 ppb
in water
2 ppb
in water
0.5 ppb
in water +
57 ppm
in food
2 ppb in
water +
57 ppm
in food
Untreated
sp. act.
+S.E.
Q
Fat head minnow,
Per cent reduction from
56 days
23.^
29-5
39-8
36.0
*f2.8
9.60
+0.20
Pimephales
118 days
36.2
36.6
5^.3
*f6.5
50.1
7-90
+p.if5
promelas.
225 days
22.3
lfl.9
36.0
52.5
7.27
+0.3^
untreated
266 days
55.2
56.6
57.2
45.0
7-55
±0.15
Specific activity expressed as )imoles P. mg protein hr"
19
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Table 5. STIMULATION (INCREASE) IN Na+-K+ATPase
OF BRAIN FROM FISHaCONTINUOUSLY EXPOSED TO DDT
DDT
concentration
57 ppm
in food
0.5 ppb
in water
2 ppb
in water
0.5 ppb
in water +
57 ppm
in food
2 ppb
in water +
57 ppm
in food
Untreated
sp. act.
+S.E.
56 days
+ 7-6
+ 0.2
+ 7.5
+10.1
+18.4
27.28
+1.84
Per cent increase over untreated
118 days 225 days 266 days
+12.5 + 1.9 +27.7
+ 9-9 +1^.5 +18.9
+10.2 + 5-7 +21.7
+ 6.k +13-5 + 6.3
+ 9.1
22.62 2*f.03 18.38
+0.6*f +0.69 +1.70
Fat head minnow, Pimephales promelas
Specific activity expressed as jomoles P. mg protein hr
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Table 6. STIMULATION (INCREASE) OF OLIGOMYCIN-INSENSITIVE
2+ a
Mg ATPase OF BRAIN FROM FISH CONTINUOUSLY EXPOSED TO DDT
DDT
concentration Per cent increase over untreated
57 ppm
in food
0.5 ppb
in water
2 ppb
in i water
0.5 ppb
in water +
57 ppm
in food
2 ppb
in water +
57 ppm
in food -
Untreated
sp. act.
+S.E.
a Fat head
56 days
+19.3
+16.7
+27.5
+26.9
+29.2
12.04
+0.85
minnow, Pimephales
118 days 225 days
+13.9 +16.9
+21.2 +14.7
+ 8.3 +11.6
+ 5-5 +23.5
+14.7
12.35 10.48
+0.79 ±0.37
promelas
266 days
+33.6
+39-6
+38.5
+36.4
8.96
+0.55
Specific activity expressed as jomoles P. mg~ protein hr~
21
-------
Table 7- PER CENT CHANGES IN ATPases OF GILL TISSUE
FROM FISH* CONTINUOUSLY EXPOSED TO DDT
DDT
concent.
57 ppm
in food
0.5 ppb
in water
2 ppb
in water
0.5 ppb
in water +
57 ppm in
food
Untreated
sp. act.
+S.E.
* (+) Values
Per cent reduction from
Na+
225 days
+0.4*
31.2
27.9
18.1
13-7
±0.93
untreated
OLIGOMYCIN
-K
266 days
35-3
30.3
12.8
13.7
10.6
±1.26
represent the enzyme
Sensitive
225 days 266
3.2 41
27.2 49
+7.2* 26
33.8 23
5-50 4
±0.32 ±0
activation
Insensitive
days 225 days 266
.3 + 4.5* 28.
.7 24.3 14.
.4 3.5 8.
.7 20.5 3.
.55 24.15 24.
.25 ±1.14 ±2.
days
6
2
7
2
36
33
a Fat head minnow, Pimephales promelas
Specific activity expressed as umoles P. mg protein hr
22
-------
999p
99
COCKROACH COXAL MUSCLE-B
MG++ATPASE
0.01
MITOCHONDRIAL
^OLIGOMYC IN-
INSENSITIVE
045 0.1 1.0
DDT CONCENTRATION (MM)
Figure 1. In vitro inhibition of Mg ATPase from cockroach
muscle homogenates treated with DDT
23
-------
SO
10
FISH BRAIN-FRACTION B
MG-H-ATPASI
0.05
DDT CONCENTRATION (MM)
Figure 2. In vitro inhibition of Mg ATPase from fish brain
homogenates treated with DDT
2k
-------
99
90
70
SO
30
— 1
FISH BRAIN -FRACTION B
MITOCHONDRIA!. Mc+*ATPASE
I
DDT
PERTHANE
0.05
0.1
1.0
CONCENTRATION(uM)
10
100
Figure J>. In vitro inhibition of mitochondrial Mg ATPase from
fish brain homogenates with DDT, 2 analogs of DDT, and
DDE, the principal metabolite
-------
99
±90
30-
FISH BRAIN-FRACTIONS
MG++ATPASI
10.71
L
MITOCHONDRIA!.
QLIGOMYCIN-
INSENSmVE
0.05 0.1
1.0
DICOFOL CONCENTRATION(MM)
10
50
Figure k. In vitro inhibition of Mg +ATPase from fish
brain homogenates treated with dicofol (Kelthane)
26
-------
99-
CO
90-
2 70
h—
CD
1 50
=5 30
u
UJ
a.
10
FISH BRAIN-B FRACTION
MITOCHONDRIA!.
ATPASE
1x10 8
MOLAR CONCENTRATION
1x10
•r-7
IxlO6
Figure 5- In vitro inhibition of Mg ATPase from fish
brain homogenates treated with tetradifon (Tedion)
-------
3
C9
s90h
o
OB
^
«70-
FISH BRAIN B FRACTION
MITOCHONDRIA!.
ATPASE
CONCENTRATION (MM)
2+
Figure 6. In vitro inhibition of Mg ATPase from fish
•
brain homogenates treated with dichlorodiphenylethanol
(DMC of Dimite) and chlorobenzilate
-------
99-
.90
FISH BRAIN B FRACTION
MITOCHONDRIA!.
ATPASE
11.9
0.1
1.0
CONCENTRATION (MM)
10
100
2-f
Figure 7« In vitro inhibition of mitochondrial Mg ATPase from
fish brain homogenates treated with ovex, Genite
and chlorbenside (Mitox)
-------
OVEX
1 \-
GENITE
S-C
CHLORBENSIDE
(MITOX")
DMC
(DIMITE")
ru M CHLOROBENZlLATE
i ' 9
O-CHjCH-O-S-O-CHjCHjCI
Cl
Cl
ARAMITE
cr
Cl Cl
PENTAC*
Figure 8. Structural formulas of chlorinated hydrocarbon
acaricides tested on ATPase system of blue gill fish
30
-------
99-
90-
70-
MITOCHONDRIAL
fATPASE
50
30
<
10
1
0-
-^^0^^^
- ^^^
\
00001
•~ "x"
^^^
^^^
MX}05&M
1 I
0-0001
»^
6.2X10"4MM
1 1 1
0-001 0-01
PUCTRAN CONCEHTRAT10H (MM)
Figure 9» In vitro inhibition of mitochondria! Mg ATPase
from brain homogenates of fish and homogenates
of two-spotted spider mites
-------
SECTION VII
REFERENCES
1. R. B. KOCH, Chlorinated hydrocarbon insecticides: Inhibition of
rabbit brain ATPase activities, J. Neurochem., 16(1969) 269.
2. R. B. KOCH, L. K. CUTKOMP and F. M. DO, Chlorinated hydrocarbon
insecticide inhibition of cockroach and honeybee brain ATPases,
Life Sci., 8(1969) 289.
3. R. B. KOCH, Inhibition of animal tissue ATPase activities by chlor-
inated hydrocarbon pesticides, Chem.-Biol. Interactions 1(1969/70) 199-
k. R. B. KOCH, L. K. CUTKOMP and F. M. DO, Chlorinated hydrocarbon
insecticide inhibition of cockroach and honey bee ATPases, Life
Sciences 8 Pt. II,(1969) 289.
5. R. B. KOCH, Fractionation of olfactory tissue homogenates, isolation
of a highly concentrated plasma membrane fraction, J. Neurochem.,
16(1969) 1A5-
6. M. E. PULLMAN, H. S. PENEFSKY, A. DATTA and E. RACKER, Partial
resolution of the enzymes catalyzing oxidative phosphorylation,
J. Biol. Chem., (1960) 235.
7. P. J. FRITZ and M. E. HAMRICK, Enzymatic analysis of adenosinetri-
phosphatase, Enzymologia, 30(.1966) 57 •
8. H. H. YAP and L. K. CUTKOMP, Activity and rhythm of ATPases in larvae
of the.mosquito, Aedes aegypti L., Life Sci., 9(1970) Part II, 1919-
9. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, Protein
measurement with the Folin phenol reagent, J. Biol. Chem., 193(1951)
265.
10. H. McILWAIN, "Chemical Exploration of the Brain", p. 15^ Elsevier, Amster-
dam, (1963)
11. D. J. FINNEY, "Probit Analysis", Cambridge University Press, Cambridge,
(1957) 318.
12. D. J. FINNEY, The meaning of bioassay, Biometrics, 21(1965) 785.
13. R. J. DAUM, Revision of two computer programs for probit analysis,
Bull. Entomol. Soc. Amer., 16: 10-15 (1970).
14. L. K. CUTKOMP, H. H. YAP, E. V. VEA and R. B. KOCH, Inhibition of oli-
gomycin-sensitive (mitochondria!) Mg +ATPase by DDT and selected
analogs in fish and insect tissues. Life Sciences 10, Part II (1971)
1201-1209.
-------
15. L. K. CUTKOMP, H. H. YAP, E. Y. CHENG, and R. B. KOCH, ATPase activi-
ty in fish tissue homogenates and inhibitory effects of DDT and
related compounds, Chem-Biol. Interactions, 3(6) (1971) k39-kk7.
16. R. B. KOCH, L. K. CUTKOMP, and H. H. YAP, Inhibition of oligomycin
sensitive and insensitive fish adenosine triphosphatase activity
by chlorinated hydrocarbon insecticides. Biochem. Pharmacol., 20
(1971) 32^3-32^5.
17. L. K. CUTKOMP, D. DESAIAH, and R. B. KOCH. The in vitro sensitivity
of fish brain ATPases to organochlorine acaricides. Life Sci.
11(23), Pt. II, 1123-1133 (1972).
18. D. DESAIAH, L. K. CUTKOMP, R. B. KOCH and H. H. YAP, Tetradifon
•p p.
(Tedion ): A Specific inhibitor of Mg dependent mitochondrial
adenosine triphosphatase acitivity. Life Sci. II, Pt. 11(1972)
389-395.
19. Y. C. CHU and L. K. CUTKOMP, The in vitro and in vivo inhibition of
ATPases in American cockroaches by chlordane, Jour. Econ. Entomol.,
6k (1971) 559-561.
20. L. K. CUTKOMP, D. DESAIAH and R. B. KOCH (Unpublished Results, 1973).
21. D. DESAIAH, L. K. CUTKOMP and R. B. KOCH. Inhibition of spider mite
ATPases by Plictran and three organochlorine acaricides. Ms sub-
mitted July, 1973.
22. H. H. YAP, D. DESAIAH, L. K. CUTKOMP and R. B. KOCH, The sensitivity
of fish ATPases to polychlorinated biphenyls, Nature, 233(1971) 61-62.
23. D. DESAIAH, L. K. CUTKOMP, H. H. YAP, and R. B. KOCH, Inhibition of
2+
oligomycin-sensitive and insensitive Mg ATPase in fish by poly-
chlorinated biphenyls. Biochem. Pharmacol., 21(1972) 857-865.
2k. R. B. KOCH, D. DESAIAH, H. H. YAP and L. K. CUTKOMP Polychlorinated
biphenyls: effect of long-term exposure on ATPase activity in fish
Pimephales promelas, Bull. Environ. Contamin. Toxicol., 7(1972) 87-92.
25. L. K. CUTKOMP, H. H. YAP, D. DESAIAH and R. B. KOCH. The sensitivity
of fish ATPases to polychlorinated biphenyls, Environmental Health
Perspectives, 1(1972) 165-168.
-------
26. D. DESAIAH, L. K. CDTKOMP and R. B. KOCH, The effect of pyrethrins
on ATPases incockroach and blue gill fish. Ms submitted July, 1973-
27. D. A. JACKSON and D. R. GARDNER, The effects of some organochlorine
pesticide analogs on Salmonid brain ATPases. Pesticide Biochem.
and Physiol, 2(1973) 377-82.
-------
SECTION VIII
PUBLICATIONS
1. Y. C. CHU and L. K. CUTKOMP, The in vitro and in vivo inhibition of
ATPases in American cockroaches by chlordane, Jour. Econ. Entomol.,
64(1971) 559-561.
2. L. K. CUTKOMP, H. H. YAP, E. Y. CHENG, and R. B. KOCH, ATPase activity
in fish tissue homogenates and inhibitory effects of DDT and related
compounds, Chem-Biol. Interactions, 3(6) (1971) 439-447.
3. R. B. KOCH, L. K. CUTKOMP, and H. H. YAP Inhibition of oligomycin
sensitive and insensitive fish adenosine triphosphatase activity
by chlorinated hydrocarbon insecticides. Biochem. Pharmacol., 20
(1971) 3243-3245.
4. H. H. YAP, D. DESAIAH, L. K. CUTKOMP and R. B. KOCH, The sensitivity
of fish ATPases to polychlorinated biphenyls, Nature, 233(1971)
61-62.
5. L. K. CUTKOMP, H. H. YAP, E. V. VEA and R. B. KOCH, Inhibition of
oligomycin-sensitive (mitochondrial) Mg ATPase by DDT and selected
•
analogs in fish and insect tissues. Life Sciences 10, Part 11(1971)
1201-1209.
6. D. DESAIAH, L. K. CUTKOMP, H. H. YAP, and R. B. KOCH, Inhibition of
oligomycin-sensitive and insensitive Mg ATPase in fish by poly-
chlorinated biphenyls. Biochem. Pharmacol., 21(1972) 857-865.
7. R. B. KOCH, D. DESAIAH, H. H. YAP and L. K. CUTKOMP, Polychlorinated
biphenyls: effect of long-term exposure on ATPase activity in fish
Pimephales promelas, Bull. Environ. Contamin. Toxicol, 7(1972) 87-92.
8. L. K. CUKTOMP, H. H. YAP, D. DESAIAH and R. B. KOCH, The sensitivity
of fish ATPases to polychlorinated biphenyls, Environmental Health
Perspectives, 1(1972) 165-168.
9. D. DESAIAH, L. K. CUTKOMP, R. B. KOCH and H. H. YAP. Tetradifon
R ?+
(Tedion ): A specific inhibitor of Mg dependent mitochondrial
adenosine triphosphatase activity. Life Sci. II, Pt. II (1972) 389-395,
-------
10. L. I. CUTKOMP, D. DESAIAH, and R. B. KXH. The in vitro sensitivity
of fish brain ATPases to organochlorine acaricides. Life Sci. II (23)
Pt. II (1972) 1123-1133.
11. D. DESAIAH, L. K. CUTKOMP and B. B. KOCH. Inhibition of spider mite
ATPases by Plictran and three organochlorine acaricides. Ms sub-
mitted July, 1973-
33
-------
SECTION IX
GLOSSARY
ADP, adenosine diphoephate
ATP, adenosine triphosphate
ATPase, ATP phosphohydrolase, E.G.3.6.1.3
BSA, bovine serum albumin
EDTA, ethylenediamine tetraacetic acid
LDH, lactic dehydrogenase
NAD, nicotinamide adenine dinucleotide phosphate
NADH, reduced nicotinamide adenine dinucleotide phosphate
PEP, phospho (enol) pyruvate
Mg +ATPase, Mg++ATP phosphohydrolase
Na+ -K+ATPase, Na+ and K+ dependent, Mg^ATP phosphohydrolase
PK, pyruvate kinase
S.E., Standard Error
*U& GOVERNMENT PRINTING OFFICE: 1974 346-319/406 1-3
39
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Report ffo.
W
4. Title A Tissue Enzyme Assay for Chlorinated Hydrocarbon
Insecticides
5. Re port Date
6.
g. Performing Orgajarittioa
Laurence K0 Cutkomp
9. Organization
t-'T' je i ffo
16030 ELZ
Department of Entomology, Fisheries, & Wildlife
University of Minnesota, St.- Paul, Minn,
JZ. Sponjor/nr Orgatdxatioa^^fafa
5. Suf p a em-try /votes
Environmental Protection Agency report number, EPA-660/2-73-027,
May
- mi3 ' ill '< o
R 801029
Type at Report and
ifi. AbsT3d certain chlorinated hydrocarbon insecticides, especially DDT and closely
related chemicals, tested at low concentrations, adversely affect the ATPase enzyme
system. DDT inhibited oligomycin-sensitive Mg^ATPase (mitochondrial) both in vitro and
in vivo. About 1 uM (1 x lO"6 M) gave 50% inhibition in fish brain and 0.5 ppb of DDT
in water inhibited about 50% of mitochondrial Mg2+ATPase. Na+-K+ATPase was not inhibite
in brain, but was inhibited in vivo in fish gills. Certain discriminating effects were
found among chlorinated hydrocarbons, particularly with respect to inhibition of Mg2+
ATPase, but the ranking of compounds by enzymic effects does not always parallel
toxicity values. Organophosphate and carbamate insecticides were ineffective. Further
research is needed both in vitro and in vivo to determine how the adverse effects on
the enzymes relate to practical interpretations of effects. The abnormally low ATPase
activity in chronically treated fish is the first report of an adverse biochemical
effect with sublethal doses of DDT. All effects appear to be primarily within the
group of insecticides and acaricides which are persistent in parts of the environment
and in organisms.
i?
7a. Descriptors Chlorinated hydrocarbon insecticides, enzyme assay.
/ 7b. Identifiers
/-.: COWRR Field & Group
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D C. 2O24O
Abstractor
W R S 1 C 1 C / 'REV .: I. N - 1 9 •> I
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