PROBLEMS IN AERIAL APPLICATION:

II. EFFECTS OF CHLORINATED.HYDROCARBONS
ON SUBSTRATE-LINKED PHOSPHORYLATION

JACK W. DAUGHERTY, Ph. D.
DANE EUGENE LACEY, B. S.
PATRICIA KORTY, B. S.

ABSTRACT

Tn a search for an explanation of an earlier finding that dieldrin decreased the rate

of methionine uptake by cardiac muscle, chickens and rats were e:
and the substrate-linked phosphorylation phenomenon was studi

degradation of glucose. Exposure to di

sed to this compound
during the anaetobic
reduced the esterification of inorganic

phceobats 50% but did not affect the production -of lactic acid. Four other chlorinated

lrocarbons, lindane,

In a recent report from this laboratory, data
were given which support the concept that
some of the hepatic changes resulting from
chlorinated-hydrocarbon intoxication could be
attributed to a reduction in active membrane
transport (1). Since this is an energy de-
pendent phenomenon, a study of the effects
of chlorinated hydrocarbons on mechanisms
involved in the synthesis of adenosine-
triphosphate (ATP) seemed a means of de-
scribing in greater detail the influence of such
compounds on biological systems,

v
EXPERIMENTAL '

White Rock chickens, obtfined locally when
1 week old, were kept in’ the laboratory on
Ralston Growena until they had attained a
weight of at least 400 g, At the time off assay-
ing the chlorinated hydtocarbon effeét, ran-
domly selected animals were weighed jand were
given dieldrin (hexachloro-epoxy-octahydro-
dimethanonaphthalene) per os, The dieldrin
had been previously dissolved in acetone, intro-

@ duced into gelatin capsules and the acetone’

allowed to evgporate before administration to

the chickens.” Dieldrin was also given by

mouth, dissolved in com ail (Mazola), with
, identical results, : j

, heptachlor. isodrin, and endrin did not produce this effect.

The male rats used in part of the study were
of the Sprague-Dawley strain and weighed in
excess of 300 g. at the time of their exposure
to the insecticide, In these tests the exposure
was accomplished by intraperitoneal injections
of dieldrin dissolved in corn oil (Mazola), Con-
trols received the oil carrier alone. The animals
received two injections, each containing 15 mg.
of the insecticide.

The present assays on the effect of the ex-
posures were carried out on 10% brain homog-
enates (in water) incubated in Warburg flasks
at 38° C. The incubation medium consisted
of those components known to support anaer-
obic glycolysis in an homogenate system (2,3).
Byain tissue was chosen for these studies as it
represents the only normal adult tissue that
can adequately phosphorylate glucose in an
homogenate system, This was of importance in
the present investigation as this type of system
was also to be used in other studies, Of equal
consideration was the fact that earlier work
had shown that the DNA content of brain
(indicative. of the content of enzymatically
active material or nucleated cells) is not
affected by exposures to chlorinated hydro-
carbons (1,4). Determinations of total nitro-
gen, some of which are included in Table 1

-1-



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EFFECT OF DIELDRIN
ON

PHOSPHATE ESTERIFICATION BY CHICKEN BRAIN

Exp. No, Animal PO, co, PO,/CO,

1 (m) Control® 3.50 3.47 1.02

{m) Test® . 1.69 2.88 0.58

II (£) Control* 3.70 3.36 1.05

(£) Test* 1.85 3.14 0.59

III (m) Control® 5.33 3.60 1.48

: {m) Test* 117 3.65 0.32

IV (£) Control* 3.31 3,45 0.96

(£) Control* 3,32 3.15 1.05

V (£) Control** 2.60 3.30 0.79

(£) Test®* 1.47 2.50 0.59

Mean Control 3.62 3.39 1.07
Values _Test 1.54 3.04 0.51

as well as
ical response

esterifica-

PO, disappearance in »M/flask/10 min.
CO, evolution in »M/flask/10 min.

* 400-500 g.

** 1300 g.

(m) male

(f), female

Studies were made on the adenosine-
triphosphatase activity under conditions similar
to those described above. There was no dis-
cernible difference between the control and
test animals (Table 4). As may be noted, “KF
actively inhibits ATPase. activity, Since KF
was included in all of the incubatign mixtures
used in the study of phosphate esterification,
it would not appear that the ATPase system
played any role in the diffgrences seen above.

Parker (11) noted a similar effect of DDT
and some of its metabolites and dieldrin on
phosphate uptake by liver mitochondrfa using
a pyruvate substrate system. It would appear
that dieldrin may have a general fncoupling
effect on phosphate esterification, whether it is
oxidatively or anaerobically supported.

It is apparent from the results of the studies
on both chickens and rats that whereas brain
glycolysis, a¥ evidenced by lactic acid pro-
duction, is not affected by the dosage levels

, used in this work, the esterification of in-

organic phosphate, and presumably the syn-

thesis of ATP, is markedly reduced, At present,
this effect does not appear to be the result of
a simple inhibition by dieldrin, per se, since no
direct in vitro effect has so. far been shown
(unpublished data). Investigations are now
being carried out to identify the active form
of the chlorinated hydrocarbon and to test the
activity of other chlorinated hydrocarbons.
From the, effectiveness of the intraperitoneal
injections it also would appear that the biologi-
tal activity of the compound does ‘not depend
on chemical alterations involved in or preceding
the process of absorption from the intestine,
At this time several other chlorinated com-
potinds of an entirely different molecular con-
figuration have been studied in a preliminary
fashion. It, is of interest’ that these com-
pounds have not been shown to inhibit phos-
phate esterification under conditions compar-
able to’those described herein. Table 3 shows
the results of typical experiments, in this case
comparing heptachlor with dieldrin, The re-
sults with lindane, isodrin, and endrin were

-3-



below, are further evidence of the refractive-
ness of brain protein content to the insecticide.
Tnorganic phosphate was determined by the
method of Fiske and SubbaRow (5); total
nitrogen by standard micro-Kjeldahl techniques
(6); protein by the method described by
Lowry et al (7); and DNA by analysis for
2-desoxyribose (8) following tissue fractiona-
tion by methods of Schneider (9) and Schneider
and Klug (10).

ye

quite obviously affected by the exposure, being r

reduced to approximately the 50% level. As may
be observed from the data given @ in Table 2,
neither sex'nor age appeared to ignificant
factors in this Of the. response to the
insecticide. It was observed, however, that
significant variations did occur between indi-
vidual chickens in terms of (1) the measurable
rate of glycolysis (in both controls as well as
test animals) and (2) the toxicological response

Taste 1 4

EFFECT OF DIELDRIN

ON
SUBSTRATE-LINKED PHOSPHORYLATION
IN
RAT BRAIN
Exp, No. Animal PO. Co, Nz PO,/CO,
I Control 4.23 38 528 lil
Test 1.87 3.8 480 0.49
Il Control 24 2.96 483 0.81
7 Test 14 3.22, 483 0.43
PO, disappearance in sM/flask/10 min.
CO, evolution in «M/flask/10 min.
N, in mg,/flask

Adenosine-triphosphatase activity was meas-
ured in an incubation mixture consisting of
Na ATP, Mg ions, KF, tris buffer (pH 7.4) and
brain homogenates. The reaction was stopped
at 30 min, (37°C) with TCA. | }

RESULTS AND DISCUSSION

In Table 2: are shown certain of the results
of exposing chickens to a single dose of 100
mg/kg of dieldrin, given 24 to 48 hours ior
to the assay. This dosage must closely ap
mate the LD., since all of the unused test
animals died within 72 hours, It may be seen
that acid production .was not significantly af-
fected by the insecticide,
detnonstrated that the acid responsible for the
evolution of CO, pvas lactic acid. It may be
concluded that the anaerobic degradation of
glucose, per se, was not influenced by the acute
exposures to dieldrin, In contrast to this, the

* ‘disappearance rate of inorganic phosphate was

Separate tests have

to a particular dose level, These variations will
undoubtedly constitute a source of continuing
difficulty in obtaining reproducible results on

different individuals and in adequately con--

trolling the dose level to obtain “predicted”
results. Similar variatioris in other areas of
(poultry) biochemistry have been observed in
othe, work (2),

Additional evidence of this effect of dieldrin
in uncoupling inorganic phosphate esterifica-
tion is given in Table 1. These data represent
the resylts of glycolysis and phosphorylation
measurements on the pooled brain homogenates
of 3 contro] and: 3 dieldrin-exposed rats in two
separate experiments. The dieldrin was given,
as described above, on the day preceding the
time of assay. ' It will be seen that the effect
produced by dieldrin exposure was identical
with that observed in chickens and that the
individual meas
those shown in Table 2.

ents closely approximate ®


brain, On the basis of the lactic acid deter-

minations, it is further shown that anaerobic
glycolysis was actually the system being
measured,

Regardless of the mode of action of dieldrin,
its effects on those biological phenomena which
are energy dependent are apparent, These find-
ings are particularly significant, in view of the
reduced rate of S-35 methionine uptake by the
heart sarcosomes of dieldrin-exposed animals
(1), since methionine is known to be actively
transported across biological membranes and
interfaces, It now becomes desirable to test
the activity of dieldrin (and other such com-
pounds) on energy dependent phenomena in
other tissues,

SUMMARY

Acute exposures of chickens and rats to the
chlorinated hydrocarbon, dieldrin, have been
shown to result in the uncoupling of inorganic
phosphate esterification during anaerobic gly-
eolysis, Acid (lactic) production is not affected.
Lindane, isodrin, endrin, and: heptachlor do
not produce this effect.

,

4.8 GOVERNMENT PRINTING OFFICE: 1963 0 - 695-543 -5-

REFERENCES

. Daugherty, J. W., Lacey, D. E., and Korty, P. “Prob-

lems in Aerial Application: I, Some Biochemical
Effects of Lindane and Dieldrin on Vertebrates,” CARI
Report 62-10, Civil Aeromedical Research Institute,
May 1962.

. Daugherty, J. W., and Herrick, C. A. J.’ Parasitol.,

38:298, 1952.

. LePage, G. A. J. Biol. Chem., 176:1009, 1948.
. Annau, E, Canad. J. Biochem. Physiol. 32:178, 1954,

. Fiske, C. H., and SubbaRow, Y. J. Biol. Chem.,

66:375, 1925.

Hawk, P. B., Oser, B. L., and Summerson, W. H.
Practical Physiological Chemistry, Blakiston, N. Y.

Lowry, O. H., Rosebrough, N. S., Farr, A. L., and
Randall, R. J. J. Biol. Chem., 193:265, 1951.

. Dische, Z. Microchemie, 8:4, 1930.
. Schneider, W. C. J. Biol. Chem., 161:293, 1945.

. Schneider, W. C., and Klug, H. L. Cancer Research,

6:691, 1946.

. Parker, V. H. Biochem. J., 77:74, 1960.


PROBLEMS IN
AERIAL APPLICATION:

Il. EFFECTS OF
CHLORINATED HYDROCARBONS ON
SUBSTRATE-LINKED “
PHOSPHORYLATION

' OKLAHOMA CITY, OKLAHOMA
MARCH 1963:
,
. U if

i

» ERALU. LIBRARY

63-4 ~
i i
; 5
i , .
} FEDERAL AVIATION AGENCY a.
: AVIATION MEDICAL SERVICE
} (OMEDICAL RESEARCH DIVISION .
' CIVIL/AEROMEDICAL RESEARCRS@MNSTITUTE |

i



practically identical with these shown for
heptachlor. While the negative findings with
lindane and heptachlor are not too surprising,
those obtained following exposure to isodrin
and particularly endrin were mildly so, since

the latter is the optical isomer of dieldrin.
Table 3 also demonstrates, on the basis of total
nitrogen and DNA, the comparability of the
test and control animals. In no experiments
to date has either of these values changed in

Taste 3

COMPARISON OF EFFECTS

OF

DIELDRIN AND HEPTACHLOR ON
PHOSPHATE ESTERIFICATION BY BRAIN

Lactic
ANIMAL PO, CO, _PO./CO, _Acid N, DNA
Rat Dieldrin
(300 g.)
Control 24 2.96 0.81 2.31 0.48 0.176
Test 14 3.22 0.43 2.58 0.48 0.171
Rat Heptachlox
(180 g.)

“ Control 23 2.68 0.85 0.52 0.128
Test 3.6 3.18 1.13 0.50 0.124
Chick Heptachlor
(1100 g.)

Control 3.0 3.18 0.94 2.21 0.44 0.118
Test 3.2 3.18 0.94 2.13 0.44 0.125
PO, disappearance in »M/flask/10 min.
CO, evolution in #M/flask/10 min.
Lactic acid in »M/flask/10 min.
N, in mg./flask
DNA in mg,/g dry weight as desoxyribose nucleic acid
° samples not estimated ”

‘ i

- .

4
i Taste 44
| ADENOSINE-TRIPHOSPHATASE
ACTIVITY OF/RAT BRAIN FOLLOWING
EXPQSURE TO DIELDRIN +
CONTROL* “TREATED*
0 Time ‘Final =, ‘Final + 0 ‘Time Final Final
> No KF _ KF Added ' No KF _ KF Added

(1) 932° 12.40°° 4.13%* 258°° 12.60%" 4.40°°
(2) 190 11.14 3.88 2.02 12.10 3.30

© ‘Two experiments run in duplicate
°° uMols of inorganic phosphate per flask

-4-

-

)



FOREWORD

The mechanism of the harmful effects of toxic substances on the per-
formance of aerial applicator personnel lies at the cellular level. Effective
field detection of early toxicity and the application of sound preventive
methods can only be achieved following an understanding of the biochemi-
cal méchanisms involved.

s



PROBLEMS IN AERIAL APPLICATION:

II. EFFECTS OF CHLORINATED HYDROCARBONS
ON SUBSTRATE-LINKED PHOSPHORYLATION

JACK W. DAUGHERTY, Ph. D.*
DANE EUGENE LACEY, B. S.
PATRICIA KORTY, B. S.

*Chief, Cytochemistry Section
‘PaaRMACOLocy-BiocHEMIsTRY BRANCH

FEDERAL AVIATION AGENCY
AVIATION MEDICAL SERVICE
AEROMEDICAL RESEARCH DIVISION *
CIVIL AEROMEDICAL RESEARCH INSTITUTE
+ OKLAHOMA CITY, OKLAHOMA

MARCH 1963



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