isolated lung. As systemic arterial blood pressure
of the table dog decreased, pulmonary artery
pressure of the isolated lung increased from 16
to 23 mm Hg. This rise indicates an increase in
total pulmonary vascular resistance since flow
is constant. Lung weight underwent a slight
increase until 15 minutes after endrin and then
decreased by 3.2 gm as pulmonary artery pressure
achieved a peak increase.

Figure 3 shows the effect on total pulmonary
resistance, hematocrit, and systemic arterial blood
pressure in the venous return preparation. Sys-
temic arterial blood pressure did not change
significantly by 20 minutes after endrin, while
pulmonary vascular resistance increased from
0.012 to 0.037. Hematocrit increased during this
period from 44% to 48% and stabilized at this
value. From 20 to 40 minutes after endrin, sys-
temic arterial pressure increased markedly and
fell to a value still above control by 60 minutes.
During this period, pulmonary vascular resist-
ance remained at an elevated value. The develop-
ment of extreme hypertension after endrin in
these succinylcholine-treated animals (noncon-
vulsing) was a consistent finding and is in marked
contrast to the early hypotension in the animals
in which convulsions are not prevented. This
striking difference is not readily explainable but
may be related to the more severe acidosis that
developed in the convulsing dogs or to the con-
vulsions,

Figure + gives mean values with standard errors
showing the effect of endrin on several parameters
in the same venous return animals of Figure 3.
Cardiac inflow (pulmonary blood flow) increased
from 70 to 83 cc/min per kg of dog by 30 minutes
after endrin as previously observed (Reins, D.
and L. B. Hinshaw, unpublished observations).
After 40 minutes, pulmonary blood flow began
to decrease and leveled at 59 cc/min per kg by
the experiment’s end. Left atrial, pulmonary-
artery, and right atrial pressure increased mark-
edly by 20 minutes post-endrin.

Ii. Discussion.

The hematocrit can increase due to an increase
in erythrocyte size and/or an increase in erythro-
cyte concentration. The concentration of red
blood cells can increase through (a) loss of plasma
water as a result of filtration; (b) addition of
erythrocytes to the circulating blood volume; or
(c) a combination of both. Results show that

17

approximately half of the rise in hematocrit after
endrin occurring in intact sham-operated dogs is
prevented by removal of the spleen, indicating
that expulsion of cell rich blood from spleen
storage’ is responsible for an appreciable portion
of the hematocrit elevation.

The hematocrit increase seen in splenectomized
dogs appears to involve other mechanisms: An
effective means for concentrating red blood cells
is by extravasation of vascular fluid, but this
does not seem to occur in the hepatosplanchnic
region as evidenced by this study and in another
report showing no evidence for loss of vascular
water in skeletal muscle (Emerson, T. E., Jr. and
Hinshaw, L. B., submitted for publication). The
pulmonary vascular bed is a possible site for loss
of vascular water and cannot be excluded as being
involved in the post-endrin hematocrit increase.
Even though lung weight fell in the isolated, per-
fused lungs, this could be due to pulmonary vas-
cular constriction following release of catechola-
mine-like agents into the blood after endrin’ with
displacement of intravascular volume. It is also
possible that in these experiments a predominant.
pre-capillary constriction occurred, and effective
capillary filtration decreased. However, this con-
dition may exist only in the lung isolated from
the heart, in which pulmonary vein orifice pres-
sure is always zero. In the venous return ex-
periments, left atrial pressure increased to a maxi-
mum mean value of 24 mm Hg, which presumably
increased pulmonary capillary pressure by an
almost equal amount. It has been demonstrated
by others that pulmonary edema results when
pulmonary venous pressure approaches or exceeds
plasma-colloid-osmotic pressure, which averaged
22.5 mm Hg in their study.?— Assuming these
data as a guide, pulmonary venous pressure in
our experiments must have approached or ex-
ceeded colloid-osmotic pressure, and some loss
of pulmonary vascular water probably occurred.

An earlier report from this laboratoryt demon-
strated a marked increase in cerebral venous and
cerebrospinal fluid pressure after endrin, which
suggests the possibility of vascular fluid loss in
the brain area. Another potential area for loss
of plasma water is through the kidney secondary
to an increased urine flow rate if endrin has an
early diuretic effect. However, this possibility
has not been adequately explored.

The development of severe acidemia should lead
to an increased hematocrit subsequent to an in-



(mm Hg)

AP

(mm Hg)

AP

Appendix

LEFT ATRIAL PRESSURE

° @—e ENDRIN
©——0 CONTROL

e ~N
24 Ne aw
e
+15
0. *
07—<_ fe - 0 - 0 - 8
+o--0-—0-—0--0--0--0
-I TM*—7 T T T T 1
RIGHT ATRIAL PRESSURE
4
24
+17 e
eA fr 7m
fe) 4 \ -2-— J
~soh-o- ohonno" e ae
-14 eit a ee
-24
-3 T $ T T T T ~T
° 10 20 30 40 50 60

TIME (MIN.)

Fieurr 3. Effect of endrin on atrial pressures in the dog (mean values are shown for
seven experimental studies and six control experiments).

38


The presence of pulmonary hypertension would
apply a greater stress to the right ventricle, but,
even in the face of this, right atrial pressure re-
mained low. The apparent adverse effect of en-
drin on the heart raises considerations regarding
possible target sites of action of this class of in-
secticides. Gowdey and coworkers’ assert that
effects of chlorinated hydrocarbon insecticides are
exerted through stimulation of central mecha-
nisms, not peripherally. Recent work in this
laboratory has shown that endrin has no direct
vascular action on the isolated perfused liver:
neither hepatic-artery nor portal-vein pressure
were altered by endrin administration.

A notable feature in the present study was the
marked rise in venous return after endrin, and
the question is raised as to the mechanism of the
increase. Endrin does not have a direct excitant
action on the heart causing its output to increase,
thus elevating venous return. The increased vol-
ume of blood returned to the right heart after
endrin originates primarily in the hepatosplanch-
nic bed, since parellel experiments performed in
this laboratory * show no increase of venous re-
turn in animals with total abdominal evisceration.
The release of stored blood in the intact dog re-
sults in a continued maintenance of a high car-
diac output. Since the released blood remains
in the active circulation, venous return is contin-
ually maintained at a higher level. The increase
in venous return is not induced by circulating
catecholamines, since adrenalectomy does not
modify the response. The slight though significant
elevation in plasma total catecholamines after
endrin in adrenalectomized dogs suggests the
active participation of the sympathetic nervous
system. Releasing reservoir stores of blood could
result from a massive sympathetic discharge
that would augment the cardiac output by in-
creasing venous return. Systemic peripheral re-
sistance markedly falls with endrin administra-
tion, which could be due to the marked rise in
blod flow. Read and coworkers,’ using an open-
chest dog preparation similar to that employed in
the present study, have shown total peripheral re-
sistance to be inversely related to blood flow on
a passive basis. The drop in resistance in the
present study could, therefore, be passively in-
duced, resulting in part. from a rise in cardiac
output, Although total vascular resistance repre-
sents net changes in resistance, some vascular
beds may be dilating and some constricting.

34

Reins and coworkers? have shown marked renal
vasconstriction, and therefore elevated resistance
in the renal bed in dogs administered endrin.

Results from this study offer support for an
active participation of the sympathoadrenal sys-
tem as suggested earlier.? The effect, however, of
greatly increased levels of catecholamines on car-
diac output as observed in the present study is
surprisingly negligible. The humoral action of
endrin is apparently potentiated by acidosis,
since maintenance of blood pH in the normal
range significantly decreases the amounts of cate-
cholamines released after endrin. It does not.
appear that hypoxia is a stimulus for catechola-
mine release, since administration of 100% oxy-
gen does not significantly reduce the elevation
of circulating catecholamines over experiments
in which endrin alone is administered. The role
of released catecholamines in this form of stress
is unknown, since their circulation in large con-
centrations appears to have little effect on any
of the measured parameters. Central-nervous
stimulation apparently accounts for the cardio-
vascular effect of endrin as well as parasympa-
thetic participation in evoking salivation and
bradycardia. These latter effects have been de-
scribed by Emerson and coworkers? after injec-
tion of endrin and by Gowdey and coworkers
following the administration of aldren. Prom-
inent central-nervous effects of the chlorinated
hydrocarbon insecticides have been documented
by Gowdey and coworkers,° Treon and co-
workers, Arena,’ Bell, and Conley."

Consistent findings in endrin poisoning are
acidosis and hypoxia. Marked convulsions seen
in the present study may have interfered with the
normal respiratory exchanges of blood gases,
since animals with endrin exhibit cyanosis. Alle-
viation of acidosis and hypoxia as performed in
the present study had no effect on lessening the
severity of the convulsions. The convulsions ap-
parently result from a direct action of endrin on
the central nervous system.

Vv. Summary.

Effects of the chlorinated-hydrocarbon insecti-
cide endrin on the cardiovascular system of the
anesthetized dog have been studied. Results in-
dicate that endrin may display both central and
peripheral actions. Hemodynamic alterations ap-
pear to result from central-nervous stimulation
and an apparent toxic action on the left ventricle.


6075 @—e ENDRIN + 0p
o——-o ENDRIN + THAM

= P>.10 P>.l0

og 507 t t

Ca 3

Ix

gs | = —_}-—_;

Ww 40-

=x # TO ee fe i
30 T T
1805

4
1604 i 4
5 54 v4
5 XZ
140-4 oN 5 5 585 abyss

v2 Se ppb
« = 120- at RE
7)
& & 100-7
w Wl t t
ra 80-
P>.10 P>.10
60-
40 tT T T T qT q qt
ry 10 «=©6©202=S So 30'—i«dO—“iéi‘—CS!
TIME (MIN.)

Figure 8. Changes in hematocrit and heart rate in dogs treated with endrin plus oxygen
and endrin plus THAM (M +SE) (numbers of experiments indicated on face of graph).

Appendix 43



‘snoaneynods sea
SJUNOUTR [RYVAL
USP “PAywpOsT -Z aUA9Iy

wor endsey out, worsnsul aFvcev ay) syuaso.der yeas Jo doy w avq aug,
JO uoIsNzuL snowaavsyuy Jo s

1} O1az JR Mesaq HOysNyt
qupuyys = sanpea uvayy

°

* ry ne 33
oO re
Z \
g a. de
> \
[4
ww
z ae 4g
! \ den
Tz
g / | [3
a
wz ve a rer 40%
2) / i [|
a Zz oa
DS 2 . ray oh do
| a3 2782
zy bass
, 3 . xeon oo °
= : mde
4 a a on oe ces [a ce eee me t [es ce ee ee ee E
o xr 26.0.6 vo ye © 2». - 0 © ° eo 00000
° a ° <7 . soho TT ~ a2 terre
° (988 %) (sw) (unw99/6H ww) ('sw6 OOI/ uw 99) (8H ww)

1OH 4HOI3M 937 7 S3ONVISISAY O14 00078 aWIT dav 900

11





ENDRIN - VENOUS RETURN
~}
T
+30
TIME (MIN)

+10

j
i

ue
J

CTITTil J T
22282 888 #88
aonvasieay Auvnownind WioL (ons 19H

rt

T
2 2
Sere ast

15
165
54

He) aay 900

Froure 3. Mean values = standard errors from five venous return experiments showing the effect of intravenous
endrin infusion on systemic arterial blood pressure (ABP), hematocrit (HCT), and pulmonary yascular resistance.
Dogs were pretreated with the muscle relaxant succinylcholine chloride. Endrin began at zero time.

21



2275
2075
185
16-4
145

Og UPTAKE
(CC/MIN/ KG)

18>
16>

hee

144

12-7 8

CO, PRODUCTION
(CC/MIN/KG)

fo)
ri

a

\

\

\

i)

\

i}

i}

\

i}

\

i}
i}
1
\
\

RQ

° 10 20 30 60
TIME (MIN.)

Frcure 4, Effect of endrin and endrin plus oxygen on oxygen uptake and carbon dioxide
production (M +SE) (numbers of experiments performed are indicated in upper frames).

Appendix 39



ADRENALECTOMIZED

fe)

CHEMICAL SURGICAL
DENERVATION DENERVATION PHENTOLAMINE PHENOXYBENZAMINE
TIME (min.) -1I0 0 10 20 30 02468 02468 0123456 ° 10 20 30
ea sgt EHHiy 7 +H HHtYy
OE
— 100
75
150
Lo £
rs £ 125
E ho

75

Ficcre 2A. Effects of denervation and adrenergic-blocking agents on adrenalectomized dogs administered endrin.
Mean changes and standard errors for systemic arterial pressure (SAP) and renal blood flow (RBEF) for each
procedure on six adrenalectomized dogs, p values for SAP changes are 0.01 for the period of endrin response

and 0.1 or greater for res)

sponses to other maneuvers. p values for RBF changes are 0,1 for each maneuyer.

p values were calculated for the final point plotted for each maneuver as compared to zero time for that ma-
neuver, Zero time in each instance indicates that measured renal blood flow had been stable for 1 to 4 minutes.

INTACT
CHEMICAL SURGICAL
DENERVATION DENERVATION PHENTOLAMINE PHENOXYBENZAMINE

TIME (min.) -I0 © 10 20 30 02468 02468 0123456 Oo 10 20 30

175

SAP
mm Hg

100 +

200

175

150

RBF
mi/min

125

100

eT

Fravre 2B, Effects of denervation and adrenergic-blocking agents on intact dogs administered endrin.
Mean changes and standard errors for five intact dogs are as described for Figure 2A. p values for SAP are

0.1 or larger for all maneuvers except the response to phenoxybenzamine where p

RBF are 0.05 for response

alue is 0.05, p values for
to endrin and phentolamine and 0.1 or larger for responses to other maneuvers.

p values were calculated as in Figure 2A.



during the entire post endrin period. Samples
were collected in oiled syringes from the femoral
artery cannula and from the pooled venous out-
flow. Oxygen uptake was then calculated from
the A-V difference using the measured outflow
(cardiac inflow) at that time.

As previously reported,! pH was found to be
decreased by endrin. It was thought possible that
changes in pH performed a role in causing con-
vulsions seen in endrin poisoning, so the organic
amine buffer THAM [Tris(hydroxymethyl)-
amino-methane, obtained from Abbott Labora-
tories, North Chicago, Illinois] was infused as an
isotonic solution (36.34 gm/7) at a rate that would
maintain blood pH constant. The average vol-
ume of THAM required by the end of the 60-
minute period was 55 cc/kg (SD +4.3).

Heart rate, pH, and hematocrit were measured
in all experiments. Plasma total catecholamine
concentrations were assayed by a semiautomated
fluorometric procedure.’ Blood samples for cate-
cholamine analyses were withdrawn at specified
intervals during a 1-hour period after endrin
in intact animals and in bilaterally adrenalecto-
mized dogs prepared as previously reported.

Blood pressures were obtained by means of ves-
sel cannulation in connection with Statham pres-
sure transducers and registered on a Sanborn
multichannel direct-writing recorder. All cardio-
vascular experiments were of an acute nature,
terminating 1 hour after endrin administration.

III. Results.

A. Systemic Vascular Responses.—Figure 1
(all figures and tables are in the Appendix)
shows the changes in mean systemic arterial
pressure, cardiac inflow, and total peripheral re-
sistance within 1 hour after intravenous admin-
istration of 8 mg/kg of endrin. Six control
animals, not given endrin, showed relatively
stable values during this period. Differences in
measured or calculated parameters were evalu-
ated for significance at 15-minute intervals after
injection of endrin. In seven experimental an-
imals, significant changes in venous return (car-
diac inflow) and total peripheral resistance were
observed within 30 minutes after endrin injec-
tion. A marked rise in cardiac inflow was ob-
served to coincide with a steady drop in resist-
ance. Mean systemic arterial pressure remained
fairly constant. Convulsions beginning within
8 to 15 minutes after injection of endrin were
observed, and extensive salivation was seen in

32

all experiments. There was no consistent corre-
lation between the onset of convulsions and the
onset of hemodynamic alterations in all experi-
ments.

B. Cardiopulmonary Responses.—Figure 2 pre-
sents findings on the effect of endrin on the pul-
monary artery pressure and pulmonary vascular
resistance. Although pulmonary artery pressure
increases significantly, pulmonary resistance is
relatively unchanged because of the large in-
crease in pulmonary blood flow (Figure 1).
Mean left atrial pressure increased markedly
within 15 minutes after endrin injection as shown
in Figure 8. Right-atrial pressure remained low
throughout most of the experimental period.

Cardiac standstill occurred in some experi-
ments in which endrin was given, and the ex-
periments were necessarily terminated. The most
critical period for development of cardiac stand-
still was observed within 25 to 30 minutes after
injection of endrin and affected about 20% of
the dogs used. To determine if endrin has a di-
rect action on the heart, a series of experiments
was performed on isolated heart-lung prepara-
tions, in which the cardiopulmonary circulation
was totally separated from the remainder of the
dog. Details of the experimental preparation
have been previously described.* Ten experi-
ments were performed at an estimated endrin
dosage of 10 mg/kg, based on the weight of
heart-lung dog. Results from these experiments
are seen in Table 2 and indicate that left-heart
failure has occurred. Left-atrial pressures rose
to high values after endrin injection, while right-
atrial pressures remained low. ‘The more sus-
tained elevated left-atrial pressures seen in the
heart-lung preparation may have been due to the
higher dose. Experiments on the totally isolated
heart support those on the intact organ inasmuch
as effects are similar though occurring at a differ-
ent time interval.

C. Metabolic Responses—Experiments were
performed to determine the effect of endrin on
oxygen uptake and carbon dioxide production,
and results are presented in Figure 4. It is seen
that control animals show little change in these
parameters, whereas injection of endrin results in
marked increases in both oxygen uptake and car-
bon dioxide production. Oxygen was continu-
ously delivered into the Starling respirator from
zero time to the termination of experiments in
the separate study depicted in Figure 4. Results


creased erythrocyte size due to the Hamburger
(bicarbonate) shift. Although mean corpuscu-
lar volume (MCV) increases in most animals, it
is difficult to accept the directional change with
great assurance. The inherent error in enumera-
tion of red cells, which is necessary to calculate
MCV, is on the order of 11% when duplicate
samples are used and can be higher. However,
the tendency for MCV to increase suggests that
some of the unaccounted rise in hematocrit in
splenectomized dogs is attributable to an increase

in red cell size. The number of red blood cells
per cubic millimeter of blood also increased in
four of five experiments, which suggest some loss
of plasma fluid from the vascular compartment
as discussed in preceding paragraphs.

The fact that systemic arterial blood pressure
and cardiac output and hence cardiac work were
below control when left atrial pressure was ele-
vated by 20 minutes post-endrin demonstrates a
failing heart. A possible cardiac action of endrin
merits further investigation.

REFERENCES

1, Emerson, T. E., Jz., Brake, C. M., and HinsHaw,

L.B.: Can. J. Physiol. Pharm. +

Srisu, R. J., MacLe. L, D,, and Visscner, M. B.:

J, Appl. Physiol. 9: 297, 1956,

3. Wert, M. H., MacLean, L. D., VisscHer, M. B,, and
Seink, W. W.: J. Clin. Invest. 35: 1191, 1956,

4, Brake, C. M., Haerson, 'T. E., JR. Witrmers, L. E.,
and Hinsuaw, L. B.: Am. J. Physiol, 207; 149, 1964.

18

5. Gunrrerors, W. G, and Muttins, G. L.: Am. J.
Physiol. 204: 35, 1963.

. Reins, D. A., Horares, D. D., and Hinsuaw, L. B.:
Can. J. Physiol. Pharm, 42 : 599, 1964.

. Happy, F. J., Castrsent, @. 8., and Visscwer, M, B.:
Am. J. Physiol. 161: 836, 1950.

. Prankerp, T. A. J.: The red cell. Thomas: Spring-
field, 111, 1961, pp. 45, 46.

n


(TPR) increased slightly in control dogs and
decreased slightly in experimental dogs (P<0.2)
at the end of 45 minutes (Figure 1). Hematocrit
and pH remained relatively constant.

During the first 30 minutes, oxygen uptake re-
mained constant for experimental animals, then
decreased from 4.5 to 2.9 ml/min/kg at 60 minutes
(P<0.2). Oxygen uptake for controls demon-
strated an over-all decrease as compared to zero
time.

E plasma level increased sixfold at 30 minutes
to 4.7-fold at 60 minutes (Figure 44), whereas
NE increased tenfold after 60 minutes (Figure
4B). No significant changes in plasma levels of
E or NE occurred in control dogs during the 60
minutes of observation (Figures 44 and 4B).
Concentration of epinephrine diminished in ad-
renal glands. No change in catecholamine con-

centrations occurred in cardiac tissue (Table 1).
Taste 1, Tissue levels of epinephrine and norepinephrine.

Epinephrine Norepinephrine
Controls ‘Treated Controls ‘Treated
Adrenals (4)* 868.6 (5) 618.7+ (4) 138.9 (5) 160.4
pe/py =SE +534 ! +305 +25.0
Heart (4) 0.158 (5) 0.4 (4) 0.516 (5) 0.500
uS/gm +SE £0,053 £0,095 +£0.059, £0,085

* Number of animals given in parentheses.
7 Differs from controls at P<0.1 level.

B. Eviscerated dogs (five control dogs. five ex-
perimental dogs). Heart rate decreased in both
control and experimental dogs of this group.
The decrease in experimental dogs was greater
than that of the controls (P<0.05 at 30 minutes).
SAP of experimental dogs increased above that
of control dogs after 20 minutes and remained
elevated for the duration of the experiment (Fig-
ure 3). Stroke volume increased from 6 to 9 ml
in experimental animals at 20, 25, and 30 minutes
(P<0.1, Figure 3). Venous return decreased in
both control and experimental dogs. Flow from
experimental animals differed maximally from
control animals at 30 minutes (P<0.2). TPR/
kg in the experimental dogs increased only
slightly above that of the control values (P<0.2
at 60 minutes). pH level of plasma from experi-
mental animals decreased until the pH was one
pH unit below that of controls (P<0.1) at 60
minutes. Hematocrit increased only slightly in
this group of dogs.

C. Towicity study (24 dogs). Results from this
study are presented in Table 2 and show the
LD,o. to be 5 mg/kg.

24

‘Tanre 2. Toxicity of endrin given intravenously to dogs.

Dose No. of Mortality ‘Mean survival
(ng/kg) animals (%) time (hr) -+SE
10 5 100 211 £0.57
5 6 100 2.75 +£0.26
3 8 xo) 20.19 £8.13

1 5 0

IV. Discussion.

Increased systemic arterial pressure has been
reported as an early effect of a lethal dose of
endrin administered to dogs immobilized with
succinylcholine chloride,'* but the mechanism of
this effect has not been previously elucidated.
Studies with the chlorinated hydrocarbon DDT,
a related insecticide, have indicated that increased
levels of catecholamines are a factor in the lethal
cardiovascular effects of this compound.” In
this study with endrin, elevation of both epine-
phrine and norepinephrine levels and probable
parasympathetic activation, make it difficult to
evaluate the exact role of the catecholamines in
the cardiovascular effects of this compound. The
high levels of epinephrine and norepinephrine
found are probably of sufficient magnitude to
cause an increased SAP through increased cir-
culating blood volume. The high levels of epine-
phrine are, apparently, not producing the changes
in heart rate that might be expected because of
an overriding effect of central nervous system
stimulation or carotid and cardiac reflexes.

Pressure in a closed system is directly propor-
tional to flow and resistance of the system. Any
change in either of these latter parameters will
effect a change in pressure. In the present study,
venous return increased with corresponding ele-
vation of cardiac output while TPR was not
appreciably altered (P<0.2 at point of greatest
divergence from controls), Evisceration of dogs
greatly diminished the increase in venous return
after endrin, indicating that the primary source
of increased circulating blood volume was visceral
vasculature. The possibility of extravascular dilu-
tion was not considered probable since the hemato-
crit tended to increase even in the eviscerated dogs
when the splenic stores of RBC were removed, as
has been reported in splenectomized dogs during
acute endrin intoxication.’

Isolated liver studies have demonstrated that
injected catecholamines cause the organ to dis-
charge stored blood," and splenic contraction has
also been shown to be caused by increased levels
of epinephrine and norepinephrine.? The level
of these substances in the circulating plasma as


ENDRIN-ISOLATED LUNG
(CONSTANT FLOW)

ENDRIN INFUSION

a NEUSION

PH —> 7.27 7.28 6.89

PAP (mm Hg)
3
|

+25
04 —s
24

|

LUNG WT. (gm)

45—-
ao

354 ¢ j— T

30

HC T(%)

150

ao

130

120

110-4

DOG A.B.P. (mm Hg)

T T T T dl
10 15 20 25 30

TIME (MIN)
Figure 2. Mean values + standard errors from five dog pump-perfused isolated lung experiments in which endrin
was infused are shown. Dog ABP = table dogs mean systemic arterial blood pressure; Het = blood hematocrit;

A\ lung wt = change in isolated lung weight from control; PAP = pulmonary artery pressure. Pulmonary blood
flow was constant. Endrin infusion began at zero time.

20

T
“5

o4
|


show that both oxygen uptake and carbon di-
oxide production are increased with oxygen ad-
ministration. Mean RQ values, however, remain
relatively unchanged in all experiments. All dogs
given endrin convulsed strongly. There was no
apparent difference in the degree of convulsions
seen in animals in each of the experiments shown
in Figure 4. Table 3 presents the mean arterial-
and venous-blood concentrations of these gases,
and it is seen that arterial and venous concentra-
tions of oxygen are very low within 30 minutes
after endrin, venous concentrations decreasing to
2 vol %. With oxygen administration, only
venous blood shows a drop in oxygen content after
endrin. The arterial concentration of carbon di-
oxide after endrin decreases at 60 minutes, while
the venous concentration is seen to vary. It was
incidentally found that arterial blood was capable
of taking up more oxygen if it was equilibrated
in a tonometer with room air.

Acidosis was observed after endrin with pH
values regularly below 7.0 at 60 minutes post-
endrin. These data are seen in Figure 5. Infu-
sion of the organic amine buffer THAM main-
tained the pH constant, but oxygen administra-
tion had no effect (Figure 5). pH was well
maintained with THAM, but changes in hemo-
dynamic parameters were in the same direction
(Figure 6). Neither oxygen administration nor
THAM infusion altered changes in mean sys-
temic arterial pressure, although cardiac flow
(venous return) increased with THAM infusion
(Figure 6). Large volumes (approximately 500
cc/experiment) of an isosmotic solution of THAM
were required to maintain pH normal. This was
considered instrumental in lowering blood vis-
cosity and increasing circulating blood volume,
which would probably effect an increase in the
cardiac inflow (see effect of THAM on circu-
lating hematocrit, below).

D. Heart Rate, Hematocrit, and pH Changes.—
Figures 5, 7, and 8 present data on hematocrit,
pH, and heart-rate changes. There is some evi-
dence of bradycardia after endrin, which is miti-
gated by both oxygen administration and THAM
infusion. Hematocrit changes are insignificant
in all instances (Figures 7 and 8) but appear to
fall with THAM infusion (Figure 8), presum-
ably because of the volumes of fluid required
to maintain pH constant. Administration of
oxygen had no effect on pH changes as nor-

33

mally observed in animals given endrin alone
(Figure 5).

E. Adrenal Response—Previous work sug-
gested participation of the adrenal medulla in the
vascular response to endrin.? Experiments were
therefore included in the present study to de-
termine plasma total catecholamine concentra-
tions in 28 dogs given endrin. Findings are pre-
sented in Figure 9. Control experiments (ani-
mals not given endrin) showed statistically in-
significant changes (P > 0.10) in mean plasma
catecholamine concentrations, Animals given en-
drin alone exhibited large mean increases in cate-
cholamine concentration from less than 4 pg/?
of plasma to over 55 pg/l (P > 0.01) by the
termination of the experiments. Increases in
concentration were significantly depressed, how-
ever, when pH was maintained constant by use of
THAM values approximating only 10 yg/2 by
60 minutes after endrin (P < 0.05, compared
with animals given endrin without THAM).
Increases in catecholamine concentration were
somewhat less (43 pg/?) when oxygen was ad-
ministered with endrin, but this difference was
not significantly different from animals given
endrin alone (P > 0.10). A series of acutely
operated bilaterally adrenalectomized dogs was
given endrin. Values of catecholamine were low-
est at zero time and increased significantly to 2.6
ng/l by 60 minutes post endrin (P < 0.01),
Hemodynamic effects of endrin in adrenalecto-
mized animals are shown in Table 4. Responses
are not significantly different from those obtained
in dogs with intact adrenal glands.

IV. Discussion.

The summary schema shown in Figure 10
helps to evaluate results obtained in these experi-
ments. Endrin injected intravenously at a dos-
age causing death in approximately 75% of the
animals appears to have at least two actions: one
on the central nervous system and one on the left
ventricle. It is not understood why endrin has a
damaging action on the left ventricle while the
right ventricle appears to be unaffected. It does
not seem feasible that the lung would release
an agent in response to endrin that would have a
depressant action in the left-ventricular muscula-
ture. Results from isolated heart studies do not
suggest that increased cardiac work caused the
left ventricle to fail, since both cardiac output and
aortic pressure remain constant after endrin.



PROBLEMS IN AERIAL APPLICATION:

III. Mechanisms of Hemoconcentration During Acute Endrin Insecticide Poisoning

I. Methods.

A. General. Tn 45 experiments, mongrel dogs of
both sexes were anesthetized intravenously with
sodium pentobarbital, 30 mg/kg. Heparin sodium,
3 mg/kg body weight, was used as anticoagulant.
Superlethal amounts of endrin insecticide, 10 mg/
kg body weight, were infused intravenously at
0.5 to 1.0 ml/min. The endrin was dissolved in
95% ethanol (approximately 30 mg/ml), The
effect of the alcohol carrier has been previously
tested and has no systemic effect on the measured
parameters at the rates used. Pressures were
obtained with Statham pressure transducers and
recorded on a Sanborn direct-writing recorder.
In all of the following groups, mean systemic
arterial blood pressure and heart rates were ob-
tained from a cannulated femoral artery. Blood
pH was determined before and after endrin with
a Beckman expanded-scale pH meter. Hemato-
crits were measured before and usually at 10-
minute intervals after endrin using a micro-
capillary centrifuge. Gross visual and histologi-
cal examination of the lungs for edema was usu-
ally made at the end of each experiment.

B. Splenectomized and eviscerated animals. To
determine whether hemoconcentration after en-
drin is associated with splenic release of erythro-
cyte rich blood or if hepatosplanchnic pooling is
involved, the spleen was removed through a mid-
line incision in 10 dogs, while total evisceration
was completed in 10 others. Endrin was then
infused into these dogs and was also given to
10 sham-operated animals.

C. Dog perfused-lung preparation. In each of
five experiments, a table dog was used to supply
blood to perfuse an isolated lung. The pulmonary
artery was connected to the femoral artery of
the table dog with tygon tubing, and constant-
flow perfusion of the lung was established with
Sigmamotor pump. The lung was removed from
the donor-animal’s body and placed on a plastic

15

tray to allow continuous weighing with a strain-
gauge weighing device.” Blood from the cut pul-
monary vein flowed by gravity into a reservoir
and was returned to a cannulated femoral vein
of the table dog by a Sigmamotor pump. Pul-
monary-artery perfusion pressure was monitored
from the inflow tubing. Pulmonary blood flow
was measured with a cylinder and stopwatch.
Endrin was infused into the pulmonary artery
of the isolated lung and passed to the table dog
in the venous blood. Any direct effect of endrin
on the lung vasculature should therefore become
evident before affecting the table dog.

D. Venous-return preparation. In contrast to the
other dogs, these animals received succinylcholine
chloride to prevent convulsions. The basic prepa-
ration has been described in detail elsewhere.
Blood was led from the cannulated superior and
inferior vena cavae through tygon tubing into
& reservoir, situated in a warming water bath,
and returned to the cannulated right atrium with
a Sigmamotor pump. ‘The azygos vein was li-
gated. Total venous return (cardiac inflow or
pulmonary blood flow) to the heart was measured
with a cylinder and stopwatch. The reservoir
level was maintained constant with the return
pump. Small-bore catheters were secured in the
pulmonary artery and left atrium through needles
that were subsequently withdrawn, and pressures
were recorded. Right-atrial pressure was recorded
from the inflow tubing. Total pulmonary vascular
resistance (pulmonary artery—left atrial pres-
sure/pulmonary blood flow) was calculated at 10-
minute intervals after endrin. All of the above
parameters were followed for 60 minutes after
endrin administration. An earlier report from
this laboratory has shown the venous-return pre-
paration to be comparably stable over this period
of time and usually longer+

E. Mean corpuscular volume. In five splenectom-
ized dogs, blood pH, duplicate hematocrit, and
duplicate red blood cell counts were determined



PROBLEMS IN AERIAL APPLICATION:
II. Peripheral Vascular Effects of the Insecticide Endrin*

I. Introduction.

A clearer understanding of the mechanism of
action of insecticides is needed in order to deal
more comprehensively with insecticide poison-
ing. This is especially true in the case of crop-
dusting pilots, who are repeatedly exposed to
insecticides. Acute exposure to chlorinated hy-
drocarbon insecticides may be fatal in animals
or man, whether absorption is by ingestion, in-
halation or dermally.»* This group of insecti-
cides includes a large number of compounds,
ranging in toxicity from DDT, which is only
moderately toxic to mammals, to endrin, a
highly toxic poison in animals and man. Until
recently, little was known of the cardiovascular
effects of endrin poisoning. Reports from this
laboratory have shown that lethal intravenous
infusion of endrin causes severe cardiovascular
alterations in the dog, including early hyperten-
sion followed by hypotension, bradycardia, in-
creased cerebrospinal fluid and cerebral venous
pressure,’ and increased renal vascular resist-
arice.° Hyperactivity of the sympathetic and
parasympathetic nervous systems also has been
indicated? The chlorinated-hydrocarbon insecti-
cides aldrin and dieldrin also cause deleterious
vascular and neurological effects in animals. +
The present investigation was undertaken to de-
lineate the peripheral vascular effects of acute and
lethal endrin poisoning in the dog.

IL. Methods.

Mongrel dogs of both sexes were intravenously
anesthetized with sodium pentobarbital, 30
mg/kg. Pressures were obtained with Statham
pressure transducers and recorded using a San-
born direct-writing recorder. Heparin sodium,
3 mg/kg body weight, was used as anticoagulant.
Ten experiments were completed as follows.
Pressures were obtained from four sites along
the vascular system of a dog forelimb using a
modification of the Haddy technique to seg-

‘With the assistance of R. HE. Hopla and C. C. Gill,

ment the limb vascular bed ;° limb-brachial-artery
orifice pressure (LAP) was taken as aortic blood
pressure; small artery pressure (SAP) was ob-
tained from a PE 50 cannula placed in a meta-
carpal artery and advanced retrogradely under
the foot pad; small vein pressure (SVP) was
measured through PE 50 tubing secured in a
digital vein with its tip pointing upstream; and
large-vein orifice pressure (LVP) was maintained
at atmospheric pressure. The forelimb was sur-
gically separated from the body except for the
bone, artery, and, in half of the series, the major
nerves. The limb was perfused at natural flow
through the intact brachial artery at the dog’s
systemic arterial blood pressure. Venous blood
flowed by gravity from the cut brachial and
cephalic veins into a plastic reservoir and was
returned to the animal through a cannulated
femoral vein by a Sigmamotor pump. The ex-
tracorporeal system consisted of plastic tygon
tubing and was primed with 100 cc of high-
molecular-weight dextran.** — Limb-blood-flow
rates (BF) were obtained with a graduated cylin-
der and stopwatch. Resistances were calculated
by dividing the pressure difference across a par-
ticular vascular segment by the total lex blood
flow (limb resistances: total=LAP-LVP/BF;
arterial=LAP-SAP/BF; small _vessel=SAP-
SVP/BF; venous=SVP-LVP/BF). Aortic
blood pressure of the animal was recorded from
a cannulated femoral artery. Hematocrits and
pH’s were determined from femoral vein blood,
Electrical activity was recorded from a flank
muscle of the dog (biceps femoris) and from
a muscle group of the forelimb being studied.
Stainless-steel needles, placed longitudinally in
the muscles 4 to 8 em apart, were utilized. Lethal
amounts of endrin insecticide, 10 mg/kg of body
weight, were infused intravenously at 0.5 to 1.0
ml/min. The ethanol solution contained ap-
proximately 25 mg/ml of endrin.

** Obtained from Cutter Laborateries, Berkeley, California.



ga

. Wert, M. H., MacLean, L. D., V!
. HINsHAW,

» Van Stryke, D. D., and Neri, J. M

REFERENCES

. Emerson, Jr. T. E, Brake, C. M., and HinsHaw,

L B.: Gan J. Physiol. Pharmacol. 42: 41-51, 1964.

. Reins, D, A., Hormes, D, D., and Hixsuaw, L. B.:

CARI Report No. 63-26, 1964,

. Rens, D.A., Hormes, D, D., and Hinsnaw, L. B.:

Can. J. Physiol. Pharmacol. 42: 599-608, 1964.

scHER, M, B,, and
Sprnx, W. W.: J. Clin. Invest. 1191-1198, 1956.
L. B., Ginpert, R, P., Kura, H., and
Visscuer, M. B.: Am. J. Physiol 185: 631-634, 1958.

: J. Pharm, and

Exptl. Therap. 138-3 ; 360-375, 1962.

30

8.

10.

i.

Emerson, Jn, 'T. E. Can. J. Physiol Pharmacol, 43:
798, 1965.

. Purtuips, F. S., Ginman, A., and CRESCcEITELL, F. N.:

J. Pharm and Exptl. Therap. 86: 222-228, 1946.

Sravinowa, W., and Riecer, J.: The Pharmacologist,
6: 198, 1964.

Hrxswaw, L. B., Rerns, D. A. Wirraers, L. E., and
Masvcer, F. D.: In press. ,

. GuNTHEROTH, W. G., and Muwrins, G. L.: Am. J.

Physiol. 204: 35-41, 1963.


7.57

L J -— - - c-]
a 7° p<oe
<0.
7.3b bd
P<0.01
Tal t 1 ;
45,
~ 40F }
2 eee
35 j}-—_-_---- =} ===
>3.9 <0.6
3ol t 1 ‘
125 <0.05
L ae a oe _ + < 9 <0
fe  1ooF a sin ene oe oo oe a er
qa NL i
w aw 75 O-—0 CONTROL IN Z
@—e EXPERIMENTAL —h—-4—- ff
50 L L 1 1 1 7
2
c.
-r 2?
ws
ee
ak
3
ge
Ww
>
150°
a 2 25h
GE
E loor
= 7k 1 1 L jl
Zz <0,2
= O3r v
Ze
& 8 o2+ == —
& a “ ~sa--8
Toot
w
SE is; <o.t <ou
ary i <ol x, <0
ag
ge lof by Me
= _—
ue ee ae ee SS a 4
S g L L i 4 Lt L 1 i rT L 4 1 1 L Jj
ir ot
5 -10-5 0 5 10 I5 20 25 30 35 40 45 50 55 60
TIME (min.)
Fieure 8. Data obtained from eviscerated control dogs in venous return studies compared to that from eviscerated
dog venous return after endrin infusion.

27



Intact dogs in these studies developed decreased
glomerular filtration rates and renal blood flow
with systemic hypertension and bradycardia dur-
ing the first hour after exposure and demon-
strated a tendency to return toward control
values with time (Figure 3).

It appears that endrin has no direct effect
on the kidney vasculature,’ since previous work
has demonstrated that endrin stimulated the sym-
patho-adrenal system, and an adrenergic block-
ing agent caused return of renal blood flows
toward control values. Animals that were adren-
alectomized did not develop the marked increase
in renal resistance seen in intact animals. Lack
of response in adrenalectomized dogs (Figure
2A) and the sharp increase in renal blood flow
when phentolamine was administered to intact
animals (Figure 2B) are evidence that changes
in renal vascular resistance are primarily due
to circulating catecholamines.

Changes in GFR in intact animals coincident
with development of hypertension and decreased
renal blood flow could also be accounted for by

circulating agents that cause afferent arteriolar
constriction.

The autoregulatory response of increased renal
resistance to systemic hypertension has been
shown to be only a fraction of the total change
seen in endrin poisoning: In the adrenalec-
tomized animals, this autoregulatory response be-
comes more significant as systemic hypertension
and bradycardia develop to the same extent in
adrenalectomized animals as in intact (Figure 1)
but with a much smaller increase in renal vas-
cular resistance (Figures 2A and 2B).

Effects of the central nervous system on the
kidney may be derived (a) directly from neural
control of the renal vascular bed or (b) indirectly
from central-nervous-system-potentiated systemic
hypertension and adrenal-gland discharge. Di-
rect neural control has a minor and variable
role as indicated by changes in blood flow dur-
ing the denervation experiments (Figure 2A
and 2B). Systemic hypertension causes an auto-
regulatory response that has previously been
shown to play a minor role in changes in renal

ENDRIN

2

ADRENAL GLANDS

ot

SYSTEMIC
+++
HYPOTENSION —_

v vv
v
AUTOREGULATION

CENTRAL NERVOUS SYSTEM

SYMPATHETIC
NERVES

y
HYPERTENSION

Figure 4, A schema summarizing the effects of endrin on the kidney.

5



SURGICAL DENERVATION
CHEMICAL DENERVATION
0

10

30.

PHENTOLAMINE
Joo co, rate om

20

GFR ml/min
yw oA
ce} fo} fo}

6

—_—

INTACT
O—© ADRENALECTOMIZED

150 7

125 7

100 +

HEART RATE
N
a
+

a
°
+

50 60 TIME (min.)

Ficuxe 3. Comparison of the mean changes and standard errors for glomerular filtration rate (GFR), filtra-
tion fraction (FFo,eq,) from clearance of creatinine, and heart rate in intact and adrenalectomized animals

administered endrin.

p values for GFR are 0.1 for all points except the 80-minute sample in intact animals, which is 0.05. p
values for FF oye; ave 0.1 or less for all points plotted. p values for heart rate are 0.1 for the 10-minute point
in both groups and the 60-miute point for intact dogs. All other points have a p value of 0.01 in both groups.

Points at the top of the figure indicate the mean time at which the procedures indicated in Figures 24 and

2B were done,

mained constant, and GFR showed only a slight
depression at an earlier time (Figure 3). Figure
1 shows the record of development of hyperten-
sion and bradycardia after endrin in typical in-
tact and adrenalectomized animals,

IV. Discussion.

Factors that may be involved in changes of
renal function after endrin poisoning are the
effects of endrin on the adrenal glands, the cen-
tral nervous system, and on the kidney directly.


before and 15 and 30 minutes after endrin. Mean
corpuscular volume was calculated by dividing
the percentage of red blood cells per 1,000 ml of
whole blood by the red cell count (erythrocytes
per cubic millimeter of blood).

II. Results.

General effects of endrin seen in all groups
include severe tonic-clonic convulsions and hyper-
excitability to noise (except in succinylcholine-
treated animals), bradycardia, copious mucoid
salivation, decreased blood pH, and_ increased
hematocrit as previously reported. Histological
examination of lung tissue in all groups after
endrin showed little evidence of pulmonary
edema; however, by gross examination, some
lungs appeared edematous.

Mean values, with standard errors, of percent
change in hematocrit from pre-endrin levels and
systemic arterial blood pressures from 10 splenec-
tomized, 10 eviscerated, and 10 sham-operated
dogs receiving lethal amounts of endrin are
graphed in Figure 1. Arterial blood pressure of
the sham-operated group failed to fall until about
20 minutes after endrin but decreased steadily
beyond this point. Blood pH decreased from
7.29 to 6.85 and 6.88 by 30 to 60 minutes, respec-
tively, after endrin. Arterial blood pressure of

the splenectomized and eviscerated groups began
to fall immediately after endrin and continued
downward to levels significantly lower (P<0.01)
than the sham-operated group by 60 minutes
post-endrin. Blood pH in the splenectomized
dogs decreased from 7.29 to 6.66 and 6.71 at 30
and 60 minutes, respectively, after endrin; blood
pH fell from 7.24 to 6.57 and 6.49, respectively,
at 30 and 60 minutes post-endrin in the eviscer-
ated group. The hematocrit increases of the
splenectomized and eviscerated groups are not
significantly different (8% increase each) and
attained a value about half as high as the sham-
operated group (14% increase) by 30 minutes
after endrin. This relationship was maintained
as the hematocrits remained relatively constant
(8%, 7% and 15% increase respectively) from
30 to 60 minutes after endrin.

Table 1 shows the effect of endrin on mean
corpuscular volume, hematocrit, red-cell count,
pH, and arterial blood pressure in five splenec-
tomized dogs. Arterial blood pressure fell, severe
acidosis developed, hematocrit increased, erythro-
cyte concentration increased slightly, and the
calculated mean corpuscular volume increased in
four of five experiments.

Figure 2 gives mean data, with standard errors,
showing the effect of endrin on the table dog and

‘Taste 1. Effect of endrin on mean corpuscular volume in splenectomized dogs.
‘Time after ABP Het RCC MCV
Exp. No. endrin (min) (mm Hg) pH (%) x10* (cu mm) (eu 4)
1 0 1% 731 45.0 7.58 59.4
15, 160 7.26 49.0 8.38 60.3
30 130 6.52 56.0 8.62 65.0
2 U 150 7.30 37.0 5.62 65.9
15 145 7.30 87.5 6.50 57.6
30 55 6.40 375 6.63 56.6
3 0 135 7.34 31.0 6.01 51.6
15 150 7.18 83.5 5.51 60.8
30 50 6.39 375 5.86 64.0
4 0 165 7.32 45.5 6.55 69.5
15 175 732 47.0 6.83 68.7
30 120 6.56 50.5 6.83, 3.9
5 0 145, 732 45.0 TAL 63.3
15 145 7.32 46.0 745 618
30 100 50.0 7.55 66.3

6.59

‘Average endrin infusion time = 9 min; ABP = mean systemic arterial blood pressure Het = blood hematocrit; RCC = erythrocyte

count; MCV = mean corpuscular volume.

16


vascular resistance.t_ This evidence indicates that
the major influence causing changes in the renal
vascular bed in response to acute endrin poison-
ing was from effects of circulating catecholamines
originating from the adrenal glands.

The schema of Figure 4 presents a summary
of ways in which endrin may affect the kidney

hemodynamically and functionally, directly
through central nervous system potentiated re-
lease of catecholamines and sympathetic nerve
discharge, or indirectly through systemic hyper-
tension and autoregulation in acute poisoning and
terminal systemic hypotension in chronic poison-
ing

REFERENCES

1. Rers, D. A., Hormes, D. D., and Hinsuaw, L. B.:
CARI Report No. 63-26 1964.

2. Exterson, T. E., Brake, C M., and Hinsxaw, L. B,:
Canadian J. Physiol. Pharm, 42: 41, 1964,

8. Sarru, H. W.: The Kidney. Oxford University Press

N.Y., NY, 1955, p. 445.


RIGHT ATRIAL PRESSURE

PULMONARY ARTERY PRESSURE

0G ABP (nna) LEFT ATRIAL PRESSURE

CARDIAC INFLOW (ee/min/thg}

imag)

(mate)

ENDRIN - VENOUS RETURN

gion meusion

we —> 160 66 96

“7

54
Ba
15:

5
a
Kite
3
15

35
4 _
5.

206
1954
1156
15
1354
115.

T T T T T ]
+10 +20 +30 +40 +50 +60

TiMe GAIN)

Figure 4. Mean values + standard errors from five venous return experiments showing the effect of intravenous en-
drin infusion on several parameters. Dog ABP ~ dog's mean systemic arterial blood pressure. Dogs were pre-
treated with the muscle relaxant succinylcholine chloride. Endrin infusion began at zero time.

22


AM 66-11

PROBLEMS IN AERIAL APPLICATION: I.-V.

L. B. Hinshaw
T. E. Emerson, Jr.
J. A. Rieger, Jr.
W. B. Stavinoha
L, A. Solomon

V. Fiorica
D. A. Reins
D. D. Holmes
Approved by Released by
Blt Hite (USitge€ so
J. Roperr Diuiz, M.D. P. V. Smerr, M.D.
Curr, Civ AERoMEDICAL Feperan Am Surgeon
Iystrrure
June 1966

FEDERAL AVIATION ADMINISTRATION
Office of Aviation Medicine



ENDRIN

intravenous

injection
v
CNS Left Ventricle
Sympathetic Adrenals Parasympathetic
nerves nerves
Heart Failure
Excessive
Increased Marked salivation Decreased
__venous increase Cardiac Output

return in plasma
catecholamines

Convulsions
(Skeletal Bradycardia

Muscles)

Vv
Decrease in

Increased effective gaseous
cardiac transport in lungs
output

hypoxia

Decrease in
vascular
resistance

Fiaure 10. Suggested schema for action of endrin in dog.

Appendix 45



Control experiments, in which only alcohol was
given, were completed for all of the preceding
groups and shoived no appreciable changes from
baseline levels.

IV. Discussion.

Results from this investigation show that fore-
limb vascular resistance increases markedly with-
in a few minutes after intravenous administra-
tion of lethal amounts of endrin insecticide. Most
of the total resistance increase is usually in the
small vessel segment, of which the arterioles con-
stitute the major resistance to blood flow.

A. Mechanisms of vascular resistance changes. It
is apparent that sympathetic innervation of limb
vessels is not necessary for the large resistance
increases following endrin administration. The
resistance patterns of the innervated and dener-
vated groups (Figures 1 and 2) are similar for
the first 10 to 15 minutes after endrin; however,
the experiments in which nerve section and phen-
tolamine injection were completed after endrin
indicate that the nerves can account for a con-
siderable portion of the total resistance increase.
Earlier experiments show that in the kidney the
increased vascular resistance that normally occurs
following endrin infusion is due primarily to
circulating catecholamine-like agents but that
the nerves also play a role° The observation
that resistance increases considerably more in
the denervated limbs than the innervated is note-
worthy. It might be expected that resistance
in the innervated limbs would increase as much
as, or more than, in the denervated limbs; how-
ever, the opposite occurs. Recall that most of the
early increase in limb resistance in both groups is
in the small vessel segment. However, resistance
in this segment begins to fall in the innervated
limb while it continues to rise in the denervated

ones at 10 to 15 minutes post-endrin. The
difference in these groups is probably due to the
high level of muscle activity in the innervated
limb and subsequent high local concentration of
vasodilator metabolites that antagonize the effects
of sympathetic nerve activity and circulating
constrictor agents. It has been shown elsewhere
that a slight local excess of metabolic breakdown
products causes an active decrease in limb resist-
ance, most of which occurs in the small vessel
segment.®

B. Mechanism of vascular fluid shifts. The dis-
proportionately large increase in pre-capillary
resistance compared to the increase of post-
capillary resistance and fall in limb blood flow
should result in a decreased capillary pressure
and consequent loss of interstitial fluid. This
is indicated in the last group of dogs, in which
limb weight falls progressively after endrin.
Most of the early weight loss can be explained
as a result of decreasing blood flow and, probably,
capillary reabsorption. The later fall in weight
when limb flow stabilizes appears due primarily
to capillary reabsorption of interstitial fluid;
however, resistance continues to increase so a fur-
ther decrease in vascular volume cannot be ex-
cluded. Since the hematocrit increases, it seems
unlikely that hemoconcentration results from loss
of vascular fluid in peripheral skeletal muscle or
skin.

The development of severe hypertension after
endrin in the nonconvulsing group compared to
the hypotension in the convulsing groups is not
readily explainable. Succinylcholine chloride
has no cardiovascular effects in the concentra-
tions used.?_ The extremely low pH’s in the con-
vulsing, spontaneously breathing dogs might play
a role in their failure to develop hypertension.” °



Five, innervated, isolated, dog-limb prepara-
tions were prepared as described above except
that the bone was severed to allow continuous
weighing of the limb with a strain-gauge weigh-
ing device. Changes of limb weight of 0.1 gram
could be determined accurately. Animals were
pretreated with the muscle relaxant succinyl-
choline chloride (Anectine, 0.5 mg/kg body
weight) to prevent convulsions that interferred
with limb weight measurements. Additional
succinylcholine was given as needed. Respiration
was accomplished by means of a Harvard con-
stant-volume respirator. Endrin insecticide was
given, and all parameters were followed for 30
minutes, at which time the limb nerves were
severed. The adrenergic blocking agent, phento-
lamine (Regitine, 5 mg), was then injected into
the brachial artery.

Ten control experiments were completed in
which the animals received only the alcohol car-
rier used in the endrin-infusion series. Five
limbs were innervated and five were denervated.

III. Results.

A. General. Severe tonic-clonic convulsions ap-
peared in all animals approximately 10 minutes
after the onset of endrin infusion, except in the
succinylcholine-treated group. Electrical activity
in the dog flank muscle of both groups reached
a mean peak frequency of 223 per minute 20 to
30 minutes after endrin. Bradycardia, copious,
mucoid salivation, and hyperexcitability to noise
also occurred.

B. Innervated forelimb experiments. Mean data,
with standard errors, before and after infusion
of endrin are presented in Figure 1. Total limb
vascular resistance increased from 2.5 to 4.6
resistance units (mm Hg/ec/min) 10 minutes
after endrin and rose to 5.2 units by 30 minutes.
Most of the early increase occurred in the small
yessel segment (small artery to small vein),
while the arterial segment contributed the major
resistance to blood flow during the later phase.
Limb blood flow decreased progressively from
approximately 20 to 6 ce/min per 100 gm. Mean
electrical activity rose from near zero to peak
frequency of 284 and 435 per minute in the dog
flank muscle and forelimb muscle, respectively.
Systemic arterial blood pressure showed an early
increase but decreased from 20 minutes post-
endrin until the end of the experiment. These
changes were accompanied by a marked fall in

blood pH from 7.21 to 6.67 and increased hem-
atocrit. Respiration was spontaneous and essen-
tially the same frquency as the convulsions.
©. Denervated forelimb experiments. Figure 2
shows mean data, with standard errors, before
and after endrin infusion, Limb-vascular-resis-
ance increases were similar to the innervated
group (Figure 1) for the first 10 to 15 minutes
after endrin. Beyond this point, limb resistance
increased steeply to high levels, most of this re-
sistance increase occurring in the small vessel seg-
ment, Limb blood flow decreased from 28 to 2
cc/min per 100 gm. Electrical activity increased
from a mean frequency of near 0 to 160 per
minute in the dog flank muscle but did not
change appreciably in the denervated forelimb.
Systemic arterial blood pressure increased ini-
tially and fell subsequently as in the preceding
group. Blood pH fell from 7.25 to 6.69 and
hematocrit increased.
D. Innervated, weighed forelimb experiments.
Figure 3 gives mean values, with standard errors,
showing the effect of endrin on leg weight and
other parameters. Arterial blood pressure be-
gan to increase at about the 15th minute follow-
ing the start of endrin infusion and continued
upward for the remainder of the experiment.
Blood flow in the isolated limb fell from 16 to
4 cc/min per 100 gms of leg and total limb
resistance increased approximately five fold, most
of which was due to an increased resistance in
the small vessel segment. Venous resistance also
increased approximately five times over control.
Concomitant with these changes was a steady loss
of leg weight (—5.5 gm) and increase in total
body hematocrit. Blood pH fell from 7.32 to 7.14,
a value appreciably higher than in the preceding
groups in which respiration was spontaneous.
Figure 4 represents mean data showing the
effect of denervation and adrenergic blockade on
limb resistances after endrin. Sectioning the
nerves to the isolated limb at the peak post-
endrin response resulted in an immediate fall in
total resistance of five resistance units, and sub-
sequent injection of phentolamine dropped total
resistance another five units. Most of the re-
sistance drop was in the small vessel segment.
Limb nerve section in control experiments de-
creased total limb vascular resistance approxi-
mately 100%, while phentolamine injected into
the same limbs did not appreciably affect limb
resistances.


Appendix

1205
100+
804
605
4074
204

SYSTEMIC ARTERIAL
PRESSURE (MM HG)

. ENDRIN (3 MG/KG)

Ved

2205
2004
1804
1607
1405
1205
100+

80-4

CARDIAC INFLOW
(CC/MIN/KG)

604
40+
204

@—e ENDRIN + 02
0-—0 ENDRIN + THAM

2.45
2.05
1.65
25
0.84
0.45

TOTAL PERIPHERAL

P<.Ol P<.0l P<.05

aN 4 4 t
\
WAS

“Rapp pe

RESISTANCE (MM HG/CC/MIN/KG)

T T T T T T 1
° 10 20 30 40 50 60
TIME (MIN.)

Freune 6. Changes in systemic hemodynamics in dogs treated with endrin plus oxygen
and endrin plus THAM (M +SE) (numbers of experiments are shown in upper frame).

Time, post-endrin Oaygen administered
(minutes) (cc/min/kg) (M +SE)
0 530 +79
10 563 £49
20 650 +119
30 744 +31
40 781 +20
50 810 +43
60 TAT £33 “
41



(ug/L PLASMA)

NOREPINEPHRINE

8- e TREATED
7- o CONTROL
6-
5-
4-
3-4
25 —
7 a
a= —---3--—--—---- 3
.@) ' ' t
fe) 30 60

TIME (MIN.)

Figure 4B. Changes in norepinephrine levels in plasma of experimental and control dogs.

29



Taste 1. Endrin toxicity studies in dogs.

Dose
(mg/kg) 1 2 13 5 10
No. animals_.___... 5 5| 8 6 5
Mean survival Permanent) 24 | 20 3 2
times (hr) survivors
Percent mortality___| 0 40 | 75 100 100

TasLe 2. Effect of endrin on isolated heart (M +SE).

A. Experimentals (five preparations)

Time ABP* RAP LAP Cardiac output

(min) (mm Hg) (mm Hg) (mm He) (co/min/kg)
0) 91 £55 6.9 +3.0 8.4 44.1 TA 45.1
20 94 46 5.2 42.1 10.3 £4.3 76 46.4
40 94 46.1 5.3 42.2 14.3 44.6 72 45.2
60 92 46.8 5.6 £23 30.0 £4.0 67 +5.8

B. Controls (five preparations)

Time ABP* RAP LAP Cardiac output
(min) (mm He) (mm He) (mm He) (co/min/ke)
0 "90 44.8 2.1 +2.0 3.6 41.2 80 2.5
20 90 £4.2 3.0 42.3 3.2 40.8 79 43.1
40 86 £7.4 3.1 £2.8 2.8 £0.8 78 43.1
60 85 47.5 3.2 43.1 3.8 £0.9 76 £4.05
*ABP=arterial blood pressure.
RAP=right atrial pressure.

left atrial pressure.

Appendix 46


(ug/L PLASMA)

EPINEPHRINE

8- ° TREATED

T- ° CONTROL

fe) 30 60
TIME (MIN.)

Fieure 4A. Changes in epinephrine levels in plasma of experimental and control dogs.

28


vascular resistance.t_ This evidence indicates that
the major influence causing changes in the renal
vascular bed in response to acute endrin poison-
ing was from effects of circulating catecholamines
originating from the adrenal glands.

The schema of Figure 4 presents a summary
of ways in which endrin may affect the kidney

hemodynamically and functionally, directly
through central nervous system potentiated re-
lease of catecholamines and sympathetic nerve
discharge, or indirectly through systemic hyper-
tension and autoregulation in acute poisoning and
terminal systemic hypotension in chronic poison-
ing

REFERENCES

1, Reins, D. A., Hotmes, D. D,, and Hinsuaw, L. B.:
CARI Report No. 63-26 1964.

2. Emerson, T. E., Brake, C M,, and Hinsuaw, L. B,:
Canadian J, Physiol. Pharm. 42: 41, 1964,

3, SmirH, H. W.: The Kidney. Oxford University Press

N.Y., N.Y., 1955, p. 445.


ENDRIN INFUSION

—

: Popob- Le

to 4 /

tit

4 HEMATOCRIT (% INCREASE)
aa

HEMATOCRITS

o4 (ZERO TIME)
—-— SHAM OPERATED 40%
—— EVISERATED 41%
----- SPLENECTOMIZED 44%

MEAN ARTERIAL BLOOD PRESSURE (mm Hg)

T T T T T T
0 10 20 30 40 50 60
TIME (MIN)

Ficure 1, Mean values + standard errors from five dogs experiments are graphed demonstrating the effects of en-
drin insecticide. A hematocrit = increase in hematocrit from control pre-endrin level. Endrin infusion began at
zero time.

19



APPENDIX

25 1205 444 fi2f
x Z 1004 ~F. —-} ~ 4 ~ St 8
re eres eC
«= 80
&=
ew 604
z a 40-4 @——e ENDRIN
eg 0---0 CONTROL
9g oe 20-4
Ho
° T T T T T T 1
160-5
1404
— jf —
el | HG
Zo
ra S 1004
8S 804
qe —3-—3- B+ ~3- 3 __§__J_-—J~_J_—
83 64 GU “be -}-}--}--}--}- -}--}--}
ge
° 407 ¢ ¢ t
204 P<.0l P<.05 P=.0l
g ° T T T T T T 7
2
7 5S 2.45
a5
ES 204
zg 1.6 els
g2 (ey) PISO E+H+
== ~--}- --f. 4
ws 124 \
ap KR.
ay 087 ALL j4-t— fe —
52 044
2 b P<.0l P<.0l pcos
.77) fe) T T T T T T 1
w ° lo 20 30 40 50 60
TIME (MIN.)

Ficure 1. Effect of endrin on systemic hemodynamics in the dog (M +SE) (numbers of

experiments are indicated in upper frame).

36


5
2a. 287 7
4
z= 4 7 [- \] 7 5 8 t
Wis 5
& = 20- a i} YH
>w 164 i s
ge 6 6 6 6 &
< —_
Re
=e 84
ac e—e ENDRIN t ‘ t
4-7 o-——o CONTROL P<.Ol P<.05 P<.05
ce) T qT T t T T 1

PULMONARY VASCULAR
RESISTANCE (MM HG/CC/MIN/KG)
o
!

T
ce] 10 20 30 40 50 60
TIME (MIN.)

Froure 2, Effect of endrin on pulmonary hemodynamics in the dog (M +SE) (numbers
of experiments are indicated in upper frame).

Appendix 387



found in the present studies is presumed sufficient
to cause this action. An increased metabolic
activity might also be expected to cause emptying
from visceral pools, but a decrease in oxygen
uptake after endrin indicates a lessened activity.
Depletion of epinephrine content of the adrenal

of dogs treated with endrin indicates the source
of the high plasma levels of this hormone. The
level of norepinephrine in the adrenal did not
change, and epinephrine and norepinephrine lev-
els in the myocardium remained unaltered.

<0.01

z 100 -
aa L 2
re °° A INKL
@ = 80F ia >
a £ 70 =
8 RS) | } <0.01 <0.02 <0
w 60 F bg gE EES
50 LL t rn 1
175 7
= 150 f jthyphitit
12 150 i A } hye
tof 125 <0.01 <0.01
7) E a v v +
E oo pimp pL tp tp
e) 7k 1 L 1 }
S o2 <o4 <0.2 <ar
gener ~-J--J--}--b-- “bob
> 801 | Pan gnng a} — re oe me a —— .
ao
a LL it it
E
wu 22.7 o-=0 CONTROL
——e EXPERIMENTAL
5 _ 2o- *—° jt Ke
ov
Ss 157 Xe
Ye iof ¥  <oo <0.01 <0.01
O35 “a + .
2 st | dmng—fa=3 <b gg fh --B- ay
° fe) LL 1 1 1 1 1 1 i 1 1 1 1 1 1 J

-10-5 O 5 10

Fieure 1.
presented directly under points for control dogs.

15 20 25 30 35 40 45 50 55 60
TIME (min.)

Comparsion of values from intact control and experimental dogs. P values at 30, 45, and 60 minutes are



INTACT DOG

= yeh yyy
200- |} ! ah
ARTERIAL athe
BLOOD PRESSURE 100-
(mm Hg) E
O-;
RENAL BLOOD FLOW = >
(cc / min) 222
ADRENALECTOMIZED DOG “TAM
AGERE HHT:
200- | EH j “+
ARTERIAL TAT Thy CST
BLOOD PRESSURE 100-"|- pase
(mm Hg) : ;
of |
RENAL BLOOD FLOW /
(ce / min.) 232 180 195 174
Time (min.) fe) 10 15 20
ENDRIN

Ficure 1. Development of bradycardia and hypertension after endrin in intact and adrenalectomized dogs.

III. Results.

A. Renal denervation experiments (five dogs).
These animals showed a marked increase in renal
resistance with decreased renal blood flow, sys-
temic hypertension (Figure 2B), and brady-
cardia (Figure 3) within 10 to 20 minutes after
infusion of endrin. Denervation of the renal
pedicle produced a variable effect on renal blood
flow. In some instances, a mild increase in flow
occurred. Injection of phentolamine into the
renal artery of these animals significantly low-
ered renal vascular resistance and restored blood
flows through the kidneys to a level nearly equal
to control values. GFR and filtration fraction

showed decreases at about 30 minutes after en-
drin, but subsequently recovered (Figure 3).

B. Renal denervation in adrenalectomized ani-
mals (sie dogs). The mean increase in systemic
arterial pressure and development of bradycardia
were similar in adrenalectomized animals and in
intact-animal experiments (above). There was
a variable effect on renal blood flow in contrast
to results obtained from animals with intact
adrenals (Figure 24). The mean effects of de-
nervation and phentolamine were less than those
observed in intact animals, although infusion
of phenoxybenzamine resulted in a marked in-
crease in blood flow., Filtration fraction re-


60
55

BOL

50
45
40
35
30
25
20

15

10

PLASMA TOTAL CATECHOLAMINES

Appendix

@—e CONTROL
A---A ENDRIN
e——a ENDRIN + 02
@—-—@ ENDRIN + THAM

-© ENDRIN +
ADRENALECTOMY &-

a

A

o
L 4

oo
“”

L 4
.7

Z
Zz -

MINUTES

Fievre 9. Effects of endrin and various treatment procedures on plasma total catecho-;
lamine concentrations (mean values).

PLASMA TOTAL CATECHOLAMINES p&/L (M =SE)

Time Endrin Endrin Adrenalee-
(min) Control Endrin plus plus tomized
oxygen THAM plus endrin
0 1.1 40.6 3.4 +3.3 3.7 43.4 1.5 41.0
(N=6) (N==6) (N= 5) (N=5)
+30 1.8 42.1 | 41.5 £389 | 26.0 420.0] 6.6 £5.2
(N= 6) (N= 6) (N=5) (N= 5)
+60 1.8 41.9 | 56.8 436.4 | 43.4 423.2] 10.3 49.6
(N=6) (N= 4) (W=4 (N=4)



60-

@——e ENDRIN
bE ©——oO CONTROL
6G 504 5
oa 6
Eo 7
4 xe i 6
= £ ao he —— dt 1 2
=x
ul 4
P>.10 P>.10
30 T T ~ 1
1807
160-7 .
6.6 6 16 76
wz '407 ‘f 6,6 sf sh-lhns j 2 aoe ae es cee y
2 STIL -3- | ~ L ~
- = 120- Ng aes ee 5 7 8 5 5
“> t oe IN
2 100+ f— ~—
& &
4
ZB 6 t t t
60-4 P>.10 P<.05 ~— P>.10
40 T T T T T T 1
fe) 10 20 30 40 50 60
TIME (MIN.)

Ficune 7. Effect of endrin on hematocrit and heart rate in dogs (M +SE) (numbers of
experiments indicated at each time of measurement),

Appendix 42


“soywatdsar auinqoa
queysuos 8 YIN pausHTdoDe sua MOL MATdsOL “PLOY ouyoNPTAuPONs yNUNEEL PsN ey WE paywaxjerd aiam spwUNY “aU UOYsNgUT eFvreAT
ayn siuasosdon yauis Jo do} 1H Uq BU, “UIE OER I HAAG WOISHIUL WLApUGT “Maoys oa uINpUD JO syrtMOLLTE [WAAAT JO VORSNzUE suouOAwATL Jo sjraye aud
‘sjusmpiedse Sop aay Wodj S1o4le pAMpUNyS F NONTLA URayE NIWOUIBAMSWOUE IBS qT [UNUTOD TEN uopnardetd qu, parwaseuUy ‘payeposy “g AMAT

—= — 3
= = 7

6.69
39%

[
i
f

TIME (MIN)

ENORIN INFUSION
7.28
30
x TOTAL
4 SMALL VESSEL
# ARTERIAL
VENOUS
1
8

DOG LIMB — NATURAL FLOW — DENERVATED

est ee te een ee

Eb peeteeantretserrr.se ws e og eege0 gseege

g sere
(1/29 /6H wus) (sw OO! /‘uw /29) (6H ww)

S3ONVLSISSY 014 00078 BNI7 dav 900

12


re

o

. Gownry, C W., Granan, A. R., SeGUIN,

REFERENCES

. Arena, J. M.: Poisoning. Chemistry-Symptoms-Treat-

ment. Thomas: Springfield, 11L, 1963.

. Emerson, T. E., Jk., Brake, C. M., and HINsHaw,

L. B.: Cardiovascular effects of the insecticide endrin.
Can. J. Physiol. Pharm, 42; 41-51, 1964.

J.J, Stay-
Ray, G W., and Wavp, R. A.: A study of the phar-
macological properties of the insecticide aldvin. Can.
J. Med. Sci, 30 3520-533, 1952.

. Gowpry, C. W., Granam, A. R., Seeury, J. J., and

Sravraky, G. W.: The pharmacological properties
of the insecticide dieldrin. Can. J. Biochem. Physiol.
32: 498-503, 1954.

. Happy, F. J, and Scorr, J. B,: Effects of flow rate,

venous pressure, metabolites and oxygen upon resist-
ance to blood flow through the dog forelimb. Cire.
Res., 15, Suppl. 1:49-59, 1964.

. Mornar, J. 1, Scorr, J. B, Prouricn, BE, D, and

Happy, F. J.: Local effects of various anions and

14

a

10.

i.

- Suitable for physiological measurements, J.

3. NEGHERBON, W. O.:

. Pace, I.

H+ on dog limb and coronary vascular resistances.
Am. J. Physiol. 203 : 125-132, 1962.

Nasu, C. W., and Hears, C.: Vascular responses to
catecholamines during respiratory changes in pF.
Am. J. Physiol. 200: 755-758, 1961.

Handbook of Toxicology. Volume

IIT: Insecticides. Saunders: Philadelphia, 1959.

and Ormstep, F.: The influence of
gas mixtures on arterial pressure and
reactivity, in “normal” and hypertensive
: 801-819, 1951.

H,
respirator
vascular
dogs. Cir

Reins, D. A., Horses, D. D., and HrnsHaw, L. B.:
Acute and chronic effects of the insecticide endrin
on renal function and renal hemodynamics. Can. J.
Physiol. Pharm. 42 : 599-608, 1964.

SwsH, R. J, MacLean, L. D., and Visscrmr, M. B.:
A device for continous recording of weight change
Appl.
Physiol. 9:297-298, 1956,


PROBLEMS IN AERIAL APPLICATION

I. Acute Effects of the Insecticide Endrin on Renal Function and Hemodynamics

I. Introduction.

Renal hemodynamic changes in the dog dur-
ing the early phase of acute intoxication by
endrin,* a chlorinated hydrocarbon insecticide,
have been reported by this laboratory.1 The
sympatho-adrenal systems have been assigned a
prominent role in renal vascular response, but
no attempt has been made to define a priority
of function. The present study was therefore
designed to evaluate the relative contribution of
the neural and humoral components of the renal
vascular response to endrin.

II. Methods.

Acute experiments were performed on five in-
tact and six adrenalectomized adult mongrel dogs,
16 to 20 kg in weight (Figure 1). Each ani-
mal served as its own control, and effects of
various maneuvers were compared to a con-
trol period of 10 to 20 minutes prior to admin-
istration of endrin. All dogs were anesthetized
with sodium pentobarbital** (30 mg/kg) ad-
ministered intravenously. Convulsions resulting
from administration of endrin were controlled
by succinylcholine chloride*** (0.5 mg/kg), and
respiration was maintained with a Starling res-
pirator using room air. Control samples were
taken after a minimum equilibration period of
30 minutes. Previous studies have shown that
vascular changes due to succinylcholine chloride
and sodium pentobarbital are minimal.* *

Endrin was administered by infusion of a
lethal dose (10 mg/kg) in alcohol (25 mg/ml)
into the blood reservoir. A priming solution of
p-aminohippurate (PAH) and creatinine was
added to the blood reservoir, and blood levels

¥ Endrin — 1,2,3,4,10,10-hexachloro-6,7,-epoxy-1,4,4a,5,6,7,8,82-
octa - hydro - 1,4, - endo-endo-5,8,-dimethanonapthalene, obtained

from Nutritional Biochemical Corp., 21010 Miles Ave., Cleveland,
Ohio.

** Nembutal, obtained from Abbott Laboratories, N. Chicago,
m.

*** Anectine,
‘Tuckahoe, N.Y.

obtained from Burroughs Wellcome Co.

were maintained by continuous infusion with a
Harvard infusion-withdrawal pump. Urine was
not collected since renal plasma flow (RPF)
and glomerular filtration rate (GFR) were deter-
mined directly from the arterial-venous differ-
ences of the clearance reagents. Renal blood flow
was measured directly with a graduated cylinder
and stop watch. Blood was returned to the
femoral vein from a reservoir via a Sigmamotor
pump. Systemic arterial pressure (SAP) was
measured from the cannulated femoral artery
with a Statham pressure transducer connected to
a Sanborn direct-writing recorder.

A. Renal denervation experiments, Five experi-
ments were performed in which the kidneys were
exposed by incision through the flank region and
the renal vein cannulated for direct-flow measure-
ments, Care was taken to avoid severing or
damaging renal nerves. Endrin was infused as
described above and, following a steady-state
response of renal blood flow, phenol was applied
to the renal pedicle. Flows were followed to
determine the effects of the chemical denervation.
Surgical denervation was then performed.
Phentolamine+ (2.5 mg) was injected into the
renal artery after flow had stabilized, and con-
secutive 30-second flows were taken for about 5
minutes.’ Phenoxybenzaminef+ (10 mg/kg in
100 ml of saline), another adrenergic blocking
agent, was added to the reservoir and infused
intravenously. The experiment was terminated
after monitoring systemic pressure and arterial
renal-blood-flow changes for a maximum of 30
minutes.

B. Renal denervation experiments on adren-
alectomized animals. Six animals were adren-
alectomized surgically, then prepared and treated
as above,

+ Regitine—obtained from Ciba Pharmaceutical Co., Summit,
NJ.

+f Dibenzyline—supplied through the courtesy of Smith,
Kline, and French,



A marked and progressive increase in venous re-
turn (cardiac output) occurs within 30 minutes
following endrin administration. Total periph-
eral resistance falls significantly and remains
low. Endrin appears to exert a toxic action on
the left ventricle; left heart failure, demon-
strated by elevated left atrial pressure regularly
occurred. Endrin produced a rise in pulmonary
artery pressure, but no changes in pulmonary
vascular resistance or right atrial pressure were
observed. ‘The effect of the insecticide in in-
creasing cardiac output primarily through in-

direct pathways was opposed by the tendency
for heart failure to occur. Animals given endrin
exhibited large increases in blood catecholamine
concentration that were significantly depressed
when pH was maintained constant with a blood
buffer. Bilateral adrenalectomy significantly de-
creased catecholamine concentrations that, how-
ever, were significantly elevated over preendrin
values. Cardiovascular alterations were not sig-
nificantly correlated with blood concentrations of
catecholomines after endrin.

REFERENCES

1, Emerson, T. E., Jr, Brake, C. M., and HinsHaw,
L. B.: Canad, J. Physiol. 42:41, 1964.

Reins, D, A, Hormes, D. D., and Hrvsuaw, L, B.:
Canad. J. Physiol. Pharm. 42: 599, 1964.

Hinsuaw, L. B. Vick, J. A, Wirrmens, L. B.,
Worrnen, D, M., Netson, D. L., and Swenson, O. P.:
Proc. Soc. Exptl. Biol. Med. 108: 24, 1961.

. WortHEeN, D. M., ANDERSON, Q. N., and HinsHaw,
L. B.: Am. J. Physiol. 202: 97, 1962.

Frorica, V.: Clin, Chim. Acta., 12: 191, 1965.

. Hinsnaw, L. B., Brake, C. M., Emerson, T. E., Jr.,
Jorpan, M. M., and Masuccr, F, D.: Am. J. Physiol.
207: 925, 1964.

Gowpry, ©. W., Grawam, A. R., Securn, J, J., and

2.

3.

35

Sravraky, G, W.: Can. J. Biochem. Physiol. 32: 498,
1954.

8, Reins, D. A., Rieger, J. A., Jv., Stavinowa, W. B.,
and Hinswaw, L. B.: Candd, J. Physiol. Pharm.
(in press).

9 Reap, ©. R., Jonson, J. A., and Kura, H.: Am. J.
Physiol. 190: 49, 1957.

10, Gowpey, C. W., Granam, A. R,, Secuin, J. J.,
Srraveaky, G. W., and Wavup, R. A.: Can. J, Med.
Sei. 30: 520, 1952.

11. Treon, J. F., Cueveranp, F. P., and Copper, J.: Agr.
Food Chem. 3: 842, 1955.

12, Arena, J. M.: N. Carolina Med. 21: 470, 1960.

13. Bett, A.: Med, J. Australia, 2:698, 1960.

14. Contry, B. E.: J. Am. Med. Assoc. 172: 2077, 1960.



PROBLEMS IN AERIAL APPLICATION: levels Release

IV. Effect of Endrin on Venus Return and Catecholamine Release

I. Introduction.

Hypertension followed by hypotension and
death have been reported in dogs poisoned with
endrin.~* The purpose of the present study was
to examine changes in cardiac output (venous
return), total peripheral resistance, and plasma
and adrenal catecholamine levels as related to the
development of hypertension in dogs acutely poi-
soned with endrin. Studies were performed in
totally perfused dogs in which conditions of
pressure and flow could be continuously monitored
and evaluated.

II. Methods.

Experiments were performed on 50 adult mon-
grel dogs of both sexes. Six dogs from the intact
group and five from the eviscerated series served
as control animals and were treated as experi-
mentals except that they received no endrin.
Convulsions ordinarily resulting from endrin in-
fusion were prevented by immobilizing animals
with succinylcholine chloride as previously des-
cribed.s Intact dogs were prepared as described
by Weil, et al.‘ in order to measure total
yenous return. Evisceration was done in one
series of experiments.® Endrin or a solvent blank
was infused into the reservoir after a minimum
equilibration period of 10 minutes.

Polyethylene catheters in the femoral artery
and portal vein were connected to Statham pres-
sure transducers. Systemic arterial pressure
(SAP) and heart rate were recorded on a San-
born direct-writing recorder. Portal vein pressure
was also recorded from intact animals but showed
no changes.

Total venous return was measured directly with
a graduated cylinder and stop watch. Stroke
volume and total peripheral resistance were cal-
culated from measured parameters. Blood samples
for determination of pH, hematocrit, and cate-
cholamines were taken immediately preceding in-
fusion of endrin and 30 and 60 minutes after
infusion. Blood samples were also taken from

23

both artery and vein at these times for O, and
CO, determinations by the method of Van Slyke
and Neil." At the end of the experiment, adrenal
glands and tissue from the left ventricle of the
heart were quickly removed and frozen with dry
ice. Estimations of epinephrine (E) and nore-
pinephrine (NE) were made by the method of
Anton and Sayre,’ using the Aminco-Bowman
spectrophotofluorometer. Recovery was estimated
with each set of analyses by carrying known
amounts of epinephrine and norepinephrine
through the entire assay procedure. Mean recov-
ery values were 85.26% (SE+4.35) for E and
73.85% (SE +2.64 for NE.

A toxicity study was also made with 24 dogs
anesthetized with sodium pentobarbital. Endrin
was given by intravenous injection, and survival
times were noted. Data from six intact control
dogs indicate that the volume of solvent (ethyl
alcohol) used produced no effect on measured
parameters.

Previous studies'** have shown that a maxi-
mum response in the parameters of interest in this
study was achieved by using an endrin dose of 10
mg/kg. Where applicable, statistical significance
was determined by applying the “t” test to the
mean of control and experimental values at a
given point in time.

Ii. Results.

Results from the various measured and calcu-
lated parameters are shown in Figures 1 and 2
(intact dog) and 8 (eviscerated dogs) and Tables
1 and 2. Control and experimental dogs were
treated identically until infusion of endrin was
begun. Data obtained at these points were pooled
and used as pretreatment values in the figures.
A. Intact dogs (10 experimental, 6 control dogs).
These dogs responded to endrin with a decreased
heart rate and increased SAP. Venous return
increased from a mean of 55 ml/min/kg in the
control period to 89 ml/min/kg (P<0.01) during
the 30 minutes. Total peripheral resistance



ACKNOWLEDGMENTS

The authors wish to express their appreciation to Joyce Markman for
her assistance in surgical procedures necessary in setting up the animals for
these experiments.

Appreciation is expressed to Dr. Francis J. Haddy, Chairman, Dept. of
Physiology, Oklahoma University Medical Center, for his review of the manu-
seript of Part III.

Qualified requestors may obtain Aviation Medical Reports from Defense Documentation
Center, The general public may purchase from Clearinghouse for Federal Scientific and
Technical Information, U.S. Dept. of Commerce, Springfield, Va. 22151


Appendix

‘Tante 8, Effect of endrin on blood concentrations of oxygen and carbon dioxide (M +SE).

Time
(min)

Control 0
+30
+60

Endrin 0
+30
+60

Endrin + 0
oxygen —- $30
+60

Taste 4. Effect of endrin following acute bilateral

Oxygen Carbon dioxide
(vol %) (vol %)

Artery Vein Artery Vein
17.13 £0.92 10.78 £1.16 26.49 £1.00 © 33,25 £1.17
16.81 £0.90 10.29 £1.25 26.63 £1.01 32,48 41.14
17.10 £0.92 10.65 1.20 24,77 +£1.28 30.44 41.32
17.31 £0.73 10.03 £0.78 26.41 +1.83 32.43 42.46
10.84 +£1.67 2.12 41.39 28.96 +2.39 37.41 42.05
11.21 £1.75 1.83 $1.12 19.97 £3.23 30,45 $1.45
14,30 £1.26 7,53 £1.85 32.35 £2.48 38,86 42.34
16.64 £1.21 4.32 $1.48 80.87 £2.15 37.23 +£5.56
14.73 £2.03 2.34 40.68 28.46 £4.61 48.56 41.11

adrenalectomy on hemodynamic parameters
in dogs (mean values, five animals).

Mean Total

Time systemic Cardiac peripheral

postendrin arterial inflow resistance

pressure
(min) (mm Hg) | (cc/min/kg) | (mm He/ce/
min/kg)

0 94 62 1.5
5 77 55 14
10 78 66 1.2
15 89 92 1.0
20 78 102 0.9
25 74 113 0.7
30 68 118 0.6
35 65 109 0.6
40 67 102 0.7
45 68 98 0.7
50 67 91 0.8
55 65 93 0.8
60 61 83 0.8

47



PROBLEMS IN AERIAL APPLICATION:

V. Effects of the Insecticide Endrin on the Cardiovascular System

I. Introduction.

Acute and chronic effects of the chlorinated
hydrocarbon insecticide endrin in dogs have been
previously reported." ? Evidence was provided
for an initial action of endrin on the central
nervous system with a subsequent sympatho-
adrenal discharge. Systemic hypertension with
marked bradycardia is an early manifestation of
endrin poisoning’? These previous studies in-
volved either multiple injections of sodium pen-
tobarbital or succinylcholine chloride for the pur-
pose of controlling convulsions during the post-
endrin period. The purpose of the present in-
vestigation was to elaborate further on the find-
ings of the earlier studies. By avoiding the re-
peated use of barbiturates or muscle relaxants,
possible effects of these agents in modifying the
cardiovascular responses to endrin were elim-
inated. The effects of endrin on the heart, pul-
monary bed, and systemic circulation were
evaluated, and the role of the adrenal glands in
the early vascular response to endrin was de-
termined.

II. Methods.

Several cardiovascular experiments with the
chlorinated hydrocarbon insecticide endrin (1, 2,
8, 4, 10, 10-hexachloro-6, 7-epoxy-1, 4, 4a, 5, 6, 7,
8, 8a-octahydro-1, 4, endo, endo-5, 8-dimethano-
aphthalene, obtained from the Nutritional Bio-
chemical Corporation, Cleveland, Ohio) were per-
formed on adult mongrel dogs intravenously
anesthetized with sodium pentobarbital (30 mg/
kg in body weight).

Previous studies with endrin employed super-
lethal doses of endrin.» ? Initial survival experi-
ments on dogs have been performed to determine
the dose of endrin to be employed. Results of
these are shown in Table 1. A dose of 3 mg/kg
was selected to be injected intravenously in all
experiments (endrin dissolved in 95% ethanol,
20 mg/ml).

31

The basic experimental preparation was one in
which total venous return was continuously
measured, and cardiac inflow was adjusted to
equal venous return.? Animals were inspired by
means of a Starling constant volume respirator,
and the chest was opened by a midsternum split-
ting incision. The right atrium was cannulated
following heparinization (3 mg/kg) and ligation
of the azygos vein. The superior and inferior
venae cavae were separately cannulated and
drained into an external reservoir placed in a
water bath to maintain normal blood tempera-
ture. Blood was returned from the reservoir
to the right atrium by a Sigmamotor pump man-
ually adjusted to maintain the reservoir level
constant (venous return equal to cardiac inflow).
Current unpublished work in this laboratory has
shown insignificant volumes of blood to be
pooled in the pulmonary circuit after endrin,
the weight of the perfused lung decreasing after
endrin. Cardiac inflow, as measured when the
plood-reservoir level remained constant, was as-
sumed equal to cardiac output in the present
study. All cardiac output data, therefore, were
derived from flow rate into the right atrium.
Total peripheral resistance was calculated by
dividing mean systemic arterial pressure by car-
diac output. Pulmonary vascular resistance was
calculated by dividing the pressure drop be-
tween pulmonary artery and left atrium by
pulmonary blood flow. Control experiments, in
which endrin was not given, were performed
in all instances. Left and right atrial pressures
were measured in both the venous return experi-
ments and in the isolated heart-lung preparation,
previously reported from this laboratory,‘ and
aortic pressure was measured.

A metabolic evaluation was performed in which
oxygen uptake and carbon dioxide production
were determined after endrin by the Van Slyke
manometric procedure and in separate experi-
ments following administration of 100% oxygen.



@—e ENDRIN

8.07 0-0 CONTROL
Peon ain °
6
= 7.07 3 re
¢t t
P<.Ol P<.Ol
6.0 T T 1
ENDRIN (3 MG/KG) ee ENDRIN + 0,
i O——oO ENDRIN + THAM
8.0-
e 5 5§ 5 5 5 5 5 5 5 4 3 4
Sf---8--—§---B--~3---5-—-9---5---9---n---3---3---8
= 704 —— OS 4
a
4 ;
P<.Ol P<.Ol
6.0 T T T t T T 7
ro) 10 20 30 40 50 60
TIME (MIN)

Ficune 5. Changes in pH in dogs treated with endrin plus oxygen and endrin plus THAM
(M +SE) (numbers of experiments at each point are indicated by numbers).

Appendix 40


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TIME (min.)

Fieure 2. Heart rate, oxygen uptake, hematocrit, and pH from intact control and dogs and intact dogs infused with
endrin during the venous return study.

26


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