was marked in three animals and present in all.

In two animals there was’ marked necrosis with
most of the fat in the mediolobular area.

Figure 4. Rat 18, Powers’ Marchi. Central vein to left.
The fat is predominately in large droplets and there
are necrotic cells near lumen of central vein. 700x

Group D. The contrast between this group
and Group © was marked. In the month since
the final dose of carbon tetrachloride, much of
the damage had been repaired. In fact, the livers
of 5 of the 8 rats were virtually normal (as far as
the fat stain revealed) and three of the five could
be considered as normal. In two animals there
was some fat both centrolobular and mediolubu-
lar. In none of the animals was there any perilo-
bular fat.

Group E. The livers of two rats were free of
fat while in the others there were occasional
scattered cells containing fat.

Ficure 5. Rat 5. Modified Weil. There is little differ-
entiation and stain is quite uniform.

Modified Weil Stain. Paraffin sections were
stained with the modified Weil stain (Figure 5)
described in a previous study (Clark, 1966).
Since the animals were not perfused and the

sinusoids: contained varying amounts of blood,

‘the method used in that report for assessing, the

staining intensity could not be used. Photomicro-
graphs were taken at a magnification of 500 on
35 mm Kodalith film. In each case a representa-
tive area was chosen visually and the same ex-
posure was used for all. The film was developed
in diluted “Dektol” (1 part. developer for 17
parts water) for 4 minutes at 24°C. Then 8 x 10
enlargements were made of these 35 mm nega-
tives. Here again, the exposure was determined
for one negative and the same exposure and ‘de-
veloping time was used for all. Then the prints
were arranged in rank order of the greyness of
the cytoplasm of the liver-cells. This was done
independently by three observers (Table 1). The
rank orders for each group were summed and
the means determined. The values.were Group
A-8.3, Group B-18.3, Group C-18.7, Group D-
22.4, Group E-22.6. This indicates that whatever
compound is stained by this method, presumably
some substance containing a secondary or tertiary
amine (Berube, Powers and Clark, 1965), the
amount is markedly increased in acute poisoning
with endrin. This substarice is not markedly in-
creased in poisoning with carbon tetrachloride.
One month is sufficient for this material to de-
crease to normal amounts. Because of possible
errors in the involved procedures used exact
quantification was impossible: Statistical studies
would be made if a microdensitometer were avail-
able. Especially interesting would be the acutely
carbon tetrachloride poisoned animals (Group C)
and the chronic endrin poisoned animals. (Group
B) ; the values found: (18.7 and 18.3 respectively)
were less than those found in Group D (one
month after carbon tetrachloride poisoning) and
in Group E (the controls) and may or may not
be significantly different.

IV. Discussion.

The easy readibility of the fat stain used
(which was developed in our laboratory, Powers,
1966) materially aided the investigation. The
changes seen in fat content of the liver after
either carbon tetrachloride poisoning or after
endrin poisoning seem completely reversible. It
should be stressed that although no animals were
lost in the three days of treatment, three were lost
in the week following cessation of treatment.
Thus, the level of the endrin was almost in the
lethal range.



TABLE 1

Group A Powers’ Cresyl Violet Clarks Loss in
Rat No. Marchi Acetate Weil Weight
1 3 PL CL 1 777 62
2 2 PL 1 16 10 16 30
3 2 PL ML 1 3°13 35
4 2 PL 2 5 6 5 18
5 2 PL i 222 31
6 3 PL 1 6 5 6 31
7 18 2 29 27 29 59
8 2 PL 1 131 23
Group B
10 0 1 27 19 25 +2
il 1SA 1 19 21 2 31
12 is 1-0 21 17 21 62
B 1A 0 89 8 21
15 18 1-0 34 34 34 32
16 18 1 4°44 54
Group C
17 3 CL 2 989 22
18 3 CL 2 33 83 33 38
19 3 CLB 2 14 15 15 33
20 3 CLC 3ML 2 23 25 23 17
21 3 CLC 3ML 2 wee oe 19
22 3 OL aan 22
23 3 CL 1 15 12 14 21
24 3 CL ween ee 16
Group D
25 2CL 0-1 22 18 22 13
26 1 CL (%) 1 20 22 18 15
27 2 CLS O41 35 35 35 17
28 18 0 24 20 24 21
29 18 0 12 14 12 16
30 0 O41 30 32 31 23
31 2CL ML 0-1 10 13 10 31
32 1 CL ML 1 25 28 26 33
Group EB
33
34 0 ° 18 24 19 +6
35 1-0 1116 11 +6
36 1 1-0 32 26 32 1
37 18 0 17 23:17 1
38 28 0 31 31 30 4
39 18 0 28 29 28 16
40 18 0 13 11 13 7
41 0 0 26 30 27 2

Summary of histological studies. The amount of lipid present was estimated on a scale
of 4: O~no fat, 1~ some fat, 2~ considerable fat, 3— most cells fat laden, The Cresyl
violet acetate stains were estimated on a scale of 3:0 —all eytoplasinic RNA in heavy clumps,
1-many cells with dispersed RNA, 2— large areas of cells with dispersed RNA. The
estimates of rank order of staining intensity by the three observers are given under the Weil
stain. ‘The loss in weight seen on the third day after the first dose of the poison is also given.
Abbreviations: PL — perilobular, ML - mediolobular, CL — centrolobular, A — some for in
von Kupffer cells, § - scattered, B — some CL necrosis, C~ considerable CL necrosis.

67-3597


plasmic RNA was confined ‘to large clumps with
the remainder of the cytoplasm unstained (Fig-
ure 2). The mediolobular regions were like the
centrolobular. The damage was scaled on a range
of 3 (Table 1). Those with RNA clumped
throughout the lobule were recorded as 0, those
with considerable dispersed RNA in the peril-
obular areas as 1 and those with marked amounts
of dispersed RNA in the perilobular areas as 2.

Fieure 1. Rat 5. Cresyl Violet Acetate.
to left.

Central vein

Cytoplasmic RNA in large clumps. 700x

Fiaure 2. Rat 5. Cresyl Violet Acetate.
RNA is diffuse with no large clumps.

Cytoplasmic
700x

Group B. ‘These were somewhat similar to
Group A but differences between zones were less
marked and two animals showed no differences.
This group resembled the control group more
than they resembled Group A.

Group C. The vacuoles in the centrolobular
areas made these regions much lighter than in the
remainder of the lobuues. In this area clumped
RNA was almost missing with cytoplasmic RNA
very diffuse. In the remainder of the lobule,
there was considerable diffuse RNA but clumped
RNA was present in most cells.

Group D. There was considerable variation in

this group; in three there was little difference be-
tween perilobular and centrolobular areas while
there were marked differences in one animal.

Group E (Control). In three animals no in-
tralobular differences were present while in the
remainder, the clumps of cytoplasmic RNA were
larger in centrolobular areas than in perilobular
areas.

Powers’ Marchi. Frozen sections were stained
with the modified Swank Davenport Marchi
(Powers, 1966). Group A—In all of these there
were fat-containing cells in the liver. These cells
were most numerous in perilobular areas (Figure
8) while in the centrolobular areas fat-containing
cells were found rarely. In one animal, the
scanty fat was scattered, in another, the medio-
lobular area was free or virtually free of fat
while there were both perilobular and centrolobu-
lar fat deposits; in another, there was fat in both
perilobular and mediolobular areas while the cen-
trolobular was almost free of fat. In contrast
to Group C there was always some scattering of
the fat containing cells and the fat was in smaller
droplets than in Group C.

Figure 8, Rat 5. Powers’ Marchi. Portal canal to left.
‘The fat is in small droplets, The centrolobular areas
were free of fat. 700x

Group B. The liver of one animal was free of
fat and, in the others, the fat present was scat-
tered. In two animals most of the fat present
was in the von Kupffer cells. It is obvious that,
in the month since the final dose of endrin, partic-
ally all the damage had been repaired. In fact,
there was very little, if any, difference (as re-
vealed by fat content) between this group and the
untreated control group.

Group C. As had been expected, the centro-
lobular areas were massively infiltrated with fat
(Figure 4) and necrosis of cells in this area


The meaning of the different distribution of fat
in poisoning with endrin and carbon tetrachloride
remains to be elucidated. Wachstein (1963)
stated that “The distribution patterns of stainable
lipids vary in various clinical and experimental
conditions. On diets deficient in certain amino
acids, particularly threonine, or in rats which are
polyphagic due to hypothalamic lesions, fat drop-
lets accumulate mainly in the cells of perilobular
areas. In contrast, in choline deficiency, accumu-
lation of lipids occurs predominantly in the cen-
tral zone of the liver lobule. In toxic liver dam-
age, stainable lipids occur likewise in central
areas.” However, he offered no explanation for
the phenomenon.

The meaning of the changés in cytoplasmic
RNA also remains to be elucidated. A tentative
explanation would be that the heavily clumped
RNA indicates a resting cell and that as the cell
becomes more active there is a dispersion of the
cytoplasmic RNA. If this is true, then the simi-
lar location (perilobular) of the fat laden cells
and those with dispersed RNA would indicate
that alterations in function as a result of endrin
poisoning occur primarily in this perilobular
area. However, there is no such localization seen
with the Weil stain. Does this indicate that at
least two widely different processes are seen in
this poisoning? Anything further would be en-
tirely speculative. Studies of the distributiion of
RNA have not been found to be very useful in
the human (Farber, Koch-Weser, Szanto and
Popper, 1951). The varying lengths of time be-
fore autopsy and the relatively poor fixation are
undoubtably the cause. In optimum cases the
RNA was reported to be in the form of, clumps
while in the others the RNA was diffuse. The
changes in RNA reported in this paper have
apparently not been seen previously. It should
be stressed that our hematoxylin and eosin slides
were of limited value and that the changes seen
were in those stained by Cresyl violet acetate.
This is probably the reason why alterations such

as we report in this paper were not reported by
Koch-Weser, Farber and Popper (1951).

There is a marked lack of correlation between
the various stains and between the stains and the
changes in weight (Table 1). Thus, rat #7 had
little fat in the liver, marked alteration in RNA,
perhaps even a decreased amount of the Weil
stainable material and a large weight loss.

Toxicology texts routinely state that in poison-
ing with halogenated hydrocarbons liver damage
oecurs. Nevertheless, the amount of liver damage
seen in our rats, which had quite massive doses
of the poison, was really minimal and completely
reversible. Our findings certainly do not suggest
that altered liver function occurred in our
animals.

Stains for reticular fibers were not made. Too
often such stains are not particularly reliable and
quantification of such data is usually of limited
meaning. Recently, a new method for staining
of reticular fibers was reported and it is planned
to check this stain and determine if quantitative
results are possible. If so, a separate report will
be made. Autoradiographic studies of distribu-
tion of labeled endrin in the poisoned animals are
planned. These will give some insight into the
possible meanings of the alterations in liver we
have reported. Also, histochemical blocking pro-
cedures will be used with the Weil stain in an
effort to elucidate the nature of this material that
increases in endrin and in dieldren (Clark, 1966)
poisoning.

Our findings that liver damage in endrin poi-
soning is minimal and reversible do not mean
that endrin and dieldren are not dangerous. They
do indicate that the liver damage is not incapaci-
tating and is not the cause of the deaths which
did occur in our animals. Rather, our findings
(Clark, 1965), (Clark, 1966, and this report) do
indicate that, despite the paucity of damage we
have seen, as yet, the primary action of these
poisons is probably on the nervous system.


AM 66-34:

PROBLEMS IN AERIAL APPLICATION:

A comparison of the acute effects of endrin and carbon tetrachloride on the

livers of rats and of the residual effects one month after poisoning

George Clark

Approved by Released by
S ht th PY S.2 a>)
J. Roperr Diz, M.D. P. V. Steerer, M.D.
Cuter, Civin Arromepicat InsriruTe Feperay Air Surgeon
July 1966

FEDERAL AVIATION AGENCY
Office of Aviation Medicine



Bo

REFERENCES

. Beruse, G. R., Powers, M. M., and Crark, Gzoraw:

Stain Techn, 40; 235-238, 1965.

. CLARK, Grorcr. In “Investigation of the effects of

specific agricultural chemicals on histological tissue.”
Senior Investigator, L. B. Hinshaw. Report submitted
6-80-65, AM 64-6 (PHARM-1) Phase 3, 1965.

. CLARK, G. OAM Report #66-5, 1966.

. Parser, E., Kocu-Weser, D., Szanto, P. B., and

Poppsr, H.: Arch. Path, 51: 399-408, 1951.

© ~

Kocu-Weser, D., Farber, E, and Popper, H.: Arch.
Path. 51: 498-509, 1951.

Koxnie, H., Groat, R. A. and Winvis, W. F.: Stain
Techn, 20: 13-22, 1945.

. Powers, M. M. and CrarK, Georce: Stain Techn.

30: 83-88, 1955.

Powers, M. M. Anat, Record. 154: 504, 1966.
Wacustern, M. In M. Romiller, The Liver: morpho-
logy, biochemistry, physiology. Academic Press, New
York. Vol. 1, p. 147, 1963.



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.


PROBLEMS IN AERIAL APPLICATION:

A comparison of the acute effects of endrin and carbon tetrachloride on the livers of rats

and of the residual effects one month after poisoning

I, Introduction.

The halogenated hydrocarbons continue to be
used as insecticides and many of these are aer-
jally applied. As poisons, they constitute a
hazard to those who handle them. The lack of
precise information on the pathological changes
induced by these compounds has seriously limited
the formulation of other than very general rules
for their use, has made it impossible to determine
limits of exposure and has made treatment only
symptomatic. In an early study~it was found
that there was (Clark, 1965), as stated by toxi-
cologists, some liver damage. It was also found
that cold acclimation (Clark, 1966) increased the
amount of liver damage. Since the liver damage
induced by carbon tetrachloride poisoning has
been extensively studied, it was thought that a
comparison of the effects on the liver of carbon
tetrachloride and of endrin would serve to par-
tially elucidate this problem.

II. Methods.

Forty-one male Charles River Rats, ranging
in weight from 410-545 gm, were divided ran-
domly into five groups. Two groups (A and B)
of eight rats each were injected intraperitoneally
once daily for three days with 8 mg/Kg of endrin
dissolved in mineral oil. Group A was sacrificed
on the day following the last injection of the
poison while Group B was sacrificed one month
later. Two Groups (C and D) of eight rats each
were injected intraperitoneally once daily for
three days with 1 ml/Kg of CCl, dissolved in
mineral oil. Group C was sacrificed on the day
following the last injection of the poison and
Group D one month later. At this time, Group
E (nine untreated rats) was also sacrificed. No
rats were lost in Groups C, D, and E, but in
Group A, one animal was dead on the morning
when the others were sacrificed and in Group B,
one animal died on the second day after cessation

of treatment with endrin and one on the fourth
day. .

The animals were anesthetized with Nembutal
and then decapitated. Portions of the liver were
immediately removed and placed in 10% acacia
formalin (Koenig et al, 1945). Blocks from each
animal were embedded in paraffin and sectioned
at 7 microns, Other blocks were frozen and
sectioned at 10 microns. The following stains
were used: Hematoxylin and Eosin, Powers’
Marchi for fat (Powers, 1966), a modified Weil
stain (Clark, 1966), and Cresyl violet acetate
(Powers and Clark, 1955).

IIL. Results.

All of the animals treated with endrin lost
weight during treatment (weight first day of
treatment. and first day after, last dose) except
one (#10) which inexplicably had a weight gain
of 2 grams, All the others lost over 2 grams
and four sst more than 50 grams—range +z to
—62 (Table 1). The three animals which died
lost the most: weight, 62-75 grams, but one animal
which survived lost 62 grams (#12) and one 59
grams (#7). All the animals treated with car-’
bon tetrachloride lost weight with a range of 13
to 88 grams. The control group mainly lost
weight (+6 to —17 grams) during the same
period.

The hematoxylin and eosin slides were of lim-
ited value but the results with the other slides
will be described in some detail and are sum-
marized in Table 1.

Cresyl violet acetate. Paraffin sections were
stained with cresyl violet acetate which. (Powers
and Clark, 1955) is a specific for nucleic acid.
Group A. In all of these there were differences
apparent between the three regions of the lobule. ~
In the perilobular areas the cytoplasmic RNA
tended to be diffuse with no large clumps (Fig-
ure 1) while in the centrolobular areas the cylo-