Taste 1. Summary of effects on liver as seen with various stains. The ratings are on a scale of 4 with 0 no
effect and 3 maximal. With PAS, the scale used was of 5 with 4 maximal and 0 least. S = scattered,

MZ = midzonal, PP = periportal.

Rat Swank Modified Sudan Nile Blue Cresyl = HandE PV
No. Davenport Marchi Swank Davenport Marchi Black B Sulfate Violet
Room-temperature and poisoned
1W 3 PP 3 PP 3 PP 1 PP 3 1 2
2W 2 PP 1 PP 2 PP 1 PP 1 0 2
3W 2 PP 3 PP 3 PP 1 PP 3 0 0
4Ww 2 PP 0 2 PP 2 PP 2 1 2
5W 2 PP 2 PP 2 PP 2 PP 2 1 1
6Ww 3 PP 3 PP 3 PP 3 PP 3 1 1
7W 1 PP-S 1 PP 28 1 PP 1 1 2
8W 2 PP 3 PP 28-PP 2 8 1 1 2
ow 2 PP 1 S-PP 18 18 3 1 2
Room-temperature controls
13 W 1s 0 28 1s 0 0 3
4.W 1s 2 PP 3 PP 2 PP 1 0 3
15 W 18 0 0 0 0 0 3
16 W 3 PP 1,PP 2 PP 1 PP 1 1 3
19 W 0 0 0 18 0 0 0
20 W 0 1 PP 2 PP 1s 0 0 0
21 W 18 0 0 0 2 0 0
22 W 2 PP 0 0 0 0 0 3
23 W 2 PP 0 0 1 0 0
Cold-adapted and poisoned
10 2 PP 0 0 0 1 1 0
20 2 PP 2 PP 2 PP 18 2 1 1
3c 1 PP 1 PP 1 PP is 1 0 2
40 2 PP 3 PP 3 PP 2 PP 1 1 1
5c 18 1 PP 1 PP 18 0 0 2
6c 28 3 MZ 1 MZ 28 2 2 1
7c 2 PP 2 PP 18 is 2 1 2
9c 2 PP 3 PP 3 PP is 2 1 1
140 2 PP 3.PP 3 PP 28 0 0 1
Cold-adapted controls
15 0 1 PP 0 0 18 0 0 0
16 C 1 PP 1 PP 1 PP 0 1 0 4
17 C 1 PP 0 1 PP 0 0 0 4
18 C 1 PP 0 0 0 0 0 3
21 0 0 0 0 0 0 4
22. 1s 0 0 0 0 0 0
23 C 1 PP 1 PP 2 PP 0 1 1 0
24.0 0 0 18 0 1 1 0
25 C 2 PP 0 1 PP 0 0 0 3

for each strain. The differences between groups
are evident with the cold-adapted nontreated
animals showing much less evidence of fatty
deposits and the room-temperature treated rats
the greatest amount. A measure of the relative
amount of fat can be made by adding the esti-
mates of fat for the groups. These values are 59
(warm treated), 40 (cold-adapted treated), 27
(warm control), and 17 (cold-adapted control).

Thus, in order of decreasing amount of fat, the
groups are warm poisoned, cold-adapted poisoned,
warm controls, and cold-adapted controls least.
It should be stressed that the livers of the
poisoned animals cannot be considered fatty livers
since the number of fat-containing cells is only
a small percentage of the total number of cells.
The effects seen with Cresyl Violet Acetate and
with H and E are similar. In the most marked



REFERENCES

1, Priest, R. J., and Horn, R. ©. Jn: Arch. Environ.
Health, 11 :361-5, 1965.

2. Haraway, D. E. Ibid., 11 :380-8, 1965.

3. CLaRK, Gxorce: In Investigation of the effects of
specific agricultural chemicals on histological tissue,
Senior investigator, HivsHaw, L. B. Report sub-
mitted 6-30-65, AM 64-6 (PHARM-1) Phase 3.

4. Koento, H., Groat, R. A., and WINDLE, W. F.: Stain
Techn., 20:18-22, 1945,

5. Powers, M. M., and CiaRK, GEORGE: Stain Techn.,
30 :83-8, 1955.

6. Davenrort, H. A.: Histological and Histochemical
Technics. Philadelphia, W. B. Saunders Co., 1960.

7. Powers, M. M.: To appear in Anat, Record, 1966.

8. Powers, M. M., and CuarxK, Grorce: Work in prog-
ress, 1966,

9. Beruse, G. R., Powers, M. M., and CrarK, GEoRGE:
Stain Techn., 40 :235-8, 1965.


25 mg of nembutal and were perfused through
the heart with acacia saline followed by acacia
formol saline.t Then the livers were excised, and
a thin slice was placed in 80% ethyl alcohol and
a much larger slice in 10% formalin.

The following routine stains were used : Sudan
Black B, Nile Blue Sulfate, periodic acid schiff
(PAS), and hematoxylin and eosin(H and E).
In addition three stains that are routine for
nervous tissue were used. These are Cresyl Violet
Acetate, which is specific for nucleic acids, Swank
Davenport Marchi, which, in the central nervous
system, is specific for unsaturated fatty acids,
and a modified Weil stain, which in nervous tissue
is specific for the secondary and tertiary amines
found in myelin.

A. aie Blue Sulfate —
1, Wash frozen sections (10 m) in distilled
water.

2. Place in 0.5 ml aqueous 1% Nile Blue Sul-
fate in a small vial for 20 minutes in an
oven maintained at 60°C.

3. Add 10 ml of 1% aqueous acid and leave
in oven for 20 minutes.

4, Wash in distilled water.

5. Mount in glycerine, wipe slide dry, and
ring cover slip with Permount.

B. Cresyl Violet Acetate—As described previ-
ously.®

C. Sudan Black B.—As described previously.*
D. H and E.—As described previously.*

E. Swank Davenport Marchi—Using diluted
Poirer solution.*

The routine method, impregnation of a 1-mm
block, was used but better results were obtained
with frozen sections.’

F. Modified Weil Stain—

1. Decerate and hydrate mounted paraffin
sections,

2. Place filled staining rack in a dry staining
dish,

3. Pour on slide 2.5 ml of a 10% alcoholic
hematoxylin diluted to 100 ml and 100 ml
of 1% ferric ammonium sulfate.

4. Thoroughly mix solutions by raising and
lowering staining rack several times. Leave
in stain for 30 minutes,

5. Wash in tap water and then in three
changes of distilled water,

6. Place in 1% ferric ammonium sulfate

solution for 1 minute.

7. Wash in three changes of distilled water.

8. Place in 0.5 M sodium oxalate solution
overnight.

9. Dehydrate, clear, and mount with Per-
mount,

III. Results.

Table 1 presents a summary of the effects seen
on the livers of these rats. The only stain that
showed a difference between the control rats on
limited diet and those on full diet was the PAS.
This was expected due to the known loss of liver
glycoyen that occurs in inanition. Unlike carbon
tetrachloride, whose effects are primarily centro-
lobular, dieldrin poisoning results in changes
located around the periportal canals. The extent
of damage (whether this represents actual
damage or is a reflection of altered function
elicited by the poison remains a subject for inves-
tigation) is indicated on a scale of 4 with 0 indi-
eating no change from control and 3 indicating
the maximal changes seen. Thus, in Figure 1 is
shown the Swank Davenport Marchi section,
which was assigned a grade of 3. The

periportal area where the fat is localized is to the

Ficure 1, Marchi Swank Davenport-stain single cell to
left with black globules lies at edge of central vein.
Numerous cells at right are in periportal region.
Area between comprises a large portion of lobule.
Dieldrin-poisoned rat.

right and the single fat-containing cell to the
left lies adjacent to the central vein of the lobule.
The intervening area comprising over one-half
of the lobule is free from fat. It is evident that
the number of cells containing appreciable
amounts of fat is quite small. With the four fat
stains there is general agreement, although there
are few cases where the same value was judged


Taste 2

Cold-adapted and treated

Rat No. Average absorbance
1c 0, 2874
2C 0, 1129
3c 0.1781
4C 0, 1739
5C 0, 2964
6C 0, 1973
7c 0, 2604
9C 0.2240
14C 0, 2351

xi=0, 2184

Cold-adapted controls

Room-temperature and treated

Rat No. Average Absorbance
1W 0, 2890
2w 0.3130
3w 0, 3036
4w 0, 3559
5W 0.2816
ow 0.3156
7w 0.3017
sw 0.2961
ow 0.3511

xx=0, 3119

Room-temperature controls

15C 0, 1823 13W 0, 3556
16C 0, 2571 14W 0, 2502
170 0. 2258 15W 0, 1905
18C 0, 1063 16W 0.1714
21C 0.0470 19W 0. 0888
220 0, 1292 20W 0, 3540
23C 0, 1604 21W 0, 1549
240 0. 2616 22W 0, 2829
250 0. 1471, 23W 0, 4494
x3=0, 1685 x4=0, 2553
Analysis of variance of absorbance of Weil-stained rat liver:
Source df ss MS F P
Tota! 3s 0, 2802
Treatment 3 0.0988
Temp 1 0.07319 0,0732 12,9 PC 0.005
Drug 1 0.02555 0.0256 4.49 PC 0.05
Temp & Drug 1 0.00007 0, 00007 <1 ns.
Error 32 0.18138 0.005668

examples, the difference between the periportal
and centrolobular areas is clearly demonstrated.
With Cresyl Violet Acetate, the cytoplasmic RNA
in the periportal areas is almost granular (Figure
2), while in the centrolobular areas, the RNA is
found in distinct clumps (Figure 8). In slides
marked 0, this clumping of the RNA extends
throughout the lobule. With H and E, differen-
ces are not as great but involve both the hema-
toxylin and the eosin. The cytoplasmic material
stained with hematoxylin in the centrolobular
area is clumped, while in the periportal areas, it
is more granular in those marked 1. In addition,
the periportal cells are redder than the centro-
lobular.

A modification of the Weil stain for myelin
sheaths was also used on formalin-fixed paraffin

liver sections, As modified,* the differentiation
of this stain is self-limiting since, while differ-
entiation requires about 8 hours, an eightfold in-
crease in differentiation time produces no further
loss in stain. The slides were read with a Photo-
volt electronic photometer. Ten readings were
taken on each section with the locations random
except that fields containing blood vessels were
not used. The averages of these readings are
found in Table 2. A factorial 2 X 2 method was
used with the results found as shown in the table
legend. It is evident that all four distributions
differ significantly from one another. The stain-
ing density is increased in the poisoned animals
and decreased in the cold-adapted animals with
no interaction between the two conditions. The
meaning of this is not known at present. By


AM 66-5

PROBLEMS IN AERIAL APPLICATION:

A COMPARISON OF THE EFFECTS OF DIELDRIN POISONING
IN COLD-ADAPTED AND ROOM-TEMPERATURE MAMMALS

George Clark
Approved by Released by
Bild Rte (PU Sgee 00
J. Roserr Drie, M.D. P. V. Srecer, M.D.
Cmer, Civin ArromepicaL InsriruTe FeprraL Air SURGEON
April 1966

FEDERAL AVIATION AGENCY
Office of Aviation Medicine



Figure 2. Cresyl Violet Acetate. Note small size of
cytoplasmic granules in dark area adjacent to portal
canal.

analogy with the use of this stain for myelin
sheaths, the functional group to which the stain
attaches is a secondary or tertiary amine and the
compound is a phospholipid. Other inferences
could be made by analogy with the results ob-
tained in the study on myelin sheaths, but these
should be verified on the liver before conclusions
are drawn. It is interesting to speculate on the
possible function of the material stained. The
amount is increased by poisoning but decreased
by cold adaptation. Is this material a necessary
substrate for detoxification of the halogenated
hydrocarbon, a “side-product” of the detoxifica-
tion, or an artifact? If it is any of these, why
should it be decreased in cold adaptation? An-
swers to these questions may give a rationale for
treatment of sublethal dosage with dieldrin and
possibly other halogenated hydrocarbons.

IV. General Discussion.

Although it should also be stressed that rank
orders of all the measures (weight loss and the
various stains) showed little correlation with one
another, some generalizations are warranted. The

Fieure 3. Cresyl Violet Acetate. Note large size of
cytoplasmic granules.

cold-adapted rats were more severely affected by
the poison than were the room-temperature rats.
Thus, three of the treated cold-adapted rats died,
while none of the treated room-temperature rats
died. The food consumption of the treated cold-
adapted rats was less and they lost more weight
than the treated room-temperature rates, The
reverse was true with the livers. There was a
greater amount of fat-in the livers of the warm
treated rats; however, the cytoplasmic RNA
showed greater alterations in the treated cold-
adapted animals than in the room-temperature
rats, and, finally, the material stained by the Weil
stain was considerably less in the cold-adapted
rats, Is all this a reflection of the altered lipid
metabolism of the cold-adapted rats or are other
factors involved? The answer to this question
would be of interest from many viewpoints be-
yond that of poisoning by an insecticide.



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:

Comparison of the Effects of Dieldrin Poisoning in Cold-Adapted

and Room-Temperature Mammals

I. Introduction.

The delivery of insecticides by air (crop dust-
ing) continues to increase. These insecticides are
poisonous to humans and constitute a hazard to
those engaged in this work. Many of these poi-
sons are halogenated hydrocarbons. Although
there has been a great amount of research on the
pathological effects of carbon tetrachloride poi-
soning and some on DDT poisoning, there is vir-
tually nothing on the pathology resulting from
poisoning by such halogenated hydrocarbons as
endrin, dieldrin, etc., except that from an over-
whelming dose. In such cases, the widespread
effects of the poison make it difficult to determine
which of the effects are directly due to the poison
and which are secondary. It is also difficult to
extrapolate from these widespread effects to what
might be expected from the smaller dosages that
would be more likely to occur in handling these
chemicals. It would also be impossible to extra-
polate from these acute effects to the possibility
of residual damage. In spite of the paucity of
information, accounts of the toxicology of the
halogenated hydrocarbons stress liver damage.
With carbon tetrachloride or trichlorethylene
poisioning, there can be no question of widespread
liver necrosis, but with other halogenated hydro-
carbons this may not be the case.’ In a prelimi-
nary study in rats to which near-fatal levels of
endrin or dieldrin were given, there was found
evidence of minor liver alterations and little else.*
In this study, it was found that the liver damage
was not only relatively light but was also quite
different from the damage produced by carbon
tetrachloride administration. Some of these
changes were indicative of altered lipid metabo-
lism. Since there are marked changes in lipid
metabolism in cold adaptation, it was thought
that the combination of cold adaptation and
dieldrin poisoning might serve to elucidate the
resulting liver alterations.

II. Materials and Methods.

Forty-nine male rats of the Charles River
strain were used. Twenty-five of these (164 to
247 grams) were placed in a room at 5°C for
4 weeks. Purina checkers and water were avail-
able as desired. On removal from the cold, all the
rats had varying, but mild, frostbitten ears. Two
animals were removed from the group; one had
bloody urine and the other a badly frostbitten
tail. The remaining 28 rats all increased in
weight during their stay in the cold. While the
others were kept in the cold, the remaining 24
male rats were kept at room temperature with
water and food as desired.

Treatment with the poison was instituted
4 days after removal from the cold. Twelve each
of the cold-adapted and room-temperature rats
were given 15 mg of dieldrin dissolved in mineral
oil intraperitoneally daily for 3 days. There were
thus four groups of animals: cold-adapted and
treated, cold-adapted controls, room-temperature
treated, and room-temperature controls.

In the earlier study,’ it was found that food
consumption of poisoned animals dropped pre-
cipitously. Because of this drop in food consump-
tion, one-half of the controls were given only the
average food consumption of the treated animals.
At this time it was also decided that, if any
animals in the treated groups died, a correspond-
ing number would be dropped from each of the
other groups. With the treated animals the ones
with the largest numbers would be dropped while
with the control groups the final (size of number
of rat) two of the rats on full diet would be
dropped and then the final ones of the animals on
limited diet. One of the treated cold-adapted
rats died within 48 hours and two more within
72 hours. Ninety-six hours after treatment began,
the treated animals were sacrificed; the controls
were sacrificed on the following day. The animals
were killed by the intraperitoneal injection of