EPA-600/1-77-006
January 1977
INTERACTION BETWEEN METHYL MERCURY AND RADIATION
EFFECTS ON NERVOUS SYSTEMS
By
Eugene W. Hupp, Dalton Day, James Hardcastle
John Hines and James Minnich
Texas Woman's University
Denton, Texas 76204
Grant No. R800282
Project Officer
Daniel F. Cahill
Experimental Biology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
.:.....«.:,;.FAL PROTECTION
M. i.
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
-------
FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
Extramural research is an important and necessary supplement to our
programs and the fulfillment of our mission. Support for the research
objectives detailed in this report resulted from a broadening scientific
view of environmental problems; specifically, the need to consider the
toxicologic effects of simultaneous exposures to multiple environmental
pollutants with common target organs.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
111
-------
ABSTRACT
The interaction between methyl mercury and ionizing radiation was
investigated in a series of experiments using rats, hamsters, and
squirrel monkeys to study the effects produced and possible mechanisms of
action. Parameters evaluated included several measurements of behavior,
brain electrical activity, lethality, blood-brain barrier permeability,
neurotransmitter and mercury concentration in various brain areas, and
brain histology.
In some cases the effects of the co-insult were less than or at least no
greater than at least one of the two insults applied alone. Nine kR was
less effective than 8 mg methyl mercury per kg of body weight plus 9 kR
in producing behavioral decrement in rats 2-U hours after radiation,
producing pyknosis of granule cells of the cerebellum in rats killed 6
hours after radiation, depressing brain norepinephrine levels, and
causing death in rats in the first 8 days after radiation. Behavior of
female squirrel monkeys was less adversely affected by 300 R plus 6 mg
methyl mercury per kg than by methyl mercury alone. Brain electrical
activity was similarly affected in rats receiving 4.5 or 9 kR, alone or
with methyl mercury. With doses of radiation in the range lethal to 50%
of the population in 30 days, the two agents were partially additive.
Possible mechanisms of action include opposite effects of the two insults
on the blood-brain barrier, with radiation increasing permeability and
methyl mercury decreasing it. Radiation may also elicit a proliferation
of peroxisome-like organelles which protect against the effects of methyl
mercury.
This report was submitted in fulfillment of Grant Number R800282 by the
Texas Woman's University under the partial sponsorship of the Environ-
mental Protection Agency. Work was completed as of February 29, 1976.
IV
-------
CONTENTS
Page
Abstract iv
List of Figures vii
List of Tables viii
Acknowledgments xii
Sections
I General Conclusions 1
II Recommendations 2
III Introduction 3
IV Methyl Mercury Effects in Rat, Hamster, and Squirrel
Monkey: Lethality, Symptoms, Brain Mercury and
Amino Acids M-
V Relative Toxicity of Various Methyl Mercury Compounds
in Rats 24-
VI Results Obtained in the Main Series of Experiments,
Methyl Mercury Dose Administered to Rats 7 Days
Before Irradiation 29
VII Results Obtained when Methyl Mercury was Administered
Immediately or 24- Hours After Head Irradiation 61
VIII Effects of Combined Insults of Methylmercuric Chloride
and X-Radiation Upon the Uptake of Sulfur-35 by the
Rat Brain 65
IX Peroxide Induced Protection Against Methylmercuric
Chloride Toxicity 73
-------
CONTENTS CONTINUED
Page
X Co-Insults Effects of Methyl Mercury and Radiation with
Different Temporal Relationships 81
XI Behavioral Observations in Squirrel Monkeys (Saimiri
Sciureus) Following Methyl Mercury Administration 94
XII Co-Insult ExperimentFemale Squirrel Monkeys 102
XIII References 107
VI
-------
FIGURES
No. Page
1. Percentage death rate of rat () and hamster (") 24 hours
after a single incremental intraperitoneal injection of
methyl mercury chloride. 9
2. Percentage death rate of rat and hamster 30 days after
a single incremental intraperitoneal dose of methyl
mercury. H
3. Formulas of methyl mercury compounds used 26
4. Experimental design for studying the effects of single and
co-insults of methylmercuric chloride and x-radiation
upon the uptake of sulfur-35 sodium sulfate by various
brain areas 66
5. Comparison of cumulative mortality (%) in male and female
rats pretreated with 1.5 percent hydrogen peroxide (HP)
or physiological saline for 5 days. The rats received
methylmercuric chloride at a dose of 10 mg per kg body
weight 48 hours after the last dose of HP or saline.
Statistical analysis by Chi square showed significant
differences between HP and saline at the 0.001 level
in males and 0.01 level in females. 76
6. A regression line analysis of the survival response to
graded doses of methylmercuric chloride exhibited by
hydrogen peroxide pretreated (open circles) and saline
pretreated (closed circles) 90-day-old female rats. 79
vn
-------
TABLES
No. Page
1. LD50 at 24 Hr S 30 Day of Methyl Mercury Chloride in Rat
and Hamster Calculated by Probit Analysis and Estimated
by Wiel's Tables 12
2. Incidence of Mortality, Symptom Production, and Brain
Mercury after Single and Multiple Intraperitoneal
Injections of Methylmercuric Chloride to Squirrel
Monkeys 13
3. Incidence of Mortality and Symptom Production in Rats
after Multiple Injections of Methyl Mercury 16
M-. Mean Total Mercury Concentrations Per Gram of Brain in
Three Areas of Rat Brain After Single and Multiple
Injections of Methyl Mercury 17
5. Mean Amino Acid Levels Per Gram of Brain in Three Areas
of Rat Brain After Treatment with Five Doses of Two Mg
of Methyl Mercury 19
6. Amino Acid Levels Per Gram of Brain in Three Areas of
Single Monkey Brain After Treatment with Single and
Multiple Doses of Methyl Mercury 20
7. Percent of Animals Dead Following Various Doses of
Three Methylmercuric Compounds 27
8. Survival Time in Days of Rats Exposed to Various
Methylmercuric Compounds Mean and (Range) 28
9. Means and Standard Deviations of Open Field Measures
of Rats Treated with Methyl Mercury and/or Gamma
Radiation 34-
Vlll
-------
TABLES CONTINUED
No. Page
10. Proportion of Animals Receiving Methyl Mercury and/or Gamma
Radiation Who Achieved at Least One Mount, Intromission
and Ejaculation 35
11. Means and Standard Deviations of Sexual Behavior Measures of
Rats Treated with Methyl Mercury and/or Gamma Radiation 36
12. Means and Standard Deviations of Sexual Behavior Measures of
Sexually Active Rats Treated with Methyl Mercury and/or
Gamma Radiation 38
13. Means and Standard Deviations of Number of Correct Responses
on Conditioned Avoidance Test 39
14-. Frequency and Amplitude of the Electroencephalogram of Rats
Exposed to Radiation and/or Methyl Mercury 42
15. Mercury Content (yg/g brain) of Various Brain Areas of Rats
Dosed with Methyl Mercury 44
16. Total, Inorganic and Organic Mercury Content of Homogenate
of Whole Brain of Rats Dosed with Methyl Mercury 45
17. Norepinephrine Analysis (ug NE/g tissue) 46
18. Percent Change from Contol in Neurotransmitters Resulting
from Various Insults 48
19. Degree of Granule Cell Degeneration from Largest to
Smallest 53
20. Observed and Expected Cell Counts for Possible Loss of
Cells 54
21. Chi-Square Values for Cell Counts 55
IX
-------
TABLES CONTINUED
No. Page
22. Mean5'5 Cell Counts and Standard Deviation for Pyknotic Granule
Cells 56
23. Accumulative Percentage Mortality in Male Rats Following
Treatment with Single and Combined Insults of 2.75 mg.
Methylmercuric Chloride (MMC) and 10,000 R X-Radiation
to the Head 62
24. Accumulative Percentage Mortality in Female Rats Following
Single and Combined Insults with 1.0 mg. Methylmercuric
Chloride Per 100 g. of Body Weight and 10,000 R X-Radia-
tion to the Head 63
25. Comparison fo Percent of Blood Concentration (PBC) Values
Obtained by Treating Rats with Varying Doses of Single
and Co-Insults of Methylmercuric Chloride and X-Radia-
tion to the Head 68
35
26. Comparison of S -Sodium Sulfate Uptake by Ten Body Tissues
in Rats Treated with Single and Co-Insults of Methylmercuric
Chloride (MMC) and X-Irradiation 70
27. Preliminary Radiation Lethality Determination 83
28. Percent Lethality at Different Levels of Insult and Different
Time Intervals Between Insults 84
29. Summary of Percent Lethality for the Co-Insult and the Single
Insult Groups 85
30. Ambulation of Rats 2 Hours After the Second Insult 86
31. Number of Rearings 2 Hours After the Second Insult 88
32. Ambulation of Rats Days 2-7 After the Second Insult 89
-------
TABLES CONTINUED
No. Page
33. Number of Hearings Days 2-7 After the Second Insult 90
3
-------
ACKNOWLEDGEMENTS
The assistance of numerous colleagues, graduate students, and technicians
who assisted in this study is gratefully acknowledged. The assistance of
graduate students Betty Hoskins, James Earhart, Mitzi Thrutchley,
Francine Joiner, Linda Forsyth, and Nancy Partlow, who participated in
the study and utilized portions of the data obtained in master's these or
doctoral dissertations, is especially acknowledged. The special
assistance of Miss Mary Cresson, who participated in many phases of the
study, and of Mrs. Susan Allen, who typed most of the final report, is
gratefully acknowledged.
XI1
«uw. «*- . its- ***
-------
The results ot these studies extend our knowledge of effects of methyl
mercury intoxication and yield additional information on possible
mechanism of action of the agent. However, much remains to be learned
in this area, especially witn resoect to possible differences in
response between species.
The results reported herein demonstrate that the interaction between
methyl mercury and ionizing radiation is complex. In some cases
(several parameters evaluated in rats within 6 hours after the second
insult, the squirrel monkey study with a relatively small radiation dose,
and the apparent activation of a protective mechanism against methyl
mercury-induced damage by radiation) the effect of the co-insult
appeared to be less or at least no greater than either insult delivered
alone; in many other cases the effects of the two agents were partially
additive, but in no case did they appear to be completely additive:
i.e., 50% of both insults together did not produce as much damage as
100% of either insult delivered alone.
-------
SECTION II
RECOMMENDATIONS
Possible interactions between agents are potentially important, and the
results obtained in this study indicate that more studies involving
multiple stresses are necessary in order to properly evaluate the
multiple stresses to which human populations are or may be exposed. We
recognize that it is not possible to measure all of the parameters which
may be affected at various times by all the possible agents, and therefore
suggest that the interaction of a limited number of agents currently
known to have the greatest potential hazard to man be investigated in
detail, with special attention to the nature of the interaction between
agents. Hopefully these results will lead to generalizations that could
be tested with a limited number of additional agents.
We believe the current data base is inadequate to set meaningful standards
for multiple stresses, and that the possible interaction between agents
should not be ignored in future standard-setting.
-------
MATERIALS AND METHODS
Male Sprague-Dawley rats aged approximately three months and having a
weight range of 275 to 320 grams (300 ± 36 g Standard Deviation)
were used in most of the experiments. Male Syrian hamsters obtained
from F2 and F3 matings for pigmentation inheritance studies had a
mean weight of 101.9 ± 14- g. The 15 squirrel monkeys were adult
males and females weighing 4-32 to 1123 grams.
These animals were obtained from and maintained under the standard
conditions of the Texas Woman's University animal colony. Two to
three monkeys, or five rats, were housed in metal cages. Hamsters
were kept in plastic cages with wire tops and corn cob bedding, five
to a cage. Rats and hamsters were fed Purina Laboratory Chow with
ad libitum water. Monkeys were fed three times a day a diet consist-
ing of Purina Monkey Chow soaked with reconstituted orange juice or
milk, fresh fruit, raisins and peanuts.''' Water was changed twice
daily. The animal rooms were maintained at 25 C with a 12 hour
light, 12 hour darkness cycle.
Methyl mercury chloride (Alfa Inorganics, Beverly, Mass.) was dis-
solved in sterile isotonic saline and administered intraperitoneally
in one or two ml of saline. Controls received a like amount of
isotonic saline. Geometrically spaced doses of 8, M-, 2, or 1 mg
per animal were first administered to groups of ten rats and hamsters
and to single monkeys. Subsequent series received doses above,
below, and intermediate to these increments.
Animals were closely watched for these first 5 hours after injection,
then observed daily for morbidity, gait, and appearance changes.
They were weighed once a week. Observations were continued for
25 days or until imminent death. At the time of sacrifice, animals
were lightly anesthetized with ether and decapitated with a Harvard
Apparatus Decapitator (Millis, Mass.). The head was dropped directly
into liquid nitrogen. This was necessary since some amino acid
levels including GABA, begin to rise within a few minutes after death.
-------
After complete freezing, the entire head was labelled and stored at -29 C
in a cold room less than four months . Tissues were fixed in formalin :
alcohol: acetic acid and stained with hematoxylin and eosin for histo-
logical examination.
The calculation of the LD50 at 24 hours and 30 days (LD5024 h
LD5C>30 £ respectively) was performed by repeated iterations of probit
analysis9 after recalculating individual animal doses to a rag/kg basis
and converting % death to probit. These values were checked by use of
Weil's10 tables.
At the time of mercury or amino acid determination, 10 to 100 mg portions
of cerebellum, brain stem, or cerebral hemisphere were removed from the
skull and weighed . The portions selected were : outer cerebral cortex
in the sensory -motor area above the corpus callosum, a central hemi-
section of cerebellum including vermis and flocculus , and pons and
medulla just below the cerebellum. The visual cortex was not included.
Matched halves were used, one half for mercury, the other for amino acid
analysis.
Total mercury was determined by flameless atomic absorption after
overnight digestion with sulfuric acid-permanganate-'- and reduction with
a stannous chloride-hydroxylamine solution1^ in a closed system. Dupli-
cate sample readings were compared to standard readings with a Perkins
Elmer Atomic Absorption Spectrophotometer set at 2537°. A standard
curve was prepared daily using the same reagents used to treat the
samples .
Amino acids were determined by two dimensional thin layer chromatography
of dinitrophenol derivatives^- on ITLC Gelman chromatographic paper.
Solvent I was toluene: pyridine: 2-chloroethanol 100:30:60 equili-
brated with 60 parts of 0.8 N ammonium hydroxide. A run required
approximately 20 minutes; after thorough drying and 90° rotation a run
in solvent II (chloroform: benzyl alcohol: glacial acetic acid 70:30:3)
required 30 minutes. Spots of GABA, glycine, glutamate, and aspartate
were identified by comparison to simultaneously performed chromatograms
of commercial DNP-amino acid preparations (Nutritional Biochemical Co.).
-------
Appropriate spots were eluted with 0.010 N NaHCC^ and read on a Perkins-
Elmer Spectrophotometer at 360 mU. Samples were automatically trans-
ferred from a fraction collector to a flow-through cuvette via a
Transferator Programmer (Gilson Medical Electronics). Micromoles of
amino acid per gram of brain were calculated.
RESULTS
CALCULATION OF LD 50 AND LD 50 3Q d
The death rates of rats and hamsters at 24 hours and 30 days after a
single intraperitoneal injection of methylmercuric chloride are shown
in the sigmoid curves of Figures 1 and 2. (An excessive number of
animals was available for this estimation because of the simultaneous
collection of tissue for biochemical analysis.) Inconsistency of the
percentage lethal response to exponentially spaced doses can be seen
and was not eliminated through repeated iterative cycles of probit
analysis on rat data. That data showed continued scatter of points
around any postulated line (chi square for linearity .2 > p > .1).
The linearity of the hamster probit curve became valid at p= .01. The
slope of the curve of response at 24 hours is much steeper for the rat
data than for the hamster. That is, the range of doses over which
approximately 50% of hamsters die is broader than that for rats. At
30 days, the response of both species was more nearly linear. The
LD50's calculated by probit analysis are presented in Table 1 with
comparison to the generally agreeing estimates obtained by use of
Weil's tables. The LD50 24 hr was 11>9 mS/kg in the rat and 22-^ mg/kg
for both two and three month old hamsters. The LD50 QQ j was 10.1 mg/kg
in the rat and 15.2 mg/kg in the hamster.
Nine squirrel monkeys were injected with methymercuric chloride to
estimate their lethal dose level (Table 2). Monkeys treated with 2 to
8 mg (3.6 to 17.0 mg/kg) did not die within 24 hours. Thus the
LD50 24 hr is greater than 17 mg/kg. Within one month, a dose of
6.4 mg/kg caused such severe debilitation, tremor, and blindness, as
to demand sacrifice; the animal would have starved to death. Heavier
animals receiving 4.8 and 5.6 mg/kg survived. Thus the LD50 OQ ^ can
be estimated as between 5.6 and 6.4 mg/kg.
SYMPTOMS OF METHYL MERCURY POISONING AFTER SINGLE AND REPEATED
INTRAPERITONEAL DOSES
The response of the three species to a single intraperitoneal treatment
in the same dosage range, was strikingly different: Rats exhibited
rapid respiratory and vascular symptoms, and did not develop motor
damage. Fatally dosed hamsters became comatose but showed no gait
abnormalities. Monkeys were not killed within 24 hours by doses (on
a mg/kg basis) fatal to 90% of the rodents, and they developed severe
neurological symptoms not seen in the rats and hamsters.
-------
Fig. 1. Percentage death rate of rat () and hamster (*) 24- hours after
a single incremental intraperitoneal injection of methyl mercury
chloride.
-------
o
LO
-------
o
s
w
w
o
o
I
(_>
Pi
w
s
H
o
o
sHivaa
11
-------
w
H
00
s
sc
O fO
^, 00
< u
H m
(5 EH
S 00
h- 1
, "]
W W
Q M
M tS
Pi
O *>H
J PQ
O Q
>H E-H
PH H 0
C | ^-*
w
S CO
M
PH CO
0 ><
> LO O J- rH LO
d" CO vH rH
^^ v~' ^^
- , --V /-x
yH LO 0
...
CO vH iH
1 1 1
O CD LO zf LO CN
. . .
CO CN rH rH iH rH
V_^ « J \ ^
.. d- CD
II LO II II
LO LD
O -H vH
TH 1 I
1 CD J-
rH CO CN CO
J* rH
O CD LO TH CO rH
rH ^^/ rH **-* TH v '
*^*\ /-^ /^
CO [> O
LO CN CO
1 1 1
O O LO CO CO O
J- CO CM TH CN CN
N*-' S^x S '
l^°\ X"*\ >^~\
O J- CN
4- CN CM
1 1 1
LO CN 3~ CO TH O
CO CO CN CN CN -x CN > rH v -
TH 10 TH
O UD CD
O CD O
CO rH
CO CN CO
(H PH
Q) (U
-P -p
w co
-pee
Pi a: !
o
LO
Q
(1)
(-;
-P
MH
O
!>i
O
tO
c^
^3
O
O
to
UH
W
-P
H
E
H
-H
0)
0
C
(j;
H
MH
C
0
O
«
4^P
LO
en
0)
f]
-p
(L)
tO
«t
CO
0)
CO
cu
CN -P
LO C
CD
-------
o
^
<£
PJ 00
J >*
CJ W
S X
co o
^r|
Pi
PH 2
< Pi
h- 1
t>H | >
Pi O*
p-) C/3
O
Pi O
W H
W
S O
M M
n 2;
co o
o E-i
>-< C_>
E-i P-l
S §
K}
"CO ^
E-
Q)
|3
W
w
H
E '
txQ
,X
tiO
E
!>>
£4
£3
O
P
CD
S
MH
o
-p
G
CD
E
&
O
CD
%>
CD
TJ
MH
O
CD
E
H
C
H
rO
m
1
CD
fn
CD
O
c_,
CD
O
TJ
G
A
H
£^
O
c
CD
f-i
CD
CD
W
CD
CO
O
Q
T?
O
pq
E
CD
-P
CO
E
H
CD
^3
CO
H
E
CD
f~l
G
o
H
-p
H
TJ
G
O
O
j
W
B
O
p
OH
E
CO
bfl
^^
bO
B
H
CO
E
H
r~t
tO
bO
B
^^\
bO
-UJ
J2
X
CD
CO
O
js;
^>
CD
i-^
G
O
S
H CN CD J-
LOCOIOOIOI i cnto 01 CN
CNO|LO|O|OI
1 1 1 1 COO TH TH
CO CN 1 CO | O 1 O 1 TH TH
O CO | CO | TH
t~-O|CN CCI 1 tDCOld- O
ocnito LOIOI !!
1 . . | . | THj-|CniTH
=t CO 1 J- TH 1 O 1 CN 1
tOC-^|LO tO| 1 CO-rH O|O
I>CNITH ooioi ot^- LOICD
1 1 . 1 . . 1 . 1 .
COCOICO -HIOI OCN tDIO
tO tO f> CO tO ft
p TJ TJ TJ TJ TJ TJ
fi CDWCDCDCDCDCD COCOtOCOCOCO
K* K*) t* t* *> *? K* K*^ K^ r>~> t*> £>) K^
tD p -r-l cO -H T-I -H -iH -H (0 CO (0 (0 tO (0
CD r^ J> "Xj [> ^ ^ J> £> TJ TJ Tj TJ TJ TJ
1 rH rH P rH r^ P
_j-tD^)LO3^l^)"J^i LOTHTHTHtOTH
LOlDCQCMCOCOCOMCO THCNCNCN J-
TJ
f^
O
P CO CO CO CO CO CO
*H ^>1 ^1 t>-) K^ K5*) ^1
CHPP^L, co rOtOfOfOtOfO
.CrC-G-GCUGeDCDCD TJT3TJTJT3TJ
G fO G G G
cncnj-CMOfnOOO j-j-j-r~-LnTH
THTHCMCNG-PCCC THTH-HTH CM
cn t~^ TH r~ d- r~ CD
TH
co to LO
oooooooepo LocNocor^
OOJ-d-OOCOCNCNOO CNiHTHTHOO
X X X X X X
LO LO LO LO LO LO
HCOCDF-OOCOOO -±COCOCNLOOO
F-CNrHtDCNCOCOtDLO OOJ-CN-THTHd-
d" -TJT tD .Ijr tO rj" LO ^t* LO z}" C*** rH -3" CT5 LO
TH
(JHp-,p-,P-,P_tp_,pHpHpL, (J-,ggP-,SP-4
CD
to co
O CD
Q CO
O
CD THCNCOJ'LOcDC^OOO^ Q -rH-HfLOCNCDCO
H C/1COCOCOCOCOCOCOCO SSSSSS
bO CD
G >
H "H
CO P-,
rH O
3 -P
0 O
H E
MH >,
**H fn
TJ O
P
H
CO
CO C
>i tO
CD £H
y/ i i
GTJ
o
B " -P
-G C
C 0 CU
H rO E
CD -P
CO P rO
E rH 0)
O CD p
-P > -P
di O
>, TJ 0)
CO C -P
CO tp,
CD cO
P P
CD O W
^ C ^l
CD CD CO
CO P TJ
0 -P
LO
" G CN
W O
>1 -rl TJ
rO -P C
TJ G tO
CD
TH -P TH
CO C TH
H
-P G
rO " 0)
CO Q)
CD -P ^
3 C -P1-
CO CD CD
to E X}
H CD
-P > G
0 0
rH E -rl
O -P
I | , f\ /-I
HH rM \J
r { ;
TJ.I G
CD H tp
O O
r-t U-l
r-1 M 1
4n O CO
H CO
PH C 0)
0 O G
m B
tO O
TJ O
CD CO
O TJ >,
O1 CD
r* Ai
C CO G
fO ft O
,c _B e
'rH C
CD -H
P-l T3
O C co
6 (0 E
JC 0
TJ -P
CD bO D.
> n s
H -H EX,
> CO CO
3
C/D C
rt3 -H
13
-------
In rats, dramatic reaction occurred within 15 minutes after adminis-
tration of 8, 4, or 3 mg per animal. They became lethargic, heads
drooping and eyes dulled. Some of the animals recovered normal color
and activity within two to three hours. Others, visibly indisting-
uishable, progressed to death. The latter showed rapid (60/minute)
abdominal breathing, postero-anterior progression of spasticity, and
loss of the ability to right and to walk. Two to three hours after
injection, respiration became more labored (20/minute), the legs flexed
tightly to the body, and the toes developed carpopedal spasm. Symptom
progression and the time of death were synchronous for all animals
treated with the same dose. Animals treated with less than 3 mg became
drowsy but did not expire.
Rats surviving the initial insult showed later general debilitation such
as weight loss,!^ abdominal bloating, coarsening and matting of the fur,
mucus laden nostrils, and decreased activity; but specific motor
changes were not seen. Upon autopsy, the liver appeared pale and en-
cased in tight strands of connective tissue. The intestine was distended
with food and gas. Other organs appeared grossly normal.
It was curious to note that all rats which survived the higher doses
of 4, 6, or 8 rag/animal and the lowest doses of 0.1 or 0.2 mg per
animal recovered from the respiratory distress described and continued
to live for one month. Intermediate doses of 2, 2.5, and 3 mg were
followed by further deaths throughout the month. The time until death
increased, and the percentage of animals dying decreased, with decreas-
ing dose.
Single doses of 0.2 to 8.0 mg of methylmercuric chloride to hamsters
simply accentuated their normal cyclic chattering, and quarreling, and
sleeping. Just after injection, the hamsters appeared drowsy; only a
mild cyanosis distinguished treated animals from the normal nap behavior
of controls dosed with saline. Most responded to poking with biting and
fussing. Deep cyanosis was not seen; a comatose condition progressed to
death about three hours after injection. Survivors showed a gradual and
steady weight loss. A few deaths occurred in each dosage level during
the following 30 day interval. No gait problems developed.
No monkeys died within the first 24 hours after a single treatment,
although the dosage levels on a rag/kg basis were comparable to those
killing 80 to 90 percent of the rats and 50% of the hamsters. (Table 2)
By 19 hours, animals dosed with 4 to 8 mg per animal (5.6 to 17 mg/kg)
became huddled and indifferent to prodding. Death ensued by 66 hours.
An animal dosed with 3 mg (6.4 mg/kg) became lethargic, clumsy, and
uncoordinated at 22 days, and deteriorated rapidly. By 24 days, she
became blind, drew her forelimbs jerkily to the mouth, and remained
rigid when handled. Sacrifice was essential. Treatment of 3 animals
with 2 or 3 mg (3.6 to 4.8 mg/kg) was followed by transient lethargy,
minor motor difficulty, and recovery to normal within 26 days.
14
-------
Four and five doses of methyl mercury were administered to series of
rats, hamsters, and monkeys to establish a dosage which would produce
symptoms within an acceptable mortality rate. In Table 3 the results
of rat series shows that a regimen of at least 5 daily doses of 2 mg
each (approximately 34 mg/kg) were required to cause the beginning of
motor symptoms (previously described as partial tucking of the hindlegs
when lifted by the tail) 12 to 15 days after the initiation of injections.
Definite signs (hindlimb tucking, paw flexion, and walking high on the
legs) were seen within three weeks after the beginning of injections in
about 40% of the survivors at the time (10-20% of the original sample).
An additional 27% of survivors exhibited moderate symptoms; the remain-
ing 33% had a normal gait. All were bloated, diarrhetic, and matted.
Four doses, or five doses of 1 mg produced no motor symptoms in an adult
or junevile (series C) rats.
Five doses of 1 mg each to hamsters produced a high mortality rate (76%)
and a low symptom rate (4%), parallelling the high death rate after a
single 5 mg dose (Fig 2). Consequently multiple two mg doses were not
administered.
The effects of repeated doses on 3 female and 3 male squirrel monkeys
are shown in Table 2. All cumulative doses from 4.7 to 20.7 mg/animal
were followed by the same degenerative pattern. The earliest appear-
ance of symptoms (toes curled, muscle spasms of the limbs,
anteroposterior rocking) occurred 5 days after injection, in a large
male. All other developed symptoms at 14- - 21 days. Deterioration of
coordination and vision was rapid.
MERCURY LEVELS IN BRAINS OF RATS AND MONKEYS
Total mercury was determined in less than 100 mg portions of sensory-
motor cortex, cerebellum, and pons and medulla of individual rats
sacrificed 21 days after treatment with methylmercuric chloride. The
levels are presented in Table 4 as mean values for groups of three rats
treated with the same dose. A range of mercury accumulation is indicated
by those means and standard deviations. A significant (at 95% confidence
limits) difference was seen between levels in animals with and without
symptoms and between both those groups and the controls. Individual
rats with motor Impairment contained 8.8 to 17.5 yg/g. cerebral hemi-
sphere, 3.7 to 18.6 yg/g cerebellum, and 4.1 to 17.5 yg/g brain stem.
There was not a significant difference (95% CL) in accumulation between
those three areas. Animals with only nonspecific symptoms of bloating
and diarrhea contained 2 to 15 yg/g cerebrum, 6 to 17 yg/g cerebellum
and 2 to 2.7 yg/g brain stem. After a single dose, the highest mercury
levels detected were less than those after multiple injections. Mercury
was detected in the brains of controls housed in the same room with
injected animals, at levels of 0.2 to 0.8 yg/g cerebral hemisphere,
and 0.0 to 0.7 yg/g brain stem.
15
-------
S3
M
^
O
M
H
O
Q
O
«
PL,
S
O
H
PL,
£*-*
CO
Q
3
J>H
E-H
M
r~]
<
PS
o
pi_(
o
w
o
55
w
M
0
55
M
.
CO
0)
rH
10
H
^
Pi
O
W
s
J
£C
H
W
s
P-H
O
CO
3
0
M
H
a
w
M
W
J
Pu
M
rH
^3
s
0
E-H
<
co
EH
ss
w
H
IT)
E
rH
fi
rH
rH
<]^
0\°
W
H H
rH tO
< E
H
D\° C
10
w
rH
10
E
H
C
<£
i |
H
<£
osp
10
Q)
s
w
g
0
(-"L,
g
>i
w
t~;
-p
H
15
C
H
rrj
tO
T^
w
l
to
TJ
Y-^
CN
P
IT)
W
£>>
(0
T)
vH
CN
LO
J-
co
CN
TH
bO
CO
rH
10
£
H
C
<£
O
55
rH
tTJ
£2
H
G
~-^
W)
o
o
LO
0
CD
O
CD
0
LO
O
LO
O
.H-
in
a>
TH
j-
0
^
^
w
'a)
Q)
is
ID
r_j
O
^1
rH
^
QJ
QJ
jj
bO
£
CN
O
CN
O
.H.-
o
CN
O
CN
O
0
O
o
01
CO
CN
O
TH
0
CO
(0
in
^
O
IP,
K*1
H
H
fO
bO
E
CN
o co j- ooo
H vH TH
O C^- tD OOO
<& u-> r-~ TH co
O iH d- OOO
in TH co TH r^
O LO CN OOO
in in in
O CO 00 OOO
CN d- CN
OOO OOO
d"
O O 00 OOO
H
t~- CN CO CO [> ID
CD CO f-- CO TH CN
f- CO TH CD 00 CN
CO CN CO CN CO TH
o d- o ooo
vH C-- LO TH TH TH
W CM CD Q W O
CO
tO
in
fq
O
HH
^
H
H
to
T)
bO
£
TH
CD
CO
0
O
O
O
0
CD
in
CD
CN
o
in
pq
CO
(0
T)
c^
O
^>
H
H
10
""O
bO
£
in
TH
16
-------
en
r,-|
Di
^
s_,
W Pi
H s
2 o
H W
s S
1 I !>-i
ffi
"z, t-1
M W
S ^
rn L_i
0
O 00
s o
S H
ej O
Pi ""O
P- 1 |
en W
O PH
H M
H H
Pi D
E-i IS
W Q
0 53
^, <
O
0 H
>H O
Pi IS
H3 M
o en
Pi
W Pi
s W
-i n,
O CNLOLOt-O
J-OOfOO iHrHJ-CN
C-^CDrHCNO -sHrHOO
rH
[>- O !-O CD C CO LO 00
OOtDC- COCncDCO
j-roLOLO CNOOO
+1 +l +i +l +l +1 +1 +l
CDCOI>vHO Lnr-vHCN
d-CNtncoo oodjj-CN
tDOCDCNO vHvHOO
H vH vH
rO rO
C35 ^}" CD C^J OJ LO jj" CO CZ3
Jd~vHOCQCO LOCNCOCO
Zf-CNLOtDO J-rHOO
H-l +1 +1 +1 +1 +1 +1 +1 +1
J- CO rH CO CO COCNOOC~~
IT^vHCxtCOrH COCSId-d-
OOOTHCOO COOJOO
rH iH rH
0
0
rH
TiJ UH
hO'H
C 0
f( -HO)
-P C ft
rC C W
bOO "H 1
H W bO S
HCUCUOO OOOO
CO *rf~* rQ C C C G C C
cococococo cocococo
rG
O
tO OOOOO COCOCOO
j-^t-^t-^fo cor^coo
bO CO CO CO CO rH
E
CN
MH
o
0)
CO W
0) OOOOO O OLOOO
O OOOOO J-CNrHO
Q rH rH vH rH CU
i |
0 b_0
^ G
rH -rH
fj-i en
rH
H
(0
P
J>^
Q C
O
T3 -H
0) "A
P 0)
[ j , (
M i i 1
H MH
H
S
G rO
-H
tO ^ T3
ft bO
-H «%
CO CU hO
+J £ C
CO (0 -H
I) C . [1 . I)
to o to
^ ft C 0
G -H W H
O C CO fi
H -H W O
-P EH
(U CO O
r-4 P^ 4 ' rn
r-| p^ -( uj
> 0 ft E
CU -P E CO O
TJ ft S E -P
E w o ft
0 >> -P B
ft CO ft ft >,
to OEw
id ft -p >,
G O O CO O
tO -P E -H
-P O bO 4n
We ni (-« ,_i
£2 w ^-i *ri
P -H O
H-l -P -H C CU
rC C C ft
C bO-rH -H CO
r^ -H MH bO G
0) rH 0) Cl) O
S en Q cq g;
0 ,Q O T3 Q)
17
-------
Hamster mercury levels were not determined since symptom production was
at such a low level. The levels of mercury in the brains of single
monkeys which had received single and multiple doses of methylmercury
are summarized in Table 2. There was little difference in level of
accumulation between brain areas at any dosage range except the highest,
5 doses of 2 mg each (20.7 rag/kg). Mercury was accumulated in all three
areas of the brain at the same levels after a single dosing. No mercury
could be detected (less than .001 ppm) in the brain tissue of a control
monkey housed in the same room with treated animals.
AMINO ACID LEVELS IN BRAIN TISSUES OF RATS AND MONKEYS
Levels of selected amino acids suspected of neurotransmitter function
were determined in 10 to 100 mg portions of the cerebral hemispheres,
cerebellum, and brain stem of rats and monkeys opposite to the areas
used for mercury determination. When 20 to 30 ul aliquotes of DNP-
derivative/extract were used as the total spot volume, amino acids
present in concentrations less than 1 yM/g of tissue were not consist-
ently visualized. The amino acids present in greater concentration,
especially glutamate, aspartate, glycine, and GABA, were easily visible
and discrete. Such spots were eluted and quantified by spectrophoto-
metric comparison to DNP-amino acid standards. A possible artifact,
DNP-hydroxide which may be present when heptane bromobenzene washing is
incomplete, was rigorously avoided by washing and by excluding all
spots with a value greater than cf 0.55 in Solvent I. (GABA Rf values
were 0.44 in Solvent I and 0.77 in Solvent II; DNP hydroxide has an Rf
of 0.60 in Solvent I and 0.85 in Solvent II according to Shank and
Aprison,1960).
In rats (Table 5) no marked differences were found in glutamate, glycine,
aspartate, or GABA levels of control and symptomatic rats and in the
three brain areas. In the monkeys, however, (Table 6), increased GABA
levels and marked reductions of glutamate, aspartate and glycine levels
were seen in all dosed brains. Glycine became so low as to be indectect-
able. An exception was found in a monkey dosed with the lowest single
dose (6.42 mg/kg); aspartate levels were normal in cerebellum. Levels
of those amino acids in control monkeys were in comparable ranges with
previously published ranges. GABA levels showed a moderate rise in
cerebral hemispheres of multiply dosed animals; in the cerebellum and
brain stem, levels were 2 to 100 times the control. Singly dosed
animals displayed only moderate rises in GABA content in the hemisphere
and inconsistent cerebellar and brain stem levels.
DISCUSSION
In our study, a range of lethal doses and some non-linearity of the
regression curve continued even though large numbers of animals were
18
-------
E-i
s
l-> ><
0 PS
ID
CO CJ
< PS
W W
PS s:
< j
>-!
W ffi
H H
PS W
K g
H
SS O
1 1
0
S S
M
< o
OS 13
pT| E-H
PJ^J f-M
O O
S CO
< u
PS CO
u o
Q
OS
w w
PH >
CO Lu,
W <~L~i
> H
H M
K-J S
Q H
M £5
O W
< S
E-i
O <
£5 W
M OS
S E-i
o
PS
s w
< E-i
W PH
S <
s:
M
LO <;
PS
,
CO
0 C
S3 <
W
Q)
H
k
0)
CO
0)
w
o
Q
hO
,X
^
bO
6
rH
td
6
H
c
0)
"^^
bO
6
o
c nd
H '.H
e o
oocn j- en j- LO
(DLncOrH COCNlvH LO^HCO CDOCNCM
. 'O ....
C^OLOrH CxIOOCN COrHO COCOvHCO
LOLOCOO COCNOO r^-Lni>CM LOOvHO
CNiLOcDLO IDt^COLO OLOLOCM O-CDCMCO
.... *. .... ....
OOOvH OOOCN OOOO OJ-rHct-
+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1
C-~l^COO t^-CO[>-O F^IOCOCO J-CMiHJ-
csicocnr^ ooj^-i>- T-HLOGOOO UDCNJ-CO
.... ..«. .... ....
OOOCOCO tD-rHCNCO OOrHO rHJ-J-CO
J-OOLO 4-OJ-CM OOO vHTHLOO
C-^COCOLO vH-rHOCN OOiXiJ- CMCOJi-vH
... .... ... ....
OrHOTH OrHOO CNrHO LOOt-ICO
+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 -H +1 +1 +1 +1
OOLOOJrH COCNOO tOvHCn OCSIt^UJ
ojc\t>-d- t^r^Lnesi TdcovHd- OCOJ-LO
«. .... ^ ... ....
COCOrHCO rHvHCOCN GCO-rHvH LOOCOJ-
TH
O
p p p p
f! ^C fl &
fl bO bO bO bO
W W -H W w -H W W -H M W -H
(UOJHO CUCPHO CUQJHO 0), >,CO C >,>,WC >,>,WC >,>,WC
COCOCOCO COCOCOCO COCOCOCO COCOCOCO
P-iOOtM fj-*CDOk-i [j-iOOPj-i UnOOtM
J^-
OOOO OOOO OOOO OOOO
.... .... .... ....
j-j-d-o j-j-j-o j-j-j-o dJJ-jj-d-j-
cococo cococo cococo -pcooococo
m
^
p
oooo oooo oooo pqoooo
.... .... ....
oooo oooo oooo ooooo
-rHvHrH rHvHx-t vHrH-rH CT-HvHrH
0) Q) -H
-P P g
n3 n3 (D <^
6 P C i
fO PH -H rfl
P fd O g
^ ft >, £
H CO H (fl
U
-------
-- 0. ,.]...- - , u,,V... - JKrM -i b.
-------
used over a six month period. The hamster death rate showed a plateau
over a range of doses. Previous reports have ranged widely; Swensson
and Ulfverscn-' tabulated oral methylmercury dicyaniinide as having a
reported LD50 of 26 and of 32 rng/kg. Ulfavarson^ lists 400 nig of methyl-
ffercury hydroxide as '-t ''2 percent LD50 dose", and Swenssonl9 reported a
lethal intraperitoneal dose of 10 mg/kg of me thy Imer curie chloride dis-
solved in oil. Feakall and Lovett^O summarized available figures as
indicating an LD50 of 30 to 150 mg/kg in rats. An LD50 for hamsters and
monkeys has not previously been stated.
Given the continuing inconsistency of results, not all of which can be
attributed to omission of information on time interval, route of admin-
istration, anion, age, and sex of the animal, one must question the
method of experimental procedure and method of LD50 estimation. Finney21 cites
studies showing all current LD50 estimation methods to produce similar
estimates over a middle range of doses. It may be that the dose re-
sponse pattern to methylmercury is in fact more complex than a simple
exponentially-linear nonthreshold relationship. More than one death
mechanism may be involved, summating at intermediate doses. Clearly
death within 24 hours was related to respiratory depression. Later,
nonspecific body thinning and bloating probably involved cumulative
inhibition of liver sulfhydryl-containing enzymes22 and brain cofactors. 3
But such inhibition cannot account for the description of brain tissue
deep in sulci as anoxic or for the greater effect on hindlimb inner-
vation^4 and activity. Further, the dose response may not be normally
distributed; methylmercury poisoning may be a threshold phenomenon in
which there are doses below which no animal is killed with the designated
time period. It may also be that rats, even from a long established
colony and strain, are not evolutionarily homogenous with respect to
methylmercury sensitivity, and susceptibility and uptake may vary as it
apparently does in humans.25
The pattern of damage to humans and other mammals is becoming well de-
fined and amenable to behavioral analysis.26 xhe initial study of Hunter
et al.27 described both classic human and animal symptoms. Slurred
speech, numbed and tingling fingers, and limb weakness progressing to
ataxia, concentric narrowing of the visual fields, fine motor disability,
and memory difficulties were seen in the first cases. Similar degenera-
tion has been described after ingestion with food28 and after respiratory
exposure of laboratory workers29 and seed dusters.30 Volunteers^l and
high fish diet consumers have reported nonspecific irritability and EEC
changes. In animals, inability of the rat to hold the head erect^2 and
increased critical fusion intensity in monkeys^ have been established
as early signs. Hunter eT al.34 described the progression in rats and
monkeys; clumsiness of the reddened cold hindlegs and weight loss were
the firct discernable changes. As the animal became moribund, the
forelegs be ;ame involved. Irritability, ataxic changers, and leg cross
when lifted L>v the tail followed sub-letnal doses." The monkey shews
^ £
degenerative patterns similar to humans."0 Surprisingly, the respiratory
distress and iiver changes have not J^esn previously commented upon.
-------
Neurological d^i.ia^e was produced in rats only by multiple doses; recnkeys
responded to single doses of greater than 6 mg. Most previous work on
rodents is in agreement.37 Only Yoshino et al. 8 obtained damage in
imrriature rats with a massive 7.5 mg/100 g body weight. It is emerging
that the immature animal shows a different pattern of damage than the
adult.39 Our pilot study too showed juvenile rats less likely to develop
gait problems than were adults. The percentage of adult rats displaying
symptoms varied from series to series, as previously reported by
Swensson and Ulfvarson.^0 Yet Klein et al.^^- found 7 doses of 10 mg/kg
to consistently produce symptoms without lethality at 14- days.
The effect of distributing the dose over a five day period varied with
the species. In rats, similar lethality was produced by 10-12 mg/kg
as a single dose and by 33 mg/kg delivered as 5 portions. Perhaps
detoxification mechanisms, especially via the kidney, are immediately
effective. In hamsters, the susceptibility to cumulative doses admin-
istered daily for five days was nearly as great as that to the dose
given as a single injection, but gait problems were rare. A squirrel
monkey was killed by 5.7 mg/kg given as five doses while another survived
a single 5.6 mg/kg. After humans ingested an estimated 3.9 mg per day
for 100 days,^2 juveniles 8-13 years old remained severely handicapped;
a 20 year old recovered some speech and mobility. Adult members of the
family remained asymptomatic. The monkey then would seem a useful if
more sensitive model for the human than is the rodent.
In the"'>th&Ke>e specites -studied, the LD50 3Q ^ays decreased with increase
'dij- body wfeiglit': hamster 15 mg'/kg,'rkt 10 mg/kg and monkey approximately
6 mg/kg. Within a species, the larger animals appeared more sensitive
than predicted on a mg/kg basis.
It is always necessary to demonstrate that mercury has in fact crossed
the blood-brain barrier and is present in animals presenting neuro-
logical symptoms attributed to its presence. In this study, mercury
was demonstrated in samples of less than 100 mg of single brain areas
of single rats and monkeys. It would be feasible to dissect free
nuclear areas in the monkey brain. In both rats and monkeys, mercury
was present in cerebral hemispheres, and in the brain stem. Variation
in content between animals was significant, but variation between areas
was not. The results of the present experiments indicating symptomatic
animals have greater than 8 yg Hg/g brain agree with previous estimates
of threshold mercury levels. Berglund and Berlin1^ reported 8 ug/g
tissue is necessary to produce symptoms; Evans et al.1*^ quote Suzuki
and Miyama as finding 10 ppm to cause head lag in mice, and Berlin
et al.^5 report 9 ug/g tissue as the calculated level producing an
effect in monkeys.
Real differences in levels of neurotransmitter-like amino acids and of
the inhibitory neurotransmitter GABA, were seen between control and
treated monkeys. But levels were not significantly different in rats.
22
-------
-IT..I Ic^c- for _ lyc'lrs--:, "r; tVv_.--. In the
free?in?, j-" liov^s, '.:l"-'.-l.;icit'-.' arid aspartate levels wer'e con-
ith the ;->revic.;; lite--j rare, It Is curious that the GABA level
01 methyimercury- tree r-.nimak were raised above those of concomitant
controls. Mercurials cause decreased synthesis in vitro of GABA,^7
presumably by inhibiting the action of glutamate decarboxylase and
stimulating the degradation of GABA by transamination with alpha-keto-
glutarate.^8 Shank and Aprison1^ however, believe glycine and GABA to
exist in at least two metabolic pools, one more active than the other.
Further analysis of defined portions of monkey brains, correlated with
behavioral changes, should be of interest.
SUMMARY
The LD50 24 hr °^ ^ethylmercuric chloride was estimated as 11.9 mg/kg in
the rat, 22.U mg/kg in the hamster, and greater than 17 mg/kg in the
squirrel monkey. At 30 days, the LD50 was 10.1 mg/kg in the rat, 15.2
mg/kg in the hamster, and estimated as 4.7 to 6.4 mg/kg in the monkey.
Motor symptoms such as hindleg tucking when lifted by the tail were
induced in the rat by 5 daily doses of 2 mg each, resulting in brain
mercury levels above 8 yg/g cerebral hemisphere or cerebellum, and more
than 6 pg/g pons and medulla. Levels of glycine, glutamate, aspartate,
and gamma-amino butyric acid (GABA) were unchanged from control levels.
In monkeys, single doses of more than 3 ing/animal and 5 doses of 0.75 mg
or more produced a neurological degenerative pattern at mercury levels
greater than 8 yg/g tissue. GABA was increased and glycine, glutamate,
and aspartate were decreased in the brain tissue. Neither single nor
repeated doses produced motor responses in the hamster prior to death.
23
-------
SECTION V
RELATIVE TOXICITY OF VARIOUS METHYL MERCURY COMPOUNDS IN RATS-
INTRODUCTION
Results presented in the previous section indicated that in our hands,
the LD5Q/30 for methylmercuric chloride is 8 to 10 mg/kg. Our persual
of the literature, which is sparse and not as well documented as it
should be, gave most LDso values ranging between 30 and 150 mg/kg for
rats - 3 to 15 times our values. Only one report on methyl mercury
cyanimide gave a value of 10 mg/kg when dissolved in oil. Another50
gives 5-6 mg of mercury/kg in mice IV or IP, and probably a similar
dose for rats.
Several factors could be responsible for the differences observed:
1) route of administration (we use the intraperitoneal because it
gives rapid absorption and mimics the oral route; others have used oral,
intravenous and subcutaneous administration), 2) age and sex of the
animals and the interval evaluated (important, but not frequently men-
tioned), 3) vehicle used (i.e., distilled H20, saline or oil have all
been used, and it may be important), H) methyl mercury compounds are
not very water soluble (some investigators have reported using con-
centrations greater than we can get in solution). Another factor we
have observed is that solutions weaken with time; one reason is inter-
action with sulfur in the stopper of the bottle. We use fresh solutions
to avoid this problem, but some workers may not be aware of this problem.
The subject of this study was the possible effect of the anion. Most
investigators have de-emphasized the importance of this possible factor
because the anion usually dissociates so that only the methyl mercury
ion is left. However, others have discussed possible differences due
to degree of dissociation and possible differences of transport of the
undissociated molecules.
"Presented by E. W. Hupp at Texas Academy of Sciences, annual meeting,
March, 1976.
-------
The three compounds and their abb.eviations used in this study are shown
in Figure 3. In a ccmpe Dative stuay > e used the molecular weights of
i-he compounds to calculate doses the. '' contained equivalent amounts of
mercury. Table 7 shows doses ba^ed on methylmercuric chloride: the
methylmercurie acetate doses were 108.6% of methylmercuric chloride and
the methylmercuric hydroxide doses were 92.6% of methylmercuric chloride.
RESULT?
Table 7 presents percent lethality, based on 10 animals per group. The
incidence of lethality was greater than that observed following methyl-
mercuric chloride in earlier studies. This may be due to the fact that
older solutions may have been used in some of the earlier studies. The
earlier LDso based on probit analysis may also have yielded an overesti-
mate. These results do not show a significant difference in lethality in
groups exposed to comparable amounts of methyl mercury in the three
compounds.
The data presented in Table 8 shows that there also was little difference
in the mean survival time of animals exposed to comparable doses of the
three compounds, although there was a tendency for the animals exposed
to the lower doses of methylmercuric hydroxide to survive somewhat longer
than animals receiving the other two compounds. These results show that
many of the differences in reported lethal doses in the literature are
not due to differences between these methyl mercury compounds.
25
-------
Figure 3. FORMULAS OF METHYL MERCURY COMPOUNDS USED
COMPOUND
ABBREVIATION
FORMULA
M.W.
Methyl-
Mercuric MMC
Chloride
Methyl-
Mercuric MMH
Hydroxide
H
j
l
H C Hg Cl
1
H
H
I
1
H C- Hg OH
i
i
H
251.1
272. 7
Methyl-
Mercuric
Acetate
MMA
H
I
O H
II !
H-C-Hg-O-C-CH
I I
H H
232.6
26
-------
SECTION VI
RESULTS OBTAINED IN THE MAIN SERIES OF EXPERIMENTS, METHYL MERCURY
DOSE ADMINISTERED TO RATS 7 DAYS BEFORE IRRADIATION
GENERAL INTRODUCTION
The animal phase of this series was conducted during the first year of
the project. Tissue analysis and interpretation of data continued in
later parts of the project. The results of evaluation of various para-
meters will be presented in separate sections. Brief literature
background for each section will be presented in that section.
GENERAL PROCEDURE
The animals used were adult (250 to 300 g) male Sprague-Dawley-derived
albino rats. Fifty to 60 animals were assigned to each of the insult
combinations listed below:
n . . Methylmercury
Combination /-,nn v j ^
mg/100 g body wt.
1 0
2 0
3 0
4 4
5 4
6 8
7 8
Radiation
kR
0.0
4.5
9.0
0.0
4.5
0.0
9.0
29
-------
Radiation was applied with a ij. S. Nuclear Corp. Gamma Irradiator at a
dose rate of approximately 500 R/min. 7 days after administration of
mercury or saline. All animals not receiving irradiation were sham-
irradiated. Methyl mercury was injected intraperitoneally. Solutions
containing either 3 or U mg methylmercuric chloride per ml of physio-
logical saline were used. Only solutions 1 to 3 days old were used, in
order to avoid deterioration of the solutions with storage. All animals
not receiving methylmercury were injected with physiological saline.
The behavioral and EEC tests, which are described in detail in succeeding
sections, were administered during the 6 hours following irradiation.
Twenty-four animals per treatment group were used in behavioral tests
and 12 other animals were used in the EEC studies. Additional animals
were assigned to combinations 6 and 7 to account for mercury-induced
lethality. The animals were killed 6 hours after irradiation.
Animals used in behavioral and EEC studies were also used to provide
tissue, with an equal number allocated to each of the following analyses:
Analysis
No. Rats from Each
Treatment Combination
GABA determination
Serotonin determination
Mercury distribution studies
Histological studies
10
10
10
10
Immediately after killing by decapitation, the heads of the animals to be
used for the biochemical studies were frozen in liquid nitrogen. The
animals used for histological studies were decapitated, then the brains
were removed and placed in fixative immediately. The biochemical and
histological analyses were performed as described in the succeeding
sections.
A. BEHAVIORAL TESTS
INTRODUCTION
Behavioral changes induced by doses of ionizing radiation have been
extensively investigated in adult animals^1 and in progeny of irradiated
females.52 Massive doses have been shown to cause an initial period of
performance decrement and incapacitation followed by a temporary recovery,
then severe incapacitation and death.
30
-------
Behavioral changes observed in humans poisoned with methyl mercury com-
pounds ^ include lack of interest, lack of concentration, despondency,
anxiety, fits of rage, and change ir. personality.^ However, only limited
experimental observation of behavioral changes in mercury poisoned animals
has been reported. °
MATERIALS AND METHODS
The subjects were 243 male Sprague-Dawley rats approximately 300 g in
weight at the beginning of the study. One hundred -sixty-one subjects
underwent both the open field and sexual behavior tests; the remaining
82 animals served only in the conditioned avoidance study. The animals
were housed in groups of 4 to 6 under a reversed 12-12 light-dark cycle
with the dark phase beginning at 6 a.m.
The open field consisted of a circular field 81 cm in diameter surrounded
by a 33 cm high wall. The field was divided by two concentric circles
and lines radiating from the center into 12 truncated triangular-shaped
areas surrounding a central circular area. The field was illuminated by
a white 100 W bulb suspended 40 cm directly above the center of the field.
Two identical sexual behavior arenas were used in the sexual behavior
tests. The arenas were plexiglass-fronted, semicircular enclosures
having a height of 28 cm and a radius of 31 cm. The arenas were placed
side by side on a table and were illuminated by two red 40 W bulbs
suspended approximately 2 m in front of the arenas.
The avoidance conditioning apparatus was a two-compartment Lehigh Valley
Electronics plexiglass shuttle box. The 46 x 20 x 18 cm box was divided
into two 23 cm long compartments by a partition extending 4.5 cm above
the floor. A "Q" light located in the end wall of each compartment 14 cm
above the floor provided visual signals and a Sonalert sound source
located in the roof of the box above the dividing partition provided an
auditory signal. The grid floor of each compartment was connected to an
AC shocker that delivered a 1 ma scrambled foot shock. All stimulus
events were electromechanically programmed. The shuttle box was contained
in a dimly lit room separated from the observer by a one-way glass
partition.
All behavior tests were conducted during the second third of the dark
phase of the light-dark cycle, and were begun 60-105 minutes after radia-
tion or sham irradiation. Data collection in all tests was conducted
under a blind procedure.
The open field test consisted of placing the subject in the central cir-
cular area of the field and observing his activity for a period of 2 min.
The following measures of emotionality and ambulation were recorded:
(a) the number of defecations, (b) the number of areas entered with all
31
-------
four feet (ambulation), and (c) the number of times the animal stood upon
his hindlegs (rearings).
Upon completion of the open field test the subject was placed in one of
the two sexual arenas and given 5-7 min to adapt to the enclosure. At
the end of the adaptation period a stimulus female was introduced to the
arena to begin the 30 min sexual behavior test. Stimulus females were
ovarectomized at approximately 100 days of age and brought into estrous
by intra-muscular injections cf 20 ug estradiol benzoate in corn oil
followed 36 hr later by 1 mg progesterone. Females were used approximate-
ly 10 hr after the progesterone injection.
During the sexual behavior sessions, two subjects were simultaneously
tested by a single observer seated approximately 2 m in front of the
arenas. The following sexual behavior measures were recorded for each
subject: (a) mount frequency (number of mounts without intromission
during the 30 min observation period), (b) intromission frequency (number
of intromissions during the 30 min observation period), (c) ejaculation
frequency (number of ejaculations during the 30 min observation period),
(d) mount latency (time from the introduction of female to the first
mount), (e) intromission latency (time from introduction of the female
to the first intromission), (f) ejaculation latency (time from introduc-
tion of female to the first ejaculation), and (g) post-ejaculatory
interval (time from first ejaculation to the next mount or intromission).
A conditioned avoidance session was begun by allowing the animal to ex-
plore the shuttle box for a 2 min period. At the end of this adaptation
period, the first trial of the conditioning sequence was initiated by the
onset of the auditory signal and the visual signal in the compartment the
subject was exploring. Ten seconds following the onset of the signals,
a 10 sec shock was administered through the grid floor of that compartment.
The signal remained on during the administration of the shock. The animal
made a correct response (avoidance) if he jumped across the partition
from the "shock" compartment to the "safe" compartment before the shock
was initiated. If the animal failed to make an avoidance response he
was allowed to escape the shock by crossing the partition after shock
onset. The signals remained on until the end of the 10 sec shock period
or until the animal made an avoidance or escape response. At the beginn-
ing of each subsequent trial, the compartment containing the subject was
designated as the "shock" compartment so that typically each compartment
alternated as the "shock" and "safe" side. Each animal was given 100
trials in a single session with a variable 20 sec interval between trials.
The number of avoidance responses in 100 trials was recorded for each
subject.
32
-------
RESULTS
OPEN FIELD TESTS
Since only 14-. 9% of the animals defecated during the open field tests, this
variable was not analyzed. The means and standard deviations of each
group for the ambulation and rearing scores are presented in Table 9. An
analysis of variance for the area and rearing variables yielded F values
significant at the £-7.64 p < .001 level. The results of the Duncan's
Multiple Range Test with unequal n_ are indicated by the superscript letters
in Table 9. It may be noted that the methyl mercury treatment alone had
little effect on both measures, as there were no differences between the
two mercury groups and controls. Both doses of radiation alone signifi-
cantly suppressed both activity measures in relation to the controls, with
the larger dose producing the greater effect. In all cases, the effect of
the co-insult was less than that of the same dose of radiation alone, but
due to the large variation within samples, these differences were not
significant at the p=.05 level.
SEXUAL BEHAVIOR TESTS
The percentage of animals in each group exhibiting at least one mount,
intromission, and ejaculation during the 30 min observation period is
presented in Table 10. A Chi-square test of independence was employed
to determine if the proportion of animals exhibiting a particular res-
ponse was the same for all treatments. This comparison was significant
at the .001 level for each of the three variables. Although neither
dose of methyl mercury presented alone produced a large reduction in
the proportion of animals exhibiting each response, both levels of
radiation alone severely reduced sexual activity, with the high radia-
tion level almost completely abolishing this behavior. As was the case
with the open field measures, co-insult treatments were less disruptive
of sexual activity than the same doses of radiation alone, although
these differences were not significant.
The means and standard deviations of the number of mounts, the number of
intromissions, and the number of ejaculations occurring during the 30 min
session for each treatment level are displayed in Table 11. The analysis
of variance for the variables mounts, intromissions, and ejaculations
yielded F_ values significant at the p < .001 level.
The results of the Duncan's Test for each of these variables are also
presented in Table 11. The controls exhibited the greatest number of
mounts, intromissions, and ejaculations, and differed from all other
groups on the latter two criteria. As was generally the case with the
previously mentioned behavior patterns, 9 kR had a greater effect in
depressing sexual behavior than 4.5 kR, and the co-insult was less
33
-------
Table 9. MEANS AND STANDARD DEVIATIONS OF OPEN FIELD MEASURES OF
RATS TREATED WITH METHYL MERCURY AND/OR GAMMA RADIATION
Methyl Mercury
Dose (mg/kg Radiation Number of
body weight) Dose (kR) Animals
0.0 0.0 22
0.0 4.5 22
0.0 9.0 21
4.0 0.0 22
4.0 4.5 22
8.0 0.0 26
8.0 9.0 26
Number
of Areas
Entered
23.95a
±8.73
17. 68b>c
±5. 65
10.76d
±9.03
23.14a
±9.67
17.86b>c
±7.40
20. 12a, b
±9.05
13.69c,d
±6.97
Number of
Rearings
7>77a,b
±5.42
4.00C
±3.19
2.57C
±2.78
8.68a
±6.62
5.14b,c
±2.95
7.73a'b
±5.17
3.88C
±4.02
a,b,c,d^eans .j_n .(-^8 same column with the same superscript do not differ at
the 5% level as tested by Duncan's test.
34
-------
Table 10. PROPORTION OF ANIMALS RECEIVING METHYL MERCURY AND/OR GAMMA
RADIATION WHO ACHIEVED AT LEAST ONE MOUNT, INTROMISSION AND EJACULATION
Methyl Mercury
Dose (mg/kg
body weight)
0.0
0.0
0.0
4.0
4.0
8.0
8.0
Radiation
Dose (kR)
0.0
4.5
9.0
0.0
4.5
0.0
9.0
Number of
Animals
22
22
21
22
22
26
26
Mounts
0.95
0.41
0.10
0.82
0.59
0.73
0.31
Intro-
Missions
0.95
0.32
0.00
0.59
0.41
0. 58
0.15
Ejacula-
tions
0.91
0.32
0.00
0.59
0.41
0. 58
0.15
35
-------
Table 11. MEANS AND STANDARD DEVIATIONS OF SEXUAL BEHAVIOR
MEASURES OF RATS TREATED WITH METHYL MERCURY AND/OR GAMMA RADIATION
Methyl Mercury
Dose (mg/kg
body weight)
0.0
0.0
0.0
4.0
4.0
8.0
8.0
Radiation Number of Number of
Dose (kR) Animals Mounts
0.0 22 20.77a
± 8.56
4.5 22 7.95c>d
±11.67
9.0 21 1.14d
± 5.01
0.0 22 17.05a»b
±14.27
4.5 22 11.09b>c
±11.18
0.0 26 15.35a»b
±12.61
9.0 26 2.62d
± 5.17
No. of
Intro-
missions
21.45
±13.99
5.59b>c»d
± 9.37
0.71d
+ 3.28
13.77a
±11.27
8.36a,b,c
± 9.34
11.27a»b
±11.03
2.81c>d
± 7.56
No. of
Ejacula-
tions
1.91
±0.92
0.73a»b
±1.16
0.0C
±0.0
1.23a
±1.11
0.73a»b
±0.93
1.04a>b
±1.04
0.35b>c
±0.89
a,b,c,d Means in the same column with the same superscript do not differ
at the 5% level of probability, as tested by Duncan's test.
36
-------
."ince the analysis presented in Ic.r le 11 included those animals failing
to exhibit the various sexual activities (i.e., having a zero frequency
for the category), additional comparisons were made to determine if the
behavior of the sexually active animals in the experimental conditions
was different from that of the active controls. An animal failing to
engage in a particular activity was therefore excluded from the com-
parsion for that sexual behavior category, thus reducing the number of
subjects in each condition included in the comparison. Although two
animals in the 9 kR alone condition were observed to mount, this con-
dition was excluded from these analyses because none of these animals
were observed to achieve an ejaculation. The means and standard devia-
tions of the frequency and latency categories for the remaining subjects
are presented in Table 12. The mean number of intromissions required
to reach the first ejaculation for each condition is also included in
this table. The analyses of variance for the number of intromissions
(F_ R =1.14), the number of ejaculations (Fj. =1.00), and the number of
intromissions to the first ejaculation (F_5 ^=1.35) categories yielded
nonsignificant outcomes (p_ < .05). The analysis of the number of mounts
variable was marginally significant (FV =2.53; p_ < .05), an effect
apparently due to the low mean mount frequency of the 8 rag/kg, 9 kR
animals. The four latency measures for the six groups were compared by
means of the Kruskal-Wallace analysis of variance by ranks. The compari-
sons for the mount latency (H_ =1.13), intromission latency (H=10.94),
and postejaculatory interval (H=5.61) failed to reach significance
(p_ < .05); however, the analysis of the ejaculation latency variable was
sTgnificant (H_ =11.35).
CONDITIONED AVOIDANCE TEST
The analysis of variance (F_=5.56; p < .001) and the subsequent Duncan's
Test indicated that the controls exhibited the greatest number of correct
conditioned avoidance responses (see Table 13), a finding consistent with
the open field and sexual behavior tests. The group receiving 8 mg
mercury and no radiation, however, did not differ significantly from the
controls but was considerably lower in the correct number of responses.
All other treatment groups were significantly lower in performance than
these two groups, but did not differ significantly from each other.
DISCUSSION
In all frequency measures except the number of rearings in the open
field, the control animals exhibited the greatest amount of activity.
Methyl mercury injections, however, did not significantly affect the
open field behavior measured in the present study, a finding that is
surprising in light of previous open field studies which indicated
methyl mercury injections reduced open field activity. Both levels of
37
-------
w
£>
M
EH
O
^C
>-"
J
^
£3)
X
w
CO
00
1
cn
W
PS
o
M
£>
<^
JX1
w
PQ
i-3
S
X
H
CO
P-i
O
C/3
o
M
H
M
W
Q
p
jyj
^
Q
2;
<;
C/>
o
2;
z
<5
w
CN
TH
0)
Q
ftJ
H
h- 1
£-H
<
M
i
<
si
<3^
0
Pi
o
"-s^
Q
j2j
<
>-"
PS
D
o
H
s
H
pr|
H
W
E
H
I-H
»-»
i-S
Q
W
H
fO
W rH
M-t
O
O
P
1 0
O -H
PH CO
-P CO
C -H
M 6
O
2n
-H
C
0
1
H
Tj
tfl
PS
>>
-P
0)
S
p
o
H
-p -p
W rti
£H H
H 3
[- \
-L-l l_J
tO
O T-i
-P W
1
£n
QJ
P H
C ra
M >
I""^
vO
P O
fO R
J 0)
W
1 C
3 O
O -H
(Tl 1 1
IU -t-1
i > CO
W H
P O
m c
J Q)
W
C
1 0
£H W
P M
P -H
M e
-P O
(0 C
J Q)
MH W
0 -P
O O
S5 S
P
O !
^
O 0)
£H W
Q) O
S Q
LO CM
LO CM
TH LO
TH
+1
O t>
CO t>
r>- co
cn CM
CM TH
+1
O CO
cn d-
CO CO
J- CN
[> CN
+1
0 CM
H I>
CM O
+1
LO CO
O CO
O cn
d- TH
CM CN
+1
CO CO
d- J-
(N CO
CM TH
+1
co r^
J- CO
cn LO
pH CN
vH TH
+1
ID [>
t> CO
H r^
CM
+1
O
o
o
o
O cn
IT-- LO
O CO
+1
cn o
CM LO
cn LO
CM CO
CO
r- o
LO J-
00 CD
cn LO
J- CN
+1
cn CD
CM O
CM O
+1
cn CM
CM cn
H d-
ID 00
H TH
+1
r- c--
LO CM
r- r^
TH
+1
d- cn
d- CM
CO LO
CN CD
H TH
H-l
zT CO
^- CM
cr> o
H TH
+1
LO
J-
o
o
CD CO
D^ O
CM d-
TH
+1
LO CM
OD O
CO cn
cn co
CM
+1
CO CO
J- LO
CO LO
J- CD
U3 TH
+1
CO cn
o 3-
CM O
+1
ID I>
CO CM
J- o
CD Cn
H TH
+1
^- [
CD ID
H 3-
CM
+1
TH LO
ID CM
CD CO
OT O
H CO
+1
CO CD
co cn
O CM
CM TH
+1
0
0
0
J^-
CO CO
CO J-
H CO
TH
+1
O LO
O CO
CO CO
CD cn
CO
CM CO
CN TH
cn CM
CM [>
cn CN
+1
CO TH
l> d-
H 0
+1
0 CO
O CO
o d-
H LO
^" J~
+1
CO CD
CO O
LO r^
TH
+1
CO TH
CN I>
cn 3-
t>- CN
TH TH
+1
CO TH
t-- cn
CO I>
TH
+1
LO
:j-
0
J-
O CO
CM CO
TH J"
H
+ 1
cn J-
r-- co
LO O
CO TH
CM TH
+1
O- LO
d- co
J- J-
CM O
CO d"
+1
O cn
00 CD
TH O
+1
CO 00
CD t>
cn co
oo cn
CM CM
+1
TH r--
co o
00 CO
TH
+1
cn LO
r- J-
LO O
CM C~-
H vH
+1
LO LO
o r-
TH cn
CM
+1
0
O
o
CO
LO (X)
CM cn
cn o
+l
CO LO
co cn
LO Jt
co 3-
CM TH
+1
O LO
O CD
C-- 3-
LO d"
CD LO
+1
LO CO
CM cn
CN O
+1
LO CM
l> 0
[> CD
C-~ cn
CM CO
+1
LO LO
CM O
00 O
TH TH
+1
O CO
LO O
CM cn
TH t^
^" LO
+1
O cn
LO TH
oo to
+1
o
cn
o
CO
38
-------
Table 13. MEANS AND STANDARD DEVIATIONS OF NUMBER OF CORRECT
RESPONSES ON CONDITIONED AVOIDANCE TEST
Methyl Mercury
Dose (rag/kg Radiation No. of Number of
body weight) Dose (kR) Animals Correct Responses
0.0
0.0
0.0
4. 0
4.0
8.0
8.0
0.0 11 25.73a
±21.54
4.5 11 6.73b
± 9.97
9.0 11 5.73b
± 6.89
0.0 10 7.0b
± 5.87
4.5 10 6.6b
± 8.50
0.0 14 17.433
±16.41
9.0 15 2 . 6b
+ 4.57
a5t>Means with the same superscript do not differ at the 5% level of
probability as tested by Duncan's test.
39
-------
gamma radiation significantly suppressed open field activity as indicated
by the areas entered and rearings variables.
Radiation also had more of an effect on sexual behavior measures than did
mercury injections; in the 9 kR alone condition, sexual activity was al-
most completely abolished. In the number of mounts variable,the animals
in both mercury conditions, though lower, were not significantly different
from controls. Mercury-injected rats, however, achieved fewer intro-
missions and ejaculations than did control animals, although there was no
difference between the low and high dose levels on these variables. The
comparisons of the sexually active animals in the experimental groups with
the active controls pointed to a remarkable similarity of behavior on
both frequency and latency variables; those animals that were sexually
active were generally not distinguishable from animals in other treatment
levels. These findings suggest that the significant treatment effects
noted in Table 11 were principally due to the almost complete suppression
of sexual behavior in some subjects and point to the wide variability of
these animals in susceptibility to radiation, mercury poisoning, and
co-insults of these agents.
The most interesting findings of the present study concerned the compari-
sons of the co-insult conditions with the radiation alone groups. In
the open field and sexual behavior tests, those animals receiving the
co-insult generally performed better than those receiving the same dose
of radiation alone. While these differences were not significant, their
consistency throughout the various tests suggests that methyl mercury in
the co-insult groups inhibited the effect of radiation upon the nervous
system as measured by the behavioral tests used in this study.
SUMMARY
Behavior of all subjects was measured by open field, sexual behavior,
and conditioned avoidance tests beginning 60-105 minutes after radiation
or sham irradiation. There was a tendency for the co-insult treatment
groups to show fewer disruptive effects than were shown by the radiation
alone groups. Although the conditioned avoidance test yielded reliable
differences between controls and all experimental conditions except for
the 8 mg MMC/kg treatment, the test did not distinguish between insult
levels. The open field and sexual behavior tests showed minimal effects
of mercury alone and significant effects of radiation alone, suggesting
that MMC in the co-insult groups inhibited the effect of radiation on
the nervous system.
-------
B. El"! Analyses
-.lectroencephalograms were rnnde on 1? animals in each of the seven treat-
ment groups 4 to 6 hours jfter the ;.oinrletion of irradiation or sham-
irradiation. The frequency and amplitude of the EEC was measured in five
or more 5-second intervals for each animal. The results are summarized
in Tahle 14. Changes revealed by this quantitative analysis are limited.
Radiation alone increased the amplitude of the EEC and caused a slight
decrease in frequency. Mercury treatment alone had little apparent effect,
no changes in amplitude were produced, but the 4 mg/kg dose appeared to
decrease the frequency while 8 mg/kg appeared to increase the frequency
of the EEC. In the low-level co-insult (4 mg/kg mercury and '-t.5 kR)
mercury apparentlv had no effect on the changes induced by irradiation,
since the means were very similar. The effect of the mercury treatment
in the high level co-insult (8 mg/kg mercury and 9 kR) is not clear-cut,
the amplitude was reduced to a value intermediate between 9 kR alone and
control, but the frequency was reduced more by the co-insult than by
radiation alone.
A subjective examination of the recordings also revealed limited changes.
Striking changes such as those elicited by a variety of stimuli, includ-
ing irradiation57 were not observed. However, some increase in "spiking"
previously described by Speck^S appeared to occur in the 9 kR group ;
these changes were less evident in the high level co-insult group.
C. DISTRIBUTION OF METHYL MERCURY IN RAT BRAINS
METHODS
The rats were killed by decapitation 6 hours after radiation. The heads
were immediately dropped into liquid nitrogen and frozen. They were
then stored at -28Cuntil the brain parts were dissected. The brain was
kept frozen by dissecting on a glass surface cooled by dry ice. Separate
brain parts were placed in pre-weighed vials, weighed and then again
frozen and stored at -28c in the storage vials until ready for digestion.
The same vial was used for weighing, storage and digestion. For some of
the samples nitric acid digestion59 was used to prepare the samples for
total mercury analysis by cold vapor atomic absorption. For the remainder
of the samples Thorpe's^0 sulfuric acid-permanganate digestion was used.
RESULTS
The samples in series A,B,C, that had received no dose of mercury showed
some residual levels of mercury even after reagent blank correction.
This residual level was higher in the case of those samples digested in
nitric acid than those digested in sulfuric acid-permanganate. In neither
case, however, did the samples show any difference dependent on brain
area or radiation treatment. Thus, these background levels were averaged
for each method, and subtracted from the individual mercury determinations
in the D,E,F,G, series animals.
41
-------
Table 14. FREQUENCY AND AMPLITUDE OF THE ELECTROENCEPHALOGRAM OF
RATS EXPOSED TO RADIATION AND/OR METHYL MERCURY
Treatment
Group
A
B
C
D
E
F
G
Rad.
kR
0.0
4.5
9.0
0.0
4.5
0.0
9.0
Methyl
Mercury
rag/kg
0.0
0.0
0.0
4.0
4.0
8.0
8.0
Electroencephalogram
Frequency
Cycles/sec.
10.9
9.3
9.9
9.2
9.2
11.7
9.1
Amplitude
uv.
73
80
90
70
80
73
80
42
-------
The results of the total mercury analysis of the brains from rats dosed
with two levels of methyl mercury and two levels of radiation are pre-
=ented in Table 15.
Methyl mercury eoT'centrations wer>, nigher in the cerebellum than the pons-
medulla or cerebral cortex regardless of mercury or radiation dosage.
Data indicates that the pons-medulla and cerebral cortex absorbed about
the same level of mercury for any one treatment. The samples that had
received 8 mg/kg dose of mercury (series F £ G) had greater mercury
concentrations by factors of 2 to 4 over those samples that had received
only the 4 mg/kg dose of mercury (series D £ E).
Radiation appeared to decrease the content of methyl mercury in all brain
sections. This decrease does appear to be related to radiation dosage.
The pons-medulla samples of series E appear to be an exception to the
above observations. There was no data to indicate that the analysis of
these samples was at fault. No explanation can be given for the anomalous
results for these samples. These total mercury analyses can be taken
as total methyl mercury since experimentation demonstrated that most of
the mercury present was in the organic form. Whole brains from two an-
imals were homogenized and 5 aliquots from each brain were analyzed for
total mercury and inorganic mercury by amodification of the method of
Magos and Clarkson.6^ By substraction the organic mercury, presumably
methyl mercury, was calculated. The results are given in Table 16.
The results of the mercury analyses provide a basis to relate the effects
observed by the various measures to mercury concentrations in the brain
responsible for these effects. They also demonstrate that most of the
methyl mercury (over 90%) injected that remains in the brain 7 days later
is still in the organic form.
D. NEUROTRANSMITTER ANALYSIS
The serotonin (5-HT) and norepinephrine (NE) levels in five brain areas
of the rat were determined using the technique of Maickel et al.^2 Tne
animals were injected with methyl mercury chloride (MMC), then seven
days later they were irradiated and six hours later sacrificed by decapi-
tation. Immediately after decapitation the heads were dropped into
liquid nitrogen and kept at -70°C until the time of analysis. The brain
areas used for analysis were the cerebral cortex, thalamus, midbrain,
pons-medulla and cerebellum. The animals were divided into seven groups:
control, 0.4 mg/kg MMC only 0.8 mg/kg MMC only, 4.5 kR only, 9.0 kR only,
0.4 mg/kg MMC plus 4.5 kR and 0.8 mg/kg MMC plus 9.0 kR.
Table 17 shows the results of these treatments on the norepinephrine
levels in the various rat brain areas. A consistent pattern is seen in
those animals treated with MMC. There is a decrease in NE with the
administration of 0.4 mg/kg MMC and an increase at 0.8 mg/kg MMC over
the 0.4 mg/kg level. Alteration of the NE levels by irradiation presents
43
-------
CO
H
s
M
<
ffl
CO
| )
o
1 ! v^"1
oi os
o
OS
0 S
'*"*x 1-3
c ^
H K
fO E-
rQ IS
i^TJ ^
\. ^H
tO M
3. IS
^" Q
H W
S CO
W O
E-i Q
^j
O CO
O H
K*-" DH
Pi
ID k-i
c_> o
Pi
W
g
LO
^ (
O
H
9
H
O O
u *
CD CT>
0 0
C_i j
CO O
o 10
W
H/ .3"
0 0
o
J- o
£*)
C_|
CX 3 W) Pi
3 U X ^
O k "-
fn 0) W) C
a s e o
H
rH 0) -P
-P >, w (0
(0 .CO -H
0) P Q t3
JH 0) CO
E-" g Pi
CO
J-
CN
O -~-
+l in
TH \H
01 v_^
TH
vH
O
LO
CN
O s~*
+1 J-
CD TH
H ^-^
un
vH
LO
cn
TH
O *~*
+1 LO
a> TH
CO ~ -
LO
o
tO
CN
CO
TH .O
s-**
o co
+1 tH
CO ^
O
r-~
.
0
X
Q)
-P
O
O
0) H
?H rd
< fc
n a)
H fn
fO Q)
^ O
PQ
CM
(D
CO
0
+ 1 LO
r- TH
CO ~_-
LO
^
CO
CO
[>
(O ^x
+1 r-
LO -H
CO v_-
vH
CN
f
H.-
vH
,
O /~-
+1 :±
C^- vH
00 *-<
(D
O
J-
CO
TH
/ ^
o d-
+ 1 TH
CD ^w'
J"
CO
0
§
H
H
1
C_4
0)
O
CN
CN
CM
O ^
+ 1 LO
LO TH
CO
cn
O
CD
LO
CO
O ^^
+1 C^
(D vH
£^ V ^
ID
^
J-
C^
TH
,
O ' ^
+1 CO
LO TH
CN ^
C
O
CO
TH
TH
/ N
O CO
+ 1 TH
TH **-'
CO
J-
0
to
H
H
T)
0)
W
C
O
PH
C
tO
0)
s
C
H
TI)
Q)
-P
G
0)
W
Q)
fn
a>
^
W
0)
! 1
P)
6
tO
w
MH
O
£4
O
-Q
R
^
G
CO
0)
CO -P
C O
0 G
H 0)
tO
H W
> -P
0) (U
o
gg
tO fi
G C
CO -H
-P
W fn
0)
G .O
m ^
6 G
-------
w
o
S
0
CJ
H
w
u
o
s
PH K"-1
O Pi
D
Ir-* O
^ PS
PI 1 fl")
H S
O i-^l
O J=H
>H H
PS W
D S
CJ
PS ffi
W H
y
CJ
M Q
S H
< CO
CD O
PS Q
O
CO
O H
0 UH
M 0
Cr) O~)O^CT)CTlCT> O">
bO
(0
OLOCDJ-CO OCDOl-O1-^
D--l>Lnr-CD CNrHd-COJ-
OOOOO THvHvHr-lTH
CN ^ f LO T~H CT) C"^ CN CO CN -^
C CT5 LO CO -Ij" CN ^H CN J" LO
ooooo VHT-HT-HT-ITH
OOOOO OOOOO
LO _~j~ C^- f o CO ^ 1 ^- CTj CO CN
C^~ CO -^t" CD CO CN f^ CN C71 CZ5
C 00 CD CO CO ^J" CO LO ^ CO
OOOOO vHr-lT-fT-lTH
xHCNCOzfLO cDr^-OOCDO
T-j
bO M
^-. ^^
hO bO
e e
J- 03
-------
H
CO
H
Pi
E
PL.
W
H
Pk
H
Pi
O
cu
H
CO
CO
H
-P
bO
^.
W
bO
CO
r-j
. *
s
rd
H
rd
H
rd
H
H
T^
CD
S
1
W
c
Pn
, t
r-j
rd
o
3
S
G
H
0)
f|
(1)
k
0
X
Q)
-P
O
c
Q)
-P
cd
0)
r.
H
H
CM
CN
LO
0
+1
OO
TH
CN
vH
TH
OO
.
O
+1
J-
CN
TH
CO
CD
J~
O
+1
LO
1>
CN
TH
LO
TH
O
+1
CD
c .
CO
o
CO
c^
oo
,
0
+1
TH
J"
TH
H
O
S
bO
S
o
o
1
Pi
t\f£
O
o
J-
co
H
^
+1
OO
OO
CD
O
CN
CO
TH
.
0
+1
TH
O
TH
CO
LO
CN
O
+1
CD
d"
TH
CD
LO
CO
o
o
+ 1
zt"
co
CO
o
jj-
J-
TH
O
+1
J-
TH
r~-
0
H
o
§
bO
£
d-
o
1
rv
0
0
_^-
oo
3"
0
+1
CM
CD
CM
LO
CN
TH
.
0
+1
CD
TH
TH
LO
O
=f
O
+1
zj-
LO
TH
TH
0
TH
0
+1
t^
=!
O
CM
CN
CM
O
+1
CN
TH
TH
H
0
S
bO
6
oo
o
Pi
y
o
0
CO
LO
I>
o
+1
TH
CM
LO
00
TH
0
+1
LO
CM
TH
^
, 1
LO
O
+ 1
00
=1"
-1
J^-
TH
TH
O
+1
00
CO
J"
0
LO
f^
LO
o
o
+1
CD
d~
CD
0
H
O
S
S
bO
6
o
0
1
Pi
V
LO
J-
cn
CN
O
+j
0
o
CM
LO
LO
TH
O
+1
CD
CD
CO
O
LO
CD
LO
0
+1
J-
oo
CN
TH
00
CD
O
O
+1
CN
CN
CO
0
OO
CM
CD
O
0
+1
CO
LO
CO
o
H
O
S
bO
6
zr
o
i
Pi
X
LO
J-
CD
en
co
o
+1
CD
LO
CN
CN
CN
CM
.
O
+1
CD
TH
TH
LO
CD
f-
0
+1
CD
3"
CM
OO
CD
J-
O
O
+1
o
LO
oo
o
CM
H
TH
.
0
+1
CO
H
CD
O
r-\
0
S
bO
e
0
o
1
X
o
CD
CO
cn
vH
0
+1
TH
CD
TH
jj-
_2j-
CM
0
+1
TH
cn
CO
0
^
CO
CD
O
+1
CD
CD
CN
^
CD
CO
O
O
+1
CM
CO
=1"
O
CN
_j-
CN
O
+1
LO
CD
cn
0
H
0
S
S
bO
S
CO
0
i
Pi
X
0
CD
to
II
cn
z
M
O
W
01
H
rd
H
CO
co
H
-P
bO
bO
CO
e
rd
H
rd
H
rd
H
H
1-TJ
LJ
0)
S
1
CO
C
P-.
H
rd
o
TJ
E
6
13
H
H
CD
_Q
CD
f-t
cu
o
X
0)
4->
fH
o
P
CD
rd
H
en
0
co
0
+1
^
LO
co
o
o
LO
TH
O
+1
CO
CD
CO
o
LO
CO
CO
0
0
+1
CO
CD
CO
o
o
o
(D
0
0
0
+1
CD
o
CN
0
^
CO
CN
O
0
+1
CM
Zj-
d~
0
o
^r]
S
bO
6
0
o
1
Pi
^
0
o
TH
O
CO
o
0
+1
CM
J-
vH
O
O
TH
CN
0
O
+1
r~
CM
H
o
CD
CO
CO
O
0
+1
CO
CO
H
0
CO
t^~
CJ)
O
0
+1
CN
TH
LO
0
0
CN
c^
CO
0
0
+1
0
r-
H
O
rH
0
IS
bO
g
d-
o
1
o
o
o
c
c
0
o
+1
CD
CD
OO
O
LO
TH
CN
O
O
+1
OO
£
vH
0
CD
TH
TH
0
+1
OO
t~-
3"
O
CM
CO
TH
0
0
+1
CN
CM
CD
o
0
^
CD
OO
0
0
+1
CD
CD
CN
O
H
O
S
S
ho
e
CO
o
1
o
0
^
CD
0
O
+1
oo
(^
LO
o
LO
TH
J-
0
o
+1
CN
TH
CN
0
LO
CO
CD
O
O
+1
J-
CN
*
O
0
CO
CN
0
O
+1
TH
[^
TH
0
TH
CD
jj-
0
0
-4-j
r-
o
CN
0
O
^r|
S
bO
E
O
o
1
Pi
LO
d-
CD
CD
3~
O
+1
CO
r-
CM
0
TH
H
OO
0
O
+1
TH
00
TH
O
0
CM
TH
0
+1
Zj~
CD
LO
O
CN
H
TH
0
O
+1
0
oo
00
o
o
^
J-
TH
0
0
+1
CN
CD
TH
0
H
O
S
S
bO
6
d-
0
i
LO
J±
co
CM
^J-
o
o
+1
CD
[~~
OO
o
CO
CD
jj-
o
0
+1
00
CO
TH
0
LO
TH
CO
O
O
+1
r^
CM
oo
O
LO
CO
00
0
0
0
+1
CD
LO
CD
0
0
H
LO
CN
0
0
+1
c^
LO
TH
O
O
S
bO
e
o
o
1
o
CD
00
0
oo
0
o
+1
CD
TH
CN
0
CO
CN
LO
0
O
+1
OO
CO
vH
O
^
CO
CO
0
o
+1
c
TH
J"
O
CO
CD
LO
O
o
o
+1
^
LO
0
o
CD
CD
H
O
O
+1
CO
LO
TH
O
H
O
S
bO
e
CO
0
1
o
CD
-------
a more complex picture; the cerebral cortex and midbrain show a decrease
at 4.5 kR and a rise at 9.0 kR over the 4.5 kR levels. The changes in
.he NE levels of animals given the co-insult do not follow a distinct
pattern. Significant changes are seen in the cerebral cortex and cere-
bellum at 4.5 mg/kg MMC plus 4.5 kR treatment.
Table 17 also shows the effects of the treatments on the 5-HT levels in
the various brain areas of the rat. The results seen in all the treat-
ment groups follow a distinct pattern. Those treated with MMC alone show
a decrease at 0.4 mg/kg and a rise over the 0.4 mg/kg at 0.8 mg/kg.
Animals treated with x-irradiation show a decreasing level of 5-HT with
increasing doses of radiation. The pattern seen in the animals receiving
the co-insult is similar to that of the irradiated animals ; for this
reason it may be that the x-irradiation is the predominate insult in these
cases. In general our levels of NE are higher than those reported in the
literature for untreated animals and somewhat lower than the values report-
ed for 5-HT.
Table 18 summarizes the percent change from untreated animals in the
levels of NE and 5-HT in the various brain areas. The lowest percent
changes in NE occurs in the cerebral cortex, cerebellum and midbrain. The
percent changes in the 5-HT levels seem to be uniformly large in all areas
of the brain. In general the serotonin seems to be affected by all treat-
ments to a greater extent than the NE.
The effects of MMC on the transmitter levels suggests that the mode of
action may invlove two mechanisms. Both NE and 5-HT show a decrease at
0.4 mg/kg MMC with a rise in the levels at 0.8 mg/kg. If one considers
the possible causes for a decrease in transmitter levels from exposure to
MMC two aspects may be considered, first is that there is a decrease in the
availability of precursors to synthesize the transmitters, and the second that
there may be a reduction in enzymes available for synthesis. Yoshino
et al.,63 found that MMC also inhibits protein synthesis. The inhibition
of protein synthesis should reduce the levels of enzymes available for
synthesis. Either of these findings might explain the reduction in
transmitter levels brought about by methyl mercury.
A number of workers have reported a decrease in the brain serotonin con-
tent resulting from x-irradiation.64 No reports are available for the
effect of x-irradiation on NE brain levels.
The effect of the co-insult on the transmitter levels clearly does not
seem to be additive for either NE or 5-HT. Data obtained for 5-HT ex-
posed to co-insult suggests that the pattern of change is similar to
x-irradiated animals, but not the methyl mercury treated animals. All
of the treatments have a considerable effect on the transmitter levels
in most brain areas. Of the two transmitters studied,5-HT has been
found to be more sensitive to the various treatments. Because of the
effects obtained on levels of both transmitters, it is suggested that
possible mechanisms of action and correlations to the neurological
changes be explored.
47
-------
to
PS
w
EH
H
g
CO
3
PS
EH
0
PS
D co
W EH
£jjj [_J^
2i co 01
H 2 -H
H 01
(-5 >i
O (0 rH
PS CD c!
g -rl
o a M
PS O rl
CM PS O)
ti (U
W C
O C? -H
2 2 a
< H 01
K EH M
O J 0
D !a
EH CO
2 W
rvj rvf
i^l l-M
O
PS
w
04
CO
rH
01
rH
(d
EH
0>
3
g
fti
H
ro
H O O CN O ro
1 1 + 1 1 1
CTl O CM CT* ^ CM
^0 ^J1 VD ^ 0^ CM
^531 ^i ^j* ^H C5 ^~^
1 1 1 1 1 1
^ r^ rH VO ^ O
kO CJl H CM CO CTl
^ CN en in ^r CN
i t l l i i
if VD O in CTi »*
&i O ^ ro ^ CJ>
^f CM in in ro CM
1 1 l 1 1 1
rH r ( rH rH rH rH
U U U U U U
g g g g g g
g g g g g g
o^ t7* cr^ o^ iji t^i
g g g g g g
^3* CO O ^* O CO
o o o o o o
1 1 1 1 1 t
PS PS PS PS PS PS
£t M rV iX X Ai
o o in in o o
CD C^ ^* ^* O^ C^
w
H
w
rH
a
c
a;
C
H
O
-P
0
^
01
w
'
01
3
g
rH
(C
,£!
EH
rH
rH
3
rQ
0)
g
W
C
O
PM
C
H
id
X!
13
H
g
g
fj
rH
rH
fll
X)
01
r<
01
U
X
.p
^
0
o
-p
01
.p
-------
E. THE EFFECT OF METHYLMERCURIC CHLORIDE AND
IONIZING RADIATION CN THE RAT BRAIN-
INTRODUCTION
Autoradlographic studies of mercury poisoning have shown preferential
accumulation of the mercury within the cytoplasm of large neurons,
particularly those in the cerebellum and brain stem.65 The cerebellar
changes seen in all species studied by Cavanagh and Chen°" have been
confined to granular cells, either as diffuse loss or in focal discrete
areas. Their findings confirmed the original observation of Hunter et
al. that in the rat degenerative lesions were almost entirely confined
to the primary sensory neurons of dorsal root and trigeminal ganglia,
and to the granular cells of the cerebellar cortex. The majority of the
literature reviewed coincided in the finding that the cerebellum was
probably the most affected area of the central nervous system. More
specifically, the granular layer was impaired more than the other cell
layers.68
Massive doses of ionizing radiation regularly and promptly bring about
characteristic morphologic alterations in certain neural tissues and in
the mesenchymal structures in and around the brain.69 it is well estab-
lished that exposure of brains of cats, monkeys, rabbits, guinea pigs,
mice, and rats to massive doses of ionizing radiation is followed
promptly by nuclear shrinkage and hyperchromatism in many of the cere-
bellar granular cells. ^ These lesions were found with increasing
frequency and Intensity after doses of 5,000 and 10,000 R, and the
cytologic changes were well established within 2 hours after radiation.71
The literature reviewed reported that both agents--methylmercuric chloride
and ionizing radiation--have a more detrimental effect on the cerebellum
than other areas of the central nervous system when administered in a
dosage large enough to produce these cytological changes. However, as of
yet it is not known what, if any, effect these two insults have on each
other.
The present study was designed to evaluate effects of the co-insult at
the light microscopy level.
PROCEDURE
Ten rats from each treatment group described previously were used for
histological observations. Immediately after decapitation, the whole
brain was removed from the cranial cavity by careful dissection with
"Portions of these data were submitted by Mitzi Short Thrutchley in
partial fulfillment of the requirements for the degree of Master of
Science in Biology in the graduate school of Texas Woman's University.
-------
bone clippers and various other dissection instruments. The whole brain
was fixed in 10% buffered formalin for 72 hours, then dehydrated in a
series of graded alcohols using a tissumaton (Fisher Scientific Apparatus)
cycle of 18 hours. The brains were embedded in paraffin (61°C) and cut
sagitally at 5 microns with an A-0 Spencer "820" microtome. Three
sections were mounted on each slide and then stained with hematoxylin-
eosin.
Of the slides prepared, five suitable sections of the brain were selected
from each group. The same sections were also used for cell counting in
the cerebellum. The different cell types were counted in an approximate
5X5 micron square in the central lobule of the cerebellar hemisphere.
The counts were averaged to obtain an estimate of the packing density of
granule cells.
All sections were analyzed with a Reichert "Zetopan" research microscope.
The brain areas examined were the cerebrum, pons, medulla, and cerebellum.
Photographs (35 mm) were taken of each group representative of the areas
affected. A Kodak high contrast copy film (Eastman Kodak Co., Rochester,
N.Y. 14650) was used because of its extreme resolution.
Cell counts of the cerebellar cells were taken of five different sections
(separate animals) of each treatment group, and were counted using a
laboratory counter (Clay Adams Division of Becton, Dickinson, and Co.,
Parsippany, N.J.) to determine if there was a loss of cells due to the
different treatments. Another cell count was taken on the granule cells
to count normal versus pyknotic, and to establish if pyknosis was inter-
mediate or complete. These cell counts were done on 5 animal sections
in each treatment group using a set number of 200 cells.
RESULTS
HISTOLOGICAL OBSERVATIONS
The gross appearance of the cerebellum, cerebrum, pons, and medulla in
the control group that received sham injection and sham irradiation
appeared normal when examined with the light microscope. In the sham
injected and 4.5 kR group there were a few pyknotic granule cells
scattered throughout the granular layer of the cerebellum, though not
as many were viewed here as in the higher radiation dosage group. The
cerebrum, pons, and medulla appeared free from change in any way.
In the animals that received sham injection and 9 kR, the cells of the
granular layer of the cerebellar cortex were altered notably by pyknosis,
thus implicating a direct effect of irradiation on nerve cells. These
pyknotic cells were present in almost all the cerebellar lobes, but
were scattered randomly rather than dense. As a rule the pyknotic cells
were isolated, but now and then were present in groups.
50
-------
The nuclei of these pyknotic cells were greatly reduced in size and
intensely hyperchromatic. The neuronal changes consisted mainly of
pyknosis and karyorrhexis of the cerebellar internal granular layer,
especially beneath the Purkinje cells.
No changes were found in Golgi cells. Purkinje cells appeared normal.
Some Purkinje cells were dark and others showed cytoplasmic chromatolysis
and vacuolation, but since these changes were also in sections from con-
trol animals, they were interpreted as artificats due to the type of
immersion fixation. In the molecular layer few changes were seen,
although some basket cells looked like empty shells and some nuclei were
rather poor in chromatin. Occasionally pyknotic nuclei cells were seen.
No lesions were detected and no perivascular cuffing by leukocytes were
observed in the cerebellar cortex. The presence of many red blood cells
was evident.
The three other areas investigatedcerebrum, pons, and medullawere not
affected by the large radiation dose. There was a possible loss of gran-
ule cells, but as far as could be determined, it was not statistically
significant. No structural changes were seen in the axons of the granule
cells.
The changes in morphology of groups given 4 rag/kg of methylmercuric
chloride (MMC) and then sham irradiated were slight. Only some granule
cells appeared pyknotic and these were very minimal. The Purkinje cells
appeared normal, as did the molecular layer. As with the previous treat-
ment groups, the cerebrum, pons, and medulla seemed free from any
cytological changes.
The smaller co-insult dosage group that was treated with 4 mg/kg MMC and
then given 4-. 5 kR was affected very little histologically. In less than
half of the slides analyzed were there any alterations, and these appear-
ed in the cerebellar cortex, usually in the granular layer. These
granular cells appeared to be in an intermediate stage undergoing cell
pyknosis, but had not completely reached this stage of cell death. In
this treatment group the other three brain areas remained normal with
no indication of alteration. It was interesting to note that in this
group with the smaller does of the insults, the combined agents produced
less cell damage than either the smaller of the individual mercury insults
or the smaller individual radiation insult.
Histologically, the group receiving the 8 mg/kg MMC and sham irradiation
has some noted alterations and these were only found in the cerebellum.
Pyknosis of isolated granule cells in the internal layer were randomly
scattered throughout the cerebellum. By comparison with the large
radiation dosage, this damage was not as pronounced as the radiation
damage. The Purkinje cells were observed to be normal as were all of the
other cell types in the cerebellum. It is interesting to note that
although the degeneration was much more extensive in the granular cell
51
-------
layer, more mercury was localized in the Purkinje cells by autoradio-
graphic studies done by Cassano et al.72
The final treatment group that received the larger doses of the co-insult,
8 mg/kg MMC and 9 kR, was surprisingly less affected than the large
mercury-treated group. As usual, the only portion of the brain affected
was the cerebellum and more specifically, the granular layer. The
destruction in this layer consisted of only intermediate pyknosis that
was previously described in the smaller co-insult group. The Purkinje
cells showed no sign of degeneration, nor did any of the other cerebellar
cell types.
In summary, the degree of granule cell degeneration in terms of treatment
groups can be seen in Table 19.
BRAIN CELL COUNT FOR POSSIBLE LOSS OF CELLS
Cell counts were taken from the anterior lobe area in the central lobule
of the cerebellum. The various brain cells counted consisted of Purkinje,
basket, granule, molecular layer cells, and granular cells in the
molecular layer. In the granular layer of the cerebellum, only a sample
number of granule cells were counted due to the vast numbers. This was
done by counting 5 oil fields of approximately 0.5 mm square, so the
number of granule cells in this study was only a representative sample
rather than the total number. The remaining four cell types were actual
numbers of cells in one lobe (anterior) that were counted on 5 different
animals and then averaged. The observed and expected cell counts for
the possible loss of cells were represented in Table 20. The experiment
tested the hypothesis that there was no difference between the various
treatment groups on the specific cell type. Therefore, a chi-square
test was calculated for each of the five different cell types and can be
seen in Table 21.
At 6 of freedom, the tabulated chi-square (X ) value at the 0.05 level
is 12.59. The expected cell count would be an average of the total
number of cells counted, since the hypothesis is testing no differences.
Since X of the Purkinje cells was less than 12.59, it was concluded that
there was no difference between treatment groups in terms of loss of
cells. However, in all of the other types of cells, the calculated X2
was larger than the tabulated X2 value, indicating that there was a
difference in number of cells between the different treatment groups.
Another cell count was made in the cerebellum on normal versus pyknotic
granular cells. The degree of pyknosis was also indicated as either
intermediate or complete pyknosis. A set number of 200 cells was counted
so cell density was not involved. Five sections of each treatment group
were counted, and their mean and standard deviation presented in Table 22.
52
-------
Table 19. DEGREE OF GRANULE CELL D
FROM LARGEST TO SMALLEST
Treatment Group
Sham injected and 9.0 kR
8.0 mg/kg MMC and sham irradiated
8.0 mg/kg MMC and 9.0 kR
Sham injected and 1.5 kR
4.0 mg/kg MMC and 4.5 kR
4.0 mg/kg MMC and sham irradiated
Sham injected and sham irradiated
53
-------
-p
c
0)
e
-p
m
fn
H
|
1 1
rfl
-P
O
H
bO
X O
bO cn
6
00 4-
bO
bO
e
00
-t-
&, bo
^ ^»
»n.
in bO
6
3- 3-
bO
bO
g
g
o
cn
P<
in
.
3"
rH
8
ii
c
o
CJ
rH 0)
H OH
0) >>
0 H
O
CO
in
CO
CN
00
3-
CN
3"
3-
CD
O
ID
CD
O
in
CN
in
3"
O
O
in
o
3"
in
u
o
-X
0)
1 1
c
H
v
fn
3"
o
in
=!
O
in
3-
0
in
3-
O
m
j.
o
in
3"
O
in
3-
O
in
n
to
t-T
CM
cn
UD
r-^
CO
H
3-
O
^J-
^
O
TH
in
rH
ID
CN
3*
TH
CO
(D
J-
TH
(N
CO
O
TH
0
ID
O
TH
II
O
-{->
0)
y
W
IT)
m
in
CN
CO
TH
in
CN
CO
H
in
CN
CO
7-t
in
CN
CO
^~*
in
CN
CO
TH
m
CN
CO
TH
in
CN
CO
TH
II
w
UD
^
CM
TH
CO
3- 3-
TH ID
TH 3-
3" 3"
00 3-
O (D
O 3-
3- 3-
ID 3-
CN ID
,
O CI)
!S t-J
^D
CN
3-
3-
U3
CN
3-
3"
ID
CN
3-
3"
ID
CN
3"
3"
UD
CN
3"
3-
tD
CN
3-
3-
CD
CN
3-
3"
II
W
CM
t^T
cn
oo
O
t-!
o
TH
CO
cn
o
TH
O
UD
m
TH
o
3-
co
TH
O
OO
H
TH
CN
O
CO
TH
CN
CN
3-
TH
II
o
c
'H to
V H
H 3
3 O
C Q)
(0 H
k O
o as
CN
00
CN
TH
CN
00
CN
TH
CN
00
CM
TH
CN
00
CN
TH
CN
OO
CN
TH
CN
00
CN
TH
CN
00
CN
TH
II
U
TJ
(U
p
0
Q)
A
-------
Cell Type Chi-Square Value
Purkinje cells
Basket cells 1".2
Granule cells 36.2
Molecular layer cells
Granule in molecular laver
-------
Table 22. MEAN" CELL COUNTS AND STANDARD DEVIATION
FOR PYKNOTIC GRANULE CELLS
Treatment
Group
Control (A)
Mean
**St. Dev.
4. 5 kR (B)
Mean
St. Dev.
9.0 kR (C)
Mean
St. Dev.
4.0 mg/kg (D)
Mean
St. Dev.
4.5 kR + 4.0
mg/kg (E)
Mean
St. Dev.
8.0 mg/kg (F)
Mean
St. Dev.
9.0 kR + 8.0
mg/kg (G)
Mean
St. Dev.
None
200.00
0.0
190.50
2.65
180.20
3.54
194.20
1.67
192.20
1.72
184.20
2.79
185.40
2.87
Degree of Pyknosis
Intermediate
9.40
2.65
10.80
3.60
6.00
1.67
7.80
1.72
9.60
2.85
14.60
2.87
Complete
8.40
4.13
6.50
3.84
"Mean of 5 sections
'"':St. Dev. = Standard Deviation
56
-------
DISCUSSION AND CONCLUSIONS
RADIATION INSULT
The extreme radiosensitivity of the granular layer of the cerebellar cortex
has been well established in this study. The granule cell was, in fact,
the most radio-vulnerable cerebellar element. Purkinje cells suffered
much less alteration than granule cells, and the incidence of their involve-
ment was also lower in this and previous studies.
Low radiosensitivity of Purkinje cells as compared with granule cells was
recognized, for example, from the observation by Hager et al.73 that
Purkinje cells in the hamster were free from change at 22 hours following
exposure to 40 kR x-irradiation, at a time when granule cells were shruken
and showed clumping of nucleoplasm and at a time when capillaries exhibited
endothelial-cell swelling. The present study used only 9 kR, and these
degenerative changes were observed in the granule cells while the Purkinje
cells remained unaltered.
There is still little knowledge of the earliest histopathologic and histo-
chemical changes occurring in the cerebellum following irradiation or in
the sequence in which the changes develop. Histochemical investigations
by Kimeldorf and Hunt?1* showed that during early radiation damage of
cerebellar tissue following high x-irradiation dose, changes occurred in
the nucleic-acid containing components of granule and Purkinje cells.
Particularly in Purkinje cells, alterations occurred in the cytoplasmic
RNA that were secondary effects due to regressive cellular alterations,
since swelling of the nerve cells was observed initially and decrease in
cytoplasmic nucleic acid content later on. The change in the structural
organization of the nuclear DNA in the pyknotic granule cells was possibly
also a secondary effect due to the increased density of the pyknotic
nuclei. But it could also have represented a primary change in the
physiochemical quality of the DNA caused by the action of ionizing radia-
tion.
In the present study, as with the studies mentioned, the granule cells
exhibited the morphological changes. Taking all the above points into
consideration, the cellular effects observed were considered to be due to
ionizing events in the cells.
METHYL MERCURY INSULT
In the present study, after the single dosages of methylmercuric chloride,
the granule cells displayed neuronal changes in the form of pyknosis and
karyorrhexis while the Purkinje and other cerebellar cells remained
unaltered. Two main types of degenerative changes have been observed in
57
-------
75
the granule cells following organic mercury poisoning. Type I:
Coagulative changescells became very dark and electron dense, probably
as a result of acute coagulative necrosis. Type II: Lucid changecells
had lost their normal chromatin pattern and appeared to electron lucid.
Miyakawa and Deshimaru^B suggested that some nuclear material may have
been lost in these cells and that the nuclear envelopes appeared to have
a compensatory thickening. These two degenerative changes that were just
described probably correspond to the light microscopic observation of dark
and light cells described by Takeuchi et al.77 in the granular layer of
the cerebellum. These dark and light cells were also observed in the
larger mercury-dosed group but not to any large extent as in the case of
the above study.
The differences in methyl mercury uptake observed between various nerve
cells could be due to the structural or metabolic properties of certain
neurons, which would, therefore, be more susceptible to the toxic action
of mercury. In this respect several cell populations with different degrees
of susceptibility may exist in a complex anatomical arrangement such as
the brain.
203
In a study using Hg, it was demonstrated that the Purkinje cells were
more heavily labelled than the granule cells; however, as was found in
the present study, the degeneration in the granular layer was much more
extensive.78 This may be due to the small size of the granule cell in
comparison with the Purkinje cell. Although the absolute content of
mercury in a granule cell was low, the concentration of mercury in a
granule cell could be actually higher than that in the Purkinje cell.
Furthermore, Kosmider^ demonstrated histochemically that the Purkinje
cells were very rich in sulfhydral groups, and that the histochemical
reaction was greatly reduced after mercury poisoning. This may suggest
that the large amounts of sulfhydral groups in the Purkinje cells may
offer a quencing effect on the mercury action inside the cell, rendering
a higher mercury tolerance.
The different responses of the neurons that were shown in this study were
probably related to the differences in the cellnlar metabolism. Likewise
the absorption, distribution, elimination, and tolerance of mercury by
different cells may vary.
81
Yoshino et al. suggested that the susceptibility of cells to organic
mercury compounds may depend upon their protein synthesizing activity-
However, mere rate of protein synthesis cannot be the critical factor
since in the cerebellum Purkinje cells were much more active in this
respect than granule cells% so this is an area needing much more investi-
gation before it is applied to the results of this study.
58
-------
CO-INSULT
Both the co-insult groups exhibited fewer cellular alterations than at
least one of their counterpart single insults. The lower co-insult group
(E) had more of an effect than its counterpart mercury single insult (D);
however, the same single dosage of radiation did more cell damage. The
larger co-insult treatment group (G) demonstrated less morphological change
than either of the larger insults alone.
The observation that the co-insult groups exhibited fewer changes than
induced by radiation alone produced two possible hypotheses. Either the
two insults had a neutralizing effect on each other or the mercury set
up a protective mechanism so as to reduce the effectiveness of the radia-
tion. Results of other studies presented in this report favor the latter
hypothesis; however, the former cannot be ruled out. Elucidation of the
mechanisms involved must await further investigation.
Whatever the reasons may be for the co-insults producing less damage than
the single insults, the study did test the hypothesis that the co-insult
would have a more deleterous effect than the single insult, and it was
proven to be incorrect.
CONCLUSIONS
The cerebellum proved to be the most susceptible area of the brain to
mercury and irradiation because of the sensitivity of some of its cells,
especially the granular cells. Why specifically the granular cells of
the cerebellum exhibited the greatest sensitivity is not known, but the
small size of the granule cell may be a strong determining factor. The
size of dose and time lapse must also have a bearing on the degree of
damage in these granular cells.
The single insult studies corresponded with the findings in the literature
with possibly only subtle differences due to the dosages and time inter-
vals. However, these studies needed to be done to link their findings
with those of the co-insult studies.
The hypothesis that co-insult treatments would have a more deterimental
effect than single insult treatments was rejected because the co-insults
had less of an effect on the cells. Because of this finding more hypo-
theses were presented: 1) The co-insults have a neutralizing effect on
each other so as to make their effects less than if administered singly
or 2) The mercury causes certain biochemical changes to set up a protective
mechanism against the radiation. According to this study, any of these
hypotheses would be feasible.
59
-------
Future work in co-insult studies is needed before any theory can be formed,
and even then it is probable that many factors are involved in each co-
insult study. The amount of time between treatments and before sacrifice
needs to be varied as well as the dosages. Many combinations of the same
type of experiment need to take place before the complete story is told.
Since there is no doubt that the cerebellum is affected, more extensive
work using electron microscopy should be done to find what is happening at
the ultrastructural level.
60
-------
SECTION v::
RESULTS OBTAINED WHEN METHYL >ZJ IVFA" ;,Af AT ':.;:;77?£_
IMMEDIATELY OR 24 HOURS A77E? hi.-.; .I.--.-' I.'.TI '!-'
A. RAT MORTALITY FOLLOWING ADMINISTRATl 3!: IT Si:, ill -.:;: ~;-i:;s:
WITH METHYLMERCURIC CHLORIDE IMMEIlATIli -7-77" v-~A3;A7:;;N
Two experiments, designed to investigate the
mortality effects of methylmercuric chloride
the albino rat, were performed. The first experiment included EC three-
month-old male rats, having a mean weight of 335.5 g and ranging between
312 g and 378 g. The second experiment included PC
female rats, ranging in weight from 226 g to 1~~ g,
In each experiment the 80 rats were divided ir.tr rour gr
taining 20 animals. One group served as a control.
irradiation treatment and an intraperitoneai injecti
saline. A second group was irradiated *ith 13,3C; r
head only. A third group received onl\- MMC ir.jecte:
in distilled water. A fourth group was treated with
In the first experiment each male rat was given l.~5 rg cf K!73 dissolved
in 1.1 ml of distilled water. The female rats used in the second exoeri-
ment were given 1 mg of MMC per 100 g of body weight. All MM? was
injected intraperitoneally immediately after irradiation or sham-
irradiation.
The results of the first experiment are summarized in 7abie 23. The
graphed results suggest the possibility that the mortality resulting
from MMC intoxication may be depressed by the effects cf x-radiation.
The results of the second experiment, involving female rate, are
summarized in Table 24. Although the results of this experiment tend
to follow the pattern observed in the first experiment, the depression
of early (Days 1-6 post treatment) MMC-inducec mortality in the co-
insult group appears less distinct in the second experiment.
61
-------
Table 23. ACCUMULATIVE PERCENTAGE MORTALITY 11-: LALE
TREATMENT WITH SINGLE AND COMBINED INSULTS CT l.~
MERCURIC CHLORIDE (MMC) AND 10,000 R X-RALIATION
Group*
Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
A (Control)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
B (MMC)
10
20
30
30
30
40
45
50
50
60
60
65
65
65
65
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
C( X-ray)
C
n
^j
\_
0
^
0
0
25
90
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
D (Co-insult)
20
20
20
20
~- r\
Z ^
2C
25
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
'Each group contained 20 rats
62
-------
Table 24. ACCUMULATIVE PERCENTAGE MORTALITY IN FEMALE RATS FOLLOWING
SINGLE AND COMBINED INSULTS WITH 1.0 mg. METHYL MERCURIC CHLORIDE PER
100 g. OF BODY WEIGHT AND 10,000 R X-RADIATION TO THE HEAD
Group"
Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
A (Control)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
B (MMC)
30
40
40
60
65
65
70
70
80
85
85
85
85
85
85
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
C (X-ray)
0
0
0
0
0
0
15
40
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
D (Co-insult)
20
35
40
50
55
60
60
65
90
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
"Each group contained 20 rats
63
-------
A possible reason for the less clear-cut effect in the second experiment
is that the average dose of MMC per animal was greater than in the first
experiment. The animals in the first experiment received approximately
C.S mg of MMC per 100 g body weight, whereas the rats in the second
experiment received 1 mg per 100 g of body weight, thus possibly over-
loading the protective mechanism elicited by the radiation.
These two experiments suggest that a protective mechanism against MMC
intoxication is triggered by the effects of x-irradiation. However,
since 10,000 R head irradiation results in one hundred percent death
by day 10 post-irradiation, any depression of MMC-induced mortality
beyond this period cannot be monitored by this type of experiment.
B. RAT MORTALITY FOLLOWING METHYLMERCURIC CHLORIDE
ALONE OR 24 HOURS AFTER X-IRRADIATION
Twenty-eight male rats weighing 324 to 377 g were used in this prelim-
inary study. Fourteen rats were injected intraperitoneally at a dose
rate of 7 mg/kg body weight with a solution containing 4 mg methyl
mercury per ml of physiological saline and received sham irradiation;
however, one received an incomplete mercury dose and was removed from
the experiment. Fourteen rats received 10,000 R of x-rays at a dose
rate of 265 R/min and methyl mercury as above.
Excess deaths were noted in the co-insult group. Four of 14 irradiated
animals died until the third day. One animal in each group died during
the third day. Additional sham-irradiated animals died on the 4th and
6th days after irradiation, but no further deaths in the co-insult group
were observed until the 7th day. All of the co-insult group were dead
by the 10th day, due to the effects of the large dose of radiation.
Further experiments will be required to establish the pattern of deaths
which occur after various doses when irradiation precedes the methyl
mercury insult. It is probable that excess deaths during the first 24
hours is due to increased blood-brain barrier permeability caused by
irradiation insult. Lack of further deaths in the co-insult group could
be due to possible protection caused by proliferation of peroxisomes (see
subsequent section in this report on this subject), or the more suscepti-
ble animals may have died within the first 24 hours following the co-
insult, while they survived for a longer period when mercury only was
administered. Replication of this experiment with larger numbers of
animals and other experiments varying the mercury and radiation doses
and the time between insults will be required to more clearly establish
the nature of the interaction of the two insults.
64
-------
SECTION VIII
EFFECTS OF COMBINED INSULTS OF METHYLMERCURIC CHLORIDE AND
X-RADIATION UPON THE UPTAKE OF SULFUR-35 BY THE RAT BRAIN*
INTRODUCTION
Studies evaluating the effects of single insults of x-radiation and
methyl mercury upon the blood-brain-barrier (BBB) have been done. Nair
and Roth82 demonstrated a BBB against sulfate, noting an increased _in_
vivo uptake of sulfate by the rat 48 hours after 10 kR head x-irradiation,
Steinwall and Olsson°3 found that the presence of methyl mercury dicyandi-
amide, as well as HgC^s brought about a change in BBB permeability,
increasing the influx of the fluorescent Evans blue-protein complex
while reducing the penetration of another indicator (Se75_selenomethio-
nine).
Results presented earlier in this report have shown that the effects of
the single insults upon brain serotonin and norepinephrine levels tended
to be neutralized in the co-insulted animals. We have also observed
that large doses of x-irradiation tended to reduce methyl mercury-induced
mortality. The present study evaluates the interaction of single and
combined doses of x-radiation and methyl mercury upon the in vivo uptake
of radioactive sulfate by various brain regions.
METHODS
Ninety 90-day-old male Sprague-Dawley rats, weighing 230 to 324 g, were
divided into 9 groups containing 10 animals per group as shown in Fig. 4.
Animals were maintained under standard laboratory conditions, housed 4
to a cage, and received food and water ad libitum. Each group received
a different dose combination of intraperitoneally injected methyl
mercury and head x-irradiation. The methyl mercury doses were either 0,
*A portion of these results were contained in a dissertation submitted by
James Earhart to North Texas State University in partial fulfillment of
the requirements for the Ph.D. degree. These results were presented by
E.W. Hupp, in a paper co-authored by Earhart and Hupp at the Radiation
Research Society meeting, June, 1976. The abstract is in press in
Radiat. Res.
65
-------
Figure M-. Experimental design for studying the effects
of single and co-insults of methylmercuric chloride and
x-radiation upon the uptake of sulfur-Cc sodiun sulfate
by various brain areas
Methylmercuric Chloride Injected
Intraperitoneally (mg/kg of Body Weight)
0 4.03 8.06
10 10 10
animals animals animals
X-Radiation
10 10 10
to Head 5 animals animals animals
in kR
10 10 10
10 animals animals animals
66
-------
4.03 or 8.06 mg/kg body weight. The x-ray doses of either 0 kR, 5 kR,
or 10 kR were delivered to unanesthetized animals by a General Electric
Maximar 250 KVP X-Ray Unit at a target distance cf 170 mm and a dose
rate of 264.5 R/minute. The filters used were O.E mm Cu and 1.0 mm Al.
During irradiation the body was shielded by a 5.- cm Thick laver of
lead. Sham-irradiated control animals were resrrai^ed for the same
length of time as animals receiving radiation. All animals were
anesthetized with pentobarbitol (40 mg/kg) and Killed by immersion in
liquid nitrogen 4-8 hours after irradiation. Five minutes prior to
sacrifice, each anesthetized animal was iniected in the femoral vein
with sulfur-35 sodium sulfate (30 yc per 100 g), based upon the findings
of Nair and Roth 84 that this dosage resulted in measurable increases of
that indicator in the rat brain at 48 hours post-irradiation. Immediate-
ly before sacrifice, 1 ml whole blood samples were taken by heart
puncture for liquid scintillation counting.
Tissue samples of caudate nucleus, cerebral cortex, thalamus, hypothala-
mus, hippocampus, inferior colliculus, medulla, cerebellum, skeletal
muscle and liver were taken for liquid scintillation counting and dry
weight determination. Frontal sections of the frozen rat brain were
sliced at intervals designed to expose each brain area to be sampled.
The sections were laid flat on the dissection plate of a dry-ice box and
desired samples were carved from the sections with a scalpel. Tissue
samples were prepared for counting after Mahlin and Lofberg^S and
counting was done on a Beckman Model LS-200 Scintillation Counter. Radio-
activity was determined for each sample and the results were expressed
as percent of the amount of radioactivity contained in the blood.^6 The..».
percent of blood concentration values (PBC values) computed for each
sample made comparisons of sulfate uptake by various tissues possible.
The PBC value for each sample is computed as follows:
CPM/mg dry wt x 102
PBC = CPM/yl whole blood
RESULTS
Percent of Blood Concentration (PBC) values for each type of sampled
tissue are tabulated in Table 25. An analysis of variance was run for
the PBC values of each tissue-type. When significant differences be-
tween PBC values were indicated, Duncan's Multiple Range Test was
performed to determine the groups between which significant differences
occurred.
Analysis of the various brain regions revealed several patterns of sul-
fate uptake following the administration of single and co-insults of
methylmercuric chloride and x-radiation. When differences were found
to exist between the single and combined insults, the co-insult effects
tended to neutralize each other. In some cases, however, one insult
tended to override the effect of the other insult. The various patterns
of neutralization are summarized below.
67
-------
w
Q
H
>< rt
m o
i_3
Q K
w o
2
H CJ
< H
EH «
m D
0 CJ
e;
w w
w g
D K)
1-3 >H
EH
H
'-^ g
CJ
m fa
ft O
w
2 EH Q
o ij <;
H D W
EH W E
< S3
04 H H
EH 1 K
3 O EH
W CJ
0 O
S3 Q EH
O S
u < a
o
Q W H
0 i-l EH
O O rt!
r3 S3 H
WHO
W <
O fa tf
O H
EH 1
S3 W X
w w
U W Q
ft! O S3
W Q <
ft
0
fri J2J
O H
^|
52 0-4
0 <
W >
H
04 5;
i< EH
ft H
g s
o
u w
EH
rtj
(*5
in
CM CJ
2
0) H
H EH
XI «C
EH 05
EH
O^ &1
& g .x
0 CJ
0 S
rH CO g
+
Cn Cn
Ctf S .V
.X oo \
0 U
0 . S
rH ^ 21
f- (J1 GI
e -x
ft< vO \
X 0 U
g
m co g
+ tT1 en
g iX
Pn OO ' .
M 0 CJ
g
m *3< g
en en
g fx
VD *\
0 CJ
- g
CO g
Cn en
g ^
oo \
0 CJ
g
^* S
K
,X
0
rH
PC^
^
in
H
O
Vt
-p
C
O
u
rH
(0
O r) 0)
H O 3
g CO
O (8 CO
+J CD -H
Ri H EH
C <
<
rr
(Ti
CN
oo
0
oo
vD
r~
CN
o
0
oo
rH
r-
rH
r-
en
CM
VO
o
00
o
in
00
CO
o
CM
H X
m 0)
JH -P
pQ r4
cu o
r) CJ
0)
u
LD
IT
CM
LTl
VO
CM
O
00
CM
r*-
'S'
CM
H
CN
CM
r~
vO
oo
en
r-
CM
i--
H
CM
CO
3
0) 0)
P rH
a o
O 3
3 S3
(0
o
oo
CM
CM
r-
en
i-H
r~
r^
H
CM
H
CM
O
t~-
rH
cn
cn
rH
O
0
00
CO
in
CM
CM
^*
rH
CQ
3
g
re
rH
fl)
£
EH
in
ro
oo
^
i-H
^T
r~
in
CN
en
in
CM
CM
CO
rH
rH
in
CN
CM
en
00
en
cn
CM
in
CO
CN
to
3
g
a
rH
re
,G
-P
Q
ft
>1
S
o
rH
OO
r^
rH
00
rH
O
oo
VD
H
00
CM
O
CN
O
00
CN
CM
CN
OO
OO
CO
CM
'I'
cn
CN
co
3
Pt
g
re
o
o
ft
ft
H
ffi
"?
00
00
r-.
in
00
H
CM
00
"vf
en
00
CM
CM
OO
^1"
m
OO
rH
cn
00
0
cn
00
00
CM
OO
co
3
H H
O 3
-H CJ
rl -H
Q) rH
n i . i
C 0
H O
^
rH
CN
i-H
vD
CM
CN
T
CM
in
CM
CN
CN
OO
CN
cn
o
CN
OO
in
CN
oo
OO
CN
CO
o
CN
re
rH
rH
3
-O
cu
g
CO
^*
i*
CN
00
00
0
vD
oo
CO
00
T
00
VD
CM
CN
cn
00
CO
CM
OO
OO
00
^J1
rH
CM
*3"
g
3
rH
H
(U
43
CU
rl
CU
U
rH O
O CN
in CN
CN
vD ^J1
oo co
in H
CN
^" m
o m
vD VD
CN
CM ^r
m cn
m rr
CN
a1 o
O CN
CO CO
rH
CM VO
VO O
[-. *}l
CN
in o
X -H
10 r3
68
-------
A summary of the effects of the single insults and co-insult combina-
tions is given in Table 26. Increases or decreases in sulfate uptake
by various tissues were categorized according to whether they were
statistically significant changes or whether they were not statistically
significant changes. The latter were arbitrarily defined as equal to or
greater than 10% of control values, although not statistically significant.
SUMMARY OF SINGLE INSULT EFFECTS
As indicated by Table 26, the x-radiation insult has a strong tendency to
cause increased uptake of sulfate by the brain. Ten kR x-irradiation, for
example, causes uptake in all brain regions monitored except the hippo-
campus and the cerebellum. The uptake of sulfate by the hippocampus
after treatment with 10 kR was the same as that in the controls, while
the cerebellum showed only a numerical decrease in uptake. Treatment
with 5 kR x-irradiation produced a similar but less pronounced pattern
"to that obtained with 10 kR x-irradiation.
Methyl mercury tends to have an effect opposite to that of x-irradiation
upon the uptake of sulfate by brain tissues, with methyl mercury gen-
erally causing a decrease in sulfate uptake. The only statistically
significant increase in sulfate uptake was indicated for the cerebral
cortex after treatment with the smaller dose of methyl mercury. This
is unexplained, especially in view of the fact that the larger dosage
causes a numerical decrease in sulfate uptake by the cerebral cortex.
SUMMARY OF CO-INSULT EFFECTS
The pattern most often seen after co-insult treatment is a neutralization
of effects. In the hypothalamus after co-insult treatment with 10 kR
and 8.06 mg methyl mercury/kg of body weight, a statistically clear case
of cancellation of effects is seen. The single insults cause uptake
changes in opposite directions: 10 kR x-irradiation resulting in a
significant increase in sulfate uptake and the larger dose of methyl
mercury resulting in a significantly reduced uptake of sulfate. The
co-insult treatment, however, produces no effect which is significantly
different from the control value. Similar, although not significant,
patterns are seen in the medulla after treatment with the largest
co-insult doses and in the caudate nucleus after co-insult treatment with
lower radiation and the higher methyl mercury doses.
X-irradiation tends to override the effect of methyl mercury in a number
of tissues. A case in point is the hippocampus, in which sulfate uptake
is significantly decreased by the larger dose of methyl mercury while
the sulfate uptake after the larger co-insult treatment is no different
from the control or the 10 kR single insult treatment uptake. Similar
patterns may be seen in the cerebellum, hypothalamus and medulla.
69
-------
s
o
M
CO E-i
H <
< M
K O
<3*
55 K
M K
M
CO 1
W X
ID
CO Q
CO 55
M <
H
/~*\
>H CJ
Q S
8 £
55 W
P4 Q
H M
Pi
>^ O
[T[ j "i
W O
X
< 0
H M
PH Pi
D D
O
W Pi
E-i W
*^H §
CM h3
r-> prj
CO E-i
H
IS 2
pS
M PH
Q O
O
CO CO
1 E-i
in 1.3
co co
55
PH M
O 1
o
55 0
O
CO Q
M 55
Pi <
OH W
S .-3
O O
0 55
M
CO
to as
CN H
M
0) 13
r\ Q
CO W
E-i H
I
H
Lf_l
o
0)
,x
tO CD
p -p
ft (0
ID 'i i
rH
Td 3
CU CO
CO 1
(OLD
CDCO
PH CO
o
Q)
Q
CU
11. 1 1
O CO
MH
(U rH
V rj
to co
-P 1
j > fO
co
MH
O
cu
rX
tO 0)
p -p
ft tO
~~) I] 1
H
"^ £3
(U W
W 1
tOi-n
CUCO
PH CO
O
C
M
^N
Pi
M
..
-P C
C 0
OJ -H
E -P
CO -H
CU T3
fn CO
E-H PH
f^
M
1
X
bO
H
CO
-P
0
55
bb
H
CO
rH
o
-p
c
0
0
CO
tO
Q)
g
cB
CO
bO
H
CO
-P
O
,
bO
H
CO
,.
bO
^^
bO
g
^^
O
s
s
4.
CO CO
« *>
O M
o as j
A A f*
W 55 H W
o co o as i_q o
ft
W
0 M
x
r .
co as i-3 co co co
j
s
A
W "
O J J
A A A
Si i "5-1
' ' A<
M
A A £p fft
M W W
j as o » M as
2
M H 55 «> M M
as as M on as as
w
E- 0
E-H A A
55 X S M
S OS
« « 55 W 55 55"
M MH E-iOOOO
A
0 O
O E-i E-i O O O
^33 0^0
55 " 0* O « E-i «
OHO OH as H
U3 O5 ID CO CO
O O O O O O O
O O CO d" CO d" J-
-1- 4-4- 4-4- 4-4-
Q O O O O O O
* »
o in o o o o to
rH TH vH
CO
P W
H 0
0) fS
CO CO PH
3 0)
ft i IH
E ' 1 i
CO CO 'H
O Td
O T3
ft C
ft tO M
H -iH
rG « CO +-1
?H f~!
1 (1) H-> CO
> o o
M -r( 55 -H
as H LH
r)
> I C
X <~* bO
0) J -P -H
-P CO CO
fn « 0)
O CO H >,
O 3 H
S D rH
rH (0 bO CO
(0 H C O
PH CO (0 'rt
/3 ,C Pi -p
Q) +J CO
PH CO -H
0) 1 - -P
o cm
1 H tO -P
O O CO
O » C
tO 3 4->
« H O O
E H ^ C
3 3
H 0) 0 bO
0) 6 . 3
xi o o
,
3 rH -H
(U O C rH
H O bO O
0 -H PH
3 PH CO -P
CO C
H 0) O
0) PH PH 0
H-> CU CO
tO MH U-i
T) C CO O
3 'rl CU
fO O dP
0 I C 0
CU TH
| I 1 f-t
-, 0)
> x; cu co
0) H CU
PH 1 U PH
rQ CO ft
X) H 3
-------
In other tissues methyl mercury tends to override the effect of x-irradia-
tion. A treatment of rat heads with 10 kR causes a significant increase
in sulfate uptake by the caudate nucleus, while the larger dose of methyl
mercury causes a numerical decrease in uptake not significantly differ-
ent from the control value. The larger co-insult causes a numerical
increase in sulfate uptake that does not differ significantly from the
control value. Similar observations are made concerning the thalamus,
inferior colliculus and cerebellum.
DISCUSSION
Insults of both x-irradiation and methyl mercury demonstrate the presence
of a BBB acting against radioactive sulfate, with irradiation disrupting
and methyl mercury enhancing the barrier effect. Our results with the
10 kR single insult corroborate the work of Nair and Roth, in which
they demonstrated that increased brain-uptake of sulfate occurs follow-
ing 10 kR head x-irradiation.
The current study demonstrated that the BBB to sulfate may be enhanced
by the intraperitoneal administration of methyl mercury. This finding
is consistent with the observations of Steinwall and Olsson^S that
methyl mercury dicyandiamide given at a dose rate of 200 mg/kg body
weight resulted in a decreased uptake of 75se_se;[_enomethionine by the
brain. That the BBB is complex is indicated by the fact that these
investigators also found an increased permeability of the cerebral blood
vessels to Evans blue dye. They obtained the same results with 75Se-
selenomethionine and Evans blue after treatment with the inorganic
mercuric chloride. This suggests that the greater toxicity of organic
mercurials may be a result of phenomena other than BBB damage.
Although the mechanism for transporting sulfate across the BBB is not
known, there are indications that in a number of both procaryotic and
eucaryotic cells sulfate-uptake is accomplished by active transport.
Since, in general, mercurials are powerful but unspecific enzyme
inhibitors,^ it seems likely that reduced sulfate-uptake in the rat
brain results from methyl mercury poisoning of an active transport
enzyme system.
The specific locality of BBB lesions caused by x-irradiation is not
known. However, if the capillary endothelium with its tight intercell-
ular junctions is the major anatomical component of the BBB, then it
is likely that the radiation-induced lesion occurs somewhere in that
structure. Maximum disruption has been found to occur at 48 hours
after irradiation, the delayed action suggesting that products such as
lipoperoxides resulting from the ionization activities of radiation are
involved in causing the barrier lesions. Possible damage is done to
either the intercellular tight junction or to some other component of
the cell membrane. Evidence for in vitro radiation damage to cancer cell
membranes through the vehicle of H202 has been reported. ^3
71
-------
Whatever the operative mechanisms involved in the BBB-altering abilities
of x-irradiation and methyl mercury, they tend to neutralize or counter-
act each other when administered as co-insults. This suggests that they
are attacking different transport pathways. It seems possible that
x-radiation may be causing anatomical leaks, while methyl mercury is
inhibiting an enzyme system mediating the transport of sulfate.
SUMMARY
This study was designed to investigate the interaction of methyl mercury
and 10 kR x-irradiation relative to the uptake of the indicator ^^S-
sodium sulfate by various regions of the brain. Several general trends
emerged. First, as previously reported in the literature, acute doses
of x-irradiation tended to cause an increase in uptake of sulfate by
the brain areas examined. Second, methyl mercury was found to have an
opposite effect upon the passage of sulfate across the BBB, decreasing
its rate of passage. Third, the general interaction pattern produced by
the co-insult was neutralization. In a few tissues a net cancellation of
effects was seen. However, more often an overriding of effects upon the
brain appears to be a typical pattern for the co-insult with methyl mer-
cury and x-irradiation.
72
-------
SECTION IX
PEROXIDE INDUCED PROTECTION AGAINST METHYLMERCURIC
CHLORIDE TOXICITY*
INTRODUCTION
Since in the results reported earlier 10 kR x-irradiation to the head
causes an increased permeability of the rat blood-brain barrier (BBS) to
S35-sodium sulfate and to the anticonvulsant drug acetazolamide,95 we
proposed that a co-insult with methyl mercury and x-irradiation might
result in a more rapid uptake of methyl mercury and thus earlier death.
However, during the period prior to total killing by x-irradiation we
observed up to 20% lower mortality for co-insulted rats than for rats
receiving only methyl mercury. The lowered mortality rate observed in
the co-insulted animals was opposite to our expectations, and suggested
that x-irradiation protects against methyl mercury. Since 10 kR to the
head caused death between days 8 and 10 after irradiation, the period
for evaluating the protective effect was limited.
A less severe insult, mimicking the x-irradiation effect, that allowed
the animals to live long enough for further investigation of the pro-
tective mechanism was needed. Lipid peroxidation occurs in irradiated
tissue homogenates^S and lipoperoxides accumulate in rat liver following
1.5 kR whole body x-irradiation.97 There is in vivo evidence that
radiation-induced peroxidation is responsible for cell damage,98 while
in vitro experiments demonstrate that radiation-produced hydrogen
peroxide can increase permeability of murine lymphoma cells. ^ Based
upon the observation that x-irradiation protects against methyl mercury
toxicity and upon evidence from the literature that radiation-induced
peroxides are responsible for the secondary effects of ionizing radiation,
we chose hydrogen peroxide (HP) for use in a pretreatment regime designed
to simulate radiation effects and to trigger the proposed protective
mechanism. Several experiments were conducted, each based upon the
results obtained in the previous experiment.
^Prepared from a manuscript to be submitted for publication. Authorship:
Earhart and Hupp
73
-------
PROCEDURE
Sprague-Dawley and Sprague-Dawley-derived rats, used in these studies,
were housed in wire-mesh cages under controlled conditions of lighting
(day-night cycle of 13-11 hrs) and temperature (22°C). Each animal was
fed standard laboratory chow and water ad libitum.
For the first study one hundred 90-day-old male and female rats were
divided into two groups of 50 each: one group received a pretreatment
with five 1 ml doses of 1.5% HP injected intraperitoneally at 24 hour
intervals and the other group received sham pretreatment with physio-
saline. Forty-eight hours after the last injection of either peroxide
or saline, each animal was given 10 mg methyl mercury per kg of rat.
The animals were observed for 30 days, and deaths recorded to the nearest
day.
In the second experiment, ninety 189 to 24-9 g female rats were randomly
divided into groups of 30 animals each. Half the rats in each group were
subjected to the hydrogen peroxide pretreatment regime, while the other
half received sham pretreatment with saline. The three groups were
intraperitoneally injected with methyl mercury at doses of 10.0, 12.5 and
15.0 mg/kg. The rats were observed for 30 days, during which time deaths
were tabulated.
In the third experiment, 217 to 283 g female rats were randomly divided
into 2 groups of 5 animals each. One group received HP pretreatment,
while the other groups received sham pretreatment with saline. Animals
were killed by decapitation 48 hours after the last dose of HP or saline.
Brains were removed and sections of the nucleus arcuatus prepared after
the technique of Srebro.^^^ Peroxisome-like organelle system-containing
glial cells were counted in 0.01337 mm^ of the nucleus arcuatus of each
rat brain.
RESULTS
The results obtained in the first experiment (Fig. 5) demonstrate that
the HP pretreatment provided significant (P < 0.01) protection against
methyl mercury-induced mortality. Eighty-four percent of the control
males and 68% of the control females died compared to 4% and 8%, res-
pectively, of the protected animals.
-------
Figure 5. Comparison of cumulative mortality (%) in male and female rats
pretreated with 1.5 percent hydrogen peroxide (HP) or physiological saline
for 5 days. The rats received methylmercuric chloride at a dose of 10 mg
per kg body weight 48 hours after the last dose of HP or saline. Statisti-
cal analysis by Chi square showed significant differences between HP and
saline at the 0.001 level in males and 0.01 level in females.
75
-------
0)
tH
o
O
H
M
3
O
f-i
0>
+J
CO
n
(%)
76
-------
Although the manner in which the peroxide-induced protective mechanism
(PIPM) operates is not understood, several observations suggest a hypo-
thesis. It is unlikely that any type of direct chemical relationship
exists between HP and metnyl mercury. Since methyl mercury was adminis-
tered 48 hours after the last dose of peroxide, the catalase activity
probably had inactivated the injected HP. If a methyl mercury-HP complex
is responsible for the PIPM, one might expect that some protection would
be achieved with a HP post-treatment regime. However, when we began
injecting methyl mercury (10 mg/kg), no reduction in mortality was
observed. Consequently, the proposed PLOS, demonstrated in the peri-
vascular glia of the periventricular regions of the brain-'-'"'-'- might be
a possible response mechanism for protecting against methyl mercury
toxicity.
Because of its high concentration of sulfhydryl groups, its perivascular
location and its proliferative response to ionizing radiation, the PLOS
appears to be suited for an important role in protection against heavy
metals. Several studies lend support to such a protective phenomenon.102
We suspected that the PLOS could offer protection only to the extent of
its level of proliferation; hydrogen peroxide or saline pretreated rats
were therefore subjected to graded doses of methyl mercury. The per-
centages of rats surviving the various doses of methyl mercury are shown
graphically in Fig. 6.
At the 10 mg/kg dose rate, for example, 93% of the HP pretreated rats
survived compared to 17% of the saline pretreated rats. This protective '\
activity of the HP pretreatment regime is apparent at each dose level.
Also, in both HP and saline pretreated groups the percentage of survivors
decreased with increasing doses of methyl mercury. A regression line
analysis of the survival data suggests that a methyl mercury dosage of
about 19.4 mg/kg is sufficient to overcome the protective effect of the
HP pretreatment compared to the 14.4 mg/kg required to kill all untreated
rats. This represents a 36% increase in protection.
In order to evaluate PLOS proliferation, a small-scale study was conducted
in which the number of PLOS containing glial cells in sections of the
nucleus -arcuatus were counted. Means of 139.4 and 183.4 were obtained
for saline and HP pretreated rats, respectively. This represents a 32%
increase in PLOS-containing glial cells following HP pretreatment, which
compares favorably with the 36% increase in protection provided by the
treatment. This supports the hypothesis that proliferation of the PLOS
is responsible for, or an indication of mechanisms responsible for, the
protection provided by HP against methyl mercury-induced lethality.
Further studies of other tissues will be necessary to substantiate the
hypothesis.
77
-------
Figure 6. A regression line analysis of the survival response to graded
doses of methylmercuric chloride exhibited by hydrogen peroxide pretreated
(open circles) and saline pretreated (closed circles) 90-day-old female rats.
78
-------
100-1
H2°2
Pretreated
7.5 10.0 12.5 15.0 17.5 20.0
Methylmercuric Chloride Dose Rate
mg/kg Body Weight
79
-------
SUMMARY
A highly significant level of protection agair^
induced mortality in rats is observed followin;
with hydrogen peroxide. An organelle system f-
glial cells of the brain is proposed as the prc
80
-------
SECTION X
CO -INSULTS EFFECTS OF METHYL MERCYRY A::
WITH DIFFERENT TEMPORAL RELATIONS:-;
INTRODUCTION
In our earlier studies of co-insult effects cf radiation and methyl
mercury, we evaluated the animals for only a few hours after the second
insult and then killed them for histological and biochemical studies and
for mercury analysis. Long-term analysis was not possible because the
radiation dose was lethal in 2-4 days following total body irradiation,
or in 8-10 days following head-only irradiation, due to damage to the
gastrointestinal system. In those studies, where two insults were used,
methyl mercury was administered 7 days before the radiation, or 7 days
before testing if used alone, to allow time for the mercury to localize
in sensitive tissues and produce an effect.
PROCEDURE
In the study to be reported here, the dose of each insult which was
lethal to approximately 50% of the animals was determined; then 100% of
this dose, 75% of this dose, and 50% of this dose was administered,
either separately or together. Various time relationships were used for
each level of insult. These relationships will be described in Table 28,
which contains all the groups. Because of limitations on the number of
animals which could be housed, treated and tested, the study was con-
ducted in three phases. Each phase contained all time relationships
but only one dose level, i.e., either 100%, 75% or 50% of the LD50/3o-
There were two runs within each phase; after comparison between runs,
data was combined for runs within phases. Because of the design, most
comparisons will be limited to comparisons between treatments within
level of dose.
The rats were tested for behavior using an open-field observation area
previously described. Briefly, the area consisted of a circular open
^Presented at Texas Academy of Sciences, March, 1976, Nancy Partlow and
E. W. Hupp, authors.
81
-------
field 81 cm in diameter surrounded by a 33 cm high wall. The field was
divided by two concentric circles and lines radiating from the center
into 12 truncated triangular-shaped areas surrounding a central circular
area. The open field test consisted of placing the subject in the
central circular area of the field and observing the subject's activity
for a period of 2 minutes. The following measures of emotionality and
ambulation were recorded: (a) the number of defecations, (b) the number
of areas entered with all four feet (ambulation), and (c) the number of
times the animal stood on its hind legs (rearings). The animals were
tested two hours after the second insult, then 2, 4, and 7 days later,
then twice weekly until 31 days after the second insult. Data collection
in all tests was conducted under a blind procedure.
RESULTS
LETHALITY
Data from which the LDsg/so for methylmercuric chloride could be deter-
mintd had been determined earlier; we did, however, have to determine the
LD5Q/30 ^OT gamma radiation, using our dosimetry and for our strain of
rat. The results are presented in Table 27. Since 4-0% of the rats
survived at doses of 725, 750 and 775 R, the dose of 750 R was selected
as the LD5Q/30 radiation dose.
The lethality observed in the various treatment groups is presented in
detail in Table 28 and summarized in Table 29. In the earlier studies
using large radiation doses, an apparent antagonism existed between the
two insults in the first few hours after the second insult, making the
co-insult less effective than either treatment alone. In contrast, the
results obtained in this study with lower doses and longer time periods
show the effects of the two insults to be partially additive. Results
for single insults were as expected, the calculated LI>50/30 yielding
40% and 70% lethality, well within the expected range for a sample size
of 10. However, 98% of the animals receiving the LD5Q/3Q of both insults
died. The groups receiving 75% of the LD50/30 of a single insult exhib-
ited 52% lethality, closely approximating an LDso- Thus the effects of
the two insults are partially additive: i.e., if fully additive, 50% of
the LDso/SO °f each insult would have resulted in 50% lethality where
only 4% was observed, while no additivity should have shown the same
effect in co-insult groups as in single insult groups. The results
presented in Table 28 indicate that within the time intervals used in
this study, the temporal relationship between doses did not affect the
mortality, so that in all cases co-insults appeared to be equally
effective .
BEHAVIOR
Ambulation scores 2 hours after the second insult are presented in Table
30. In the groups receiving 100% of the LD5Q/30, mercury alone or
radiation alone produced some reduction in ambulation. In co- insulted
82
-------
Table 27. PRELIMINARY RADIATION LETHALITY DETERMINATION
Dose Number % Survival
in R of Rats at 30 days
900 10 0
800 20 5
775 10 40
750 10 40
725 10 40
700 20 60
83
-------
Table 28. PERCENT LETHALITY AT DIFFERENT LEVELS OF INSULT
AND DIFFERENT TIME INTERVALS BETWEEN INSULTS
Treatment"
R-7d-Hg
Hg-7d-R
Hg-4d-R
R-4d-Hg
R-ld-Hg
Hg-ld-R
Hg-15m-R
R-15m-Hg
Hg only
R only
Control
100
100
100
100
90
90
100
100
100
40
70
0
% of LD50/30
75
70
50
60
50
40
40
60
50
0
0
0
50
0
20
10
0
0
0
0
0
0
0
0
refers to radiation, the second value the interval in days (d) or
minutes (m) between insults, Hg refers to mercury treatment. This format
is used in all succeeding tables.
-------
Table 29. SUMMARY OF PERCENT lZr
THE CO-INSULT AND THE SINGLE -N:
Treatment 100 75 50
All co-insult 98 52 i+
Hg only 40 0 0
Rad only 70 0 0
Control 0 00
85
-------
Table 30. AMBULATION OF RATS 2 HOURS AFTER THE SECOND INSULT
Treatment
R-7d-Hg
Hg-7d-R
Hg-4d-R
R-4d-Hg
R-ld-Hg
Hg-ld-R
Hg-15ra-R
R-15m-Hg
Hg only
R only
Control
% of
100
1
1
1
1
2
1
1
1
4.
0.
6.
1.
1.
0.
7.
9.
7.
6.
21.
4
5
4
8
3
5
6
5
9
7
6
1
1
2
1
LD
75
0.
8.
5.
0.
7.
21.
1
1
6.
0.
8.
22.
28.
50/30
0
0
0
0
7
0
3
4
5
7
2
1
1
2
1
1
1
1
1
1
1
1
50
2.
6.
2.
0.
0.
8.
2.
1.
5.
7.
8.
2
7
0
5
7
7
9
4
5
5
7
86
-------
animals, when radiation was the second insult, ambulation was similar
to that in the group receiving radiation alone; when mercury was the
second insult, the depression was much greater than either insult
produced alone. This relationship was observed even in the group where
only 15 minutes intervened between insults. A similar pattern was
observed in the animals receiving 75% or 50% of the LD50, except at the
50% level of radiation alone or radiation as the second insult, where
ambulation did not decrease except in the case of the 15 minute interval
between insults. The results show this measure of behavior to be more
sensitive in detecting effects of the insults than lethality data. This
measure also differentiated between the two insults with regard to the
severity of the defect in the lowest dose group, and showed that the
temporal relationship between the insults affected the results obtained.
Data on the number of rearings in the tests conducted 2 hours after the
second insult is presented in Table 31. The effect on the number of
rearings generally agrees with the ambulation data, although in most
cases the differences are less marked. Again, 50% or 75% of the
was nearly as effective as 100% of the
The ambulation scores and the number of rearings in tests conducted 2-7
days after the second insult are presented in Tables 32 and 33. All of
the animals in the 100% of the LDso group irradiated 7 days before mercury
administration died in the period 2-7 days after the second insult, and
provided insufficient data to tabulate. The effects on ambulation
observed in this time period in the animals receiving 100% of the LD5Q/30
were very similar to those observed 2 hours after the second insult. In
the lower dose groups, effects were less pronounced. In the 75% of
LD5Q/30 groups radiation alone had a slight effect with a very similar,
somewhat lower, activity occurring in the mercury alone and co-insult
groups. The depression in activity was less than that observed at 2 hours.
It should be recalled that these groups showing similar activity during
this time period later exhibited differences in mortality, with 52% of
the co-insulted animals dying while none of the mercury only treated
animals died. The ambulation scores for the animals receiving 50% of the
LD5Q/30 doses did not differ significantly from the control scores.
As was the case 2 hours after treatment, effects on rearings were similar
to but less marked than effects on ambulation. Only the groups receiving
100% of the two insults 7 or 4 days apart exhibited a significantly
decreased number of rearings during this time period.
The ambulation scores and the number of rearings for the entire 31-day
test period are presented in Tables 34 and 35. Since nearly all the co-
insulted animals receiving 100% of the LDso/30 and 52% of tne co-insulted
animals died during the test period, removal of animals from the test
groups by death may have influenced group means. In most cases, the
31-day mean ambulation score for the animals receiving 100% of the
LD5Q/30 was very similar to that observed 2 hours after the second insult.
87
-------
Table 31. NUMBER OF HEARINGS 2 HOURS AFTER THE SECOND INSULT
% of LD50/3
Treatment
R-7d-Hg
Hg-7d-R
Hg-i4d-R
R-4d-Hg
R-ld-Hg
Hg-ld-R
Hg-15m-R
R-15m-Hg
Hg only
R only
Control
1
2
3
2
3
3
5
3
1
3
3
4
00
. 4
. 1
. 8
.1
. 8
. 2
. 5
. 9
.1
. 5
. 0
7
3.
7.
8.
2.
5.
7.
3.
7.
4.
8.
9.
5
7
9
3
7
5
0
7
3
5
4
1
0
9
7
9
2
5
8
5
4
7
6
10
50
. 5
. 6
. 6
. 3
. 5
.1
. 7
.1
.3
. 0
. 6
88
-------
Table 32. AMBULATION OF RATS DAYS 2-7
AFTER THE SECOND INSULT
Treatment
R-7d-Hg
Hg-7d-R
Hg-4d-R
R-^d-Hg
R-ld-Hg
Hg-ld-R
Hg-15m-R
R-15m-Hg
Hg only
R only
Control
1
1
10
9.
3.
0.
24.
1
1
1
1
1
2
T" *
5.
2.
9.
*.
5.
0
.
0
2
5
2
5
9
9
7
7
0
% of
2
2
2
LD
75
1.
2.
3.
21.
1
2
1
1
2
2
2
9.
2.
8.
9.
2.
6.
9.
50/30
4
1
6
2
3
2
1
5
6
9
9
2
1
1
1
1
50
0.
7.
7.
9.
9.
21.
23.
1
2
2
1
9.
1.
2.
9.
7
3
4
0
6
6
0
7
8
1
1
89
-------
Table 33. NUMBER OF HEARINGS DAYS 2-7
AFTER THE SECOND INSULT
% of LD50/3
Treatment
R-7d-Hg
Hg-7d-R
Hg-4d-R
R-4d-Hg
R-ld-Hg
Hg-ld-R
Hg-15m-R
R-15m-Hg
Hg only
R only
Control
1
2.
1 .
1.
2.
2.
5.
3.
3.
2.
2.
00
0
3
14
8
0
4
3
7
5
9
7
6.
6.
5.
5.
5.
5.
6.
9.
4.
6.
6.
5
5
6
8
4
5
4
3
0
9
8
1
0
9
6
7
4
6
6
8
11
7
6
5
50
. 1
. 5
.0
.9
. 7
. 8
. 5
. 2
. 0
.1
.9
90
-------
Table 34. MEAN AMBULATION SCORES
FOR THE 30 DAY TEST PERIOD
% of LD50/30
Treatment
R-7d-Hg
Hg-7d-R
Hg-4d-R
R-4d-Hg
R-ld-Hg
Hg-ld-R
Hg-15m-R
R-15m-Hg
1
4
9
14
14
20
16
16
12
00
. 4*
.8*
.0*
. 5
. 8
.0*
.3*
.1*
7
24
26
28
23
27
26
21
24
5
. 0
. 9
. 9
. 4
. 0
. 5
. 9
. 9
5
25
25
22
24
24
27
23
25
0
. 6
.4
. 5
.1
. 4
. 0
. 6
.4
Hg only 21.5 29.0 28.2
R only 17.2 31.2 24. 0
Control 25.5 34.3 22.9
"Data on 1 week or less
91
-------
Table 35. MEAN NUMBER OF HEARINGS
FOR THE 30 DAY TEST PERIOD
% of LD
Treatment
R-7
Hg-
Hg-
R-4
R-l
Hg-
Hg-
R-l
d-Hg
7d-R
4d-R
d-Hg
d-Hg
Id-R
15ra-R
5m-Hg
10
2.
2.
1.
3.
6.
2.
4.
2.
0
4"
6*
7.*.
"
4
4
8*
9 *
9*
75
6.
12.
10.
8.
8.
9.
8.
12.
50/30
2
7
4
3
1
5
7
1
50
10.
11 .
11.
8.
9.
9.
10.
9.
4
7
9
1
1
3
6
6
Hg only 4.2 10.6 9.7
R only 3.7 9.8 8.2
Control 4.1 12.0 8.9
*Basedon7daysorless
92
-------
Due to recovery with time after treatment, the 31-day mean for the
animals receiving 75% of the 1*050/30 exhibited less effect of the treat-
ment than in the earlier time periods previously discussed. Ambulation
in the groups exposed to 50% of the LD50/3o did not differ significantly
from that of the controls.
Results on the number of rearings for the entire 31-day period are more
difficult to interpret. Results in the 100% and 50% groups are similar
to those on ambulation, although somewhat more variable. The 75% group
exhibit more variability and an apparently greater depression in some
co-insult groups. However, a careful examination of the data indicated
the apparent difference was due to an increase in the number of rearings
as the experiment progressed (probably due to greater familiarity with
the testing apparatus) while some groups did not exhibit this difference
due to death of 50% or more of the animals in the group. Thus data on
the number of rearings appears to be less reliable and less useful than
the ambulation data.
CONCLUSIONS
The two insults tested in this study are partially additive in that 100%
of the LDso °f the two insults applied separately resulted in 98% lethal-
ity and 75% of the 1^50/30 resulted in 52% lethality. Behavioral tests,
especially ambulation scores during the first 7 days after the second
insult, are more sensitive indicators of damage than lethality data.
The behavioral observations were also able to discriminate between
groups with regard to which insult was applied first while lethality
results did not. However, the ambulation scores of the co-insulted groups
exhibiting approximately 50% lethality following 75% of the LDso/30
a single insult were similar to the groups receiving mercury only, in
which no deaths occurred.
93
-------
SECTION XI
BEHAVIORAL OBSERVATIONS IN SQUIRREL MONKEYS (SAIMIRI SCIUREUS)
FOLLOWING METHYL MERCURY ADMINISTRATION
INTRODUCTION
A limited number of studies were conducted on squirrel monkeys (Saimiri
sciureus) in order to compare the results obtained in rats with those
obtained in an experimental primate. The lethality results obtained
in preliminary experiments have been presented earlier, as that seemed
the most appropriate location for those data. This section contains
the results of several behavioral studies. The first series involved
methyl mercury as a single insult, since data on this insult alone on
squirrel monkeys was not available at the time the study was initiated.
The final study involved the co-insult of the two agents under investi-
gation.
Subtle distrubances of behavior appearing before any clinical symptoms
have been attributed to methyl mercury poisoning.102 In order to quan-
titate such variable parameters, measurements of the conditioned response
have been utilized. These behavioral tests serve not only to indicate
disorders in the brain, but also to give insight to the state of the
whole organism.
Although fetal and newborn animals show more susceptibility to mercury
exposure, reports of variable degrees of intellectual and emotional
disturbance-'-^^ indicate serious effects in the adult. Due to conflicting
published conclusions as to whether mercury exposure affects only motor
and visual skills while leaving intellectual skills undamaged-^ 5 the
study was designed to observe behavioral changes in Saimiri sciureus
resulting from exposure to methylmercuric chloride. General behavior
observations were executed in order to find subtle behavioral changes
after exposure to methyl mercury. Behavior was quantitatively measured
in a Wisconson General Test Apparatus (WGTA) in order to test the
hypothesis that changes in conditioned responses occur before any overt
pathological effects are perceptible. Electrophysiological measure-
ments were made to investigate possible electrically measurable
differences between sighted and blind animals.
"From a manuscript by Francine Joiner and E. W. Hupp, submitted to
Environmental Research.
94
-------
SUBJECTS
The subjects were four female and 12 male squirrel monkeys. The four
females (Group I) were jungle born and of an undetermined, advanced age
of approximately 10 years. Four of the males (Group II) were born in
the colony at Texas Woman's University and the remaining eight (Group III)
were young adult males, jungle born.
INJECTION PROCEDURE
Females were injected intraperitoneally three times over a period of 27
months with 1 ml saline or 4.0, 3.0, 2.0 mg/animal of methylmercuric
chloride. The intervals between doses were 17 and 10 months; each
animal received the same dosage each time. Males were injected once
with 1 ml saline or 6.0, 4.5, or 3.0 mg/kg methylmercuric chloride. The
four colony-born males (Group II) were studied a year before experi-
mentation with the eight jungle born males (Group III) began.
BEHAVIORAL OBSERVATIONS
GENERAL. A tape recorder counting off 60 15-second intervals was used
to mark the time period in which each behavioral action of the animals
was recorded.
WGTA. Males were trained to a criterion of 20 correct responses out of
25 in a modified WGTA. Animals learned to recognize four pairs of visual
discriminanda differing in shape and color for a raisin reward. Pre-
injection and postinjection data consisting of three parameters, latency,
duration, and correctness of response were measured from 15 trials per
day per animal.
FOOD CONSUMPTION
All pre-feeding food weights and post-feeding weights of remains were
recorded in order to estimate daily intake of food. These data were
recorded for the females and the four colony-born males. An 18-hour
food deprivation schedule was enforced during training and testing of
male animals in the WGTA.
ELECTROPHYSIOLOGY
Electroencephalograms (EEC) and electroretinograms (ERG) were obtained
with an E £ M physiograph. Transistorized amplifiers (type 7070 channel
amplifier and type 7171 high-gain coupler) were used with the high fre-
quency filter set at 30 Hz for both EEC and ERG. The time constant was
0.3 for EEG and 3.2 for ERG. Animals were injected with Sodium Pento-
barbitol of doses sufficient to produce an anesthesia level obliterating
voluntary muscular movement. During a 15 minute period of dark adapta-
tion, drops of a mydriatic, epinephrine, and a topical optic anesthetic
were applied to the right eye of each animal. After attaining topical
95
-------
anesthesia and pupil dilation, the ERG electrode was applied. The elec-
trode was a modified version of that used by Ogden and Van Dyk-1-^ for
ERG recording in human infants and young children. The electrode was
constructed from a filament of platinum wire :-'.^:. -\- ~er~-\~: rr.d her*
in circular fashion to form a smooth ending. - -- ; :~--s -? --. . _ : > r.eal
wick was fashioned over the electrode. The elr rf ::<- wa; Z-T~.^~ inserted
ander the eyelid and the eyelid was then ta^el : . _. " '.oti. rri-.^la-
tion was achieved by manual manipulation of ar, o:a;ji jc:~er. -- reveal
brief intervals of light from a 6 volt lamp. L reference needle elec-
trode was placed subcutaneously over the surer-1:: Liar" ~,r;n. 7.1.^ needle
electrodes were inserted subcutaneously to recorc frr-r r:-: t-r-.Doral area
to the medial occipital region. Electrocarcirzra" (I.",") ':as also
monitored.
RESULTS
BEHAVIORAL OBSERVATIONS
GENERAL. An Average Daily Activity Level cor.-isrir.r c,r a zaily average
of all behavioral patterns requiring movement T.;a; calc-i.arez for Groups I
and II. The females (Group I) showed decreases in activity (Table 36)
paralleling signs of mercury poisoning but nor acrrrac':.:rg the severity
of decrease in activity of the affected males (Table ?"). The extent
of decrease in activity for the two high-dose nales was directly appli-
cable to the amount of mercury per kg of body weight received by each
animal with the level of activity decreasing the most with the highest
dosage animal.
WGTA. Of the Group II animals, animal #1 (6.0 mg/kg) and animal #2 (4.5
mg/kg) showed the only changes in WGTA data. Their changes, coinciding
with clinical signs of poisoning, showed decreases in correct responses
and increases in both latency and duration. Results froir Group III
concurred with decreases in correct responses in latency and duration
for animals 1A (6.0 mg/kg) and 3B (4.5 mg/kg). WGTA changes occurred
only in conjunction with clinical signs of illness.
FOOD CONSUMPTION
Females showed no continuing change in food consumption until the decrease
for the high-dose animal after the third injection (Table 38). Two
males (6.0 mg/kg and 4.5 mg/kg) had significant decreases in food con-
sumption after a single injection (Table 39).
SIGNS OF MERCURY POISONING
Vomiting and temporary anorexia were observed in most of the treated
animals immediately after injection. Although both males and females
showed apathy, weakness, and fatigue, females showed continuing attention
to stimuli even to the day of death. Males showed severe lack of
96
-------
Table 36. AVERAGE DAILY ACTIVITY LEVEL FOR FIVE
30 DAY PERIODS - GROUP I: FEMALES
An. #
1
2
3
4
mg/kg Run la Run 2b Run 3C Run 4d Run 5e
6.5 205.8 177.9 152.1 140.8 138.1
4.8 155.9 188.2 210.7 139.0 135.0
3.6 205.6 196.8 204.8 172.6 166.5".''
0.0 206.4 172.1 217.6 166.3 159.1
a!6 months after first dose,immediately before second dose
bimmediately after second dose
C4 months after second dose
^9 months after second dose, immediately before third dose
eimmediately after third dose
97
-------
Table 37. AVERAGE DAILY ACTIVITY LEVEL
FOR TWO 30 DAY PERIODS - GROUP II: MALES
Animal # mg/kg Preinjection Postinjection
1
2
3
4
6.0
4.5
3.0
0.0
231.5
246.5
298.7
239.3
92.0
130.2
279.5
201.0
98
-------
Table 38. AVERAGE FOOD CONSUMPTION ?EF
DAY IN GRAMS: GROUP I, FEMALE;
Animal #
1
2
3
4
rag/kg
6.4
5.1
3.8
0.0
Run
54.
37.
40.
44.
la
8
2
1
7
Run 2^ F.U" 0 - ^ ^ r! 4"^
55.7 56.4 20.8
31.9 49.0 38.6
35.5 35.5 50.9
34.6 48.6 51.1
a!6 months after first dose, immediately before second dose
bimmediately after second dose
C4 months after second dose
^immediately after third dose
99
-------
Table 39. AVERAGE FOOD CONSUMPTION PER
DAY IN GRAMS: GROUP II, MALES
Animal # mg/kg Preinjection Run Postinjection Run
1 6.0 86.2 23.2
2 4.5 54.8 29.4
3 3.0 85.0 81.7
4 0.0 66.2 63.0
100
-------
Interest progressing to coma. Two males in Group II became completely
blind; one survived and one died. In Group III, both males from the
6.0 nig/kg treatment group became blind before death.
Other signs observed were intention tremors, fine and gross motor
incoordination, ataxia, spasticity, paralysis, frequent licking of
extremities, and fits of anger. Bleeding from the sutures of the
skull was observed in many animals during necropsy.
ELECTROPHYSIOLOGY
All blind animals as well as sighted animals had well defined ERG
responses to photic stimulation. Simultaneous changes in EEG were
observed for all animals except the blind ones, although responses
varied in intensity. Severe tremoring of ill animals were evidenced in
the electric recordings even in deep anethesia.
DISCUSSION
Although two animals per treatment group in Group III allowed for
statistical measurement, the behavioral measurements did not lend to
reliable statistical deduction. The variance between subjects in the
same treatment group was so great that any combination of the data
cancelled out the effects observed. Evidently there were individual
differences in response to methyl mercury poisoning or the response
was modified by some unnoted factor. Significant change in the indices
measured correlated with the clinically observed symptoms of methyl
mercury poisoning. If the animals did not show symptoms of sickness,
changes in the variables of the experiment were not observed. The
measurements of general activity and food consumption served as
competent measures of illness in the animals. Loss of appetite and
diminished activity indicated onset of illness.
The electrophysiological investigation showed that blindness was not
attributable to impaired retinal function. The corresponding measures
of EEG stimulation gave an interesting research direction to follow.
Direct recording from visual cortex would serve future investigation.
Although the data for ill animals showed changes in responses in the
WGTA, there was no sufficient evidence for decreased memory function
except in the state of severe illness. Failure to perform in this case
may have been due to the deteriorated general condition of the subject,
rather than due to failure to remember the task. Therefore, it was
concluded that a exposure to methyl mercury does not permanently alter
learning performances in adult Squirrel monkeys.
101
-------
SECTION XII
COINSULT EXPERIMENTFEMALE SQUIRREL MONKEYS
INTRODUCTION
This study was conducted to extend the preceding experiments involving
mercury only to an experiment involving mercury and radiation as
co-insults. The study was also designed to extend previous studies
which we have conducted with rats to one using squirrel monkeys. A
limited number of studies have been reported for either insult used
alone, but to our knowledge, no coinsult studies with monkeys have
been done.
PROCEDURE
BEHAVIORAL OBSERVATIONS
The monkeys were subjected to the same training and testing regime in
the WGTA as described for the preceding studies. A total of 12 monkeys
began initial training, and 11 reached the criteria of 20 correct
responses out of 25. Following training, the monkeys were tested for
8 days pre-treatment and 30 days post-treatment.
TREATMENT PROCEDURE
Whole-body radiation was applied with a General Electric X-ray unit
operated at 250 KVp, 15 ma, 0.5 mm cu, 1.0 mm Al filtration at a dose
rate of 55 R/min. Methylmercuric chloride in physiological saline
(3 mg/ml) was injected intraperitoneally. In the coinsult groups,
methylmercury was administered within 10 minutes of the end of
irradiation. Each group except Group 6 contained two monkeys.
Ideally, all blocks in Table 4-0 should have been filled; however, the
number of animals and amount of time available for testing limited
the experiment to the groups shown. These groups we selected as
those expected to yield the greatest amount of information.
102
-------
Table 40. DOSES OF METHYL MERCURY CHLORIDE
PER KILOGRAM OF BODY WEIGHT AND RADIATION IN R
Radiation Dose
High (300) Low (150)
Control (0)
OJ
w
o
0
0)
s
High
6 mg/kg Group 1
Low
3 mg/kg
Control Group
Group 6
Group 2
Group 3
Group 5
103
-------
RESULTS
All animals administered methyl mercury, alone or as a coinsult, vomited
within 2 hours of administration of the agent. Anoxeria of 24-72 hours
duration was also observed. The animals were lethargic and exhibited
general signs of discomfort during this period. This was followed by
a prompt return to normal behavior. Activity of the animals receiving
radiation alone could not be distinguished from that of the controls.
Only one animal died; one female which received the high dose of methyl
mercury alone began exhibiting typical symptoms of the terminal syndrome
described earlier (Hoskins and Hupp) and died 2 days later, 7 days after
treatment.
The number of correct responses obtained in the WGTA was reduced in all
animals that received the high dose of methyl mercury, either alone or
as a coinsult, the first day after treatment. One of the two females
which received the low coinsult also had a decreased number of correct
responses at this time. The two females that received methyl mercury
only continued to have fewer correct responses. The female that died
performed normally 4- days after treatment, then failed to perform sub-
sequent days until death while exhibiting obvious signs of methylmercury-
induced illness. The female that survived returned to normal by 9 days
after treatment. No significant depression in the number of correct
responses was observed in the other groups after the first day. For an
unexplained reason, one of the two controls stopped performing at all a
few days after treatment of the treated animals.
The latency (period of time for the animal to choose a stimulus object,
beginning when the tray touched the transfer cage and ending when the
animal touched a stimulus object with his hand) appeared to be affected
for a longer period of time than the number of correct responses. The
animals receiving the coinsult had increased latency for 12 and 13 days;
the animal that survived the mercury only had increased latency for 7
days while the one that died had increased latency all the days she
performed. Thus, in contrast to the number of correct responses, latency
seemed to be affected more in the coinsulted animals.
One of the animals receiving the low coinsult had increased latencies for
9 days after treatment, though no significant changes were observed
in the other one receiving this treatment. One of the two animals
receiving 300 R radiation only had a slight increase in latency on
days 1, 2, 13, and 14 after treatment with the other showing no
change; the one animal receiving 150 R also showed no change. One
control exhibited very constant latencies, while the one that ceased
to perform had increased latencies for the last several days that she
performed.
104
-------
The duration of response (from the end of latency to when the subject
put the raisin in her mouth) was generally similar to that for the
number of correct responses but the data seemed to be more variable
than for the other two parameters. In agreement with the other two
paramenters, the two groups that received the large dose of methyl-
mercury showed the only consistent treatment effect. One animal
receiving the coinsult generally had increased duration of response
through 20 days after treatment, then returned to pre-treatment values;
the other had increased duration through day 12, then generally had
some increase through day 25. The animal surviving the high mercury
treatment generally had increased duration for 8 days after treatment,
while the decedent had increased duration all the days that she
responded following treatment.
One female receiving the low coinsult had increased duration of
response through day 5, then returned to pre-treatment values, while
the other member of this group showed no change. One animal receiving
300 R had several days with high duration of response, but exhibited
no consistent pattern; the other did not show any change, responding
with the greatest consistency shown for this parameter of any of the
animals tested. One control and the animal receiving 150 R only had
very consistent responses; as with the latency responses, the control
that quit responding had increased duration for several days before
completely stopping.
DISCUSSION
Response of the mercury-treated females was similar to that of other
monkeys tested previously. As previously observed, some variation
existed in groups receiving the same treatment. In this case, marked
differences were observed in the group receiving the high mercury dose
and in the low coinsult group. In the former, one female died while
the other recovered and exhibited no persistent symptoms. In the latter
group, one female exhibited changes for a few days and then returned to
normal while the other showed no effect.
The results obtained in this and previous studies indicate varying
thresholds for the various effects produced by methylmercury, with
individual differences causing different thresholds. The sensitivity
for the various effects would appear to be vomiting > transient
lethargy > anorexia > performance decrement in WGTA > reduced general
activity > blindness > death. With regard to the latter two
parameters, some animals receiving a dose that was lethal to one or
more animals receiving a similar dose became blind while others did
not. In the case of females, no blind animals were produced, while
several males became blind.
105
-------
Coinsult effects were generally similar to those observed in rats. In
this study, the coinsult groups performed very similarly to those
receiving the same dose of mercury only. In some parameters measured,
the high coinsult groups appeared slightly more adversely affected
than mercury only; for other parameters, the reverse appeared to be
the case. Sample size was too small to draw extensive conclusions
from with regard to lethality; no major additivity of the two insults
is possible, however, since both animals receiving the high coinsult
survived while only one of the two mercury-only animals survived.
Based on this and previous studies, 6 mg methylmercuric chloride/kg
body weight appears to be approximately an LDj-Q dose. The LD5Q for
radiation has not been accurately determined; based on previous
hematology studies in this laboratory, the 300 R dose was considered
barely sublethal. Thus if significant additivity of the two insults
exists, one or both animals receiving the high coinsult should have
died.
The behavior of the control animal which failed to continue to perform
is unexplained. A total of 25 animals have been tested in our apparatus
under the same conditions, and this is the only one which has failed to
continue to perform. The test procedure may thus considered to be
very reliable.
106
-------
REFERENCES
1. Matsumoto, H. , G. Koya, and T. Takeuchi, "Fetal Minamata Disease:
A Neuropathological Study of Two Cases of Intrauterine Intoxication
by a Methylmercury Compound," J_. Neuropath. Exp. Neurol. , 29, pp
563-574 (1965),
2. Pierce, R. E., J. F. Thompson, W. H. Likosky, L. N. Nickey, W. F.
Barthel, and A. F. Hinman, "Alkyl Mercury Poisoning in Humans:
Report of an Outbreak," JAMA, 220, pp 1439-14-1+2 (1972); Bakir, P.,
S. F. Damluji, L. Amin-Zaki, M. Murtadha, A. Khalidi, N. Y. Al-Rawi,
S. Tikriti, H. I. Dhahir, T. W. Clarkson, J. C. Smith, and R. A.
Doherty, "Methylmercury Poisoning in Iraq: An Interuniversity
Report," Sci. , 181, pp 230-241 (1973).
3. Hunter, D. R., R. Bomford, and D. S. Russell, "Poisoning by Methyl
Mercury Compounds," Quart. J_. Med. , 9, pp 193-213 (1940); Klein, R. ,
S. P. Herman, P. E. Brubaker, G. W. Lucier, and M. R. Krigman,
"A Model of Acute Methylmercury Intoxication in Rats," Arch. Path.,
93, pp 408-418 (1972); Berlin, M., C. A. Grant, J. Hellberg, J.
Hellstrom, and A. Schiitz, "Neurotoxicity of Methylmercury in
Squirrel Monkeys," Arch. Environ. Health, 30, pp 340-348 (1975).
4. Bergland, F. , and M. Berlin, "Risk of Methylmercury Cumulation in
Man and Mammals and the Relation Between Body Burden of Methyl-
mercury and Toxic Effects." In: Chemical Fallout, Miller, M. W.,
and G. G. Berg (ed.). Springfield, 111., C. C. Thomas, 1969.
pp 258-273.
5. Miller, M. W., and G. G Berg, ed., "Chemical Fallout: Current
Research on Persistent Pesticides." Springfield, 111., C. C.
Thomas, 1969. 531 pp; Nelson, D., ed., "Hazards of Mercury: Special
Reports to Secretary's Pesticide Advisory Committee, Department of
Health, Education and Welfare," November, 1970, Environ. Res., 4,
pp 1-69; Wallace, R. A., W. Fulkerson, W. D. Shultz, and W. S. Lyon,
"Mercury in the Environment: The Human Element," Oak Ridge, Oak
Ridge Natl. Lab NSF-EPI, 61 pp; Clarkson, T. W., "The Pharmacology
of Mercury Compounds," Ann Rev. Pharmacol. , 12 pp 375-406 (1972);
D'ltri, F. M., "The Environmental Mercury Problem." Cleveland,
Ohio, CRC Press, The Chemical Rubber Co., 1972. 124 pp.; Friberg, L. ,
107
-------
and J. Vostal, "Mercury in the Environment: An Epidemiological and
Toxicological Appraisal." Cleveland, Ohio, CRC Press, The Chemical
Rubber Co., 1972. 215 pp.
6. Peakall, D. B., and R. J. Lovett, "Mercury: Its Occurrence and
Effects in the Ecosystem," Biosci., 22, pp 20-25 (1972).
7. Hupp, E. W., "Reproduction in an Indoor Squirrel Monkey Colony,"
VII Intern. Kongr. Fortplanzung, Muchen, Vol. 3, pp 2313-2320 (1972).
8. Shank, R. P., and M. H. Aprison, "Method for Multiple Analyses of
Concentration and Specific Radioactivity of Individual Amino Acids
in Nervous Tissue Extracts," Analyt. Biochem., 35, pp 136-145 (1970).
9. Finney, D. J., Statistical Methods in Biological Assay, 2nd ed.,
New York, Hafner Publishing Co., 1964. 668 pp.
10. Weil, C. S., "Tables for Convenient Calculation of Median-Effective
Dose (LD 50 or ED 50) and Instructions in Their Use," Biometrics,
8, pp 249-263 (1952).
11. Hatch, R., and W. L. Ott, "Determination of Submicrogram Quantities
of Mercury by Atomic Absorption Spectrophotometry," Anal. Chem.,
40, pp 2085-2087 (1968).
12. Thorpe, V. A., "Determination of Mercury in Food Products and
Biological Fluids by Aeration and Flameless Atomic Absorption
Spectrophotometry," J_. Assoc. Of fie. Anal. Chem., 54, pp 206-210
(1971).
13. Hoover, W. L., J. Melton, and P. A. Howard, "Determination of Trace
Amounts of Mercury in Foods by Flameless Atomic Absorption,"
J_. Assn. Of fie. Anal. Chem., 54, pp 860-865 (1971).
14. Shank, R. P., and M. H. Aprison, "Method for Multiple Analyses of
Concentration and Specific Radioactivity of Individual Amino Acids
in Nervous Tissue Extracts," Analyt. Biochem., 35, pp 136-145 (1970).
15. Miyakawa, T., and M. Deshimuru, "Electron Microscopic Study of
Experimentally Induced Poisoning due to Organic Mercury Compounds:
Mechanism of Development of the Morbid Change," Acta Neuropath.
(Berlin), 14, pp 126-136 (1969); Klein, R., S. P. Herman, P. E.
Brubaker, G. W. Lucier, and M. R. Krigman, "A Model of Acute Methyl-
mercury Intoxication in Rats," Arch. Path., 93, pp 408-418 (1972).
16. Shank, R. P., and M. H. Aprison, "Method for Multiple Analyses of
Concentration and Specific Radioactivity of Individual Amino Acids
in Nervous Tissue Extracts," Analyt. Biochem., 35 pp 136-145 (1970);
108
-------
"Postmortem Changes in the Content and Specific Radio-
17.
Activity of Several Amino Acids in Four Areas of the Rat Brain,"
J_. Neurobiol. , 2, pp 145-151 (1971); Shaw, R.K., and J. D. Heine,
"Ninhydrin-positive Substances Present in Different Areas of Normal
Rat Brain," £. Neurochem. , 12, pp 151-155 (1965); Berl, S., and
H. Waelsch, "Determination of Glutamic Acid, Glutamine, Glutathione,
and X-amino Butyric Acid and Their Distribution in Brain Tissue,"
J_. Neurochem. , 3, pp 161-169 (1958); Kandera, J., G. Le Levi, and
A. Lajtha, "Control of Cerebral Metabolite Levels. II: Amino
Acid Uptake and Levels in Various Areas of Rat Brain," Arch. Biochem.
Biophys. , 126, pp 249-260 (1968).
Swensson, A., and U. Ulfavarson, "Toxicology of Mercury Compounds
Used as Fungicides." Occup. Hlth. Rev. , 15, pp 5-11 (1963).
18. Ulfvarson, U. , "The Effect of the Size of the Dose on the Distri-
bution and Excretion of Mercury in Rats after Single Intravenous
Injection of Various Mercury Compounds," Toxicol. Appl. Pharmacol . ,
15, pp 517-524 (1969).
19. Swensson, A., "Investigation on the Toxicity of Some Organic Mercury
Compounds Used as Seed Disinfectants," Acta Med. Scand. , 143,
pp 365-384 (1952).
20. Peakall, D. B., and R. J. Lovett, 1972, op.cit.
21. Finney, D. J. , 1964, op. cit.
22. Webb, J. L. , "Mercurials." In: Enzyme and Metabolic Inhibitors ,
Vol. II . New York, Academic Press, 1966. pp 729-1070.
23. Paterson, R. A., and D. R. Usher, with tech. asst. of R. K. Biswas
and J. Sreter, "Acute Toxicity of Methylmercury on Glycolytic
Intermediates and Adenine Nucleotides of Rat Brain," Life Sci. ,
10, pp 121-128 (1971).
24. Chang, L. W. , and H. A. Hartmann, "Ultrastructure Studies of the
Nervous Systems after Mercury Intoxication. I: Pathological
Changes in the Nerve Cell Bodies," Acta Neuropath. (Berlin),
20, pp 122-138 (1972).
25. Birke, G. , A. G. Johnels, L. 0. Plantin, B. Sjostrand, S. Skerfving,
and T. Westermark, "Studies on Humans Exposed to Methylmercury
Through Fish Consumption," Arch. Environ . Health., 25, pp 77-91
(1972).
26. Spyker, J. M. , "Assessing the Impact of Low Level Chemicals of
Development: Behavioral and Latent Effects," Fed. Proc . , 34,
109
-------
pp 1835-184-4 (1975); Evans, H. L. , V. G. Lathies, and B. Weiss,
"Behavioral Effects of Mercury and Methylmercury," Fed. Proc.,
34, pp 1858-1967 (1975).
27. Hunter, D. R., R. Bomford, and D. S. Russell, "Poisoning by Methyl
Mercury Compounds," Quart. J_. Med. , 9, pp 193-213 (194-0).
28. Curley, A., V. A. Sedlak, E. F. Girling, R. E. Hawk, W. F. Barthel,
P. E. Pierce, and W. H. Likosky, "Organic Mercury Identified as
the Cause of Poisoning in Humans and Hogs," Sci., 172, pp 65-67
(1971); Pierce, R. E., J. F. Thompson, W. H. Likosky, L. N. Nickey,
W. F. Barthel, and A. F. Hinman, "Alkyl Mercury Poisoning in Humans:
Report of an Outbreak," JAMA, 220, pp 1439-1442 (1972).
29. Hook, 0., K. D. Lundgren, and A. Swensson, "On Alkyl Mercury Poison-
ing with Description of Two Cases," Acta Med. Scand., 150, pp 131-
137 (1954).
30. Taylor, W., H. A. Guirgis, and W. K. Stewart, "Investigation of a
Population Exposed to Organomercurial Seed Dressing," Arch. Environ.
Health, 19, pp 505-509 (1969).
0
31. Falk, R., J. 0. Ekman, U. Greitz, and B. Aberg, "Whole Body Measure-
ments on the Distribution of Mercury-203 in Humans after Oral Intake
of Methylradiomercury Nitrate," Acta Radio!. Ther. Phys., 9, pp 55-
72 (1970); Miettinen, J. K., T. Rahola, T. Hattala, K. Rissanen, and
M. Tillander, "Retention and Excretion of Hg-203 Labelled Methyl
Mercury in Man after Oral Administration of Methyl Mercury Bio-
logically Incorporated into Fish and Muscle Protein," Scand. J_. Clin.
Inves. (Suppl. 113), 25 p 97 (1970). ~
32. Evans, H. L., V. G. Lathies, and B. Weiss, "Behavioral Effects of
Mercury and Methylmercury," Fed. Proc., 34, pp 1858-1967 (1975).
33. Berlin, M., C. A. Grant, J. Hellberg, J. Hellstrom, and A. Schiitz,
"Neurotoxicity of Methylmercury in Squirrel Monkeys," Arch. Environ.
Health, 30, pp 340-348 (1975).
34. Hunter, D. R., R. Bromford, and D. S. Russell, "Poisoning by Methyl
Merucry Compounds," Quart. J_. Med. , 9, pp 193-213 (1940).
35. Miyakawa, T., and M. Deshimuru, "Electron Microscopic Study of
Experimentally Induced Poisoning due to Organic Mercury Compounds:
Mechanism of Development of the Morbid Change," Acta Neuropath.
(Berlin), 14, pp 126-136 (1969); Klein, R., S. P. Herman, P. E.
Brubaker, G. W. Lucier, and M. R. Krigman, "A Model of Acute Methyl-
mercury Intoxication in Rats," Arch. Path., 93, pp 408-418 (1972).
110
-------
36. Berlin, M., C. A. Grant, J. Hellberg, J. Hellstrom, and A. Schutz,
"Neurotoxicity of Methylmercury in Squirrel Monkeys," Arch. Environ.
Health, 30, pp 340-348 (1975).
37. Miyakawa, T., and M. Deshimuru, "Electron Microscopic Study of
Experimentally Induced Poisoning due to Organic Mercury Compounds:
Mechanism of Development of the Morbid Change," Acta Neuropath.,
(Berlin), 14, pp 126-136 (1969); Pekkanen, T. J., and M. Sandholm,
"Effect of Experimental Methyl Mercury Poisoning on Number of
Sulfhydryl (SH) Groups in Brain, Liver, and Muscle of Rat," Acta
Vet. Scand., 12, pp 551-559 (1971); Swensson, A., and U. Ulfvarson,
"Distribution and Excretion of Mercury Compounds over a Long Time
After a Single Injection," Acta Pharm. Toxicol., 26 pp 273-283
(1968).
38. Yoshino, T., T. Mozai, and K. Nakao, "Distribution of Mercury in
the Brain and Its Subcellular Units in Experimental Organic Mercury
Poisonings," J_. Neurochem., 13, pp 3970-4006 (1966a); ,
"Biochemical Changes in the Brain of Rats Poisoned with an Alkyl-
mercury Compound with Special Reference to the Inhibition of Protein
Synthesis in Brain Cortex Slices," J_. Neurochem., 13, pp 1223-1230
(1966b).
39. Spyker, J. M., "Assessing the Impact of Low Level Chemicals of
Development: Behavioral and Latent Effects," Fed. Proc., 34,
pp 1835-1844 (1975).
40. Swensson, A., and U. Ulfvarson, "Distribution and Excretion of
Mercury Compounds over a Long Time after a Single Injection,"
Acta Pharm. Toxicol., 26, pp 273-283 (1968).
41. Klein, R. S. P. Herman, P. E. Brubaker, G. W. Lucier, and M. R.
Krigman, "A Model of Acute Methylmercury Intoxication in Rats,"
Arch. Path., 93, pp 408-418 (1972).
42. Pierce, R. E., J. F. Thompson, W. H. Likosky, L. N. Nickey, W. F.
Barthel, and A. F. Hinman, "Alkyl Mercury Poisoning in Humans:
Report of an Outbreak," JAMA. 220, pp 1439-1442 (1972).
43. Bergland, F., and M. Berlin, 1969, op. cit.
44. Evans, H. L., V. G. Lathies, and B. Weiss, 1975, op. cit.
45. Berlin, M., C. A. Grant, et. al., 1975, op. cit.
46. Spyker, J. M., 1975, op. cit.
47. Webb, J. L., 1966, op. cit.
Ill
-------
48. Rogers, K. S., T. E. Thompson, and L. Hellerman, "The Effects of
Silver Ions, Organic Mercurials and pH on the Dissociation of
Bovine Liver Glutamate Dehydrogenase," Biochem. Biophys. Acta.,
64, pp 202-204 (1962).
49. Shank, R. P., and M. H. Aprison, 1971, op. cit.
50. Friberg, L., and J. Vostel, "Mercury in the Environment," CRC
Press, The Chemical Rubber Co., Cleveland, Ohio, 1972. pp 93-107.
51. Gould, M. N., and M. B. Yatbin, "The Effects of X-Irradiation on
the Early Stages of the Memory System in Rats," Physiol. Behav.,
11, pp 177-179 (1973); Casarett, A. P., "Swim-Tank Measurement of
Radiation-Induced Behavioral Incapacitation," Psychol. Rep., 33,
Vol. 3, pp 731-736 (1973).
52. Roberts, J. M., "Prenatal Chemical Protection Against Radiation-
Induced Learning Deficiencies," Radiation Res., 49, pp 311-321 (1972)
53. Casarett, A. P., and C. L. Comar, "Incapacitation and Performance
Decrement in Rats Following Split Doses of Fission Spectrum Radia-
tion," Radiation Res., 53, pp 455-461 (1973).
54. Curley, A., V. A. Sedlak, E. F. Girling, R. E. Hawk, W. F. Barthel,
P. E. Pierce, and W. H. Likosky, "Organic Mercury Identified as
the Cause of Poisoning in Humans and Hogs," Sci., 172, pp 65-67
(1971).
55. Bryce-Smith, D., "Behavioral Effects of Lead and Other Heavy Metal
Pollutants," Chem. Br., 8, pp 240-243 (1973).
56. Post, E. M., M. G. Yang, J. A. King, and V. L. Snager, "Behavioral
Changes of Young Rats Force-Fed Methyl Mercury Chloride," P_-S_.E_.B_.
M_. , 143, pp 1113-1116 (1973); Spyker, J. M. , J. B. Sparber, and
A. M. Goldberg, "Subtle Consequences of Methyl Mercury Exposure:
Behavioral Deviations in Off-Spring of Treated Mothers," Sci.,
pp 621-623 (1972).
57. Klemm, W. R., Animal Electroencephalography, New York, Academic
Press, 1969. pp 269; Garcia, J., N. A. Buchwald, G. Bach-Y-Rita,
B. H. Feder, and R. R. Koelling, "Electroencephalographic Response
to Ionizing Radiation," Sci., 140, pp 289-350 (1963).
58. Speck, L. B., "Effects of X-Irradiation on Rat Electroencephalogram
and Brain Serotonin," J. Neurochem., 9, pp 573-574 (1962).
112
-------
59. Hoover, W. L., J. Melton, and P- A. Howard, "Determination of Trace
Amounts of Mercury in Foods by Flameless Atomic Absorption," J_. Assn.
Offic. Anal. Chem., 54, pp 86G-B65 (1971).
60. Thorpe, V. A., "Determination of Mercury in Food Products and
Biological Fluids by Aerations and Flameless Atomic Absorption
Spectrophotometry," J_. Assn. Offic. Anal. Chem. , 54, pp 206-210
(1971).
61. Magos, L., and T. W. Clarkson, "Atomic Absorption Determination of
Total Inorganic and Organic Mercury in Blood," J_. Assn. Offic.Anal.
Chem. , 55, pp 966-971 (1972). ~~
62. Maikel, R. P., T. J. Cox, J. Saillant, and F. P. Miller, "A Method
for the Determination of Serotonin and Norepinephrine in Discrete
Areas of Rat Brain," Int. J_. Neuropharm. , 7, pp 275 (1968).
63. Yoshino, Y., M. Toshiyi, and N. Kiku, "Biochemical Changes in the
Brain in Rats Poisoned with an Alkyl Mercury Compound with Special
References to Inhibition of Protein Synthesis in Brain Cortex
Slices," £. Neurochem., 13, pp 1223-1230 (1966).
64. Hines, J. F., and J. R. Lott, "Oxygen Uptake and Serotonin Levels
in Various Rat Brain Tissues," Texas J. Sci., 16, Vol. 4, pp 484
(1964); Speck, L. B., 1962, op. cit.; Palaic, D., and Z. Supek,
"Drug-Induced Changes of the Metabolism of 5-Hydroxytryptamine in
the Brain of X-Ray Treated Rats," J_. Neurochem. , 12, pp 329-333
(1965).
65. Davis, L. J. Wands, S. Weiss, D. Price, and E. Girling, "Central
Nervous System Intoxication from Mercurous Chloride Laxatives,"
Arch. Neur., 30, pp 428-431 (1974).
66. Cavanagh, J. B., and F. C. K. Chen, "The Effects of Methyl-Mercury-
Dicyandiamide on the Peripheral Nerves and Spinal Cord of Rats,"
A eta Neuropath. (Berlin), 19, Vol. 3, pp 208-215 (1971).
67. Hunter, D., and D. S. Russell, "Focal Cerebral and Cerebellar Atrophy
in a Human Subject due to Organic Mercury Compounds," J\ Neur.
Neurosurg. Psychiat., 17, pp 235-241 (1954). ~~
68. For a more detailed review of the literature on histological obser-
vations, see the thesis by Thrutchley.
69. Hicks, S. P., and K. A. Wright, "Variation of Pathological Responses
to Radiation with Time Intensity Factors," Am. J_. Clin. Path. , 24,
pp 77 (1954); Haymaker, W. G. L. Laqueur, W. J.~~H. Nauta, J. E.
Pickering, J. C. Sloper, and F. Vogel, "The Effects of Gamma Radia-
tion on the Central Nervous System and Pituitary Gland of Macaque
Monkeys," J. Neuropath. Exper. Neur., 17, pp 12 (1958); Vogel, F. S. ,
113
-------
C. G. Hoak, J. C. Sloper, and W. Haymaker, "The Induction of Acute
Morphological Changes in the Central Nervous System and Pituitary
Body of Macaque Monkeys by Cobalt 60 (Gamma) Radiation," J_. Neuro-
path. Exper. Neur., 17, pp 138 (1958), Kimeldorf, D. J., "and E. L.
Hunt, "Neurophysiological Effects of Ionizing Radiation." In:
Ionizing Radiation: Neural Function and Behavior, New York,
Academic Press, 1965.
70. Vogel, R. S., "Effects of High-Dose Gamma Radiation on the Brain and
on Individual Neurons." In: Response of the Nervous System to
Ionizing Radiation, Haley, T. J., and R. S. Snider (ed.). New York,
Academic Press, 1962.
71. Ibid.
72. Cassano, G. B., D. L. Viola, B. Ghetti, and L. Amaducci, "The
Distribution of Inhaled Mercury Vapors in Brain of Rats and Mice,"
£ Neuropath. Exper. Neur., 28, pp 308-320 (1969).
73. Hager, H., W. Hirschberger, and A. Breir, "Electron Microscope
Observations of the X-Irradiated Central Nervous System." In:
Response of the Nervous System to Ionizing Radiation, Haley, T. J.,
and R. S. Snider (ed.).New York, Academic Press, 1962.
74. Kimeldorf, D. J. , and E. L. Hunt, "Neurophysiological Effects of
Ionizing Radiation." In: Ionizing Radiation: Neural Function
and Behavior, New York, Academic Press, 1965.
75. Takeuchi, T. , G. Koya, M. Sasaki, Y. Hirata, K. Fukimoto, T. Miya-
zaki, and J. Ogi, "An Experimental Pathological Study on the Etiology
of Minamata's Disease, Especially the Role of Methylmercuric Sul-
fide," Kumamoto Med. £., 16, pp 713-735 (1962); Brown, W. J., and
N. Yoshida, "Organic Mercurial Encephalopathy. An Experimental
Electron Microscope Study," Advanc. Neur. Sci. (Tokoyo), 9, pp 34-42
(1965).
76. Miyakawa, T., and M. Deshimaru, "Electron Microscopical Study of
Experimentally Induced Poisoning due to Organic Mercury Compound:
Mechanism of Development of the Morbid Change," Acta Neuropath.
(Berlin), 14, pp 126-136 (1969).
77. Takeuchi, T., et al., 1962, op. cit.
78. Chang, L. W., and H. A. Hartmann, "Ultrastructural Studies of the
Nervous System after Mercury Intoxication. I: Pathological Changes
in Nerve Cell Bodies," Acta Neuropath. (Berlin), 20 pp 122-138 (1972).
114
-------
79. Kosmider, S., "The Pathogenic Mechanisms of Experimental Poisoning
by Mercury Vapor," Arch. Immune1. et Ther. Exp., 13, pp 204-239
(1965).
80. Chang, L. W., and H. A. Hartmann, "Blood-Brain Barrier Dysfunction
in Experimental Mercury Intoxication," Acta Neuropath. (Berlin),
21, pp 179-184 (1972).
81. Yoshino, Y., T. Mozai, and K. Nakao, "Distribution of Mercury in
the Brain and Its Subcellular Units in Experimental Organic
Mercury Poisonings," J_. Neurochem. , 13, pp 397-406 (1966).
82. Nair, V., and L. J. Roth, "Effect of X-Irradiation and Certain
Other Treatments on Blood-Brain Barrier Permeability," Rad. Res.,
23, pp 249-261 (1964).
83. Steinwall, Oskar, and Yngve Olsson, "Impairment of the Blood-Brain
Barrier in Mercury Poisoning," Acta Neurol. Scandinav., 45,
pp 351-361 (1969).
84. Nair, V., and L. J. Roth, 1964, op. cit.
85. Mahlin, D. T., and R. T. Lofberg, "Determination of Several Iso-
topes in Tissue by Wet Oxidations." In: The Current Status of
Liquid Scintillation Counting, Bransome, Edwin D. Jr. (ed.). New
York, Grune and Staton, 1970. Chapter: 22, pp 212-221.
86. Nair, V., and L. J. Roth, 1964, op. cit.
87. Ibid.
88. Steinwall, Oskar, and Yngve Olsson, 1969, op. cit.
89. McCready, R. G. L., and G. A. Din, "Active Sulfate Transport in
Saccharomyces cerevisiae," FEES Lett., 38, Vol. 3, pp 361-363 (1973).
90. Clarkson, T. W., "The Biological Properties and Distribution of
Mercury," Proc. Biochem. Soc., 130, pp 61-63 (1972).
91. Holman, B. L., "The Blood-Brain Barrier: Anatomy and Physiology,"
Progr. Nucl. Med., 1, pp 236-248 (1972).
92. Nair, V., and L. J. Roth, 1964, op. cit.; Nair, V., et_ al. , 1964,
op. cit.
115
-------
93. Rosenberg, Howard M., and Eleanor Matthews, "Short-Term Effects of
Ionizing Radiation on Volume-Regulation of Murine Lymphoma Cells
In Vitro. Evidence of the Involvement of Hydrogen Peroxide," Int.
~ Radiat. Biol., 23, Vol. 1, pp 91-94 (1973).
94. Nair, V., and L. J. Roth, 1964, op. cit.
95. Davis, Elizabeth A., and E. D. Wills, "Effects of Dietary Anti-
oxidants on Lipid Peroxide Formation in Animal Tissues after
Whole-Body Irradiation," Int. J_. Radiat. Biol. , 22, Vol. 1,
p 23 (1972).
96. Ahlers, I., Eva Ahlersova, Anna Sedlakova, and M. Praslieka,
"Tissue Lipids in Lethally X-Irradiated Rats," Folia Biol. (Praha),
19, p 124 (1972).
97. Ershoff, B. H., and C. W. Steers, "Antioxidants and Survival Time
of Mice Exposed to Multiple Sublethal Doses of X-Irradiation,"
Proc. Soc. Exp. Biol. Med., 104, p 274 (1960).
98. Rosenberg, Howard M., and Eleanor Matthews, 1973, op. cit.
99. Srebro, Z., "A Comparative and Experimental Study of the Gomori
Positive Glia," Folia Biol., 17, Vol. 3, p 177 (1969);
"The Ependyma, the Cysteine-Rich Complex-Containing Periventri-
cular Glia, and the Subfornical Organ in Normal and X-Irradiated
Rats and Mice," Folia Biol., 18, Vol. 4, p 327 (1970);
and T. Cichocki, "A System of Periventricular Glia in Brain
Characterized by Large Peroxisome-Like Cell Organelles," Acta
Histochem., 41, p 108 (1971).
100. Srebro, Z. , 1969, op. cit.; , 1970, op. cit.;
A. Slebodzinshi, and E. Szirmai, "Radiation Disease in Hypo- and
Hyperthyreotic Rats," Agressologie, II, p 343 (1970); ,
and T. Cichocki, 1971, op. cit.; , H. Lach, and E.
Szirmai, "A Novel Aspect of the Response of the Central Nervous
System to Irradiation: The Peroxidase-Positive Glia," Rad. Res. ,
50, p 65 (1972).
101. Wislocki, George B., and Elizabeth H. Leduc, "Vital Staining of
the Hematoencephalic Barrier by Silver Nitrate and Trypen Blue and
Cytological Comparisons of Neurohypophysis, Pineal Body, Area
Postrema, Intercolumnar Tubercle and Supraoptic Crest," J_. Comp.
Neurol., 96, p 371 (1952); van Breeman, V. L., and C. D.~Clemente,
"Silver Deposition in the Central Nervous System and the Hematoen-
cephalic Barrier Studied with the Electron Microscope," J_. Bio-
physic and Biochem. Cytol., I, No. 2, p 161 (1954); Hammond, A. L. ,
116
-------
"Mercury in the Environment: Natural and Human Factors," Sci.,
171, p 788 (1971); Rustam, H., and T. Hamdi, "Methyl Mercury"
Poisoning in Iraq. A Neurological Study," Brain, 97, p 499 (1974);
Yoshino, Y., T. Mozai, and K. Nakao, 1966, op. cit.
102. Bryce-Smith, D., 1973, op. cit.
103. Hughes, R., R. Belser, and C. W. Brett, "Behavioral Impairment
Produced by Exposure to Subclinical Amounts of Methylmercury
Chloride," Environ. Res., 10, pp 54-58 (1975).
104. Yahr, M. D., "Classification of Dementia-Producing Diseases,"
Med. Cl. N. Am., 56, pp 1407-1418 (1972).
105. Hellberg, Jan, and Mats Nystrom, "The Influence of Methylmercury
Exposure on Learning-Set Behavior of Squirrel Monkeys," Psy. Res.
Bull., 12, pp 1-9 (1972); Hellberg, J., "Neurotoxic Effects of
Methylmercury on Squirrel Monkeys. Behavior and Function Disor-
ders," Nord. Hyg. Tidskr., 53, pp 5-10 (1972); Post, E. M., M. G.
Yang, J. A. King, and V. L. Sanger, "Behavioral Changes of Young
Rats Force-Fed Methyl Mercury Chloride," P. .§_ E.-B_. M_. ? m35 pp
1116 (1973); Spyker, J. M., J. B. Sparber, and A. M. Goldberg,
"Subtle Consequences of Methylmercury Exposure: Behavioral
Deviations in Offspring of Treated Mothers," Sci., pp 621-623
(1972).
106. Ogden, T. E., "A Technique for ERG Recording in Infants and
Young Children," Vision Res., 14, p 305 (1974).
117
-------
TECHNICAL REPORT DATA
(Please read Instructions on rue reverse before completing)
1. REPORT NO.
EPA-600/1-77-006
3 RECIPIENT'S ACCESSIOI*NO.
4 TH LE AND SUBTITLE
INTERACTION BETWEEN METHYL MERCURY AND RADIATION
EFFECTS ON NERVOUS SYSTEMS
5. REPORT DATE
January 1977
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
Eugene W. Hupp, Dalton Day, James Ilardcastle, John
Mines, James Minnich
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Texas Woman's University
Denton, Texas 76204
10. PROGRAM ELEMENT NO.
1FA628
11. CONTRACT/GRANT NO.
R800282
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
15. SUPPLEMENTARY NOTES
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
16. ABSTRACT
The interaction between methyl mercury and ionizing radiation was investigated
in a series of experiments using rats, hamsters, and squirrel monkeys to study the
effects produced and possible mechanisms of action. Parameters evaluated included
several measurements of behavior, brain electrical activity, lethality, blood-brain
barrier permeability, neurotransmitter and mercury concentration in various brain
areas, and brain histology.
In some cases the effects of the co-insult were less than or at least no greater
than at least one of the two insults applied alone.
Possible mechanisms of action include opposite effects of the two insults on
the blood-brain barrier, with radiation increasing permeability and methyl mercury
decreasing it. Radiation may also elicit a proliferation of peroxisome-like
organelles which protect against the effects of methyl mercury.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Radiation effects
Stress (physiology)
Ionizing Radiation
Methyl mercury
06 R, F
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21 NO. OF PAGES
130
20 SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
118
-------
|