concentration in the bone marrow was lower than in the blood, A
high concentration of mercury was observed in the nasal and oral
mucosa, the pharynx, esophagus, and pancreas, After 20 minutes,
Ostlund®® found that all tissues except the fat deposits, brown fat,
and the central nervous system contained mercury in the form of a
non-volatile metabolite. The fat tissues contained only dimethyl
mercury,

At one hour, Ostlund®® found a decrease in the total body concentra-
tion although the kidney, liver, and adrenal cortex concentrations
had increased. The mercury concentration in the kidney, gall bladder,
liver, and fat were the same, The concentration in the fat had
decreased in relation to the blood, The nasal, pharyngeal, oral,

and esophageal mucosa concentrations remained unchanged while the
gastric and intestinal mucosas showed a marked accumulation of
mercury. An increase in mercury concentration was observed in the
central nervous system and hypophysis. All organs contained non-
volatile metabolites of dimethyl mercury,

After four hours, the mercury content of the fat remained higher than
that of the blood. The bronchi, liver, kidneys, and adrenal cortex
contained higher concentrations than the fat, A small concentration
was observed in the muscles, A few areas of the intestines showed a
high mercury content while the remainder of them showed a concentra-
tion comparable to the blood. All organs contained non-volatile
metabolites except the fat and central nervous system which still
contained mercury in the dimethyl form.

After 16 hours, the distribution of dimethyl mercury and non-volatile
metabolites was the same as after four hours. The highest concentra-
tions were observed in the kidney, liver, and nasal mucosa. The
muscles, intestines, spleen, and lungs showed concentrations of
mercury comparable to the blood. No mercury accumulation was seen
in the bronchi. The concentration in the upper digestive tract had
decreased by 16 hours, and a moderate mercury uptake was observed

in the lens and hypophysis. ‘The fat tissue and tissue of the central
nervous system both showed mercury concentrations less than that
found in the blood. No mercury was detected in the connective
tissue or compact bone,

By 24 hours, the nasal mucosa and inner cortex of the kidney were
only tissues containing large amounts of mercury. Differences in
the mercury concentration in most structures had disappeared. Less
than the average concentration was seen in the central nervous
system and fat. After four days, the central nervous system concen-
tration had increased, and uptake by the hair follicles was observed,
Uptake in the basal part of the hair occurred by 16 days. The
mercury concentration of the central nervous system was the same as
that of the blood by this time.

13


cytological effect of different mercurials, At high concentration,
fixation of cells results, At lower, but still high concentrations,
extensive cell death occurs, C-mitosis results from exposure to
still lower concentrations, Spindle abnormalities such as multi-
polar mitosis may result from concentrations too low to cause
c-mitosis., Methyl mercury and methoxyethyl mercury caused similar
effects,2°® Piskesjo'°® described the effects of different concen~
trations. At 200-2000 x 10°° M all mitosis showed lethal effects,
Some showed signs of heterochromaty, At 10-100 x 107° M, c-mitosis
dominated. The control level was reached at 1 x 10°° M. Ahmed and
Grant?°® observed c-mitosis in Vicia faba and Trandescantia root
tips after exposure to methyl mercury, Polyploid and multinucleate
cells were observed but chromosome fragments were absent.

The toxic threshold is characterized by partial inactivation of the
spindle.?°® The threshold is least for methyl mercury, ethyl mercury
is next, methoxyethyl, and butyl mercury is highest. Ramel+? reported
a threshold of 8 x 10°’ M at 72 hours for phenyl mercury. He stated
that the threshold for organic mercurials is .005 of that for
inorganic mercurials.

Ramel and Magnusson?°” have also reported effects on chromosome
segregation, They found that high but non-lethal concentrations

gave rise to abnormal wing positions in emerging Drosophila. Higher
concentrations cause reduced mobility or failure to emerge, As 25
mg/L methyl mercury, phenyl mercury, and methoxyethyl mercury,
exceptional female offspring resulted. Twice as high a dose yielded
some exceptional males, He found that methyl and phenyl mercury were
similar in levels required to cause exceptional offspring, but five times
as much methoxyethyl was required, Female exceptions were usually in
the form of xxy exceptions, Male xo exceptions were found, The
effect is apparently primary disjunction at the first mitosis.

MITOCHONDRIA
Lehninger*°® suggested that mercury may be an important agent in
mitochondrial leakage since it causes swelling in the mitochondrial
membrane, The swelling may be induced by changes in the secondary
or tertiary structure of proteins in the mitochondrial membrane
resulting from the binding of mercuric ions to membrane sulfhydryl
groups. The increase in membrane permeability caused by the swelling

results in leakage of pyridine nucleotides. Only oxidized NAD is
lost.

34


greatly to the development of gross symptoms. Among these are heart
muscle damage and changes in corneal appearance, In addition,
studies of blood uptake have given insight into the transport and
subsequent distribution of different mercurials,


chlorophylls, in algae is inhibited by mercurials and may be another
factor in reduced photosynthesis.??”

RATIONALE FOR THE ACTION OF MERCURIALS
Hughes*®® suggested that since the fundamental reaction of mercury
is with thiols, the differences in distribution and organ effects

is dependent upon this reaction. He discussed the differences in
affinity for sulfhydryl groups dependent on chemical species present,
large organic functional groups, and charges, These factors serve

to direct the mercurial to specific sulfhydryl groups. Peakall and
Lincer?®° have suggested a more complex sequence of events. They
found reduced numbers of sulfhydryl groups in brain and liver tissues
but not in muscle, They suggested that the reduction of sulfhydryl
groups was not solely dependent on binding by mercury but also on
inhibition of glutathione reductase which interconverts sulfhydryl
groups and disulfide linkages, Glutathion reductase activity is
negligible in muscle, The work of Pekkanen and Sandholm!*° supported
this hypothesis. In addition, in living organisms, distribution is
dependent on transport of mercurials and barriers to mercurial uptake
by specific organs. Since the blood is rich in thiols, it can trans-
port large amounts of mercurials. Five to ten percent of the thiol
content of the plasma is small diffusible compounds. These can
diffuse into cells, When in contact with membranes, the differing
lipid solubilities among mercurials may determine which dissolve in
lipid membranes. Some mercurials can then dissociate from the thiol
and pass through the membrane, Hughes!**® suggested this as a
rationale for the ready diffusion of methyl mercury. Larger
mercurials or bivalently charged ones would not pass so easily
through membranes. Thus Hughes suggested that actual pathological
effects on human organs is due to the real concentration of mercury
in the organs.

Several researchers studying the greater resistance to mercurialism
of sucrose-fed rats over chow-fed rats?1, ?%°,1°° illustrated Hughes‘
rationale of effect. Surtshin and Yagi®! found that sucrose- and
chow-fed rats three hours after injection had the same mercury
content in the kidney. However, the sucrose-fed had a higher
content in the soluble fraction and less in the nuclear and granular
fractions than the chow-fed. They suggested that the increased
resistance could be related to the decreased binding in the renal
nuclei and mitochondria. Thus distribution depended on binding to
thiols. A greater sulfhydryl group content was found in the soluble
fraction of sucrose-fed rats, By binding in the soluble fraction
rather than in the granular fractions, some protection was afforded
the granular fraction,

IN VIVO EFFECTS--RATIONALE FOR SPECIFIC ORGAN DAMAGE

Webb (Chapt. 7)°° commented on tissue homogenate effects as opposed
to enzyme effects resulting from the action of mercurials on enzymes

39


Methyl mercurials are typically distributed rather evenly among
organs in rats.®° Berlin and Ullberg®* traced the distribution
after single intravenous injections of methyl mercuric dicyandiamide
(0.5 mg/kg) in mice. They found that soon after injection (up to
one hour) the greatest concentration of mercury was in the blood.
They noticed early accumulation in the kidney (especially in the
cortex)’, the liver, pancreas, mucosa of the alimentary canal, the
gall bladder, .and the urinary bladder, After four hours, the con-
centration in the blood was still high. Mercury was first found

in the muscles after four hours. The liver accumulation had also
reached its maximum at this time, and mercury was found in the intes-
tinal contents. By the end of the first day after injection, mercury
was rather uniformly distributed. However, none was found in the
bony. skeleton and little in the central nervous system. Some accumu-
lation was seen in the lacrimal and salivary glands, The kidney and
liver concentrations were still well above the average body concen-
tration. On the fourth, eighth, and sixteenth days, Berlin and
Ullberg** reported a decline of mercury concentration by a factor of
two to four in all organs except the brain and spinal cord where

the mercury content more than doubled. After sixteen days, the
brain concentration was exceeded only by that of the colonic mucosa
and renal cortex. Swensson et ales also found that methyl mercury
was fairly uniformly distributed. They found mercury in the kidney
after 32 days. Swensson and Ulfvarson”® stated that the distribution
in poultry is the same as in rats.

Some investigators have considered transfer to the fetus and fetal
distribution. Berlin and Ullberg®* found earliest traces in the
fetus of mice after four hours. By the end of the first day after
injection of the mother, the fetal concentration was equal to that
of the mother. The distribution within the fetus was the same as in
the mother except that more mercury was seen in the fetal skin.
Suzuki et at?” reported even distribution between the head and body
of the fetus, They also found a similar concentration in the
placenta, However, less'mercury was found in the amniotic membrane,
They found that the quantity of mercury transferred to a mouse
litter was proportional to the number of fetuses in the litter.

The main routes of excretion of intravenously injected methyl
mercury are the feces and urine. Ostlund?® showed that negligible
amounts of mercury were exhaled by mice while 40% was excreted in the
feces and 14% in the urine for the first seven days after injection.
Swensson and Ulfvarson®® found that only 20% of the injected dose
was excreted by all routes in the first ten days after injection in
poultry. Swensson and Ulfvarson®® approximated the excretion rate
in poultry to be 3.7 x 10°° day? (ug Hg/kg wt).

Swensson and Ulfvarson®® and Berglund and Berlin®® stated that the
excretion rate of methyl mercury fitted an exponential relationship
if extremely toxic values were not included. Ostlund®® found a

7


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45


ethyl mercury was detected in the tissues as such for slightly longer
periods of time. In addition, they showed that rats injected intra-
muscularly contain more ethyl mercury in the kidney and blood and
less in the liver. They hypothesized that the liver was the main
detoxification reservoir for poultry and the kidney for mice,

Rucker and Amend®® studied the retention of ethyl mercury in fish
exposed to mercury in the water. The mercury content of the gill of
rainbow trout rose rapidly the first two hours after a single exposure
and declined the next 24. After two weeks it slowly reached a back-
ground level. The blood level rose as the gill concentration declined
and reached a maximum at 32 hours. The blood level began to decline
by the third day and attained a background level after six to eight
weeks. The mercury level of the liver rose gradually and reached a
peak by the third day. It dropped slowly and did not approach back-
ground level until the 20th week after exposure, The mercury level
in the kidneys rose during the first 24 hours, then declined slightly
during the next seven days, It reached its maximum at three weeks
and declined slowly thereafter.

Rucker and Amend®® also investigated the distribution in trout after
multiple exposures to ethyl mercury chloride. The mercury level in
the gills was always low before daily exposure and high afterwards.
The blood level showed a steady increase through the 10th day of
exposure, The kidney and liver steadily accumulated mercury. Their
concentrations remained lower than that of the blood, but they still
accumulated mercury through the last treatment. During twelve
weekly exposures, they found that the blood level increased through
the 10th week and then declined. It did not return to a background
level until 17 weeks after treatment stopped. The liver mercury
content rose as long as exposure continued. After discontinuation

of the treatment, the level declined; however, mercury was still
present the 33rd week after exposures had ended, The kidneys showed
a steady increase during the first three weeks, then a sharp increase,
Mercury content of the kidneys continued to increase until one week
after exposure had ended. The content then declined but was still
high the 44th week after exposure. The mercury content of the muscle
was always lowest. It reached a maximum during the 12th week and
declined to background by the 17th week.

Mercuric Salts

Friberg et al.o* described the distribution of subcutaneously
injected mercuric chloride in rabbits, Highest concentration was in
the kidney and the second highest was in the liver. Only small
amounts of mercury were found in the brain, Swensson et al.®° found
that mercury was rapidly fixed to the kidneys of rats reaching a
maximum concentration 24 hours after injection. Swensson and
Ulfvarson®® showed that 40% of the intravenously injected mercuric

7


SECTION VI

BIOCHEMICAL EFFECTS

The action of mercurials at different levels of organization (i.e.,
the organism, the tissue, the cell) could be explained through an
understanding of the effects of mercurials on cellular enzymes and
enzyme systems if adequate knowledge were available. The purpose of
this section is to clarify enzymatic effects both in terms of
possible action of mercurials on enzymes in general and their effects
on specific groups of enzymes, Cases in which organ damage can be
related to biochemical changes resulting from mercurial exposure will
be discussed, More important, however, is the understanding of the
problems in generalizing from molecular changes to tissue damage
which can be gained from review of biochemical effects.

GENERAL EFFECTS

Webb (Chapt. 4)?°° has discussed reactions between enzymes and
mercurials, Chapters 4 and 7 of his book provide the most complete
overview of mercurial inhibition available. First, he emphasized
the variety of groups of chemical compounds which can react with
mercurials, Low molecular weight thiols (i.e., co-factors and amino
acids), non-enzyme proteins, and enzymes can all be affected by
mercurials. Thus, energy metabolism, cell structural proteins, and
a variety of cellular processes could be inhibited by mercurials.
However, the reactivity of mercurials with sulfhydryl groups and the
cellular consequences of these reactions depend on a number of
factors.

Differential activity of sulfhydryl groups and changes in reactivity
following denaturization are among the factors important to the
reaction between mercurials and sulfhydryl groups. Sulfhydryl groups
involved in the tertiary structure of the protein may not be readily
available to react with mercurials, Disulfide linkages are less
reactive with mercurials than sulfhydryl groups. Different reac-
tivities of free sulfhydryl groups may be influenced by several
factors, Steric interference or electrostatic interaction may impede
a reaction. The ionization state may influence reactivity with
particular sulfhydryl groups. Once the mercurial reacts with a
sulfhydryl group other factors become important in determining the
effect of the mercurial on the enzyme's functional properties, The
mercurial can combine with a sulfhydryl group at an active site on
the enzyme. If the mercurial combined with a sulfhydryl group
vicinal to the active site, the other group to which the mercurial

is bound could sterically or electrostaticaliy interfere with the
@pproach of the enzyme substrate to the active site. The reacted
sulfhydryl groups could be involved with maintaining the enzyme

35


CONTENTS

ABSTRACT . 1. 1 eee ee ee ee

ACKNOWLEDGMENTS .......

Sections

‘I CONCLUSIONS .......

II INTRODUCTION .........

III ORGANISM RESPONSE TO MERCURIALS
SYMPTOMS . 2... eee eee

UPTAKE, RETENTION, EXCRETION,

Methyl Mercury .......
Dimethyl Mercury ......
Phenyl Mercury ......
Methoxyethyl Mercury ...
Ethyl Mercury .......

Mercuric Salts .......

Metallic Mercury ...

Comparison of Mercurials ..

IV ORGAN AND TISSUE EFFECTS ...
CENTRAL NERVOUS SYSTEM ...
KIDNEY «6... ee ee eee
LIVER... ee ee ee ee
HEART 2. 1 1 6 ee ee ee ee

BLOOD... ew ew ee eee

iii

BIOTRANSFORMATI ON

12
14
16
16
17
18
19
23
23
25
26
27

27


5.92-11 for those exposed to protein-bound methyl mercury, When the
ratio of mercury concentration in blood components is calculated as
red cell to plasma mercury content, a ratio of 0.625 results from
administration of metallic mercury and 27-53 is found in subjects
exposed to alkyl mercurials., Berlin et al.°? reported higher red
cell to plasma mercury concentrations in vapor exposed mammals than
in mammals injected intravenously with a mercuric salt, Sixty-seven
to eighty-four percent of the mercury was bound in red cells in the
former case while the latter only showed 25-31%. No differences in
blood binding of mercuric salts was observed for subcutaneous vs.
intravenous injection.®”

Kudsk®® observed a high rate of mercury vapor uptake by the plasma
during the first hour after exposure, Fifty percent of the total
mercury in the blood is located in the plasma during the first hour.
During subsequent hours the plasma uptake is only 10-15% of that of
the whole blood. Therefore, 85-90% of the mercury taken up after the
first hour is probably oxidized in the erythrocytes.°* Magos’®
described two phases of uptake of mercury vapor-a fast phase followed
by a slow, steady increase. The percentage absorbed was linearly
related to hemoglobin concentration.

The difference in mercury uptake in erythrocytes for different
mercurials appears to be due to the stability of alkyl mercurials
bound to hemoglobin, Takeda et al.®? found that ethyl mercury was
bound to hemoglobin as ethyl mercuric cysteine by mercaptide linkages.
Within 30 minutes after exposure, most of the mercury was bound
inside the erthyrocytes, Once combined, release was difficult
because of the high stability of the bond, Suzuki et al,°° suggested
a similar explanation for the elevation of alkyl mercury in red

cells of the umbilical cord and fetus over that of the maternal
blood,

Clarkson? and Kudsk®® discussed factors that could influence the
uptake of mercury vapor. Clarkson?! found that an increase in
oxygen accelerated mercury uptake and stated that the vapor was

being oxidized to the ionic form. After studying the influence of a
number of compounds on mercury vapor uptake, Kudsk®® found a possible
relationship between uptake, oxidation, and the coupled glutathione
and carbohydrate metabolism.

In addition to investigations of uptake and distribution within the
blood, some researchers have been interested in the effects of
mercurials on blood components. Lindahl and Hell®* found hemolysis
of fish erythrocytes. The half-time of hemolysis for fish incubated
in 0.5 x 10°* M phenyl mercury at 20 C was 55 minutes. No hemolysis
was observed at 0.3 x 10°° M. Benesch and Benesch?* investigated
half-times of hemolysis for Salyrgan, a diuretic, and phenyl mercury.
A half-time of hemolysis of 103 minutes was found for the former and

28


Ostlund®** also described fetal uptake in pregnant mice, As early as
five minutes after injection or inhalation, he observed moderate
mercury uptake in the ovaries and oviducts= though no fetal uptake
was seen, The placenta showed a mercury concentration comparable it»
that of the blood. By one hour, the fetuses showed marked mercury
uptake in their nasal, oral and esophageal mucosa, and bronchi.
Moderate uptake was observed in the liver. Only the placenta con-
tained dimethyl mercury as the sole mercury form. Fetal tissues
contained non-volatile metabolites of dimethyl mercury, The mercury
concentration of the fetuses was approximately the same as the
maternal blood by 16 hours. No accumulation was observed in the
bronchi, nasal, oral or pharyngeal mucosae though the lens showed
marked uptake.

Ostlund** also measured excretion of injected and inhaled dimethyl
mercury. He found two phases in the excretion rate curve, a rapid
phase during the first six hours and a slow phase. The rapid phase
corresponded to the time required for exhalation of dimethyl mercury.
Ten percent of the body burden of mercury remained after the rapid
phase of excretion was completed. The half-time of the slowly
excreted metabolite was 9,5 days. The half-life of a similar dose of
methyl mercury is 8,9 days. Ostlund suggested that this slowly
excreted metabolite is methyl mercury. A rapid excretion phase of
six hours was also observed with inhalation as the route of admin-
istration. The half-time of elimination of the slow phase was

seven days, Ostlund concluded that the excretion rate is the same
for the two routes of entry.

Ostlund®® described the mercury present in mice after administration
of dimethyl mercury as volatile or non-volatile metabolites. He
showed that the volatile exhaled portion was dimethyl mercury. The
even distribution pattern of the non-volatile portion gave support
to the hypothesis that the non-volatile portion is methyl mercury.
Thin-layer chromatography confirmed this hypothesis. The only
organic metabolite of dimethyl mercury is methyl mercury.

Phenyl Mercury

Several investigations of the distribution of phenyl mercurials after
single intravenous injections have been conducted, Swensson et a1.®>
determined the mercury content of the blood, brain, liver, and
kidneys at specified times after injection into rats, After three
hours, the mercury content of the blood was the highest of the four
analyzed but steadily declined during the following hours, The kidney
contained the second highest concentration though it was much lower
than the blood, The brain and liver contained the same amount of
phenyl mercury (less than ten percent of the blood concentration).

By the end of 24 hours, the kidney content had risen sharply,
increasing to 125 times the 3-hour value. The liver content was 10
times its 3-hour value.

14


found that more mercury was accumulated in the methyl form. The
blood concentration was 100 times greater than for inorganic mercury.°?
Ulfvarson®® found greater retention of methyl mercury than methoxy-
ethyl mercury and mercuric nitrate in the brain, Swensson et al, 2, 8S
also found a higher ontent of organic mercurials in the blood. They
found greater blood retention with alkyl than phenyl mercurials, The
liver showed greater accumulation with alkyl mercurials.°® Renal
values were twice as high as for the inorganic form. Friberg®? found
that rats given inorganic mercury excreted twice as much in the feces
and more than 20 times as much in the urine as those given methyl
mercury, Though the percentage loss from the parts of the brain were
the same for the two mercury compounds, mercuric chloride content
diminished more rapidly than methyl mercury. Rates of loss from the
liver and spleen were greater for inorganic mercury although the
difference was not significant.

Swensson and Ulfvarson®® compared injections of phenyl mercury,
methyl mercury, mercuric chloride, and methoxyethyl mercury in
poultry. The brain content in all cases was 5 to 10 times higher
than the blood content. The brain concentration for methyl mercury
was highest of all, They found that the relative distribution among
the different organs remained constant except for the kidney, The
kidney concentration of phenyl mercury decreased with time but, in
relation to other organs, its concentration increased, The rela-
tionship was found to be variable with mercuric nitrate. Swensson
and Ulfvarson®* stated that the general distribution pattern
revealed a uniform distribution for methyl mercury. All other
mercurials showed low blood and muscle concentration with high
concentration in the kidney and liver, Testing excretion rates,
they found that methoxyethyl mercury has the fastest (10% remaining
after 10 days), phenyl mercury the next (20% remaining), mercuric
nitrate (40% remaining) next, and methyl mercury the slowest (80%
remaining). As previously mentioned, Swensson and Ulfvarson®®
characterized the excretion vs. time curve as an exponential function
for methyl mercury and as a hyperbolic function for all others.

Friberg et a1.®° also noted a similarity between phenyl mercury and
an inorganic mercuric salt. For both compounds, they found the
greatest concentration in the kidneys with the liver containing the
second highest concentration, Only small amounts were found in the
brain, The concentrations for both compounds were of the same order
of magnitude, Mercuric nitrate concentration in the kidney provided
an exception to this distribution pattern, On day 6 its content was
higher than day 1. By day 40, it was 60% of the day one concentra-
tion, Readings successively fell for phenyl mercury and were about
20% of the day 1 concentration by day 40, Friberg et al.®° found
that distribution patterns within organs were similar for the two
compounds except for the liver, Phenyl mercury showed even distri-
bution within the liver while mercuric nitrate showed concentration

20


remained constant for 96 hours. The total mercury in the kidney
increased for 24 hours and then remained constant for 96 hours.

After three hours the phenyl mercury content of the liver declined,
and after 12 hours the phenyl mercury content of the kidney decreased.
Only about half of the kidney mercury was in the form of phenyl
mercury.

Several researchers have studied dosage effects of phenyl mercury.
Miller et al. S° found that liver and kidney concentrations were
proportional to dose in birds, Ulfvarson®” found some changes in
distribution in rats when the dose was increased by a factor of ten.
He found that excretion rate was only slightly affected. The blood
concentration was much higher than expected while the liver contained
less than anticipated. By the 24th day, all mice showed a lower
mercury concentration than expected. The increased rate of excretion
reflects a saturation phenomenon at the higher dosage.®*

Methoxyethy] Mercury

Ulfvarson®* described the distribution of subcutaneously injected
methoxyethyl mercury in rats, Three days after injection he found
the highest concentrations in the kidneys and liver. The blood and
muscles showed similar concentrations and were several times lower
in mercury concentration. The brain only showed a trace of mercury.
Ten to 20 days after intravenous injection in poultry, Swensson and
Ulfvarson®® found that kidney concentration was higher than liver
concentration but this difference appeared to decrease with time.
The brain concentration was the third highest and muscle, fourth.
The mercury concentration of the blood was low.

Swensson and Ulfvarson®® found that 90% of the methoxyethyl mercury
was excreted by the 10th day after intravenous injection in poultry.
They described excretion vs, time as a hyperbolic function. They
found an excretion rate of 7.1 x 107° day~ 1 (ug mercury/kg body
weight)7?

Ethyl Mercury

Miller et al.°* studied ethyl mercury distribution after oral doses
and intramuscular injections in poultry. They investigated the
distribution of mercury and the percent remaining as ethyl mercury,
The liver consistently retained the highest concentration of mercury,
the kidney second, and the blood lowest. Ethyl mercury was present
in the liver for 21 days though the percentage of total mercury in
the form of ethyl mercury declined after 10 days, Ethyl mercury was
present in the kidney for 14 days though it declined after two days.
It was present in the blood seven days, The percent as ethyl mercury
declined after two days. Miller et al.°* found similar distribution
and metabolism of ethyl mercury after oral administration. However,

16


28 for the latter, Hemolytic action of alkyl mercurials varies in
the following way from most active to least: n-butyl, n-propyl,
ethyl, methyl.°* It was suggested that injury to the erythrocyte
cell membrane could result from interaction between the mercurials
and the Lipophilic components of the membrane. Benesch and Benesch??
found that larger amounts of mercurials (Salyrgan and phenyl mercury)
were required to cause hemolysis than to form a monolayer on the
membrane surface, They suggested that hemolysis resulted from
structural changes caused by reaction between membrane sulfhydryl
groups and mercurials,

Lindahl and Hell®* reported tissue injuries in gills of fish

exposed 10-40 minutes in 0.3 x 10°° M phenyl mercury. After 10
minutes exposure the outer epithelial layer separated from the gill
tissue because of dissolution of the basement layer, By 40 minutes
a decrease in circulation in the secondary gills was observed. After
49 minutes, only the phalanges of the pillar of secondary filaments
remained, Mucous was found on the gill filaments. They found that
the gill filaments were highly penetrable to phenyl mercury. Rucker
and Amend®® observed hyperplasia of the gills of trout and salmon
exposed to ethyl mercury.

Lindahl and Hell®* measured respiration in secondary gill filaments
to assess effects of mercurials on gill function. They found that
oxygen consumption of the gills was reduced by 30% whether the
exposure period was 40 or 60 minutes. The secondary gill filaments
composed about 35% of the total gill filaments. They suggested that
the functional and structural changes of the gill were the ultimate
cause of fish death since it prevented the fish from obtaining
adequate oxygen.

EYE LENS

Kipling'® suggested that local absorption of mercury could cause
mercurialentis. The condition has two phases, The first involves a
greyish granular discoloration of the anterior capsule of the lens.
In the second, the anterior lens blooms and, when observed through a
slit lamp, appears brownish-grey, deep rose-brown, pinkish copper,
or golden, These characteristics occur at low levels where other
evidence of mercury exposure does not. In addition, a band-shaped
opacity of the cornea may occur. Individuals showing this type of
lesion usually already have mercurialentis., Stal lines have also
been attributed to mercury.

SUMMARY
The neurological and renal disturbances observed in living organisms
have been shown to result from extensive brain and renal damage,

Other tissue effects have been observed which do not contribute as

29


loss of C-14 was not the result of an exchange reaction, Norseth and
Clarkson®* observed biotransformation of methyl mercury in rats.
Small amounts of inorganic mercury were found in blood plasma the
first to the tenth days after injection (0.004-0.009 g/ml). Equal
amounts were found in the red cells with inorganic mercury comprised
0.2% of the mercury, The brain contained mainly organo-mercurials at
all times tested, One to four percent inorganic mercury was found
in the brain. Inorganic mercury increased slowly in the kidney
reaching a maximum at 28-30 days. On the 16th day after injection,
inorganic mercury accounted for one-half of the total mercury in the
kidney and on the 30th day, 90%. In the liver, 33% of the mercury
was in an inorganic form by the 25th day and 50% by the 50th day.
Fifty percent of the mercury in the feces was in the inorganic form
by the 10th day, With increasing time after injection, the pattern
of distribution of the released mercury approached that of injected
salts (accumulation in the kidney). Biotransformation is dose
dependent with the percent of the original dose which is transformed
decreasing with increasing dose.°®

Norseth and Clarkson®* stated that organic mercurials manifested
different properties than inorganic because of the stability of the
C-Hg bond. The slow rate of excretion in rats possibly is due to
the trapping of methyl mercury in the blood cells and the slow rate
of metabolism of this compound. Therefore, biotransformation of
methyl mercury to inorganic mercury is important in the excretion of
the former compound,®*, 5© 5°

A number of factors could influence the uptake, distribution, and
excretion of methyl mercury. Studies have included the effects of
ionic vs. protein-bound methyl mercury and dosage effects on uptake,
distribution, and excretion. Ostlund’® has studied a number of
other factors such as previous exposure, sex, and activity.

Miettinen et al.*® investigated the effects of different forms of
methyl mercury (ionic or protein-bound) on excretion of methyl
mercury. They found an average half-time of 76 days for protein-
bound methyl mercury. This value is close to the range for methyl
mercuric nitrate (65-75 days). Miettinen et al.*® and Jarvenpaa et
al.*© showed insignificant differences in half-time of excretion for
protein-bound and ionic methyl mercury in several aquatic vertebrates,

Several investigators have contributed to knowledge of dosage effects,
Ulfvarson®” stated that the distribution and excretion of methyl
mercury is independent of dose. Piper et al.#? found that the
percentage distribution among organs was constant for different
dosage levels. In contrast, Ostlund®® found that distribution was
not proportional to dose, The percentage of dose/gram of tissue
increased in blood corpuscles with increasing dose, although plasma
concentration was the same at all doses. The brain concentration was

11


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SECTION III

ORGANISM RESPONSE TO MERCURIALS

SYMPTOMS

Mercury poisoning manifests itself in different ways for different
routes of administration, kinds of mercury compounds, and duration
of exposure. Some of the information is represented by single
examples of unusual routes of entry. However, much information has
been supplied by studies of industrially exposed populations and
inhabitants of Minimata, Japan, These situations have supplied
adequate numbers to determine predominant symptoms of poisoning by
different mercurials. In addition, animal studies have supplied
preliminary evidence of concentrations of mercurials necessary to
cause neurological symptoms.

Mercurials can enter the body through inhalation, subcutaneous or
intravenous injection, cutaneous application, or ingestion. Each
route of entry can exhibit unique symptoms unobtainable from any

other mode of administration. For example, subcutaneous injection

of metallic mercury causes deep lesions in the area of injection.}* 14 1°
Application of mercuric chloride or metallic mercury to the skin may
cause dermatitis, discoloration of the fingernails, systemic poisoning,
purpura, petechiae!® and skin lesions.® Corrosion of the conjunctiva
and cornea may result from eye contact. Ingestion of mercuric chloride
causes a burning metallic taste, thirst, and soreness of the pharynx.?®
One-half gram of mercuric chloride can be a lethal dose causing

shock, ulceration of the gastro-intestinal tract, and destruction of
the tubular epithelium of the kidney.!” Mercury fulminate applied

to the skin may cause dermatitis and, to the eye, conjunctivitis

and edema of the lid.1® Mercury oxycyanide when locally applied

gives rise to corrosion of the membranes and when ingested, abdominal
pain, vomiting, and diarrhea. Phenylmercuric acetate applied to the
skin causes effects similar to burning.'® Vapour exposure may cause
mercurialentis,»® Organic mercurials affect the central nervous
system while inorganic forms primarily affect kidney function, Methyl
mercury, mercurial diuretics, and phenylmercuric acetate will be
considered under organic mercurials, Inorganic mercurials reviewed
will include mercuric chloride, metallic mercury, mercury fulminate,
and mercury oxycyanide,

The most frequent route of administration of methyl mercury is
ingestion of protein bound methyl mercury in food items. The most
extensive discussion of the clinical effects of methyl mercury resulted
from data on victims of "Minimata disease", The earliest symptoms
include fatigue, headache, and reduction in powers of concentration
and memory.?® A latent period of two to seven months may be required

3


a similar observation for phenyl mercury and mercuric chloride in
rabbits,

Histological observations after death due to methyl mercurialism
revealed ishemic atrophy resulting from fatty and hyaline degenera-
tion of the cortical arterioles.®? In addition, fatty degeneration
affected renal tubules, especially the loops of Henle in the renal
medulla,°? Klein et al.®* found degeneration of the distal convoluted
tubule. Tokuomi® also observed fatty degeneration of parenchymatous
cells, Johnson and Koumides'* and Klein et al.?? observed epithelial
necrosis of the renal proximal convoluted tubules after metallic
mercury injection methyl mercury ingestion, respectively, The
tubules contained eosinophilic debris, Spherical masses in the lumen
of tubules containing ribosomes, smooth endoplasmic reticulum,
cytoplasm, and sometimes a microbody have been observed in mice
exposed to methyl mercury ,°* Kanzantis et al.?” reported similar
results, Johnson and Koumides'* observed exudate around many
glomeruli and the distal tubules contained altered red cells.
Glomeruli of mice studied by Fowler®? were normal, Kanzantis et al.?’
and Klein et al. 22 found schlerotic changes in some glomerili. Their
histochemical analysis revealed an abundance of lipids in the proximal
convoluted tubule epithelium, Areas of infiltration with round cells
were found in the interstitium, Kanzantis et al.?” reported an
absence or suppression of alkaline phosphatase and abnormal succinate
dehydrogenase distribution, They observed no enzyme activity in the
proximal convoluted tubule though some was observed in the distal
convoluted tubules and ascending loops of Henle.

Conn et al, 85 attempted to explain the cause of renal failure, They
stated that changes in renal circulation were the basic cause of
acute renal insufficiency. They questioned whether persistent
ishemia was responsible for the development of renal insufficiency
or whether intrarenal blood shunts, which may operate during

periods of renal inadequacy, play a role in the development or
continuation of renal insufficiency. In tests with dogs, they found
that the renal circulatory bed ranged from normal in dogs with normal
amounts of urine to 40-50% of normal in anuric dogs, Intrarenal
distribution of blood flow was normal, Oxygen consumption and para-
amino hippurate were reduced in all animals showing reduced urine
flow, Tubular degeneration and necrosis were observed, Conn et al. 86
stated that reduction in para-amino hippurate and oxygen consumption
were closely correlated with these morphological changes. However,
oliguria or anuria can be better correlated with renal circulatory
impairment. They found no evidence of intrarenal shunts. They
stated that the change in urine flow is dependent on renal ishemia
and nephron damage.

LIVER

Friberg et al.®° reported that mercuric salts were accumulated
unevenly in the liver with greater localization around connective

26


SECTION IV

ORGAN AND TISSUE EFFECTS

Studies of the effects of mercurials on tissues resulted primarily
from the Minimata incident. Subsequent investigations on animals
other than man have given rise to knowledge of the intra-organ
distribution and physiological changes at the tissue level such as
changes in tissue respiration, Since marked neurological signs

have been observed, the central nervous system has been the focus

of many investigations, The blood has also been extensively studied
in order to elucidate differences in transport of different mercurials,
The liver, kidneys, gill, heart muscle, and eye lens have also been
included in tissue studies of the effects of mercurials.

CENTRAL NERVOUS SYSTEM

Berlin and Ullberg®* found that the distribution of methyl mercury
following single intravenous injections in mice was fairly uniform
within the brain, This differed from the heterogeneous distribution
resulting from the injection of mercuric salts. They found that the
hippocampus and gray matter of the cerebellum take up the most methyl
mercury. As in inorganic mercury poisoning, the gray substance has
a higher concentration than the white matter. Aberg et al.®? found
a localization in the cerebellum in man exposed to low methyl mercury
levels. Yoshino et al.®+ found that high mercury in the cerebellum
was inconsistent, He found highest mercury content in the calcarine
areas, Piper et al.*? found the highest concentration of methyl
mercury in the cerebrum and the lowest in the spinal cord of pigs.
After a single oral administration, Berlin and Ullberg** found that
distribution in the cerebellum, brainstem, and spinal cord of mice
was fairly constant from days 7-35. The concentration in the
cerebrum varied more than other tissues with an apparent preferential
retention up to the 13th day, After subcutaneous and intravenous
injections of mercury sublimate in rabbits, Miyama et al.®” observed
the highest mercury concentration in the brainstem for both routes,
The cerebellum was generally second in concentration, Either the
cerebral cortex or hippocampus was lowest in mercury concentration.

Berlin et al.°? studying several mammals (rats, rabbits, monkeys)
compared distribution in the brain of vapor-exposed vs. injected
mercury salts. In mammals exposed to mercury vapor, they found more
mercury in the gray matter than the white matter. The nucleus
dentatus in the cerebellum, nucleus olivarius inferior in the brain
stem, the choroid plexus, and the nucleus subthalamicus all showed
marked mercury uptake, The collicus superior also appeared to show
accumulation, In the cerebral cortex, different uptake was observed

23


ACKNOWLEDGMENTS

The efforts of the following individuals who reviewed the manu-
script are acknowledged with sincere thanks: Dr. Robert Beyers

and Mr. Henry J, Kania, Savannah River Ecology Laboratory, Aiken,
South Carolina; Dr. James O'Hara, Sargent Lundy, Chicago, Illinois;
Mrs, Patricia Murphy; Dr. Walter M. Sanders III, U. S. Environmental
Protection Agency, Southeast Environmental Research Laboratory,
Athens, Georgia; Dr, James Schindler and Dr. Robert Taylor, Univer-
sity of Georgia, Athens, Georgia.

The author was supported by Research Grant R-800510 from the
U. S. Environmental Protection Agency to the University of Georgia
during the preparation of this review.


EYE LENS . 6 6 ee ee ee ee ee ee
V CELLULAR EFFECTS... . 2. 2 we

INTRACELLULAR DISTRIBUTION... ...

CELL MORPHOLOGY ... 1. 1 ee eee
CELL MEMBRANE . . 1. ee ee eee
CHROMOSOMES . 1... 1 ee ee ee ee
MITOCHONDRIA . 2 2 ee ee ee ee ee
VI BIOCHEMICAL EFFECTS .. 2... 2 ee eee
GENERAL EFFECTS .. 1. ee eee eee
CHEMICAL PROPERTIES--ORGANIC MERCURIALS VS.
INORGANIC MERCURIALS ... 2... wu
INHIBITION BY MERCURIALS ........

RATIONALE FOR THE ACTION OF MERCURIALS .

IN VIVO EFFECTS--RATIONALE FOR SPECIFIC
ORGAN DAMAGE . 6 6 ee ee ee eee

VIL REFERENCES... 1 ee ee ee ee ee

iv

31
31
32
33

33

35

35

36

36

39

39

43


a constant percentage of dose, Muscular tissue showed a lower
percentage uptake with increasing dose as did the liver. The
percentage concentration was constant over all dosage levels.
Ulfvarson®” stated that the half-time of excretion increases with
decreasing dose. Ostlund®® experimentally found the opposite. He
showed that in mice the half-time of excretion increased with
increasing dose and suggested that this change could have been
caused by increasing inhibition of sulfhydryl enzymes at higher
levels.

Ostlund®® compared the effect of sex and swimming activity on
excretion rate of methyl mercury and found no difference for mice.
Miettinen et al.46 found no effect of sex on excretion of methyl
mercury for man, In addition, Ostlund®® found that trace doses of
radioactive mercury administered after large doses of non-radio-
active methyl mercury distributed in the same way as doses of trace
amounts, He suggested that as the larger dose was excreted, the
first sites to which it was bound were made available to the trace
dose administered later. Increasing time of exposure results in
increased concentration of methyl mercury; therefore, increase in
mercury concentration with increasing age or size has been observed
in many organisms 5% 5% 60 61, 62

Dimethyl Mercury

Ostlund®® studied the distribution of dimethyl mercury in mice, He
found no difference due to route of administration (inhalation or
intravenous injection), In both cases, he observed a rapid reduc-
tion in blood concentration with simultaneous deposition of dimethyl
mercury in fat (within five minutes after administration). Five
minutes after administration, the bronchi and nasal mucosa were high
in mercury concentration. Moderate concentrations of dimethyl
mercury were observed in the liver and kidney, though the renal
cortex had a high mercury concentration, A concentration similar to
that in the blood was seen in the spleen, lungs (except bronchi),
and intestines, Moderate uptake was observed in the salivary glands
after five minutes, The mercury concentration varied for different
parts of the central nervous system after five minutes, Mercury
content of the cerebral cortex and cerebellum were lower than that
in the blood, After five minutes, most of the mercury present was
in the form of dimethyl mercury, The brain stem and medulla oblongata
showed concentrations similar to that of the blood. The concentra-
tions of the hypophysis and the spinal cord were similar to that of
the liver.

At 20 minutes, the mercury concentration of the blood was low and
the concentration in the fat, high. Concentration of mercury was
increasing in the liver, kidney, and the red pulp of the spleen.

The gall bladder showed the same concentration as the liver. The

12


SECTION II

INTRODUCTION

The presence of mercury has been known since prehistoric time. It
was first used c. 1000 A.D. as a medication for skin diseases.
Ulrich Ellenborg made the earliest survey of mercury's toxic pro-
perties in 1473.1 The use of mercurials has extended from pharma-
ceuticals to agriculture and industry resulting in the broad
dissemination of mercury into the environment.

Agricultural and industrial applications have given rise to the
recent problem of mercury pollution. The first serious incident
resulting from industrial mercury discharges occurred in Minimata,
Japan, in the form of an undefined neurological disorder. The first
victim was reported in 1933 but the problem eventually affected many
individuals of the Minimata Bay and River areas reaching epidemic
proportions in 1953.° A similar but smaller outbreak occurred in
1965 in Niigata, Japan.® The mercury problem was next discovered in
Sweden, although it never caused the health menace observed in Japan.
The first evidence for the detrimental effect of mercurials on the
Swedish environment appeared in 1955 when a decrease in the numbers
of predaceous and seed-eating birds was observed.* Borg et al.*
attributed this decline to the use of seed dressings. Westermark
was the first to note abnormally high levels of mercury in Swedish
fish.© Interest in mercury in America began in March, 1970, when
the Canadian Fisheries Department closed a commercial fishery on
Lake St. Clair because of high mercury content in the fish. Several
waterways in the United States have since been closed to fishing
because of high mercury levels,”

The detrimental impact of mercurials on natural populations and
individual species has stimulated investigation of the mercury
problem at other levels of biological organization. Medical des-
criptions of symptoms and organ histopathology are numerous ,”» ® °
Cellular distribution,'° genetic effects,+? and enzyme responses?”
to mercury have all added to the knowledge of the physiological
effects of mercury. The effects at each level of organization must
be related to more refined levels in order to determine the actual
physiological responses to mercurials., Only when biochemical
effects can be related to clinical symptoms in the whole organism
will the effects of mercurials on populations and communities be
fully understood,

The aim of this paper is to bring together information concerning
the effects of mercurials at all levels of organization: organism,
organ, tissue, cell, and molecule, It is hoped that a review of
the effects of mercurials at each of these levels will clarify the
total physiological impact of mercury.

2


nitrate was retained in poultry 10 days after injection, Their
excretion data fit a hyperbolic curve with a rate constant of

1.9 x 10°* day"? (ug mercury/kg body weight)"?, Swensson et a1.°°
showed that 2.3% of intravenously injected mercuric nitrate was
excreted within four hours after injection. Large amounts were

also accumulated in the colon indicating appreciable fecal excretion.

Though it appears that the two routes of administration (intravenous
and subcutaneous injections) give rise to the same distribution
pattern, Miyama et al.°” has shown some differences between these

two routes of injection, He sacrificed rabbits 4 and 24 hours after
injection, The mercury content of the brain was always higher for
subcutaneously injected animals. At four hours the amount of mercury
in the brain of subcutaneously injected rabbits was twice that found
in animals receiving intravenous injection. In contrast, the mercury
content of the liver was always lower in subcutaneously injected
rabbits, Except for the fourth hour, the mercury concentration of the
kidney was higher in animals receiving subcutaneous injections.
Mercury excretion in the urine was higher in animals intravenously
injected though no significant difference in fecal mercury was shown
for the two routes of injection.

With increasing dose of intravenously injected mercuric nitrate,
Ulfvarson®® ©” observed a saturation phenomenon in rats, The rate
of excretion was probably increased with increasing dose. Body
components contained less mercury than expected at increasing dosage
levels, Thus, relative distribution and excretion were influenced

by the size of dose,

Metallic Mercury

Several researchers have become interested in the effects of exposure
to mercury vapor since this route of administration is common for
individuals industrially exposed. Kudsk®°® suggested that uptake
occurs at the alveoli of the lungs by simple diffusion. Berlin et al. 3°?
in investigating the distribution of mercury vapor in rats, rabbits,
and monkeys, found no difference in species. The body distribution
was approximately the same as that found for injected mercuric salts;
however, the mercury content of the brain of vapor-exposed animals
was 10 times that of animals exposed to the mercuric salt, They
emphasized that differences in brain uptake are not due to the
exchange in the alveoli but in differences in blood cell/plasma
ratios for the two mercurial forms, No difference in rate of elimina-
tion from the body was observed for the two forms.

Magos”° studied the intravenous injection of metallic mercury and
mercuric chloride in rats, He found that loss by exhalation started
immediately and lasted 15 seconds. Much less mercuric chloride was
lost than injected mercury during this period of time. More metallic

18


Ecological Research Series



not especially sensitive to mercurials. Brubaker et alt found
enhanced incorporation of amino acids in kidney exposed to methyl
mercury and suggested induction of protein synthesis by methyl
mercury.

Paterson and Usher!®® studied the effect of methyl mercury on
glycolytic intermediates of the rat brain. They studied glucose-1-
phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1-6-
diphosphate, phosphoenolpyruvate, pyruvate, dihydroxyacetone
phosphate and the adenine nucleotides (ATP, ADP, and AMP). Dosage
levels of 0.05, 0.5, and 5 mg/g were used. The glycolytic inter-
mediates were statistically different from the controls at the two
higher dosage levels. Two distinct patterns were apparent. At the
highest level, a 13-17% increase in glucose-l-phosphate, dihydroxy-
acetone phosphate, glyceraldehyde-3-phosphate, and 2- and 3-phospho-
glycerate was observed. An 18% increase in a-glycerophosphate was
observed at the two higher dosage levels. Other differences were
less significant when compared to controls than the highest group.
At 0,05 mg/g glucose-1-phosphate was reduced and 3-phosphoglycerate
was increased. The a-glycerophosphate was the same as the control.
The ratio of ATP to ADP and AMP was lowered with the middle and high
dose when compared to their control but raised with the low dose.
These differences were not statistically significant, Paterson and
Usher!®® stated that the accumulation of glyceraldehyde-3-phosphate,
3-phosphoglycerate, and 2-phosphoglycerate indicated inhibition of
enzymes toward the end of the glycolytic path, They suggested that
phosphoglycerate mutase, enolase, pyruvate kinase, and pyruvate
dehydrogenase were probably the affected enzymes, Thus a decrease
in ATP was observed. They discussed the possible alternate path if
the glycolytic metabolites were transferred to the mitochondrion
for oxidation--the alternate path was the dihydroxyacetone/a-glycero
phosphate shuttle. The increase in a-glycerophosphate levels
indicated that the alternate path was operating on the glycolytic
intermediates; however, since a-glycerophosphate was the same for
the two higher mercurial levels, the shuttle was probably operating
maximally. They concluded that mercurials have an acute effect on
glycolytic intermediates,

The biochemical basis for kidney damage appears to be inhibition of
energy-transforming cycles. Shore and Shore!®° measured uptake of
whole kidney homogenates. At four hours after exposure, only 20-30%
inhibition was observed. By twelve hours after injection of the
rats, some showed complete inhibition of oxygen consumption while
others retained partial activity. Complete inhibition of the tri-
carboxylic acid cycle was observed by 24 hours, Oxidative phos-
phorylation was reduced by 90% by six hours after injection. The
a-ketoglutarate dehydrogenase system was inhibited more than
succinate dehydrogenase or cytochrome oxidase. Phosphorylation was
inhibited more than oxidation, Shore and Shore?®® suggested that

41


acute fulminate, the patient lapses into unconsciousness and shows
involuntary movements marked by agitation and shouting. Death due

to pneumonia and high fever usually follows shortly. In the third
form, chronic with irritation, the symptoms are similar to the common
form but in later stages the patient shows psychic excitement and
pyramidal symptoms. Some show frequent convulsions. Individuals
with the fourth form, chronic with contractions, show common symptoms,
but the joints become fixed by contractures in abnormal positions.
All individuals Tokuomi reviewed showing the last two forms died.

Organic mercurial diuretics are administered intravenously and sensi-
tization to them may give rise to toxic or allergenic reactions.
Immediate effects may include pallor, stertorous respiration, jerky
movements and collapse, Although no immediate effects are apparent,
one or two hours after injection the patient may develop chills,
fever, dypsnea, asthmatic attacks, pulmonary edema, and prostration.
This condition may lead to death. After several tolerated injections,
an additional one may yield giddiness, transitory dypsnea, orthopnea,
apprehension, substernal pain, brachycardia, syncope, fall in blood
pressure, collapse, and cyanosis. The patient may be drowsy, mentally
confused, or even delirous. The allergenic reaction may include
chills, fever, cutaneous eruptions, and exfoliative dermatitis.
Mercurial diuretics may cause tubular nephrosis.,

The most common clinical characteristics of poisoning by inorganic
mercury salts are tremor and erethism.® Tremor is first present
only during excitement. It is first observed in the hands, then the
eyelids, tongue, arms, cheeks, head, and eventually the legs are
involved, A blue line on the gums may appear, and salivation some-
times increases. Stomatitis and gingivitis with ulceration of the
mucous membranes occur but are not common,

Von Oettingen!® indicated that ingestion of mercuric chloride is not
usually followed by evidence of nervous system involvement as in
alkyl mercurials. He stated that prolonged use could lead to chronic
poisoning with sudden fever, glandular swelling, scarlatiniform
eruptions, edema, anuria, and possible nephrosis. Kanzantis et al.°°
indicated that albuminuria and the nephrotic syndrome may result from
prolonged mercury exposure. The nephrotic syndrome manifests itself
with heavy albuminuria, hypoalbuminaemia, and hypercholesterolemia.
The albuminuria may be due in part to protein excretion with cyto-
plasmic blebs from the proximal tubule instead of glomerular
malfunction ,*+

Von Oettingen?® discussed the effects of metallic mercury including
two paths of entry, the skin and the gastro-intestinal tract. The
skin may show inflammatory reactions such as eczema, petechial
hemorrhages, and excessive perspiration. The fingernails could show
desquammation and dystrophy. Subcutaneous globules of mercury may
cause granulomas, Disturbances to the gastro-intestinal tract result

5


before nervous disorders become apparent.!* The early neurological
symptoms in acute or subacute cases include numbness of the extremi-
ties and lips, slurred speech, unsteady gait, deafness, concentric
constriction of the visual field, tremor, and slight mental disturb-
ances,» °° These symptoms occurred in 70-100% of the cases reported
in Minimata.® Muscle involvement often leads to spasticity and
rigidity with muscle stretch reflexes exaggerated or depressed.” °
Hypethesia and convulsions are sometimes present.% ° Tremors include
choreoathetosis, myoclonus, and coarse resting and action tremors.®
Mental disturbances range from increased emotional lability to the
mental confusion of the seriously ill. The former is characterized
by alternating euphoria and depression and the latter by periods of
drowsiness and stupor interchanging with periods of shouting and
restlessness.” Children under ten years appear to be more suscep-
tible, and exposed fetuses may develop cerebral palsy symptoms at
birth.” In severely affected patients, generalized muscle wasting
may occur with the patient becoming terminally comatose. Death
usually results from intercurrent infection, pneumonia, or inanition.

Symptomatic behavioral changes have been observed in animals exposed
to methyl mercury, Behavioral changes in fish include increased
difficulty in maintaining their balance, irregular respiration,”°
and impairment of learning.*+ Mice exhibit a characteristic hind-
limb crossing behavior after exposure to methyl mercury”? and, at
less critical levels, show changes in swimming behavior.*° Chicks
also show impaired learning when exposed to low levels of mercury
as embryos.* More acute dosage levels cause impaired muscular
coordination,?°

Little is known of methyl mercury levels required to cause neuro-
logical symptoms, Berglund and Berlin®® have reported that the body
burden at which a toxic level is reached in the brain varies with
species, However, they have hypothesized that a level of eight
micrograms per gram of brain is required to elicit neurological signs
in man, Suzuki?” has indicated that a peak concentration of twenty
micrograms per gram wet brain tissue is the threshold of neurological
symptoms. At a peak of thirty micrograms per gram of wet brain
tissue, death follows neurological signs, Skerfving®® and Hammond?®
gave a 0.2 ug/g whole blood as a minimal blood concentration for

neurotoxic exposure. This is equivalent to ingesting 4 ug/Kg weight
daily.

Tokuomi® divided methyl mercury poisoning into four groups based on
its course and prognosis. The first is the common form with the more
prevalent symptoms indicated earlier (numbness of extremities,
slurred speech, unsteady gait, deafness, constriction of visual
field, tremor, mental disturbances), Although some cases later
develop characteristics of other forms, the course of the common
type is slow improvement with some sequelae. In the second type,

4


133,

134,

135.

136.

137.

138.

Surtshin, A., M. Audia, and H. L. White. Effect of Prior
Injections of HgCl, on Renal Pickup of Mercury from Subsequent
Injection, Amer, J. Physiol. 195:150-152, 1958.

Hell, C. E. B., and P, E, Lindahl. Effects of Phenylmercuric
Hydroxide on Energy Metabolism of Rat Liver and Brain. Oikos.
22:30-36, 1971.

Brubaker, P., G. Lucier, and R. Klein. The Effects of Methyl
Mercury on Protein Synthesis in Rat Liver. Biochem. Biophys.
Res. Comm. 44(6):1552-1558, 1971.

Paterson, R. A., and D. R. Usher. Acute Toxicity of Methyl
Mercury on Glycolytic Intermediates and Adenine Nucleotides
of Rat Brain, Life Sci. 10:121-128, 1971.

Goodman, L. S., and A. Gelman. Mechanisms of Diuretic Action.
In: The Pharmacological Basis of Diuretics. New York,
Macmillan Co., 1955. p. 847-848.

Hirsch, G. Inhibition of Renal Organic Ion Transport by
Methyl Mercury. Environ. Physiol. 1:51-54, 1971.

54 SUS. GOVERNMENT PRINTING OFFICE:1974 546-318/361 1-3


of sulfhydryl groups does not yield complete inhibition, and (6) in-
hibition occurs without the reaction of sulfhydryl groups.

Citing several authors work, Webb?°° summarized several enzymes which
fit the first type. Inhibition was proportional to the number of
sulfhydryl groups reacted in several dehydrogenases (lactate, malate,
3-phosphoglyceralde, succinate) and pyrophosphatase, Several enzymes
which have single groups at their active center appear to be inhibited
by type 1 inhibitors, Webb included glycerol phosphate dehydrogenase
and ficin in this group. Sanner and Pih1??° discussed papain. They
found a single reactive sulfhydryl group essential to the enzyme's
activity and inhibition was of the type 1, Fasella and Hammes!?*
concluded that sulfhydryl groups were not actually involved in the
activity of hexokinase after titrating the enzyme with a mercurial.
Webb?°° therefore placed this in the second category--enzymes in
which the reaction of sulfhydryl groups does not yield inhibition.
Webb also placed enolase and catalase in this group. He included
ATP-ase, alcohol dehydrogenase, aldolase, B-amylase, phosphorylase,
rodanase, urease, and xanthin oxidase in group 3 in which inhibition
occurs after a specific number of sulfhydryl groups are bound.
Gilmour and Gilbert}? studying rabbit myosin, found activation up

to a concentration of 3 M mercury/10° g myosin, Higher concentra-
tions yielded inhibition. DNA is also not affected until a critical
level of bound sulfhydryl groups is reached.!}° The fourth type in
which inhibition is complete before all groups react is shown by
cytochrome c-reductase, and malate dehydrogenase, Rajagopalan et
al,11* studied hepatic aldehyde oxidase and found that two sulf-
hydryl groups per mole reacted rapidly with p-mercuribenzoate
resulting in inactivation of the enzyme, Therefore, Webb*°°

included aldehyde oxidase of rabbit liver in this category. In
addition, Marshall et al.11® found that carbamyl phosphate synthetase
is greatly inhibited before sulfhydryl groups are saturated, Several
enzymes show inhibition proportional to the number of sulfhydryl
groups reacted but are not totally inhibited when all react. Webb?°9
has included lactate dehydrogenase from pig muscle, phosphogluco-
mutase and phosphorylase in the group. The last possible type is

not clearly demonstrated by any enzyme,}°?

The different rates at which inhibition occurs are dependent on
several factors, Webb (Chapt. 7)}°° discussed these. The more
slowly developing inhibition could be caused by less available sulf-
hydryl groups reacting or secondary denaturation. The differences
in time required for inhibition (i.e., 1-2 minutes for succinate
dehydrogenase inhibition vs. 5-20 minutes for enolase) are caused
by the following factors: (1) relationship between sulfhydryl
groups and enzyme activity, (2) different reactivities of different
sulfhydryl groups, (3) tendency to undergo structural changes which
could lead to inactivation.

37


the anterior end of calcarine fissures.» ®* Yoshino"?* found that
the calcarine fissure was also the most prominent area of effect in
dogs. Hunter and Russel1®® found a great loss of neurons with the
most severely affected showing only gliosis. The remaining neurons
were small and many were distorted, In addition, they reported no

fat in the granule cells in the cortex or white matter, The sub-
cortical white matter was also reduced, They found that the oligo-
dendroglial cells were in stages of acute swelling. Hunter and
Russel1°* reported for both macro- and microscopic investigations

that the severity of atrophy and cell destruction in the area

striata decreased with approach to the occipital pole. They observed
the foci of cortical atrophy in the left precentral, the right
post-central, and left superior temporal gyri. The hypothalmus,
brain stem and basal ganglia were not affected. In contrast, McAlpine
and Araki? and Kurland et al.® reported changes of varying degrees in
the basal ganglia, brainstem, and hypothalmus.

Several workers reported similar changes in the cerebellum, Hunter
and Russell®® observed a loss of cells in the granular layer. The
Purkinje cells were spared and the molecular layer narrowed. Some
gliosis was observed in the molecular layer, The changes were most
marked in the depths of the sulci. Yoshino®} and Kim®* found similar
changes in dogs and mylinating cultures, respectively. Only Hunter
and Russell®* described changes in Purkinje cell morphology. They
found some lay abnormally high in the molecular layer with the main
dendrites oblique, horizontal or directed towards the depths of the
cortex, Basket and climbing fibers were absent in severely affected
cells, Stellate bodies formed at the terminal ends of fibers, They
attributed gross ataxia to these alterations in the cerebellar
cortex.

Tokuomi® characterized the histological changes in three groups.
First, regressive changes included the conspicuous changes in the
cerebellum and calcarine cortex revealed by cell loss or spongy
appearance of the cortex and white matter in severe cases, Second,
progressive changes included the proliferation of glial cells, The
third group of changes included disturbances of circulation charac-
terized by hemorrhages in the gray and white matter, perivascular
edema, and cystic dilation around blood vessels.

Lindahl and Hell®* investigated a functional parameter of brain
damage--respiration of brain slices. They exposed fish to 0.3 x 107° M
phenyl mercury. A small inhibition of respiration was observed in
brain slices when succinate was provided as substrate,

KIDNEY

Berlin and Ullberg®* found early accumulation of methyl mercury and
mercuric chloride in the renal cortex in mice, Friberg et al.®° made

25


disruption of phosphorylation could be more important to the cycle's
breakdown than direct inhibition of Krebs' cycle enzymes. They
stated that it was difficult to determine the cause of renal failure,
but that without energy supplied by the tricarboxylic acid cycle,

the kidney could not function, They also indicated that mercury may
be transferred from site to site several hours after maximal kidney
concentration, Maximal kidney concentration occurs at three hours
and maximal inhibition at 24 hours.

Mercurial diuretics apparently act through the suppression of energy
providing enzyme systems thus depressing tubular resorptive mechanisms
relying on active transport.t®” 1°® Goodman and Gelman?®” suggested
that resorption of chloride ions is specifically blocked while Hirsch!*®
thought it is a general depression of tubular function,

Hell and Lindah1'** studied the effects of phenyl mercury on the
energy metabolism of liver slices, In vitro reduction of mitochon-
drial oxidation of a-ketoglutarate and succinate, oxygen consumption,
and the P/O ratio were observed. No effects were observed in liver
slices of rats exposed in vivo. In vitro vs. in vivo inhibition of
several liver enzymes has been compared. Fifty percent inhibition
of alkaline phosphotase resulted from exposure to 107° M mercuric
ion. In vivo exposure to 0.23 mg/L (the median tolerance limit for
96 hours) resulted in a slight but significant increase in activity.
Acid phosphatase showed 40% inhibition at 10°* M, A slight, but a
significant decrease was observed in vivo.

42


in different cortical layers with the ganglionic higher than others.
In the cerebellar cortex, the Purkinje and granular cells appeared
to accumulate more mercury than the molecular layer. The periphery
of the dense zone showed higher accumulation. Some differences in
distribution due to route of administration were observed. The
choroid plexus and pia mater were higher when compared to the rest
of the brain in injected animals. The area postrema showed greater
accumulation than the rest of the brain for injected but not for
vapor exposed animals.

Extensive histological examinations of human tissue have been made
by several investigators. Tokuomi? stated that the distinct general
features of methyl mercurialism were diffuse encephalpathy with
cerebellar and cerebral cortex involvement and effects of pyramidal
and extrapyramidal tracts with disturbances of peripheral nerves.
His macroscopic observations revealed cerebral swelling and
turbidity of the meninges in acute cases. Chronic cases showed
atrophy of the brain with increase in surrounding fluid. Convolu-
tional atrophy was especially apparent around the medial aspects of
the occipital lobes and the anterior ends of both calcarine fissures.
The cerebellum was also atrophied and the gray matter thin. Kurland
et al.® observed scattered punctate hemorrhages in addition to the
characteristics reported by Tokuomi.? McAlpine and Araki? also
reported edema and occasional hemorrhages. Hunter and Russell1®?
macroscopically observed a few flecks of atheroma in the main
cerebral arteries. In addition to the features reported above,
Hunter and Russell found that the left optic nerve was slightly
divided at the chiasma and the right was slightly flattened. They
found occasional small foci of atrophy in the cortex of the Rolandic
area and the frontal and temporal lobes. The normal cortical stria~-
tion of Gennari were absent. They also described atrophy of folia
in the depths of the sulci in the lateral lobes of the cerebellum,
The flocculli and dentate nuclei were not affected.

Fimreite and Karstad®® observed different characteristics after acute
exposure of red-tailed hawks to methyl mercury. The primary site of
damage was the spinal cord instead of the brain. They observed
swelling of the axons and myelin of the myelinated nerves of the
spinal cord. The nerve root and dorsal root ganglia were infiltrated
with heterophils, Klein et al.?? observed similar characteristics in
the sciatic nerve, Lesions were greater in the spinal cord and
reduced cerebrally,?®

Microscopic examination of brain tissue from humans whose death
resulted from methyl mercurialism revealed many signs of damage.
Hunter and Russell's®* microscopic examination of the cerebrum
showed that the cortex of the area striata was atrophied in both
hemispheres, They attributed constriction of the visual field to
gross atrophy of the area striata, The atrophy was greatest about

24


SECTION I

CONCLUSIONS

Studies of mercurial poisoning show that gross symptoms can be
related to tissue damage. In turn, investigations of cellular changes
reveal that tissue damage results from the morphological changes
occurring in cells, The changes at the cellular level are more
subtle in the early stages of damage, and early changes precede
evidence of tissue damage. Thus, cellular changes provide a more
subtle index of damage due to mercurial exposure. Relating morpho-
logical changes at the levels of the organism, tissue, and cell to
biochemical effects is more difficult, Caution is stressed in trying
to understand mercurial poisoning in terms of biochemical damage.

The cell membrane, non-enzymatic cellular protein, and outer cell
layers provide protection to the inner cells or organs, Nevertheless,
researchers have theorized on probable biochemical effects which lead
to the more apparent symptoms of mercurialism. Damage to Krebs’
cycle enzymes and thus to the energy obtaining mechanism of the
kidney, severely limits the kidney's ability to actively resorb

ions, In addition, possible damage to both protein synthesis and
Krebs' cycle enzymes in the brain could result in the extensive
tissue damage observed in alkyl mercurial poisoning. Thus, bio-
chemical changes can be related to other levels of organization and
ultimately to the organism as the basis of the morphological and
behavioral changes which characterize mercurialism,


After four days the kidney content had fallen to one-fourth its
24-hour value and the liver content had declined to less than one-
sixth of its former value. The blood had decreased to one-third
its previous content, but the brain content remained the same,
After 16 days the mercury content of all tissues measured remained
the same with the exception of the blood content which had declined
sharply. After 32 days only small amounts of mercury were found in
all tissues,

Swensson et al. 8° also studied the mercury distribution in dogs at
four hours after intravenous injection. The organs tested included
the liver,kidney, brain, cerebellum and colon. The kidney contained
the highest concentration, and the liver and colon contained the
next highest concentrations, Each contained approximately one-ninth
the concentration in the kidney. The brain and cerebellum contained
the lowest amount. Together they had accumulated approximately
one-fortieth of the concentration found in the kidney. In addition,
Swensson et al, 85 found that 4.3% of the mercury was excreted in the
urine within four hours after injection.

Swensson and Ulifvarson®® investigated the distribution of phenyl
mercury in poultry. They measured mercury content of the brain,
kidney, liver, muscles, and blood 10 and 20 days after injection.
After 10 days they found the highest concentration in the kidneys
and liver, The muscle contained about one-third that found in the
kidneys or liver, The brain contained even less, and the blood
showed the lowest concentration. After 20 days, the kidney had a
greater concentration than the liver, and the brain contained over
three times more mercury than the muscle. In addition, they found
that the distribution between different organs was constant following
injection with the exception of the kidney content which increased
in relation to the other organs with time although its absolute
mercury content decreased. They observed a similar distribution
pattern in dogs.

Measurements of excretion of intravenously-injected phenyl mercury
revealed that 20% of phenyl mercury hydroxide remained in the body
10 days after injection.*® The excretion data for phenyl mercury
fit a hyperbolic function with a rate constant of 1.1 x 10°* day?
(ug mercury/kg body weight)7?

Ulfvarson®® studied the distribution of single subcutaneous injec-
tions of phenyl mercury in rats, Three days after injection he
found the highest concentration of mercury in the kidneys and the
second highest in the liver. The blood, muscle, and brain were low
in mercury concentration.

Intramuscular injection of phenyl mercury in birds was studied by
Miller et al.® They found that the total mercury in the liver

15


109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

Webb, J. L. Enzyme and Metabolic Inhibitors. Vol. II.
Malonate, Analogs, Dehydroacetate, Sulfhydryl Reagents, 0-
iodobenzoate, Mercurials. New York, Academic Press, 1966.
Chapts. 4 and 7.

Sanner, R., and A, Pihl, Studies on the Active SH Groups of
Papain and on the Mechanism of Papain Activation by Thiols,
J. Biol, Chem, 238(1):165-171, 1963.

Fasella, P., and G, G. Hammes. Studies of the Enzyme Hexo-
kinase, IV. The Role of Sulfhydryl Groups. Arch, Biochem.
Biophys. 100:295-297, 1963.

Gilmour, D., and M., Gilbert. The Binding of p-Chloromercuri-
benzoate by Myosin. Arch, Biochem, Biophys. 93:605-616, 1961.

Gruenwedel, D. W., and D. S. Lu. Changes in the Sedimentation
Characteristics of DNA due to Methyl Mercuration. Biochem.
Biophys. Res. Comm. 40(3):542-547, 1970.

Rajagopalan, K. V., I. Fridovich, and P. Handler. Hepatic
Aldehyde Oxidase, J. Biol. Chem. 237(3):922-928, 1962.

Marshall, M., P. Mitzenberg, P. Cohen. Physical and Kinetic
Properties of Carbamyl Phosphate Synthetase from Frog Liver.
J. Biol. Chem, 236(8):2229-2237, 1961.

Kreke, C. W. Inhibition of Trypsin Sulfhydryl Reagents:
Selective Toxicities of Organic Mercury Compounds. J. Pharm,
Sci. 58(4):457-459, 1969.

Van Eys, J., B. J. Nuende, and M. K. Patterson, Jr. J. Biol.
Chem. 234(9):2308-2316, 1959.

Nishida, M., and K. L. Yielding. Alterations in Catalytic

and Regulatory Properties of Glutamate Dehydrogenase Resulting
from Reaction with One Molecule of C-14 Labelled Methyl Mercuric
Iodide, Arch, Biochem. Biophys. 141:409-415, 1970.

Bitensky, M. W., K. L. Yielding, and G. M, Tomkins. Reciprocal
Changes in Alanine and Glutamate Dehydrogenase Activities after
Exposure of Crystalline Bovine L-glutamate Dehydrogenase to
Organic Mercury, J. Biol. Chem. 240(2):663-667, 1965,

Smalt, M. A., C. W. Kreke, and E, 8. Cook, Inhibition of

Enzymes by Phenylmercury Compounds. J. Biol. Chem, 224:
999-1004, 1958.

52


Ellis and Fang?® compared distributions in liver cells versus kidney
cells, phenyl mercury versus mercuric ions, dosage effect, and effect
of time after incubation, They observed a difference in nuclear and
soluble fractions between kidney and liver cells. No differences in
the mitochondrial and microsomal fractions between kidney and liver
cells was observed, Changes in distribution were not observed at
increasing dosage levels for either, In contrast, Rao et al.°”
observed a change in distribution with substrate concentration, When
dosage was increased, the nuclear and microsomal fractions showed
little change; however, the mitochondria showed a greater percentage
uptake and the soluble fraction, a lower percentage uptake, Accumu-
lation was dosage dependent.°° °7 No difference in distribution with
increasing time after exposure were observed. Comparison of the two
mercurials revealed that greater uptake of phenyl mercury than
mercuric ions occurred in all fractions after the first six hours.
Phenyl mercury was accumulated and lost more rapidly. Ellis and
Fang’® suggested that the greater toxicity of phenyl mercury was due
to its greater uptake and storage.

Other variables such as mercuric concentration, incubation time, and
temperature could influence mercurial distribution. Rao et al.®”
studied these factors for phenyl mercury and a mercuric salt. Mercury
uptake increased with time of incubation for both mercurials; however,
longer exposure time resulted in a greater uptake by mitochondria of
phenyl mercury than of the mercuric salt. They also suggested, as
did Ellis and Fang,°° that this difference in uptake could be respon-
sible for the difference in toxicity of these compounds. Initially

a rapid phase of uptake was observed followed by a slower phase. A
non-linear increase in mercury accumulation with increasing tempera-
ture from 3-37°C was observed for both mercurials., At 37°C a
reduction in uptake was observed for phenyl mercury but not for
mercuric chloride. Ellis and Fang®® suggested that the phenyl
mercury might damage the absorption mechanism,

CELL MORPHOLOGY

Miyakawa and Deshimaru®® followed the microscopic pathological
changes in brain tissue after mercurial exposure. They observed
changes in occurrence of intracellular organelles, At 12-13 days,
though no pathological changes were observed in brain tissues,
lysosomes were more numerous in the granule cells bordering the deep
sulcus of the cerebellar vermis. By 19-20 days this granule layer
showed marked localized changes, At this time the Purkinje and Golgi
cells showed an increase in lysosomes. The more prominent changes
observed in the granule cells included shrunken nuclei and peripheral
vacuolation, Kim®* observed similar changes in a myelinating culture.
A progression of more minute changes were observed using microscopy.
The ribosomes disappeared and the intranuclear substance increased,
Observations revealed intracellular pathological changes of two

32


types. In the first, the nuclear membrane gradually concentrated with
disappearance of the cell organelles such as the golgi apparatus and
the ribosomes, In the second, the nuclear membrane degenerated and
the intranuclear substance streamed out. The first was more frequently
observed, In both cases, the mitochondria appeared to be the most
resistant organelles,

CELL MEMBRANE

Rothstein’? stated that the cell membrane is the most probable site
of damage rather than the enzymes within the cell. He described two
aspects of the action of heavy metals on the membranes--chemical
reaction with groups on the membrane and resulting physiological
disturbances, During the rapid phase of mercury uptake by cells, the
mercury binds to the membrane. The reaction is reversible and can
inhibit phenomena associated with the surface enzymes, permeability
barrier, alteration of bioelectric potential, and changes in surface
transport systems. A slower, only partially reversible phase of
me~cury uptake, indicates incorporation of mercury within the cell,
Evidence of the effect on the first phase of uptake can be obtained
by looking at inhibition of glucose transport. Evidence of second
inhibition can be gained from respiration studies, Demis and Roth-
stein’©° described the application of this approach to the inhibition
of rat diaphragm. The extreme action of mercury on the membrane is
irreversible breakdown. The increase in permeability resulting from
membrane breakdown is an all or none phenomenon for individual
cells.?°1 When mercurials bind to the membrane in large enough
quantity, a threshold stress level is reached and the cell membrane
breaks down,?% 102

CHROMOS OMES

Reported effects of mercurials on chromosomes are chromosomal
breakage, radiomimetic effects, and c-mitosis. Skerfving et al.?°?
examined chromosomes of humans exposed to methyl mercury by eating
fish. They found a significant rank correlation between the fre-
quency of cells with chromosome breaks and mercury concentration,
They were unable to show a significant increase in polyploidy and
aneuploidy in humans, Fiskesjo'°® stated that alkyl mercurials

were not good inducers of polyploidy because of their great toxicity.
Ramel!? also found chromosome bridges and fragments in Allium after
exposure to 0,25 x 10°° M phenyl and methyl mercury.

Other authors have used c-mitosis as an index of chromosome damage,
Ramel}* described c-mitosis as strong contraction of the chromosome
with delayed division of the centromere which gives rise to a cross-
like chromosome configuration. They have described the appearance
of cells after different concentrations of mercurials and the
threshold of c-mitosis, Ramel!?»2°* generally described the

33


62.

63.

64,

65.

66.

67.

68.

69.

70.

71.

72.

73.

Underdahl, B., and Hastein. Mercury in Fish and Water from
a Ruier and a Fyord in the Kragerf Region, South Norway.
Oikos, 22:101-105, 1971.

Miller, V. L., G. E. Bearse, T, S. Russell, and E, Csonka.
Absorption, Distribution, and Excretion of Phenyl Mercuric
Acetate. Toxicol, Appl. Pharmacol. 3:459-468, 1960.

Miller, V. L., P. A. Klavano, A. C, Jerstad, and E, Csonka.
Absorption, Distribution, and Excretion of Ethylmercuric
Chloride. Toxicol, and Appl. Pharmacol. 3:459-468, 1961.

Rucker, R. R., and D, F. Amend. Absorption and Retention of
Organic Mercurials by Rainbow Trout and Chinook and Sockeye
Salmon, Prog. Fish Culturist. 31(4):197-201, 1969.

Friberg, L., E. Odeblad, and S, Forssman, Distribution of
Two Mercury Compounds in Rabbits after a Single Subcutaneous
Injection, A.M.A. Arch, Indus, Health, 16:163-168, 1957.

Miyama, T., M. Murakami, T. Suzuki, and H. Katsunuma.
Retention of Mercury in the Brain of Rabbit after Intravenous
or Subcutaneous Injection of Sublimate. Ind, Health. 6:107-
115, 1968.

Kudsk, F. N, Uptake of Mercury Vapour in Blood in vivo and
in vitro from Hg-containing Air, Acta Pharmacol. Toxicol.
27:149-169, 1969.

Berlin, M., J. Fazackerley, and G. Nordberg. The Uptake of
Mercury in the Brains of Mammals Exposed to Mercury Vapour
and to Mercuric Salts. Arch. Environ, Health, 18:719-729,
1969.

Magos, L. Uptake of Mercury by the Brain, Brit. J. Indus,
Med, 25:315-318, 1968.

Magos, L. Transport of Elemental Mercury by the Blood.
Naunyn-Schmiedebergs Arch, Pharmak. Exp. Path, 259(2):183,
1968.

Magos, L. Mercury-blood Interaction and Mercury Uptake by
the Brain after Vapor Exposure. Environ. Res. 1(4):323-337,
1967.

Kiwimae, A., A, Swensson, U. Ulfvarson, and G. Westoo. Methyl
Mercury Compounds in Eggs from Hens after Oral Administration
of Mercury Compounds, Agri. Food Chem, 17(5):1014-1016, 1969.

48


in areas of connective tissue, In general, Friberg stated that
deposition was similar but that the inorganic form disappeared more
slowly from the kidney.

Swensson et al. compared phenyl mercury, methyl mercury, and
mercuric nitrate, They found that mercuric nitrate had the greatest
tendency to become fixed in the kidneys in rats, Phenyl mercury
accumulated in lower concentration. Methyl mercury accumulated least
and was more evenly distributed among organs. They stated that 10
times as much methyl mercury as mercuric nitrate accumulated in the
brain and 2.5 times as much methyl mercury as phenyl mercury.

Blood content immediately after injection was greater for mercuric
nitrate, phenyl mercury was second, and methyl mercury, least. In
accordance with data previously presented, the excretion rate was
very slow for methyl mercury. During the first four hours, 4.3% of
the phenyl mercury was excreted, 2.3% of mercuric nitrate, and only
0.33% (maximum found) of methyl mercury.

Suzuki et a1.°” compared placental transfer of different mercurials.
They found the largest transfer with methyl mercury and the least
with phenyl mercury. Mercuric chloride was transferred in a quantity
twice that of phenyl mercuric acetate but only one-tenth that of
methyl mercuric acetate, Methyl mercury was fairly uniformly dis-
tributed within the fetus and placenta though a lesser amount was
found in the amniotic membrane. Mercuric chloride and phenyl
mercuric acetate were retained uniformly within the fetus but in
higher concentrations in the amniotic membrane and placenta. The
placenta therefore appeared to provide a barrier to the passage of
mercuric chloride and phenyl mercuric acetate but not to methyl
mercuric acetate, The ratio of the mercury content of the maternal
blood: placenta: fetus was as follows: mercuric chloride, 1:19:0.4;
phenyl mercuric acetate, 1:4,5:0.3; methyl mercuric acetate, 1:19:2.1.
There appeared to be a difference in binding and release of mercurial
from the placenta determined by chemical structure.®” Suzuki et a1.°”
suggested that water solubility may play some part in the retention
on the amniotic membrane,

A unique type of biotransformation has been observed with some
mercurials~in vivo methylation. Kiwimae et al.”% investigated
mercury transfer to eggs and methylation. They found that phenyl
mercuric hydroxide, mercuric nitrate, and methoxyethyl mercuric
hydroxide were all partially transformed to methyl mercury in hens.
The methylation process, however, was not rapid or complete. The
mercury concentration in the hen eggs was influenced by rate of
excretion and the mercury compound. Initially, the total mercury
concentration in the eggs increased rapidly. After one or two
months the rate of increase in the eggs was only moderate. The egg
concentration was dependent on the level in the food. The order of
accumulation of mercurial from highest to lowest was as follows:

21


96.

97.

98.

99,

100.

101.

102,

103.

104,

105.

106.

107.

108,

Ellis, R. W., and S. C. Fang. Elimination, Tissue Accumula-
tion, and Cellular Incorporation of Mercury in Rats Receiving
an Oral Dose of 203-Hg Labelled Phenylmercuric Acetate.
Toxicol, Appl. Pharmacol, 11:104-113, 1967.

Rao, A. V., E. Fallin, and S. C. Fang. Comparative Study of
Uptake and Cellular Distribution of 203-Hg Labelled Phenyl
Mercuric Acetate and Mercuric Acetate by Pea Roots, Plant
Physiol. 41:443-446, 1966.

Miyakawa, T., and M. Deshimaru, Electron Microscopical Study
of Experimentally Induced Poisoning due to Organic Mercury
Compounds, Acta Neuropath. 14:126-136, 1969.

Rothstein, A. Cell Membrane as Site of Action of Heavy Metals.
Fedn. Proc. Fedn, Amer, Soc, Exp. Biol, 18:1026-1035, 1959.

Demis, D., and A, Rothstein. Relationship of the Cell Surface
to Metabolism, XII. Effects of Mercury and Copper on Glucose
Uptake and Respiration of Rat Diaphragm, Am. J. Physiol.
180:566-574, 1955.

Passow, H., and A, Rothstein. The Binding of Mercury by Yeast
Cell in Relation to Changes in Permeability. J. Gen. Physiol.
43:621-633, 1960.

Skerfving, S., K. Hannson, and J. Lindsten, Chromosome
Breakage in Humans Exposed to Methyl Mercury through Fish
Consumption, Arch, Environ, Health, 21:133-139, 1970.

Fiskesjo, G. Some Results from Allium Tests with Organic
Mercury Halogenides, Hereditas. 62:314-322, 1969,

Ramel, C. Genetic Effects of Organic Mercury Compounds, Oikos
(Suppl.) 9:35-37, 1967.

Fiskesjo, G. The Effect of Two Organic Mercury Compounds on
Human Leukocytes in vitro, Hereditas, 64:142-146, 1970.

Ahmed, M., and W, Grant. Cytological Effects of the Mercurial
Fungicide Panogen 15 on Tradescantia and Vicia faba Root Tips.
Mutation Res, 14:391-396, 1962,

Ramel, C., and J, Magnusson. Genetic Effects of Organic
Mercury Compounds. II, Chromosome Segregation in Drosophila
melanogaster, Hereditas, 61:231-254, 1969.

Lehninger, A. L. The Mitochondrion; The Molecular Basis of
Structure and Function, New York, W. A. Benjamin, Inc., 1964,
p. 148 and 186,

51


ABSTRACT

Literature on the biological effects of mercurials was reviewed
with the purpose of understanding impact on individual organisms in
terms of biochemical or cellular damage. Mercurialism is manifested
primarily in kidney or brain damage in animals and in growth reduc-
tion in plants. Exposure to inorganic mercury compounds usually
results in kidney damage while alkyl mercurialism is characterized
by brain damage; however, some degree of both kidney and neurological
injury results from exposure to either category of mercurials.
Kidney injury is due apparently to damage of Kreb's cycle enzymes,
thus reducing available energy to actively resorb ions. Impaired
protein synthesis as well as reduction in activity of Kreb's cycle
enzymes may be important in brain damage resulting from mercury
poisoning. Photosynthetic damage is apparently the biochemical
basis of mercurial effects observed in plants.

This report was submitted in partial fulfillment of Project Number
R800510 by the Savannah River Ecology Laboratory, University of
Georgia, under the sponsorship of the Environmental Protection
Agency.

ii


of the Krebs cycle, Webb stated that many enzymes of the tri-
carboxylic acid cycle are sensitive to mercurials, However, work
with homogenates, mitochondria, etc., have shown that the non-
enzyme proteins provide considerable protection so that the actual |
effect on the activity of the Krebs cycle is not great, Rothstein®?
emphasized the protection provided by the membrane.

Several researchers have attempted to determine the cause of the
severe pathological changes in certain brain cells. Hughes??®
suggested that mercurials do not actually block neuron function but
block their metabolism, Hell and Lindah1!** observed marked inhibi-
tion of mitochondrial oxidation of a-ketoglutarate and succinate
after in vitro exposure to phenyl mercury, However, rats subjected
in vivo to phenyl mercury showed no differences in mitochondrial
oxidation, respiration, or aerobic glycolysis when compared to the
control rats. The rats could have still been in the latent period
prior to manifesting neurological symptoms, or the absence of effect
could have been due to non-enzymatic protein and membrane protection.

Yoshino et al.® studied changes in the brain of rats after in vivo
exposure to ah yl mercury. They considered both rats in the latent
period and after the manifestation of neurological symptoms. They
found marked reduction of parameters measured only in rats showing
neurological symptoms. Oxygen consumption was reduced 37%. No
difference in anaerobic lactic acid formation was observed in rats
with or without neurological symptoms. A 27% decrease in aerobic
lactic acid formation was observed in rats manifesting neurological
symptoms. In addition, they measured the activities of several
sulfhydryl-dependent enzymes in five major areas of the brain, In
control rats, succinate dehydrogenase and ATP-ase showed significant
differences in activity among the five areas tested while aldolase
did not, The activities of succinate dehydrogenase and ATP-ase were
lower in the white matter than in other areas, No change in enzyme
activities was observed during the latent period, Succinate dehydro-
genase was significantly reduced in rats showing neurological
symptoms though no difference in the rate of decrease was observed
among the five areas. Only a slight decrease (not statistically
significant) was observed in ATP-ase activity of rats showing
neurological symptoms vs. those not. No change in aldolase activity
was observed, One process, protein synthesis, was inhibited during
the latent period, Leucine incorporation was reduced 57% during the
latent period and 42% after neurological symptoms were apparent.
Yoshino et al.°?” suggested that the disturbance of respiration
would be delayed until the supply of protein became insufficient for
the maintenance of cell life. This would require several days since
the average half-life of protein is 14 days, Though damage to
succinate dehydrogenase and Krebs' cycle may be important in reduced
oxygen consumption of the brain, their study indicated that the main
biochemical involvement is with reactions essential for protein
synthesis, In contrast, Webb*°® stated that protein synthesis was

40


Few enzymes are resistant to mercury. Webb (Chapt. 7)}°° reviewed
many articles and found several enzymes inhibited less than 10%.
Among these were most alkaline phosphatases, many proteases (i.e.,
trypsin'?®) and peptidases, some pyrophosphatases, most RNA-ases and
DNA-ases, He stated that most enzymes or enzyme groups should be
discussed more specifically. The basic organization of Webb will
be used since he dealt with the more important enzymes about which
much information was known.

Many electron transport enzymes are sensitive to mercurials, Van
Eys et al.??” observed 93% inhibition of glycerol phosphate dehydro-
genase with 1 x 10°° M p-chloromercuribenzoate and 100% inhibition
with 1 x 10°* M.?® Yeast alcohol dehydrogenase was inhibited 50% by
1.5 x 10°” p-chloromercuribenzoate, Conversely, 10°° M meth yl
mercury stimulated L-glutamate dehydrogenase’?® 21° though it inhibited
alanine dehydrogenase.**® Nishida and Yielding'!® hypothesized that
a shift in enzyme conformation caused the inhibition effect. The
sensitivity of pre-cytochrome enzymes of the electron transport
chain suggests that these are the sites of mercurial action,1°°
However, Webb (Chapt. 7)?°° developed his discussion further and
showed that the cytochrome system is not immune to mercurial action,
Cytochrome oxidase is inhibited 81-98% at 160 x 107° M of a variety
of mercurials.?®° Lucier et al.}®? and Lucier et al.+°* have inves-
tigated changes in a liver cytochrome resulting from methyl mercury
exposure, They have found that mercury can stimulate synthesis of
cytochrome 450 while increasing degradation yields a decrease in the
total amount of cytochrome 450 present. In addition, Webb?°° stated
that cytochrome inhibition was not reversed by thiols. Thus, the
electron transport system is sensitive to mercurials due to the
inhibition of a number of the enzymes of the electron transport chain.

Webb (Chapt. 7)*°° stated that mercurials were not effective
uncouplers of oxidative phosphorylation. Shore and Shore!?° observed
a reduction in oxidative phosphorylation in rat-kidney mitochondrial
system after injection of 3 mg mercuric chloride/kg body weight.

Webb (Chapt, 7)*°® summarized studies of the effects of mercurials
on photosynthesis, He stated that the Hill reaction was very
sensitive. San Pietro and Lang!** studied pyridine nucleotide
reductase which catalyses the transfer of electrons from the photo-
lytic system to the pyridine nucleotides, They observed 50%
inhibition at 1.2 x 10°° M p-chloromercuribenzoate and 90% inhibition
at 1.6 x 10° mM, Photophosphorylation is not as greatly inhibited,
Kahn and Jagendorph'®® studied an enzyme from spinach chloroplasts
which probably functions in photophosphorylation. It was inhibited
completely at 10°* M mercuric ions. Harriss et al.t®© studying
algae showed that small quantities of mercurials result in measurable
effects on algal photosynthesis and 50 ppb can cause cessation of
photosynthesis in some algae. Lipid biosynthesis, including

38


similar relationship in mice. He considered-the mercury accumulated
in the fur and skin as mercury excreted, Swensson et al.®® found
that excretion of methyl mercury began almost immediately after
injection and then decreased nearly parallel with blood concentration.

Since ingested methyl mercury must enter the body by absorption through
the gastro-intestinal tract, uptake rates have been investigated.
Berglund and Berlin? stated that greater than 90% of the ingested
methyl mercury is absorbed in the digestive tract for rats and man,
Aberg et al.°® indicated that the main uptake of absorbed mercury in
the organs of the abdominal cavity, Accumulation follows an
exponential curve .?5 9

Distribution of single doses of orally administered methyl mercur:
compounds have been described in several organisms. Aberg et al.
described distribution in man, They found mercury in the blood
fifteen minutes after administration and concentration in the blood
corpuscles was already ten times that of the plasma, The maximum
blood concentration was reached three to six hours after administra-
tion. The corpuscle to plasma ratio remained the same for 24 days.
The main uptake was in the abdominal cavity with localization in the
liver. Some uptake was observed in the head with possible localiza-
tion in the cerebellum. Glomski and Brody*® demonstrated the
cerebellar localization more clearly. The pons usually attained the
second highest mercury concentration in their study while the white
matter was lowest in mercury concentration, After 40-50 days, only
0.12% of the dose/gram was found in the hair. No mercury was ever
detected in the sperm,

Piper et al.*+ studied the distribution of an oral dose in pigs.

They found diffuse distribution with a small dose, The largest
concentration was generally found in the renal cortex (19.3%) and

the second highest in the liver (13.6%). The next highest concentra-
tion varied among the renal medulla (6.8%), psoas muscle (6.0%), and
the cerebrum (5.9%). Klein et al.?? and Newberne et al.*? also
observed the highest concentration in the kidney after ingestion of
methyl mercury by rats while Fimreite and Karstad®° observed the
highest level in the liver of hawks. The liver and kidneys are sites
of highest concentration in duck and seal.*° Even when small doses
were given, mercury was observed on day 32,*7 Piper et al.*+ found
that the distribution of methyl mercury was stabilized by day 7 after
administration and remained so until day 35, the last day they
sampled, The cerebrum had the largest concentration of the central
nervous system, and the spinal cord had the smallest. The distribu-
tion in the cerebellum, brainstem, and spinal cord was fairly constant
from days 7 to 35, The cerebrum preferentially retained mercury
until day 13,

The distributions of mercury in plants and invertebrates have been
studied little. Vernberg and O'Hara** have provided some information

8


85.

86.

87.

88.

89,

90.

91.

92,

93.

94,

95,

Conn, H. L., L. Wilds, J. Helwig. A Study of the Renal
Circulation, Tubular Function and Morphology, and Urinary
Volume and Composition in Dogs Following Mercury Poisoning
and Transfusion of Human Blood, J. Clin, Invest. 33:732-741,
1954,

Arrhenius, E. Effects of Organic Mercury Compounds on the
Detoxification Mechanism of Liver Cells in vitro. Oikos
Suppl. 9:32-35, 1967.

Malek, P., J. Kolc, M. Vrana, and Z, Blazek. Effect of Mercury
Derivatives of Fluorescein on the Fribrillation Threshold in
Experimental Myocardial Infarction, Experentia, 27:273, 1971.

Suzuki, T., T. Miyama, H. Katsunuma, Mercury Contents in the
Red Cells, Plasma, Urine, and Hair from Workers Exposed to
Mercury Vapor, Ind, Health, 8:39-48, 1970.

Takeda, Y., T. Kunyi, T. Terao, and T. Ulsito, Mercury
Compounds in the Blood of Rats Treated with Ethylmercuric
Chloride, Toxicol. Appl. Pharmacol. 13:165-173, 1968.

Suzuki, T., T. Miyama, and H. Katsunuma., Comparison of
Mercury Contents in Maternal Blood, Umbilical Cord Blood,
and Placental Tissues. Bull. Environ, Contam, Toxicol.
5(6):502-508, 1971.

Clarkson, T. W. Isotope Exchange Methods in Studies of the

Biotransformation of Organo-mercurial Compounds in Experimental
Animals, In: Chemical Fallout, Miller, M. W., and G. G. Berg
(ed.). Springfield, Charles C. Thomas Pub., 1969. Chapt. 13.

Kudsk, F, N. Factors Influencing the in vitro Uptake of
Mercury Vapour in Blood, Acta Pharmacol. Toxicol, 27:161-172,
1969,

Benesch, R., and R. Benesch. Relation Between Erythrocyte
Integrity and Sulfhydryl Groups, Arch. Biochem. Biophys.
48:38-42, 1954.

Suzuki, T., H. Yoshikawa, and H, Matsushita. Haemolytic
Action of Alkyl Mercuric Compounds and Their Interaction with
a Phospholipid Monolayer, Indus. Health. 8:48-53, 1970.

Norseth, T, The Intracellular Distribution of Mercury in

Rat Liver after Methoxyethyl Mercury Intoxication, Biochem.
Pharmacol, 16:1645-1654, 1967.

50


Much longer half-times were found in some aquatic species, Jarvenpaa
et al.2° citing their earlier work, stated a half-time of 100-170
days for pike, flounder, and eel, In a later experiment, they found
a half-time of 600 days for these same fish. After oral doses of
protein-bound and ionic methyl mercury, they observed half-times of
780 and 700 days, respectively, for flounder, 750 and 640 for pike,
910 and 1030 for eel. Miettinen et al.®°* studied half-times of
elimination for pike and rainbow trout. Half of the ionic methyl
mercury was eliminated in 94 days while 110 days was required for
protein-bound methyl mercury. The proteinate vs. ionic forms

showed insignificant differences in half-times.7°% 4° Jarvenpaa

et al.*© summarized by stating a half-time of approximately two

years for the bulk of methyl mercury ingested by these organisms,
Miettinen et al.®° found that the half-times of excretion for pike,
flounder, and perch varied from 112-490 days for different experi-
ments, They observed a faster excretion rate for the bulk of the
mercury in a mollusc (11-50 days). The half-time of excretion for
the slower excretion phase in the crayfish was 144-297 days, The
following half-time of elimination were recorded by Miettinen et al;?0P
Serranus scriba, 267 days; Carcinus maenas, 400 days; Tapes decussatus,
481 days; Mytilus galloprovincialis, 1000 days. They found that the
fast component of elimination comprised only 4-6% of the total mercury
present for the first two species and about 20% for the last two,

The resorption of methyl mercury excreted into bile may be one

reason for the prolonged biological half-life of methyl mercury in
animals ,°2

Friberg®* discussed the distribution of multiple subcutaneous
injections of methyl mercury. Doses were given once daily for seven
days, One day after the initial series of injections, 33% of the
total body content was found in the blood and 33% in the kidney.
Ten percent was found in the liver, 16% in the spleen, and 2% in
each of the following: cerebrum, cerebellum, and brain stem. By
the 16th day, the blood concentration had fallen to half that seen
one day after the injections ceased, The cerebral concentration
was still 75% of the first value on the 16th day. On the 16th day
after injections ceased, the cerebellum and the brain stem were
reduced to 80% of the first day's value, The kidney had 90% of the
first day's value, and spleen and liver had 50%. No excretion or
half-time data were given,

During the passage of methyl mercury through an organism, it can be
transformed to inorganic mercury. This transformation is not
dependent on the bacteria inhabiting the gut of vertebrates ,®°
Ostlund*® observed biotransformation of a methyl mercurial containing
both C-14 and Hg-203. By observing C-14 exhalation, he found that
4,2% of the given dose was transformed on the first day, 1.2% on

the second, and 0,3-0.9% every day from the third to the tenth.

Since the ratio of C-14 to Hg-203 in the body did not change, the

10


EPA-660/3-73-022
February 1974

A REVIEW OF THE

PHYSIOLOGICAL IMPACT OF MERCURTALS

By

M. Catherine Ferens

University of Georgia
Athens, Georgia

‘Project R800510
Program Element 1BA023

Project Officer

Dr. Harvey W. Holm
Southeast Environmental Research Laboratory
U. S. Environmental Protection Agency
College Station Road
Athens, Georgia 30601

Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D. C. 20460

For sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C, 20402 - Price $1


SECTION V

CELLULAR EFFECTS

Many investigators have attempted to understand the effects of
mercurials on tissues by determining the extent of cell damage and
the interaction between mercurials and cellular components, Cellular
distribution of mercurials, their effects on cell morphology and
cellular components (i.e., chromosomes, membranes, mitochondira) are
topics researchers have considered.

INTRACELLULAR DISTRIBUTION

Intracellular distribution has been studied by several investigators.
Kanzantis’” suggested that the lysosome may be one of the main
organelles which concentrate mercurials, Norseth®® found 26.6% of
the cell mercury in the mitochondrial fraction, 36% in the lysosomes
and perioxisomes, and 37.4% in the microsomes after the cells were
exposed to methoxyethyl mercury. Using a multi-term equation which
included data from marker enzymes, he found 11.7% of the cell Hg in
the microsomes, 3.5% in the nuclear material and 1.6% in the super-
natant, In a similar report,'° he compared mercuric chloride, methyl
mercury, and methoxyethyl mercury. After mercuric chloride exposure,
lysosomes showed the highest concentration. The mitochondria were
second, and the microsomes contained the least. The microsomal
fraction was greater with methyl mercury. Distribution was more
even with methoxyethyl mercury. The microsomal and lysosome-
peroxisome fractions contained approximately equal percentages.

The mitochondrial percentages were lower.

Comparisons of the distribution of mercurials with increased time
after exposure have been made, Norseth?® reported distribution vs.
time for mercuric chloride, At one hour after exposure of the
liver, the mitochondria and microsomes contained equal percentages
of mercury. The lysosomes contained less than half that amount.
After one day, all three categories contained approximately equal
percentages. By four days, the lysosomes and peroxisomes contained
the highest percentage of the liver mercury content. The mitochon-
dria contained the second highest and the microsomes least, with
about half the percentage of the lysosomes, Yoshino®1* studied the
intracellular distribution of methyl mercury with increasing time
after exposure of brain cells. At six hours, the highest concentra-
tion was found in the mitochondria and the second highest in the
microsomes, The supernatant contained the least mercury. After day
one, the mitochondria contained a slightly higher percentage than
the other two and the microsomal and supernatant contained equal
percentages, When mercury/mg N was calculated, Yoshino®!* found the
same pattern of distribution at six hours. At day one, no difference
in mercury/mg N was observed.

31


mercury than mercuric ions was found in the lungs, brain, and heart
30 seconds after exposure, Only 6% of the injected dose of metallic
mercury was retained in the blood while 45% of injected salt was
retained, Magos’? suggested that this difference in blood content
was influenced more by diffusion than by oxidation of metallic
mercury, The ratio of brain mercury content/blood mercury content
was 10 times higher for metallic mercury.”° Thirty seconds after
injection, body and blood content were approximately equal for
metallic mercury while 11 times as much mercuric chloride was found
in the blood as outside the blood.”° After 30 seconds the mercury
content of the blood fell slowly. By this time both injected mercury
and mercuric ion were in the same oxidized state,

Magos’? discussed retention of mercury vapor in white mice at
different dose levels. The uptake was dependent on degree of
exposure for all organs examined. Twenty-four hours after exposure,
the kidneys showed the highest content (12.36% of the body burden).
The fur and skin were slightly lower in content (11.62%). The
liver contained 6,02% and the lungs, 4.62% of the total concentra-
tion, The brain contained 2,84% of the body burden. The smallest
amounts of mercury were detected in the heart (0.83%) and blood
(0.42%). The heart and lungs lost mercury more rapidly than the
body average while the blood and brain lost it more slowly. After
eight days the brain and kidney had almost equal percentages of the
body burden.

In summary, different routes of administration give similar distri-
bution and excretion patterns for each mercurial with the exception
of metallic mercury. Birds, dogs, rabbits, rats, mice, and man
also show similar distribution and excretion patterns for specific
mercurials though the main reservoirs of rats and poultry are
different for some mercurials, However, some aquatic vertebrates
exhibited markedly different excretion rates though among different
fishes, the excretion rate was similar. Phylogenetically related
species appear to follow similar patterns of methyl mercury elimina-
tion with some dependency on mode of entry and temperature.”° In
addition, dosage levels appear to influence excretion rates for most
mercurials, Though the distribution pattern is not changed with
increased dose, the percentage retained in each organ is frequently
less than anticipated at higher dosage levels. The last major
factor influencing uptake, distribution, and excretion of mercurials
is the type of mercurial, This is perhaps more significant than
other factors since uptake, distribution, and excretion are all
influenced by the type of mercurial, The significance of this factor
necessitates further discussion.

Comparison of Mercurials

Several basic differences in distribution of mercurials are apparent.
Friberg®? compared an inorganic mercury salt to methyl mercury, He

19


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency,
nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.


10,

11.

SECTION VIL

REFERENCES

Holmstedt, B. The Toxicology and Metabolism of the Different
Mercury Compounds. A Short Historical Survey. Oikos Suppl.
9:25, 1967.

Tokuomi, H. Medical Aspects of Minimata Disease, Rev. Intern.
Oceanogr. Med. Tome XIII-XIV:5-35, 1969.

Sumino, K. Analysis of Organic Mercury Compounds by Gas
Chromatography. Pt. 1: Analytical and Extraction Method of
Organic Mercury Compounds, Kobe J. Med. Sci. 14:115-130, 1968.

Borg, K., H. Wanntorp, K. Erne, and E, Hanko, Mercury
Poisoning in Swedish Wildlife. J. Appl. Ecol. 3 (Suppl.):
171-172, 1966.

Westoo, G. Methyl Mercury Compounds in Animal Foods. In:
Chemical Fallout, Miller, M. W., and G. G. Berg (ed.).
Springfield, Charles C., Thomas Pub., 1969, Chapt. 5.

Anonymous. Mercury in Water Supplies. J. Amer. Water Works.
62(5):285, 1970.

Zeller, H., and J. Finger. Investigation of Mercury Pollution
in the Southeastern U. S. (Tenth Ann, Environ. and Water
Resources Engineering Conf. Vanderbilt Univ., Nashville.
1971.)

Kurland, L, H., S. N, Faro, and H. Siedler. Minimata Disease:
The Outbreak of a Neurologic Disorder in Minimata, Japan, and
Its Relationship to the Ingestion of Seafood Contaminated by

Mercuric Compounds. World Neurol. 1:370-395, 1960.

McAlpine, D., and S. Araki. Minimata Disease: An Unusual
Neurological Disorder Caused by Contaminated Fish. Lancet.
2:629-631, 1958.

Norseth, T. Studies of Intracellular Distribution of Mercury.
In: Chemical Fallout, Miller, M. W., and G. G. Berg (ed.).
Springfield, Charles C. Thomas Pub., 1969. Chapt. 21.

Ramel, C. Genetic Effects of Organic Mercury Compounds, I.
Cytological Investigations on Allium Roots. Hereditas,
61: 208-230, 1969.

43


tissue, bile ducts, and the portal veins. Phenyl mercury showed a
more even distribution but with more accumulation around the portal
veins, Berlin and Ullberg®* found uniform liver distribution with
methyl mercury.

Arrhenius®® studied the effects of organic mercury compounds on the
detoxification mechanism of the liver, He described a detoxification
system in which the first step was oxygenation of dimethyl aniline
yielding dimethylaniline-n-oxide as an intermediate. In the second
step, oxygen was transferred to one of the methyl groups yielding
formaldehyde and monomethyl aniline. He found that 1.0 mM organic
mercury inhibits detoxification greater than 80%. 0.10 mM organic
mercury yields preferential inhibition of the second step, Leakage
products are larger when this preferential effect is observed. The
significance of this type of mercurial effect comes from the
potentially hazardous effect of these leakage products.

Lindahl and Hel1®* measured some functional parameters of mercurial
effect on liver slices, They found that oxygen consumption increased
15% when fish were exposed to 1 mg/L for 40 minutes, In addition,
they found that the glycolytic rate was inhibited under both aerobic
and anaerobic conditions,

HEART

Few observations of the effects of mercurials on heart muscle have
been made. Johnson and Koumides'* found many foci of inflammatory
cells and necrosis of myocardial fibers in a human subject who had
died from an injection of metallic mercury, Hunter and Russe11°?
reported subendocardial fibrosis replacing muscle in individuals
suffering from methyl mercurialism, Malek et al.®” observed rapid
accumulation and long term retention in ishemic heart muscle although
the same did not occur in healthy heart tissue exposed to a mercurial,
They also observed a rise in the heart fibrillation threshold in
ishemic tissue resulting from application of a mercury fluorescein
derivative. Granular changes have been observed in the cytoplasm of
the myofibrils of the heart and in smooth muscle cells of some blood
vessels.°°

BLOOD

A marked difference in the distribution of different mercurials
between blood and blood plasma has been observed, Swensson et a
studied the distribution of methyl mercury and inorganic mercury in
the blood, They found a plasma to whole blood mercury content ratio
of 0.10 for methyl mercury and 0.80 for inorganic compounds, Suzuki
et al. 87 showed a similar trend for alkyl vs. inorganic mercurials,
The mean ratio for workers exposed to metallic mercury was 1.3. A

ratio of 14-27 was found for subjects exposed to alkyl mercury and

a.35

27


structure. Changes in enzyme structure resulting from the reaction
between sulfhydryl groups and mercurials produce a continuum of
events ranging from initial reversible reactions between sulfhydryl
groups and mercurials to the unfolding of the polypeptide helix
which alters the reactivity of the remaining sulfhydryl groups and
finally causes precipitation of the protein,

Several physical factors can also influence mercurial reaction with
sulfhydryl groups and subsequent inhibition, Hydrogen ion concen-
tration effects the ionization of sulfhydryl groups and thus the
competition between the hydrogen and mercuric ions for the sulfur,
Several different species of mercurials derived from the one
injected are found, For example, injection of mercuric chloride
yields mercuric ions, mercuric chloride, mercuric hydroxychloride,
and mercuric hydroxide. The pH alters hydroxyl ion concentration
and thus influences the quantities of each chemical species found.
Protein charge is influenced by pH; therefore, changes in attraction
or repulsion of different mercurials could occur at different pH
values, Protein reaction with mercurials is usually increased as
pH is reduced. The pH determines the state of aggregation of
protein-mercurial complexes and the rate of secondary denaturation
of the protein. Changes in pH may not affect the reactivity of
different sulfhydryl groups in the same way.

CHEMICAL PROPERTIES--ORGANIC MERCURIALS VS. INORGANIC MERCURIALS

Studies of organismal, tissue and cellular effects have suggested
that organic mercurials act differently from inorganic ones. Webb
(Chapt, 7)*°° discussed important differences in chemical properties.
Mercuric chloride can react with two ligands while organic mercurials
can only react with one. Organic mercurials have a lower water
solubility than inorganic ones, Unsubstituted aryl and alkyl
mercurials are more lipid soluble than mercuric chloride, thus,
enhancing their tissue penetrability. Their bond configurations are
different. Mercuric chloride is linear while organic C-Hg-X have a
bond angle of 130° or greater. Organic mercurials have a greater
molecular size. Therefore, steric factors may impede their reaction
with sulfhydryl groups. Affinities for ligands are somewhat less
for organic mercurials than for mercuric ions.

INHIBITION BY MERCURIALS

Webb (Chapt. 7)1°° has discussed several categories of mercurial
inhibition, The division has been based on the number of sulfhydryl
groups reacted before inhibition occurs. He discussed the following
types: (1) inhibition runs parallel to sulfhydryl groups reacted,
(2) reaction of sulfhydryl groups does not yield inhibition, (3) in-
hibition occurs only after a set number of sulfhydryl groups have
reacted, (4) inhibition is complete before all groups react, (5) in-
hibition is parallel to reaction of sulfhydryl groups but saturation

36


methyl mercury, phenyl mercury, and methoxyethyl mercury. The concen-
tration between yolk and white also varied with the mercurial, Only
methyl mercury showed a higher concentration in the white. With
methyl mercuric hydroxide, the influence of increase in dose effect
was linear, a four-fold increase in dose yielded a four-fold increase
in mercury concentration of the white. With mercuric nitrate and
phenyl mercuric hydroxide, the dose level only slightly affected the
concentration in the white. The content of the white doubled with a
four-fold increase in dose for methoxyethyl mercury. The yolk showed
a four-fold increase in mercury concentration for a four-fold increase
in dose for all mercurials tested. The percentage of mercury as
methyl mercury in the whites one week after feeding began was 81-94%,
This remained constant during feeding but fell rapidly after feeding
stopped. For other compounds, the mercury content of the whites was
less affected by dose than the yolk. No particular equilibrium
between whites and yolks appeared to exist. Methyl mercury was the
primary constituent of the whites from hens given any one of the
compounds, The concentration varied with the compound given but was
lowest when methyl mercury was given. Greater than half of all the
compounds given were converted to methyl mercury by the time they
reached the egg. Methyl mercury was also found in the organs.

Greater than 50% of the mercury in the muscle and blood was methyl
mercury. Only small percentages of methyl mercury were found in the
kidney and liver for all mercurials tested. Microbial methylation by
specific bacteria and fungi or in mud sediments has been investigated
by several researchers,” 7°) 7677578 7° Genetic connection between
methionine biosynthesis and mercury resistance has been demonstrated.®°
Lander’® has shown that methyl mercury synthesis involves several
steps in methionine biosynthesis and provides a mechanism for the
occurrence of methyl mercury products in organisms not exposed to
methyl mercury.

Ulfvarson®” investigated the difference in dosage effects among
mercury compounds. Only with methyl mercury was the relative
distribution and excretion of mercury unaffected by dose, With
methoxyethyl mercury an increase in dose by a factor of 10 resulted
in organ concentration lower than expected, Mercuric nitrate also
showed lower values than expected. Therefore, all but methyl mercury
showed a saturation phenomenon.

In summary, all parameters of mercurial distribution (i.e., organ
retention, placental transfer, egg transfer, etc.) revealed that the
most marked difference of mercurials is between methyl mercury and
other mercurials, Ethyl mercury has been placed in the same category
as methyl mercury.*” Though quantitative differences in distribution
pattern can be shown between mercuric nitrate, phenyl mercury, and
methoxyethyl mercury, they all show similar characteristics, For
example, dosage response shows a similar pattern, the excretion
pattern follows the same curve, and placental transfer is blocked,
Thus, alkyl mercurials stand out among other mercurials and markedly
influence uptake, distribution, and excretion characteristics,

22


in hunger or lack of appetite, the latter resulting in weight loss.
Increased salivation and a metallic taste in the mouth may be noted.
The gingiva may be spongy and ulcerated. The teeth may erode and
fall out. Stomatitis may develop to an ulcerative or gangrenous
state, Rhinitis, anosmia, and necrosis of the maxilla can occur
rarely. The nervous system is eventually involved giving rise to

the neuromotor and nervous disturbances previously described. Renal
involvement can occur with nephritis, polyuria leading to anuria, and
uremia,

Mercury fulminate, when applied to the skin, may yield a form of
dermatitis which is difficult to heal. When applied to the eyelids,
it causes conjunctivitis, Inhalation or ingestion can cause chronic
nercury poisoning,

Mercury oxycyanide applied locally can result in the corrosion of
membranes and, after absorption, systemic effects. Ingestion causes
abdominal pain, vomiting, diarrhea and eventually death. Prolonged
cases may involve kidney damage,®

In summary, prolonged exposure to all forms of mercury can lead to
mercury poisoning. The two major responses to mercury poisoning
involve neurological and renal disturbances, The former is
characteristic of organic mercurial poisoning where liver and renal
damage are of relatively little significance.** The latter is
characteristic of inorganic mercurial poisoning.*?

UPTAKE, RETENTION, EXCRETION, BIOTRANSFORMATION

Many investigators have studied the uptake, retention, distribution,
and excretion of various mercurials. Methyl, dimethyl, phenyl, and
methoxyethyl mercurials are most frequently studied organic forms,
Several researchers have also included ethyl mercurials in their
work. Inorganic salts (mercuric nitrate, mercuric chloride, mercuric
hydroxide) and mercury vapor are the most frequently studied inorganic
mercurials, Data on various routes of entry and dosage levels are
included in the literature. In addition, several researchers have
investigated biotransformation of organic mercurials, the in vivo
formation of mercuric ions from metallic mercury, and in vivo
methylation of inorganic mercurials,

Methyl Mercury

Studies of the distribution and excretion of methyl mercury have
considered single and multiple exposure from several routes of entry
(oral, intravenous and subcutaneous injections). Mammals (pigs,
mice, rats), poultry, and aquatic vertebrates (eels, flounders, pike)
have been studied,


12.

13.

14,

15.

16.

7,

18,

19.

20.

21.

22,

23,

2.

Jackim, E., J. M. Hamlin, and S. Sonis, Effects of Metal
Poisoning on Five Liver Enzymes in the Killifish (Fundulus

heteroclitus). J. Fisheries Res. Bd, Can, 27:383-390, 1970.

Hill, D. M. Self-administration of Mercury by Subcutaneous
Injection, Brit. Med. J. 1:342-343, 1967.

Johnson, H. R. M., and 0. Koumides. Unusual Case of Mercury
Poisoning. Brit. Med. J. 1:340-341, 1967.

Von Oettingen, W. F. Poisoning: A Guide to Clinical Diagnosis
and Treatment, Philadelphia, W. B. Saunders, 1958. p. 416-422

Cummings, J. N. Poisoning by Mercury, In: Heavy Metals and
the Brain, Oxford, Oxford Sci. Pub., 1959. Chapt. 10.

Kanzantis, G. Chronic Mercury Poisoning-Clinical Aspects.
Ann. Occup. Hyg. 8:65-71, 1965.

Whitehead, K. P. Organic Mercury Compounds. Ann. Occup. Hyg.
8:85-89, 1965.

Kipling, M. D. Mercury and the Eye. Ann. Occup. Hyg. 8:81-83,
1965.

a, Miettinen, V., E, Blankenstein, K, Rissanen, M,. Tillandek,
J. Miettinen, and M, Valtonen, Preliminary Study on the
Distribution and Effects of Two Chem. Forus of Methyl Mercury
in Pike and Rainbow Trout. (FAO Tech. Conf. on Mar, Poll.
Rome. 1970.) b. Miettinen, J., M. Heyraud, and S. Keckes.
Mercury, a Hydrospheric Pollutant: Biological Half-time of
Methyl Mercury in Four Med. Species: A Fish, a Crab, and Two
Molluscs. (FAO Tech. Conf. on Mar. Poll. Rome. 1970.)

Weir, P., and C. Hine. Effects of Various Metals on
Behavior of Goldfish. Arch, Environ, Health, 20:45-51, 1970.

Klein, R., S, Herman, P. Brubaker, G, Lucier, M. Krigman,
A Model of Acute Methyl Mercury Intoxication in Rats. Arch.
Pathol, 93:408-418, 1972.

Spyker, J., S. Sparber, and A. Goldberg. Subtle Consequences
of Methyl Mercury Exposure: Behavioral Deviations in Off-
spring of Treated Mothers. Science. 177:621-623, 1972.

Rosenthal, E., and S. Sparber. Methyl Mercury Dicyandiamide:

Retardation of Detour Learning in Chicks Hatched from Injected
Eggs. Life Sci. 11:883-892, 1972.

44


37.

38.

39,

40.

41.

42,

43,

4h.

45.

46.

47.

48,

49,

Suzuki, T., N. Matsumoto, T. Miyama, and H. Katsunuma.
Placental Transfer of Mercuric Chloride, Phenyl Mercury Acetate
and Methyl Mercury Acetate in Mice, Ind, Health. 5:149-155, 1967.

Ostlund, K. Studies on the Metabolism of Methyl Mercury and
Dimethyl Mercury in Mice. Acta Pharmacol. and Toxicol. 27(Suppl.)
1:1-132, 1969,

Aberg, B., L. Ekman, R. Falk, U. Greitz, G. Persson, and J. Snihs.

Metabolism of Methyl Mercury (Hg-203) Compounds in Man. Arch.
Environ, Health. 19(4):478-484, 1969.

Glomski, C., and H, Brody. Distribution and Concentration of
Mercury in Autopsy Specimens of Human Brain. Nature, 232:
200-201, 1971.

Piper, R. C., V. L. Miller, and E. 0. Dickinson. Toxicity and
Distribution of Mercury in Pigs with Acute Methylmercurialism.
Am. J. Vet. Res, 32(2):263-273, 1971.

Newberne, M., 0. Glaser, L. Friedman, Chronic Exposure of
Rats to Methyl Mercury in Fish Protein. Nature. 237(5349):
40-41, 1972.

Jones, A., Y. Jones, W. Stewart. Mercury in Marine Organisms
of Tay Region, Nature. 238:164-165, 1972.

Vernberg, W., and J. O'Hara, Temperature Salinity Stress and
Mercury Uptake in the Fiddler Crab, Uca prigilator. J. Fish.
Res, Bd, Canada, 29(10):1491-1494, 1972.

Jarvenpaa, T., M. Tillander, and J. K. Miettinen. Methyl
Mercury: Half-time of Elimination in Flounder, Pike, and Eel.
Suomen Kemistilehti B. 43:439-442, 1970.

Miettinen, J., T, Rahola, T, Hattula, K. Rissanen, and M.
Tillander, Elimination of 203ng - Methyl Mercury in Man,
Ann. Clin. Res, 2:116-122, 1971.

Loutit, J. Investigation of the Mating System of Pseudomonas
aeruginosia straini. VI. Mercury Resistance Associated with
Sex Factor (FP). Genet. Res., Camb. 16:179-184, 1970.

Forrester, C., K. Ketchen, and C. Wong. Mercury Content of
Spiny Dogfish (Squalus acanthias) in the Straight of Georgia,
British Columbia, J. Fish. Res, Bd, Canada, 29(10):1487-1490,
1972,

Gaskin, D., K. Ishida, and R, Frank. Mercury in Harbour

Porpoises (Phocoena phocoena) from the Bay of Fundy Region.
J. Fish, Res. Bd, Canada, 29(11):1644-1646, 1972.

46


on the fiddler crab, Gill tissue accumulated more than the hepato-
pancreas and most uptake in the gill tissue occurred during the
first 24 hours of exposure. The hepatopancreas concentration rose
throughout the period studied, Uptake was the same under a variety
of salinity and temperature combinations.

Several algae and molluscs living in water receiving an industrial
effluent have been investigated.*® Thallose algae (Porphyra, Ulva)
took up more mercury than other types. The highest mercury concen-
tration in Fucus was found in the stipe and holdfast, Mercury con-
centration in the thallus gradually increased toward the apex. Jones
et al.*® also considered several molluscs (Littorina, a herbivore;
Mytilus, a suspension feeder; and Nucella, a carnivore). Appreciable
mercury was present at all trophic levels, In Mytilus, the ctenida
contained the greatest concentration and appeared to be the area of
uptake and release,

The main route of excretion of ingested methyl mercury is the feces,
the same route found for injected methyl mercury, Aberg et a1.°?
found that in man 13-14% of the ingested dose was excreted in the
first ten days after administration, The urine contained only 0.18-
0.27% in a comparable period. After 49 days, 33-35% of the dose had
been excreted in the feces while only 3.3% had passed in the urine,
Aberg et al.°® stated fecal and urinary excretion were the only two
significant routes, In the flounder, pike, and eel, Jarvenpaa et
al.*® observed slow excretion from the 10th to the 100th day. They
found some indication of rapid loss during the second to the fourth
days, They calculated elimination, including vomiting, for the
first few days for protein-bound ingested methyl mercury and ionic
methyl mercury, The pike lost ten percent of the former and five
percent of the latter. The eels lost 17% of both. The flounder lost
33% of the protein-bound and 12% of the ionic form, Miettinen et al,*°
studied the elimination of protein-bound methyl mercury in man, In
the first three to four days, six percent had been eliminated in the
feces and a small amount in the urine. With increases in time, they
found a less marked difference in the quantity of mercury excreted
through the two routes.

Half-times of excretion have been recorded for these same species,
Miettinen et al.*® gave an average half-time of 65-75 days for either
protein-bound or ionic methyl mercury in man, Aberg et al.°° gave a
similar range (70-74 days). Miettinen et al.*° found a mean half-

time of 71 days for women and 79 days for men. These latter values
were not significantly different. However, other differences in
response of mercurials associated with sex factor have been observed.
Loutit*” found that Pseudomonas aeruginosa with the (FP) sex factor
were more resistant to HgCl,. A higher concentration of mercury is
present in male dogfish sharks*® and rats*® while male and female
harbour porpoises contain the same concentration of mercury.*® Fowler®1
observed more marked histological changes in the kidneys of female mice.

9