The Toxicology of Radioactive Substances. Volume 5

188 Pages·1970·7.39 MB·English

by  A. A. Letavet and E. B. Kurlyandskaya (Eds.)

THE TOXICOLOGY OF
RADIOACTIVE
SUBSTANCES
VOLUME 5
Zinc-65
Edited by
A. A. LETAVET
and
E. B. KURLYANDSKAYA
Translated by
A. CROZY
Translation edited by
G. W. DOLPHIN
UNITED KINGDOM ATOMIC ENERGY AUTHORITY
Authority Health and Safety Branch
Radiological Protection Division, Harwell
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First English edition 1970
Library of Congress Catalog Card No. 61-9783
This is a translation of the original Russian
published in 1965 by Medgiz, Moscow
Printed in Hungary
08 013414 9
TOXICOLOGY OF RADIOACTIVE ZINC-65
E. B. KURLYANDSKAYA
STUDY of the chronic action of the radioisotope 65Zn is of great practical
and theoretical importance. The practical interest of such investigations
arises from its use in certain branches of industry (metallurgy, engineer­
ing) for tracing technological processes. In addition, it is now known that
all the brass parts of cyclotrons are covered with a layer of 65Zn formed
by the action of neutrons on copper. According to the findings of Cohn
et al. (1961) cyclotron workers were found to have about 0-2 per cent
of the maximum permissible content of 65Zn in the body. According to
the findings of the same authors some reactor workers were also found
to have 65Zn in an amount of 40-120 £JiCi/kg weight. Swanberg (1962)
found that in some persons working near a reactor the amount of 65Zn
reached 41 \LC\.
A considerable amount of radiozinc has been found in the water of the
Columbia River (U.S.A.) which receives the wastes of the Hanford reac­
tors and also in river plants, molluscs, etc. (Watson, Davis and Hanson,
1961; and others).
Fairly high concentrations of 65Zn have been found in the pasture grass
and milk of cows in the Columbia River district. The liver, bone and hair
of these animals contained 65Zn in a larger amount than did the meat (Per­
kins and Nielsen, 1959). 65Zn was found in 12 persons who used for drink­
ing the water of the Columbia River. However, its amount in persons
whose diet included meat, milk and water from this region was less than
0-01 per cent of the permissible body level for this isotope. Thus, the possi­
bility of entry of 65Zn into the human body has become quite real in the
last few years.
The theoretical importance of these investigations and the biological
effects of radiozinc is connected with the fact that its stable isotope is a
trace element constituent of vitally important structures: proteins, en­
zymes, hormones, etc. The role of zinc in the vital activity of the body has
now been fairly well studied. The human body takes in about 16 mg zinc
and requirements for it are fully met by the zinc content of foodstuffs.
Lack of zinc in the animal feeds leads to retardation of growth, disturbance
2 The Toxicology of Radioactive Substances
in reproductive functions and changes in metabolism, chiefly of proteins
and carbohydrates.
Zinc is of great importance in the activity of various enzymes. In some,
zinc is a specific component of the enzyme molecule which deprived of
zinc loses its biological activity, such as carbonic anhydrase. According
to Scott and Fisher (1938) the crystalline preparation of this enzyme con­
tains for 16-5 per cent nitrogen 0-22 per cent zinc. It has been shown that
glutamic dehydrogenase is also a zinc metal enzyme. Per gram protein
this enzyme contains as much as 333 mg zinc (Vallee et al., 1955). Accord­
ing to Vallee et al. (1956) zinc also forms part of the molecule of a number
of other dehydrogenases (lactic dehydrogenase of the rabbit muscle,
alcohol dehydrogenase of beef liver, etc.).
For some enzymes although zinc is not a specific component it may as
part of ionic exchange replace the natural metal component (phospha-
tases, anolase, chymohexase, certain polypeptidases, carnosinase, etc.). The
catalytic properties of zinc as a metal have also been repeatedly confirmed
in various investigations. It is known that zinc like heavy metals may acti­
vate or depress enzymatic activity. Zinc has an effect on the activity of a
number of hormones including sex hormones and also gonadotrophin and
other pituitary hormones. It has been shown that crystalline preparations
of insulin always contain zinc although its role as a constituent of the
molecule of insulin is still under discussion. The close link of zinc with a
number of enzymes and hormones is one of the main reasons for its influ­
ence on the various aspects of metabolism of proteins, carbohydrates and
fats and on redox and other processes.
We could give considerably more information on the biological role of
zinc but this is not the special purpose of the present paper. Nevertheless,
starting from such a multi-faceted function of zinc in the living body it
may be assumed that the localization of radioactive zinc, moving with its
stable analogue, in certain organs and tissues, enzyme and hormone mole­
cules, etc., maybe the cause of changes in the bodv which appear on chron­
ic entry of radiozinc into the body. These changes will differ from those
produced by other isotopes as occurs on prolonged administration to
rabbits of G0Co, 5yFe, and so on.
The radioactive isotope of zinc, 05Zn, has a fairly long half-life (T =
1/2
245 days) and possesses a complex spectrum of emanation. About 97 — 98-5
per cent of its decays occur through capture by the nucleus of the orbital
electron from the K level (K capture) and subsequent emission of charac­
teristic X-radiation with an energy of 0-008 MeV (a detailed account of the
radiations from 65Zn is given in the paper by Avrunina in the present
volume).
We studied the chronic effect of 65Zn, following daily (6 times a week)
Toxicology of Radioactive Zinc-65 3
oral administration in the form of the chloride 65ZnCl . Unfortunately,
2
we were not able to carry out the complete set of investigations with the
preparation 65Zn without carrier. The amount of 65Zn at our disposal
without a carrier enabled us to carry out with this preparation only one
series of investigations and this only for administration to rabbits in a dose
of 1 (jiCi/mg daily for 5 months.
In order to pinpoint the role of stable zinc in the reaction of the body
to the administration of 65Zn with carrier we set up a control group
the animals of which received only stable zinc in an amount equal to its
entry in the first group (0-1 mg/kg). We did not introduce other control
animals with a smaller amount of carrier on the grounds that the group
of rabbits receiving 1 [JiCi/kg zinc without a carrier was a control for the
group of animals which received 65Zn and 0-01 mg/kg zinc to the same
radioactivity level. For group No. 3 (0-1 [xCi/kg 65Zn and 0-001 mg/kg
stable zinc) we considered it possible not to have a special control since
the amount of zinc received by this group was considerably less than the
natural intake in the food for rabbits. In line with the findings presented by
Voinar (1960), 100 g of oats contains 1*9 mg zinc and 200 g of beet and
other vegetables 1-86 mg (the food ration for our test animals). In addition,
the rabbits in the winter received 100 g of hay and in the summer 500 g
of fresh grass also containing zinc.
We used in all 78 rabbits introduced into the experiment at the age of
4-6 months and of the same weight (about 2-5 kg) but of different sex
(in the test and controls respectively). These animals were distributed
over the following groups.
First (20 rabbits) received daily, 10 [JiCi/kg 65Zn (the preparation con­
tained stable zinc in an amount of 0-1 mg/kg).
Second (9 rabbits) received daily, 1 [JiCi/kg 65Zn (0-01 mg/kg stable
carrier).
Third (12 rabbits) received daily, 0-1 (JiCi/kg 65Zn and 0-001 mg/kg stable
zinc.
Fourth (11 rabbits) received daily, 1 (i.Ci/kg 65Zn without carrier.
Fifth (13 rabbits) received daily, stable zinc in an amount of 0-1 mg/kg.
Sixth (13 rabbits) biological controls.
All the investigations, the results of which are outlined in the present
book, were carried out on the same animals under the control of vital
dosimetry and absolute dosimetry of the organs after sacrifice made at
different times after the start of administration.
In some experiments additional groups were introduced in line with
the aims of the experiment as will be discussed in the individual papers.
In the work of Khristov investigations were also carried out on white
rats.
4 The Toxicology of Radioactive Substances
It should be noted that for comparative analysis of the biological effects
the observation periods were kept uniform.
To elucidate the features of the chronic effect of radioactive zinc the
following problems were studied.
1. The metabolism (distribution, excretion) of 65Zn in the body fol­
lowing of single and chronic administration.
2. The effect of 65Zn on the bioelectrical activity of the brain.
3. The effect of 65Zn on cardiac activity by ECG investigations.
4. The effect of 65Zn on peripheral blood and haemopoiesis.
5. The effect of 65Zn on certain aspects of protein and carbohydrate
metabolism.
6. Changes in immunological reactivity and immunogenesis.
7. The morphological changes in organs and tissues.
Analysis of the findings showed that 65Zn is distributed in the body in a
relatively diffuse fashion with affinity for certain organs. Thus, according
to the findings of Khristov for white rats following single oral, intra-
tracheal and subcutaneous administration, the differential absorption coeffi­
cient (d.a.c.) is highest for the pituitary, pancreas and liver, less for the
kidneys, spleen and bones, while for the intestines, lungs, heart and supra-
renals the d.a.c. is lowest. The findings obtained by Khristov correspond
to the published findings obtained for single administration of 65Zn
(Miiller, 1945; Ballou and Thompson, 1961; and others). Similar patterns
were established by Avrunina in rabbits following prolonged chronic
oral intake of 65Zn but a notable feature was the comparatively low (as
compared with 60Co and 59Fe) retention of 65Zn in the gastro-intestinal
tract and contents (about 35-40 per cent of the total activity in the body).
Equilibrium in the accumulation of 65Zn in the body following daily
intake was set up in the rabbits in a period of 1*5-3 months from the start
of administration and subsequently its contents in the organs varied within
relatively narrow limits.
A certain discrepancy was noted in the distribution of 65Zn with and
without carrier. In the absence of carrier the concentration of zinc in the
brain and the bones was higher than in the presence of carrier.
After establishment of equilibrium the absorbed doses were as follows:
in the first group, in the rabbit liver 0*78 rad/day and in the whole body
0*45 rad/day; in the second group, in the liver 0-065 and in the body 0-043
rad/day; in the third group 0-0044 and in the fourth 0-031 rad/day. The
total dose in the first group for 3-16 months' observation was respectively
41-258 rad, in the second in 3-16 months 3-0-20-7 rad, in the third in 16
months 2-4 rad and in the fourth group in 5 months 5-2 rad.
What were the changes found in the rabbits of the different groups
receiving radiozinc daily for 16-19 months?
Toxicology of Radioactive Zinc-65 5
Three to four weeks after the start of chronic administration in the
rabbits of the first group (10 jxCi/kg) changes were found in the bio-
currents of the brain expressed in the EEG (Ovakimov) manifest in extension
of the repetition ranges to the right in response to rhythmic photostimula-
tion. These changes in imposed rhythm were of an unstable character
giving way from time to time to periods of normal EEG traces. At later
times of exposure the EEG did not deviate from normal despite progressive
changes in other organs and systems, which indicates the high sensitivity
of the CNS which responds early to the action of low radiation doses
and points to its high compensatory powers on exposure to noxious
factors of the external environment. Similar changes are also observed
on exposure to 59Fe (Ginzburg, 1967).
Changes in the biocurrents of the heart in the ECG appeared after 4-6
months (Ovakimov and Bibikhin) when there were already frank changes
in the haemopoietic organs and certain metabolic processes. In line with the
latter, changes were found in the T wave now related to trophic processes
in the heart muscle. The use of functional pharmacological tests (adminis­
tration of adrenaline, smelling of ammonia) revealed a decline in sensitiv­
ity to ammonia (olfactory-cardiac reflex) in animals receiving radiozinc
in a dose of 10 (JiCi/kg and to a lesser extent 1 fiCi/kg. The findings obtained
concur with those previously established by Saitanov (1963, 1967) and
Golovshchikova (1963) showing a rise in the excitability of the centres
of the vagus nerves on chronic exposure to 60Co and 59Fe. Thus, in rela­
tion to the bioelectrical activity of the brain and heart, the effect of radio­
zinc does not differ from other radionuclides and this effect is a common
factor.
Frank changes were seen in the peripheral blood and organs of haemo­
poiesis. Comparison of the findings obtained on administration of 10 (JiCi/kg
of 65Zn with carrier and also administration of stable zinc alone shows
that the reaction of the animals to the administration of 10 |i,Ci/kg of 65Zn
represented the combined effect of radioactive and stable zinc. However,
it must be stressed that in relation to some reactions these two isotopes
behaved as synergists, in others, as antagonists. If we compare their effect
on erythropoiesis it will be seen that the total amount of haemoglobin
and its content per erythrocyte in animals receiving 10 (JiCi/kg of 65Zn pro­
gressively fell and according to Beloborodova and Red'kina these animals
eventually developed hypochromia of the erythrocytes. In rabbits given
daily stable zinc the amount of haemoglobin appreciably rose in the same
way as did its content per erythrocyte.
Change in the size and shape of the erythrocytes was noted in equal
measure both for radioactive and stable zinc. In the animals of both groups
microspherocytosis was observed giving way to gradual increase in dia-
6 The Toxicology of Radioactive Substances
meter and volume. Both zinc isotopes produced changes in the ratio of
the numbers of reticulocytes in the bone marrow and peripheral blood,
which enabled Beloborodova and Red'kina to postulate increase in the
voiding of reticulocytes by the bone marrow. Detailed analysis of the
changes in the red blood is given in the papers of these authors in the pres­
ent book.
The changes in erythropoiesis after administration of 1 [JiCi/kg of 65Zn
were considerably less marked. In this case, at the start of the experiment
reticulocytosis was somewhat more obvious in the peripheral blood as
compared with the picture for 10 [xCi/kg of 65Zn. The number of bone mar­
row reticulocytes subsequently fell somewhat. Following administration
of 65Zn without carrier in the same amount these phenomena in the early
stages of exposure (up to 5 months) were not noted, so confirming the
previous assumption that stable zinc increases release of reticulocytes by
the bone marrow. The picture of the bone marrow in the rabbits receiving
65Zn without carrier shows certain changes in the erythrogram which may
be related to accelerated processes of erythroblast maturation.
Of particular interest are those changes found in rabbits in the periph­
eral blood and bone marrow on administration of 0*1 [JiCi/kg of 65Zn when
the absorbed body dose was 0-004 rad/day. During the first three months
of administration some signs of suppression of erythropoiesis were noted:
reduction in the number of erythroblasts and reticulocytes in the bone
marrow. This suppression could not be attributed to stable zinc because
of its insignificant amount (0*001 mg/kg) but after 6-12 months the whole
of medullary haemopoiesis was activated, as described in detail in the paper
by Red'kina.
Thus, a high dose of 65Zn (10 (JiCi/kg) depresses erythropoiesis which
is not true for 1 (JiCi/kg. Following administration of 0*1 [xCi/kg, there was
stimulation of the red series of the bone marrow. This is also true of other
isotopes, e.g. 35S, when certain intermediate doses produce only individual
signs of depression of haemopoiesis but do not have a stimulatory effect
characteristic of very low doses of radioactive isotopes. Thus, it appears
that these doses have little biological effect.
Depression of lymphopoiesis was observed only after administration
of 65Zn in an amount of 10 (JtCi/kg and was manifest in gradually develop­
ing lymphopenia. After exposure to stable zinc there was only short­
lived lymphocytosis in the initial period of treatment. After administration
of zinc both with and without carrier in a dose of 1 [JiCi/kg no changes were
noticed in the lymphocyte count. There were some qualitative, essentially
degenerative, changes in the lymphocytes in the peripheral blood. In
animals given 0-1 (j,Ci/kg, lymphocytosis appeared periodically.
A notable feature was the high sensitivity of the granulocyte series of
Toxicology of Radioactive Zinc-65 7
the bone marrow to exposure to 65Zn. Although after administration of
stable zinc (0*1 mg/kg) only the initial stages of neutrophil maturation
in the bone marrow were accelerated while in the peripheral blood there
was some increase in the absolute number of neutrophils without a shift
to the left, with 65Zn the changes in neutrophils were more sharply marked.
As well as accelerated maturation of neutrophils at the myelocyte and
promyelocyte stage, there was considerable delay in maturation of the
neutrophils at the rod-nuclear stage. In the peripheral blood, together
with increase in the total number of neutrophils, the nucleus of the neutro­
phils displayed a shift to the left. It is important to note that in the first
and second groups the effect was very similar despite the tenfold difference
in doses.
After administration of 1 (JiCi/kg 65Zn without carrier the relative and
absolute number of juvenile cells of the neutrophil series increased gradu­
ally in the bone marrow despite the brevity of exposure. In the same way
as in the preceding groups, the absolute number of neutrophils in the pe­
ripheral blood rose. The intensified release into the blood of rod nuclear
neutrophils produced a shift to the left in the nuclear formula of the neutro­
phils. Similar findings in relation to neutrophil leucopoiesis were obtained
after administration of 65Zn in an amount of 0-1 [JiCi/kg.
Thus, in relation to neutrophil leucopoiesis the picture observed was
characteristic of 65Zn. This distinctive pattern of change is also underlined
by the fact that it was manifest for all doses of 65Zn with and without
carrier. The stable isotope of zinc disturbed the processes of maturation
in the neutrophils much more weakly. It is possible that the changes found
were connected with the relatively high content of zinc in the neutrophils.
It is known that one leucocyte contains 25 times more zinc than one
erythrocyte (Vallee and Gibson, 1948; Wolff, 1956). It may also be sup­
posed that its preferential incorporation into the granules of the neutro­
phils rich in ribonucleic acids changes the cell metabolism and disturbs
differentiation.
Changes in the protein fractions of the blood serum were found only
in groups of rabbits receiving 10 (JiCi/kg 65Zn and 0-1 mg/kg stable zinc.
These changes were manifest in a significant fall in the a, ft and y-globulin
fractions appearing nine months after the start of administration. By the
twelfth month these disturbances had returned to normal but again ap­
peared on immunization and reimmunization of rabbits (Orlyanskaya). It
appears that when the changes run along the same lines radiozinc deepens
the effect of stable zinc. 65Zn in a dose of 1 [id/kg with and without stable
carrier in the same way as a dose of 0-1 [xCi/kg did not produce significant
changes in the protein fractions.
No fundamental changes were seen in carbohydrate metabolism (Orlyan-
8 The Toxicology of Radioactive Substances
skaya) although the role of zinc in this process led us to expect them. The
constancy of the blood sugar level observed indicates that the sugar-fixing
function of the liver remains unchanged for all the doses of radiozinc and
its stable analogue employed by us. The only change noted was in the
character of the sugar curve following administration of glucose in rabbits
of the first group especially at late times during chronic exposure. In the
rabbits of this group there was an absence of rise in the sugar level, as
compared with the physiological control and stable zinc groups, 60 min
after administration of glucose. This was possibly associated with the
stimulating effect of low doses of 65Zn on the insulin apparatus.
Distinct changes in neutrophil leucopoiesis made it necessary to study
the physiological properties of the neutrophils. These investigations were
carried out by Volkova in animals receiving 0*1 [JiCi/kg 65Zn. Phasic changes
were observed in the phagocytic activity of the blood neutrophils. In phase
I (from the third to the fifth month) there was a moderate fall in the in-
gestive function of the neutrophils and the phagocytic capacity of the
blood. The intensity of ingestion by the neutrophils was not outside the
limits of physiological variation.
In phase II the ingestive function of the phagocytes was sharply acti­
vated as a result of increase in the number of absorbed microbes. Phase
III began in the eighth month and was characterized by slight fluctuations
in the phagocytic activity of the neutrophils against a background of
quite high phagocytic capacity of the blood due to sustained neutro-
phylesis. Immunization of the animals carried out as functional tests on the
state of immunity produced in this phase a further fall in the phagocytic
activity of the neutrophils.
Investigation of certain other aspects of immunological reactivity car­
ried out by Filatov showed considerable changes in the rabbits of the
first and second groups. Thus, study of the effect of 65Zn on unspecific
immunity factors, namely, changes in serum complement of the burdened
rabbits showed considerable fall in the complement activity for the ani­
mals of both groups. Immunization of the rabbits with antigens from the
rat liver carried out 12 months from the start of daily administration of
radiozinc in an amount of 10 and 1 (JiCi/kg brought to light considerable
depression of the processes of formation of complement-fixing antibodies
while in rabbits of the first group changes in immunogenesis appeared
earlier and were more marked than in animals of the second group. In
the rabbits of the third group (0-1 (xCi/kg) only a certain delay in formation
of complement-fixing antibodies was noted. All the same, in these animals
comparative study of accumulation of antibodies by the method of com­
plement-fixation and the ring-precipitation test revealed a change in the
avidity of the antibodies for antigen in the rabbits receiving 65Zn.