1
Morphohistological Changes in Liver, Spleen and Thymus Under Action
of Cu (II) Chelate of Ethyl Ether Salicylidene DL- β- phenyl- α- alanine and
Its Composition with ZnO in Presence of Subcutaneous Tumor
Gasparyan H.V¹·², Arakelova E. R³, Ghazaryan S. H.¹·²
1. Yerevan Medical Institute after Mehrabyan
2. The Scientific-Technological Centre of Organic and Pharmaceutical Chemistry of NAS RA
3. State Engineering University of Armenia
Key words: liver, spleen, thymus, aminoacid chelates, tumor, hystology
Introduction
It’s common knowledge from literature that derivatives of several amino acids have marked
antioxidant activity and are able to inhibit development of tumor tissues in organism,
meanwhile most of these amino acids moderately decrease toxicity of a number of antitumor
preparations, which, in its turn, leads to the preservation of histostructure and integrity of
inner organs in different pathological situations. /1, 2, 15/
Besides, from literature we can find out that several chelates are able to regulate various
physiological processes progressing in cells and tissues of live organism thus changing the
direction of proliferation and differentiation in several organs, and in its turn leading to
increase in immune-protective abilities of the organism in general /3/.
In this viewpoint, complex compounds of amino acids with various transition metals are of
special interest, as they possess some biological activities /anti-inflammatory, phagocytal,
antivirus/ etc /4/.
At present antitumor and anti-metastatic properties of these derivative amino acids have also
been detected /5.6/.
The presented data wouldn’t be sufficient, if not to take into consideration, in case of effect
on tumor with therapeutic agents /medicine, radiation etc/, it’s important to determine the
character of morphological changes caused in the organs and organ systems, which, in its
turn, allows to judge about therapeutic intervention.
Studying tumor before and in the process of treatment gives precise notion about the effect
of therapeutic agent. Consequently, in our opinion, the studies of effect of new amino acids
and their chelates on morphofunctional state of organs and organ-systems in the organism is
one of the perspective directions in chemotherapy of malignant formations.
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Based on these notions, complex compounds of Cu (II) chelate of ethyl ether salicylidene
DL-β-phenyl-α-alanine and its composition with ZnO are of special interest, as these
compounds cause oppression of growth of subcutaneous tumor /7/.
To determine morphofunctional state of tumor tissues and studies organs, hematoxylin-eosin
staining is usually enough in fine cuts filled with paraffin.
Taking into consideration the abovementioned literature sources, the studies of
morphofunctional state of liver, thymus, spleen, as well as the dynamics of change in
histostructure in ‘clean’ subcutaneous tumor, is one of the most important problems in
modern medicine. The importance of histological studies of the aforementioned organs is
because in different pathological conditions they more completely reveal immune
mechanisms of the organism, as well as sensitivity to chemotherapeutic effects.
Materials and Methods
Under experiment tasks, we took biopsy samples from the following groups:
1. Intact group – healthy animals
2. Control group – presence of subcutaneous tumor
3. Experimental group – animals with tumors who received chemical compound Cu (II)
chelate of ethyl ether salicylidene DL- β-phenyl-α-alanine.
4. Experimental group – animals with tumors who received under analogical conditions
chemical compound Cu (II) chelate of ethyl ether salicylidene DL- β- phenyl- α- alanine and
its composition with ZnO.
After dissection and visual examination of internal organs of animals, all studied organs
were fixed in spirit, formalin to conduct further histological, histochemical, and
morphometrical studies. Biopsy materials were taken from ‘pure’ tumor in Groups 2, 3 and 4.
The obtained microtome cuts were stained with hematoxylin-eosin by Van-Gizon technique
to assess common morphological changes in liver, thymus, spleen and nature of tumor tissue
of white mice /8/. Nucleocytoplasmic ratio of tumor tissue is determined with the help of the
morphometric ocular grid of G.G. Avtandilov /9/.
The histomorphological and morphometric methods mentioned by us are sufficiently
informative to assess dynamics of changes in the studied organs in experimental animals.
Histomorphological studies in the tissues of aforementioned organs were conducted with the
help of "Jeneval" microscope. Used objectives were as follows: GF Plan 25 x / 0.50 – 0.17A/,
GF – Plan 40x / 0.65 ~ 0.17A/ , oc. GF - PW 10x, which includes re-equipped digital photo
camera CANON 400 D with 8 megapixel matrix connected with microscope and computer
with a system of adapters of the same model.
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RESULTS AND DISCUSSION
It’s known that hepatocyte synthesizes a large amount of substances of protein and nonprotein nature playing a definite role in the metabolism of all substances especially for
antitumor preparations, it actively takes part in the processes of detoxication and excretion of
endogenous and exogenous factors from the organism taking part in LP (Lipid peroxidation)
processes etc /10,11/.
It can be concluded from the aforementioned data that liver plays an important role for the
support of general homeostasis of the organism. Based on this, the studies of its morphohistological characteristics under current experimental conditions are rather topical.
Histological studies showed that both the control group /Group 2/ and experimental animals
having received Cu (II) chelate of ethyl ether salicylidene DL- β- phenyl- α- alanine /Group
3/, hepatic parenchyma undergoes significant morphohistological disturbances. Fissures have
been expanded, where nidi of lymphohystiocyte infiltrations of different sizes are detected in
the stroma of organism.
In the acini and portals of triads of hepatic parenchyma, phenomena of ballooning
degeneration and isosmotic swelling have been detected in the animals of Group 3. Like
Group 2, Group 3 infiltrating nidi of lymphoid nature are often observed in separate lobules
of hepatic parenchyma /Fig. 2 and 3/.
Another morphological picture has been detected in hepatic parenchyma under the effect of
Cu (II) chelate of ethyl ether salicylidene DL-β- phenyl-α- alanine with ZnO. As compared
with Group 3, the histological picture in the hepatic tissue comparatively normalizes in
animals having received Cu (II) chelate of ethyl ether salicylidene DL- β- phenyl- α- alanine
with ZnO. In most cases, the structure of hepatic tissue is preserved, nevertheless, processes
of karyorrhexis and plasmatosis are simultaneously observed. It should be noted that they a
little are worse in quantity and quality than the indicators in Groups 2 and 3. The number of
karyolyses and karyopyknosis hepatocytes has relatively reduced in peripheral and central
parts of the organ, as well as nidi of infiltrations of lymphoid nature.
Reduction in destructive and dystrophic processes in hepatic parenchyma has been observed
/Figure 1/.
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Fig. 1 / with ZnO/
Fig. 2 /Control/
Fig. 3 / compounds without ZnO
Liver in hematoxylin-eosin staining, oc.10, ob. 25
It’s known that marked involutional processes occur in thymus in case of different shapes of
cancer, that is the formation and preservation of immunological functions decreases leading
to immunological deficiency /12/.
Besides, it’s important to cite that the whole process of developing Т-lymphocytes from stem
cells of the marrow is carried out through thymus, as well as humoral and cellular immunity
of the organism is disturbed in case of immunological deficiency /13, 16/. Hereby it could be
concluded there are compounds, which can help to preserve or recover histostructure of the
thymus, and they have important significance for the recovery of protective mechanisms of
organism.
Histological studies showed that as compared with Group 3 /Fig. 5/, destructive and
dystrophic changes have relatively been suppressed in the group having received Cu (II)
chelate of ethyl ether salicylidene DL- β- phenyl- α- alanine with ZnO. Organ lobules are
easily differentiable, involutional processes reduce /Fig. 6/. Macrophage elements are
frequently observed, which witness the activation of reticular cells of thymus, as these cells
produce specific proteins for maturing thymocytes in definite stages /14/.
Besides, nidi of destroying red pulp are often observed in the thymus with the presence of
lymphoid infiltration etc.
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Fig. 4 /Control/
Fig. 5 /without ZnO/
Fig. 6 /compounds with ZnO/
Thymus in hematoxylin-eosin staining, oc.10, ob.25
Almost analogical picture is observed in case of histological studies of spleen. In the spleen of
Groups 2 and 3 /Fig. 7,8/ together with marked histomorphological changes of histostructure
of this organ under the effect of Cu (II) chelate of ethyl ether salicylidene DL-β-phenyl-αalanine with ZnO, there is mainly tendency towards recovery, though nidi of destroying red
pulp are recorded in spleen pulps and sinuses, and sections of leukemic infiltrations in
sinuses /Fig. 9/.
Fig. 7 /Control/
Fig. 8 /without ZnO/
Fig. 9 /compounds with ZnO/
Spleen in hematoxylin-eosin staining, oc.10, ob.25
The obtained histological data enable us to judge that under the effect of Cu (II) chelate of
ethyl ether salicylidene DL- β-phenyl-α-alanine with ZnO normalization of histostructure
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occurs, as well as metabolic state of hepatocytes, thymocytes and cells of spleen tissue
normalize thus, to some extent, increasing immune protection of the organism in general.
Together with the obtained data, radically contrary data were obtained when studying
morphohistological characteristics of subcutaneous tumor under analogical experimental
conditions.
Studies showed that as compared with control group /Group 2 – Fig. 10/, experimental
compounds result in significant decrease in parenchymatous elements, meanwhile observing
destruction of the tumor stroma itself. Unlike control group, both groups are marked with
destruction of tumor histostructure, infiltration processes become more frequent, nidi of
lipophanerosis are detected. Nidi of lymphoid infiltrations, edemas, necroses, haemorrhages
etc are often observed in all experimental groups /Fig. 11, 12/. It can be concluded that under
the effect of Cu (II) chelate of ethyl ether salicylidene DL-β-phenyl-α-alanine with ZnO,
marked destruction of tumor tissue occurs, thereby proving that this chemical compound
possesses antitumor activity.
Fig. 10 /Control/
Fig. 11 /without ZnO/
Fig. 12 /compounds with ZnO/
Tumor tissue, in hematoxylin-eosin staining, oc.10, ob.25
It’s the first time we attempted to metrically assess the effect of studied compounds on the
morphofunctional state of tumor tissue through morphometric ocular grid of G.G.
Avtandilov. As parenchymatous (aggressive) cells mainly dominate in tumor tissue, the
decrease in their amount in various situations may be qualified as suppression of tumor tissue
activity.
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Taking into consideration the aforementioned, it can be definitely stated that the coefficient
of ratio matching points on parenchymatous elements and points matched on tissue stroma
enables to assess the functional state of tumor tissue.
Micrometric studies showed that PSC /parenchyma-stroma coefficient/ of the control group
significantly exceeds the PSC of Groups 3 and 4, thus once again proving the moderate
antitumor activities of selected compounds / Table 1/.
Parenchymal-stromal coefficient of tumor tissue
20
18
17,8±0.01
The average number of cells
16
14±0.01
14
13,8±0.01
11,2±0.02
12
11±0.02
10
Parenchymatous elements
7,2±0.01
8
stroma
6
4
2
0
K
K=2.47
Chel
K=1.3
Chel+ZnO
K=1.2
The table shows, if the PSC of the control group makes up 2.47 ± 0.02 /conditionally-100%/,
the coefficient of Groups 3 and 4 makes up 1.27± 0.001 and 1.23± 0.001 /51,4% and 49.8%/,
respectively.
Thus, morphometric data showed, if both compounds have almost the same effect on tumor
tissue, histological studies of liver, thymus and spleen showed favourable effect of Cu (II)
Chelate of ethyl ether salicylidene DL- β- phenyl- α- alanine with ZnO on the histostructure
of the studied organs, thereby proving its relatively low-toxic effect than that Cu (II) chelate
of ethyl ether salicylidene DL- β-phenyl-α-alanine.
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