НОФАЛЬ А.Е., ПОТАПОВ А.С., ХЛЕБНИКОВ А.И.

5. Donatia, A.; Ventrutia, A.; Cavallinia,G.; Masinia, treated with the CYP2E1 inducer pyrazole . J. Physiol.
M.; Vittorinia, S.; Chantretb, I.; Codognob, P.; and Gastro. and Liver Physiol., (289)308.
Bergamini, E. (2008): In vivo effect of an antilipolytic 12. Locci C. T. and Bergamini, E. (1982): Peroxisomal
drug (3,5-dimethylpyrazole) on autophagic proteolysis enzyme activities in regenerating liver of rats. Bull.
and autophagy-related gene expression in rat liver. Res. Exp. Med., 181(3): 225-230.
Biochem. and Biophys. Res. Commun., 366 : 786– 13. Locci C. T. and Bergamini, E. (1983): Effects of
792. antilipolytic agents on peroxisomal beta-oxidation of
6. Lillie, R.D. and Fulmer, H.M. (1976): fatty acids in rat liver. Biochem. Pharmacol., (32)11:
Histopathological Technique and Practical 1807-1809.
Histochemistry. 4th edn. New York, Mc Graw Hill. 14. Bergamini, E.; Tata, V. D.E.; Loccr Cubeddu, T.,
7. Pearse, A. G. E. (1972): Histochemistry, Theoretical Masiello, P. and Pollera, M. (1987): Increased
and Applied, 3rd end., vol. 2. Churchill Livingstone. degradation in rat liver induced by antilipolytic
London. agents:A model for studying autophagy and protein
8. Potapov, A. S.; Nudnova, E. A.; Domina, G. A.; degradation in Liver. Exp. and mol. pathol., 46: 114-
Kirpotina, L. N.; Quinn, M. T.; Khlebnikov, A. I. и 122.
Schepetkin, I. A. (2009): Synthesis, characterization 15. Donati, A. (2006): The involvement of
and potent superoxide dismutase-like activity of novel macroautophagy in aging and anti-aging interventions,
bis(pyrazole)–2,2-bipyridyl mixed ligand copper(II) Mol. Aspects Med., 27: 455–470.
complexes. Complexes // Dalton Trans.; 4488-4498. 16. Lu, Y. and Cederbaum, A. I. (2006):
9. Wilson, W.L. and Bottiglieri, N.G. (1962): Phase I Enhancement by pyrazole of lipopolysaccharide-
studies with pyrazole. Can. Chem., 21: 137-141. induced liver injury in mice: role of cytochrome P450
10. Lelbach, W.K. (1969): Liver cell nercrosis in rats 2E1 and 2A5. Hepatol., (44)1:263-274.
after prolonged ethanol ingestion under the influence 17. Bergamini, E. and Segal, H.L. (1987): Effects of
of an alcoholdehydrogenase inhibitor. Experientia, 25: Antilipolytic drugs on hepatic peroxisomes. Biol. and
816-818. Med.., 295-303.
11. Yongke, Lu.; Xiaodong, W. and Cederbaum, A. I.
(2005): Lipopolysaccharide-induced liver injury in rats

TOXICOLOGICAL ASSESSMENT OF EFFECT OF 1,5-BIS(3,5-

DIMETHYLPYRAZOL-1-YL)-3-OXAPENTANE-DIACETATOCOPPER
ON ALBINO RAT LIVER
Nofal A., Potaрov A.S., Khlebnikov A.I.

The present study provides evidence that 1,5-Bis (3,5-dimethylpyrazol-1-yl)-3-oxapentane-

diacetatocopper has a toxicological and histopathological effect on the liver. The present study re-
vealed many histopathological alterations on the liver; inflammatory infiltration, marked vacuolated
cytoplasm, congested blood vessels, hemorrhage, pyknotic and binucleated cells, as well as, some of
the degenerated cells showed karyorhexis, pyknosis and necrosis. Sera of animals treated with 1,5-
Bis (3,5-dimethylpyrazol-1-yl)-3-oxapentane-diacetatocopper revealed a significant decrease in glu-
cose and albumin.
Ключевые слова: histopathological alterations, glucose and albumin.

INTRODUCTION tilipolytic agent (one of agents that inhib-

its lipolysis). 3,5-Dimethylpyrazole markedly de-
The liver is a vital organ, essential for life. pressed plasma fatty acid and blood sugar after
Its functions center mostly around taking up 15 minutes to 3 hours of its administration [2].
molecules and macromolecules from the blood, The administration of antilipolytic drugs 3,5-
enzymatically modifying them, and eventually dimethylpyrazole to rats induced significant de-
returning them to the bloodstream in different crease in plasma fatty acid in 15 min, glucose
forms for distribution to the body's cells and tis- and insulin [3].
sues [1]. The administration of rat by antilipolytic
3,5-Dimethylpyrazole as a hypoglycemic drugs (3,5 dimethylpyrazole at dose 12mg/Kg
agent (one of various agents that decrease the body weight) revealed that the peroxisomal en-
level of glucose in the blood and are used in the zyme activities decreased significantly in 2-3
treatment of diabetes mellitus) and as an-
38 ПОЛЗУНОВСКИЙ ВЕСТНИК № 3 2014
ТОКСИКОЛОГИЧЕСКОЕ ИССЛЕДОВАНИЕ ВЛИЯНИЯ 1,5-БИС(3,5-ДИМЕТИЛПИРАЗОЛ-1-ИЛ)-3-
ОКСАПЕНТАН-ДИАЦЕТАТОМЕДИ НА ПЕЧЕНЬ БЕЛЫХ КРЫС

hours which increased degradation of liver pro- and stirring the mixture for 24 hours at room
teins [4]. temperature. Green crystals formed were filtered
Bergamini et al. [5], showed similar results and dried in vacuo.
of elevated activities of alanine aminotransferase 1,5-Bis(3,5-Dimethylpyrazol-1-yl)-3-oxa-
(ALT) and aspartate aminotransferase (AST) pentanediacetatocopper (C18H28N4O5-Cu) was
when rats administered with 3,5- dissolved in 0.9% mammalian saline (9 gm so-
dimethylpyrazole at dose 12 mg/kg body weight. dium chloride dissolved in 1000 ml distilled wa-
Either LPS-induced liver injury in mice was en- ter) and injected intraperitoneally (ip) at dose 12
hanced by pyrazole, as indicated by pathological mg/kg body weight /day [3].
changes and increases in ALT and AST. LPS-
induced oxidative stress was also enhanced EXPERIMENTAL DESIGN
by pyrazole [6].
Treatment of rats with pyrazole elevated The animals were divided into 2 groups.
the hepatic microsomal dimethylnitrosamine de- 1- Control group:
methylase activity (DMNd) by several fold [7]. Animals of this group (24 rats) were main-
Pyrazole administration increased the toxicity of tained on normal diet throughout the whole ex-
dimethylnitrosamine when measured as a 50% perimental period. They were sacrificed after
lethal dose or as a histopathological effect on different times parallel to that of treated groups.
the liver [8]. 2- Group 2:
The administration of antilipolytic drug (3,5 Animals of this group (24 rats) were in-
dimethylpyrazole at dose 12 mg/kg body weight) jected daily for 6 weeks intraperitoneally (ip) by
revealed as early as 30 min many vacuolated 1,5-Bis (3,5-Dimethylpyrazol-1-yl)-3-oxapentane-
lysosomes at the electron microscopic level and diacetatocopper freshly dissolved in saline (12
autophagic vacuoles are observed in the liver mg/kg bw/ day). Animals were then sacrificed 2,
cells after 1 hour. After 1 hr and 45 min, vacu- 4 and 6 weeks after beginning of the treatment,
oles often contain recognizable peroxisome [4]. 8 animals in each period were sacrificed 2 hr
Antilipolytic drugs induced both autophagic pro- after injection.
teolysis and higher expression of an autopagy Histological preparation
related gene. The effect of antilipolytic drug on For light microscopic studies, immediately
autophagy gene expression might not be secon- after sacrification, liver was removed carefully
dary to the stimulation of autophagic proteolysis and quickly fixed in 10% neutral formalin for 24
[3]. hr, washed in running tap water, fixed and stored
in 70% ethyl alcohol. Tissue pieces were dehy-
MATERIAL AND METHODS drated in ascending series of ethyl alcohol (70%,
80%, 90% and two changes 100%), cleared in
Animals two changes of xylene and embedded in molten
Healthy adult male albino rats (Rattus paraplast paraffin (mp. 50-58 oC). Sections of 5
norvegius), approximately three months old and microns thickness were cut using rotary micro-
weight (120 ± 5) g were used in the present tome (Leica, Model Rm 2125, Germany), and
study. The animals were kept under constant mounted on clean slides without using any ad-
condition of temperature for at least two weeks hesive medium. For histological examination,
before the experimental period. Animals were sections were stained with Ehrlich's haematoxy-
maintained on a standard diet, manufactured lin and counterstained with eosin [10].
especially for laboratory purposes, obtained from Biochemical analysis
Atimida Company for national development. Wa- Blood samples were taken from portal vein
ter was available ad libitum. Animals were kept and left to coagulate at 37ºC in incubator, centri-
under constant temperature (30±2Cº) and the fugated at 174 g/min for 15 min (1550 r.p.m) with
humidity was 45±5%with 12:12 light-dark cycle. centrifuge (Shanghai Surgical Instruments Fac-
Chemical used tory, Model 9-1). Sera were stored at -20Cº until
The ligand 1,5-bis (3,5-dimethylpyrazol-1- further analysis. Specimens from control and all
yl)-3-oxapentane was prepared following a pre- treated groups were obtained for examination
viously described procedure [9]. after 2, 4 and 6 weeks from starting the experi-
1,5-Bis-(3,5-dimethylpyrazol-1-yl)-3- ment.
oxapentane (C18H28N4O5-Cu) was prepared by Serum albumin
adding a solution of 1,5-bis(3,5-dimethylpyrazol- Serum albumin was determined according
1-yl)-3-oxapentane (0.262 g, 1 mmol) in 2 ml of the method of Doumas et al. [11].
acetone to a suspension of Cu(CH3Coo)2 H2O
(0.199 g, 1 mmol) in 2 ml of the same solvent
ПОЛЗУНОВСКИЙ ВЕСТНИК № 3 2014 39
 НОФАЛЬ А.Е., ПОТАПОВ А.С., ХЛЕБНИКОВ А.И.

Serum glucose cells showed nuclei with signs of karyolysis,

Serum glucose was determined according pyknosis and necrosis.
the method of Bergmeyer et al. [12]. Biochemical results
Statistical analysis Serum Albumin (g/dL).
All biochemical results were expressed as Intraperitoneal administration of 1,5-
mean ± standard error. The results were ana- Bis(3,5-Dimethylpyrazol-1-yl)-3-oxapentane-
lyzed statistically utilizing computer program diacetatocopper to rats revealed that biochemi-
(Excel) with two levels of significance at P≤0.05 cal determination of albumin after 2, 4 and 6
& P≤0.01 denote low and high significant weeks of injection clearly exhibited significant
changes from control, respectively. decrease compared with control group.
Serum glucose (mg/dl).
RESULTS Intraperitoneal administration of 1,5-
Bis(3,5-Dimethylpyrazol-1-yl)-3-oxapentanedi-
Histological observations acetatocopper to rats revealed that biochemical
The hepatocytes of control rats arranged in determination of serum glucose after 2, 4 and 6
strands around the central vein with one or two weeks of injection clearly exhibited significant
spherical nuclei and eosinophilic cytoplasm. decrease compared with control group.
Blood sinusoids are occupied by phagocytic
Kupffer cells. DISCUSSION
Animals treated with 1,5-Bis(3,5-
Dimethylpyrazol-1-yl)-3-oxapentane-diacetato- Complex 1,5-bis(3,5-dimethylpyrazol-1-yl)-
copper intraperitoneally at dose 12 mg/kg 3-oxapentane diacetatocopper was prepared by
showed many histological abnormalities the reaction of ligand and copper(II) acetate
throughout the whole experimental periods. Liver monohydrate in acetone solution similary to pre-
sections after 2 weeks of treatment, exhibited viously reported procedure for copper nitrate
abnormal arrangement of hepatic strands, in- complexes [13]. The proposed structure for this
flammatory infiltration around widened blood compound was confirmed by UV-Vis and IR
vessel, few condensed cells, binucleated cells spectroscopy, molar conductivity measurements
and degenerated liver cells were observed with and elemental analysis data.
enlarged congested blood vessel. Moreover, Our results indicated that treating rats with
slight widened blood sinusoids, slight cytoplas- 1,5-Bis (3,5-Dimethylpyrazol-1-yl)-3-oxapentane-
mic vacuolation, activated enlarged Kupffer cells diacetatocopper caused many histopathological
and fatty degeneration were. alterations in the liver structures, inflammatory
Liver sections after 4 weeks of treatment infiltration, swelling and marked enlarged vacuo-
manifested loss of normal hepatic architecture. lated cytoplasm in cells, congestion of blood
Inflammatory infiltration was observed, the bile vessels, hemorrhage, pyknotic cells and binu-
ductule was surrounded by inflammatory cells cleated cells, as well as, some of the degener-
and fibrosis with collagen deposition. Few Kup- ated cells showed karyorhexis, pyknosis and
pfer cells and widened enlarged sinusoids were area of necrosis. Large spaces were detected in
seen. Congested blood vessel and increase in some areas due to degeneration of cells. Wide
central vein size with hemorrhage were ob- spread area of necrosis and hyperplasia. Simi-
served. Steatosis, condensed cells and binucle- larly, Rats injected intraperitoneally with pyrazole
ated cells also were seen. Large spaces were at dose 200 mg/kg body wt, once per day for 2
detected in some areas due to degeneration of days, pyrazole produced swelling of mitochon-
hepatocytes. dria and induced liver histopathology & liver in-
Liver sections after 6 weeks of the same jury [14].
previous treatment, showed severe changes In human beings primary toxicity of pyra-
including disarrangement of hepatic strands and zole was found in the liver, kidney and bone
loss of normal hepatic structure. Dense lympho- marrow [15]. Livers of rats dying 6 to 11 days
cytic infiltration around the central vein and dark after the administration of pyrazole showed ex-
stained hepatocytic nuclei indicating cell pykno- tensive centrolobular necrosis with inflammatory
sis. Swelling and marked enlarged vacuolated reactions in the parenchyma as well as fatty infil-
cytoplasm in parenchymal cells with condensed tration of the surviving cells [16].
nuclei were noticed. Congested blood vessel Known effects of antilipolytic agents (3,5-
with hemorrhage, pyknotic cells and binucleated dimethylpyrazole at dose 12 mg/kg body weight)
cells. Some of the degenerated cells showed may be related to features of rat liver autophagy
karyorhexis and other degenerated cells lost [17]. The administration of antilipolytic drug (3,5-
their boundaries. Wide spread area of necrosis dimethylpyrazole at dose 12 mg/kg body weight)
and hyperplasia within the liver lobules and revealed as early as 30 min many vacuolated
around central veins and the portal tracts. Most lysosomes at the electron microscopic level and
40 ПОЛЗУНОВСКИЙ ВЕСТНИК № 3 2014
ТОКСИКОЛОГИЧЕСКОЕ ИССЛЕДОВАНИЕ ВЛИЯНИЯ 1,5-БИС(3,5-ДИМЕТИЛПИРАЗОЛ-1-ИЛ)-3-
ОКСАПЕНТАН-ДИАЦЕТАТОМЕДИ НА ПЕЧЕНЬ БЕЛЫХ КРЫС

autophagic vacuoles are observed in the liver model for studying autophagy and protein degradation
cells after 1 hour. After 1 hr and 45 min, vacu- in Liver. Exp. and mol. pathol., 46: 114- 122.
oles often contain recognizable peroxisome [3]. 6. Lu, Y. and Cederbaum, A. I. (2006): Enhancement
by pyrazole of lipopolysaccharide-induced liver injury
Examination of sera of animals treated with
in mice: role of cytochrome P450 2E1 and 2A5.
1,5-Bis(3,5-dimethylpyrazol-1-yl)-3-oxapentane- Hepatol., (44)1:263-274.
diacetatocopper in the present study revealed a 7. Yityoong, Y. Tu.; June S.; Katherine F. L. and
significant decrease in serum glucose and albu- Chung S. Y.(1981): Pyrazole-induced cytochrome P-
min. Several investigators also obtained similar 450 in rat liver microsomes: An isozyme with high
results. In this concern, treatment of fasting rats affinity for dimethylnitrosamine. Biochem. and Bio-
with antilipolytic drugs (either 3,5-dimethyl- phys. Res.Commun., (103) 3: 905–912.
pyrazole at dose 12 mg/kg body weight) resulted 8. Ritva, P. E.; Mandy, M. R.; Emily, H. and Carolyn,
B. (1983): Pyrazole Effects on Mutagenicity and Tox-
in a decrease in free fatty acid and glucose
icity of Dimethylnitrosamine in Wistar Rats. Can.
plasma levels within 5-10 min and in a significant Res., 43: 496.
increase in the plasma glucagon to insulin ratio 9. Potapov, A.S.; Domina, G.A.; Domina, G.A.;
within 15 min. [18]. The administration of an- Khlebnikov, A.I., Ogorodnikov, V.D. (2007): Facile
tilipolytic drugs (3,5-dimethylpyrazole at dose 12 synthesis of flexible Bis(pyrazol-1-yl) alkane and re-
mg/Kg body weight) decreased the concentra- lated ligands in a superbasic medium // Eur. J. Org.
tion of free fatty acids, glucose and insulin in rat Chem.; 5112-5116.
blood plasma (by 10-15 min), and peroxisomal 10. Lillie, R.D. and Fulmer, H.M. (1976): Histopa-
th
thological Technique and Practical Histochemistry. 4
enzyme activities decrease significantly in 2-3
edn. New York, Mc Graw Hill.
hours which play an important role in the control 11. Doumas, B.T; Watsone, A.W. and Biggs, H.G.
of lipid metabolism [2]. (1971): Albumin standards and the measurement of
Stem cells may be stimulated and give rise serum albumin with bromcresol green. Clin. Chem.
to oval cell proliferation in periportal areas, dur- Acta., 31(1): 87-96.
ing chemically induced hepatocarcinogenesis in 12. Bergmeyer, H.U.; Bernt, E.; Schmidt, F. and
rat [19]. Lindros et al. [20] reported that chronic Stork, H. (1974): D-Glucose determination with
treatment rats with the higher dose of hexokinase and glucose-6-phosphate dehydro-
genase. New York: 1196–1201.
4-methylpyrazole seemed to cause a relative 13. Potapov, A.S.; Nudnova, E.A.; Domina, G.A.;
decrease in the liver protein content. Kirpotina, L.N.; Quinn, M.T.; Khlebnikov, A.I.,
The administration of antilipolytic agents Schepetkin, I.A. (2009): Synthesis, characterization
(3,5-dimethylpyrazole at dose 12 mg/kg body and potent superoxide dismutase-like activity of novel
weight) can trigger autophagy and intracellular bis(pyrazole)–2,2-bipyridyl mixed ligand copper(II)
degradation of proteins in rat liver cells. Auto- complexes. Complexes // Dalton Trans.; 4488-4498.
phagic vacuoles are formed in a few minutes 14. Yongke, Lu.; Xiaodong, W. and Cederbaum, A. I.
and their number increases thereafter to reach (2005): Lipopolysaccharide-induced liver injury in rats
treated with the CYP2E1 inducer pyrazole . J. Physiol.
the highest values in a few hours [3]. Gastro. and Liver Physiol., (289)308.
In conclusion, 1,5-bis (3,5-dimethylpyrazol- 15. Wilson, W.L. and Bottiglieri, N.G. (1962): Phase I
1-yl)-3-oxapentane-diacetatocopper has a toxi- studies with pyrazole. Can. Chem., 21: 137-141.
cological effect in blood and liver of albino rats. 16. Lelbach, W.K. (1969): Liver cell nercrosis in rats
after prolonged ethanol ingestion under the influence
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