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Corresponding author: reznichenko6531@gmail.com
Hepatoprotective effect of hypoxenum against exogenous
toxicosis of white rats
Aleksei Reznichenko*, Svetlana Vodianitskaia, Iana Masalykina, and Andrei Manokhin
Belgorod State Agricultural University named after V.Y. Gorin, Belgorod, 308503, Russia
Abstract. Any disturbances in the organism caused by infection, drug administration, vaccination, etc., are
accompanied with disturbance of liver functions. Various xenobiotics entering animal organisms with food
or water have the highest hepatotoxicity. Thus, an important trend in modern research is a search for
substances increasing liver capacity against pathological activities, strengthening its detergent actions. The
goal of this research was to study hepatoprotector properties of hypoxenum using the experimental acute
toxic hepatitis model in white rats. The acute toxic hepatitis was caused by injecting the rats abdominally
with tetrachloromethane in medical paraffin at a dose of 0.4 ml per 100 g of live weight once a day for
3 days. This resulted in disturbance of hepatocytes’ cytoplasmic membranes, accompanied with an
increased rate of transamination enzymes and alkaline phosphotase, as well as an acute drop in glucose and
total protein. Administration of hypoxenum stopped this pathological process. After the administration of
the preparation, the animal’s body weight increased, activity of transamination enzymes and alkaline
phosphotase returned to physiological norm, protein and glucose content increased, general physiological
condition of the white rats improved. Thus, hypoxenum may be administered to animals as a
hepatoprotector at a dose of 50.0 mg/kg of live weight.
1 Introduction
Currently,
there is an active ongoing search for
substances increasing liver capacity against
pathological activities, strengthening its detergent
actions.
The most commonly, liver damage is observed
in large farms where high concentration of animals
requires continuous use of antibacterial preparations,
vaccines and other drugs aimed at preventing
development of infectious diseases in animals [1, 2].
Multiplicity of liver functions leads to the fact that a
disturbance in any kind of metabolism affects this organ,
leading to cell damage and development of a new, more
severe pathological process or complicating the main
condition.
Recently, negative impact of a number of medical
preparations on liver was discovered, whose
hepatotoxicity acutely increases with their
biotransformation due to formation of active metabolites.
It has been established that poisoning with xenobiotics
and metabolites of hormones and proteins accumulated
in the body causes toxicosis and promotes increased
intensity of lipid peroxidation (LPO).
Hepatoprotective effect may be seen from
preparations that improve metabolic processes in the
body, as well as those inhibiting lipid peroxidation
and having antihypoxic activity
[3, 4].
Studies of toxic damage to liver attracted attention
from many scientists [5–7]. Their general opinion is that
deep dystrophic changes in the liver appear under the
influence of hepatotoxic factors against the background
of deficiency of biologically active substances. In
particular, toxic substances entering the body with food
and being formed inside the body due to indigestion and
disturbance of intermediary metabolism have direct
influence on hepatocytes when they find their way into
blood and reach the animal’s liver . Depending on the
amount and duration of their admission to the organ’s
parenchyma, activity of oxidizing enzymes reduces,
glycogen level drops sharply, fatty liver infiltration
develops, cytolysis of liver cells with subsequent
necrosis are observed. Excessive accumulation of toxins
in the body and inability of physiological detoxification
systems to ensure their effective removal lead to
endogenic intoxication [8, 9].
According to S.B. Matveev, medium molecular
weight substances are universal biochemical markers of
endogenic toxicosis. Such substances are represented by
intermediate and end products of normal and disrupted
protein and lipid metabolism; they accumulate in the
body in amounts exceeding normal concentrations and
are products of free radical lipid peroxidation,
intermediary metabolism, medium molecular weight
peptides [10].
In case of liver damage independent of its etiology,
the leading pathomorphological symptom is structural
cell damage, leading to increased permeability and (or)
destruction of hepatocyte membranes and membrabes of
various organellas, development of hyperenzymemia of
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 27, 00079 (2020) https://doi.org/10.1051/bioconf/20202700079
FIES 2020
mitochondrial enzyme aspartate aminotransferase and
cytoplasmic enzyme alanine aminotransferase [ 11].
In recent years, a strong case has been presented for
the fact that lipid peroxidation processes is an important
mechanism in hepatocyte damage and/or progression of
chronic diffuse liver diseases. Currently, there is an
active ongoing search for substances increasing liver
capacity against pathological activities, strengthening its
detergent actions by means of increasing the activity of
cytolytic enzymes and cytochrome Р-450, facilitating
restoration of liver functions after various damages [12].
Hepatoprotective effect may be seen from
preparations that improve metabolic processes in the
body, as well as those inhibiting lipid peroxidation and
having antihypoxic activity [13].
Due to that, the authors tested hypoxenum, a
preparation with antioxidant properties.
The goal of this research was to study
hepatoprotective properties of hypoxenum using the
experimental acute toxic hepatitis model in white rats.
The following tasks were involved in attaining the set
goal:
• to induce toxic hepatitis in white rats by
administration of tetrachloromethane;
• to establish hepatoprotective effect of hypoxenum
and compare it with that of heptran.
2 Material and method of analysis
The acute toxic hepatitis was caused by injecting the rats
abdominally with tetrachloromethane in medical paraffin
at a dose of 0.4 ml per 100 g of live weight once a day
for 3 days [14].
Hypoxenum and heptran were employed for
treatment of the animals.
Effectiveness of the preparations was evaluated by
changes in body mass, clinical conditions of the white
rats and biochemical indicators of blood serum; the latter
were determined with biochemical analyzer.
Research data were subjected to mathematical
processing according to N. A.Plokhinsky [15],
calculating average values (М), coefficients of variation
(m) and significance test value (р). Differences were
held significant at р<0.05.
Hypoxenum (sodium salt of [poly-(2,5-
dyhydroxyphenylen)]-4-tiosulphonic acid) is a black
powder, odorless or with a weak specific odor. The
preparation is produced by Petrokhim (Belgorod,
Russia).
Heptran is a complex preparation that has
hepatoprotective and antistress activity, 1 ml of heptran
contains 50 mg of carnitine, 200 mg of magnesium
sulphate, 220 mg of sorbiton, 30 mg of
cyanocobalamine, 8 mg of calcium pantotenate, 20 mg
of nicotineamid, auxiliary and forming substances.
3 Test results and discussion
Four groups of 6 white outbred male rats with a weight
of 160–180 g each were selected for the experiment.
The first group was control, others were experimental
ones. The first group did not receive any preparations
(intact animals). The rats of the second, third and fourth
group were administered with a 50 % emulsion of
tetrachloromethane in petroleum jelly by introperitoneal
injection in a dose of 4.0 ml/kg of live weight for three
days. Immediately after ending administration of
tetrachloromethane, the animals in the third group were
administered hypoxenum for 7 days, at a dose of 50
mg/kg of live weight. The fourth group was administered
heptran at a dose of 1 ml/l of water for the same period
of time
The design of the experiment is shown in Table 1.
Table 1. Design of the experiment
Groups Administered
preparation Amount of
Control 1
_
experimental 2
tetrachloromethane 4.0 ml/kg of
live weight
3 hypoxenum +
tetrachloromethane 50 mg/kg of
live weight
4 Heptran +
tetrachloromethane 1 ml/l of
water
Blood sampling took place on days 7 and 14 after the
beginning of the experiment.
Body weight of the experimental animals (Table 2)
was monitored before administration of
tetrachloromethane, after finishing its administration, as
well as in days 7 and 14 (end of the experiment).
Table 2. Changes in body weight of rats
Indicators Groups
1 2 3 4
Control
tetrachloromethane tetrachloromethane +
hypoxenum tetrachloromethane +
heptran
Before administering
tetrachloromethane, g 162.6±3.7 164.2±4.1 166.1±5.0 163.2±3.8
After administering
tetrachloromethane, g 162.8±3.3 157.3±2.8 156.2±4.1 154.2±3.1
Day 7. 173.1±2.9 150.4±3.2 167.7±3.9 166.2±2.4
Day 14, (before culling), g 181.2±3.9 157.4±3.1 186.2±3.9 180.1±2.9
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From the data in the table, it is evident that a gradual
decrease in weight began in the second group right after
administration of tetrachloromethane. At that, its
maximum values were reached in Day 7 of the
experiment, witnessing to development of toxic
hepatitis; after that, the body weight started a slow
increase and by the end of the experiment, it was 4.1 %
lower than the initial values.
In the third experimental group, hypoxenum had a
positive effect on animal organisms. So, administration of
the preparation not only stopped reduction in weight of the
animals after development of toxic hepatitis, but facilitated
its increase – in the end of the experiment the weight of the
animals exceeded the initial values by 12.2 %.
In the fourth experimental group, the body weight of
the animals after administration of heptran exceeded the
initial values by 10.3 % by the end of the experiment,
witnessing to hepatoprotective action of the preparation.
Biochemical indicators of the animal blood are given
in Table 3.
Table 3. Biochemical parameters of rat blood
Indicators Groups
1 2 3 4
Control
tetrachloromethane hypoxenum
tetrachloromethane Heptran +
tetrachloromethane
7 days after administration of preparations
AST units/l 188.7±5.36 222.6±5.36** 193.6±5.44 197.1±5.39
AST units/l 98.3±5.36 119.1±5.21* 101.3±5.13 104.4±5.43
Albumines, g/l 30.3±1.69 29.7±1.89 34.2±0.67 33.8±1.89
Total protein, g/l 67.6±1.23 63.8±1.02* 69.8±1.37 68.8±1.29
Urea, mmol/l 6.74±0.57 8.33±0.65 6.69±0.73 7.92±0.49
Creatinine, mg/dl 0.48±0.32 0.59±0.35 0.46±0.39 0.49±0.38
Bilirubin, mg/dl 0.50±0.17 0.72±0.19 0.51±0.12 0.53±0.14
Cholesterol, mmol/l 1.14±0.15 1.69±0.16* 1.28±0.20 1.32±0.21
Glucose, mmol/l 9.27±0.78 6.24±0.82* 9.13±1.11 9.24±0.97
Alkaline phosphotase, units/l
350.0±4.87 392.9±4.89** 352.6±5.18 357.1±5.22
In the end of the experiment
AST units/l 178.7±6.32 203.2±6.29* 180.9±6.89 181.3±5.76
AST units/l 92.8±5.47 109.6±5.58* 94.4±4.35 97.6±5.29
Albumines, g/l 32.7±1.29 30.4±0.96 38.1±1.39* 36.7±1.14
Total protein, g/l 68.9±1.45 66.0±1.58 72.3±1.40 72.7±1.51
Urea, Mmol/l 6.79±0.57 7.14±0.69 6.47±0.64 7.11±0.65
Creatinine, mg/dl 0.48±0.22 0.50±0.39 0.54±0.31 0.49±0.36
Bilirubin, mg/dl 0.56±0.19 0.59±0.15 0.53±0.09 0.58±0.31
Cholesterol, mmol/l 1.23±0.26 1.57±0.21 1.27±0.23 1.30±0.29
Glucose, Mmol/l 9.26±0.98 5.92±0.96* 9.97±1.11 8.96±1.05
Alkaline phosphotase, u/L 351.8±5.21 380.0±4.96* 350.8±5.29 357.3±5.49
Note * – р <0.05
From the data in the table, it is evident that
administration of tetrachloromethane led to damage to
cytoplasmic membranes of hepatocytes, reflected in an
increased activity of transamination enzymes in blood
serum: in the second group, 7 days after the
administration, activity of aspartate aminotranspherase
and alanine aminotranspherase increased by 17.9 and
21.1 % respectively. In the end of the experiment, this
increase amounted to 13.7 and 18.1 %, respectively (in
all cases р< 0.01–0.05).
As it is known, increased activity of transamination
enzymes in blood serum is an objective indicator of
damage to liver parenchyma. In healthy animals, enzyme
concentration inside hepatocytes is significantly higher
than in blood serum. When hepatocytes are damaged,
this serum-cell gradient is acutely disrupted. Cytolysis of
liver parenchyma is accompanied with increase in
permeability of hepatocyte cell membranes and
membranes of cell organellas; at that, cytoplasmic,
mitochondrial and lysosomic enzymes are released in
circulation [13].
Administration of hypoxenum stopped this
pathological process. For instance, in the third
experimental group, activity of aspartate
aminotranspherase and alanine aminotranspherase
increased only by 2.6 and 3.1 % respectively on Day 7 of
the experiment. By the end of the experiment, activity of
these enzymes was increased by 1.7 and 1.8 %, however
this difference with control was found to be statistically
insignificant.
As for heptran, its activity was also effective
throughout the experiment. For instance, on Day 7, there
was an increase in activity of aspartate
aminotranspherase and alanine aminotranspherase by 4.4
and 6.2 %. In the end of the experiment activity of these
enzymes exceeded the controls by 1.5 and 5.2 %
(р>0.05).
After the rats were administered tetrachloromethane,
in the second group there was observed a reduction of
total protein content in the blood serum by 5.6 % (Day 7)
and by 4.2 % (in the end of the experiment), however,
only the change at Day 7 was statistically significant
(р<0.05).
It means that tetrachloromethane disturbs metabolism
of individual amino acids in the body. As it is well
known, liver takes an active part in metabolism of
3
BIO Web of Conferences 27, 00079 (2020) https://doi.org/10.1051/bioconf/20202700079
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methionine. When it is damaged, the mechanism of
transsulfuration is disturbed, resulting in a reduced
content of cysteine and glutathione in blood. The latter is
an important link in the hepatocytes’ antioxidant system.
In case of ССl
4
-induced hepatitis, hypoproteinemia is
observed as a result of inhibited protein synthesis and
degradation of amino acids.
In the third and fourth experimental group, after
administration of hypoxenum and heptran, the protein
level was even somewhat high, however, these changes
were not deemed statistically significant.
The research data demonstrate that the studied
preparations normalize protein synthesis in liver
parenchyma, previously inhibited in toxic hepatitis,
improve deactivation of protein breakdown products,
prevent development of endogenic toxicosis.
Administration of tetrachloromethane to rats caused a
sharp reduction of glucose content in the blood serum of
the animals in the second group by 32.7 % by Day 7 and
by 36.1 % by Day 14 in comparison with controls. It
means that development of the toxic hepatitis involves a
hypoglycaemia against the background of a sharp drop in
glycogen content in the liver as a result of inhibited
glyconeogenesis and amplified glycogenolysis. At that,
synthesis of insulinases that degrade insulin is disrupted.
After administration of hypoxenum in the third
group, the glucose level first reduced somewhat (by
1.5 % on Day 7) and increased by 7.6 % in the end of the
experiment. However, these changes were deemed
statistically insignificant.
After administration of heptran, fluctuations of
glucose content in blood serum were small and did not
show statistical difference from control.
Thus, both preparations are effective in preventing
development of hypoglycaemia.
As for alkaline phosphotase, after administration of
tetrachloromethane, its content in blood serum of the
second group of animals increased by 12.3 % on the Day
7 and by 8.0 % in the end of the experiment in
comparison with the controls (р<0.05), witnessing to
damaged liver parenchyma. As it is known, alkaline
phosphotase is an excretory enzyme; it is a complex of
isoenzymes of various tissues with prevalence of
enzymes of liver, osseal and intestinal origin.
After administration of hypoxenum and heptran in
third and fourth experimental groups, alkaline
phosphotase was within the physiological norm and
showed no practical difference from that of control
animals.
Thus, the research confirmed that hypoxenum and
heptran have a hepatoprotective action on animal
organism. Both studied preparations arrested the
development of a toxic hepatitis in rats, reflected in
decrease in the level of transamination enzymes and
alkaline phosphotase, and increase in the content of
protein and glucose in blood serum. This is an evidence
of restored functioning of hepatocytes and normalized
functioning of the organism.
4 Conclusion
As a result of the research, hypoxenum may be
recommended as an effective hepatoprotector
preparation for farm animals. Recommended dosage is
50.0 mg/kg of live weight; recommended route of
administration is with water.
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