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УДК 615:099.031 DOI 10.33632/1998-698Х.2021-1-9-14
ACETAMINOPHEN INDUCED HEPATITIS IN LABORATORY ANIMALS
V.O. Dombrovskiy, V.I. Egorov - Candidate of Biological Sciences, I.I. Idiyatov - Candidate of Biological Sciences
FSBSI "Federal Center for Toxicological, Radiation and Biological Safety", 420075, Kazan Scientific town-2, e-mail: vnivi@mail.ru.
Toxic hepatitis is common in human and veterinary medicine. One of the methods for modeling hepatitis is the administration of acetaminophen. In human medicine Acetaminophen overdose is the most frequent cause of ALF (Acute Liver Failure). Acute toxic hepatitis caused by acetaminophen reversible process, however chronic acetaminophen hepatitis, judging by the
change in the relative size of the liver and its structure, as well as the level of serum enzymes, is an irreversible process that leads to deep systemic damage to the organism.
Keywords: acetaminophen, acute hepatitis, chronic hepatitis.
Introduction. The liver is all the important functions of the body, its functions are multifaceted, but the most important is detoxification and bile synthesis. The liver is the main target organ of drugs. The liver, as the "main laboratory of the body", is constantly confronted with many potentially toxic substances that enter the body in a variety of ways, mainly through the gastrointestinal tract. There are no drugs that under certain conditions do not cause liver damage. After metabolic transformations, some of them become toxic. Hepatotoxic factors are various xenobiotics that enter the body with food, water, and air. These include antibiotics, cytostatics, pesticides, mycotoxins, etc [12,15].
Liver diseases in animals occupy one of the leading places in the list of diseases from the total number of non-infectious pathologies. Animal husbandry suffers significant economic damage from hepatopathies due to the death of animals, decreased productivity, reproductive ability, resistance, the development of many infectious and non-communicable diseases against this background, as well as an increase in direct material costs for therapeutic and preventive measures [2].
The study of the diverse aspects of the pathology of hepatic function in diseases of various etiologies is especially important due to the fact that, due to the exceptional compensatory potentials of this organ, their clinical manifestations are often detected already at the stage of severe morphofunctional disorders, often not amenable to reverse development. This increases the relevance of studying pathogenesis, developing methods for early diagnosis and correction of the functional state of the liver as the main organ for stabilizing the body's homeostasis in normal and pathological conditions [8].
The liver is the largest gland of the body, performing many functions necessary for maintaining life, playing a major role in the metabolism (biosynthesis, secretion, detoxification and excretion) of various substances. One of the main functions of the liver is the
barrier - the neutralization of poisons and toxins, as well as protecting the body from pathogenic bacteria. The organ has great functional capabilities, but the liver is a highly aerobic, oxygen-dependent tissue, which leads to high sensitivity of hepatocytes to the damaging effects of various toxicants. The response of the body to external negative effects is a condition called oxidative stress - an increase in oxidative reactions in organs and tissues, induced by harmful environmental factors, accompanied by an increase in the concentration of free radical forms of metabolites that disrupt metabolic and energy processes in the body, as well as leading to destruction cell membranes and pathologically altered cell functions, including hepatocytes. Pathological changes in the liver affect all organs and systems, provoking the occurrence of diseases of various nosologies [10].
Liver failure in model animals can be caused in various ways. These include: 1) surgical routes - hepatectomy and primary vessels; 2) toxic liver damage (carbon tetrachloride, thioacetamide, dipin, etc.); 3) special diets (choline-deficient, etc.); 4) a combination of hepatotoxins with hepato-carcinogens that suppress the proliferation of hepatocytes; 5) a combination of retrorcin (DNA-binding pyrrolisidine alkaloid) with hepatectomy; 6) genetic models. Modeling of toxic drug hepatitis is a necessary component of preclinical studies of hepatoprotective and antioxidant drugs, which are used not only in the practice of a veterinarian, but also in humane medicine [14].
In acute liver failure syndrome, the biochemical blood parameters, especially the catalytic activity of organspecific enzymes (alkaline pho-sphatase, cholinesterase, alanine transferase, aspa-rtate transferase, lactate dehydrogenase), most dramatically change [9].
Some drugs that are rapidly metabolized and lead to the formation of toxic intermediate products - metabolites that accumulate in tissues in large quantities, destroy its structural components.
In this regard, it can be assumed that medicinal substances in tissues should cause
reverse processes and use compensatory possibilities that lead to gross structural deformations.
Such drugs include non-steroidal antiinflammatory drugs (NSAIDs) that inhibit cyclooxygenase-2 (COX-2) and reduce prostaglandin synthesis. These substances include acetaminophen (APAP) [16]. Paracetamol, also called acetaminophen or 4-hydroxyacetanilide or N-acetyl-para-aminophenol (APAP), being a non-steroidal anti-inflammatory drug (NSAID), was first synthesized in 1878 by Morse and first used clinically by von Mering in 1887 [5]. In human medicine Acetaminophen overdose is the most frequent cause of ALF (Acute Liver Failure) in the USA [6]. Therapeutic doses are safe, however toxic metabolites occurring during overdose cause severe organ toxicity [11].
Unlike NSAIDs, whose mechanism of action is associated with the blockade of the active center of cyclooxygenase, the ligand of which is arachidonic acid, acetaminophen is able to inhibit the synthesis of prostaglandins, but only in conditions of low concentration of peroxidases. Accordingly, acetaminophen is not active in the focus of inflammation, where the level of peroxidases is high, but effective in the brain under conditions of a low concentration of this class of enzymes [1].
Acetaminophen (APAP) is a well-known analgesic and antipyretic drug. It is considered to be safe when administered within its therapeutic range, in humans and some animals, but in cases of acute intoxication, hepatotoxicity can occur. APAP overdose is the leading cause of acute liver failure in the northern hemisphere [19]. Therefore paracetamol, is one of the best-known experimental models of hepatotoxicity [13].
The toxicity of acetaminophen is due to the interaction of its reactive metabolite N-acetyl-p-benzokquinone with liver proteins and a decrease in the level of glutathione in hepatocytes [4]. Acetaminophen - induced liver damage is associated mainly with excessive formation of reactive oxygen and nitrogen forms forming the reactive intermediate N-acetyl-p-benzoqu-inoneimine. More than 80% of the administered dose of acetaminophen is easily detoxified in the liver by glucuronidation or sulfation. In the processes of detoxification of N-acetyl-p-benzoquinoneimine, conjugates of reduced glutathione are involved. When excess metabolite is produced [16].
APAP causes a fatal hepatic necrosis and hepatic failure in overdose along with oxidative stress such as lipid, DNA and protein peroxidation, significant decrease in hepatic GSH (reduced glutathione (GSH)) levels, and alteration in the antioxidant enzyme system, decrease in the activity of hepatic d-aminolevulinic acid dehydratase), and an increase in the various inflammatory cytokines [5].
After high doses of APAP, the enzyme systems which metabolize APAP via conjugation with sulfate or glucuronide become saturated and the formation of the toxic intermediate metabolite "N-acetyl-p-benzoquinoneimine" (NAPQI) increases. NAPQI causes rapid depletion of the limited glutathione (GSH) stores of the liver, irreversibly binds to hepatocytes and causes liver necrosis [5].
APAP hepatotoxicity is dependent on cytochrome P450 (CYP) enzymes that metabolize APAP to the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). At a therapeutic dose, around 90% of APAP undergoes glucuronidation and sulfation before excretion, while 5-10% of APAP is metabolized by CYP enzymes (mainly the CYP2E1 subtype) to form NAPQI, which is normally detoxified and excreted in the urine by conjugation with glutathione (GSH) [11].
Acetaminophen toxicosis is usually associated with single acute ingestion, and the primary target organs affected are the liver and the red blood cells in dogs and cats respectively. Because signs can progress rapidly with acute acetaminophen overdose, administration of N-acetylcysteine is always recommended, even when the history is unclear [3]. With a single injection of acetaminophen in a dose exceeding 300 mg 1 kg of body weight, significant changes in the liver are observed in a day, leading to the development of acute liver failure [7,17].
The widely known hepatotoxicity of acetaminophen in acute overdoses, while variants of its toxicity, especially pathogenetic structural and functional changes poorly studied [8,11].
In addition to liver damage in case of acetaminophen poisoning, a nephrotoxic effect can also occur. Potential mechanisms of nephro-toxicity in an overdose of APAP, due to cytochrome P450, as well as prostagla-ndinsy-nthetase and the enzyme N-deacetylase.
Materials and methods. The study was conducted on the basis of the Department of
Toxicology of the Federal Center for Toxico-logical, Radiation and Biological Safety (Federal Center for Toxicological, Radiation and Biological Safety ), Kazan. Acute hepatitis was simulated in an experiment on laboratory animals; for this, 12 of white rats were selected, divided by the principle of analogues into 3 groups. The first served as a biological control, animals of the second group received intra-gastrically acetami-nophen at a dose of 1000 mg / kg body weight in the form of an aqueous suspension twice with an interval of 24 hours. Rats of the third group received acetaminophen at a dose of 500 mg / kg, twice a week, for a month.
The action of acetaminophen was monitored the clinical condition of experimental animals, by analyzing the content of hepatic enzymes in the blood serum (aspartate amino-transferase (AST), alanine aminotra-nsferase
(ALT), pathomorphological picture, and also determining the relative mass of the liver. Blood sampling was performed on days 7 and 30 after the start of the experiment. Decapitation was performed 30 days after the start of the experiment.
Statistical processing of the obtained digital material was carried out by the method of variation statistics using the Microsoft Excel program.
Results. During the study period, the clinical condition of white rats of the acute hepatitis group did not differ from the control, while the animals of the third group significantly lost weight by the end of the experiment, and inhibition and experimental apathy of the experimental subjects were observed. Feed and water intake were consistent with physic-ological needs. No deaths were noted
Table 1 - Relative liver mass of white rats with acute and chronic hepatitis induced by acetaminophen_
Index Group
I II III
Relative liver mass, mg / g 30,05±0,89 32,45±044 39,05±0,68*
Note: * - differences wit h control are significant wit 1 accuracy p < 0.05
A tendency toward an increase in the mass of the liver of experimental groups two and four was noted. Thus, the relative liver mass of animals receiving acetaminophen chronically was 1.3 times higher than the control, and 1.08 times higher in the acute experience group.
Against the background of acute toxic hepatitis, the liver mass had no significant differences in comparison with the control (Table 1).The results of the study of the content of liver enzymes in the blood serum of experimental rats are shown in table 2 and 3.
Table 2 - The results of a biochemical study of blood serum on the 7th day
Index Group
I II III
AST, u/l 107,83±5,44 631,38±39,87* 285,725±14,12*
ALT, u/l 58,95±4,17 192,2±3,2* 99,75±0,65*
Note: * - differences with control are significant with an accuracy of p < 0.05.
In comparison with the control group, the activity of aspartate aminotransferase in animals of the second group significantly increased in comparison with the control by
485,5%, alanine aminotransferase - by 226%. The activity of liver enzymes of the third group also increased: ALT - by 164.97%, AST - by 69,2%.
Table 3 - The results of a biochemical study of blood serum on the 30th day after the first seed.
Index Group
I II III
AST, u/l i20,0±2,39 274,30±24,00* 3i7,55±i0,69*
ALT, u/l 52,1±4,32 112,25±9,02* 200,03±7,41*
Note: * - differences with control are significant with an accuracy of p < 0.05.
Thus, the activity of aspartate aspartate aminotransferaseo in animals of the second group significantly increased in comparison with the control by 128.5%, alanine aminotransferase - by 115.4%. In the third group, an increase in the level of liver enzymes AST was also noted - by 164.4%, ALT - 283.9%.
When decapitating animals, the presence of yellowness of the organs of the gastrointestinal tract was detected in the third group of experimental animals to a greater extent, in the third group - to a lesser extent. In animals of the biological control group, the liver had a dark cherry color, while the organ of the third group, against the background of acetaminophen poisoning, was painted in light brown colors, with focal granularity. The
liver of rats of the second group was cherry in color with foci of enlightenment.
Conclusion. Based on the studies, it can be concluded that acute toxic hepatitis can be caused by a single injection of acetaminophen at a dose of 1000 mg / kg. Based on the data of biochemical and pathomorphological studies, acute toxic hepatitis is a reversible process, even without treatment, since 21 days after the administration of acetaminophen, hepatic enzymes significantly decreased, relative to the 7th day of the experiment. Chronic acetaminophen hepatitis, judging by the change in the relative size of the liver and its structure, as well as the level of serum enzymes, is an irreversible process that leads to deep systemic damage to the body.
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УДК 619:615.849:636.92:616.993.192.1 DOI 10.33632/1998-698Х.2021-1-14-19
КОМБИНИРОВАННОЕ ВОЗДЕЙСТВИЕ у-ИЗЛУЧЕНИЯ И ЭЙМЕРИЙ НА ОРГАНИЗМ КРОЛИКОВ
А.М. Идрисов- кандидат ветеринарных наук, доцент, Т.Р. Гайнутдинов- кандидат биологических наук, К.Н. Вагин - кандидат биологических наук, Н.М. Василевский. -доктор ветеринарных наук, профессор, Ф.Х. Калимуллин - кандидат биологических наук, Е.Н. Майорова - кандидат биологических наук, Г.И. Рахматуллина - кандидат
биологических наук.
ФГБНУ «Федеральный центр токсикологической, радиационной и биологической безопасности» 420075, г. Казань Научный городок-2;
e-mail: vnivi@mail.ru.
