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7PHARMACEUTICS
BILE FORMATION FUNCTION OF LIVER IN CASESS OF ANTI-TUBERCULOSIS DRUGS
AFFECTION IN RATS
Garlitska N.
Ph.D., Associate Professor, Department of General Chemistry, Faculty of Pharmacy, I. Horbachevsky Ter-
nopil National Medical University, Ternopil, Ukraine
Fira L.
DSc, Professor, Head of Pharmacy Department, Educational Scientific Institute of Postgraduate Education,
I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine
Kachur O.
Ph.D., Assistant Professor, Department of General Chemistry, Faculty of Pharmacy, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine https://doi.org/10.5281/zenodo.6778468
Abstract
The disorders in hepatocytes plasma membranes permeability were defined by the increased alkaline phos-phatase activity in blood serum which was decreased in the liver. It was determined that total bilirubin and bile acids content in blood serum of the affected animals increased. It influenced hepatocytes excretion in bile capillaries and caused cholestasis and revenues decrease in bile. The most pronounced disorders of bile formation function in cases of isoniazid and rifampicin affection was evidenced in the organism of immature and senior animals in comparison with mature animals.
Keywords: isoniazid, rifampicin, bile formation function.
Introduction. Biliary function is a vital function of the liver, which results from the sequential vectorial transport of endogenous and exogenous substrates through three compartments [17]. Bile is produced by hepatocytes and it is then modified by the cholangio-cytes lining the bile ducts. The production and secretion of bile require active transport systems within hepatocytes and cholangiocytes in addition to a structurally and functionally intact biliary tree. Initially, hepatocytes produce bile by secreting conjugated bilirubin, bile salts, cholesterol, phospholipids, proteins, ions, and water into their canaliculi [2].
Hepatocellular insufficiency syndrome is characterized by hyperbilirubinemia due to its unconjugated fraction [14]. Hyperbilirubinemia indicates impaired absorption, conjugation and excretion of bilirubin in the bile, as a toxic damage of liver occurs parenchymal jaundice [23].
Recent studies indicate the existence of a strong correlation between hepatic injury and oxidant stress in experimental animals treated with anti-tuberculosis drugs [13, 15]. Since all the drugs used in the treatment of tuberculosis are shown to have hepatotoxic effects, studies have been performed to prevent or reduce the toxicity by the use of natural herbal drugs and/or synthetic compounds, without interfering with the therapeutic actions of the drugs.
Rifampicin, isoniazid, pyrazinamide and etham-butol are first line drugs used for the treatment of tuberculosis. According to the Centre of monitoring of adverse reactions of drugs, isoniazid - 29.2%, rifampicin - 26.7%, capreomycin - 17.1%, ethambutol - 10.2% dominate among monopreparations in high incidence of the adverse reactions in world [3]. Thus, the risk of the development of hepatitis increases in patients who take rifampicin together with isoniazid. In this case
hepatitis incidence is 5-8%. During the isoniazid monotherapy, the incidence of hepatitis is 1.2%, but during the rifampin monotherapy - 0.3% [3].
According to the researches [16, 24] the hepato-toxicity of isoniazid may be developed in two ways:
1. The accumulation of free radicals with activation of lipid peroxidation and the formation of reactive metabolites: acetylisoniazid, hydrazine, monoacetylhy-drazine;
2. The increased activity of N-acetylisoniazid by N-hydroxylation and formation of acetyl radical and acetyl carbonium ion.
The metabolism of acetyl hydrazine and microsomal monooxygenases cause hepatotoxic effect as a result of the covalent addition of acetyl groups to the liver proteins [16], which manifest as temporary asymptomatic increase of transaminases activity. The hepato-toxicity of rifampicin is also due to the formation of toxic metabolites as a result of its deacetylation in the liver that leads to hepatocytes dystrophy [18, 20].
Several in vivo studies found that rifampicin plus isoniazid induced hepatocyte apoptosis in rodent animals [7, 19]. The mechanism through which rifampicin induces liver injury remains obscure. An earlier study demonstrates that oxidative stress in the mitochondria is involved in the pathogenesis of rifampicin plus iso-niazid-induced apoptotic liver cell injury in mice [6].
The objective of the study was to investigate the bile formation function of liver in different age groups of rats that were intoxication by isoniazid and rifam-picin.
Materials and methods. The experiments were conducted on outbred white male rats of three age groups: the 1st group -immature (3-month-old animals, 90-110 g in weight); 2nd group -mature (6-month-old animals, 150-170 g in weight); and the 3rd group -senile
(18-months-old animals, 280-300 g in weight). They were kept on a standard diet at the vivarium of Ternopil National Medical University.
The sustentation and manipulations of animals were carried out according to the "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes" [10].
All experimental animals of each age group were divided into two groups: the control group - intact animals (injected with a physiological solution); the experimental group - animals that were administered isonia-zid and rifampicin. In the each study group 6 animals were selected.
The experimental toxic affection of animals was simulated by combined effect of isoniazid and rifam-picin. Isoniazid and rifampicin were administered intra-gastrically in aqueous solution to animals every day, 0.05 g/kg and 0.25 g/kg accordingly, during the 7th and 14th days. Euthanasia was performed by means of thiopental sodium (25 mg/kg) on the 7th and 14th day from the first day of anti-tuberculosis drugs administration.
The study of liver homogenate and blood serum was performed. The blood was taken from the heart of animals by centrifugation at 3000 rpm during 30 min. The obtained blood serum (a sedimentary liquid) was used for researches. Selected liver (250 mg) was used to obtain the homogenate by the method of differential homogenization; it was used after previous perfusion in physiological solution.
The activity of liver enzyme markers was determined by the alkaline phosphatase activity (ALP) (the reagents of OOO NPP Filisit- Diagnostics, Ukraine) in blood serum and liver homogenate. Estimation of the alkaline phosphatase activity was based on the property of the enzyme to hydrolyse the etheric bond in P-glyc-erophosphate and eliminate phosphoric acid. Phosphorus was determined by colorimetric method due to the reaction with molybdenum reagent in the presence of a reducing eikonogen or ascorbic acid. The product of reaction was molybdenum blue; its colour intensity was directly proportional to the amount of phosphorus in the simple evaluation of the enzyme activity [21].
The bile formation function of liver in animals was defined by the content of total bilirubin (TB) and bile acids (BAs) in blood serum. The total bilirubin content
was determined by caffeine reagent, which together with diazotized sulphanilic acid formed pink-purple colour of azobilirubin. The colour intensity of this solution was directly proportional to the concentration of total bilirubin in the sample. Evaluation of total biliru-bin in blood serum was performed by the calibration graph, mmol / L [21]. Determination of bile acids content was based on the reaction of colour products formation by condensation, which interacted with bile acids and oxymethyl furfural. These solutions were obtained from fructose. They are the products of hydrolysis by adding concentrated sulfuric acid to sucrose. The bile acids content was evaluated by the calibration graph due to the tauroholic acid content, g / L [21].
The processing of statistical data was carried out in the SPSS-22 software package. The distribution of data is analyzed according to Kolmogorov-Smirnov's criterion of normality. The obtained values had a non-parametric distribution, so the difference between the groups was analyzed according to the Student's t-crite-rion and the non-parametric Wilcoxon's criterion for the connected samples. The criterion x2 was used to evaluate the difference between categorical data. The difference of probability values was p>0.95 (P significance level). Differences were considered probable at p<0.05. [9]
Results and discussion. The metabolism of acetyl hydrazine and microsomal monooxygenases cause hepatotoxic effect as a result of the covalent addition of acetyl groups to the liver proteins [22]. The hepatotox-icity of rifampicin is also due to the formation of toxic metabolites as a result of its deacetylation in the liver that leads to hepatocytes dystrophy [6].
Based on the above, we studied alkaline phospha-tase activity in blood serum and liver of the affected rats (Table 1). Alkaline phosphatases are a group of isoenzymes, located on the outer layer of the cell membrane; they catalyze the hydrolysis of organic phosphate esters present in the extracellular space. Although alkaline phosphatases are present in different body tissues and have different physiochemical properties, they are true isoenzymes because they catalyze the same reaction. In the liver, alkaline phosphatase is cytosolic and present in the canalicular membrane of the hepatocyte [5].
Table 1
Alkaline phosphatase activity in blood serum (nmol / s-L) and liver (nmol / s-g) of rats affected by isoniazid and rifampicin (M±m)
Research material Group of animals Age group of animals
immature mature senior
Research duration, days
7th 14th 7th 14th 7th 14th
Control group 1924.64±113.32 2405.80±159.13 3007.25±240.58
Blood serum
Experimental 3247.83± 3849.28± 4210.15± 4540.94± 4330.43± 4781.52±
group 192.09* 206.60* 159.13* 142.96* 208.35* 137.81*
Control group 739.78±30.90 1338.23±54.21 1705.11±74.21
Liver
Experimental 562.35± 496.20± 941.27± 836.01± 1341.23± 1235.14±
group 19.44* 20.17* 38.82* 35.68* 30.79* 16.64*
Note: here and in the following tables * - significant differences between the animals of intact controls and the affected animals, p< 0.05.
On the 7th day of anti-tuberculosis drugs administration ALP activity increased in blood serum by 69% in immature animals in comparison with the control group (p<0.05), by 75% - in mature animals and by 44% - in senior animals. The senior animals proved to be the least sensitive. We evidenced the highest ALP activity in blood serum at the end of research in the immature animals, which was 200% in comparison with intact animals (p<0.05).
In liver of the experimental group, this enzyme decreased in the immature rats by 24% on the 7th day of the experiment and by 33% on the 14th day in comparison with the control group (p<0.05), in the mature rats - by 30% and 38%, in the senior rats - by 21% and 28%, respectively (Table 1). The lowest alkaline phosphatase activity was on the 14th day of research in the mature animals after the effect of isoniazid and rifam-picin (836.01 ± 35.68) nmol / (sg) that is in 1.6 times lower than in the control group (1338.23 ± 54.21 nmol / (s-g)).
Increased hepatic enzyme activity demonstrably parallels the rise in serum alkaline phosphatase activity; this occurs primarily due to increased translation of the mRNA of alkaline phosphatase and increased secretion of alkaline phosphatase into serum via canalicular leakage into the hepatic sinusoid [12]. Studies report that vesicles containing alkaline phosphatase, and many
such enzymes bound to the sinusoidal membranes, are found in the serum of patients with cholestasis. Because alkaline phosphatase is newly synthesized in response to biliary obstruction, its serum level may be normal in the early phase of acute biliary obstruction even when the serum aminotransferases are already at their peak [8].
It was determined that increase in alkaline phos-phatase activity in blood serum of all age rats' groups after the administration of anti-tuberculosis drugs into their bodies cause the release of ALP out of the damaged hepatocytes as well as the restoration of its synthesis in bile tubules. We consider that this dynamic activity of alkaline phosphatase may evidence the development of hepatocytes destruction and intrahepatic cholestasis caused by liver architectonics damage and possible development of cirrhosis.
We evidenced a significant increase (p <0.05) in total bilirubin (TB) content in blood serum of all age animals in comparison with control rats (Table 2). Bilirubin is a breakdown product of hem, which is released from red blood cell lysis. Serum bilirubin level represents hepatic synthetic and excretory function well; hence, most well-recognized prognostic models including child-pugh score and model for end-stage liver disease score have serum TB as a component [11].
Table 2
Total bilirubin content in blood serum (jimol / L) of rats affected by isoniazid and __rifampicin (M±m)_
Group of animals Age group of animals
immature mature senior
Research duration, days
7th 14th 7th 14th 7th 14th
Control group 12.19±0.55 12.49±0.47 15.09±0.78
Experimental group 13.38±0.69 18.68±1.50* 16.53±1.17* 17.53±1.61* 18.97±1.12* 19.25±1.27*
The TB content in blood serum was increased by 10% in the immature animals, by 32% - in the mature animals and by 26% - in the senior animals in comparison with the intact control (p<0.05) on the 7th day of the experiment. The combined action of antituberculosis drugs had led to an even greater increase of TB content already on the 14th day of the experiment. It was 153% in animals of the immature age, in animals of the mature age - 140%, and in animals of the senior age -128% (Table 2). The most susceptible to isoniazid and rifampicin were immature rats; the TB content in blood serum exceeded the level of the intact control (p<0.05) by 53% till the end of the experiment.
It was established that hepatotoxicity of the metabolites of isoniazid and rifampicin caused the lipid peroxidation of hepatocyte biomembranes and bile formation dysfunction. Rifampicin can also inhibit the glucuronil-transaminases and cause bilirubin metabolism disorders and jaundice [7]. Increase in total biliru-bin content under the influence of the toxicants evidenced the damage of cell membranes and erythrocytes predominantly and decrease in hemolysis as well as liver excretory dysfunction.
The researched results of bile acids (BAs) content in blood serum of all age rats are presented in Table 3. BAs are synthesized from cholesterol by hepatocytes and conjugated with either taurine or glycine. The accumulation of cytotoxic BAs can induce an inflammatory response and trigger hepatocyte apoptosis and necrosis. If left untreated, cholestasis will cause liver damage, liver fibrosis, cirrhosis, and even liver failure [4].
On the 7th day of research the BAs content increased by 42% in the immature animals, by 54% - in the mature animals and by 50% - in the senior animals if compared with the animals of the intact control (p<0.05).
On the 14th day in the immature and the mature animals the BAs content increased in 1.71 and 1.76 times, respectively, in comparison with animals of the intact control (p<0.05). The maximum increase of BAs content in blood serum was recorded in the senior animals on the 14th day of research. It was 180% in comparison with the control group (p<0.05).
Table 3
Bile acids content in blood serum (g / L) of rats affected by isoniazid and rifampicin (M±m)_
Group of animals Age group of animals
immature mature senior
Research duration, days
7th 14th 7th 14th 7th 14th
Control group 6.95±0.43 9.48±0.58 12.02±0.64
Experimental group 9.90±0.49* 11.89±0.55* 14.60±0.8* 16.69±0.65* 18.09±0.87* 21.59±0.84*
It was established, that in case of drug-induced hepatitis the intestines and liver suffer from the affection, which was caused by the disorders of biosynthesis and hepatoenteral circulation of bile acids. The increase in bile acids content in blood serum of affected animals may have the toxic effect on hepatocyte mitochondria that caused increase in ions permeability to internal membrane of mitochondria, ions swelling and release of cytochrome C into cytosol as well as cells apoptosis. The immature and senior animals were the most sensitive to bile formation function after administration of isoniazid and rifampicin.
Conclusions. We determined the increase of alkaline phosphatase activity in blood serum and its decrease in liver. It proved the toxic effect of anti-tuberculosis drugs on liver of all age animals. It evidences the development of hepatocytes destruction and intra-hepatic cholestasis caused by liver architectonics damage and possible development of cirrhosis. It was characterized by accumulation of bile acids and total biliru-bin as well as other bile components in blood that could inhibit the synthesis of components complement in hepatocytes. The most pronounced disorders of bile formation function in cases of isoniazid and rifampicin affection was evidenced in the organism of immature and senior animals in comparison with mature animals.
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