AcETAmINOPhEN EffEcT ON fREE RAdIcAL OXIdATION INdIcEs IN RATs WITh TYPE 2 dIAbETEs mELLITUs

Background. Acetaminophen is a drug used to relieve pain syndrome. It is used both independently and in composition of combined drugs. Type 2 diabetes is an age-related disease that is associated with a violation of insulin synthesis by pancreas. Objective. The aim of the research was to study the effect of acetaminophen on major free radical oxidation indices of rats with type 2 diabetes mellitus in time dynamics. Methods. We conducted two series of experiments. The first series comprised rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions. The second series involved rats with type 2 diabetes mellitus and acetaminophen administration at a dose of 55 mg/kg for the period of 7 days. Results. Administration of acetaminophen for rats with type 2 diabetes mellitus caused the increase in the content of malondialdehyde, diene and triene conjugates and Schiff bases in blood plasma and malondialdehyde, diene and triene conjugates in liver homogenate. The maximum increase in these indices was observed on the first day of the experiment. Gradually these indices decreased on the 3rd, 5th and 7th days of the experiment. Conclusions. Free radical oxidation increased in both series of the experiment. This process developed in rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions more intensively, than in rats with type 2 diabetes mellitus and administration of acetaminophen at the highest therapeutic dose during 7 days.


Introduction
Acetaminophen is a drug used to relieve pain syndrome.It is used both independently and in composition of combined drugs.A long time it was considered to be the safest drug among the group of analgesics/antipyretics [1,2].Acetaminophen has a relatively low toxicity in therapeutic doses.However, a conscious and often uncontrolled administration of high doses of the drug causes complications that sometimes can lead to death due to hepatic insuffi ciency [3,4,5].
Type 2 diabetes is one of the most common diseases, every year its frequency is steadily increasing.The prevalence of diabetes is associated with changes in environmental factors, especially the populations (genetic, demographic), the concentration of risk factors in the populations (increased body weight, arterial hypertension, cardiovascular diseases, lipid metabolism disorders, etc.).Considering all above, the aim of our research was to study free radical oxidation activity in rats with type 2 diabetes mellitus and acetaminophen toxic lesions.

Methods
The experiments were carried out on white rats weighing 180-220 g on a standard diet and free access to water in vivarium.
We conducted two series of experiments.In the first series toxic lesion was caused by a single intragastric administration of acetami nophen suspension in 2% starch solution for the animals at a dose of 1250 mg/kg (1/2 LD 50 ).In the second series the suspension of acetami nophen in a 2% starch solution at a dose of 55 mg/kg was managed, which corresponds to the highest therapeutic dose during 7 days.Non-genetic form of experimental type 2 dia be tes mellitus was modeled by Islam S., Choi H. method [7,8], that is, a single intraperitoneal administration of streptozotocin solution at a dose of 65 mg/kg (Sigma, USA) to the rats, which was diluted by citrate buffer (pH 4.5) with the previous (15 minutes ahead) intraperitoneal nicotinamide administration at a dose of 230 mg/kg.The rats with the same body weight, which were given the same amount of solvent (citrate buffer pH 4.5), were used as the control group.
In the first series of the experiment, the rats were divided into 4 groups: the 1 st group was the intact (control); the 2 nd group involved a single acetaminophen administration; the 3 rd group comprised the animals with type 2 diabetes mellitus caused by streptozotocin administration; and the 4 th group contained the rats with a single administration of acetaminophen after streptozotocin administration.In the second series of the experiment, the rats were divided into 4 groups: the 1 st group was the intact (control); the 2 nd group was with acetaminophen administration during 7 days; the 3 rd group were the animals with type 2 diabetes mellitus caused by streptozotocin administration; the 4 th group were the rats with administration of acetaminophen during 7 days after streptozotocin administration.
The animals were removed from the experiment on the 5 th , 3 rd , 5 th and 7 th days after last acetaminophen administration by euthanasia under thiopental anaesthesia.All experiments on rats were carried out according to The Guideline Principles for the Care and Use of Laboratory Animals [10].
Evaluation of the content of diene and triene conjugates was carried out by the method [6].Determination of malondialdehyde content was carried out by the method [9].Determination of the contents of the Schiff bases was carried

Results
The content of malondialdehyde, diene and triene conjugates and Schiff bases increased in animals with lesions caused by acetaminophen and type 2 diabetes mellitus.
As presented in Table 1, the content of malondialdehyde in blood plasma increased by 229.2% on the 1 st day of the experiment in the 2 nd group of experimental animals, it increased by 95.5% in the 3 rd group of animals.The maximum of this index increase (in 5.21 times) was observed in the animals with type 2 diabetes mellitus and acute acetaminophen toxic lesions (the 4 th group).This index decreased on the 3 rd and 5 th days of the experiment.The maximal decrease of malondialdehyde content was observed on the 7 th day of the experiment in all groups of animals.
The content of diene and triene conjugates in blood plasma of the animals with single acetaminophen administration (the 2 nd group) increased in 4.11 and 3.36 times on the 1 st day of the experiment to compare with the control animals.These indices increased by 62.8% and 104.8% in the 3 rd group of animals with streptozotocin action.The content of diene conjugates increased in 3.43 times, and triene conjugates in 6.81 times in the animals with type 2 diabetes mellitus and acute acetaminophen toxic lesions (the 4 th group).These indices decreased on the 3 rd , 5 th and 7 th days of the experiment.
The content of Schiff bases increased in 3.4 times on the 1 st day of the experiment in the 2 nd group of experimental animals after acetaminophen administration.This index increased by 101.6% in the 3 rd group of animals with streptozotocin administration.The maximal increase of Schiff bases in 5.26 times was observed in the 4 th group of animals with type 2 diabetes mellitus and single acetaminophen administration.
According to the results (Table 2), the content of malondialdehyde in liver homogenate increased in 4.76 times on the 1 st day of the experiment in the 2 nd group of experimental animals to compare with the control animals, and by 60.8% in the 3 rd group of animals.The maximum of this index increase (in 5.7 times) was observed in the 4 th group of animals.The content of diene and triene conjugates in the experimental animals increased the most in the 4 th group of animals on the 1 st day of the experiment in 6.76 and 6.56 times respectively.These indices increased on the 1 st day of the experiment in 5.88 and 4.85 times in the 2 nd group, and in 2.99 and 3.63 times in the 3 rd group respectively.
The content of malondialdehyde, diene and triene conjugates, Schiff bases increased on the 1 st day of the experiment in blood plasma (Table 3) and liver homogenates (Table 4).These indices decreased on the 3 rd , 5 th , 7 th days in all series of the experiment.These changes were less pronounced than in the 1 st series of the experiment.
In the 2 nd group of experimental animals with acetaminophen administration during 7 days, the content of malondialdehyde in blood plasma on the 1 st day of the experiment increased by 126.5%, in liver homogenate -by 138.6%; diene and triene conjugates in blood plasma increased by 105.9% and 142.3%, in liver homogenate increased in 3.0 and 3.2 times; Schiff bases increased by 134.3% in blood plasma to compare with control animals.
In animals with type 2 diabetes and acetaminophen administration during 7 days (the 4 th group), malondialdehyde in blood plasma increased the most in 4.13 times on the 1 st day of the experiment, it increased in 4.9 times in lever homogenate; the content of diene and triene conjugates increased in blood plasma in 3.1 and 3.2 times, in 5.7 and 4.2 times in liver homogenate; Schiff bases increased in 4.1 times in blood plasma.

Discussion
The activation of free radical processes is universal mechanism in case of toxic action of the vast majority of toxic agents.The mechanism of cell damage by free radical metabolites, which are formed as a result of large number of biocidal xenobiotics biotransformation, including acetaminophen, involves their ability to initiate processes of lipid peroxidation and oxidation modification of proteins, covalently bind with bio-macromolecules (proteins, nucleic acids, lipids) and generate reactive oxygen intermediates (ROI), which are highly toxic and capable to initiate new chains of free radical reactions.The severity of damage effect of free radicals depends on the intensity of their production and functional ability of antioxidant system.As the result of metabolic transformations of acetaminophen, as proved by a number of researchers [1,2], free radical metabolites, as well as reactive oxygen intermediates are formed; the damage by acetaminophen is accompanied by the intensification of free radical processes.Detoxification system is exhausted due to hyperglycaemia induced by streptozotocin injections, and reactive metabolites of acetaminophen prove even more toxic damage, the intensification of reactive oxygen intermediates formation and activation of lipid peroxidation processes in particular.

Table 1 . Dynamics of content of malondialdehyde, diene and triene conjugates and Schiff bases in blood plasma of rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions (M±m; n=10)
[12,he method[12, 13].Quantitative indices were processed statistically.The results of the experiment were processed by means of statistical program Statistica [11] using parametric Student's t test and Wilcoxon signed-rank test for non-parametric statistical hypothesis test.Changes were considered significant at р≤0.05.
Notes: here and in the following tables p 1 -significant difference compare with control animals; p 2 -significant difference compare with the animals, which were administered with acetaminophen.out