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dOI 10.11603/IJMMR.2413-6077.2017.2.8372

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

О. B. Furka, І. B. Ivanusa, М. М. Mykhalkiv, І. М. Klishch
I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE

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.

KEY WORDS: acetaminophen; malondialdehyde; diene and triene conjugates; Schiff bases; diabetes 
mellitus.

Corresponding author: Olha Furka, Department of Medical 
Biology, I. Horbachevsky Ternopil State Medical University, 
2 Yu. Slovatskoho, Ternopil, Ukraine, 46001 
Phone number: +380352252584
E-mail: furkaob@tdmu.edu.ua

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 some-
times 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 asso-
ciated with changes in environmental factors, 
especially the populations (genetic, demo-
graphic), 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 LD50). 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 

International Journal of Medicine and Medical Research 
2017, Volume 3, Issue 2, p. 45–50
copyright © 2017, TSMU, All Rights Reserved

О. B. Furka et al.



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iSSn 2413-6077. iJmmR 2017 Vol. 3 issue 2

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 1st group was 
the intact (control); the 2nd group involved a 
single acetaminophen administration; the 3rd 
group comprised the animals with type 2 
diabetes mellitus caused by streptozotocin ad-
ministration; and the 4th group contained the 
rats with a single administration of acetami-
nophen after streptozotocin administration. In 
the second series of the experiment, the rats 
were divided into 4 groups: the 1st group was 
the intact (control); the 2nd group was with 
acetaminophen administration during 7 days; 

the 3rd group were the animals with type 2 
diabetes mellitus caused by streptozotocin 
administration; the 4th group were the rats with 
administration of acetaminophen during 7 days 
after streptozotocin administration.

The animals were removed from the expe-
riment on the 5th, 3rd, 5th and 7th days after last 
acetaminophen administration by euthanasia 
under thiopental anaesthesia. All experiments 
on rats were carried out according to The Gui-
deline Principles for the Care and Use of Labo-
ratory Animals [10].

Evaluation of the content of diene and trie-
ne 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 

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)

Group  
of animals

Time after acetaminophen administration (days)
Content of 1st day 3rd day 5th day 7th day

Control
n=10

MDA, μmol/l 7.48±0.47
DC, U/l 1.12±0.03
ТC, U/l 0.58±0.01

Schiff bases, U/l 2.53±0.33
Acetaminophen 

(single)
n=10

MDA, μmol/l 24.62±1.42 
p1<0.001

22.63±1.11 
p1<0.001

21.79±1.12 
p1<0.001

21.47±1.13 
p1<0.001

DC, U/l 4.62±0.49 
p1<0.001

4.47±0.41 
p1<0.001

3.42±0.48 
p1<0.001

3.11±0.47 
p1<0.001

ТC, U/l 1.97±0.37 
p1<0.001

1.80±0.24 
p1<0.001

1.76±0.32 
p1<0.001

1.65±0.31 
p1<0.001

Schiff bases, U/l 8.61±0.77 
p1<0.001

8.04±0.71 
p1<0.001

7.79±0.65 
p1<0.001

7.60±0.62 
p1<0.001

Type 2 DM 
n=10

MDA, μmol/l 14.62±0.92 
p1<0.001

14.22±1.02 
p1<0.001

14.03±0.69 
p1<0.001

13.56±0.65 
p1<0.001

DC, U/l 1.831±0.24 
p1<0.001

1.79±0.31 
p1<0.001

1.72±0.28 
p1<0.001

1.66±0.31 
p1<0.001

ТC, U/l 1.20±0.18 
p1<0.001

1.17±0.23 
p1<0.001

1.08±0.16 
p1<0.001

0.95±0.18 
p1<0.001

Schiff bases, U/l 5.10±0.47 
p1<0.001

5.01±0.59 
p1<0.001

4.87±0.44 
p1<0.001

4.63±0.52 
p1<0.001

Acetaminophen 
(rats with  

Type 2 DM) 
n=10

MDA, μmol/l 39.01±1.12 
p1<0.001
p2<0.001

38.72±0.91 
p1<0.001
p2<0.001

38.13±1.19 
p1<0.001
p2<0.001

37.58±1.22 
p1<0.001
p2<0.001

DC, U/l 3.86±0.28 
p1<0.001
p2<0.001

3.70±0.50 
p1<0.001
p2<0.001

3.38±0.51 
p1<0.001
p2<0.001

3.14±0.52 
p1<0.001
p2<0.001

ТC, U/l 4.00±0.39 
p1<0.001
p2<0.001

3.91±0.49 
p1<0.001
p2<0.001

3.77±0.54 
p1<0.001
p2<0.001

3.53±0.48 
p1<0.001
p2<0.001

Schiff bases, U/l 13.33±1.15 
p1<0.001
p2<0.001

13.20±0.93 
p1<0.001
p2<0.001

12.98±0.97 
p1<0.001
p2<0.001

12.81±0.61 
p1<0.001
p2<0.001

Notes: here and in the following tables
p1 – significant difference compare with control animals;
p2 – significant difference compare with the animals, which were administered with acetaminophen.

О. B. Furka et al.



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out by the method [12, 13]. Quantitative indices 
were processed statistically. The results of the 
experiment were processed by means of sta-
tistical 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.

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 1st day of the experiment in the 
2nd group of experimental animals, it increased 
by 95.5% in the 3rd group of animals. The max-
imum of this index increase (in 5.21 times) was 
observed in the animals with type 2 diabetes 
mellitus and acute acetaminophen toxic lesions 
(the 4th group). This index decreased on the 3rd 
and 5th days of the experiment. The maximal 
decrease of malondialdehyde content was ob-
served on the 7th 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 2nd group) 

increased in 4.11 and 3.36 times on the 1st day 
of the experiment to compare with the control 
animals. These indices increased by 62.8% and 
104.8% in the 3rd group of animals with strep-
tozotocin action. The content of diene conju-
gates increased in 3.43 times, and triene con-
jugates in 6.81 times in the animals with type 2 
diabetes mellitus and acute acetaminophen 
toxic lesions (the 4th group). These indices de-
creased on the 3rd, 5th and 7th days of the ex-
periment.

The content of Schiff bases increased in 3.4 
times on the 1st day of the experiment in the 2nd 
group of experimental animals after acetami-
nophen administration. This index increased 
by 101.6% in the 3rd group of animals with 
streptozotocin administration. The maximal 
increase of Schiff bases in 5.26 times was ob-
served in the 4th group of animals with type 2 
diabetes mellitus and single acetaminophen 
administration.

According to the results (Table 2), the con-
tent of malondialdehyde in liver homogenate 
increased in 4.76 times on the 1st day of the 
experiment in the 2nd group of experimental 
animals to compare with the control animals, 
and by 60.8% in the 3rd group of animals. The 
maximum of this index increase (in 5.7 times) 
was observed in the 4th group of animals.

Table 2. Dynamics of content of malondialdehyde, diene and triene conjugates in liver homoge-
nate of rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions (M±m; n=10)

Group of animals
Time after acetaminophen administration (days)

Content of 1st day 3rd day 5th day 7th day
Control

n=10
MDA, μmol/l 13.67±1.16

DC, U/l 7.83±0.26
ТC, U/l 3.69±0.16

Acetaminophen 
(single)

n=10

MDA, μmol/l 65.10±3.57
p1<0.001

64.51±3.50
p1<0.001

63.67±2.41
p1<0.001

62.63±2.11
p1<0.001

DC, U/l 46.09±1.43
p1<0.001

44.21±2.07
p1<0.001

39.47±1.72
p1<0.001

38.94±2.06
p1<0.001

ТC, U/l 17.93±0.78
p1<0.001

17.18±1.56
p1<0.001

16.14±1.69
p1<0.001

15.38±1.28
p1<0.001

Type 2 DM 
n=10

MDA, μmol/l 21.98±1.80
p1<0.001

21.55±2.04
p1<0.001

20.79±1.92
p1<0.001

20.19±2.08
p1<0.001

DC, U/l 23.44±1.00
p1<0.001

21.66±1.50
p1<0.001

20.47±1.13
p1<0.001

20.19±1.70
p1<0.001

ТC, U/l 13.43±0.58
p1<0.001

12.78±1.37
p1<0.001

12.02±1.04
p1<0.001

11.47±1.11
p1<0.001

Acetaminophen 
(rats with  

type 2 DM) 
n=10

MDA, μmol/l 77.94±2.43
p1<0.001
p2<0.001

77.31±4.14
p1<0.001
p2<0.001

76.53±2.00
p1<0.001
p2<0.001

75.70±2.09
p1<0.001
p2<0.001

DC, U/l 52.98±1.03
p1<0.001
p2<0.001

51.25±1.89
p1<0.001
p2<0.001

51.03±2.27
p1<0.001
p2<0.001

47.34±2.34
p1<0.001
p2<0.001

ТC, U/l 24.24±1.49
p1<0.001
p2<0.001

23.56±1.45
p1<0.001
p2<0.001

22.94±1.67
p1<0.001
p2<0.001

22.01±1.64
p1<0.001
p2<0.001



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The content of diene and triene conjugates 
in the experimental animals increased the most 
in the 4th group of animals on the 1st day of the 
experiment in 6.76 and 6.56 times respectively. 
These indices increased on the 1st day of the 
experiment in 5.88 and 4.85 times in the 2nd 
group, and in 2.99 and 3.63 times in the 3rd 
group respectively.

The content of malondialdehyde, diene and 
triene conjugates, Schiff bases increased on the 
1st day of the experiment in blood plasma (Tab-
le 3) and liver homogenates (Table 4). These 
indices decreased on the 3rd, 5th, 7th days in all 
series of the experiment. These changes were 
less pronounced than in the 1st series of the 
experiment.

In the 2nd group of experimental animals 
with acetaminophen administration during 7 
days, the content of malondialdehyde in blood 
plasma on the 1st 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 4th group), malondialdehyde in blood 
plasma increased the most in 4.13 times on the 
1st 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, 

Table 3. Dynamics of content of malondialdehyde, diene and triene conjugates and Schiff bases 
in blood plasma of rats with type 2 diabetes mellitus and acetaminophen administration  

at a dose of 55 mg/kg during 7 days (M±m; n=10)

Group of animals
Time after acetaminophen administration (days)

Content of 1st day 3rd day 5th day 7th day
Control

n=10
MDA, μmol/l 7.48±0.47

DC, U/l 1.12±0.03
ТC, U/l 0.58±0.01

Schiff bases, U/l 2.53±0.33
Acetaminophen

(7 days)
n=10

MDA, μmol/l 16.94±0.94 
p1<0.001

16.54±0.99 
p1<0.001

16.12±1.04 
p1<0.001

15.77±0.96 
p1<0.001

DC, U/l 2.31±0.29 
p1<0.001

2.24±0.20 
p1<0.001

2.00±0.21 
p1<0.001

1.83±0.21 
p1<0.001

ТC, U/l 1.42±0.07 
p1<0.001

1.24±0.21 
p1<0.001

1.19±0.20 
p1<0.001

1.17±0.28 
p1<0.001

Schiff bases, U/l 5.93±0.64 
p1<0.001

5.75±0.71 
p1<0.001

5.58±0.77 
p1<0.001

5.43±0.65 
p1<0.001

Type 2 DM 
n=10

MDA, μmol/l 14.62±0.92 
p1<0.001

14.22±1.02 
p1<0.001

14.03±0.69 
p1<0.001

13.56±0.65 
p1<0.001

DC, U/l 1.831±0.24 
p1<0.001

1.79±0.31 
p1<0.001

1.72±0.28 
p1<0.001

1.66±0.31 
p1<0.001

ТC, U/l 1.20±0.18 
p1<0.001

1.17±0.23 
p1<0.001

1.08±0.16 
p1<0.001

0.95±0.18 
p1<0.001

Schiff bases, U/l 5.10 ± 0.47 
p1<0.001

5.01 ± 0.59 
p1<0.001

4.87 ± 0.44 
p1<0.001

4.63 ± 0.52 
p1<0.001

Acetaminophen 
(rats with  

Type 2 DM) 
n=10

MDA, μmol/l 30.90±0.55 
p1<0.001
p2<0.001

30.54±0.73 
p1<0.001
p2<0.001

29.91±0.87 
p1<0.001
p2<0.001

29.40±1.31 
p1<0.001
p2<0.001

DC, U/l 3.52±0.21 
p1<0.001
p2<0.001

3.25±0.45 
p1<0.001
p2<0.001

2.85±0.37 
p1<0.001
p2<0.001

2.46±0.39 
p1<0.001
p2<0.001

ТC, U/l 1.88±0.33 
p1<0.001
p2<0.001

1.83±0.33 
p1<0.001
p2<0.001

1.76±0.36 
p1<0.001
p2<0.001

1.63±0.30 
p1<0.001
p2<0.001

Schiff bases, U/l 10.60±0.77 
p1<0.001
p2<0.001

10.41±0.82 
p1<0.001
p2<0.001

10.22±0.82 
p1<0.001
p2<0.001

10.03±0.68 
p1<0.001
p2<0.001

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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 transfor-
mations of acetaminophen, as proved by a 
number of researchers [1, 2], free radical meta-
bolites, 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 meta-
bolites of acetaminophen prove even more 
toxic damage, the intensification of reactive 
oxygen intermediates formation and activation 
of lipid peroxidation processes in particular. 

Table 4. Dynamics of content of malondialdehyde, diene and triene conjugates and Schiff bases  
in liver homogenate of rats with type 2 diabetes mellitus and acetaminophen administration  

at a dose of 55 mg/kg during 7 days (M±m; n=10)

Group of animals
Time after acetaminophen administration (days)

Content of 1st day 3rd day 5th day 7th day
Control

n=10
MDA, μmol/l 13.67±1.16

DC, U/l 7.83±0.26
ТC, U/l 3.69±0.16

Acetaminophen
(7 days)

n=10

MDA, μmol/l 32.62±1.75 
p1<0.001

31.83±2.78 
p1<0.001

31.34±1.63 
p1<0.001

29.79±1.84 
p1<0.001

DC, U/l 23.63±1.60 
p1<0.001

23.45±1.42 
p1<0.001

21.43±1.40 
p1<0.001

20.82±1.74 
p1<0.001

ТC, U/l 12.02±0.79 
p1<0.001

11.22±0.90 
p1<0.001

10.91±0.99 
p1<0.001

10.03±0.90 
p1<0.001

Type 2 DM 
n=10

MDA, μmol/l 21.98±1.80 
p1<0.001

21.55±2.04 
p1<0.001

20.79±1.92 
p1<0.001

20.19±2.08 
p1<0.001

DC, U/l 23.44±1.00 
p1<0.001

21.66±1.50 
p1<0.001

20.47±1.13 
p1<0.001

20.19±1.70 
p1<0.001

ТC, U/l 13.43±0.58 
p1<0.001

12.78±1.37 
p1<0.001

12.02±1.04 
p1<0.001

11.47±1.11 
p1<0.001

Acetaminophen 
(rats with  

Type 2 DM) 
n=10

MDA, μmol/l 67.00±1.78 
p1<0.001
p2<0.001

66.54±2.63 
p1<0.001
p2<0.001

65.66±2.20 
p1<0.001
p2<0.001

65.43±2.18 
p1<0.001
p2<0.001

DC, U/l 44.75±1.14 
p1<0.001
p2<0.001

42.88±1.55 
p1<0.001
p2<0.001

41.51±1.67 
p1<0.001
p2<0.001

39.14±1.70 
p1<0.001
p2<0.001

ТC, U/l 15.62±1.12 
p1<0.001
p2<0.001

15.19±1.26 
p1<0.001
p2<0.001

14.92±1.25 
p1<0.001
p2<0.001

14.42±1.25 
p1<0.001
p2<0.001

Consequently, we can assert that the radical 
oxidation processes in the rats with type 2 
diabetes and acetaminophen action increases 
significantly that may cause excessive formation 
of free radicals and violation of their neutra-
lization.

Conclusions
Acute lesions by acetaminophen (1/2 LD50) 

of animals with type 2 diabetes causes sig-
nificant increase of lipids peroxidation processes 
in comparison with the animals without the 
simulated pathological process, and non-
diabetic animals, which were modeled by 
acetaminophen poisoning, as indicated by the 
increase in the concentration of diene and 
triene conjugates, TBA-active products, and 
Schiff bases.

The administration of acetaminophen at a 
higher therapeutic dose for the rats with hyper-
glycemia during 7 days was also accompanied 
by a significant increase in the concentration 
of lipid peroxidation products, but less pro-
nounced than in cases of acute lesions.



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Received: 2017-10-11

О. B. Furka et al.