Pharmaceutical Biology

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Paracetamol-induced nephrotoxicity and oxidative

stress in rats: the protective role of Nigella sativa

Dogukan Canayakin, Yasin Bayir, Nurcan Kilic Baygutalp, Esen Sezen

Karaoglan, Hasan Tarik Atmaca, Fatma Betul Kocak Ozgeris, Mevlut Sait
Keles & Zekai Halici

To cite this article: Dogukan Canayakin, Yasin Bayir, Nurcan Kilic Baygutalp, Esen Sezen
Karaoglan, Hasan Tarik Atmaca, Fatma Betul Kocak Ozgeris, Mevlut Sait Keles & Zekai Halici
(2016) Paracetamol-induced nephrotoxicity and oxidative stress in rats: the protective role of
Nigella�sativa, Pharmaceutical Biology, 54:10, 2082-2091, DOI: 10.3109/13880209.2016.1145701

To link to this article: https://doi.org/10.3109/13880209.2016.1145701

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PHARMACEUTICAL BIOLOGY, 2016
VOL. 54, NO. 10, 2082–2091
http://dx.doi.org/10.3109/13880209.2016.1145701

RESEARCH ARTICLE

Paracetamol-induced nephrotoxicity and oxidative stress in rats: the protective

role of Nigella sativa
Dogukan Canayakina, Yasin Bayira, Nurcan Kilic Baygutalpa, Esen Sezen Karaoglanb, Hasan Tarik Atmacac,
Fatma Betul Kocak Ozgerisd, Mevlut Sait Kelesd and Zekai Halicie
a
Department of Biochemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey; bDepartment of Pharmaceutical Botany, Faculty of
Pharmacy, Ataturk University, Erzurum, Turkey; cDepartment of Pathology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale,
Turkey; dDepartment of Biochemistry, Faculty of Medicine, Ataturk University, Erzurum, Turkey; eDepartment of Pharmacology, Faculty of
Medicine, Ataturk University, Erzurum, Turkey

ABSTRACT ARTICLE HISTORY

Context Nigella sativa L. (Ranunculaceae) (NS) is traditionally used to treat many conditions such as Received 23 March 2015
inflammation. Accepted 19 January 2016
Objective This study evaluates the effects of NS seeds ethanol extract in paracetamol-induced Revised 27 October 2015
acute nephrotoxicity in rats. Published online 22
Materials and methods Forty-eight female Wistar Albino rats were divided into eight groups: February 2016
I ¼ sham; II ¼ sham + 1000 mg/kg NS; III ¼ sham + 140 mg/kg (N-acetyl cysteine) NAC; IV ¼ 2 g/kg KEYWORDS
paracetamol; V ¼ 2 g/kg paracetamol + 140 mg/kg NAC; VI, VII and VIII ¼ 2 g/kg paracetamol + 250, Glutathione; kidney; malon-
500 and 1000 mg/kg NS, respectively. Paracetamol administration (oral) was carried out 1 h after NS dialdehyde; superoxide
and NAC administrations (oral), and all animals were sacrificed 24 h later. dismutase
Results Paracetamol administration significantly increased serum urea (88.05 U/L) and creatinine
(0.80 U/L) when compared with the sham group (49.80 and 0.31 U/L, respectively). However, serum
urea level was reduced to 65.60, 56.00 and 54.18 U/L, with 250, 500 and 1000 mg/kg doses of the
extract, respectively. Also, serum creatinine level was reduced to 0.64, 0.57 and 0.52 U/L with 250,
500 and 1000 mg/kg doses of the extract, respectively. NS administration increased superoxide
dismutase and glutathione, and decreased malondialdehyde levels in the kidneys. Kidney
histopathological examinations showed that NS administration antagonized paracetamol-induced
kidney pathological damage.
Discussion and conclusions The results suggest NS has a significant nephroprotective activity on
paracetamol-induced nephrotoxicity. It may be suggested that the antiinflammatory and antioxidant
effects of NS ethanolic extract originated from different compounds of its black seeds.

Introduction
Paracetamol (acetaminophen) is one of the most
commonly used analgesic–antipyretic drugs worldwide,
and in most countries, it is available without a
prescription. Paracetamol overdose is known to cause
hepatotoxicity and numerous studies about paraceta-
mol-induced hepatotoxicity and its mechanisms are
available in the literature. Significant paracetamol-
induced hepatotoxicity usually triggers nephrotoxicity.
Renal insufficiency is reported to occur in 1–2% of
patients exposed to paracetamol toxicity (Prescott
1983). After oral administration, about 63% of para-
cetamol is metabolized via glucuronidation and 34% via
sulphation primarily in the liver. The water-soluble
metabolites consisting of these metabolic pathways are
excreted via the kidney. N-Acetyl-p-benzoquinone
(NAPQI) is a reactive intermediate that occurs when
oxidization of 55% percent of paracetamol takes place
by the microsomal P-450 enzyme system. NAPQI is
detoxified by intracellular glutathione (GSH) in thera-
peutic doses (Dahlin et al. 1984). Accordingly, NAPQI
has been implicated as responsible metabolite of
paracetamol toxicity (Waring 2012). In paracetamol-
overdosing cases, glutathione stores are depleted, and a
rapid increase in the concentration of NAPQI causes
necrosis (Bessems & Vermeulen 2001). Additionally
this condition results in the production of massive
metabolites, causing large amounts of unbound reactive
species. N-Acetyl-cysteine (NAC) is an antidote cur-
rently being clinically used for paracetamol toxicity

CONTACT Dr. Yasin Bayir yasinbayir@hotmail.com Department of Biochemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum, Turkey
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

(Flanagan & Meredith 1991). NAC metabolizes to
cysteine and increases the intracellular GSH level
(Dilger & Baker 2007). Paracetamol toxicity creates
acute tubular necrosis, which is one of the main causes
of acute renal failure (Blantz 1996). Serum urea and
creatinine levels may be indicators of acute tubular
necrosis induced by paracetamol (Cobden et al. 1982;
Blantz 1996). Free radicals are produced by exposure to
drug toxicity in an organism, and oxidative damage
plays an important role in paracetamol-induced hepa-
torenal injuries (Das et al. 2010; Kheradpezhouh et al.
2010). Therefore, natural compounds having antioxi-
dant activity could be used for alternative treatments of
paracetamol toxicity.
Nigella sativa L. (Ranunculaceae) (NS), commonly
known as black seed, is a prominent herb among various
medicinal plants, with a rich historical background and
wide spectrum of pharmacological potential. NS seeds
are widely used for many diseases throughout the world,
including bronchitis, asthma, cough, diarrhoea, rheuma-
tism, influenza and skin disorders. Additionally, it has
been widely used as an antihypertensive, liver tonics,
diuretics, digestive, antidiarrhoeal, appetite stimulant,
antianxietic and antibacterial agents (Abdel-Sater 2009;
Boskabady et al. 2010; Abel-Salam 2012; Ahmad et al.
2013; Bin Sayeed et al. 2014). Many active compounds
have been isolated in different varieties of black seed thus
far. Some of the most important active compounds are
thymoquinone (30–48%), thymohydroquinone, dithy-
moquinone, p-cymene, carvacrol, a-pinene, thymol,
quercetin, kaempferol and quercetin-3 (Al-Jassir 1992;
Ahmad et al. 2013). Today, numerous clinical and
animal investigations conducted on NS have explored
important pharmacological actions, including antidia-
betic, anticancer, immunomodulator, analgesic, anti-
microbial, antiinflammatory, spasmolytic,
bronchodilator, hepatoprotective, renoprotective, gastro-
protective and antioxidant properties (Kokoska et al.
2008; Ravindran et al. 2010; Ashraf et al. 2011;
Effenberger-Neidnicht & Schobert 2011; Ahmad et al.
2013). Studies have shown that NS ethanol extract and
its major compounds quercetin, kaempferol and so forth
have strong antioxidant properties and palliative effects
on inflammation (Hadjzadeh et al. 2007; Dwarampudi
et al. 2012). We have previously reported the antioxidant
effect and anti-inflammatory activity of NS ethanol
extract (Bayir et al. 2012a, 2012b). In the literature, there
is no experimental study about protective effects of NS in
paracetamol-induced nephrotoxicity. In this study, we
investigated the potential reno-protective effects of NS
ethanol extract in rats with paracetamol-induced
nephrotoxicity.
PHARMACEUTICAL BIOLOGY 2083
Materials and methods
Animals
The study was approved by and conducted in accordance
with the Institutional Animal Care and Use Committee
at Ataturk University under protocol no. 31.05.2013/361.
In total, 48 adult (6-month-old) female Wistar albino
rats weighing 200 ± 20 g obtained from the Ataturk
University Experimental Animal Laboratory (ATADEM)
were used. All animals were housed in facilities approved
by international guidelines.
Drugs
Paracetamol
A paracetamol suspension was prepared in 1% carboxy-
methyl-cellulose in saline phosphate buffer, and animals
were administered 2 g/kg of this suspension orally by
gastric tube (1 mL of suspension contains 0.4 g of
paracetamol) (Parasetamol, Doga Drug Stock LLC,
Zekeriyaköy, Istanbul).
N-Acetylcysteine (NAC)
NAC (600 mg, Mentopini Hermesarzneimittel Gmbh,
Münich, Germany) was provided and a solution was
prepared in 0.9% saline, pH 7.0. Groups 3 and 5 were
administered 140 mg/kg of this NAC solution orally by
gastric tube.
Thiopental sodium
Thiopental sodium (I.E. Ulugay, Topkapı, Istanbul) was
provided and used for intraperitoneal (i.p.) euthanasia at
a 50 mg/kg dose.
Chemicals
All other chemicals were of the highest analytical grade
commercially available.
Preparation and administration of the N. sativa
extract
NS seeds were purchased from a local herbal shop in
Erzurum, Turkey, at 2013 February. The seeds were
botanically identified and authenticated by Dr. Esen
Sezen Karaoglan. The herbarium specimen is deposited
in the Department of Pharmaceutical Botany, Faculty of
Pharmacy, Ataturk University at 4 ± 2  C. The seeds
(100 g) were cleaned, dried, mechanically powdered and
extracted with 300 mL of 96% ethanol three times. The
2084 D. CANAYAKIN ET AL.
obtained extracts were filtered and combined, and the
solvent was evaporated under reduced pressure using a
rotary, which yielded a blackish-brown liquid concen-
trate (yield 25%) that was kept in a domestic refrigerator
at 4  C. The doses of NS (250, 500 and 1000 mg/kg) were
selected from described values in the literature
(Hadjzadeh et al. 2007; Al-Naqeep et al. 2011;
Hadjzadeh et al. 2011). The extract was emulsified in
water and administered to rats using an oral gavage.
Experimental design
The rats were randomly divided into the following eight
groups, with each group containing six rats: (I) sham
operation group, (II) sham operation + 1000 mg/kg NS
group, (III) sham operation + 140 mg/kg NAC group,
(IV) 2 g/kg paracetamol group, (V) 2 g/kg paraceta-
mol + 140 mg/kg NAC group, (VI) 2 g/kg paraceta-
mol + 250 mg/kg NS group; (VII) 2 g/kg
paracetamol + 500 mg/kg NS group and (VIII) 2 g/kg
paracetamol + 1000 mg/kg NS group (Table 1). The
groups were housed separately in different cages.
Experimental modelling
During the acclimatization period, the rats were fed a
diet of standard commercial rat pellets and fresh water
ad libitum and maintained under constant conditions
(temperature: 22 ± 1  C, humidity: 40–60% and con-
trolled lighting using a 14 h:10 h light-dark cycle for a
week prior to the experiments). Sixteen hours before the
experiment, the animals were allowed water only. In view
of diurnal enzyme activity variations, animals were
sacrificed at a fixed time. All surgical procedures were
performed under sterile conditions. The paracetamol
dose to induce nephrotoxicity was selected from the
values described in the literature (Kuralay et al. 1998;
Chattopadhyay 2003). The rats were deprived of food
after paracetamol administration but had free access to
food and water 4 h after paracetamol administration
until they were sacrificed. In addition, 24 h after
paracetamol administration, the experiment was
Table 1. Experimental protocols in the eight nephrotoxicity groups.
Group
I Sham
II Sham + NS
III Sham + NAC
IV Paracetamol
V Paracetamol + NAC
VI Paracetamol + NS 250 mg/kg
VII Paracetamol + NS 500 mg/kg
VIII Paracetamol + NS 1000 mg/kg
NS: Nigella sativa L. ethanol extract; NAC: N-acetylcysteine.
terminated by thiopental sodium at a 50 mg/kg dose
and all animals were then sacrificed. The kidneys were
rapidly removed and washed in ice-cold saline in all
groups. The organs were labelled and stored at 80  C
until the biochemical analyses were conducted. Cardiac
blood samples were collected immediately and
transferred to the laboratory from all groups, and
the sera were obtained to determine biochemical
parameters.
Biochemical investigations
Biochemical investigation of kidney tissues
After macroscopic analyses, rat tissues were kept at
80  C. All tissue samples from each rat were first
perfused with PBS/heparin and then grinded in liquid
nitrogen using a tissue lyser II grinding Jar Set. As well,
0.1 g was weighed and then treated with 4.5 mL of an
appropriate buffer. This mixture was homogenized on
ice using an Ultra-Turrax homogenizer for 5 min.
Homogenates were filtered and centrifuged using a
refrigerator centrifuge at 4  C. These supernatants were
then used to determine SOD and MDA levels with highly
sensitive ELISA kits (Cayman Chemical SOD assay kit
item number 706002, Cell Biolabs Oxi SelectTM TBARS
Assay STA-330 Kit, Cell Biolabs, San Diego, CA,
respectively). Measurements were performed according
to the manufacturers’ instructions. The GSH levels in the
kidney tissues were measured using the method created
by Sedlak and Lindsay (1968), according to the modified
methods, with an ELISA reader. The average absorb-
ances of each sample and standard were calculated. A
standard curve was plotted and an equation was
obtained from the absorbances of standards. All assays
were carried out at room temperature in duplicate.
Linear SOD, GSH and MDA concentrations were
calculated according to this equation. The results of the
SOD, GSH and MDA levels in the tissues were expressed
as, respectively, U/mg protein, nmol/mg protein and
nmol/mg protein. All data were presented as the
mean ± standard deviation (SD) results based on per
mg protein.
Orally water 2 ml + sham operation
Orally NS 1000 mg/kg + sham operation
Orally NAC 140 mg/kg + sham operation
Orally paracetamol 2 g/kg
Orally paracetamol 2 g/kg + NAC 140 mg/kg
Orally paracetamol 2 g/kg + NS 250 mg/kg
Orally paracetamol 2 g/kg + NS 500 mg/kg
Orally paracetamol 2 g/kg + NS 1000 mg/kg
 PHARMACEUTICAL BIOLOGY 2085

Biochemical investigation of serum drug, NAC. The serum levels of urea and creatinine were
found to be lower in the NS treatment groups (NS 250,
Serum urea and creatinine levels were measured using
NS 500 and NS 1000 mg/kg, groups) (p50.05). The
the commercial kits (BEN) (Biochemical Enterprise,
extract at a dose of 1000 mg/kg had a significantly higher
Milano, Italy) BK 151 and CR280 in an automatic
protective effect on nephrotoxicity than standards at 250
biochemical analyzer (ChemWell, Palm City, FL) at
and 500 mg/kg; in fact, the protective effect at this dose
340 nm and 510 nm, respectively.
was significantly higher than the effect of NAC
(p50.05).
Histological examination The administration of paracetamol caused increments
The kidneys of rats in all groups were fixed in 10% neutral in both serum urea and creatinine levels. The serum urea
formalin for 48–72 h. Then, tissues were trimmed and level, which increased with paracetamol treatment to
processed for a routine histopathological examination. 88.05 U/L, was reduced to 65.60, 56.00 and 54.18 U/L,
Tissues were embedded into paraffin wax and 4–5 mm with 250, 500 and 1000 mg/kg doses of the extract,
thick sections were cut. All tissue sections were stained respectively. The serum creatinine level, which increased
with Haematoxylin and Eosin (H&E) and then examined to 0.80 U/L by paracetamol administration, was reduced
under a light microscope (Olympus BX51, Tokyo, Japan). to 0.64, 0.57 and 0.52 U/L with 250, 500 and 1000 mg/kg
Renal lesions in rats were assessed according to Zhang doses of the extract, respectively (Table 2).
et al. (2008) and graded into five categories by utilizing a
scale of 0–5, where 0 ¼ normal histology and 1 ¼ tubular Biochemical results for oxidant and antioxidant
epithelial cell degeneration, without significant necrosis/ levels of kidney tissue in rats
apoptosis; 2–5 ¼525%,550%,575% and 75% of the
tubules showed tubular epithelial cell necrosis/apoptosis, The activity of SOD and the levels of MDA and GSH in
respectively, accompanied by other concomitant alterations. the paracetamol toxicity in rat kidneys are shown in
Table 3. SOD activity and the GSH level were found to
be decreased and the MDA level was found to be
Statistical analysis significantly increased in the paracetamol group com-
Data obtained from the serum urea and creatinine levels, pared with the sham group (p50.05).
tissue SOD activity and GSH and MDA levels were NS administration restored GSH and SOD and
subjected to a one-way analysis of variance (ANOVA) decreased MDA levels in kidneys (Table 3). The NS
followed by Duncan’s multiple range test (DMRT). extract showed a dose-dependent inhibition compared
Differences among the groups were obtained using the with the standard drug, NAC. The extract at a dose of
Duncan multiple comparison and were considered 1000 mg/kg had significantly higher scavenging activity
significant at p50.05. All results were expressed as on kidneys than standards at 250 and 500 mg/kg; in
mean ± SD using the IBM SPSS Statistics 19 software addition, the scavenging activity at this dose was
(SPSS Inc., Chicago, IL). Results of the histological score significantly higher than the effect of NAC (p50.05).
were compared between groups using a one-way
ANOVA and Tukey’s multiple comparison tests. Results of histopathological examinations
Histological statistical analyses and graphs were prepared
using GraphPad Prism version 4.0 (GraphPad Software, Histopathological scores of rat kidneys in all groups are
La Jolla, CA), and p50.05 was considered statistically presented in Figure 1. Kidneys of the control group
significant.
Table 2. Effects of NS treatments on changes in paracetamol-
induced nephrotoxicity in rats.
Results Group Urea (U/L) Creatinine (U/L)
a
The effects of NS on serum urea and creatinine Sham 49.80 ± 11.91 0.31 ± 0.08a
Sham + NS 1000 mg/kg 48.64 ± 5.57a 0.37 ± 0.11ab
levels Sham + NAC 140 mg/kg 46.30 ± 12.72a 0.38 ± 0.09ab
Paracetamol 2 g/kg 88.05 ± 25.69c 0.80 ± 0.21d
Serum urea and creatinine levels were found to be higher Paracetamol 2 g/kg + NAC 140 mg/kg 59.86 ± 12.67ab 0.55 ± 0.25c
in the paracetamol group when compared with the sham Paracetamol 2 g/kg + NS 250 mg/kg 65.60 ± 11.57b 0.64 ± 0.16c
Paracetamol 2 g/kg + NS 500 mg/kg 56.00 ± 14.52ab 0.57 ± 0.07c
group (p50.05). NS administration dramatically Paracetamol 2 g/kg + NS 1000 mg/kg 54.18 ± 8.50ab 0.52 ± 0.11bc
reduced serum urea and creatinine levels. The NS extract
showed a dose-dependent protective effect on paraceta- Results are means ± SD. Means in the same column with the same
superscript are not significantly different compared with the Duncan test
mol-induced nephrotoxicity compared with the standard (p ¼ 0.05). NS: Nigella sativa L. ethanol extract; NAC: N-acetylcysteine.
2086 D. CANAYAKIN ET AL.

Table 3. Effects of NS treatments on tissue SOD activity and GSH and MDA levels in paracetamol-induced
nephrotoxicity in rats.
Group SOD (U/mg protein) GSH (nmol/mg protein) MDA (nmol/mg protein)
b bc
Sham 44.97 ± 8.98 2.39 ± 0.43 1.80 ± 0.68a
Sham + NS 1000 mg/kg 45.69 ± 10.62b 2.61 ± 0.54c 1.76 ± 0.40a
Sham + NAC 140 mg/kg 41.22 ± 10.95b 2.46 ± 0.65c 2.08 ± 0.76ab
Paracetamol 2 g/kg 23.31 ± 6.62a 1.32 ± 0.42a 4.06 ± 0.29d
Paracetamol 2 g/kg + NAC 140 mg/kg 36.31 ± 13.25b 2.08 ± 0.47bc 2.81 ± 0.74bc
Paracetamol 2 g/kg + NS 250 mg/kg 25.79 ± 8.79a 1.85 ± 0.40b 3.16 ± 1.59c
Paracetamol 2 g/kg + NS 500 mg/kg 39.40 ± 8.91b 2.08 ± 0.59bc 3.23 ± 0.58c
Paracetamol 2 g/kg + NS 1000 mg/kg 46.54 ± 9.11b 2.26 ± 0.51bc 2.26 ± 0.53ab

Results are means ± SD. Means in the same column with the same superscript are not significantly different compared to the
Duncan test (p ¼ 0.05). NS: Nigella sativa L. ethanol extract; NAC: N-acetylcysteine.

Figure 1. Histopathological score differences in groups. *Indicates that histopathological score significantly greater than the sham
group, #indicates that histopathological score is significantly lower than the paracetamol 2 g/kg group, p 5 0.05 was considered
statistically significant.

(Figure 2A) animals showed a normal histologic morph-
ology. The sham + NS 1000 mg/kg group (Figure 2B) and
the sham + NAC 140 mg/kg group (Figure 2C) animal
kidneys were observed to be similar to those of the
control group. The paracetamol 2 g/kg + NS 1000 mg/kg
group (Figure 2H) and the paracetamol 2 g/kg + NAC
140 mg/kg group (Figure 2G) had similar morphologies
to that of the normal group, and interstitial extravasation
of red blood cells (RBCs) and vascular congestion could
not be observed. Restoration of the apical brush border
and the narrow lumina was detected in most of the
tubules.
The histopathology of the kidneys of the paracetamol-
treated group (Figure 2E) showed severe proximal
tubular distortion with enlarged lumina. Signs of necro-
sis, vacuolization, epithelial degeneration and pyknotic
nuclei, in addition to the partial to complete loss of the
luminal brush border, were observed. Hyaline casts,
desquamated cells and necrotic cell debris were seen in
the lumina of some tubules. Extravasation of RBCs and
congested blood vessels were also encountered in the
interstitium of the renal cortex. Stained sections of the
paracetamol 2 g/kg + NS 250 mg/kg group (Figure 2D)
and the paracetamol 2 g/kg + NS 500 mg/kg group
(Figure 2F) maintained a better morphology with a
marked reduction in the extent of proximal tubular
necrosis. The presence of mild tubular epithelial
vacuolization, luminal cast formation and cell desquam-
ations were hardly detected.
Discussion
Although paracetamol, with the alternative name acet-
aminophen, has a reasonable safety profile in therapeutic
 PHARMACEUTICAL BIOLOGY 2087

Figure 2. (A) Normal structure of kidney in control group. Haematoxylin and eosin. Magnification 200. (B) Stained sections of kidney
of sham + NS 1000 mg/kg group. Slight degenerations were seen. Haematoxylin and eosin. Magnification 200. (C) Stained sections
of kidney of sham + NAC 140 mg/kg group. Similar appearance to the control group. Haematoxylin and eosin. Magnification 200.
(D) Stained sections of kidney of paracetamol 2 g/kg + NS 250 mg/kg group. Haematoxylin and eosin. Magnification 200.
(E) Paracetamol-administered rat kidney. Severe necrosis, congestion and extravasation of red blood cells were seen. Haematoxylin
and eosin. Magnification 200. (F) Stained sections of kidney of paracetamol 2 g/kg + NS 500 mg/kg. Slightly to mild degenerations
were seen. Haematoxylin and eosin. Magnification 200. (G) Paracetamol 2 g/kg + NAC 140 mg/kg group. Structures of kidney were
similar to the control group. Haematoxylin and eosin. Magnification 200. (H) Paracetamol 2 g/kg + NS 1000 mg/kg group. Structures
of kidney were similar to the control group. Haematoxylin and eosin. Magnification 100.

doses, its overdose remains the most important cause of
liver injury and even death in many parts of the world
among all drug toxicities (Larson et al. 2005; Karakus
et al. 2013; Yayla et al. 2014). Hepatotoxic and nephro-
toxic effects of paracetamol overdose occur by a complex
sequence of events (Hinson et al. 2010). Acetaminophen-
induced nephrotoxicity becomes evident after
hepatotoxicity in most cases, but the occurrence of
renal tubular damage and acute renal failure, even in the
absence of liver injury, should not be ignored (Eguia &
Materson 1997).
Although the hepatotoxic mechanism of paracetamol
overdose is well described, there is little evidence of the
nephrotoxic mechanism of paracetamol. Based on the
2088 D. CANAYAKIN ET AL.
clinical and animal studies, some of the potential
mechanisms of nephrotoxicity are the cytochrome P-
450 pathway, prostaglandin synthesis and N-deacetylase
enzymes (Bessems & Vermeulen 2001). At high doses of
paracetamol, the reactive derivative of paracetamol
occurred by the P-450-dependent metabolism, NAPQI
could not be detoxified by GSH and depleted GSH levels
will cause liver necrosis, leakage of hepatocellular
contents into the blood and liver failure (Eguia &
Materson 1997; Mugford & Tarloff 1997; Karakus et al.
2013). Loss of glutathione with an increased formation of
reactive oxygen species and increased oxidative stress are
essential events of the toxicity mechanism. In association
with these essential events, many inflammatory medi-
ators, such as certain cytokines and chemokines, are
released by various mechanisms, and these mediators
can modify the toxicity (Hinson et al. 2010). Free radicals
can stimulate lipid peroxidation and play a key role in
drug toxicities (Kehrer 1993). As a result of this lipid
peroxidation, MDA levels increase and this reflects tissue
damage (Draper & Hadley 1990). SOD and GSH are key
antioxidant defence system enzymes to detoxify reactive
molecules or repair the resulting damage in the cell
(Whidden et al. 2011).
Acetyl cysteine has long been recognized as an
effective antidote of paracetamol overdose and it is
routinely used to minimize and repair hepatotoxicity in
overdose cases. By having rich cysteine sources, acetyl
cysteine provides cysteine to stimulate the replenishment
of GSH, and increased GSH plays a key role in the
detoxification of NAPQI (Massey & Racz 1981; Dawson
et al. 1984). The antioxidant effects of NS and its
biologically active components quercetin and kaempferol
have been demonstrated in a large number of studies on
different organs (Kokoska et al. 2008; Ashraf et al. 2011;
Bourgou et al. 2012).
In this study, we demonstrated that NS ethanol
extract plays an important role in paracetamol-induced
nephrotoxicity. In the assessment of kidney injury,
levels of urea and creatinine in serum should be first
determined. These enzymes are very sensitive markers
employed in the diagnosis of kidney diseases. Serum
urea and creatinine levels may be indicators of acute
tubular necrosis caused by paracetamol toxicity
(Cobden et al. 1982; Blantz 1996). In our study,
paracetamol administration altered serum urea and
creatinine levels, indicating nephrotoxicity. These
results were in accordance with previous studies
(Anthony et al. 2012; Saxena et al. 2012). The
administration of NS as a nephroprotective alternative
decreased these altered values up to the serum levels of
control groups. In fact, the highest serum urea and
creatinine reductions were observed using NS at a dose
of 1000 mg/kg, and this reduction was statistically
significantly higher than that of NAC administration.
These results have shown that NS improves the
nephrotoxic effects of paracetamol.
It is well known that SOD, GSH and MDA are
important indicators of the antioxidant capacity of the
body that resulted in protection against the damage
caused by oxidative stress. In the present study, we have
investigated alterations to the tissue MDA level and
reductions in the tissue GSH level and SOD activity as
markers of the oxidative stress process following the oral
administration of paracetamol. The increase in lipid
peroxidation was accompanied by a significant reduction
in the GSH level. GSH is a tripeptide, that is, found in
many mammalian tissues and is an important free-
radical scavenger and scavenger of NAPQI, which is a
reactive intermediate of paracetamol (Yayla et al. 2014).
It plays an important role in the antioxidant defence
system and removes free-radical species, such as hydro-
gen peroxide and superoxide radicals, and it maintains
membrane protein thiols. The decreased SOD and GSH
levels and the increased MDA levels were in accordance
with previous research studying paracetamol-induced
nephrotoxicity (Ghosh & Sil 2007; Yousef et al. 2010;
Abdul Hamid et al. 2012). Through NS administration,
tissue MDA levels decreased and GSH levels and SOD
activity increased, showing the antioxidant activity of the
extract. These results suggest that NS reduces the
oxidative stress in kidneys. We can say that NS
prevented the depletion of antioxidant enzymes, includ-
ing GSH, in the present study. This suggestion is
supported by histopathologic findings, indicating that
the administration of the NS extract significantly
prevented necrosis and congestion, as well as reduced
the degree of vascular dilatation. Previous studies
suggesting potential antioxidant capacity of NS extracts
support our findings (Havakhah et al. 2014; Mollazadeh
& Hosseinzadeh 2014). Also, thymoquinone, which is
the main ingredient of NS, supplementation reverses
lead-induced oxidative stress in adult rat testes
(Mabrouk & Ben Cheikh 2015). Previously Nagi et al.
(2010) suggested that thymoquinone reverses acetamino-
phen-induced oxidative stress, nitric oxide production
and energy decline in mice liver. In previous studies,
thymol, thymoquinone and other active ingredients of
NS acted as scavenger of superoxide, hydroxyl radical
and singlet molecular oxygen (Kruk et al. 2000; Nagi &
Mansour 2000; Badary et al. 2003). Thymoquinone
supplementation increases the expression of antioxidant
genes, superoxide dismutase, catalase and glutathione
peroxidase (Ismail et al. 2010). Thus, NS extract may
reduce oxidative stress through direct antioxidant effect
of its ingredients as well as induction of endogenous

antioxidant enzymes leading to the prevention of
paracetamol-induced nephrotoxicity.
Our results show that the selected dose of paraceta-
mol-induced nephrotoxicity in biochemical and histo-
pathologic aspects. The administration of NS
significantly reduced the toxic effects of paracetamol
on the kidneys in a dose-dependent manner. The
nephroprotective properties of NS may be related to its
positive effects on the antioxidant system. It is concluded
that NS can protect kidneys from the damage caused by
paracetamol overdose and might be a potential thera-
peutic candidate against paracetamol-induced acute
nephrotoxicity.
Conclusion
In light of these observations, we found that the
therapeutic administration of NS prevented paraceta-
mol-induced oxidative stress changes. It can be specu-
lated that the role of the ethanoic NS extract in
preventing the formation of paracetamol-induced
nephrotoxicity, as seen in the present study, is in part
due to the anti-inflammatory and antioxidant effects of
the different compounds (thymoquinone, qurecetin,
kaempferol and so forth) of the black seeds. These
compounds may interfere with antioxidant and anti-
inflammatory mechanisms, which may be crucial to
reducing paracetamol-induced nephrotoxicity. Further
prospective and randomized clinical controlled trials are
required to understand better this novel candidate for
use in treating paracetamol nephrotoxicity and reducing
paracetamol-related mortality.
The use of NS seeds for centuries as a condiment in
Turkish dishes, without side effects, and the desirable
amount of effective substances present in the seeds
encourage consideration of NS as a new and natural
medicinal source.
Acknowledgements
This research is included in Master’s thesis of Dogukan
Canayakin and was conducted in the Laboratory of
Biochemistry, Laboratory of Pharmacology at Ataturk
University, Faculty of Pharmacy and Medicine, 25240
Erzurum/Turkey. The second author (Y. Bayir) wishes
to thank Professor Ahmet Cakir for his kind support in the
study.
Disclosure statement
None of the authors has a commercial or financial interest or
other relationship with the manufacturers of pharmaceuticals,
laboratory supplies and/or medical devices or with commercial
providers of medically related services.
PHARMACEUTICAL BIOLOGY 2089
Funding information
This study was supported by the Research fund of Atatürk
University (BAP-2012/077).
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