Paracetamol-induced nephrotoxicity and oxidative stress in rats: the protective role of Nigella sativa

Abstract

Context Nigella sativa L. (Ranunculaceae) (NS) is traditionally used to treat many conditions such as inflammation.

Objective This study evaluates the effects of NS seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats.

Materials and methods Forty-eight female Wistar Albino rats were divided into eight groups: I = sham; II = sham + 1000 mg/kg NS; III = sham + 140 mg/kg (N-acetyl cysteine) NAC; IV = 2 g/kg paracetamol; V = 2 g/kg paracetamol + 140 mg/kg NAC; VI, VII and VIII = 2 g/kg paracetamol + 250, 500 and 1000 mg/kg NS, respectively. Paracetamol administration (oral) was carried out 1 h after NS and NAC administrations (oral), and all animals were sacrificed 24 h later.

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.

Keywords:

• Glutathione
• kidney
• malondialdehyde
• superoxide dismutase

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 paracetamol-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 paracetamol 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 <5% percent of paracetamol takes place by the microsomal P-450 enzyme system. NAPQI is detoxified by intracellular glutathione (GSH) in therapeutic 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 currently being clinically used for paracetamol toxicity (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 hepatorenal injuries (Das et al. 2010; Kheradpezhouh et al. 2010). Therefore, natural compounds having antioxidant 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, rheumatism, 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, dithymoquinone, p-cymene, carvacrol, α-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 antidiabetic, anticancer, immunomodulator, analgesic, antimicrobial, antiinflammatory, spasmolytic, bronchodilator, hepatoprotective, renoprotective, gastroprotective 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.

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 obtained extracts were filtered and combined, and the solvent was evaporated under reduced pressure using a rotary, which yielded a blackish-brown liquid concentrate (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 paracetamol + 140 mg/kg NAC group, (VI) 2 g/kg paracetamol + 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.

Table 1. Experimental protocols in the eight nephrotoxicity groups.

Group
I	Sham	Orally water 2 ml + sham operation
II	Sham + NS	Orally NS 1000 mg/kg + sham operation
III	Sham + NAC	Orally NAC 140 mg/kg + sham operation
IV	Paracetamol	Orally paracetamol 2 g/kg
V	Paracetamol + NAC	Orally paracetamol 2 g/kg + NAC 140 mg/kg
VI	Paracetamol + NS 250 mg/kg	Orally paracetamol 2 g/kg + NS 250 mg/kg
VII	Paracetamol + NS 500 mg/kg	Orally paracetamol 2 g/kg + NS 500 mg/kg
VIII	Paracetamol + NS 1000 mg/kg	Orally paracetamol 2 g/kg + NS 1000 mg/kg

NS: Nigella sativa L. ethanol extract; NAC: N-acetylcysteine.

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 controlled 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 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 Select™ 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 absorbances 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.

Biochemical investigation of serum

Serum urea and creatinine levels were measured using the commercial kits (BEN) (Biochemical Enterprise, Milano, Italy) BK 151 and CR280 in an automatic biochemical analyzer (ChemWell, Palm City, FL) at 340 nm and 510 nm, respectively.

Histological examination

The kidneys of rats in all groups were fixed in 10% neutral formalin for 48–72 h. Then, tissues were trimmed and processed for a routine histopathological examination. Tissues were embedded into paraffin wax and 4–5 μm thick sections were cut. All tissue sections were stained with Haematoxylin and Eosin (H&E) and then examined under a light microscope (Olympus BX51, Tokyo, Japan).

Renal lesions in rats were assessed according to Zhang et al. (2008) and graded into five categories by utilizing a scale of 0–5, where 0 = normal histology and 1 = tubular epithelial cell degeneration, without significant necrosis/apoptosis; 2–5 = <25%, <50%, <75% and ≥75% of the tubules showed tubular epithelial cell necrosis/apoptosis, respectively, accompanied by other concomitant alterations.

Statistical analysis

Data obtained from the serum urea and creatinine levels, tissue SOD activity and GSH and MDA levels were subjected to a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). Differences among the groups were obtained using the Duncan multiple comparison and were considered significant at p < 0.05. All results were expressed as mean ± SD using the IBM SPSS Statistics 19 software (SPSS Inc., Chicago, IL). Results of the histological score were compared between groups using a one-way ANOVA and Tukey’s multiple comparison tests. Histological statistical analyses and graphs were prepared using GraphPad Prism version 4.0 (GraphPad Software, La Jolla, CA), and p < 0.05 was considered statistically significant.

Results

The effects of NS on serum urea and creatinine levels

Serum urea and creatinine levels were found to be higher in the paracetamol group when compared with the sham group (p < 0.05). NS administration dramatically reduced serum urea and creatinine levels. The NS extract showed a dose-dependent protective effect on paracetamol-induced nephrotoxicity compared with the standard drug, NAC. The serum levels of urea and creatinine were found to be lower in the NS treatment groups (NS 250, NS 500 and NS 1000 mg/kg, groups) (p < 0.05). The extract at a dose of 1000 mg/kg had a significantly higher protective effect on nephrotoxicity than standards at 250 and 500 mg/kg; in fact, the protective effect at this dose was significantly higher than the effect of NAC (p < 0.05).

The administration of paracetamol caused increments in both serum urea and creatinine levels. The serum urea level, which increased with paracetamol treatment to 88.05 U/L, was reduced to 65.60, 56.00 and 54.18 U/L, with 250, 500 and 1000 mg/kg doses of the extract, respectively. The serum creatinine level, which increased to 0.80 U/L by paracetamol administration, was reduced to 0.64, 0.57 and 0.52 U/L with 250, 500 and 1000 mg/kg doses of the extract, respectively (Table 2).

Table 2. Effects of NS treatments on changes in paracetamol-induced nephrotoxicity in rats.

Group	Urea (U/L)	Creatinine (U/L)
Sham	49.80 ± 11.91^a	0.31 ± 0.08^a
Sham + NS 1000 mg/kg	48.64 ± 5.57^a	0.37 ± 0.11^ab
Sham + NAC 140 mg/kg	46.30 ± 12.72^a	0.38 ± 0.09^ab
Paracetamol 2 g/kg	88.05 ± 25.69^c	0.80 ± 0.21^d
Paracetamol 2 g/kg + NAC 140 mg/kg	59.86 ± 12.67^ab	0.55 ± 0.25^c
Paracetamol 2 g/kg + NS 250 mg/kg	65.60 ± 11.57^b	0.64 ± 0.16^c
Paracetamol 2 g/kg + NS 500 mg/kg	56.00 ± 14.52^ab	0.57 ± 0.07^c
Paracetamol 2 g/kg + NS 1000 mg/kg	54.18 ± 8.50^ab	0.52 ± 0.11^bc

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

Biochemical results for oxidant and antioxidant levels of kidney tissue in rats

The activity of SOD and the levels of MDA and GSH in 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 significantly increased in the paracetamol group compared with the sham group (p < 0.05).

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)
Sham	44.97 ± 8.98^b	2.39 ± 0.43^bc	1.80 ± 0.68^a
Sham + NS 1000 mg/kg	45.69 ± 10.62^b	2.61 ± 0.54^c	1.76 ± 0.40^a
Sham + NAC 140 mg/kg	41.22 ± 10.95^b	2.46 ± 0.65^c	2.08 ± 0.76^ab
Paracetamol 2 g/kg	23.31 ± 6.62^a	1.32 ± 0.42^a	4.06 ± 0.29^d
Paracetamol 2 g/kg + NAC 140 mg/kg	36.31 ± 13.25^b	2.08 ± 0.47^bc	2.81 ± 0.74^bc
Paracetamol 2 g/kg + NS 250 mg/kg	25.79 ± 8.79^a	1.85 ± 0.40^b	3.16 ± 1.59^c
Paracetamol 2 g/kg + NS 500 mg/kg	39.40 ± 8.91^b	2.08 ± 0.59^bc	3.23 ± 0.58^c
Paracetamol 2 g/kg + NS 1000 mg/kg	46.54 ± 9.11^b	2.26 ± 0.51^bc	2.26 ± 0.53^ab

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

NS administration restored GSH and SOD and decreased MDA levels in kidneys (Table 3). The NS extract showed a dose-dependent inhibition compared with the standard drug, NAC. The extract at a dose of 1000 mg/kg had significantly higher scavenging activity on kidneys than standards at 250 and 500 mg/kg; in addition, the scavenging activity at this dose was significantly higher than the effect of NAC (p < 0.05).

Results of histopathological examinations

Histopathological scores of rat kidneys in all groups are presented in Figure 1. Kidneys of the control group (Figure 2A) animals showed a normal histologic morphology. 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.

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 < 0.05 was considered statistically significant.

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×.

The histopathology of the kidneys of the paracetamol-treated group (Figure 2E) showed severe proximal tubular distortion with enlarged lumina. Signs of necrosis, 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 desquamations were hardly detected.

Discussion

Although paracetamol, with the alternative name acetaminophen, has a reasonable safety profile in therapeutic 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 nephrotoxic 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 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 mediators, 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 hydrogen 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, including 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 acetaminophen-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 paracetamol-induced nephrotoxicity in biochemical and histopathologic 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 therapeutic candidate against paracetamol-induced acute nephrotoxicity.

Conclusion

In light of these observations, we found that the therapeutic administration of NS prevented paracetamol-induced oxidative stress changes. It can be speculated 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.

Funding information

This study was supported by the Research fund of Atatürk University (BAP-2012/077).

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.