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Abstract

Hepatotoxicity is a major dose-limiting side effect of CP chemotherapy besides nephrotoxicity and gastrointestinal dysfunction. TQ, a principal Nigella sativa seed oil constituent, has been shown to improve hepatic functions in various in vivo models of acute hepatic injury. In view of this, the present study aimed to evaluate the effect of TQ against CP-induced hepatotoxicity. Rats were divided into four experimental groups; control, CP, CP+TQ and TQ. Animals in CP+TQ and TQ groups were administered TQ (1.5 mg/kg bwt, orally), with or without a single hepatotoxic dose of CP (6 mg/kg bwt, i.p.) respectively, for 14 days before and four days following the CP treatment. CP induced an upsurge in serum ALT and AST activities, indicating liver injury, as also confirmed by the histopathological findings. CP caused significant alterations in the activities of membrane marker enzymes, carbohydrate metabolic enzymes, and the enzymatic and nonenzymatic components of the antioxidant defense system. TQ supplementation ameliorated all these adverse biochemical and histological changes in CP-treated rats. Thus, TQ may have excellent scope for clinical applications in combating CP-induced hepatic pathophysiology.

Keywords

Cisplatin thymoquinone carbohydrate metabolism antioxidant system hepatotoxicity

Introduction

The liver is a vital organ that plays a significant role in the body’s metabolism of xenobiotics, and that renders it particularly susceptible to injury by chemicals/drugs to which humans are frequently exposed. Among soft tissues, the liver is found to be the largest repository of various orally administered drugs. Cisplatin (cis-diamminedichloroplatinum II, CP) is one of the most valuable cancer chemotherapy drugs, however its severe side effects and consequent systemic toxicity greatly limits its therapeutic utility.^1–3 Although extensive studies have demonstrated CP-induced nephrotoxicity in both clinical and animal researches, unfortunately, very limited studies have focused on CP-induced liver damage, and advances in developing potent hepatoprotective strategies/agents are still lacking. CP is known to induce pronounced structural and functional alterations in the hepatic tissue. CP-induced histopathological aberrations are manifested as inflammatory infiltration, dissolution of hepatic cord, dilated sinusoids, and extensive disorganization of hepatocytes.^4,5

Increasing evidence indicates that CP induces an overwhelmed generation of reactive oxygen species (ROS)/free radicals and depletes endogenous antioxidant reserves leading to a condition known as oxidative stress. CP-induced ROS react with cellular macromolecules (nucleic acid, proteins, and lipids), causing extensive liver damage.^1,5 Thus, the combinatorial strategies utilizing phytochemicals with potent antioxidant properties may prove beneficial in the amelioration of CP-induced deleterious hepatic alterations. One such phytochemical is thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone; C₁₀H₁₂O₂; molecular weight: 164.2 g/mol). Thymoquinone is a redox-active quinone, primarily obtained from the volatile oil of Nigella sativa seeds. TQ, as a bioactive component, is also found in several other medicinal plants belonging to genera Monarda, Juniperus, and Thymus.^6–8

TQ exhibits a broad spectrum of pharmacological and therapeutic properties/activities, particularly antioxidant, antidiabetic, antitumor, anti-inflammatory, cardioprotective, nephroprotective and hepatoprotective.^9,10 TQ exists in tautomeric forms, including the enol form (reduced) and the keto form (oxidized) or as mixtures of both the states. The keto form contributes to a significant fraction (∼90%) and is primarily responsible for its inherent pharmacological properties.¹¹ TQ is a potent antioxidant agent, and its scavenging activity against a wide array of free radicals and ROS is well documented.¹² Moreover, owing to its high lipophilicity, TQ can easily cross the membrane lipid bilayers and thus has easy accessibility to subcellular compartments which, further facilitates its ROS/free radical scavenging activity. Previous studies have also shown the metabolic conversion of TQ into thymohydroquinone (DHTQ) and glutathionylated dihydrothymoquinone (DHTQ-GS) via. enzymatic and nonenzymatic reduction, respectively. These reduced metabolites of TQ were found to act as more potent free radical scavengers than TQ itself.¹³

Several experimental rat/mice models exhibited substantial ameliorative effect upon TQ consumption against the hepatotoxicity induced by a variety of free radical generating agents such as carbon tetrachloride (CCl₄),¹⁴ acetaminophen,¹² cadmium,¹⁵ lead,¹⁶ cyclophosphamide,¹⁷ tamoxifen,¹⁸ cypermethrin,¹⁹ aflatoxin B1 (AFB1)²⁰ and methotrexate.²¹ Moreover, TQ has been reported to potentiate the plasma and liver antioxidant capacities and enhance the expression of liver antioxidant genes in hypocholesterolemic rats.²² Studies by Al-Malki and Sayed have shown TQ to reduce cisplatin-induced inflammation in the hepatic tissue.²³ Owing to its unique effect on physiological functions, TQ is considered an important biological response modifier (BRM) that can diminish the side effects of anticancer drugs while at the same time increasing their efficacy.²⁴ TQ has been shown to synergize the antitumor activity of CP against non-small cell lung carcinoma²⁵ and Ehrlich ascites carcinoma (EAC) while ameliorating its nephrotoxic side effects.²⁶

We have recently reported that oral TQ supplementation protects against acute as well as chronic CP treatment-induced renal and gastrointestinal toxicities.^6,7,27,28 To the best of our knowledge, the possible ameliorative efficacy of TQ against CP-induced perturbations in hepatic metabolic functions and redox status has not yet been investigated. Considering the potential clinical use of CP and numerous pharmacological properties of TQ, the present study evaluates the preventive/protective potential of TQ against CP treatment-induced acute liver dysfunction.

Material and methods

Chemicals and drugs

Cisplatin (product code: P4394) and thymoquinone (product code: 274666; purity 98%) were purchased from Sigma-Aldrich Chemical Corp. (St. Louis, MO, USA). All other chemicals used were of analytical grade and were purchased either from Sigma Chemical Corp. or from SRL (Mumbai, India).

Diet

Standard rat pellet diet was obtained from Aashirwaad industries, Chandigarh (1544, Sector 38-B, Chandigarh, India).

Ethical approval

Animal experiments were permitted by the Ministry of Environment, Forests and Climate Change, Government of India under registration no. 714/GO/Re/02/CPCSEA issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) dated 29th October 2014 and approved by the Institutional Animal Ethics Committee (IAEC) of the Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India.

Experimental design

Adult male Wistar rats, weighing approximately 150 g were used to conduct the present study. The animals were purchased from National Institute of Biologicals (NIB), New Delhi. Prior to use, rats were acclimatized for 7 days in 12 h:12 h light and dark illumination cycles. The animals were kept in polycarbonate cages in a well-ventilated room maintained at 26 ± 2°C and 50–60% relative humidity and fed standard rodent diet supplied by Aashirwad Industries, Chandigarh, India, with free access to water. Rats were randomized into 4 groups with six rats per group, as mentioned below:

Group 1—Control (C), received 0.9% normal saline

Group 2—CP alone (CP), received a single intraperitoneal injection of CP (6 mg/kg bwt) in 0.9% normal saline

Group 3—Thymoquinone alone (TQ), received TQ (1.5 mg/kg bwt, orally) during the entire duration of the experiment

Group 4—CP-treated + TQ administered (CP+TQ), received TQ (1.5 mg/kg bwt, orally) for 14 days before and four days following a single CP injection.

Animals in all four groups were sacrificed on the fifth day after a single CP injection, under light diethyl ether anesthesia. Blood samples were collected from overnight fasted rats, and the liver was removed and processed for histopathology and the preparation of homogenate as described below.

Liver homogenate preparation

The liver was homogenized in 0.1 M Tris–HCl buffer (pH 7.5) by a glass–Teflon homogenizer (Thomas PA, USA) by passing five pulses; at 4°C to make a 10% (w/v) homogenate. The homogenate was then subjected to high-speed Ultra-Turrex Kunkel homogenizer (Type T-25, Janke & Kunkel GMBH & Co., KG. Staufen) for three pulses of 30 s each with an interval of 30 s between each stroke. One part of the homogenate was saved at −20°C for estimation of GSH, total-SH, and lipid peroxidation, while the other part was centrifuged at 2000 rpm for 10 min at 4°C in a high-speed Remi centrifuge (Remi motors, Mumbai, India) to remove the cell debris. The supernatant was saved in aliquots and stored at −20°C for analyses of membrane, carbohydrate metabolism and free radical scavenging enzymes.

Biochemical analysis

Blood samples were centrifuged at 2000 × g for 10 min and sera were separated. Serum samples were deproteinated with 3% trichloroacetic acid in a ratio of 1:3, left for 10 min and then centrifuged at 2000 × g for 10 min. The precipitate obtained was used to quantitate total phospholipids (PLs). Cholesterol (Chl) levels were determined directly in serum samples by method of Zlatkis and Boyle.²⁹ Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined in serum by Reitman and Frankel method using kit from span diagnostics.

Assay of liver carbohydrate metabolic enzymes

The activities of the enzymes involving oxidation of NADH or reduction of NADP were determined spectrophotometrically on UV-1700 (Pharma Spec, Shimadzu Corp., Japan) fixed for 340 nm using 3 ml of assay buffer in a 1-cm cuvette at room temperature (28–30°C). The enzyme activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) were assayed as described by Khundmiri et al.³⁰ Hexokinase (HK) was estimated by the method of Crane and Sols³¹ and the remaining glucose was measured by method of Nelson.³²

Assay of membrane enzymes and lysosomal marker enzyme

The activities of membrane marker enzymes viz. alkaline phosphatase (ALP), γ-glutamyl transferase (GGTase), leucine aminopeptidase (LAP), and lysosomal marker enzyme viz. acid phosphatase (ACPase) were determined as described by Farooq et al.³³

Assay of enzymes involved in free radical scavenging

Superoxide dismutase (SOD) was assayed by the method of Marklund and Marklund.³⁴ Catalase (CAT) and glutathione peroxidase (GSH-Px) activities were determined by the method of Giri et al.³⁵ and Flohe and Gunzler,³⁶ respectively. The activities of thioredoxin reductase (TR) and glutathione-S-transferase (GST) were determined using 5,5′-dithiobisnitrobenzoic acid and 1-chloro-2-4-dinitrobenzene as substrates, respectively.^37,38 Glutathione reductase (GR) was assayed from the oxidation of NADPH to NADP⁺ in the presence of oxidized glutathione.³⁹

Thiobarbituric acid reactive substances (TBARS), total –SH group and GSH estimation

TBARS (LPO products) were measured as malondialdehyde (MDA) equivalents as described by Ohkawa et al.⁴⁰ Total-SH and GSH were determined after reaction with 5,5′-dithiobisnitrobenzoic acid by the method of Sedlak and Lindsay⁴¹ and Jollow et al.,⁴² respectively.

Haematoxylin-eosin (HE) histopathology

The liver was cut into pieces and kept in Bouin’s fluid, and embedded in paraffin. Transverse tissue sections of 5 µm thickness were cut with a rotary microtome and stained with hematoxylin and eosin (HE). Light microscopic observations were made under Zeiss microscope (Carl Zeiss model Axioskop 40 FL). Ten sections from four rats of each group were quantified with auto measure software (Rel 4.8), and the total area of 200 hepatocytes cells was analyzed. Histopathological changes, in particular, hepatocellular degeneration, hepatocellular vacuolation, nuclear pyknosis, and sinusoidal alterations, were recorded at the initial magnification of 400×. The semi-quantitative data of histological scores is expressed as normal (−), moderate (+), high (++), Very high (+++).

Statistical analysis

All data are expressed as mean ± SEM for at least three different preparations. Statistical evaluation was conducted by one-way ANOVA using origin 6.1 software. A probability level of p < 0.05 was selected as indicating statistical significance. Most of the changes between various groups were compared with control values for better understanding and clarity.

Results

The effect of TQ supplementation against the CP-induced acute deleterious alterations in rat liver was evaluated. Liver homogenate was prepared from all the four experimental groups and was used to determine various parameters, including the activities/levels of hepatocyte membrane enzymes, carbohydrate metabolic enzymes and both the enzymatic and nonenzymatic parameters of the hepatic antioxidant defense system. The effect of TQ was also examined on CP-induced histopathological aberrations.

Biochemical parameters

As can be seen from the data (Table 1), CP treatment caused a significant increase in ALT (+63%) and AST (+59%) and Chl (+41%) and PLs (+25%) levels as compared to the control group, showing the induction of hepatotoxicity. In contrast, TQ supplementation before and following CP treatment significantly prevented the CP-induced increase in activities/levels of ALT, AST, Chl and PLs. Thus, TQ administration to CP-treated (CP+TQ) rats lowered the severity of various CP-elicited hepatotoxic alterations in serum parameters. However, TQ supplementation alone caused a significant decrease in the activity of AST and Chl levels, while activity/level of ALT and PL were similar to that of control.

Table 1.

Effect of TQ with and without CP treatment on serum parameters.

Parameters/groups	ALT (IU/L)	AST (IU/L)	Cholesterol (mg/dl)	Phospholipid (mg/dl)
Control	43.78 ± 2.23	28.31 ± 0.86	88.26 ± 2.51	221.4 ± 5.98
CP	71.33 ± 2.78* (+63%)	44.94 ± 1.32* (+59%)	124.50 ± 3.22* (+41%)	295.7 ± 6.33* (+25%)
CP+TQ	50.51 ± 2.53** (+15%)	25.62 ± 0.81** (−10%)	93.53 ± 3.05** (+6%)	229.1 ± 6.49** (+3%)
TQ	37.66 ± 1.40 (−14%)	21.04 ± 0.67* (−26%)	70.32 ± 2.14* (−20%)	217.85 ± 7.34 (−2%)

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated; ALT: alanine aminotransferases; AST: aspartate aminotransferases.

Results are mean ± SEM for six different samples (n = 6) in each group.

Values in parenthesis represent percent change from control.

* Significantly different from control.

** Significantly different from CP at p < 0.05 by one way ANOVA.

Histopathological alterations in the liver

Histopathological observations of liver sections (Figure 1) showed extensive damage by CP treatment. Lobules with necrotic hepatocytes, voluminous sinusoids, nuclear pyknosis, and an enlarged central vein were obvious in CP-treated rats (Figure 1(B)). The combination group, CP+TQ, showed noticeable preservation of histological appearance (Figure 1(D)). Clear sinusoids, intact hepatocytes, and no apparent necrosis were observed in the CP+TQ group of rats. Moreover, control (Figure 1(A)) and TQ supplemented (Figure 1(C)) rats showed normal architecture of the liver.

Figure 1.

Histopathology of rat liver showing sinusoids (↑), hepatocytes (↑) and central vein (↑) in control (A) with normal histoarchitecture of hepatocytes, intact central vein, and clear sinusoids whereas, in CP-treated group (B) lobules with necrotic hepatocytes, voluminous sinusoids, nuclear pyknosis, and an enlarged central vein were apparent. TQ administered group (C) shows well-preserved conditions of hepatocytes, sinusoids, and intact central vein, very similar to that of control. CP+TQ group (D) shows relatively intact histoarchitecture, clear sinusoids, and no apparent necrosis. H & E stain, initial magnification ×400 scale bar = 50 µm.

The histopathological damage scores from the histological images of different experimental groups (Table 2) suggest high degeneration of hepatocytes, hepatocellular vacuolation, and nuclear pyknosis in the CP-treated group. However, the scores indicate relatively clear sinusoids, no obvious nuclear pyknosis and moderately intact hepatocytes in the CP+TQ combination group. Control and TQ alone group scores indicate somewhat intact histoarchitecture with clear sinusoids and intact hepatocytes.

Table 2.

The histopathological damage score in different experimental groups.

Parameters	Control	CP	CP+TQ	TQ
Hepatocellular degeneration	−	+++	+	−
Hepatocellular vacuolation	−	+++	+	−
Nuclear pyknosis	−	+++	−	−
Clear Sinusoid	+++	+	+++	+++

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated.

The semi-quantitative data is expressed as normal (−), moderate (+), high (++), very high (+++).

Membrane enzymes and biomarker enzyme of lysosomes

The effect of CP and TQ alone and their combination (CP+TQ) was determined on the activities of ALP, GGTase, and LAP (membrane enzymes) and ACPase (lysosomal enzymes) in liver homogenates prepared from different experimental groups. CP treatment causes significant decline in the specific activities of ALP (−32%), GGTase (−37%), and LAP (−53%), whereas the activity of ACPase was increased significantly (+58%) in liver homogenate. However, TQ administration before and following CP treatment significantly reduced CP-induced membrane damage as indicated by significant attenuation of CP-induced decline in membrane enzyme activities (Table 3). Moreover, the administration of TQ to CP-treated rats significantly ameliorated the CP-induced increase in the activity of ACPase. However, supplementation of TQ alone to the rats did not affect membrane enzymes’ activities while caused a significant decline in the activity of ACPase in liver homogenate.

Table 3.

Effect of TQ with and without CP treatment on membrane and lysosomal enzymes in liver homogenates.

Enzymes/groups	ALP (µmol/mg protein/h)	GGTase (µmol/mg protein/h)	LAP (µmol/mg protein/h)	ACPase (µmol/mg protein/h)
Control	6.71 ± 0.221	2.58 ± 0.051	10.72 ± 0.313	1.55 ± 0.032
CP	4.59 ± 0.089* (−32%)	1.62 ± 0.022* (−37%)	5.06 ± 0.148* (−53%)	2.45 ± 0.047* (+58%)
CP+TQ	5.83 ± 0.112** (−13%)	2.29 ± 0.017** (−11%)	9.08 ± 0.531** (−15%)	1.49 ± 0.026** (−4%)
TQ	6.85 ± 0.163 (+2%)	2.43 ± 0.044 (−6%)	11.70 ± 0.625 (+9%)	1.19 ± 0.066* (−23%)

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated; ALP: alkaline phosphatase; GGTase: γ-glutamyl transferase; LAP: leucine aminopeptidase; ACPase: acid phosphatase.

Results are expressed as mean ± SEM of six different samples (n = 6) in each group.

Values in parenthesis represent percent change from control.

* Significantly different from control.

** Significantly different from CP at p < 0.05 by one way ANOVA.

Carbohydrate metabolism

The effect of CP and TQ alone and in combination (CP+TQ) was determined on the activities of various enzymes of carbohydrate metabolism viz. HK and LDH (glycolysis), MDH (citric acid cycle), G6Pase and FBPase (gluconeogenesis), ME, and G6PDH (pentose phosphate pathway) in liver homogenates. As shown in Table 4, CP treatment caused a significant increase in the activities of LDH (+57%), HK (+45%) and ME (+61%) while MDH (−32%), G6Pase (−37%), FBPase (−31%) and G6PDH (−64%) activities decreased profoundly in the liver homogenate. In contrast, CP-elicited increase in LDH. HK and ME activities was prevented significantly in the liver tissue upon TQ administration. Furthermore, TQ administration before and following CP treatment also prevented CP-induced decline in MDH, G6Pase, FBPase and G6PDH activities in liver homogenates. However, no significant change was observed in the activities of these enzymes in TQ alone group except MDH and G6PDH which were increased significantly by TQ.

Table 4.

Effect of TQ with and without CP treatment on carbohydrate metabolic enzymes in liver homogenates.

Enzymes groups	LDH (µmol/mg protein/h)	MDH (µmol/mg protein/h)	HK (µmol/mg protein/h)	G6Pase (µmol/mg protein/h)	FBPase (µmol/mg protein/h)	G6PDH (µmol/mg protein/h)	ME (µmol/mg protein/h)
Control	0.58 ± 0.024	1.27 ± 0.044	1.09 ± 0.038	0.671 ± 0.008	2.75 ± 0.037	0.061 ± 0.007	0.054 ± 0.005
CP	0.91 ± 0.016* (+57%)	0.86 ± 0.015* (−32%)	1.58 ± 0.042* (+45%)	0.42 ± 0.013* (−37%)	1.91 ± 0.113* (−31%)	0.022 ± 0.009* (−64%)	0.087 ± 0.005* (+61%)
CP+TQ	0.66 ± 0.035** (+14%)	1.21 ± 0.027** (−5%)	1.25 ± 0.063** (+15%)	0.589 ± 0.016** (−12%)	2.33 ± 0.127** (−15%)	0.053 ± 0.006** (−13%)	0.068 ± 0.003** (+26%)
TQ	0.52 ± 0.037 (−10%)	1.51 ± 0.047* (+19%)	1.03 ± 0.125 (−6%)	0.693 ± 0.011 (+3%)	2.99 ± 0.15 (+9%)	0.072 ± 0.004* (+18%)	0.049 ± 0.003 (−9%)

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated; HK: hexokinase; LDH: lactate dehydrogenase; MDH: malate dehydrogenase; G6Pase: glucose-6-phosphatase; FBPase: fructose-1,6-bisphosphatase; G6PDH: glucose-6-phosphate dehydrogenase; and ME: malic enzyme.

Results are expressed as mean ± SEM of six different samples (n = 6) in each group.

Values in parenthesis represent percent change from control.

* Significantly different from control.

** Significantly different from CP at p < 0.05 by one way ANOVA.

Antioxidant defense parameters

Antioxidant defense parameters are potential biomarkers to determine the physiological state of the cell, tissue or organ. To evaluate the antioxidant effect of administration of TQ against the oxidative stress induced by CP, the activities/levels of various enzymatic and nonenzymatic parameters of oxidative stress were determined in liver homogenates of all the four experimental groups i.e. C, CP, CP+TQ and TQ alone.

LPO measured in terms of thiobarbituric acid reactive substances (TBARS), reported as malondialdehyde (MDA) levels, was significantly enhanced (+90%) in the CP-treated rats accompanied by a significant decline in GSH (−33%) and total SH (−49%) levels in the rat liver (Table 5). However, TQ supplementation prior to and following CP treatment significantly prevented CP-induced increase in MDA and decrease in GSH and total SH levels in rat liver.

Table 5.

Effect of TQ with and without CP treatment on LPO, total SH and GSH levels in liver homogenates.

Enzyme groups	Lipid peroxidation (nmols/gm tissue)	Total SH (µmols/gm tissue)	GSH (µmol/gm tissue)
Control	97.67 ± 4.26	17.40 ± 0.56	0.68 ± 0.012
CP	185.35 ± 5.41* (+90%)	11.72 ± 0.45* (−33%)	0.35 ± 0.008* (−49%)
CP+TQ	115.54 ± 3.18** (+18%)	15.79 ± 0.54** (−9%)	0.48 ± 0.015** (−29%)
TQ	83.44 ± 3.54 (−15%)	18.05 ± 0.63 (+4%)	0.61 ± 0.023 (−10%)

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated; LPO: lipid peroxidation; Total-SH: sulphydryl groups; and GSH: glutathione.

Results are expressed as mean ± SEM of six different samples (n = 6) in each group.

Values in parentheses represent percent change from control.

* Significantly different from control.

** Significantly different from CP at p < 0.05 by one way ANOVA.

The effect of CP and TQ alone and their combination (CP+TQ) was also determined on the activities of various antioxidant enzymes in the rat liver. As shown in Table 6, CP treatment caused a marked decline in the activities of SOD (−42%), CAT (−48%), GSH-Px (−39%), GR (−57%), TR (−48%), and GST (−40%) in the liver homogenate. Administration of TQ prior to and following CP treatment markedly reduced the severity of CP-induced oxidative damage as indicated by significant amelioration of CP-induced decline in the activities of all major antioxidant enzymes including, SOD, CAT, GSH-Px, GR, TR, and GST in liver. Activities of these enzymes were not affected significantly in TQ alone group except CAT and GST which were increased significantly by TQ.

Table 6.

Effect of TQ with and without CP treatment on enzymatic antioxidant parameters in liver homogenates.

Parameters groups	SOD (µmol/mg protein/min)	CAT (µmols/mg protein/min)	GSH-Px (µmols/mg protein/min)	GST (µmols/mg protein/min)	GR (µmols/mg protein/min)	TR (µmols/mg protein/min)
Control	53.35 ± 2.41	19.97 ± 1.28	0.152 ± 0.011	0.341 ± 0.015	0.081 ± 0.005	1.054 ± 0.112
CP	31.02 ± 1.83* (−42%)	10.46 ± 0.772* (−48%)	0.093 ± 0.007* (−39%)	0.206 ± 0.017* (−40%)	0.035 ± 0.002* (−57%)	0.552 ± 0.106* (−48%)
CP+TQ	46.33 ± 1.69** (−13%)	17.79 ± 0.629** (−11%)	0.138 ± 0.012** (−9%)	0.294 ± 0.023** (−14%)	0.073 ± 0.008** (−10%)	0.918 ± 0.074** (−13%)
TQ	59.42 ± 2.09 (+11%)	24.03 ± 1.417* (+20%)	0.172 ± 0.022 (+13%)	0.399 ± 0.014* (+17%)	0.088 ± 0.01 (+9%)	1.033 ± 0.137 (−2%)

CP: cisplatin treated; TQ: Thymoquinone administered; CP+TQ: Thymoquinone administered + cisplatin treated; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; GST: glutathione-S-transferase; GR: glutathione reductase; and TR: thioredoxin reductase.

Results are expressed as mean ± SEM of six different samples (n = 6) in each group.

Values in parentheses represent percent change from control.

* Significantly different from control.

** Significantly different from CP at p < 0.05 by one way ANOVA.

Discussion

One of the major pathophysiologic event in CP-induced hepatic dysfunction is the elevated generation of free radicals and ROS leading to disruption of the oxidant-antioxidant balance. Since the past few decades, plant-derived natural substances/nutrients that exhibit strong antioxidant properties have become a part of therapeutic strategies for the amelioration of drug/chemical-induced toxicities.^6,3,28 In this regard, TQ, the principal bioactive constituent of Nigella sativa seed oil, has shown potential therapeutic benefits in a wide variety of oxidative stress related ailments.⁹

In view of the implications of oxidative stress in CP-induced hepatic pathophysiology and TQ’s noteworthy effectiveness against oxidative stress-mediated pathologies, the present study was undertaken to investigate the possible protective efficacy of TQ against CP-induced acute liver damage. A single dose of CP treatment produced a typical hepatic damage pattern, as recorded by elevated serum levels of transaminases viz. ALT and AST, the classical diagnostic indicators of hepatotoxicity. Levels of serum cholesterol and PLs were also increased significantly upon CP treatment. These consequences suggest impairment in hepatic functions in CP-treated rats. Histopathological examination of the liver of all four experimental groups further corroborated the biochemical findings. It showed degeneration and vacuolation of hepatocytes along with nuclear pyknosis in the liver of CP-treated rats, while TQ supplementation to the CP-treated rats abrogated the CP-induced alterations in the liver histoarchitecture. Furthermore, CP-elicited increase in serum levels of ALT, AST, cholesterol, and PLs were significantly prevented in TQ administered animals.

Clinically, the hepatocyte membrane is highly susceptible to drug/chemical-induced injury.⁴³ The functional integrity of the membrane lining the hepatocytes was assessed by monitoring the activities of several membrane marker enzymes viz. ALP, GGTase, and LAP in the liver homogenates. In agreement with the previous studies, our results show that activities of all the membrane marker enzymes were declined significantly by single-dose CP treatment, suggesting severe damage to the membrane of the hepatocytes (Table 3). The observed decrease in the membrane enzyme activities could be due to the reaction of membrane-associated PUFA with CP generated ROS/free radicals, and that could have initiated the autocatalytic lipid peroxidation reaction and disrupted the structural integrity of hepatocyte membrane that eventually lead to the loss of membrane enzymes. Another possibility is the oxidative modification of the enzymes either directly by CP or by CP generated free radicals and ROS, as reported previously.¹ On the contrary, CP-induced decline in the membrane enzyme activities was significantly prevented upon oral administration of TQ, indicating the restoration of membrane integrity. The observed ameliorative effect of TQ on membrane enzyme activities could be attributed to the redox properties of its benzoquinone ring structure and its unrestricted crossing over of biological barriers. CP-induced enzyme inactivation and enhanced autocatalytic LPO could have been directly prevented by TQ owing to its inherent free radical scavenging and antioxidant properties.¹⁰ Decline in the loss or leakage of active enzyme molecules from hepatocytes into the blood, as evidenced by the observed decline in serum ALT and AST activities in the CP+TQ group, suggest an overall restoration of membrane integrity upon TQ supplementation. Furthermore, a significant increase in the activity of ACPase was observed in the liver homogenates of CP-treated rats (Table 3). This increase could be due to the CP-induced morphological alterations in size and number of lysosomes in tissues, as suggested previously.⁴⁴ However, oral gavage of TQ prior to and following CP treatment caused a reversal of CP-induced alteration in ACPase activity (Table 3).

Since the liver is a major organ that coordinates various carbohydrate metabolic pathways to meet the cellular energy demands hence, it was imperative to assess the effect of CP on hepatic energy metabolism and to examine if TQ could restore the metabolic capacity of the liver in CP-treated rats. The activities of various enzymes related to carbohydrate metabolism were analyzed in liver homogenates prepared from the different experimental groups of rats. CP treatment caused a significant decline in the activity of the citric acid cycle enzyme, MDH, with a concurrent increase in the activities of LDH and HK (glycolytic enzymes) (Table 4). A marked decrease in MDH activity suggests impairment in aerobic metabolism of glucose while elevated LDH activity signifies increased dependence on anaerobic glycolysis for energy generation. This could be due to CP-induced mitochondrial damage, thereby leading to less ATP production and consequent perturbation of hepatic metabolic functions. However, TQ administration significantly abolished/reduced the CP-induced increase in LDH and HK and decrease in MDH activity, suggesting the reestablishment of energy dependence on aerobic metabolism and/or improvement of mitochondrial functions in the liver. Earlier studies have demonstrated TQ supplementation to upregulate MDH genes in the leukocytes of streptozotocin (STZ) induced diabetic rats.⁴⁵

The liver, being a major site of gluconeogenesis, plays a vital role in the homeostatic regulation of blood glucose levels. CP treatment caused a significant decline in the activities of G6Pase and FBPase, the key gluconeogenic enzymes, indicating declined hepatic production of glucose via gluconeogenesis. Reduction in the oxaloacetate levels, a major glucose precursor, due to CP-induced decline in MDH activity could be the possible reason for the lowered rate of gluconeogenesis in CP-treated rats. G6PDH is a key enzyme of the pentose phosphate pathway that oxidizes glucose or glucose-6-phosphate to 6-phosphogluconate and generates NADPH, which, in turn, provides reducing equivalents for various anabolic reactions and protects the cell from ROS-induced oxidative damage. In addition, the NADP malic enzyme (ME) that belongs to a family of oxidoreductases and oxidizes malate to pyruvate, is another vital source of NADPH in the cells. The activities of these major NADPH generating enzymes (G6PDH and ME) were reciprocally affected by CP treatment. A substantial decline in G6PDH activity could be a consequence of mitochondrial dysfunction or due to direct oxidation of functional -SH groups at the enzyme’s active site by CP generated free radicals/ROS. Thus, the lesser generation of NADPH due to decreased G6PDH activity could have been compensated by enhanced activity of ME in the liver. However, TQ supplementation resulted in an overall improvement of hepatic metabolism, as evidenced by higher activities of G6PDH and gluconeogenic enzymes in the CP+TQ combination group compared to the CP alone group. This could be attributed to the fact that TQ through redox transition of its benzoquinone ring structure could have directly scavenged/neutralized the CP generated free radicals/ROS before they reached their cellular targets, including the metabolic enzymes.¹³

CP-induced ROS/free radical generation and enhanced LPO leading to oxidative stress has been demonstrated as the primary causative factor in CP-induced liver dysfunction.¹ The well-developed hepatic antioxidant defense armory comprises both enzymatic (SOD, CAT, GSH-Px, TR, GR, GST) and nonenzymatic (GSH and total-SH) components that protect the hepatocytes from xenobiotics/drug-induced oxidative damage. Earlier studies have reported the depletion of endogenous antioxidant (enzymatic and nonenzymatic) reserves and increase in the markers of oxidative stress, including peroxidation of membrane lipids in liver and other tissues upon CP treatment.^7,2,28 In line with previous reports, a single dose of CP treatment caused excessive LPO as evident by high levels of malondialdehyde in the liver (Table 5). GSH, the most abundant nonprotein thiol that helps maintain the cells’ reducing environment, was decreased in CP-treated rats. The decline in total –SH content and GSH levels in the liver could be due to direct oxidation of –SH groups by CP generated ROS or preferential binding of CP to the thiol groups.⁶ However, CP-induced hepatic oxidative stress was significantly attenuated when animals received TQ via oral gavage both before and after CP treatment, and that could be attributed to the potent antioxidant and free radical scavenging activities of TQ.

Various enzymatic components of the antioxidant system function to protect the liver from drug/chemical-induced ROS including superoxide anion (O₂^·−), hydroxyl radical (·OH), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), and peroxyl radicals, by transforming these species to substantially less reactive and more tolerable forms. However, CP-induced oxidative stress occurs due to weakened endogenous antioxidant defense armory in the liver, as reported previously.¹ In the present study, a single dose CP treatment was found to cause a marked decline in the activities of SOD, CAT, and GSH-Px in the hepatic tissue, and that, in turn, would have enhanced the vulnerability of the liver to oxidative damage (Table 6). This decrease could be due to the direct binding of CP to -SH groups of functional cysteine residues or due to direct oxidation of these active site cysteine thiols groups by CP generated free radicals/ROS, as suggested earlier.⁴⁶ CP has been known to deplete the essential cofactors viz. Cu/Zn and Se which are essentially required for the activities of SOD and GSH-Px, respectively.⁴⁷ Thus, another possible reason for the observed decline in the activities of SOD and GSH-Px could be the depletion of Cu/Zn and Se, respectively, from the hepatocytes. However, TQ supplementation to CP-treated rats prevented CP-induced suppression in the activities of these antiperoxidative enzymes in the liver. Studies by Ismail et al. have shown that TQ administration increases the expression of SOD, CAT, and GSH-Px genes in rat liver.⁴⁷

GR and GST are the major GSH dependent enzymes that function in defense against oxidative stress. GR regenerates GSH by converting oxidized form (GSSH) to the reduced form (GSH) by utilizing the reducing power of NADPH supplied by G6PDH reaction. Furthermore, GST helps in the detoxification of xenobiotics/drugs by conjugating them to GSH via its sulfhydryl (-SH) group. A single dose of CP caused a significant decline in the activities of these GSH dependent enzymes (Table 6). The possible reason for the marked decline in the activities of GR and GST could be the reduced availability of their substrate i.e. GSH, in the liver. The activity of another enzyme, a major disulfide reductase, was also declined in the liver homogenates of CP-treated rats. Reduced activities of GR and TR could be due to the reduced activity of G6PDH, since, NADPH supplied by G6PDH is an essential coenzyme required for both TR and GR activities. However, TQ supplementation prior to and following CP treatment significantly prevented the CP-induced decline in TR, GR and GST activities in liver (Table 6). Orally gavaged TQ attenuated the CP-induced oxidative stress in liver by suppressing the augmentation of LPO and restoring the activities/levels of all the major antioxidant enzymes and hepatic nonenzymatic antioxidants, thus strengthening the endogenous antioxidant defense.

Conclusions

In conclusion, the results of the current study suggest that CP-induced ROS and free radicals contributed to the hepatotoxic effects of a single dose CP treatment in Wistar rats. TQ supplementation considerably alleviated the CP-induced disruption of membrane integrity and impairment in metabolic and redox status of liver as evident by improved membrane enzyme activities, hepatic metabolism and strengthened antioxidant defense in CP+TQ combination group. The observed ameliorative effects of TQ against CP-induced deleterious alterations on the hepatic structure and functions could be attributed to the potent free radical scavenging activity and antioxidant properties of TQ that could have lowered the CP-induced oxidative stress in hepatic tissue. Thus TQ, as a functional food or as a combinatorial nutraceutical may have a potential for clinical application to abrogate the accompanying hepatotoxic side effects in CP chemotherapy.

Footnotes

Acknowledgments

Financial support to the Department of Biochemistry from the UGC-SAP-DRS III, DST-FIST and DBT-PURSE programmes is gratefully acknowledged. The authors gratefully acknowledge University Grants Commission (UGC), New Delhi for the award of senior research fellowship (under MANF scheme) to FS and ZF. TA is the recipient of senior research fellowship from DBT (Department of Biotechnology), Government of India.

Authors’ contributions

FS wrote the manuscript and FK reviewed and edited it. FK, FS and ZF conceived and designed the research. FS, ZF and TA conducted the biochemical studies. SA and IP conducted and analyzed the histopathology data. All authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

F Khan

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Thymoquinone supplementation ameliorates cisplatin-induced hepatic pathophysiology

Abstract

Keywords

Introduction

Material and methods

Chemicals and drugs

Diet

Ethical approval

Experimental design

Liver homogenate preparation

Biochemical analysis

Assay of liver carbohydrate metabolic enzymes

Assay of membrane enzymes and lysosomal marker enzyme

Assay of enzymes involved in free radical scavenging

Thiobarbituric acid reactive substances (TBARS), total –SH group and GSH estimation

Haematoxylin-eosin (HE) histopathology

Statistical analysis

Results

Biochemical parameters

Histopathological alterations in the liver

Membrane enzymes and biomarker enzyme of lysosomes

Carbohydrate metabolism

Antioxidant defense parameters

Discussion

Conclusions

Footnotes

Acknowledgments

Authors’ contributions

Declaration of conflicting interests

Funding

ORCID iD

References