Biochemical alterations in diclofenac-treated rats: Effect of selenium on oxidative stress,inflammation,and hematological changes

Abstract

We investigated the effect of selenium (Sel), a trace element in diclofenac sodium (DCF), nonsteroidal anti-inflammatory drugs-induced hepatic and renal toxicities in adult rats. Five experimental groups namely control, DCF (10 mg/kg), Sel (0.125 mg/kg), DCF + Sel (0.125 mg/kg), and DCF + Sel (0.25 mg/kg) consisting of 10 rats each were orally treated for 7 consecutive days. Following killing, biomarkers of hepatic and renal toxicities, antioxidant enzyme levels, myeloperoxidase activity, nitric oxide levels, reactive oxygen and nitrogen species (RONS), and lipid peroxidation (LPO) were analyzed spectrophotometrically. Further, the concentration of tumor necrosis factor alpha (TNF-α) was assessed using enzyme-linked immunosorbent assay, and hematological indices: white blood cells (WBC), lymphocytes, and neutrophils and eosinophil counts. Results indicated that DCF-induced increases in biomarkers of hepatic and renal toxicity were significantly (p < 0.05) lessened in serum of rats co-exposed to DCF and Sel in a dose-dependent manner. DCF mediated decrease in antioxidant status, and increases in RONS, LPO, and TNF-α levels were reduced (p < 0.05) in the liver and kidney of rats co-exposed to DCF and Sel. Additionally, Sel reduced hematological abnormalities associated with DCF treatment. Light microscopic examination showed that the severity of histopathological lesions induced by DCF was lessened in rats co-exposed to DCF and Sel. Taken together, Sel supplementation mitigated DCF-induced oxidative stress, inflammation, and hematological abnormalities in the liver and kidney of treated rats.

Keywords

Diclofenac selenium oxidative stress inflammation drug toxicity

Introduction

Diclofenac sodium (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) extensively consumed^1
–3 and ubiquitous globally.⁴ The number of patients taking NSAIDs exceed over 30 million on a daily basis,^5,6 with over a million prescription written in the United States annually.⁷ Various types of NSAIDs have been produced and withdrawn from the market due to their adverse effects on the liver.⁸ Combined with antimicrobial drugs, NSAIDs are the major cause of drug-induced liver damage.^8,9 DCF is used as analgesics, antipyretics, and in the management of inflammatory pains associated with osteoarthritis, musculoskeletal injuries, and rheumatoid arthritis,^10,11 as a prescription drug or readily available for purchase as an over-the-counter (OTC) medication.^12,13 The ready availability of DFC as an OTC easily encourages its misuse with a huge propensity for abuse, consequent addiction that can result in drug-induced hepatic and renal injury subject to dosage and exposure duration. DCF exerts its therapeutic effect via nonselective inhibition of prostaglandin biosynthesis,^14,15 and it is safe when administered at therapeutic dose¹¹; higher doses of DCF administered for long period lead to hepato-, nephron-, and bone marrow toxicity and enteropathy that results in gastrointestinal bleeding, ulceration, fulminant hepatics failure, aplastic anemia, and acute kidney injury.^11,16 In the liver, DCF is metabolized to 4-hydroxydiclofenac (4′-OH-DCF) by CYP2C9 and a minor metabolite, 5-hydroxydiclofenac (5′-OH-DCF), by CYP 3A4^17,18 and other hydroxylated forms, which further undergo phase II reaction such as glucuronidation and sulfation prior to excretion in the urine (65%) and bile (35%).¹⁰ 4′-OH-DCF and 5′-OH-DCF can further be metabolized into highly reactive benzoquinone imines^19,20 by cytochrome CYP450 2C9. This reactive intermediate selectively reacts with protein cysteine¹⁹ causing damage to rats’ livers. DCF is metabolically activated predominantly by CYP 3A4 in human liver microsomes and forms protein adducts.²¹ Increase in DCF concentration leads to increase in the activities of hepatic transaminases, lactate dehydrogenase (LDH), and DCF reactive metabolites.¹⁷ Inhibition of CYPs is reported to confer protection on rat hepatocytes²⁰; however, DCF quinoneimines have been shown to be inactivated by glutathione-S-transferases (GSTs),²² NAD(P)H:quinone oxidoreductase1 by conjugation with glutathione (GSH), and reduction reaction.^20,23,24

Earlier studies attribute the mechanisms of DCF toxicity to induction of oxidative stress, mitochondrial damage, and the interaction of its reactive metabolite with cellular macromolecules, leading to alteration of proteins integrity.^16,25 Low intracellular level of nicotinamide adenine dinucleotide (NADH), GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and other reducing agents potentiate the binding of DCF to macromolecules and important hepatic proteins.²¹ Chemical agents that could lessen the toxic effect of DCF that enzymatically enhance its metabolism, conjugation, and biodisposition may reverse DCF adverse effects. Selenium (Sel) is a trace element and essential component of selenoproteins (GPx and thioredoxin reductase)²⁶ required for endogenous antioxidant enzyme balance, which modulate intracellular redox status.^27,28 Selenoprotein protects cells against oxidative stress–induced damages.²⁹ Here, we investigate the potential benefit of Sel supplementation in mitigating DCF-induced hepatic and renal toxicities, biochemical, and histological alteration in albino Wistar rats after acute exposure for 1 week. To our knowledge, studies to address DCF toxicity in this specific manner are spares in the scientific literature. Ultimately, there is the potential of developing safer drugs or identifying supplementation in the short term to reduce DCF-induced damages and promote overall global public health.

Materials and methods

Animal protocol

Fifty rats of Wistar strain weighing between 140 g and 200 g obtained from the animal house of the Department of Pharmacy, College of Medical Science, University of Ibadan, Nigeria, were used for this study. The animals were allowed to acclimatize for 2 weeks and were fed with rat pellets and water ad libitum throughout the experiment. The rats were subjected to natural photoperiod of 12-h light/12-h dark and adequately cared for according to the conditions stated in the “Guide for the Care and Use of Laboratory Animals” published by the National Institute of Health. Moreover, the University of Ibadan Ethical Committee performed the study in line with the US NAS guidelines and authorization.

Chemicals

Diclofenac sodium was purchased from Hovid Bhd, Malaysia. Reagents assay kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alanine phosphate, l-γ-glutamyltransferase, creatinine, urea, and LDH activities were products of Randox Laboratories Limited (Crumlin, UK). Other reagents such as Sel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate trihydrate, trichloroacetic acid, thiobarbituric acid, 5′,5′-dithiobis-2-nitrobenzoic acid, 1-chloro-2,4-dinitrobenzene, GSH, and epinephrine were procured from Sigma Chemical Co. (St Louis, Missouri, USA). Enzyme-linked immunosorbent assay (ELISA) kits for the assessment of tumor necrosis factor alpha (TNF-α) activity were purchased from Elabscience Biotechnology Company (Beijing, China).

Experimental design

Animals were acclimatized for 2 weeks and were randomly assigned to five experimental groups of 10 rats each and were treated by gavage for 7 consecutive days as follows:

Group 1 (control): no treatment.

Group 2 (DCF): treated with 10 mg/kg of diclofenac sodium.

Group 3 (Sel): treated with 0.125 mg/kg of Sel.

Group 4 (DCF and Sel-1): co-treated with DCF (10 mg/kg) and Sel (0.125 mg/kg).

Group 5 (DCF and Sel-2): co-treated with DCF (10 mg/kg) and Sel (0.25 mg/kg).

The doses of DCF and Sel used in the present investigation were chosen from our pilot study and in line with previously published doses.^11,30,31 At the end of treatment period, the final body weights of the rats were recorded, prior to collection of blood by retro-orbital venous plexus puncture into plain tubes and euthanize by cervical dislocation. Rat serum was subsequently obtained by centrifugation of the clotted blood at 3000 × g for 10 min. The sera were kept frozen at −20°C until needed for biochemical analysis of liver and kidney function biomarkers.

Assay of liver and kidney function indices

Analyses of serum activities of AST, ALT, alkaline phosphatase (ALP), LDH, gamma glutamyl transferase (GGT), and creatinine and urea levels were performed using available commercial kits from Randox Laboratories Limited.

Assessment of oxidative stress indices

Liver and kidney samples of experimental rats were homogenized in 0.1 M phosphate buffer (pH 7.4) using a Teflon homogenizer. The resulting tissue homogenates were then centrifuged at 12,000 × g for 15 min at 4°C using a cold centrifuge to obtain the post mitochondrial fraction, which was used for the assessment of oxidative stress and inflammation biomarkers. Hepatic and renal protein concentration was assessed according to the method described by Bradford.³² Superoxide dismutase (SOD) activity was assessed in line with the method described by Misra and Fridovich,³³ and sample aliquots were added to 2.5 ml of 0.05 M carbonate buffer (pH 10.2) followed by 0.3 ml of adrenaline. The increase in absorbance at 480 nm was observed every 30 s for 150 s. Activity of catalase (CAT) was assessed according to the method described by Clairborne³⁴ based on the loss of absorbance observed at 240 nm as CAT splits hydrogen peroxide. Change in absorbance was read at 240 nm every minute for 5 min. Activity of GST was measured in line with the method of Habig et al.,³⁵ whereas GPx activity was assessed according to the established method.³⁶ GSH level was assessed according to the method described by Jollow et al.,³⁷ whereas lipid peroxidation (LPO) was assayed as malondialdehyde in line with the established method.³⁸

RONS detection

Hepatic and renal reactive oxygen and nitrogen species (RONS) production was quantified according to established protocol which is based on the RONS-dependent oxidation of 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) to dichlorofluorescin (DCFsc).^39,40 Briefly, the reaction mixture consisted of 10 µL of the sample, 150 µL of 0.1 M potassium phosphate buffer (pH 7.4), 35 µL of distilled water, and 5 µL of DCFH-DA (200 µM, final concentration 5 µM). The fluorescence emission of (DCFsc) resulting from DCFH-DA oxidation was analyzed for 10 min (30-s interval) at 488 nm excitation and 525 nm emission wavelengths using a SpectraMax plate reader (Molecular Devices, San Jose, California, USA). The rate of DCFsc formation was expressed in percentage of control group.

Assay of pro-inflammatory biomarker

Hepatic and renal nitric oxide (NO) level was assessed using Griess reagent according to established protocol.⁴¹ Briefly, the reaction mixture consisting of equal volume of sample and Griess reagent was incubated for 15 min before the absorbance was evaluated at 540 nm. NO level was determined according to Green et al.,⁴¹ by measuring the tissue nitrites content, the stable end products of NO. The nitrites content was obtained by extrapolation using a standard sodium nitrite curve. The level of NO extrapolated from the standard curve and then expressed as units/mg protein. Moreover, myeloperoxidase (MPO) activity was evaluated according to the method described by Granell et al.⁴² Additionally, TNF-α concentration was evaluated using commercially available ELISA kits (Elabscience Biotechnology Company) with the aid of a SpectraMax plate reader (Molecular Devices) as stated in the manufacturer’s manual.

Histopathological examination of liver and kidney

Liver and kidney samples were fixed using 10% phosphate buffered formalin for 3 days. The samples were embedded in paraffin after dehydration procedures. Tissue sections of 4–5 µm were cut using a microtome before staining with hematoxylin and eosin. The tissue histology was examined under a light microscope and the histopathological aberrations scored by pathologists who were blinded to the treatment groups.

Statistical analysis

Results were analyzed using the one-way analysis of variance and the post hoc Bonferroni test with the aid of GRAPHPAD PRISM 5 (GraphPad Software, La Jolla, California, USA) to ascertain significant differences in the treatment groups. p Values less than 0.05 were considered to be significant.

Results

Sel improves final body weight gain and relative organ weights in DCF-treated rats

Exposure to Sel alone did not result in treatment-related changes, whereas administration of DCF alone decreased body weight gain, increased relative kidney weights, and significantly (p < 0.05) increased relative liver weight compared to control (Table 1). DCF and Sel co-treated rats exhibited increase in body weight gain, reduction in relative liver and kidney weight compared to DCF alone–treated rats.

Table 1.

Changes in organo-somatic indices in rats treated with diclofenac and selenium for 1 week.

	IBW (g)	FBW (g)	Weight gain (g)	RLW (g)	RKW (g)
Control	130.1 ± 13.74	133.0 ± 13.86	±2.90	2.61 ± 0.47	0.62 ± 0.04
DCF (10 mg/kg)	158.7 ± 17.83	149.3 ± 24.31	±9.41	3.28 ± 0.48*	0.64 ± 0.02*
Sel (0.125 mg/kg)	158.0 ± 18.82	161.2 ± 17.54	±3.20	2.67 ± 0.47	0.55 ± 0.03
DCF + Sel (0.125 mg/kg)	157.7 ± 22.56	152.6 ± 21.61	±5.10	3.10 ± 0.53	0.63 ± 0.13
DCF + Sel (0.25 mg/kg)	158.3 ± 17.88	174.2 ± 33.48	±15.90	2.99 ± 0.15	0.59 ± 0.04

DCF: diclofenac; Sel: selenium; IBW: initial body weight; FBW: final body weight; RLW: relative liver weight; RKW: relative kidney weight; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Sel reduces biomarkers of hepatic and renal function in DCF-treated rats

As presented in Table 2 and 3, serum activities of hepatic transaminases (AST, ALT, ALP, GGT) and LDH were increased (p < 0.05) in rats treated with DCF alone. Rats co-treated with DCF and Sel exhibited reduced (p < 0.05) transaminase and LDH activities compared to DCF alone–treated rats. In addition, biomarkers of renal function (urea and creatinine) levels were increased (p < 0.05) in DCF alone–treated rats when compared to control, whereas they were markedly reduced in rats co-treated with DCF and Sel when compared to DCF alone–treated rats.

Table 2.

Serum levels of biomarkers of hepatic function in rats co-treated with diclofenac and selenium for 1 week.

	Liver function
	AST (IU/L)	ALT (IU/L)	ALP (IU/L)	LDH (IU/L)	GGT (IU/L)
Control	661.6 ± 26	267.98 ± 1.40	362.12 ± 53.5	10.66 ± 2.5	6.59 ± 0.2
DCF (10 mg/kg)	784.1 ± 16^a	295.43 ± 2.98^a	720.77 ± 51.3^a	14.98 ± 1.3^a	12.31 ± 1.0^a
Sel (0.125 mg/kg)	670.7 ± 17	256.51 ± 9.11	381.06 ± 10.5	6.1 ± 0.95	5.70 ± 1.9
DCF + Sel (0.125 mg/kg)	712.5 ± 12^b	272.64 ± 11^b	694.97 ± 50^b	6.3 ± 1.1^b	4.93 ± 0.1^b
DCF + Sel (0.25 mg/kg)	675.0 ± 31^b	263.8 ± 1.4^b	682.96 ± 12^b	3.2 ± 1.1^b	5.62 ± 1.3^b

DCF: diclofenac; Sel: selenium; AST: aspartate aminotransferase; ALT: alanine amino transferase; ALP: alkaline phosphatase; LDH: lactate dehydrogenase;

GGT: gamma glutamyl transferase; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Table 3.

Serum levels of biomarkers of renal function in rats co-treated with diclofenac and selenium for 1 week.

	Kidney function
	Urea (mmol/L)	Crea.(mmol/L)
Control	4.11 ± 0.1	25.74 ± 4
DCF (10 mg/kg)	9.50 ± 1.3^a	59.98 ± 12^a
Sel (0.125 mg/kg)	4.47 ± 0.6	25.59 ± 5.1
DCF + Sel (0.125 mg/kg)	6.83 ± 1.5^b	52.13 ± 3.4
DCF + Sel (0.25 mg/kg)	5.32 ± 0.7^b	64.78 ± 4.0

DCF: diclofenac; Sel: selenium; CREA: creatinine; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Sel improves hepatic and renal antioxidant status in DCF-treated rats

Treatment of rats with DCF alone resulted in decrease (p < 0.05) in SOD, CAT, and GPX activities in the liver and kidney of treated rats when compared with the control (Tables 4 and 5). Increased (p < 0.05) GST activities were observed in the liver, but decreased (p < 0.05) in the kidney of DCF alone–treated rats compared to control. The decrease in these antioxidant enzymes was improved (p < 0.05) in rats co-treated with DCF and Sel compared to rats treated with DCF alone. Administration of DCF alone resulted in decrease (p < 0.05) in GSH level, whereas it markedly increased RONS and LPO levels in the liver and kidney of treated rats compared to control rats. Rats co-treated with DCF and Sel exhibited increase (p < 0.05) in GSH level and decrease in RONS and LPO levels compared with DCF alone–treated rats (Table 6).

Table 4.

Antioxidant enzyme levels in the liver of rats following diclofenac and selenium treatment for 1 week.

	Liver
	SOD	Cat	GPx	GST
Control	9.72 ± 0.41	6.62 ± 0.36	8.78 ± 0.28	0.35 ± 0.15
DCF (10 mg/kg)	4.35 ± 0.30^a	2.46 ± 0.18^a	3.92 ± 0.01^a	2.73 ± 0.80^a
Sel (0.125 mg/kg)	6.07 ± 0.13	7.13 ± 0.17	5.01 ± 0.23	0.37 ± 0.20
DCF + Sel (0.125 mg/kg)	5.17 ± 0.13^b	5.22 ± 0.82^b	4.96 ± 0.01^b	1.31 ± 0.48^b
DCF + Sel (0.25 mg/kg)	4.61 ± 0.74	3.27 ± 0.85^b	4.59 ± 0.17^b	1.18 ± 0.19^b

DCF: diclofenac; Sel: selenium; SOD: superoxide dismutase (nmole epinephrine oxidized/min/mg protein); CAT: catalase (µmol H₂O₂ consumed/min/mg protein); GPx: glutathione peroxidase (µmol of residual GSH/mg protein); GST: glutathione-s-transferase (µmol CDNB-GSH complex formed/min/mg protein); SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Table 5.

Antioxidant enzyme levels in kidney of rats following diclofenac and selenium treatment for 1 week.

	Kidney
	SOD	Cat	GPx	GST
Control	2.61 ± 0.07	1.20 ± 0.01	2.41 ± 0.01	2.19 ± 0.17
DCF (10 mg/kg)	2.35 ± 0.08^a	0.42 ± 0.08^a	2.26 ± 0.07^a	1.09 ± 0.27^a
Sel (0.125 mg/kg)	2.61 ± 0.08	1.31 ± 0.42	2.50 ± 0.05	2.39 ± 0.04
DCF + Sel (0.125 mg/kg)	2.35 ± 0.04	0.64 ± 0.04	2.36 ± 0.02^b	1.87 ± 0.21^b
DCF + Sel (0.25 mg/kg)	2.45 ± 0.07^b	0.84 ± 0.08^b	2.44 ± 0.10^b	1.99 ± 0.24^b

DCF: diclofenac; Sel: selenium; SOD: superoxide dismutase (nmol epinephrine oxidized/min/mg protein); CAT: catalase (µmol H₂O₂ consumed/min/mg protein); GPx: glutathione peroxidase (µmol of residual GSH/mg protein); GST: glutathione-s-transferase (µmol CDNB-GSH complex formed/min/mg protein); SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Table 6.

Effects of selenium on GSH, RONS, LPO, NO, and MPO activities in the liver and kidney of rats treated with DCF for 1 week.

	GSH (µmol/mg protein)	RONS (DFC % fold over control)	LPO (µmol MDA/mg protein)	NO (U/mg protein)	MPO (U/mg protein)
Liver
Control	5.03 ± 0.70	100.00 ± 10.75	18.22 ± 0.30	4.71 ± 0.17	1.24 ± 0.32
DCF (10 mg/kg)	2.69 ± 0.55^a	156.19 ± 8.36^a	28.62 ± 4.63^a	7.24 ± 0.90^a	2.10 ± 0.92^a
Sel (0.125 mg/kg)	3.13 ± 0.11	103.43 ± 14.92	18.77 ± 0.81	5.03 ± 0.48	1.26 ± 0.37
DCF + Sel (0.125 mg/kg)	4.00 ± 0.14^b	126.61 ± 2.31^b	23.87 ± 0.79^b	5.75 ± 0.98^b	1.57 ± 0.03^b
DCF + Sel (0.25 mg/kg)	5.49 ± 0.65^b	171.03 ± 24.49	22.28 ± 2.59^b	6.92 ± 0.41	0.85 ± 0.10^b
Kidney
Control	1.53 ± 0.15	100.00 ± 8.36	0.54 ± 0.06	0.21 ± 0.10	1.19 ± 0.26
DCF (10 mg/kg)	0.42 ± 0.29^a	161.56 ± 13.69^a	1.04 ± 0.13^a	0.79 ± 0.23^a	1.70 ± 0.19^a
Sel (0.125 mg/kg)	1.31 ± 0.21	120.15 ± 14.68	0.60 ± 0.19	0.28 ± 0.04	1.09 ± 0.35
DCF + Sel (0.125 mg/kg)	0.84 ± 0.05^b	104.41 ± 31.9^b	0.70 ± 0.09^b	0.50 ± 0.09^b	2.75 ± 0.16^b
DCF + Sel (0.25 mg/kg)	0.85 ± 0.22^b	134.38 ± 6.46^b	0.79 ± 0.07^b	0.61 ± 0.09^b	1.46 ± 0.48

DCF: diclofenac; Sel: selenium; DCF alone. GSH: glutathione; RONS: reactive oxygen and nitrogen species; LPO: lipid peroxidation; NO: nitric oxide; MPO: myeloperoxidase; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Sel suppresses pro-inflammatory biomarker in DCF-treated rats

DCF treatment alone resulted in increased (p < 0.05) NO and TNF-α levels and MPO activity in the liver and kidney compared with control rats. Administration of Sel at 0.125 and 0.25 mg/kg reduced (p < 0.05) NO and TNF-α levels as well as MPO activities in the liver and kidney compared to DCF alone–treated rats (Tables 6 and 7).

Table 7.

TNF-α levels in the liver and kidney of rats treated with diclofenac and selenium for 1 week.

TNF-α (pg/mL)	Liver	Kidney
Control	1.72 ± 0.17	2.19 ± 0.05
DCF (10 mg/kg)	3.32 ± 0.10^a	3.80 ± 0.24^a
Sel (0.125 mg/kg)	1.94 ± 0.11	2.24 ± 0.11
DCF + Sel (0.125 mg/kg)	2.36 ± 0.09^b	3.54 ± 0.07
DCF + Sel (0.25 mg/kg)	2.30 ± 0.03^b	2.63 ± 0.07^b

TNF-α: tumor necrosis factor-α; DCF: diclofenac; Sel: selenium; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Sel ameliorates histopathological lesions in the liver and kidney of DCF-treated rats

Microscopic examination revealed normal architecture of the liver and kidney of control rats ((a) upper and lower pane). Kidney from rats treated with DCF alone showed severe congestion of the interstitium, whereas kidney of Sel alone–treated rats showed no visible lesions ((b) and (c) respectively lower panel). Liver from rats treated with DCF alone showed mild congestion of the portal area, Sel treatment alone showed moderate portal congestion ((b) and (c) upper panel). DCF-induced histopathological lesions were alleviated in hepatic and renal cells of rats co-treated with DCF and Sel ((d) and (e) upper and lower panel), respectively (Figure 1).

Figure 1.

Representative photomicrographs of the liver (top panel) and kidney (lower panel) from diclofenac (DCF) and Sel treated rats. (a) Control sections showed normal morphology; (b) DCF alone resulted in congestion of the portal area; whereas (c) Sel alone very mild portal congestion. Co-exposure to DCF and Sel reversed DCF-induced alterations in hepatic architecture and periportal cellular infiltration, (d and e) in a dose dependent manner. Kidney of rats treated with DCF alone showed severe congestion of the interstitium, whereas Sel alone presented normal kidney morphology. The kidney of rats co-exposed to DCF and Sel (0.125 and 0.25mg/kg) exhibited (d and e). mild cellular infiltration of the interstitium and no visible lesions, respectively (original magnification ×400). DCF: diclofenac; Sel: selenium.

Sel improves hematological indices in DCF-treated rats

Lymphocytes (LYMPH) and Neutrophils (NEUT) counts were reduced (p < 0.05) in DCF alone–treated rats compared to control, whereas white blood cells (WBC) counts increased (p < 0.05) compared to control (Table 8). There was no difference (p < 0.05) in the level of eosinophils when compared to control. LYMPH and NEUT counts increased (p < 0.05) in rats co-treated with DCF and Sel compared to DCF alone–treated rats, whereas the level of WBC was decreased (p < 0.05) compared to control.

Table 8.

Effect of diclofenac and selenium treatment on hematological parameters in rats with for 1 week.

	Total WBC (10⁹/L)	Neutrophils (%)	Lymphocytes (%)	Eosinophil (%)
Control	5.28 ± 0.89	44.00 ± 5.66	55.33 ± 5.03	2.20 ± 0.45
DCF (10 mg/kg)	8.23 ± 0.15^a	20.00 ± 1.41^a	73.00 ± 5.66^a	1.67 ± 0.58
Sel (0.125 mg/kg)	6.83 ± 0.15	43.25 ± 3.95	51.75 ± 2.36	2.00 ± 0.00
DCF + Sel (0.125 mg/kg)	8.55 ± 0.78	34.00 ± 2.65^b	59.67 ± 0.58^b	1.67 ± 0.58
DCF + Sel (0.25 mg/kg)	7.45 ± 0.07^b	34.00 ± 2.83^b	66.00 ± 2.83^b	2.00 ± 0.00

DCF: diclofenac; Sel: selenium; WBC: white blood cells; SD: standard deviation.

Values are expressed as means ± SD of 10 rats.

^a p < 0.05 versus control.

^b p < 0.05 versus DCF alone.

Discussion

DCF is safe at therapeutic doses and can be toxic in humans and animals when administered in large doses. Toxcities associated with DCF are as a result of its reactive metabolites, 4′-OH-DCF and 5′-OH-DCF^19,22,43 and the highly reactive benzoquinone imines,^19,20 conjugation with reduced GSH and inactivated by GSTs,^23,24 enhances DCF-metabolite excretion⁴⁴ with subsequent reduction in DCF toxicity. Induction of biodisposition enzyme containing Sel and enhancement of cellular antioxidant status will therefore reduce DCF-associated toxicities. Our findings on Sel and DCF co-administration buttress this hypothesis of the potential protective effect of Sel (important for the production of selenoproteins) in a dose-dependent manner. At a high dose of Sel (0.25 mg/kg), exposure to DCF was less toxic (0.125 mg/kg). DCF treatment alone resulted in body weight changes (p < 0.05) and was reversed by Sel supplementation in treated rat. These changes in body weight can be attributed to hepatomegaly⁴⁵ in response to DCF hepatotoxic effect. Hepatic transaminases (AST, ALT, GGT, ALP) and LDH are widely used as biomarkers for hepatic injury, serum levels of transaminases were elevated (p < 0.05) in rats treated with DCF, and as previously reported,^10,11,46,47 leading to release of enzymes into the blood stream. Conversely, rats co-treated with Sel demonstrably ameliorate DCF-induced hepatic injury dose-dependently, evaluated by transaminases levels in serum.^48,49

Renal function marker enzymes such as urea and creatinine are metabolic waste products primarily excreted in the urine. They are important in the diagnosis of kidney injury. Hence, increases (p < 0.05) in serum urea and creatinine levels in rats treated with DCF alone signify renal dysfunction as previously reported,^50,51 which have shown nephrotoxicity of high-dose DCF treatment.^11,50 Excretion and accumulation of conjugates have been implicated in renal function and end-stage renal diseases.⁵⁰ Reduction (p < 0.05) in serum urea and creatinine levels in Sel and DCF co-treated rats reflects the protective role of Sel in DCF-induced renal dysfunction.

Endogenous antioxidant defense system plays an important role in the protection of cells from RONS-mediated oxidative injury. Decreases (p < 0.05) in endogenous antioxidant enzymes (SOD, CAT, GST, GPx) activities as well as GSH level were observed in the liver and kidney of DCF-treated rats as previously observed.^22,47 Reduction in antioxidant function may have resulted from accumulation of cytotoxic metabolites in the renal and hepatic tissues of rats exposed to DCF.²² However, increased (p < 0.05) SOD, CAT, GST GPx activities in Sel and DCF co-treated rats showed the protective augmentation of Sel against DCF-induced oxidative stress via enhancement of antioxidant enzymes activities in line with previous findings.^48,52
–54 GSH acts as a cofactor of several detoxifying enzymes (e.g. GPx, GST), scavenging intracellular free radicals.^55,56 Depletion of GSH in hepatic and renal tissues indicates GSH utilization,⁵⁷ which subsequently leads to hepatic and renal toxicity in the DCF-treated rats. Sel supplementation, however, reversed this trend in a dose-dependent manner and in line with previous studies.⁵⁷

Furthermore, increase (p < 0.05) in RONS and LPO observed in DCF-treated rats is indicative of RONS production and oxidative cellular damage, respectively, in the liver and kidney of rats. Elevated LPO is reflective of oxidative deterioration of polyunsaturated lipid^58
–60 induced by generated RONS.^61,62 Sel has been reported to mitigate LPO.⁶³ Decreases (p < 0.05) in RONS and LPO levels in rats co-exposed to Sel and DCF reveal the effects of Sel on DCF-mediated oxidative damage in the liver and kidney of treated rats. Thus, the mechanism of DCF-induced oxidative damage involves overwhelming protective antioxidant activities of SOD, CAT, GPx GST, and GSH, as well as enhancement of RONS production and deteriorative LPO in the liver and kidney of treated rats. However, Sel’s ameliorative effect may be related to its incorporation in selenoproteins and enhancing overall cellular antioxidant activities.

DCF treatment triggered an increase (p < 0.05) in MPO activity as well as the level of NO and pro-inflammatory cytokines TNF-α in the liver and kidney of treated rats. MPO is a critical effector of inflammation, abundantly expressed in NEUT, monocytes, and macrophages.⁶⁴ Activated MPO uses hydrogen peroxides to generate deleterious hypochlorite during tissue inflammation.⁶⁵ DCF acts synergistically with pro-inflammatory cytokine to increase inducible nitric oxide and NO production via nuclear factor kappa-B (NF-κB) signaling, which induces cell damage.⁶⁶ Increase in hepatic and renal NO, MPO, and TNF-α levels in rats treated with DCF alone may contribute to hepatic and renal damage by inducing inflammatory and nitrosative stress responses. The apparent decreases in MPO activity, NO, and TNF-α levels in rats co-exposed to Sel and DCF demonstrated the anti-inflammatory effect of Sel. DCF induced increases in WBC and LYMPH count but reduced NEUT, eosinophil’s level in treated rats as previously reported.⁶⁷ Although, Ibanez et al.⁶⁸ reported that DCF is associated with significant risk of neutropenia, corroborating our findings, and may probably be due to immune modulatory effects. DCF-induced increases in WBC and LYMPH count were however lowered (p < 0.05) by Sel co-treatment in an inflammatory environment.

Histological examination of the liver and kidney of treated rats indicated changes that corroborated our biochemical findings. The cellular integrity of hepatic and renal tissues was significantly distorted by DCF. Co-exposure of Sel and DCF to rats protected against DCF-induced histological changes is evident by limited alteration of the histoarchitecture of the liver and renal tissues compared to the DCF alone–treated rats. Physical examination of the kidney of the DCF-treated rats reveals a pale color compared to control.

Taken together, our findings demonstrated that exposure to DCF elicits hepatic and renal injury in rats via mechanism involving induction of oxidative stress and inflammation. Sel supplementation, on the other hand, relieved DCF-induced hepatic and renal toxicity and attenuated hematological abnormalities. Decreased hepatic and renal tissue injuries were also observed in rats co-treated with Sel, enhanced overall antioxidant status, and suppressed pro-inflammatory cytokines in rats exposed to DCF. It can be concluded that Sel supplementation can help in mitigating drug-induced hepatic and renal injury and improve drug safety.

Supplemental material

Supplemental Material, Manuscript_result_(1) - Biochemical alterations in diclofenac-treated rats: Effect of selenium on oxidative stress, inflammation, and hematological changes

Supplemental Material, Manuscript_result_(1) for Biochemical alterations in diclofenac-treated rats: Effect of selenium on oxidative stress, inflammation, and hematological changes by Solomon E Owumi and Uche J Dim in Toxicology Research and Application

Footnotes

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

Solomon E Owumi

Supplemental material

Supplemental material for this article is available online.

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