Biomedicine & Pharmacotherapy 96 (2017) 905–911

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Antioxidant and anti-inflammatory effects of virgin coconut oil T

supplementation abrogate acute chemotherapy oxidative nephrotoxicity
induced by anticancer drug methotrexate in rats
⁎
Ademola C. Famurewaa, , Patrick M. Ajab, Ekenechukwu K. Maduagwunab,
Chima A. Ekeleme-Egedigwec, Odomero G. Ufebea, Sharon O. Azubuike-Osud
a
Department of Medical Biochemistry, Faculty of Basic Medical Sciences, Federal University, Ndufu-Alike, Ikwo, Abakaliki, Ebonyi State, Nigeria
b
Department of Biochemistry, Faculty of Biological Sciences, Ebonyi State University, Abakaliki, Nigeria
c
Department of Chemistry/Biochemistry/Molecular Biology, Faculty of Science, Federal University, Ndufu-Alike, Ikwo, Ebonyi State, Nigeria
d
Department of Physiology, Faculty of Basic Medical Sciences, Federal University, Ndufu-Alike, Ikwo, Abakaliki, Ebonyi State, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Methotrexate (MTX) is an efficacious anticancer agent constrained in clinical use due to its toxicity
Methotrexate on non-targeted tissue, a considerable source of worry to clinicians. Because the toxicity is associated with
Virgin coconut oil oxidative stress and inflammation, the study explored antioxidant and anti-inflammatory effect of virgin coconut
Nephrotoxicity oil (VCO) supplementation in nephrotoxicity induced by MTX in rats.
Oxidative stress
Methods: Rats were randomized into 4 groups (n = 6) as follows: Control group; MTX group injected with single
Chemotherapy
dose of MTX (20 mg/kg, ip) on day 14; VCO (5%) + MTX and VCO (15%) + MTX groups were pre-treated with
VCO diet and injected with single dose of MTX (20 mg/kg, ip) on day 14. After 3 days of MTX injection, serum
kidney markers, renal activities of antioxidant enzymes and glutathione (GSH) content were determined. Lipid
peroxidation level and inflammatory markers- interleukin-6 (IL-6), nitric oxide (NO) and C-reactive protein
(CRP) were estimated in kidney. Histopathological alterations were examined for kidney damage.
Results: MTX nephrotoxicity was evidenced by markedly elevated serum renal markers along with significant
decreases in renal GSH and activities of antioxidant enzymes confirmed by histopathology. Lipid peroxidation
level, IL-6, NO and CRP markedly increased compared to control. VCO supplementation prior to MTX injection
attenuated MTX-induced oxidative nephrotoxicity via prominent increases in GSH and antioxidant enzyme ac-
tivities in a dose-dependent manner. The renal inflammatory markers and MDA depleted considerably compared
to MTX control group. Histopathological alterations were mitigated to confirm the biochemical indices.
Conclusion: VCO supplementation demonstrates nephroprotective activity by attenuating MTX oxidative ne-
phrotoxicity via antioxidant and anti-inflammatory activities in kidney. Our results suggested that VCO may
benefit cancer patients on MTX chemotherapy against kidney injury.

1. Introduction treatment discontinuation [4–6]. The anticancer mechanism of MTX

Methotrexate (MTX) is an antimetabolite anticancer agent used in
treatment of malignancies and non-neoplastic inflammatory diseases
such as rheumatoid arthritis and psoariasis [1–3]. It is targeted at in-
ducing apoptosis in cancer cells; its widespread use and administration
over a wide dose range has promoted MTX chemotherapy for breast
cancer, acute lymphoblastic leukemia and lung cancer [2]. However,
MTX chemotherapy evokes several side effects of clinical concern which
has limited its application for cancer treatment [2]. In some cases, it
could cause severe organ toxicity necessitating dose reduction or
involves inhibition of enzymes synthesizing activated folate to alter
DNA replication and cell division [7].
Nephrotoxicity is one of the most prominent toxicities induced by
MTX [4,5]. MTX renal toxicity is associated with precipitation of MTX
and tubular necrosis [8]. The impairment results in delayed MTX
elimination and accumulation in blood resulting in systemic toxicity
[9,10]. Although the underlying mechanism of MTX-induced renal
damage is not clearly elucidated; however, accumulating evidence has
implicated oxidative stress and inflammation as possible culprits in-
volved in MTX-induced renal damage [1,8]. Literature suggests that

⁎
Corresponding author.
E-mail address: ademola.famurewa@funai.edu.ng (A.C. Famurewa).

https://doi.org/10.1016/j.biopha.2017.12.008
Received 15 October 2017; Received in revised form 23 November 2017; Accepted 4 December 2017
0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
A.C. Famurewa et al.
inflammatory mediators such as IL-6, tumor necrosis factor-α (TNF-α)
and expression of nuclear factor-kappa B (NF-кB) may be involved in
MTX hepatorenal damage [8]. Oxidative stress is well implicated in
tissue damage via depletion of antioxidant defense systems [11,12].
Experimental studies indicate that acute MTX triggers renal damage via
alterations in antioxidant defense systems in rat models [1,13,14].
There is evidence indicating that MTX inhibits NADP malic enzymes
and decreases availability of NADPH essential for recovery of GSH from
GSSG in cellular redox homeostasis [15]. The available agent against
MTX toxicity is leucovorin medication and there are controversies that
it may compromise anticancer efficacy of MTX [16]. The clinical use of
folate supplementation to salvage normal cells from MTX toxicity may
alter anticancer efficacy of MTX [16]. Research reports show that nat-
ural products contain bioactive compounds known as natural anti-
oxidants may mitigate oxidative damage induced by MTX che-
motherapy. Therefore, natural strategies that could reduce its
nephrotoxicity without compromising its anticancer action is highly
warranted.
In recent times, research interest has emerged to explore the bene-
ficial health effect of natural products. The plant oil VCO from Cocos
nucifera is emerging as functional food oil possessing beneficial health
effects against oxidative damage and side effects of chemotherapy [17].
Phytochemical analyses found VCO to contain potent natural phenolic
antioxidants such as flavonoids and phenolic acids [18,19]. Systematic
investigations report that wet method of producing VCO from fresh and
mature kernel devoid of chemical refining, bleaching and deodorizing
conserves VCO bioactive phenolic compounds [19,20]. Recent evi-
dences suggest that the various antioxidant health effects of VCO may
be attributed to the bioactive phenolic compounds in the natural oil
[21–23]. Recently, VCO has shown a number of antioxidant and
pharmacological activities in pre-clinical studies [17,24,25]. However,
the health effect of VCO supplementation in MTX-induced ne-
phrotoxicity remains to be reported. We sought to explore the possible
antioxidant and nephroprotective effects of VCO supplementation
against MTX-induced nephrotoxicity and pro-inflammation in rats.
2. Materials and methods
2.1. Drug and chemicals
Methotrexate used in the present study was purchased from the
Morningside Healthcare Ltd, Leicester, UK. The kits used for biochem-
ical assays of kidney function parameters were obtained from Randox
Laboratory Ltd., UK. Thiobarbituric acid (TBA) was purchased from Hi
Media Laboratories Pvt. Ltd., India. Commercial kits for nitric oxide,
interleukin-6 and C-reactive protein were purchased from R&D
Systems, USA and TULIP DIAGNOSTICS, respectively. All other re-
agents used were obtained commercially and of analytical grade.
2.2. Animals
Twenty-four male Wistar rats (7 weeks old) were purchased from
the Veterinary Department, University of Nigeria, Nsukka, Enugu State,
Nigeria. The animals were kept under natural lighting condition (12 h
light/12 h dark) at temperature (25 °C ± 3 °C), and were allowed free
access to pelleted commercial rodent chow (Vital Feeds Nigeria Ltd.,
Jos, Nigeria) and distilled water ad libitum. The rats were acclimatized
two weeks preceding treatment protocols and were handled in humane
manner according to the approved animal experimental procedures
given by the National Research Council, Guide for the Care and the Use
of Laboratory Animals [26].
2.3. Preparation of virgin coconut oil and diet
The wet extraction method according to Nevin and Rajamohan [23]
was used to obtain VCO in this study. Mature coconuts (Cocos nucifera
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Biomedicine & Pharmacotherapy 96 (2017) 905–911
L.) were purchased from commercial central market in Abakaliki,
Ebonyi State and used for the extraction of VCO. The coconut meat
(edible part) obtained from the mature coconuts was ground to produce
viscous slurry. About 400 ml of water was added to the slurry, sieved
with cheesecloth to obtain creamy coconut milk, which was left
standing for 48 h to separate the creamy top and water layers. The top
layer was carefully removed and subjected to mild heating (50 °C) to
remove moisture. The heating thus separates the oil which was gently
scooped out and filtered into an air-tight container. This oil thus pre-
pared without refining, bleaching and deodorizing was used for the
current study. The VCO supplemented diets (5% and 15% w/w) was
prepared thus: VCO of 5 g and 15 g were separately added to the normal
rat chow of 95 g and 85 g respectively, and mixed thoroughly. The
preparation of the VCO diets was done on regular demand.
2.4. Experimental design
Following 2 weeks acclimatization, the animals were randomly as-
signed into 4 groups (n = 6 rats per group), and the treatment was as
follows:
Group 1 (Normal control): received clean water (5 ml/kg)
Group 2 (MTX control): received MTX (20 mg/kg bw, ip) on day 14
only to induce renal damage [5]
Group 3 (5% VCO + MTX): received 5% VCO supplemented diet
(w/w) for 17 days + MTX (20 mg/kg bw, ip) on day 14 only
Group 4 (15% VCO + MTX): received 15% VCO supplemented diet
(w/w) for 17 days + MTX (20 mg/kg bw, ip) on day 14 only
All animals (fasted) were anaesthetized with ether and sacrificed 3
days after the MTX ip administration to obtain blood collected via retro-
orbital venous plexus by capillary tubes into plain sample bottles. The
blood samples were centrifuged (3000g for 15 min) to separate serum
used for analyses of kidney function markers. The left side kidney was
dissected out, washed in cold saline solution, dried with tissue paper
and weighed. The kidney tissue was minced and homogenized in 0.1M
phosphate buffered saline (1:5 w/v, pH 6.4) and centrifuged (3500g for
20 min). The supernatant obtained was used for analyses of lipid per-
oxidation, antioxidant enzyme activities, reduced glutathione and pro-
inflammatory markers. The remaining kidney sample was fixed in 10%
buffered formalin for histopathological examination.
2.5. Biochemical analyses
The renal function markers (uric acid, urea and creatinine) were
estimated in serum stored at 4 °C using commercial kits by RANDOX.
The renal activity of SOD was assayed by the method of Arthur and
Boyne [27]. The activity of CAT in the kidney was assayed by the
method of Sinha [28], while GPx was assayed according to the method
of Paglia and Valentine [29]. The renal GSH level was determined by
the method of Exner et al. [30]. Lipid peroxidation marker was esti-
mated by measuring spectrophotometrically the level of thiobarbituric
acid-reactive substances (TBARS) expressed as malondialdehyde (MDA)
as described by Wallin et al. [31]. Nitric oxide (NO) was estimated
using commercial kit containing Griess reagents (R&D Systems, USA)
following the method of Green et al. [32]. The renal level of interleukin-
6 (IL-6) was analyzed by ELISA method with commercially available
assay kit for rats (R&D Systems Inc., USA), and C-reactive protein by the
method of Andersen and McCarthy [33] as described in the kit (TULIP
DIAGNOSTICS).
2.6. Histopathological study of the kidney
Qualitative analysis of kidney was carried out; the kidney samples
were fixed for 48 h in 10% phosphate buffered formalin and dehydrated
in graded alcohol, cleared in xylene and embedded in molten wax.
Sections of the tissue (5 μm thick) were prepared by using a rotary
microtome and stained with haematoxylin and eosin (H&E) dye. It was
A.C. Famurewa et al. Biomedicine & Pharmacotherapy 96 (2017) 905–911

Table 1 group. The kidney index was calculated as a ratio (%) of the kidney
Effect of VCO supplementation on body weight (at sacrifice), absolute kidney weight and weight (g) to body weight (g).
relative kidney weight of MTX-treated rats.

Group Body weight (g) Kidney Kidney index 3.2. Effect of VCO supplementation on MTX-induced renal dysfunction
weight (g) (%)
Initial Final Table 2 depicts estimation of serum urea, creatinine and uric acid
levels as markers of renal function. Levels of these markers were sig-
Control 128.4 ± 3.0 161.8 ± 4.3 0.65 ± 0.03 2.80 ± 0.11
MTX 121.2 ± 2.9 136.2 ± 2.3* 0.58 ± 0.03 2.52 ± 0.15 nificantly elevated (P < 0.05) in the MTX-treated animals compared to
VCO (5%) + MTX 114.2 ± 2.1 147.2 ± 2.9 0.50 ± 0.02 2.50 ± 0.14 control group. Pretreatment with VCO supplementation (5% and 15%)
VCO (15%) + MTX 115.4 ± 4.4 138.9 ± 3.2 0.55 ± 0.04 2.51 ± 0.18 in MTX-intoxicated rats reversed the renal dysfunction shown by sig-
nificant decreases in the renal function marker levels dose-dependently
VCO: Virgin coconut oil; MTX: Methotrexate; Values are mean + SEM (6 rats/group).
in comparison to rats treated with MTX alone (P < 0.05). Although,
* Significant when compared to control group in the same column (P < 0.05).
creatinine level in VCO (5%) + MTX group was significantly higher
compared to control (P > 0.05). It was observed that 15% VCO diet
Table 2 markedly reduced creatinine level than 5% VCO diet in this study.
Effect of VCO supplementation on serum markers of renal function in MTX-treated rats.

3.3. Effect of VCO supplementation on MTX-induced oxidative stress

Group Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl)
markers
Control 17.40 ± 0.43 0.42 ± 0.01 6.02 ± 0.27
MTX 28.60 ± 3.43* 0.89 ± 0.04 7.52 ± 0.17* Assessment of oxidative stress and lipid peroxidation was carried
VCO (5%) + MTX 19.42 ± 0.96# 0.73 ± 0.02#,* 6.21 ± 0.27#
out by measuring renal SOD, CAT, and GPx activities, GSH and MDA
VCO (15%) + MTX 17.25 ± 0.80# 0.57 ± 0.03#,@ 5.82 ± 0.25#
content measured as TBARS. Table 3 shows the activities of the anti-
VCO: Virgin coconut oil; MTX: Methotrexate; Values are mean + SEM (6 rats/group). oxidant enzymes prominently (P < 0.05) depressed in kidney tissue of
* P < 0.05: significant when compared to control group in the same column. MTX-treated rats compared to the control group. In addition, renal
#
P < 0.05: significant when compared to MTX group in the same column. content of GSH reduced considerably, while MDA level significantly
@
P < 0.05: significant when compared to VCO (5%) + MTX group in the same increased. In contrast, dietary supplementation of VCO (5% and 15%)
column.
significantly increased the renal activities of these enzymes compared
to the MTX-treated group (P < 0.05). Also, it was observed that VCO
mounted for light microscopic examination. The sections were then supplementation markedly attenuated alterations in GSH and MDA
viewed and photomicrographs taken using a Motic™ 9.0 megapixels compared to MTX-treated group.
microscope camera at ×400 magnifications.
3.4. Effect of VCO supplementation on MTX-induced pro-inflammation
2.7. Statistical analyses
The effects of VCO supplementation on renal levels of IL-6, CRP and
The results were expressed as the mean ± SEM (6 rats/group). NO are shown in Figs. 1–3. The levels of IL-6, CRP and NO were sig-
Statistical analyses were performed with SPSS version 22.0 for windows nificantly (P < 0.05) elevated in kidneys of MTX-treated rats com-
(SPSS Inc., Chicago, IL, USA) using analysis of variance (ANOVA) fol- pared to normal control rats. Conversely, pretreatment with VCO sup-
lowed by post-hoc Tukey test. Values were considered statistically plemented diet prominently reduced (P < 0.05) the renal levels of pro-
significant when p < 0.05 inflammatory markers in comparison to MTX-treated rats. The IL-6,
CRP and NO levels between the VCO + MTX groups were comparable,
3. Results and to normal control (P > 0.05) in this study.

3.1. Effect of VCO supplementation on body weight and kidney weight 3.5. Renal histology and histopathology

Table 1 presents the effect of VCO supplemented diet on body
weight, kidney weight and relative kidney weight of rats. The body
weight of rats in MTX group (20 mg/kg) significantly (P < 0.05) re-
duced at sacrifice compared to normal control group. Dietary supple-
mentation of VCO to MTX-treated rats improved the body weight al-
though insignificantly when compared with rats in MTX group
(P > 0.05). However, administration of MTX alone insignificantly
decreased the kidney weight and relative kidney weight. Similarly, the
rats in VCO + MTX groups demonstrated insignificant decreases
(P > 0.05) in kidney weight and kidney index compared with the MTX
The effect of VCO supplementation on the renal histology of the
MTX-treated rats is presented in Fig. 4. Histopathological study of
kidney in control group showed a normal renal structure with normal
glomeruli (G) in their Bowman’s capsule (white arrow) surrounded by
normal renal tubules (black arrow). MTX group showed histopatholo-
gical changes with degenerative and desquamated necrotic tubules
(white arrow). The affected swollen tubules showed epithelial cells with
nuclear pyknosis (blue) and karyorrhexis. On the contrary, the pre-
treatment and concurrent VCO supplementation (5% and 15%) in MTX-
treated rats was able to restore MTX-induced histopathological

Table 3
Effect of VCO supplementation on renal oxidative stress markers- SOD, CAT, and GPx (U/mg protein), GSH (mg/g protein) and MDA (nmol/mg protein) of MTX-treated rats.

Group SOD CAT GPx GSH M DA

Control 1.15 ± 0.01 3.22 ± 0.08 232.4 ± 11.9 6.36 ± 0.22 3.49 ± 0.09
MTX 0.97 ± 0.03* 1.50 ± 0.15* 27.6 ± 3.63* 2.58 ± 0.14* 4.24 ± 0.09*
VCO (5%) + MTX 1.11 ± 0.01# 1.96 ± 0.15 94.7 ± 6.70# 3.22 ± 0.11# 3.38 ± 0.11#
VCO (15%) + MTX 1.14 ± 0.01# 2.93 ± 0.13# 115.3 ± 4.24# 3.64 ± 0.19# 3.43 ± 0.23#

VCO: Virgin coconut oil; MTX: Methotrexate; Values are mean + SEM (6 rats/group).
* P < 0.05: significant when compared to control group in the same column.
#
P < 0.05: significant when compared to MTX group in the same column.

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A.C. Famurewa et al.
alterations in a dose-dependent manner.
4. Discussion
There is currently an unmet need for effective management of side
effect of MTX chemotherapy. Although published papers have high-
lighted non-targeted organ toxicity of MTX, MTX-induced ne-
phrotoxicity remains a source of worry to clinicians, thus limiting MTX
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Biomedicine & Pharmacotherapy 96 (2017) 905–911
Fig. 1. Effect of VCO supplementation on renal level of interleukin-6 (IL-6)
in MTX-administered rats. VCO = virgin coconut oil;
MTX = Methotrexate. Values are expressed as mean ± SEM (n = 6).
*
Significant when compared to control (P < 0.05); #significant com-
pared to MTX control (P < 0.05).
use in clinical practice [9]. A natural approach to block renal MTX
damage would have beneficial clinical impact on the safety of the drug.
We hereby investigated the antioxidant and anti-inflammatory poten-
tials of VCO supplementation against pro-inflammation and oxidative
nephrotoxicity induced by anticancer drug MTX in rats.
In the present investigation, MTX induced nephrotoxicity in rats
indicated by elevated serum creatinine, urea and uric acid as compared
to normal control group. The MTX-induced oxidative stress in the
Fig. 2. Effect of VCO supplementation on renal level of nitric oxide (NO)
in MTX-administered rats. VCO = virgin coconut oil;
MTX = Methotrexate. Values are expressed as mean ± SEM (n = 6).
*
Significant when compared to control (P < 0.05); #significant com-
pared to MTX control (P < 0.05).
Fig. 3. Effect of VCO supplementation on renal level of C-reactive protein
(CRP) in MTX-administered rats. VCO = virgin coconut oil;
MTX = Methotrexate. Values are expressed as mean ± SEM (n = 6).
*
Significant when compared to control (P < 0.05); #significant com-
pared to MTX control (P < 0.05).
A.C. Famurewa et al.
current study was found to be associated with profound increases in
renal levels of pro-inflammatory markers confirmed by histopatholo-
gical observations. Alterations in renal function markers in MTX-treated
rats highlight a tubular dysfunction supported by recent reports [6,8].
Some studies have shown that MTX induces functional impairment in
kidneys associated with the precipitation of MTX in renal tubules [8].
The kidneys are a major elimination pathway for many antineoplastic
drugs and their metabolites, and the tubules are vulnerable to damage
[10]. Our histological analysis observed tubular damage by MTX. Other
studies attributed the tubular damage to cystic dilation and in-
flammatory infiltrations in glomeruli [34]. Interestingly, our observa-
tions support the report by Erboga et al [1] that MTX precipitation may
cause renal tubular and glomerular degeneration and necrosis. There-
fore, MTX-induced damage to tubular and glomerular integrity could
result in significantly increased serum levels of creatinine, urea and uric
acid in this study. Interestingly, the pretreatment with VCO supple-
mented diet (5% and 15%) in VCO + MTX groups improved renal
function and attenuated the increased levels of renal markers compared
to MTX group. The restorative impact of VCO diet was dose-dependent
showing higher beneficial health effect with 15% VCO supplemented
diet in this study. The current observation demonstrates ne-
phroprotective capacity of VCO against nephrotoxicity of MTX in rats.
The present results are consistent with previous findings that VCO diet
modulated renal toxicity induced by diabetes and cyclophosphamide in
animal models [24,35].
The mechanism underlying MTX nephrotoxicity is not well eluci-
dated in the current literature. Chemotherapy triggers production of
reactive oxygen species (ROS) or reactive nitrogen species (RNS) and a
variety of inflammatory responses [35]. ROS generation and the con-
sequent oxidative stress have been implicated in MTX nephrotoxicity
[5,13]. Furthermore, a recent study suggests the involvement of pro-
inflammation in MTX-induced renal impairment [5]. In our study, we
have observed, in consonance with the reports of previous studies, that
acute MTX administration to rats induced oxidative stress demonstrated
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Biomedicine & Pharmacotherapy 96 (2017) 905–911
Fig. 4. Histological photomicrograph of rat kidney
400×. Control: normal histology of kidney; MTX
group: tubular degeneration and glomerular damage
indicated by necrosis of epithelial cells shown by
arrows; VCO supplementation prevented necrosis
and tubular degeneration in VCO + MTX groups in a
dose-dependent manner showing near normal ap-
pearance of glomeruli and tubules.
by considerable renal decrease in SOD, CAT and GPx activities [1,3,17].
In addition, MTX treatment markedly reduced renal GSH, an important
non-enzymatic antioxidant that protect against ROS, while lipid per-
oxidation index, MDA, increased significantly. The cellular defense
mechanism against ROS is provided by GSH, SOD, CAT and GPx. SOD is
the primary scavenger of superoxide radical by converting it to H2O2
and H2O. The concerted activity of GPx converts peroxide radicals and
other ROS to H2O2 and H2O, while CAT converts H2O2 to H2O and O2.
The significant attrition in antioxidant enzyme activities could be ex-
plained by their exhaustion due to the persistent attack of ROS pro-
duction induced by MTX [37]. Studies suggest that depletion of sulf-
hydryl groups (eSH groups) in GSH and other proteins is the
prerequisite for ROS generation [38]. Several studies indicate that MTX
treatment indirectly decreases cellular GSH level via inhibition of
NADPH synthesis essential for recovery of GSH from oxidized glu-
tathione (GSSG) in cellular redox homeostasis [15,34]. In agreement
with this in the present study, the markedly decreased GSH content in
MTX toxicity could lead to increased susceptibility of renal tissue to
ROS damage. An important ROS damage is the peroxidation of poly-
unsaturated fatty acid. ROS attacks polyunsaturated fatty acids in cell
membrane to initiate lipid peroxidation. In the current study, MTX-in-
duced lipid peroxidation is shown by prominent elevation in renal
content of lipid peroxidation marker, MDA, in MTX-control rats [36].
The histopathological observation, desquamation of renal epithelial
cells, is related to peroxidation of basement membranes in MTX-control
rats. Our results strongly suggest that the impaired antioxidant defense
associated with oxidative stress contribute to MTX nephrotoxicity in
this study.
Although a number of antioxidant agents have demonstrated ben-
eficial efficacy against MTX toxicity, the potential of natural oil such as
VCO in MTX nephrotoxicity remains unexplored. The mechanism of
anticancer action of MTX is inhibition of folate synthesis. Leucovorin
(5-formyl tetrahydrofolate) is a medication used to circumvent toxicity
of MTX in normal cells. However, the intriguing report that the clinical
A.C. Famurewa et al.
use of leucovorin may undercut anticancer effect of MTX [16] warrants
emergence of agent with natural origin. We report here, for the first
time, that VCO supplemented diet (5% and 15%) significantly elevated
GSH, SOD, CAT and GPx activities and also abrogated increase in MDA
levels in the kidney. Consistent with our biochemical findings, the VCO
diet alleviated the histopathological lesions observed in MTX group
comparable to near normal. In line with our findings, recent studies
suggest that VCO improves antioxidant defenses for enhancement of
memory and also promotes resistance against cardiotoxicity and testi-
culotoxicity in various animal models [39–41]. VCO contains bioactive
compounds with promising antioxidant and pharmacological activities.
Phytochemical analysis found potent flavonoids and phenolic acids
mainly ferulic and p-coumaric acids in VCO [19]. The polyphenols in
VCO could combat MTX-induced oxidative stress to restore renal ac-
tivity of SOD, CAT, GPx and GSH level in VCO + MTX groups [19]. The
restoration of enzymatic and non-enzymatic antioxidants averted the
course of lipid peroxidation demonstrated by reduced level of MDA.
The current findings support previous conclusions that the VCO anti-
oxidant activity could be due to its antioxidant phenolics [19].
Activation of pro-inflammatory responses is also effective in the
current MTX-induced kidney damage [42]. Present study illustrated
that MTX administration significantly elevates renal levels of IL-6, CRP
and NO compared to normal control. Oxidative stress has been im-
plicated to commence or amplify inflammation via activation of several
transcription factors associated with inflammatory mechanisms [36].
The redox-sensitive transcription factor, NF-кB, is critical and is well
known to trigger pro-inflammatory cytokines and inducible nitric oxide
synthase (iNOS). Activation NF-кB by MTX-induced oxidative stress
could trigger cascades of inflammatory responses [6,36]. Our findings
corroborate recent reports that MTX provokes hepatorenal and sciatic
nerve pro-inflammation evident by considerably increased levels of
TNF-α, IL-1β, NF-кB and iNOS [6,8,43]. We suggest that MTX-induced
oxidative stress-mediated pro-inflammation ultimately resulted in sig-
nificant level of IL-6 which subsequently stimulates acute phase protein
CRP observed in this study [44]. Furthermore, inflammatory processes
activate iNOS thus exacerbate NO production. Aberrant release of NO
has been demonstrated to underlie pathophysiology of various diseases
including diabetes and nephrotoxicity [45]. The markedly increased
level of NO may be involved promoting oxidative stress and MTX ne-
phrotoxicity in this study [44]. These findings show that oxidative
stress and pro-inflammation play an active role in MTX nephrotoxicity.
We found that dietary VCO supplementation in VCO + MTX groups
reversed the pro-inflammation by marked decrease in IL-6, CRP and NO
levels in kidney dose-dependently, demonstrating anti-inflammatory
activity of VCO. Our present study gives supporting evidence to pre-
vious reports that the use of agents with antioxidant property can
modulate oxidative stress and pro-inflammation. Similar anti-in-
flammatory effects of VCO and its phenolics were also observed in
models of inflammation, arthritis and in vitro studies [46–48].
5. Conclusion
In conclusion, the study suggests nephroprotective effect of VCO
supplementation in rats via its antioxidant and anti-inflammatory
properties. The beneficial health effect may be afforded by the poly-
phenols in VCO. Clinical studies would be worthy to further consider
VCO supplementation as a potential nephroprotective adjuvant strategy
against MTX nephrotoxicity in cancer patients.
Conflict of interest
The authors declare no conflict of interest
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