Amelioration of Doxorubicin-Induced Liver And Kidney Toxicities By Nicorandil

Abstract

Antineoplastic agents are widely used in cancer chemotherapy, despite causing organ toxicity. This study was conducted to investigate the ameliorative properties of nicorandil alone and co-administered with prednisolone and diltiazem on doxorubicin-induced hepato-and nephro-toxicities in rats. Seventy female Wistar rats were treated for 16 days as follows: GI: normal saline (10 ml/kg; normal control); GII: normal saline (doxorubicin control); GIII: gallic acid (200 mg/kg); GIV-VI: nicorandil (0.22, 0.43 and 0.86 mg/kg respectively); GVII: diltiazem (3.43 mg/kg); GVIII: diltiazem + nicorandil (0.43 mg/kg); IX: prednisolone (0.57 mg/kg); and GX: prednisolone + nicorandil (0.43 mg/kg). Doxorubicin (40 mg/kg) was administered on day 14 i.p. to animals in GII-X. Nicorandil significantly (p<0.05) decreased alanine aminotransferase (ALT), aspartate aminotransferase (AST), renal creatinine, renal and hepatic malondialdehyde (MDA), and increased hepatic and renal catalase (CAT) and superoxide dismutase (SOD), compared to those administered doxorubicin alone. Co-administration of nicorandil with pred-nisolone and diltiazem significantly increased catalase, glutathione and superoxide dismutase, and decreased malondialdehyde, compared with the doxorubicin-only group. In conclusion, nicorandil decreased renal and hepatic markers of injury and increased enzymatic and non-en-zymatic antioxidants. Co-administration with the calcium channel blocker/phospholipase A 2 inhibitor did not elicit superior protective effect.

Full text

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
ABSTRACT
Antineoplastic agents are widely used in cancer chemotherapy, despite causing organ toxici-
ty. This study was conducted to investigate the ameliorative properties of nicorandil alone and
co-administered with prednisolone and diltiazem on doxorubicin-induced hepato- and nephro-
toxicities in rats. Seventy female Wistar rats were treated for 16 days as follows: GI: normal sa-
line (10 ml/kg; normal control); GII: normal saline (doxorubicin control); GIII: gallic acid (200
mg/kg); GIV-VI: nicorandil (0.22, 0.43 and 0.86 mg/kg respectively); GVII: diltiazem (3.43
mg/kg); GVIII: diltiazem + nicorandil (0.43 mg/kg); IX: prednisolone (0.57 mg/kg); and GX:
prednisolone + nicorandil (0.43 mg/kg). Doxorubicin (40 mg/kg) was administered on day 14
i.p. to animals in GII-X. Nicorandil signicantly (p<0.05) decreased alanine aminotransferase
(ALT), aspartate aminotransferase (AST), renal creatinine, renal and hepatic malondialdehyde
(MDA), and increased hepatic and renal catalase (CAT) and superoxide dismutase (SOD),
compared to those administered doxorubicin alone. Co-administration of nicorandil with pred-
nisolone and diltiazem signicantly increased catalase, glutathione and superoxide dismutase,
and decreased malondialdehyde, compared with the doxorubicin-only group. In conclusion,
nicorandil decreased renal and hepatic markers of injury and increased enzymatic and non-en-
zymatic antioxidants. Co-administration with the calcium channel blocker/phospholipase A2
inhibitor did not elicit superior protective effect.
Key words: Antineoplastic, Hepatotoxicity, Nephrotoxicity
INTRODUCTION
The liver and kidneys are vital organs responsible for detoxication through drug biotrans-
AMELIORATION OF DOXORUBICIN-INDUCED LIVER AND
KIDNEY TOXICITIES BY NICORANDIL ALONE AND CO-
ADMINISTERED WITH PREDNISOLONE AND DILTIAZEM
1*Abidemi J. Akindele, 1,2Kennedy I. Amagon, 1Gboyega T. Ekundayo,
3Dhirendra Singh, 4Daniel D. Osiagwu.
Afliation:
1* Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Medical
Sciences, College of Medicine, University of Lagos, Idi-Araba Campus, P.M.B. 12003,
Lagos, Nigeria.
1,2 Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Jos, Nigeria.
3 Department of Pharmacology, Shakambhari Institute of Higher Education and Technology,
Roorkee, Uttarakhand, India.
4 Department of Anatomic and Molecular Pathology, Faculty of Basic Medical Sciences,
College of Medicine, University of Lagos, Idi-Araba Campus, P.M.B. 12003, Lagos, Nigeria.
Corresponding author: 1*Abidemi J. Akindele
Email: ajakindele@cmul.edu.ng, jakindele@unilag.edu.ng

6PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
formation and elimination respectively. In the process of performing these essential roles, liver
and kidney cells are exposed to toxic (reactive) and non-toxic (unreactive) products of metabo-
lism. Considering the vital roles the liver and kidneys play, therapeutic and even sub-therapeu-
tic doses of drugs over a long period of administration can lead to nephro- and hepatotoxicity
(Fauci et al., 2015). Liver failure (acute) has been largely associated with drug-induced liver
toxicity (Lee and Senior, 2005; Wang et al., 2008). Cases of toxicity to the kidneys due to med-
ications account for about 60% of cases of acute injury to the kidneys, accounting for substan-
tial morbidity and mortality worldwide (Schetz et al., 2005).
One of the major problems associated with cancer chemotherapeutic agents relates to se-
lectivity, as rapidly dividing normal body cells are also affected as well as the targeted cancer
cells. A signicant number of anticancer drugs generate reactive oxygen species (ROS). Doxo-
rubicin is indicated for various kinds of cancers, e.g. hematological malignancies, primary
bone and soft tissue sarcomas, Hodgkin’s disease, cervical, uterine, lung, breast and ovarian
cancers (Chang et al., 2011; Thippeswamy et al., 2011). Its use has been associated with testic-
ular, hematological, renal, cardiac, hepatic and pulmonary toxicities (Injac et al., 2008; Mohan
et al., 2010).
Phospholipase A2 activation, through various mechanisms, has been implicated in various
forms of cytotoxicity (Cummings et al., 2000). Prednisolone is a corticosteroid which inhib-
its phospholipase A2 activity, thus elicit anti-inammatory and immunosuppressive activities,
among other established pharmacological actions. It mitigates symptoms and improves histob-
iochemical aberrations in various liver diseases (Mukherjee and Mukherjee, 2009; Czaja and
Manns, 2010; Fede et al., 2012).
Cell death and cytotoxic effects of drugs have been linked with increased intracellular calci-
um level (Farghali et al., 2000), a reason for which antagonism of calcium has been explored
in the mitigation of cytotoxicity. Diltiazem, indicated for arrhythmias, angina pectoris and
hypertension, has been explored for this purpose (Miura and Miki, 2003).
Nicorandil is mainly indicated for chronic angina pectoris, with studies showing that its
mechanism of action results in K+ efux and inactivation of voltage-gated calcium channels,
causing a reduction in free intracellular Ca2+ (Nakae et al., 2009). Since a number of cell deaths
have been attributed to an elevation in the intracellular calcium level (Kristian and Siesjo,
1998), nicorandil may be promising in preventing and/or ameliorating hepatotoxicity and neph-
rotoxicity. This hepatoprotective property of nicorandil was demonstrated by Taye et al. (2008)
who reported on the drug’s ability to protect the liver against carbon tetrachloride-induced hep-
atotoxicity in rats. Furthermore, nicorandil has been demonstrated to inhibit ischemia-reperfu-
sion-induced apoptosis and endoplasmic reticulum stress (Wu et al., 2015).
Gallic acid (GA) is a phenolic compound, known to possess radical scavenging activity and
protect against many diseases like cancer, diabetes and cardiovascular diseases, where oxida-
tive stress has been implicated (Kaur et al., 2005). Findings from numerous in vivo and in vitro
investigations give credence to its antiproliferative effect (Babu et al., 2016).
We investigated the effect of administration of nicorandil alone and in combination with a
calcium channel blocker (diltiazem) and a phospholipase A2 inhibitor (prednisolone) on an-

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
ti-cancer agent (doxorubicin)-induced hepato- and nephrotoxicity in this study.
MATERIALS AND METHODS
Animals
Wistar rats (female; 150-200 g) were procured from the College of Medicine, University of
Lagos, Laboratory Animal Centre, Nigeria. They were kept in a room maintained at 25oC with
relative humidity of 40-75% and 12 h light/dark cycle. Rodent pellet diet (Livestock Feeds
PLC, Lagos, Nigeria) and water were made freely available to the animals. The acclimatization
of rats before the start of experimental procedures was for 2 weeks.
Ethical approval was obtained from the Institutional Animal Care and Use (IACU) Commit-
tee/Ofce of Laboratory Animal Welfare (OLAW) of the Faculty of Pharmaceutical Sciences/
Pharmacology, University of Jos, Nigeria (Approval Ref. UJ/FPS/F17-00379 dated 3/1/2019).
Procedures followed the National Research Council (US) guidelines on experimental animals
use (National Research Council (US) Committee for the Update of the Guide for the Care and
Use of Laboratory Animals, 2011).
Female rats only were used in this study based on the assertion that they are more sensitive
to toxicity and are not more variable than male rats (Lipnick et al., 1995; OECD, 2000; Becker
et al., 2016).
Drugs and Chemicals
These include doxorubicin (Celon Laboratories Ltd., Gajularamaram, India), nicorandil
(Rivopharm UK Ltd., London, UK), gallic acid (Sigma Chemical Co., St. Louis, MO, USA),
prednisolone (Hovid Berhad, Malaysia), formalin (Unique Pharmaceuticals, Sango-Ota, Nige-
ria), and diltiazem (Sano-Aventis, S.p.A., Milan, Italy).
Treatment
Female Wistar rats, randomly allotted into ten groups with seven rats in each group, were
treated as enumerated below: Group I: Normal saline (10 ml/kg; normal control); Group II:
Normal saline (10 ml/kg; doxorubicin control); Group III: Gallic acid (200 mg/kg); Groups IV-
VI: Nicorandil (0.22, 0.43 and 0.86 mg/kg); Group VII: Diltiazem (3.43 mg/kg); Group VIII:
Diltiazem (3.43 mg/kg) and Nicorandil (0.43 mg/kg); Group IX: Prednisolone (0.57 mg/kg);
and Group X: Prednisolone (0.57 mg/kg) and Nicorandil (0.43 mg/kg).
Rats in Groups I to X were treated p.o. as outlined above for 16 days. Doxorubicin (40 mg/
kg) was given i.p. on day 14 to the rats in Groups II-X, 2 h post-treatment according the method
of Rashid et al. (2013). Doses of diltiazem and prednisolone used in this study were based on
the outcomes from a previous study by Akindele et al. (2014).
Haematological and Biochemical Analysis
On day 17, rats were made unconscious by inhaled anesthesia and blood was collected via
cardiac puncture using a 5-ml syringe attached to a needle. Blood was collected into heparin-
ized (1.5-2 ml) and plain (1.5-2 ml) sample bottles for haematological and biochemical esti-
mations respectively. An automated haematological analyzer was used to determine full blood
count on samples obtained from animals in the various groups; parameters determined include
platelets (PLT), white blood cell (WBC), haemoglobin (HB) and red blood cell (RBC). The

8PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
sera generated from blood samples after coagulation and centrifugation were used for estima-
tion of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransfer-
ase (ALT), total protein, triglycerides (TG), albumin, cholesterol, urea, low density lipoprotein
(LDL), creatinine and high-density lipoprotein (HDL) (Burtis et al., 2011).
Measurement of Antioxidant Parameters
Hepatic and renal (homogenates) levels of catalase (CAT), glutathione peroxidase (GPx),
superoxide dismutase (SOD), reduced glutathione (GSH) and malondialdehyde (MDA) were
determined using established protocols (Habbu et al., 2008; Gasparovic et al., 2013).
Histopathological Analysis
Representative samples of the liver and kidneys, harvested after rats were sacriced, were
xed in 10% formo-saline for histopathological analysis. The tissues were duly processed - de-
hydrated in graded alcohol, embedded in parafn, cut into thick sections (4-5 µm), and stained
with hematoxylin-eosin. Photomicroscopically, slides were viewed using ×40, ×100, and ×400
objectives (Habbu et al., 2008; Girish et al., 2009).
Statistical Analysis
Data presented as mean ± standard error of mean (SEM) were analysed (GraphPad Software
Inc., CA, USA; Prism 5) using one-way ANOVA with Turkey’s post-hoc test. p<0.05 was ad-
judged signicant.
RESULTS
Effect on Biochemical Parameters
Doxorubicin caused signicant (p<0.05) elevation in ALP, ALT and AST levels compared
with the normal control (Table 1). Co-administration of diltiazem plus prednisolone signi-
cantly (p<0.05) decreased ALT level compared with doxorubicin control group. Nicorandil
co-administered with diltiazem and nicorandil co-administered with prednisolone signicantly
(p<0.05) decreased AST and ALT concentrations respectively, compared with those of doxo-
rubicin control group. Doxorubicin elicited signicant (p<0.05) diminution in albumin level
relative to normal control. However, values in all other groups were statistically comparable
(p˃0.05) to normal control. The same observation was made in respect of LDH level versus
normal control.
From the results, creatinine level in animals administered doxorubicin only was signicantly
(p<0.05) elevated versus normal control (Table 1). There was signicant (p<0.05) diminution
in the concentration of creatinine in all the doses of nicorandil, and with nicorandil co-admin-
istered with diltiazem and prednisolone groups when compared with the group that received
doxorubicin alone.
Effect on Hepatic Antioxidant Indices
Doxorubicin elicited signicant (p<0.05) diminution in the levels of CAT, SOD, GSH
and GPx, and signicant (p<0.05) elevation in MDA level versus normal control (Table 2).
Nicorandil (0.43 mg/kg) alone and when co-administered with diltiazem/prednisolone signi-
cantly (p<0.05) reversed these effects versus doxorubicin control.
Effect on Renal Antioxidant Indices
Doxorubicin signicantly (p<0.05) diminished the renal levels of CAT, GSH, SOD and GPx,

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
and elicited signicant (p<0.05) elevation in MDA level versus normal control (Table 3). Gallic
acid, nicorandil (all doses), diltiazem/prednisolone and co-administration of both agents with
nicorandil (0.43 mg/kg) signicantly (p<0.05) reversed these trend of antioxidant indices elic-
ited in the doxorubicin control group.
Effect on Lipid Parameters
Doxorubicin elicited signicant (p<0.05) elevation in cholesterol and triglycerides levels ver-
sus normal control. Nicorandil at the dose of 0.22 mg/kg signicantly (p<0.05) diminished the
cholesterol level versus doxorubicin control. When co-administered with diltiazem, nicorandil
caused signicant (p<0.05) elevation of low-density lipoprotein level versus doxorubicin con-
trol (Table 4).
Table 1: Effect of Nicorandil Alone and Co-Administered with Diltiazem and Predniso-
lone (in the Presence of Doxorubicin) on Biochemical Parameters
Treatment Dose
(mg/kg)
AST
(U/L)
ALT
(U/L)
ALP
(U/L)
ALB
(mg/L)
LDH
(U/L)
Urea
(mmol/L)
Creatinine
(µmol/L)
Total Pro-
tein (mg/L)
Normal saline 10 (ml/
kg)
99.97
±
16.52
89.32
±
19.98
103.45
±
13.15
30.76
±
1.93
0.31
±
0.06
10.02
±
1.15
41.64
±
4.26
72.73±2.37
Normal saline
+ Doxoru-
bicin
10 (ml/
kg)
40
499.50
±
153.75*
312.21
±
95.51*
184.48
±
31.20*
22.96
±
2.73*
0.36
±
0.04
11.70
±
2.39
105.77
±
11.25*
67.48±2.02
Gallic acid +
Doxorubicin 200
40
163.08
±
40.12#
171.77
±
43.14#
91.72
±
15.73
32.52
±
1.37
1.30
±
0.43
11.74
±
1.74
98.08
±
6.33#
69.11±3.15
Nicorandil +
Doxorubicin 0.22
40
211.20
±
31.84#
107.38
±
43.82#
75.86
±
21.66
30.74
±
0.71
0.70
±
0.26
14.62
±
1.44
56.67
±
6.44#+
74.01±5.28
Nicorandil +
Doxorubicin 0.43
40
188.96
±
24.55#
81.62
±
24.20#
165.00
±
40.88+*
34.56
±
2.55
0.65
±
0.23
8.74
±
0.28
48.75
±
2.46#+
78.37±5.33
Nicorandil +
Doxorubicin 0.86
40
262.00
±
57.90#
241.18
±
76.45#
136.78
±
20.76
36.52
±
2.17
0.50
±
0.12
8.72
±
1.22
49.20
±
5.05#+
74.06±6.30
Diltiazem +
Doxorubicin 3.43
40
366.03
±
62.25
148.28
±
40.96#
108.48
±
17.54
27.86
±
3.00
0.71
±
0.14
15.60
±
3.74
48.96
±
3.63#+
62.18±4.44
Diltiazem +
Nicorandil +
Doxorubicin
3.43
0.43
40
262.52
±
24.02#*
165.48
±
31.31#
77.26
±
4.33
29.90
±
2.93
0.96
±
0.23
12.92
±
4.35
47.52
±
4.08#+
62.14±4.39
Prednisolone
+ Doxoru-
bicin
0.57
40
348.55
±
167.11
254.76
±
84.91*#
100.82
±
22.29
25.80
±
2.52
0.48
±
0.09
13.86
±
2.49
92.55
±
14.02
61.46±1.29
Prednisolone
+ Nicorandil
+ Doxoru-
bicin
0.57
0.43
40
244.07
±
33.15#
278.92
±
53.28*#
112.20
±
28.63
32.58
±
3.42
0.47
±
0.10
13.96
±
2.47
78.20
±
10.19#
63.81±9.92
Results are expressed as mean ± SEM. *p<0.05 when compared with the control group; #p<0.05
when compared with toxicant group; +p<0.05 when compared with gallic acid group (One-way
ANOVA followed by Turkey’s multiple comparison test). ALB – Albumin; LDH – Lactate
dehydrogenase.

10 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Table 2: Effect of Nicorandil Alone and Co-Administered with Diltiazem and Predniso-
lone (in the Presence of Doxorubicin) on Liver Antioxidant Indices
Treatment Dose (mg/kg)
CAT
(U/mg)
SOD
(U/mg)
GSH
(U/mg)
GPx
(U/mg)
MDA
(nmol/g)
Normal saline 10 (ml/kg) 27.46±2.87 8.58±0.57 0.88±0.15 323.55±27.52 7.61±0.85
Normal saline +
Doxorubicin
10 (ml/kg)
40 12.71±1.18*4.77±0.73*0.23±0.32*112.96±22.66*17.49±1.16*
Gallic acid +
Doxorubicin
200
40 33.45±4.30 9.16±0.63#1.09±0.63#112.22±5.49*#5.67±0.87#
Nicorandil + Doxo-
rubicin
0.22
40 12.46±1.93+6.77±2.16+0.65±0.09 112.93±1.20*17.49±2.62*
Nicorandil + Doxo-
rubicin
0.43
40 20.01±4.74+4.96±0.52*+ 0.48±0.09 251.04±16.42+10.05±1.37#
Nicorandil + Doxo-
rubicin
0.86
40 21.45±7.99#+ 9.98±0.52#0.23±0.05*+ 113.50±1.04*16.05±1.74*
Diltiazem + Doxo-
rubicin
3.43
40 24.79±1.97#+ 5.00±0.71*0.39±0.14+257.03±17.93#+ 5.85±1.28#
Diltiazem +
Nicorandil + Doxo-
rubicin
3.43
0.43
40
24.55±1.55#+ 9.79±0.64#+ 0.26±0.03*+ 107.67±1.89*+ 5.70±1.40#
Prednisolone +
Doxorubicin
0.57
40 12.47±1.05 7.77±1.00#1.53±0.33*# 278.89±63.72 5.63±2.27#
Prednisolone +
Nicorandil + Doxo-
rubicin
0.57
0.43
40
21.42±0.81#+ 4.46±1.00*+ 1.49±0.46#176.33±36.51*6.24±3.05#
Results are expressed as mean ± SEM. *p<0.05 when compared with the control group; #p<0.05
when compared with toxicant group; +p<0.05 when compared with gallic acid group (One-way
ANOVA followed by Turkey’s multiple comparison test).
Table 3: Effect of Nicorandil Alone and in Combination with Prednisolone and Diltiazem
on Renal Antioxidant Indices
Treatment Dose (mg/kg) CAT
(U/mg) SOD
(U/mg) GSH
(U/mg) GPx
(U/mg) MDA
(nmol/g)
Normal saline 10 (ml/kg) 39.09 ±4.19 5.51±0.59 0.35 ±0.04 180.73±14.29 6.00±1.08
Normal saline +
Doxorubicin 10 (ml/kg)
40 15.05±1.10*3.73±0.60*0.19±0.05*88.47±4.77*13.80±2.37*
Gallic acid + Doxo-
rubicin 200
40 54.07±3.57*# 7.39±0.97*# 0.20±0.03*90.60±4.55*4.41±0.73#
Nicorandil + Doxo-
rubicin 0.22
40 23.95±5.71*#+ 3.71±0.27*+ 0.18±0.03*126.84±14.38*7.19±1.17#
Nicorandil + Doxo-
rubicin 0.43
40 24.25±5.60*+ 4.75±0.37+0.20±0.05*89.13±3.20*7.06±1.25#
Nicorandil + Doxo-
rubicin 0.86
40 18.54±4.01*+ 3.91±0.74+0.20±0.04*129.15±23.89*13.48±0.88*#+
Diltiazem + Doxoru-
bicin 3.43
40 30.57±4.64*#+ 4.01±0.32+0.17±0.02 104.86±2.46*3.98±0.79#
Diltiazem +
Nicorandil + Doxo-
rubicin
3.43
0.43
40
30.22±3.17+4.02±0.35+0.23±0.04 104.41±9.30*3.96±0.75#
Prednisolone + Doxo-
rubicin 0.57
40 15.10±0.98*+ 3.48±0.38*+ 0.37±0.08#+ 135.90±28.65*# 8.32±1.93#
Prednisolone +
Nicorandil + Doxo-
rubicin
0.57
0.43
40
28.97±3.39#+ 6.12±0.98#+ 0.22±0.04 138.41±21.16*# 6.01±0.35#
Results are expressed as mean ± SEM. *p<0.05 when compared with the control group; #p<0.05
when compared with toxicant group; +p<0.05 when compared with gallic acid group (One-way
ANOVA followed by Turkey’s multiple comparison test).

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Table 4: Effect of Nicorandil Alone and Co-Administered with Diltiazem and Predniso-
lone (in the Presence of Doxorubicin) on Lipid Parameters
Treatment Dose (mg/kg)
HDL
(mg/dL)
LDL
(mg/dL)
Cholesterol
(mg/dL)
Triglycerides
(mg/dL)
Normal saline 10 (ml/kg) 0.68±0.07 0.31±0.06 0.61±0.06 0.48±0.04
Normal saline + Doxo-
rubicin
10 (ml/kg)
40 1.25±0.08 0.36±0.04 1.86±0.27*0.96±0.12*
Gallic acid + Doxoru-
bicin
200
40 1.20±0.16 1.30±0.43*# 2.37±0.80*0.92±0.14
Nicorandil + Doxoru-
bicin
0.22
40 0.88±0.22 0.70±0.25+1.15±0.04#0.76±0.10
Nicorandil + Doxoru-
bicin
0.43
40 1.37±0.12 0.65±0.23+1.46±0.26 0.98±0.21*
Nicorandil + Doxoru-
bicin
0.86
40 1.20±0.10 0.50±0.12+1.33±0.15 0.76±0.14
Diltiazem + Doxorubicin 3.43
40 1 .80±0.37 0.71±0.14+2.25±0.40*1.12±0.19*
Diltiazem + Nicorandil +
Doxorubicin
3.43
0.43
40
1.52±0.64 0.96±0.23*+# 2.12±0.68*1.17±0.28*
Prednisolone + Doxo-
rubicin
0.57
40 1.34±0.05 0.48±0.09+1.15±0.12#1.05±0.13*
Prednisolone +
Nicorandil + Doxoru-
bicin
0.57
0.43
40
1.42±0.17 0.47±0.10+1.18±0.13#0.98±0.17*
Results are expressed as mean ± SEM. *p<0.05 when compared with the control group; #p<0.05
when compared with toxicant group; +p<0.05 when compared with gallic acid group (One-way
ANOVA followed by Turkey’s multiple comparison test)
Effect on Haematological Parameters
Doxorubicin produced signicant (p<0.05) decline in WBC, RBC and PLT levels versus nor-
mal control (Table 5). Administration of gallic acid, nicorandil (all doses), diltiazem/prednis-
olone alone and co-administered with nicorandil (0.43 mg/kg) signicantly (p<0.05) reversed
the diminution in RBC level induced by doxorubicin.
Histopathological Analysis
Figure 1(A-F) shows the representative photomicrographs of liver sections of the different
experimental groups. The doxorubicin control group showed steatosis in comparison with the
normal control and intervention groups which revealed normal hepatocellular architecture.
Figure 2(A-F) shows the representative photomicrographs of kidney sections of the differ-
ent experimental groups. The doxorubicin control group showed cortical necrosis and thin
glomeruli basement membrane unlike the normal control which revealed normal renocellular
architecture. Like the normal control, the nicorandil, diltiazem and prednisolone (individual
and combination) groups displayed normal renocellular architecture.

12 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Table 5: Effect of Nicorandil Alone and Co-Administered with Diltiazem and Predniso-
lone (in the Presence of Doxorubicin) on Haematological Parameters in Rats
Treatment Dose (mg/kg) WBC (103/µL) RBC (106/µL) Platelets (103/µL) HB (g/dL)
Normal saline 10 (ml/kg) 8.98±1.18 7.05±0.29 777.60±60.39 13.68±0.59
Normal saline +
Doxorubicin
10 (ml/kg)
40 2.42±0.66*5.20±0.50*269.20±36.43*12.20±1.16
Gallic acid + Doxo-
rubicin
200
40 4.28±1.48*6.39±0.39#360.50±36.14*14.00±1.16
Nicorandil + Doxo-
rubicin
0.22
40 1.65±0.62*7.29±0.29#310.00±48.31*14.27±0.63
Nicorandil + Doxo-
rubicin
0.43
40 2.96±1.10*6.62±0.32#308.60±36.56*13.30±0.76
Nicorandil + Doxo-
rubicin
0.86
40 2.76±1.05*6.64±0.34#234.60±41.83*14.52±1.23
Diltiazem + Doxoru-
bicin
3.43
40 3.24±0.43*6.56±0.24#304.50±62.57*12.18±0.91
Diltiazem +
Nicorandil + Doxo-
rubicin
3.43
0.43
40
2.86±0.36*6.59±0.45#292.00±40.68*10.88±0.68
Prednisolone + Doxo-
rubicin
0.57
40 3.40±0.94*6.88±0.45#298.40±87.40*13.42±1.05
Prednisolone +
Nicorandil + Doxo-
rubicin
0.57
0.43
40
4.82±1.68*6.51±0.55#243.20±45.94*11.48±1.35
Results are expressed as mean ± SEM. *p<0.05 when compared with the control group; #p<0.05
when compared with toxicant group (One-way ANOVA followed by Turkey’s multiple com-
parison test).

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Figure 1: Photomicrographs of liver sections showing effect of nicorandil alone and co-ad-
ministered with prednisolone and diltiazem (in the presence of doxorubicin). A. Showing
normal architecture (control, treated with normal saline 10 ml/kg p.o.); B. Showing ste-
atosis (treated with doxorubicin 40 mg/kg i.p.); C. Showing normal architecture (treat-
ed with doxorubicin 40 mg/kg i.p. and gallic acid 200 mg/kg p.o.); D. Showing normal
architecture (treated with doxorubicin 40 mg/kg i.p. and nicorandil 0.43 mg/kg p.o.); E.
Showing normal architecture (treated with doxorubicin 40 mg/kg i.p., diltiazem 3.43 mg/
kg p.o. and nicorandil 0.43 mg/kg p.o.); and F. Showing normal architecture (treated with
doxorubicin, 40 mg/kg i.p. prednisolone, 0.57 mg/kg p.o. and nicorandil, 0.43 mg/kg p.o.).
H&E stain, × 400.

14 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Figure 2: Photomicrographs of kidney sections showing effect of nicorandil alone and
co-administered with prednisolone and diltiazem (in the presence of doxorubicin). A.
Showing normal architecture (control, treated with normal saline 10 ml/kg p.o.); B.
Showing Cortical necrosis (Black arrows) and glomeruli basement membrane (red ar-
rows) (treated with doxorubicin 40 mg/kg i.p.); C. Showing normal architecture (treat-
ed with doxorubicin 40 mg/kg i.p. and gallic acid 200 mg/kg p.o.); D. Showing normal
architecture (treated with doxorubicin 40 mg/kg i.p. and nicorandil 0.43 mg/kg p.o.); E.
Showing normal architecture (treated with doxorubicin 40 mg/kg i.p. and diltiazem 3.43
mg/kg p.o. and nicorandil 0.43 mg/kg p.o.); and F. Showing normal architecture (treated
with doxorubicin 40 mg/kg i.p. and prednisolone 0.57 mg/kg p.o. and nicorandil 0.43 mg/
kg p.o.). H&E stain, × 400.

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PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
DISCUSSION
The development of new drugs or drug repurposing for the management of drug-induced
nephro- and hepatotoxicity remains relevant in order to reduce the risks associated with clini-
cal use of antineoplastic agents. Increase in ALT (liver explicit) and AST levels occurs due to
injured tissues seepage caused by hepatocellular necrosis (Ozer et al., 2008), while elevation in
the level of ALP emanates from overproduction and release in blood as a result of hepatobiliary
injury and cholestasis (Ramaiah, 2007). In essence, increase in these liver enzymes levels; as
well as total bilirubin and lactate dehydrogenase (LDH) (Okwa et al., 2013) suggests damage
to hepatic cells. This relates with results from this present study where administration of doxo-
rubicin was shown to signicantly increase concentrations of serum AST, ALT, ALP and LDH,
as well as diminution in albumin and total protein concentrations - consequences of diminished
synthetic capacity because of hepatic dysfunction (Thapa and Walia, 2007).
In this study, nicorandil signicantly reduced doxorubicin-induced elevated levels of liver
enzymes (AST and ALT) following injury, suggesting hepatoprotective property. Diltiazem in
the presence of doxorubicin caused signicant reduction in the level of ALT. The level of ALT
in this case was lower than that of its combination with nicorandil, but higher compared to the
value for nicorandil in the presence of doxorubicin. Diltiazem did not elicit signicant change
in the level of AST relative to the doxorubicin group, but its combination with nicorandil
caused signicant reduction in the level of AST compared to the doxorubicin group. The level
of AST in this case (diltiazem plus nicorandil) was higher relative to that elicited by nicorandil
administered in the presence of doxorubicin. Bojani et al. (2009) reported the ameliorative
effect of diltiazem on toxicants induced injury. In that study, diltiazem was shown to prevent
monosodium glutamate toxicity on histology of the ovaries of Wistar rats. The ndings in this
study in respect of ALT, a more specic liver enzyme, suggest that diltiazem administered with
nicorandil may not offer any advantage in terms of hepatoprotective effect when compared to
administration of nicorandil alone. However, unlike in this case, Humphrey (1998) reported
synergistic relationship between nicorandil and diltiazem in terms of cardiovascular and phar-
macokinetic interactions. Nicorandil in the presence of doxorubicin elicited better (lower) AST
and ALT levels compared with the respective values in the group that received a combination
of nicorandil and prednisolone in the presence of doxorubicin. It can therefore be posited that
co-administration of nicorandil with prednisolone does not have any advantage in terms of
hepatoprotective effect relative to nicorandil alone. Agbo-Godeau et al. (1998) reported that
co-administration of nicorandil and prednisolone may cause risk elevation in respect of gastric/
intestinal ulceration/bleeding in humans. The ndings in this study showed that the ALT value
in the group that received the combination of prednisolone and nicorandil in the presence of
doxorubicin was higher compared to the group that received prednisolone in the presence of
doxorubicin, and much higher than the group that received nicorandil in the presence of doxo-
rubicin.
Our results showed that doxorubicin elicited signicant elevation in creatinine level versus
normal control. Urea was increased too, though in an insignicant manner. This result cor-
relates with reported cases of nephrotoxicity due to doxorubicin (Injac et al., 2008; Mohan
et al., 2010). In this present study, nicorandil was shown to reduce creatinine concentration
following injury relative to the animals administered doxorubicin only. In fact, levels were
signicantly lower compared to the group administered the standard drug, gallic acid. This re-
duction is a clear indication of the nephroprotective property of nicorandil. Diltiazem elicited

16 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
signicant diminution in creatinine concentrations versus normal control and standard antiox-
idant groups. This observed effect with diltiazem can be attributed to its antioxidant property
(Deray, 1999; Filipets and Gozhenko, 2014). The result from the present study did not show a
signicant difference in urea, creatinine and total protein when nicorandil was co-administered
with diltiazem in one group and with prednisolone in another group, compared to animals
administered nicorandil alone. Thus, co-administration of these drugs cannot be said to offer
better protection to the kidney than administration of nicorandil alone.
Oxidative stress results in diminution in the level of endogenous antioxidants and consequent
increase in MDA level. Decrease in GSH concentration is also associated with enhanced lip-
id peroxidation. Glutathione evacuates hydrogen peroxide, superoxide radicals and other free
radical species, and preserves protein thiols of membranes (Prakash, 2001). Diminution in CAT
activity permits assimilation of superoxide radical and hydrogen peroxide which is deleterious
(Chance and Greenstein, 1992). Results from this present study support this, as signicant
MDA elevation and decreased CAT, SOD, GSH and GPx levels were recorded after adminis-
tration of doxorubicin in both liver and kidneys, relative to the control group. In the kidneys
and liver, signicant reduction in MDA level was observed after nicorandil administration
(doxorubicin presence), relative to the animals administered doxorubicin only. This ability of
nicorandil to reduce lipid peroxidation and free radical formation correlates with reports of
other researchers (Teshima et al., 2003; Tanabe et al., 2012). The excessive generation of ROS,
concomitant with the reduction of antioxidant defense activities in the liver, is closely related
to the induction and progression of hepatic cell death (Hadi et al., 2012; Akbulut et al., 2014).
Results from this study showed that nicorandil followed by administration of doxorubicin
increased CAT, SOD, GSH and GPx, though in an insignicant manner (in both liver and
kidney), compared to the group of animals administered doxorubicin alone. This indicates the
ability of nicorandil to stimulate in vivo antioxidant activity, thus enhancing hepatoprotection.
This protective property of nicorandil correlates with a study conducted by Kong et al. (2015)
where nicorandil reduced apoptosis and decreased oxidative stress. Diltiazem and predniso-
lone, alone and co-administered with nicorandil did not cause signicant difference in MDA
level versus nicorandil alone. This clearly shows that co-administering nicorandil with either
diltiazem or prednisolone has no overall advantage over administering nicorandil alone.
Increase in TG, CHOL, LDL and a decrease in HDL are indicative of injury/damage to cells
of the liver, as observed by Ahmad et al. (2014). This trend is similar to that observed in this
study where the administration of doxorubicin caused a decrease in HDL and total protein, as
well as an increase in levels of LDL, CHOL and TG. An indication of reversal or protection
would be an increase in TP and HDL, and a decrease in LDL, CHOL and TG. Administration
of nicorandil (all doses) was able to achieve this. Co-administration of diltiazem and nicorandil
did not produce signicant difference in terms of a better reversal of the indices of toxicity,
compared to that produced by nicorandil alone.
Doxorubicin elicits dose-related reduction in haematocrit, haemoglobin and RBC levels
(Henderson et al., 1982; Badylak et al., 1985), similar to results from this present study where
WBC, RBC, platelets and haemoglobin levels decreased in a signicant manner following
doxorubicin administration, compared to animals administered normal saline only. Another
study by Dorgalaleh (2013), revealed a decrease in haematological parameters like RBC counts,

17
PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
platelets and HB in renal failure. Nicorandil (all the doses), diltiazem/prednisolone alone and
in combination with nicorandil caused increase in RBC level compared with the doxorubicin
(toxicant) group. This effect may be attributed to the protective effect of nicorandil, as previ-
ously discussed. However, diltiazem administered alone and co-administered with nicorandil
was observed to increase WBC better than in the group administered nicorandil alone, though
in an insignicant manner.
The ability of free radicals to cause liver and kidney injury is well established (Rahman,
2007; Anwar and Mohamed, 2015) and correlates with histopathological observations in re-
spect of the liver and kidneys of rats in this study. As observed in this study, the representative
liver of the doxorubicin control group revealed steatosis (fatty deposits). This supports ndings
from our biochemical analysis where doxorubicin was observed to cause injury. Normal he-
patic architecture manifested in representative liver of rats given gallic acid plus doxorubicin
or nicorandil plus doxorubicin, an indication that gallic acid and nicorandil were able to pro-
tect the hepatic cells against injury. The representative liver and kidneys of rats administered
nicorandil combined with diltiazem or prednisolone showed normal hepatic and renal architec-
tures respectively.
CONCLUSION
In conclusion, doxorubin-induced toxicity to the liver, kidneys and antioxidant system was
ameliorated by nicorandil. Co-administration of nicorandil with diltiazem/prednisolone did not
however elicit superior protection at the doses used.
CONFLICT OF INTEREST
None to declare; funding was not received from any not-for-prot, commercial, or public entity.
ACKNOWLEDGEMENT
Dr. Margaret Sodiya (Pharmacognosy Department, University of Lagos, Nigeria) generous-
ly provided gallic acid. Mr. Sunday Adenekan (Biochemistry Department of same institution)
provided technical assistance.
AUTHOR CONTRIBUTIONS
Abidemi J. Akindele - Conception, design, interpretation and manuscript writing
Kennedy I. Amagon - Execution, interpretation and manuscript writing
Gboyega T. Ekundayo - Execution and manuscript writing
Dhirendra Singh - Conception and design
Daniel D. Osiagwu - Execution and interpretation
REFERENCES
Agbo-Godeau, S., Joly, P., Lauret, P., Szpirglas, R. & Szpirglas, H. (1998). Association of
major aphthous ulcers and nicorandil. Lancet 352, 1598-1599.
Ahmad, D., Gulfraz, M., Ahmad, M.S., Nazir, H., Gul, H. & Asif, S. (2014). Protective action
of Taraxacum ofcinale on CCl4 induced hepatotoxicity in rats. Afr. J. Pharm. Pharmacol. 8,
775-780.
Akbulut, S., Elbe, H., Eris, C., Dogan, Z., Toprak, G., Otan, E., Erdemli, E. & Turkoz, Y.

18 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
(2014). Cytoprotective effects of amifostine, ascorbic acid and N-acetylcysteine against meth-
otrexate-induced hepatotoxicity in rats. World J. Gastroenterol. 20, 10158-10165.
Akindele, A.J., Adeneye, A.A., Olatoye, F. & Benebo, A.S. (2014). Protective effect of se-
lected calcium channel blockers and prednisolone, a phospholipase-A2 inhibitor, against gen-
tamicin and carbon tetrachloride-induced nephrotoxicity. Hum. Exp. Toxicol. 33, 831-846.
Anwar, M.M. & Mohamed, N.E. (2015). Amelioration of liver and kidney functions disor-
ders induced by sodium nitrate in rats using wheat germ oil. J. Rad. Res. Appl. Sci. 8, 77-83.
Babu, R., Sowjanya, N. & Sarvamangala, D. (2016). Production of gallic acid - A short re-
view. Int. J. Sci. Res. Method. 4, 125-132.
Badylak, S.F., Van Vleet, J.F., Herman, E.H., Ferrans, V.J. & Myers, C.E. (1985). Poikilocy-
tosis in dogs with chronic doxorubicin toxicosis. Am. J. Vet. Res. 46, 505-508.
Becker, J.B., Becker, J.B., Prendergast, B.J. & Liang, J.W. (2016). Female rats are not more
variable than male rats: a meta-analysis of neuroscience studies. Biol. Sex Differ. 7: 34.
Bojani, V., Bojani, Z., Najman, S., Savi, T., Jakovljevi, V., Najman, S. & Janci, S.
(2009). Diltiazem prevention of toxic effects of monosodium glutamate on ovaries in rats. Gen
Physiol. Biophys. 28 Spec No, 149-154.
Burtis, C.A., Ashwood, E.R. & Bruns, D.E. (2011). Tietz Textbook of Clinical Chemistry and
Molecular Diagnostics; 5th edition. Saunders, p. 2238.
Chance, B. & Greenstein, D.S. (1992). The mechanism of catalase actions-steady state anal-
ysis. Arch. Biochem. Biophys. 37, 301-339.
Chang, Y.L., Lee, H.J. & Liu, S.T. (2011). Different roles of p53 in the regulation of DNA
damage caused by 1,2 heteroannelated anthraquinones and doxorubicin. Int. J. Biochem. Cell
Biol. 43, 1720-1728.
Cummings, B.S., McHowat, J. & Schnellmann, R.G. (2000). Phospholipase A2s in cell inju-
ry and death. J. Pharmacol. Exp. Ther. 294, 793-799.
Czaja, A.J. & Manns, M.P. (2010). Advances in the diagnosis, pathogenesis, and manage-
ment of autoimmune hepatitis. Gastroenterology 139, 58-72.
Deray, G. (1999). Nephroprotective effect of calcium antagonists. Presse Med. 28, 1667-1670.
Dorgalaleh, A., Mahmudi, M., Tabibian, S., Khatib, Z.K., Tamaddon, G.H., Moghaddam,
E.S., Bamedi, T., Alizadeh, S. & Moradi, E. (2013). Anemia and thrombocytopenia in acute
and chronic renal failure. Int. J. Hematol. Oncol. Stem Cell Res. 7, 34-39.
Farghali, H., Kmonickova, E., Lotkova, H. & Martinek, J. (2000). Evaluation of calcium
channel blockers as potential hepatoprotective agents in oxidative stress injury of perfused
hepatocytes. Physiol. Res. 49, 261-268.

19
PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Fauci, A.S., Braunwald, E. & Kasper, D.L. (2015). Harrison’s Principle of Internal Medicine.
19th ed. United States.
Fede, G., Spadaro, L., Tomaselli, T., Privitera, G., Germani, G., Tsochatzis, E., Thomas, M.,
Bouloux, P.M., Burroughs, A.K. & Purrello, F. (2012). Adrenocortical dysfunction in liver dis-
ease: a systematic review. Hepatology 55, 1282-1291.
Filipets, N.D. & Gozhenko, A.I. (2014). Comparative assessment of nephroprotective prop-
erties of potassium and calcium channel modulators in experimental renal injury. Eksp. Klin.
Farmakol. 77, 10-12.
Gasparovic, A.C., Jaganjac, M., Mihaljevic, B., Sunjic, S.B. & Zarkovic, N. (2013). Assays
for the measurement of lipid peroxidation. Methods Mol. Biol. 965, 283-296.
Girish, C., Koner, B.C., Jayanthi, S., Ramachandra Rao, K., Rajesh, B. & Pradhan SC.
(2009). Hepatoprotective activity of picroliv, curcumin and ellagic acid compared to silymarin
on paracetamol induced liver toxicity in mice. Fundam. Clin. Pharmacol. 23, 735-745.
Habbu, P.V., Shastry, R.A., Mahadevan, K.M., Joshi, H. & Das, S.K. (2008). Hepatoprotec-
tive and antioxidant effects of Argyreia speciosa in rats. Afr. J. Trad. Complement Altern. Med.
5, 158-164.
Hadi, N.R., Al-Amran, F.G. & Swadi, A. (2012). Metformin ameliorates methotrexate-in-
duced hepatotoxicity. J. Pharmacol. Pharmacother. 3, 248-253.
Henderson, B.M., Dougherty, W.J., James, V.C., Tilley, L.P. & Noble, J.F. (1982). Safety
assessment of a new anticancer compound, mitoxantrone, in beagle dogs: comparison with
doxorubicin. I. Clinical observations. Cancer Treat. Rep. 66, 1139-1143.
Humphrey, S.J. (1998). Cardiovascular and pharmacokinetic interactions between nicorandil
and adjunctive propranolol, atenolol or diltiazem in conscious dogs. Methods Find Exp. Clin.
Pharmacol. 20, 779-791.
Injac, R., Boskovic, M. & Perse, M. (2008). Acute doxorubicin nephrotoxicity in rats with
malignant neoplasm can be successfully treated with fullerenol C60(OH)24 via suppression of
oxidative stress. Pharmacol. Res. 60, 742-749.
Kaur, S., Michael, H., Arora, S., Harkonen, P.L. & Kumar, S. (2005). The in vitro cytotoxic
and apoptotic activity of Triphala -an Indian herbal drug. J Ethnopharmacol. 97, 15-20.
Kong, J.J., Zhang, D.D., Li, P., Wei, C.Y., Yu, H.J., Zhang, H., Zhang, W., Wang, Y.F. & Cao,
Y.P. (2015). Nicorandil inhibits oxidative stress and amyloid-β precursor protein processing in
SH-SY5Y cells overexpressing APPsw. Int. J. Clin. Exp. Med. 8, 1966-1975.
Kristian, T. & Siesjo, B.K. (1998). Calcium in ischemic cell death. Stroke 29, 705-718.
Lee, W.M. & Senior, J.R. (2005). Recognizing drug induced liver injury: current problems,
possible solutions. Toxicol. Pathol. 33, 155-64.

20 PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Lipnick, R.L., Cotruvo, J.A., Hill, R.N., Bruce, R.D., Stitzel, K.A., Walker, A.P., Chu, I.,
Goddard, M., Segal, L., Springer, J.A. & Myers, R.C. (1995). Comparison of the up-and down,
conventional LD50, and xed dose acute toxicity procedures. Food Chem. Toxicol. 33, 223-231.
Miura, T. & Miki, T. (2003). ATP-sensitive K+ channel openers: old drugs with new clinical
benets for the heart. Curr. Vasc. Pharmacol. 1, 251-258.
Mohan, M., Kamble, S., Gandhi, P. & Kasture, S. (2010). Protective effect of Solanum tor-
vum on doxorubicin-induced nephrotoxicity in rats. Food Chem. Toxicol. 4, 436-440.
Mukherjee, S. & Mukherjee, U. (2009). A comprehensive review of immunosuppression
used for liver transplantation. J. Transplant. 2009, 701464. doi: 10.1155/2009/701464.
Nakae, I., Matsumoto, T., Horie, H., Yokohama, H., Omura, T., Minai, K., Matsui, T., Noza-
wa, M., Takahashi, M., Sugimoto, Y., Ito, M., Izumi, M., Nakamura, Y., Mitsunami, K. &
Kinoshita, M. (2000). Effects of intravenous nicorandil on coronary circulation in humans:
plasma concentration and action mechanism. J. Cardiovasc. Pharmacol. 35, 919-925.
National Research Council (US) Committee for the Update of the Guide for the Care and Use
of Laboratory Animals (2011). Guide for the Care and Use of Laboratory Animals; 8th edition.
Washington (DC): National Academies Press (US).
OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical
Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation Environmen-
tal Health and Safety Monograph Series on Testing and Assessment No. 19.
Okwa, I.B., Akindele, A.J., Agbaje, E.O., Oshinuga, O.T., Anunobi, C.C. & Adeyemi, O.O.
(2013). Effect of subclinical, clinical and supraclinical doses of calcium channel blockers on
models of drug-induced hepatotoxicity in rats. EXCLI. J. 2, 231-250.
Ozer, J., Ratner, M., Shaw, M., Bailey, W. & Schomaker, S. (2008). The current state of se-
rum biomarkers of hepatotoxicity. Toxicol. 245, 194-205.
Prakash, J., Gupta, S.K. & Singh, N. (2001). Chemopreventive activity of Withania somnif-
era in experimentally induced brosarcoma tumours in Swiss albino rats. Phytother. Res. 15,
200-204.
Rahman, K. (2007). Studies on free radicals, antioxidants, and co-factors. Clin. Interv. Aging.
2, 219-236.
Ramaiah, S.K. (2007). A toxicologist guide to the diagnostic interpretation of hepatic bio-
chemical parameters. Food Chem. Toxicol. 45, 1551-1557.
Rashid, S., Ali, N., Nafees, S., Ahmad, S.T., Arjumand, W., Hasan, S.K. & Sultana, S. (2013).
Alleviation of doxorubicin-induced nephrotoxicity and hepatotoxicity by chrysin in Wistar
rats. Toxicol. Mech. Methods 23, 337-345.
Schetz, M., Dasta, J., Goldstein, S. & Golper, T. (2005). Drug-induced acute kidney injury.

21
PROCEEDINGS OF THE NIGERIAN ACADEMY OF SCIENCE VOLUME 12, 2019
Curr. Opin. Crit. Care 11, 555-565.
Tanabe, K., Lanaspa, M.A., Kitagawa, W., Rivard, C.J., Miyazaki, M., Klawitter, J., Schrein-
er, G.F., Saleem, M.A., Mathieson, P.W., Makino, H., Johnson, R.J. & Nakagawa, T. (2012).
Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-decient mouse.
Am. J. Physiol. Renal Physiol. 302, 1151-1160.
Taye, A., El-Moselhy, M.A. & Morsy, M.A. (2008). Hepatoprotective effect of nicorandil
against carbon tetrachloride-induced hepatotoxicity in rats. El-Minia Med. Bull. 19, 312-324.
Teshima, Y., Akao, M., Baumgartner, W.A. & Marbán, E. (2003). Nicorandil prevents oxida-
tive stress-induced apoptosis in neurons by activating mitochondrial ATP-sensitive potassium
channels. Brain Res. 990, 45-50.
Thapa, B.R. & Walia, A. (2007). Liver function tests and their interpretation. Indian J Pedi-
atr. 74, 663-671.
Thippeswamy, A.H., Shirodkar, A. & Koti, B.C. (2011). Protective role of Phyllantus niruri
extract in doxorubicin-induced myocardial toxicity in rats. Indian J. Pharmacol. 43, 31-35.
Wang, N., Li, P., Wang, Y., Peng, W., Wu, Z. & Tan, S. (2008). Hepatoprotective effect of
Hypericum japonicum extract and its fractions. J. Ethnopharmacol. 116, 1-6.
Wu, H., Ye, M., Yang, J., Ding, J., Yang, J., Dong, W. & Wang, X. (2015). Nicorandil protects
the heart from ischemia/reperfusion injury by attenuating endoplasmic reticulum response-in-
duced apoptosis through PI3K/Akt signaling pathway. Cell Physiol. Biochem. 35, 2320-2332.