ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 47
OBJECTIVE: To show the protective effects of taxifolin
on cisplatin-induced oxidative renal injury.
STUDY DESIGN: Rats were divided into 3 groups: the
healthy group, the cisplatin group (2.5 mg/kg cisplatin
only), and the taxifolin-cisplatin group (50 mg/kg taxi-
folin+2.5 mg/kg cisplatin). The rats were sacrificed after
14 days, and the kidneys were removed for examination
of malondialdehyde (MDA), total glutathione (tGSH),
and super oxide dismutase (SOD) levels. Blood urea
nitrogen (BUN) and creatinine levels were measured in
the blood samples taken before the animals were sacri-
ficed. Kidney tissues were examined histopathologically.
Finally, biochemical and histopathological results of the
groups were compared.
RESULTS: Cisplatin significantly increased MDA and
decreased tGSH and SOD in the rat kidney tissue
(p<0.001). Additionally, cisplatin caused an increase in
serum creatinine and BUN levels (p<0.001). Taxifolin
prevented the cisplatin-induced increase in MDA and
decrease in tGSH and SOD (p<0.001). It also decreased
serum creatinine and BUN levels significantly as com-
pared with the cisplatin group (p<0.001). In the cis-
platin group microscopic examinations showed obvious
kidney damage including dilation, severe necrosis, and
degenerative changes. Taxifolin prior to cisplatin mark-
edly ameliorated these changes.
CONCLUSION: We have demonstrated for the first
time that taxifolin can prevent cisplatin-induced neph-
rotoxicity. (Anal Quant Cytopathol Histpathol 2019;
41:47–54)
Keywords: antioxidants, cisplatin, flavonoids, kid­
ney, nephrotoxicity, oxidative stress, taxifolin.
Cisplatin is an alkylating agent first approved by
the end of the 1970s for use in ovarian cancer and
then used widely all around the world especial­
ly for solid tumors such as head, neck, testicular,
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4102-0047/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Protective Effect of Taxifolin on Cisplatin-
Induced Nephrotoxicity in Rats
Ali Veysel Kara, M.D., Mehmet Naci Aldemir, M.D., Fatih Ozcicek, M.D.,
Renad Mammadov, M.D., Gulce Naz Yazıcı, M.D., Mukadder Sunar, M.D., and
Mine Gulaboglu, Ph.D.
From the Departments of Nephrology, Medical Oncology, Internal Medicine, Pharmacology, Histology and Embryology, and Anatomy,
Faculty of Medicine, Erzincan University, Erzincan, Turkey; and the Department of Biochemistry, Faculty of Pharmacy, Ataturk Univer­
sity, Erzurum, Turkey.
Dr. Kara is Nephrologist, Department of Nephrology, Faculty of Medicine, Erzincan University.
Dr. Aldemir is Oncologist, Department of Medical Oncology, Faculty of Medicine, Erzincan University.
Dr. Ozcicek is Associate Professor, Department of Internal Medicine, Faculty of Medicine, Erzincan University.
Dr. Mammadov is Pharmacologist, Department of Pharmacology, Faculty of Medicine, Erzincan University.
Dr. Yazıcı is Histologist, Department of Histology and Embryology, Faculty of Medicine, Erzincan University.
Dr. Sunar is Anatomist, Department of Anatomy, Faculty of Medicine, Erzincan University.
Dr. Gulaboglu is Professor, Department of Biochemistry, Faculty of Pharmacy, Ataturk University.
Address correspondence to: Ali Veysel Kara, M.D., Department of Nephrology, Faculty of Medicine, Erzincan University, 24030 Erzin­
can, Turkey (aliveyselkara@hotmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. ovarian, lung, and bladder cancer. However, the
use of cisplatin as a chemotherapeutic agent is
frequently restricted by numerous side effects
such as neurotoxicity, ototoxicity, and particular­
ly nephrotoxicity.1 There are a number of clinical
presentations which can be seen due to cispla­
tin nephrotoxicity, such as acute kidney injury,
Fanconi-like syndrome, distal renal tubular acido­
sis, hypomagnesemia, renal concentration defects,
etc. But acute kidney injury is the most serious
and relatively common side effect, which can be
seen in nearly 30% of patients after a single dose.2
Although the complete understanding of the mech­
anisms involved in cisplatin nephrotoxicity is not
clear, it is thought that cisplatin-induced nephro­
toxicity is a complex process involving oxidative
stress, apoptosis, mitochondrial dysfunction, DNA
damage, and inflammation.3 In view of this, many
agents have been used to prevent nephrotoxicity,
interacting with at least one of these underly­
ing mechanisms, and especially oxidative stress.
Antioxidants such as Vitamin C, vitamin E, sele­
nium, alpha lipoic acid, super oxide dismutase,
glutathione, and plant-based agents have been
evaluated.4 Several beneficial effects were noted in
in vitro and in vivo mice studies.5 Flavonoids are
one of the most commonly studied plant-based
agents because they are widely distributed and
are thought to have health-promoting properties
due to their high antioxidant capacity.6 Dihydro­
flavonols, a group of flavonoids, display differ­
ent biological activities including reactive oxygen
species (ROS) scavenging and metal-binding activ­
ities. Taxifolin (3,5,7,3’,4’-pentahydroxy-flavanone
or 2,3-dihydroquercetin) is one of the most com­
mon dihydroflavonols and a minor component in
complex preparations such as silymarin, pycnoge­
nol, and venoruton.7 In nature it is found in French
maritime bark, Douglas fir bark, and Siberian larch
wood and also citrus fruits, grapes, olive oil, and
onions.8 It can be easily extracted and used. Sev-
eral studies show its role in the inhibition of free
radical formation at key stages of apoptosis and
to correct cerebral ischemia-reperfusion injury.5,9
Taxifolin was also found to exhibit anticancer and
neuroprotective effects.10-12 In a recent study by
Zhou and colleagues, the high antioxidant cap-
acity of taxifolin was demonstrated.13
These indicate that taxifolin has potential as an
antioxidant agent. However, no study has been re-
ported on the protective effects of taxifolin against
cisplatin-induced oxidative renal injury. In this
study we aimed to show the protective effects of
taxifolin on cisplatin-induced oxidative renal injury.
Materials and Methods
Animals
All the animals used in this study were obtained
from the Ataturk University Medical Experiment-
al Application and Research Center. A total 30 al-
bino Wistar male rats weighing between 280 and
295 g were randomly selected. Before the experi­
ment the rats were divided into 3 groups with 10
rats in each group, and the animals were housed
and fed in the pharmacology laboratory at room
temperature (22°C). Animal experiments were per­
formed in accordance with the National Guide-
lines for the Use and Care of Laboratory Animals
and were approved by the local animal ethics
committee of Ataturk University, Erzurum, Tur­
key (Ethics Committee Number 2018/09, dated
09.08.2018).
Chemical Agents Used in the Experiment
Cisplatin vials (50 mg/100 mL; Cisplatin-Ebewe)
were provided by Liba-Turkey, thiopental sodium
(Pental sodium) was provided by I
·
.E. Ulagay Tur­
key, and Taxifolin was provided by Evalar-Russia.
Experimental Groups
Experimental animals were divided into the fol­
lowing 3 groups: healthy group, cisplatin group
(given 2.5 mg/kg cisplatin), and taxifolin+cisplat­
in group (given 50 mg/kg taxifolin+2.5 mg/kg
cisplatin).
Experimental Procedure
Taxifolin, 50 mg/kg, was administered to the taxi­
folin+cisplatin group (n=10) via oral gavage. Dis­
tilled water was given to the healthy (n=10) and
cisplatin (n=10) groups as the solvent via oral
gavage. One hour after the administration of tax­
ifolin and distilled water, 2.5 mg/kg cisplatin
was injected intraperitoneally to the rats in the
taxifolin+cisplatin and cisplatin groups. Taxifolin,
cisplatin, and distilled water were administered
at the indicated doses and volumes mentioned
above once daily for 14 days. At the end of that
period all rats were sacrificed by high dose anes­
thesia (50 mg/kg thiopental) and the kidneys were
removed. Malondialdehyde (MDA), total gluta­
thione (tGSH), and super oxide dismutase (SOD)
levels were measured in the removed kidney tis­
sues. Additionally, blood urea nitrogen (BUN) and
48 Analytical and Quantitative Cytopathology and Histopathology®
Kara et al

3. creatinine levels were measured in the blood sam­
ples taken before the animals were sacrificed. Kid­
ney tissues were examined histopathologically.
Finally, biochemical and histopathological results
of the groups were compared.
Biochemical Analysis
Preparing the Samples. 25 mg of the tissue was ho-
mogenized using a solution of 1.15% KCl (Merck,
Germany). The homogenate was centrifuged at
4,000 rpm for 30 minutes at +4°C. Supernatants
were then used for nitric oxide (NO) and MDA
measurements. Tissues (25 mg) taken for tGSH
analysis were washed with isotonic sodium
chloride (I
·
.E. Ulagay, Turkey) and subsequently
brought to 2 mL total volume with phosphate
buffer solution (0.213 g NaH2PO4.2H2O [Merck,
Germany]+1.563 g Na2HPO4.2H2O [Merck]+0.038
g EDTA (Sigma-Aldrich, Germany)+100 mL dH2O,
pH=7.4), and then were homogenized in an icy
environment. After that, the tissues were centri­
fuged at 1,000 rpm for 15 minutes at a tempera-
ture of +4°C. The supernatant was used as the
sample for analysis. The protein concentration of
the supernatant was measured using the method
described by Bradford.14
MDA Analysis
MDA measurements were based on the meth­
od used by Ohkawa et al, involving spectropho-
tometric measurement of absorbance of the pink-
colored complex formed by thiobarbituric acid
and MDA.15 The serum/tissue homogenate sam-
ple (0.1 mL) was added to a solution containing
0.2 mL of 80 g/L sodium dodecyl sulfate, 1.5
mL of 200 g/L acetic acid, 1.5 mL of 8 g/L 2-
thiobarbiturate, and 0.3 mL distilled water. The
mixture was incubated at 95°C for 1 hour. Upon
cooling, 5 mL of n-butanol:pyridine (15:1) was
added. The mixture was vortexed for 1 minute
and centrifuged for 30 minutes at 4,000 rpm. The
absorbance of the supernatant was measured at
532 nm. The standard curve was obtained by using
1,1,3,3-tetramethoxypropane.15
tGSH Analysis
Analysis was made according to the method
described by Sedlak and Lindsay. DTNB (5, 5’-
dithiobis [2-nitrobenzoic acid]) disulfide is chro­
mogenic in the medium, and DTNB is reduced
easily by sulfhydryl groups. The yellow color
produced during the reduction is measured by
spectrophotometer at 412 nm. For measurement, a
cocktail solution (5.85 mL 100 mM Na-phosphate
buffer, 2.8 mL 1 mM DTNB, 3.75 mL 1 mM
NADPH, and 80 µL 625 U/L glutathione reduc­
tase was prepared. Before measurement, 0.1 mL
meta-phosphoric acid was added to 0.1 mL se-
rum/tissue homogenate and centrifuged for 2
minutes at 2,000 rpm for deproteinization. The
0.15 mL cocktail solution was added to 50 µL of
supernatant. The standard curve was obtained by
using glutathione disulfide.16
SOD Analysis
Measurements were performed according to the
method of Sun et al.17 When xanthine is con-
verted into uric acid by xanthine oxidase, SOD
forms. If nitro blue tetrazolium (NBT) is added to
this reaction, SOD reacts with NBT and a purple-
colored formazan dye occurs. The sample was
weighed and homogenized in 2 mL of 20 mmol/L
phosphate buffer containing 10 mmol/L EDTA at
pH 7.8. The sample was centrifuged at 6,000 rpm
for 10 minutes and then the brilliant supernatant
was used as assay sample. The measurement mix­
ture containing 2,450 μL measurement mixture
(0.3 mmol/L xanthine, 0.6 mmol/L EDTA, 150
μmol/L NBT, 0.4 mol/L Na2CO3, and 1 g/L bo-
vine serum albumin), 500 μL supernatant, and
50 μL xanthine oxidase (167 U/L) was vortexed.
Then it was incubated for 10 min. At the end of
the reaction, formazan occurred. The absorbance
of the purple-colored formazan was measured at
560 nm. As more of the enzyme exists, the least
O2− radical that reacts with NBT occurs.
Serum Creatinine Measurement
Quantitative determination of serum creatinine
levels was determined by spectrophotometry in
a Roche Cobas 8000 auto analyzer. This kinetic
colorimetric test is based on the Jaffe procedure.
Creatinine reaction with picrate in alkaline solu-
tion forms a yellow-orange complex. This com-
plex was measured at a wavelength of 505 nm.
The rate of dye formation is proportional to the
concentration of creatinine in the sample. In the
test, “rate-blanking” was used to minimize biliru­
bin interference. Serum or plasma were corrected
with −26 μmol/L (−0.3 mg/dL) to correct non­
specific reaction caused by serum/plasma pseudo-
creatinine chromogens, including proteins and
ketones. Creatinine+picric acid→(Alkaline pH)
yellow-orange complex.
Volume 41, Number 2/April 2019 49
Effect of Taxifolin on Cisplatin Nephrotoxicity

4. Serum BUN Measurement
Quantitative determination of serum urea level
was performed by spectrophotometry in a Roche
Cobas 8000 auto analyser. BUN was calculated
with the equation of BUN=urea*0.48. Kinetic
test was made with urease and glutamate dehy­
drogenase. Urea is hydrolyzed by urease, and
ammonium and carbonate are formed (urea+
2 H2O→[urease] 2 NH4
+). In the second reaction,
L-glutamate is produced by the reaction of 2-
oxoglutarate and ammonium in the presence of
glutamate dehydrogenase (GLDH) and NADH
(NH4
++2-oxoglutarate+NADH→[GLDH] L-
glutamate+NAD++H2O). The rate of decrease in
the NADH concentration is directly proportional
to the urea concentration in the specimen and is
measured photometrically (340 nm wavelength).
Histopathological Examination
All of the tissue samples were first identified in a
10% formaldehyde solution for light microscope
assessment. Following the identification process,
tissue samples were washed under tap water in
cassettes for 24 hours. Samples were then treated
with conventional grade alcohol (70%, 80%, 90%,
and 100%) to remove the water within tissues. Tis­
sues were then passed through xylol and embed­
ded in paraffin. Four-to-five micron sections were
cut from the paraffin blocks, and hematoxylin-
eosin staining was administered. Their photos
were taken following the Olympus DP2-SAL
firmware program assessment. Histopathological
assessment was carried out by the pathologist
blinded to the study groups.
Statistical Analysis
Statistical analyses were carried out using the
Statistical Package for the Social Sciences, Win-
dows version 18.0 (SPSS, Chicago, Illinois, USA).
Descriptive statistics for each variable were deter­
mined. Normality of the data distribution was
assessed with the Kolmogorov-Smirnov test. Re­
sults for continuous variables were demonstrated
as mean±standard deviation of the mean (mean±
SD). Significance of differences between the
groups was determined using one-way ANOVA
test followed by Tukey’s post hoc test. A p value
<0.05 was considered significant.
Results
Biochemical Results
Mean and median levels of parameters are shown
in Table I. Serum BUN and creatinine levels were
significantly higher in the cisplatin group as com­
pared with the healthy and taxifolin+cisplatin
groups (p<0.001) (Figure 1). There were no signif­
icant differences between the healthy and taxifo­
lin+cisplatin groups in terms of BUN and creat­
inine (p=0.158 and p=0.811, respectively). MDA
level was significantly higher in the cisplatin group
as compared with the healthy and taxifolin+cis­
platin groups (p<0.001). SOD and tGSH levels
were significantly lower in the cisplatin group
as compared with the healthy and taxifolin+cis­
platin groups (p<0.001). There were no statisti­
cally significant differences between the healthy
and taxifolin+cisplatin groups in terms of MDA,
SOD, and tGSH (p=0.057, p=0.067, and p=0.060,
respectively) (Figure 2).
Histopathological Results
According to the conducted microscopic evalua­
tions of kidney tissue, the glomerular structure
and morphology, proximal and distal tubules were
50 Analytical and Quantitative Cytopathology and Histopathology®
Kara et al
Table I Biochemical Results of the Study
Healthy group (n=10) Cisplatin group (n=10) Taxifolin+cisplatin group (n=10)
Mean±SD Median Mean±SD Median Mean±SD Median
(Min-Max) (Min-Max) (Min-Max)
MDA (nmol/g protein) 3.07±0.37 3.0 (2.5–3.7) 9.13±0.49a 9.05 (8.5–9.9) 3.51±0.35b
3.6 (3.0–4.0)
tGSH (µmol/g protein) 8.16±0.61 8.15 (7.1–9.1) 2.72±0.52a 2.75 (2.0–3.4) 7.57±0.51b
7.4 (6.9–8.5)
SOD (U/mg protein) 15.9±2.8 16.5 (12–20) 6.3±0.83a 6.1 (5.1–7.9) 13.7±2.21b 14 (10–17)
BUN (mg/dL) 48±7 49 (39–56)
246±11a 243 (234–265) 58±7b 59 (48–66)
Creatinine (mg/dL) 0.85±0.08 0.83 (0.77–1.0) 2.78±0.26a 2.8 (2.4–3.1) 0.91±0.05b 0.90 (0.85–0.98)
Data are the mean±SD of 10 rats in each group.
ap<0.01 compared to healthy group.
bp<0.01 cisplatin group.
BUN = blood urea nitrogen, MDA = malondialdehyde, SOD = super oxide dismutase, tGSH = total glutathione.

5. normal in the control group (Figure 3A). In the cis­
platin group, microscopic examinations showed
obvious kidney damage including dilation, severe
necrosis, and degenerative changes in epithelium
of the tubule lumens and congestion in blood
vessels. Glomeruli of kidney tissue were dilated,
following proximal tubules showed vacuolation;
distal tubules were mostly necrotic, and the cells
present in their lumen were seen (Figure 3B). Con­
gested blood vessels were outstanding across the
tissue (Figure 3C). In rats treated with taxifolin
prior to cisplatin, marked amelioration of both
glomeruli and tubules of renal tissue was ob-
served and congestion was decreased in blood ves­
sels (Figure 3D).
Discussion
Cisplatin as a potent chemotherapeutic drug wide­
ly used for the treatment of many solid tumors.
But its potent nephrotoxicity, which occurs in a
significant percentage of patients, is the main lim­
iting factor for clinical usage and has been a mat-
ter of concern since nearly its introduction.18 There
are several pathogenesis pathways suspected for
cisplatin nephrotoxicity. These are oxidative and
nitrosative stress, activation of glutathione S-
transferase, apoptosis, renal vasoconstriction, in­
flammation, inhibition of protein synthesis, and
renal histomorphological damage.19 Oxidative
stress is the most frequent and typically accused
among these pathways. Cisplatin causes ROS
production by the disrupted respiratory chain
and results in mitochondrial dysfunction. ROS in
renal cells also reduces the antioxidant enzyme
activity and intracellular concentrations of anti-
oxidants by reacting with thiol-containing anti­
oxidant molecules such as glutathione.20 So, the
majority of studies for prevention of cisplatin
nephrotoxicity have been focused on antioxidant
therapy.
Particularly, plant-based antioxidants are given
more consideration because they may detoxify
ROS without affecting antineoplastic efficacy of
cisplatin.21 Flavonoids make up one of the main
groups of plant phenolic antioxidants and have
high chelating properties.22 They are widely found
in leaves and flowers of plants and also abun­
dantly found in foods and beverages made from
plants.23 Taxifolin (3,5,7,3,4-pentahydroxy flava­
none or dihydroquercetin) is a flavanolol which is
a subclass of flavonoids and is abundantly found
in citrus fruits, onion, olive oil, etc.24
Topal and colleagues showed that taxifolin had
a marked antioxidant, radical scavenging, and
chelating activity and concluded that it could be
used in the food and pharmaceutical industry
for delaying formation of toxic oxidation prod­
ucts.25 Also, Shubina et al showed the antioxidant
Volume 41, Number 2/April 2019 51
Effect of Taxifolin on Cisplatin Nephrotoxicity
Figure 1 (A) The effects of taxifolin on creatinine levels in rats given cisplatin. Bars are mean±SD. The taxifolin+cisplatin and healthy
groups are compared with the cisplatin group. *p<0.001. (B) The effects of taxifolin on BUN levels in rats given cisplatin. Bars are
mean±SD. The taxifolin+cisplatin and healthy groups are compared with the cisplatin group.
_____
**p<0.001.
BUN = blood urea nitrogen, CG = cisplatin group, HG = healthy group, TCG = taxifolin+cisplatin group.

6. and iron-chelating properties of taxifolin.26 The
strong radical scavenger effect of taxifolin was
also shown by Li et al.27 Recent studies also
showed that taxifolin has anticancer properties10,28
and antibacterial action.29 In a recent study by
Zhao et al it was concluded that taxifolin may
mitigate the effects of streptozotocin-induced dia­
betes in rats.30 Rehman et al showed that taxifolin
can prevent postprandial hyperglycemia due to its
antioxidant and anti-inflammatory properties.31
Although there are several studies which have
shown that flavonoids can prevent cisplatin neph­
rotoxicity, there is no study with taxifolin as a
sole agent. In our study, the results of biochemical
tests showed that cisplatin causes oxidative stress
in rat kidneys. MDA was increased as an oxidant
agent, and tGSH and SOD were decreased as an­
tioxidant agents. As a result of kidney damage,
serum BUN and creatinine levels were also in-
creased. Obvious kidney damage including dila­
tion, severe necrosis, and degenerative changes in
the epithelium of the tubule lumens and conges-
tion in blood vessels were also shown by micro­
scopic examination after cisplatin therapy. Treat­
ment with taxifolin prior to cisplatin reversed
oxidative damage in kidneys and prevented acute
kidney injury and histopathological damage due
to cisplatin.
52 Analytical and Quantitative Cytopathology and Histopathology®
Kara et al
Figure 2 (A) The effects of taxifolin on MDA levels in rats given cisplatin. Bars are mean±SD. The taxifolin+cisplatin and healthy groups
are compared with the cisplatin group. (B) The effects of taxifolin on tGSH levels in rats given cisplatin. Bars are mean+SD. The
taxifolin+cisplatin and healthy groups are compared with the cisplatin group. (C) The effects of taxifolin on SOD levels in rats given
cisplatin. Bars are mean±SD. The taxifolin+cisplatin and healthy groups are compared with the cisplatin group.
_____
*p<0.001.
CG = cisplatin group, HG = healthy group, MDA = malondialdehyde, SOD = super oxide dismutase, TCG = taxifolin+cisplatin group, tGSH = total
glutathione.

7. In conclusion, we have demonstrated for the first
time that taxifolin can prevent cisplatin-induced
nephrotoxicity due to its high antioxidant cap-
acity. We believe that our study will inspire more
comprehensive clinical studies.
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Volume 41, Number 2/April 2019 53
Effect of Taxifolin on Cisplatin Nephrotoxicity
Figure 3
(A) Hematoxylin-eosin staining
in kidney tissue in the control
group. = glomerulus,
P = proximal tubule, D = distal
tubule, ×200.
(B) Hematoxylin-eosin staining
in kidney tissue in the
cisplatin group. =
glomerulus, P = vacuolation in
proximal tubule, D = necrotic
distal tubule, « = dilation in
glomerulus, Ø = congestion in
blood vessels, ×200.
(C) Hematoxylin-eosin staining
in kidney tissue in the
cisplatin group. =
glomerulus, P = vacuolation in
proximal tubule, D = necrotic
distal tubule, Ø = congestion
in blood vessels, ×200.
(D) Hematoxylin-eosin staining
in kidney tissue in the
taxifolin+cisplatin treated
group. = glomerulus,
P = proximal tubule, D = distal
tubule, ×200.

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