Biomedicine & Pharmacotherapy 100 (2018) 575–582

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

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

The protective effect of astaxanthin against cisplatin-induced nephrotoxicity T

in rats
⁎
Gorkem Akcaa, Huseyin Erena, , Levent Tumkayab, Tolga Mercantepeb, Mustafa Ozan Horsanalic,
Ezgi Devecid, Eyup Dila, Adnan Yilmaze
a
Recep Tayyip Erdogan University School of Medicine, Urology Department, Rize, Turkey
b
Recep Tayyip Erdogan University School of Medicine, Histology and Embryology Department, Rize, Turkey
c
Recep Tayyip Erdogan University Training and Research Hospital, Urology Department, Rize, Turkey
d
Recep Tayyip Erdogan University School of Medicine, Rize, Turkey
e
Recep Tayyip Erdogan University School of Medicine, Biochemistry Department, Rize, Turkey

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

Keywords: Purpose: The aim of this experimental study was to investigate the antioxidant effects of astaxanthin against
Astaxanthin cisplatin-induced nephrotoxicity in rats.
Antioxidant Methods: Forty-eight male Sprague-Dawley rats weighing 264.83 ± 7.39 g were randomly divided into six
Cisplatin groups of eight animals each. These were constituted as control, olive oil control, astaxanthin control, cisplatin
Nephrotoxicity
control, 16 mg/kg cisplatin & 25 mg/kg astaxanthin and 16 mg/kg cisplatin & 75 mg/kg astaxanthin groups.
Oxidant
Biochemical evaluation was performed by measuring blood urea nitrogen, serum creatinine, total oxidant status
and total antioxidant status. Renal corpuscle, proximal and distal tubules areas (μm2) were calculated for his-
topathological evaluation, and Caspase-3 staining was performed for immunohistochemical evaluation.
Results: Cisplatin reduced total antioxidant status levels and increased blood urea nitrogen, serum creatinine,
total oxidant status, and Caspase-3 levels. It also caused dilatation, vacuolization, and loss of tubular epithelial
cells in the proximal and distal tubules, and glomerular degeneration and edema were determined in kidney
tissue (p < 0.05). Administration of 25 mg and 75 mg astaxanthin increased total antioxidant status levels,
reduced blood urea nitrogen, serum creatinine, total oxidant status, and Caspase-3, and ameliorated degen-
erative distal and proximal tubules, glomerular degeneration and edema in kidney tissue (p < 0.05).
Conclusions: The nephrotoxic effect of cisplatin was diminished by the antioxidant effect of astaxanthin.

1. Introduction
Antineoplastic chemotherapeutic agents have been used for many
years, but because these agents disrupt the cell cycle, both normal and
neoplastic cells are affected by them [1]. Cisplatin is a platinum-based
drug and a useful chemotherapeutic agent in various malignancies,
including solid malignant tumours of the head, neck, bladder, testis,
ovary, prostate, cervix, oesophagus and lung [2]. Cisplatin has several
side effects, such as nausea and vomiting, nephrotoxicity, neurotoxicity,
ototoxicity and, rarely, ocular toxicity [3]. Nephrotoxicity is the most
frequent and dose-limiting side effect of cisplatin therapy, and irre-
versible kidney damage occurs in one-third of patients despite protec-
tive measures [1,2]. Nephrotoxic effects occur in several ways, in-
cluding via oxidative stress, inflammation, fibrogenesis, mitochondrial
damage, apoptosis and necrosis [4,5]. Cisplatin accumulates most in the
S3 segment of the proximal tubules, followed by the distal collecting
tubules and the S1 segment of the proximal tubules [6].
Exposure to oxidant radicals is increasing steadily in industrialised
societies. Oxidative stress plays an active role in acute kidney injury
induced by cisplatin administration. Cisplatin specifically increases the
production of hydroxyl radicals, potent free radicals that consume in-
tracellular antioxidant stores and affect directly cell components, such
as lipids, proteins and DNA, thus impairing the cellular structure [7,8].
Reactive oxygen species (ROS) involved in cisplatin nephrotoxicity in-
clude superoxide anion, hydrogen peroxide, hydroxyl radical and re-
active nitrogen species, such as peroxynitrite and nitric oxide [9–11].
Cisplatin also inhibits antioxidant enzymes; hence, the renal activities
of superoxide dismutase (SOD), glutathione peroxidase (GPx),

⁎
Corresponding author.
E-mail addresses: gakca7@hotmail.com (G. Akca), huseyin.eren@erdogan.edu.tr (H. Eren), levent.tumkaya@erdogan.edu.tr (L. Tumkaya),
tolga.mercantepe@erdogan.edu.tr (T. Mercantepe), drozanhorsanali@yahoo.com (M.O. Horsanali), ezgidevec@gmail.com (E. Deveci), eyup.dil@erdogan.edu.tr (E. Dil),
adnan.yilmaz@erdogan.edu.tr (A. Yilmaz).

https://doi.org/10.1016/j.biopha.2018.02.042
Received 1 January 2018; Received in revised form 11 February 2018; Accepted 13 February 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

glutathione reductase, total antioxidant status (TAS) and catalase de-

crease markedly [12,13]. Malondialdehyde (MDA) and total oxidant
status (TOS) levels also increase as a result of damage to antioxidant
systems [13–15]. TAS is an effective assay in both human and animal
models for the measurement of serum antioxidative stress [16,17]. Its
use as an antioxidant in the literature is increasing steadily [18]. TAS
and TOS are also easy-to-perform, stable, reliable, sensitive and in-
expensive methods for measuring total antioxidant capacity [19].
Carotenoids, an antioxidant group, include over 700 organic, so-
luble substances produced by phytoplankton, algae, plants and limited
numbers of fungi and bacteria [20]. Astaxanthin is a highly antioxidant,
anti-inflammatory, carotenoid pigment, and it is considered the stron-
gest and safest known antioxidant in nature. It is obtained from the
microalgae Haematococcus pluvialis [21]. Astaxanthin exhibits anti- Fig. 1. Schematic experimental timeline.

oxidant activities through two pathways: it scavenges free radicals and

protects against chain reactions that may be caused by free radicals or it
terminates chain reactions that may already have taken place [22]. Due
to its beneficial effects on human health, astaxanthin is widely used as a
nutritional supplement and antioxidant [23,24]. The U.S. Food and
Drug Administration (FDA) approves the use of astaxanthin as a human
dietary supplement.
This study employed biochemical, histopathological and im-
munohistochemical methods to investigate the antioxidant effects of
astaxanthin against cisplatin nephrotoxicity under experimental con-
ditions.
2. Materials and methods
Forty-eight male Sprague Dawley rats aged 3–5 months and
weighing 264.83 ± 7.39 g were procured from the Recep Tayyip
Erdogan University Animal Care and Research Unit (Rize, Turkey). All
animals received care according to the criteria outlined in the ‘Guide for
the Care and Use of Laboratory Animals’ prepared by the National
Academy of Sciences and published by the National Institutes of Health.
Approval for the study was granted by the Recep Tayyip Erdogan
University (Rize, Turkey) Animal Ethical Committee (No. 2016/32).
2.1. Study design
was the 16 mg/kg cisplatin and 75 mg/kg astaxanthin group (n = 8),
having received 75 mg/kg astaxanthin intraperitoneally daily for eight
days and a single 16 mg/kg dose of cisplatin intraperitoneally on the
fifth day (Fig. 1).
2.2. Biochemical analysis procedure
The kidneys were removed from the abdominal cavities. Phosphate
buffered saline PBS), a water-based salt solution containing sodium
phosphate dibasic, sodium chloride and sodium phosphate monobasic,
was then prepared for kidney tissues (pH: 7.4). The specimens were
washed in ice-cold PBS and weighed. The ratio of tissue weight to
homogenisation buffer was 1:10. Specimens were homogenised in PBS
for one min. The homogenates were centrifuged at 2717 g for 20 min.
TAS and TOS levels were measured using a commercial kit (RL0024,
Rel Assay Diagnostics, Turkey). The TAS results were expressed as
Trolox Equivalent/L and the TOS results as μmol H2O2 Equivalent/L.
Blood samples were collected from all rats for biochemical evalua-
tion. Blood was separated by centrifugation at 1739 g for 15 min at 4 °C.
Levels of blood urea nitrogen (BUN) and serum creatinine were de-
termined using a standard AutoAnalyzer (Architect c16000
AutoAnalyzer, Abbott Diagnostics, Waltham, Massachusetts, USA). The
results were expressed as mg/dL [28–30].

Animals were kept in Recep Tayyip Erdogan University Animal Care

and Research Unit (Rize, Turkey). All animals were maintained and fed
in a sterile experimental animal unit environment having 55–60% hu-
midity, at a temperature of 22 ± 3 °C, and 12-h light:12-h light-dark
cycle. Rats were allowed ad libitum access to commercially available
standard rat chow (Bayramoğlu Feed and Flour Industry Trading
Corporation Erzurum, Turkey) and tap water throughout the experi-
ment. After sufficient time had been allowed for adaptation to the la-
boratory conditions, the experimental animals were divided randomly
into six groups.
Anesthesia was administered to all groups with 50 mg/kg in-
traperitoneal ketamine hydrochloride (Ketalar ®, Eczacibası Parke-
Davis, Istanbul, Turkey) and 10 mg/kg intraperitoneal Xylazine HCl
(Alfazyne ®, Alfasan International BV Woerden, Holland).
All groups received oral distilled water daily for eight days. Group 1,
the control group (n = 8), received no drug injections except for an-
aesthetics. Group 2 acted as the olive oil control group (n = 8). Olive oil
was used for dissolving astaxanthin in Groups 3–6. Group 2 received
intra-peritoneal olive oil only for eight days [25]. Group 3, the astax-
anthin control group (n = 8), received only intra-peritoneal astaxanthin
75 mg/kg dissolved in olive oil [26]. Group 4 acted as the cisplatin only
group (n = 8) and received a single 16 mg/kg dose of cisplatin (Cis-
platin DBL 100 mg/100 ml vial, Orna Ilac, Istanbul) intraperitoneally
on the fifth day of the study [27]. Group 5 represented the 16 mg/kg
cisplatin and 25 mg/kg astaxanthin group (n = 8), having received
25 mg/kg astaxanthin intraperitoneally daily for eight days and a single
16 mg/kg dose of cisplatin intraperitoneally on the fifth day. Group 6
2.3. Histopathological analysis procedure
Kidneys were fixed in 10% neutral formaldehyde. Specimens were
then dehydrated in an ascending alcohol series. Next, all sections were
cleared in xylene and embedded in paraffin using routine laboratory
methods. They were stained with hematoxylin and eosin (H&E) and
analysed by two histologists blinded to the study groups under a light
microscope (Leica DM6200-Germany). Photographs were taken with an
Olympus DP20 camera.
2.4. Quantitative analysis
The renal corpuscle, proximal and distal tubule areas (μm2) were
measured using the Olympus DP2-BSW (Ver.2.1 to Ver.2.2, Build 6212,
Olympus Corporation, Tokyo, Japan) software system. This consists of a
camera (Olympus DP20, Olympus Corporation, Tokyo, Japan) attached
to a light microscope (Leica DM6200, Germany) and a computer with a
software system. H&E (Darmstadt, Germany)-stained sections were
placed onto the microscope tray, and their sectional boundaries were
determined using this programme (40x objective lens). Once the area
was identified, distinct and separate frames were assessed by two
blinded histopathologists (Fig. 2).
2.5. Immunohistochemistry (IHC) analysis procedure
Sections were deparaffinised and treated with proteinase K solution

576
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

Table 1
Kidney tissue biochemical results.

Group Kidney

TAS TOS

Control 8.69 ± 0.19** 26.07 ± 0.71**

Olive oil 9.16 ± 0.15** 24.81 ± 0.4**
Astaxanthin 75 mg 12.60 ± 0.7** 25.06 ± 0.50**
Cisplatin 6.92 ± 0.75* 34.13 ± 0.88*
Cisplatin + Astaxanthin 25 mg 10.47 ± 0.82** 23.02 ± 0.92**
Cisplatin + Astaxanthin 75 mg 11.13 ± 0.34** 22.20 ± 0.78**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

3. Results

3.1. Biochemical results

2
Fig. 2. The renal corpuscle, proximal and distal tubule areas (μm ) were measured using
the Olympus DP2-BSW (Ver.2.1 to Ver.2.2, Build 6212, Olympus Corporation, Tokyo,
Japan) software system.
(20 μg/mL in PBS, Abcam, UK), washed with distilled water and placed
in 3% hydrogen peroxide. After several washes with PBS (10x, pH 6.0,
ab128983, Abcam, UK), the sections were placed into an equilibration
buffer. Sections were incubated with Caspase-3 (1:200, Abcam Rabbit
polyclonal to active Caspase-3, Abcam, UK). After washing with PBS, all
sections were incubated in a secondary antibody (Rabbit Specific HRP/
DAB [ABC] Detection IHC kit, ab64218, Abcam, UK) and subsequently
incubated in anti-digoxigenin-peroxidase. The reaction was revealed
with 0.06% 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical,
St. Louis, MO) in PBS. Finally, all sections were counterstained with
Harris hematoxylin.
TAS and TOS were used as markers for demonstrating the anti-
oxidant effect of astaxanthin and the oxidative effect of cisplatin, re-
spectively. Following cisplatin administration, we observed increased
TOS levels and decreased TAS levels (p < 0.05). Astaxanthin 75 mg
alone did not affect TOS levels, but it did increase TAS levels
(p < 0.05). While a single dose of cisplatin lowered TAS and aug-
mented TOS concentrations, a combination of cisplatin and astaxanthin
increased TAS levels and reduced TOS levels (p < 0.05) (Table 1)
(Fig. 3).
Cisplatin treatment increased BUN and serum creatinine levels
(p < 0.05) (Table 2) (Fig. 3), and the astaxanthin-treated groups ex-
hibited decreased BUN and serum creatinine levels (p < 0.05)
(Table 2) (Fig. 3).
3.2. Histopathological results

2.6. Stereological analysis 3.2.1. Light microscopy results

Mean numerical Caspase-3-positive cell densities were calculated
using the fractional method, a stereological technique involving an
unbiased counting frame. The Stereo Investigator (MicroBrightField
9.0, Colchester, VT, CA, USA) software system was used. This system
consists of a camera attached to a light microscope, a motorised system
equipped with a microscope tray and a computer with a software
system. IHC-Caspase-3-stained sections were placed on the microscope
tray, and their sectional boundaries were determined using this pro-
gramme. After determining the area, separate and distinct frames were
identified by systematic random sampling of the sections, in line with
the rules of space fragmentation with the step interval of the x- and y-
axes. Measurements in 20 different selected areas in all groups were
taken following the method described by Mercantepe [31].
2.7. Statistical analysis
Data were analysed using the SPSS Statistics 20.0 (IBM Inc.,
Chicago, USA) software for statistical calculations. Biochemical vari-
ables were expressed as mean ± standard deviation. Differences be-
tween the groups were tested using the one-way analysis of variance
(ANOVA) followed by the Tukey HSD test; p values < 0.05 were con-
sidered statistically significant. The renal corpuscle, proximal and distal
tubule areas (μm2) and Caspase-3-positive cell numerical density values
were expressed as the mean ± standard deviation, and differences
between the groups were tested using the one-way ANOVA followed by
the Duncan test; p values < 0.05 were regarded as significant.
Light microscopic sections from the renal tissue of rats from the
control group exhibited regular Bowman’s capsules with normal his-
tological structural features (Fig. 4A and B). The olive oil and astax-
anthin 75 mg groups also exhibited normal tubules, and their Bowman’s
capsules had regular margins with no additional pathology (Fig. 4C–F).
On the other hand, light microscopic sections from the cisplatin group
exhibited damage in the renal cortex and medulla (Fig. 4G). We also
observed the loss of tubular epithelial cells and common tubular dila-
tations (Fig. 4H). Furthermore, we detected a cast formation in the
proximal and distal tubules, as well as capillary congestions in the
peritubular areas (Fig. 4G–H). In the cisplatin + astaxanthin 25 mg-
treated group, the Bowman’s capsules and proximal and distal tubules
exhibited regular morphologies. Levels of glomerular degeneration and
tubular epithelial cell injury were also lower than those of the cisplatin
group (Fig. 4I and J). The cisplatin + astaxanthin 75 mg-treated group
also exhibited regular Bowman’s capsules and proximal and distal tu-
bules, similar to those of the astaxanthin 25 mg-treated group (Fig. 4K
and L).
3.2.2. Immunohistochemical (IHC) results
Sections from the control, olive oil only and astaxanthin 75 mg
groups were Caspase-3 negative (Fig. 5A–F). However, we observed
greater Caspase-3 staining of proximal and distal tubular epithelial cells
in the cisplatin group compared to the control group (p < 0.05)
(Table 3) (Fig. 5G and H). The astaxanthin 25 mg and astaxanthin
75 mg groups exhibited less staining for the Caspase-3 expression than
the cisplatin only group (p < 0.05) (Table 3) (Fig. 5I–L).
3.2.3. Semi-quantitative analysis results
Higher Bowman’s capsule and proximal and distal tubular area

577
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

Fig. 3. Biochemical analysis graphs.

A: Total Antioxidan Status. B: Total Oxidan Status. C:Blood Urea Nitrogen. D: Serum Creatinine. *P < 0.05, #P < 0.05; Control vs Cisplatin group, ##
P < 0.05 Cis + Asta 25 mg vs
Cisplatin group, ###P < 0.05 Cis + Asta 75 mg vs Cisplatin group.

Table 2 first to demonstrate nephrotoxicity using cisplatin and second to pre-

Serum biochemical parameters. vent this effect using astaxanthin, an antioxidant agent. Although cis-
platin treatment increased BUN, serum creatinine and TOS levels and
Group
lowered TAS levels, 25 mg and 75 astaxanthin treatment combined with
Bun (mg/dL) Creatinine (mg/dL) cisplatin reduced BUN, serum creatinine and TOS levels and increased
**
TAS levels. There were no statistically significant differences between
Control 40.14 ± 1.95 0.412 ± 0.016**
the 25 mg and 75 mg astaxanthin treatment groups in terms of BUN,
Olive oil 39.42 ± 2.63** 0.395 ± 0.263**
Astaxanthin 75 mg 42.14 ± 1.95** 0.424 ± 0.019** serum creatinine, TAS or TOS concentrations. These biochemical results
Cisplatin 44.28 ± 1.88b* 0.451 ± 0.02* show that astaxanthin protects the kidneys from cisplatin-induced ne-
Cisplatin + Astaxanthin 25 mg 38.57 ± 1.71** 0.394 ± 0.02** phrotoxicity.
Cisplatin + Astaxanthin 75 mg 36.42 ± 1.98** 0.380 ± 0.023** Cisplatin is widely used for the tumours of several organs, such as
the head-neck region, bladder, testis, ovary, prostate, cervix and lung
* p ˂ 0.05 when compared to the control group.
** p ˂ 0.05 when compared to the cisplatin group.
[1,33]. Frequent use results in severe adverse effects, such as ne-
phrotoxicity, neurotoxicity, ototoxicity and, rarely, ocular toxicity [34].
measurements were found in the cisplatin group compared to the Nephrotoxicity, the most common side effect limiting cisplatin dosages,
control group (p < 0.05). The cisplatin + astaxanthin 25 mg and the is observed in almost 30% of patients [35,36]. Due to its low molecular
cisplatin + astaxanthin 75 mg groups exhibited smaller Bowman’s weight, cisplatin permeates the glomerular basal membrane easily and
capsule and proximal and distal tubular area measurements than the accumulates in the proximal tubular inner medulla and outer cortices
cisplatin only group (p < 0.05) (Tables 4–6). [37]. Pabla et al. reported a reduced glomerular filtration rate, in-
creased serum creatinine and decreased magnesium and potassium le-
4. Discussion vels [38]. The pathophysiological effect of cisplatin on the kidneys is
exerted in the form of apoptosis and necrosis in proximal tubule cells
Several studies have investigated the effect of antioxidants on ne- via oxidative stress, inflammation and vasoconstriction of the renal
phrotoxicity [30,32]. However, to the best of our knowledge this is the vascular structure [39,40]. Tubular cell death was identified as the
first experimental study to show the antioxidant effect of astaxanthin on main underlying histopathological characteristic of nephrotoxicity in
cisplatin-induced nephrotoxicity. The main purposes of this study were one experimental study [41]. Histopathological studies have

578
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

Fig. 4. Light microscopic photographs from kidney tissue sections stained with H&
E.
A (x200)-B (x00): Control Group; Healthy kidney tissue from the control group.
Glomerules (GL) and Bowman’s capsules (bs) exhibit regular structures. The prox-
imal (ps) and distal tubules (dt) are typical. Mesangial cells (green arrow) also
exhibit a normal structure. A normal proximal tubule epithelium cell with a brush
border (blue arrow). C (x40)-D (x400): Olive oil Group; Glomerules (GL).
Bowman’s capsule space (bs) with a regular structure. The proximal (ps) and distal
tubules (dt) are typical. Mesangial cells (green arrow) exhibit a normal structure. A
normal proximal tubule epithelium cell with a brush border (blue arrow). E (x200)-
F (x400): Astaxanthin 75 mg Group; Glomerules (GL). A regular Bowman’s cap-
sule space (bs). The proximal (ps) and distal tubules (dt) are typical. Mesangial cells
(green arrow) exhibit a normal structure. A normal proximal tubule epithelium cell
with a brush border (blue arrow). G (x40)-H (x400): Cisplatin Group; Damaged
kidney tissue from the cisplatin group. Degenerative glomeruli (arrow) and glo-
merular amyloidosis (green arrow) can be seen. Necrotic tubular cells and frequent
tubular dilatations are present (comet arrow), together with a tubular cyst forma-
tion (arrowhead). Significantly damaged cells can be seen in the tubules (spiral
arrow). Peritubules can be seen in congenital capillaries. I (x200)-J (x400):
Cisplatin + Astaxanthin 25 mg Group; Glomerulus with a regular structure (gl).
The proximal (pt) and distal tubules (dt) are typical. Bowman’s capsule (arrow). K
(x100)-L (x400): Cisplatin + Astaxanthin 75 mg Group; Glomerulus with a reg-
ular structure (gl). The proximal (pt) and distal tubules (dt) are have a stranded
structure. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

demonstrated interstitial haemorrhage, atrophy, interstitial inflamma- of tubular cells, tubular dilatations and peritubular capillary conges-
tion and tubular dilatation [42,43]. Our histopathological findings were tions in the cisplatin group. In terms of histopathological features, both
similar among the control, olive oil and astaxanthin 75 mg groups. In the cisplatin + astaxanthin 25 mg and cisplatin + astaxanthin 75 mg
contrast, we observed oxidative damage in the cortex and medulla, loss groups exhibited a protective effect against cisplatin-induced toxicity

579
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

Fig. 5. Light microscope image of kidney tissue by light microscopy. Caspase-3 staining.
A (x200)-B (x400): Control Group; Healthy morphological structures in the kidneys. Caspase-3-negative proximal and distal tubular epithelial cells (arrow). C (x200)-D (x400): Olive Oil
Group; E (x200)-F (x400): Astaxanthin 75 mg Group; G (x20)-H (x400): Cisplatin Group; Samples exhibit proximal and distal tubular epithelial cell positivity (arrow). I (x200)-J
(x400): Cisplatin + Astaxanthin 25 mg Group; Proximal and distal tubular epithelial cells (arrow) are immunologically negative. K (x200)-L (x400): Cisplatin + Astaxanthin 75 mg
Group; Proximal and distal tubular epithelial cells (arrow) are immunologically negative.

580
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

Table 3
Caspase-3 positive nuemarical density measurement (mm3) data.

Group Caspase-3 positive nuemarical density measurement (mm3) data (Mean ± Standart Deviation)

Control 3.17 ± 1.72**

Olive oil 2.5 ± 1.64**
Astaxanthin 75 mg 7.67 ± 3.01**
Cisplatin 110.33 ± 12.58*
Cisplatin + Astaxanthin 25 mg 15.00 ± 4.73**
Cisplatin + Astaxanthin 75 mg 15.17 ± 6.27**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 4
Renal corpuscle area measurement (μm2) data.

Group Renal corpuscle area measurement (Mean ± Standart Deviation)

Control 4840.15 ± 676.28**

Olive oil 4986.76 ± 551.44**
Astaxanthin 75 mg 5093.14 ± 568.26**
Cisplatin 10414.61 ± 2712.56*
Cisplatin + Astaxanthin 25 mg 4373.14 ± 904.04**
Cisplatin + Astaxanthin 75 mg 4935.31 ± 154.165**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 5
Proximal tubules area measurement (μm2) data.

Group Proximal tubules area measurement (μm2) data (Mean ± Standart Deviation)

Control 1466.16 ± 153.49**

Olive oil 1591.28 ± 256.01**
Astaxanthin 75 mg 1364.83 ± 256.00**
Cisplatin 3452.18 ± 1058.12*
Cisplatin + Astaxanthin 25 mg 1496.54 ± 123.32**
Cisplatin + Astaxanthin 75 mg 1701.1723 ± 427.15**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 6
Distal tubules area measurement (μm2) data.

Group Distal tubules area measurement (μm2) data (Mean ± Standart Deviation)

Control 2492.52 ± 344.78**

Olive oil 2362.15 ± 359.304**
Astaxanthin 75 mg 2299.48 ± 322.09**
Cisplatin 4798.49 ± 1848.47*
Cisplatin + Astaxanthin 25 mg 1508.07 ± 311.16**
Cisplatin + Astaxanthin 75 mg 2255.53 ± 459.22**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

compared to the cisplatin only group. anti-carcinogenic, anti-angiogenic, neuroprotective, im-

Although the mechanisms underlying the nephrotoxic effects of
cisplatin have not yet been elucidated fully, oxidative stress seems to
play a critical role in oxidative damage, while hypoxia and mitochon-
drial damage are implicated in the pathogenesis of cisplatin ne-
phrotoxicity [44]. Previous studies have observed indicators of renal
oxidative damage, such as increased MDA and TOS and reduced glu-
tathione (GSH), TAS and SOD levels in cisplatin-induced ne-
phrotoxicities [13,45]. Verma described both TAS and TOS as useful
assays for evaluating oxidative stress [13]. Ali showed in a review study
that oxidative damage plays an essential role in the pathogenesis of
nephrotoxicity, as well as reported that a large number of antioxidant
agents has been investigated in terms of prevention [46]. Astaxanthin, a
carotenoid pigment obtained from the microalgae Haematococcus plu-
vialis, is a potent antioxidant with anti-inflammatory, anti-apoptotic,
munomodulator, anti-diabetic and anti-obesity activities [47–49]. Yeh
et al. showed that astaxanthin reduces retinal oxidative stress in
streptozocin-induced diabetic rats [50]. Similarly, Mosaad reported
that astaxanthin protects the kidneys from gentamicin-induced ne-
phrotoxicity [51].
Faubel et al. reported that cisplatin-induced nephrotoxicity causes
apoptosis in renal tubular epithelial cells, in addition to acute tubular
necrosis [52]. Caspase-3 activation is regarded as an irreversible
terminal event before cell death, and it is used as a marker of apoptosis
[52]. In our study, we also determined that cisplatin causes apoptosis in
renal tubular epithelial cells (p < 0.05) (Table 3) (Fig. 5G and H).
Another experimental study showed that astaxanthin reduces oxidative
stress via cytochrome C, Caspase-9 and Caspase-3 in early acute renal
failure [53]. Similarly, we determined that astaxanthin reduces the

581
G. Akca et al.
apoptosis caused by cisplatin by lowering the Caspase-3 expression in
renal tubular epithelial cells (p < 0.05) (Table 3) (Fig. 5I–L).
Astaxanthin has not previously been used in the context of cisplatin
nephrotoxicity. Our findings show it prevents such nephrotoxicity.
Decreased Bowman’s capsules and proximal and distal tubular areas
were observed in both the cisplatin + astaxanthin 25 mg and the cis-
platin + astaxanthin 75 mg groups upon histopathological analysis
compared with the cisplatin group. We observed less staining for the
Caspase-3 expression upon immunohistochemical examination in all
groups except for the cisplatin only group. All the biochemical, histo-
pathological and immunohistochemical data obtained in our study
support the idea that astaxanthin exhibits an antioxidant effect that
protects the kidney from oxidative stress.
This study has a number of limitations, the principal being that it
constitutes a pilot study of the protective effect of astaxanthin against
cisplatin-induced nephrotoxicity. Further assays, such as MDA, GSH or
SOD, could be used to evaluate oxidant and antioxidant effects. The
mechanism of the molecular effect of astaxanthin, especially in terms of
antioxidant enzymes and proteins, must also be elucidated through
further studies.
5. Conclusion
Exposure to oxidant radicals has been increasing steadily in in-
dustrialised societies. The use of oxidants in foodstuffs or in pharma-
ceuticals has prompted researchers to investigate antioxidant sub-
stances. This experimental study revealed the antioxidant effect of
astaxanthin, an inexpensive and easily accessible chemical. We think
beneficial antioxidant effects can be obtained by adding astaxanthin to
the diets of patients exposed to oxidant stress.
Conflict of interest
Authors declare no conflict of interest.
References
[1] N.M. Elsherbiny, M.A. Eladl, M.M. Al-Gayyar, Renal protective effects of arjunolic
acid in a cisplatin-induced nephrotoxicity model, Cytokine 77 (2016) 26–34.
[2] D. Lebwohl, R. Canetta, Clinical development of platinum complexes in cancer
therapy: an historical perspective and an update, Eur. J. Cancer 34 (10) (1998)
1522–1534.
[3] M.E. Cooley, L. Davis, J. Abrahm, Cisplatin: a clinical review part II-nursing as-
sessment and management of side effects of cisplatin, Cancer Nurs. 17 (4) (1994)
283–293.
[4] X. Yao, et al., Cisplatin nephrotoxicity: a review, Am. J. Med. Sci. 334 (2) (2007)
115–124.
[5] S. Gullans, L. Mandel, Coupling of energy to transport in proximal and distal ne-
phron, Kidney: Physiol. Pathophysiol. 3 (1992) 445–482.
[6] R. Kröning, A. Lichtenstein, G. Nagami, Sulfur-containing amino acids decrease
cisplatin cytotoxicity and uptake in renal tubule epithelial cell lines, Cancer
Chemother. Pharm. 45 (1) (2000) 43–49.
[7] D.M. Townsend, et al., Metabolism of cisplatin to a nephrotoxin in proximal tubule
cells, J. Am. Soc. Nephrol. 14 (1) (2003) 1–10.
[8] H. Masuda, T. Tanaka, U. Takahama, Cisplatin generates superoxide anion by in-
teraction with DNA in a cell-free system, Biochem. Biophys. Res. Commun. 203 (2)
(1994) 1175–1180.
[9] C.A. Davis, H.S. Nick, A. Agarwal, Manganese superoxide dismutase attenuates
cisplatin-induced renal injury: importance of superoxide, J. Am. Soc. Nephrol. 12
(12) (2001) 2683–2690.
[10] G. Kadikoylu, et al., The effects of desferrioxamine on cisplatininduced lipid per-
oxidation and the activities of antioxidant enzymes in rat kidneys, Hum. Exp.
Toxicol. 23 (1) (2004) 29–34.
[11] Y.I. Chirino, R. Hernández-Pando, J. Pedraza-Chaverrí, Peroxynitrite decomposition
catalyst ameliorates renal damage and protein nitration in cisplatin-induced ne-
phrotoxicity in rats, BMC Pharmacol. 4 (1) (2004) 20.
[12] O.A. Badary, et al., Naringenin attenuates cisplatin nephrotoxicity in rats, Life Sci.
76 (18) (2005) 2125–2135.
[13] P.K. Verma, et al., Total antioxidant and oxidant status of plasma and renal tissue of
cisplatin-induced nephrotoxic rats: protection by floral extracts of Calendula offici-
nalis Linn, Ren. Fail. 38 (1) (2016) 142–150.
[14] I.K. Mohan, et al., Protection against cisplatin-induced nephrotoxicity by Spirulina
in rats, Cancer Chemother. Pharm. 58 (6) (2006) 802.
[15] M. Nazıroǧlu, A. Karaoğlu, A.O. Aksoy, Selenium and high dose vitamin E admin-
istration protects cisplatin-induced oxidative damage to renal, liver and lens tissues
in rats, Toxicology 195 (2) (2004) 221–230.
582
Biomedicine & Pharmacotherapy 100 (2018) 575–582
[16] E.H. Jansen, T. Ruskovska, Comparative analysis of serum (anti) oxidative status
parаmeters in healthy persons, Int. J. Mol. Sci. 14 (3) (2013) 6106–6115.
[17] C.P. Rubio, et al., Spectrophotometric assays for total antioxidant capacity (TAC) in
dog serum: an update, BMC Vet. Res. 12 (1) (2016) 166.
[18] R. Apak, et al., Total antioxidant capacity assay of human serum using copper (II)-
neocuproine as chromogenic oxidant: the CUPRAC method, Free Radic. Res. 39 (9)
(2005) 949–961.
[19] O. Erel, A novel automated direct measurement method for total antioxidant ca-
pacity using a new generation, more stable ABTS radical cation, Clin. Biochem. 37
(4) (2004) 277–285.
[20] P. Astorg, Food carotenoids and cancer prevention: an overview of current research,
Trends Food Sci. Technol. 8 (12) (1997) 406–413.
[21] H.D. Choi, et al., Effects of astaxanthin on oxidative stress in overweight and obese
adults, Phytother. Res. 25 (12) (2011) 1813–1818.
[22] Y. Chen, et al., Screening and characterization of astaxanthin-hyperproducing
mutants of Haematococcus pluvialis, Biotechnol. Lett. 25 (7) (2003) 527–529.
[23] M. Anderson, Method of inhibiting 5α-reductase with astaxanthin. 2001, Google
Patents.
[24] M. Guerin, M.E. Huntley, M. Olaizola, Haematococcus astaxanthin: applications for
human health and nutrition, Trends Biotechnol. 21 (5) (2003) 210–216.
[25] L.-S. Wu, et al., SENP1 attenuates the liver fibrosis through down-regulating the
expression of SMAD2, Biochem. Biophys. Res. Commun. 495 (1) (2017) 755–760.
[26] H. Turkez, F. Geyikoglu, M.I. Yousef, Beneficial effect of astaxanthin on 2,3,7,8-
tetrachlorodibenzo-p-dioxin-induced liver injury in rats, Toxicol. Ind. Health 29 (7)
(2013) 591–599.
[27] S.H. Kim, et al., Xanthorrhizol has a potential to attenuate the high dose cisplatin-
induced nephrotoxicity in mice, Food Chem. Toxicol. 43 (1) (2005) 117–122.
[28] A. Yilmaz, et al., Lasting hepatotoxic effects of prenatal mobile phone exposure, J.
Maternal-Fetal Neonatal Med. 30 (11) (2017) 1355–1359.
[29] O. Erel, A novel automated method to measure total antioxidant response against
potent free radical reactions, Clin. Biochem. 37 (2) (2004) 112–119.
[30] F. Geyikoglu, et al., Effect of oleuropein against chemotherapy drug-induced his-
tological changes, oxidative stress, and DNA damages in rat kidney injury, J. Food
Drug Anal. 25 (2) (2017) 447–459.
[31] T. Mercantepe, et al., Protective effects of estrogen and bortezomib in kidney tissue
of post-menopausal rats: an ultrastructural study, Ren. Fail. 38 (7) (2016)
1129–1135.
[32] R.E. Salmas, et al., Effects of propolis, caffeic acid phenethyl ester, and pollen on
renal injury in hypertensive rat: an experimental and theoretical approach, Cell
Biochem. Funct. 35 (6) (2017) 304–314.
[33] E. Cvitkovic, et al., Improvement of cis‐dichlorodiammineplatinum (NSC 119875):
therapeutic index in an animal model, Cancer 39 (4) (1977) 1357–1361.
[34] M.E. Cooley, et al., Cisplatin: a clinical review part I-current uses of cisplatin and
administration guidelines, Cancer Nurs. 17 (3) (1994) 173–184.
[35] E.A. Hartshorn, A.J. Anand, B. Bashey, Newer insights into cisplatin nephrotoxicity,
Ann. Pharmacother. 27 (12) (1993) 1519–1525.
[36] Z. Farooqui, et al., Protective effect of Nigella sativa oil on cisplatin induced ne-
phrotoxicity and oxidative damage in rat kidney, Biomed. Pharmacother. 85 (2017)
7–15.
[37] M. Kuhlmann, G. Burkhardt, H. Köhler, Insights into potential cellular mechanisms
of cisplatin nephrotoxicity and their clinical application, Nephrol. Dial. Transplant.
12 (12) (1997) 2478–2480.
[38] N. Pabla, Z. Dong, Cisplatin nephrotoxicity: mechanisms and renoprotective stra-
tegies, Kidney Int. 73 (9) (2008) 994–1007.
[39] T. Karasawa, P.S. Steyger, An integrated view of cisplatin-induced nephrotoxicity
and ototoxicity, Toxicol. Lett. 237 (3) (2015) 219–227.
[40] D. Huang, et al., Targeting Oct2 and P53: formononetin prevents cisplatin-induced
acute kidney injury, Toxicol. Appl. Pharmacol. 326 (2017) 15–24.
[41] G.-S. Oh, et al., Cisplatin-induced kidney dysfunction and perspectives on im-
proving treatment strategies, Electrolytes Blood Press. 12 (2) (2014) 55–65.
[42] C. Gandhi, R. Zalawadia, R. Balaraman, Nebivolol reduces experimentally induced
warm renal ischemia reperfusion injury in rats, Ren. Fail. 30 (9) (2008) 921–930.
[43] Ç. Akgüllü, et al., The usefulness of carvedilol and nebivolol in preventing contrast
nephropathy in rats, Ren. Fail. 37 (3) (2015) 511–517.
[44] X. Yao, et al., Cisplatin nephrotoxicity: a review, Am. J. Med. Sci. 334 (2) (2007)
115–124.
[45] Z.-N. Ma, et al., Nephroprotective effects of saponins from leaves of Panax quin-
quefolius against cisplatin-induced acute kidney injury, Int. J. Mol. Sci. 18 (7)
(2017) 1407.
[46] B.H. Ali, M.S. Al Moundhri, Agents ameliorating or augmenting the nephrotoxicity
of cisplatin and other platinum compounds: a review of some recent research, Food
Chem. Toxicol. 44 (8) (2006) 1173–1183.
[47] G. Hussein, et al., Antihypertensive and neuroprotective effects of astaxanthin in
experimental animals, Biol. Pharm. Bull. 28 (1) (2005) 47–52.
[48] M. Ikeuchi, et al., Effects of astaxanthin in obese mice fed a high-fat diet, Biosci.
Biotechnol. Biochem. 71 (4) (2007) 893–899.
[49] Y. Naito, et al., Prevention of diabetic nephropathy by treatment with astaxanthin
in diabetic db/db mice, Biofactors 20 (1) (2004) 49–59.
[50] P.-T. Yeh, et al., Astaxanthin inhibits expression of retinal oxidative stress and in-
flammatory mediators in streptozotocin-induced diabetic rats, PLoS One 11 (1)
(2016) e0146438.
[51] Y.O. Mosaad, N.Abd El Khalik Gobba, M.A. Hussein, Astaxanthin; a promising
protector against gentamicin-induced nephrotoxicity in rats, Curr. Pharm.
Biotechnol. 17 (13) (2016) 1189–1197.
[52] S. Faubel, et al., Caspase-1–deficient mice are protected against cisplatin-induced
apoptosis and acute tubular necrosis, Kidney Int. 66 (6) (2004) 2202–2213.
[53] S.-X. Guo, et al., Astaxanthin attenuates early acute kidney injury following severe
burns in rats by ameliorating oxidative stress and mitochondrial-related apoptosis,
Mar. Drugs 13 (4) (2015) 2105–2123.