Pakistan J. Zool., vol. 50(3), pp 1157-1170, 2018.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.3.1157.1170

Comparative Effects of Radon Inhalation
According to Mouse Strain and Cisplatin Dosein a Cisplatin-induced Renal Damage Model
Kaori Sasaoka1, Takahiro Kataoka1, Norie Kanzaki1, Yusuke Kobashi1,
Akihiro Sakoda2, Yuu Ishimori2 and Kiyonori Yamaoka1,*
1
Graduate School of Health Sciences, Okayama University, 5-1 Shikata-cho,
2-chome, Kita-ku, Okayama 700-8558, Japan
2
Ningyo-toge Environmental Engineering Center, Japan Atomic Energy Agency,
1550 Kamisaibara, Kagamino-cho, Tomata-gun, Okayama 708-0698, Japan
ABSTRACT
Cisplatin (CDDP) is widely used for treating solid cancers; however, it induces nephrotoxicity causedby oxidative stress. Here, we investigated whether radon inhalation has different effects against CDDPinduced renal injury in two mouse strains differing in radiosensitivity, and determined the appropriate
dose of CDDP combined with radon inhalation for highly radiosensitive mice. CDDP was administered at
20 mg/kg weight to C57BL/6J and BALB/c mice after radon inhalation at 1000 Bq/m3 and 2000 Bq/m3 for
24 h. Radon inhalation had a slight positive effect against CDDP toxicity in C57BL/6J mice with respect
to improved hair condition, whereas radon inhalation exacerbated CDDP-induced toxicity in BALB/cmice, such as a decrease in hair condition, higher creatinine levels, and decreased antioxidant contents(catalase and glutathione). When BALB/c mice were treated with a lower dose of CDDP (15 mg/kg) after
1000 Bq/m3 radon inhalation, the creatinine level was reduced and the superoxide dismutase content wasincreased, suggesting that this combination might have a protective effect against the CDDP-induced
renal damage. The supportive effect of radon inhalation shows its good potential as a candidate treatment
to alleviate CDDP-induced renal damage in veterinary medicine.

INTRODUCTION

C

isplatin (cis-diamminedichloroplatinum II, CDDP)
is a widely used chemotherapeutic agent for various
solid organ tumors such as tumors of the head, neck, testis,ovaries, and bladder (Ϛetin et al., 2006; Rana et al., 2016;
Santos et al., 2007). CDDP is well known to be taken
up by the proximal tubule cells and accumulates in the
mitochondria of these cells (Hanigan and Devarajan, 2003;
Noori and Mahboob, 2010; Prabhu et al., 2013; Uehara etal., 2011; Yao et al., 2007). However, CDDP has a toxic
effect by covalently binding to DNA (Hato et al., 2014).
Although CDDP also has ototoxicity, gastrotoxicity, andmyelosuppression, the main dose-limiting toxicity of the
drug rests in this nephrotoxicity. Oxidative stress has been
proposed as a key contributing factor to CDDP-induced
nephrotoxicity (Ozkok and Edelstein, 2014).
Oxidative stress represents an imbalance between* Corresponding author: yamaoka@md.okayama-u.ac.jp0030-9923/2018/0003-1157 $ 9.00/0
Copyright 2018 Zoological Society of Pakistan

Article Information
Received 23 June 2017
Revised 30 July 2017
Accepted 02 October 2017
Available online 25 April 2018
Authors’ Contribution
KS, TK and KY designed the study.
KS, NK, YK, TK, YI and AS collected
samples and analyzed data. KS, TK
and KY wrote the article.
Key words
Cisplatin, Oxidative stress, Renal
injury, Radon, Mice strain.

the production of reactive oxygen species (ROS) and theirremoval by antioxidant substances (Galle, 2001). ROS
such as superoxide (O2-) are caused by continuous oxygen
consumption, and are produced within the mitochondria;
antioxidant substances such as superoxide dismutase(SOD), catalase (Cat), and glutathione (GSH) eliminate
these ROS (Hanigan and Devarajan, 2003; Kruidering etal., 1997). However, the uptake of CDDP in cells and theirsubsequent accumulation in the mitochondria induces adecrease in the activities of SOD, Cat, and GSH; moreover,intracellular GSH levels are further decreased or depleted
owing to the rapid reaction with CDDP to promote its
transformation to highly reactive forms (Ozkok andEdelstein, 2014; Uehara et al., 2011). This phenomenon
contributes to the accumulation of ROS, leading to astate of oxidative stress within the cell (Pabla and Dong,
2008). Furthermore, CDDP has been suggested to induce
mitochondrial dysfunction, and thus ROS are generated
from the respiratory chains of these malfunctioning
mitochondria (Kawai et al., 2006; Oh et al., 2016; Ozkok
and Edelstein, 2014; Pabla and Dong, 2008).
Radon (222 Rn) is a natural radioactive gas that emits

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K. Sasaoka et al.

α-rays and is absorbed in the body through the lungs via
respiration as well as through the skin (Kataoka et al.,2016). Radon is delivered to many tissues through thebloodstream and throughout the body (Nishiyama et al.,2013), and is lipo-soluble with a high amount of energy
release (Yamato et al., 2013). At the Misasa Medical
Center of Okayama University Hospital, radon-based
therapy has been performed using a concentration of 2000
Bq/m3 and 42°C at room temperature (Sasaoka et al.,2016). Indications of this therapy are respiratory diseases
such as bronchial asthma, pain-related diseases such as
osteoarthritis, and digestive diseases such as chronicpancreatitis and gastroenteritis.
We previously reported that radon inhalation could
enhance antioxidant function in various organs in mice,such as the brain, lung, pancreas, liver, and kidney(Kataoka et al., 2011). Moreover, radon inhalation was
found to mitigate some ROS-related diseases, including
the effective prevention and treatment of the renal injury
related to streptozotocin-induced type 1 diabetes and ofhepatic and renal injuries induced by carbon tetrachloride(Kataoka et al., 2011; Nishiyama et al., 2015). Therefore,radon inhalation might help to suppress the renal damage
induced by CDDP owing to its antioxidant functions.
Chronic kidney diseases are observed with a highprevalence in geriatric dogs and cats at 0.4–1.5% and 10%,respectively (Finch et al., 2016; Fiocchi et al., 2017). Thus,
the widespread use of CDDP in the treatment of various
solid tumors in these animals, as well as in humans, isassociated with a problematic issue of nephrotoxicity(Knapp et al., 1987, 1988). In addition, there are several
types of dog breeds (approximately 400) and cat breeds(over 40) (Parker, 2012; Vapalahti, 2016), which differ
with respect to their risk of developing certain diseases
such as cardiovascular, hepatic, kidney, and bone and joint
diseases. Therefore, these strain-specific risks and effects
of drug administration should be taken into considerationin veterinary practice.
In addition, various mouse strains have been used foranimal experiments, such as C57BL/6J, BALB/c, ICR,
and Swiss albino mice (Maliakel et al., 2008; Sung etal., 2008), and strain-specific differences with respect toradio-sensitivity have been documented: C57BL/6J miceshow relatively low sensitivity, whereas BALB/c miceshow relatively high sensitivity (Onoue, 1960). However,no study has yet directly assessed the difference in thissensitivity between strains with respect to the effectsof radon inhalation on the CDDP-induced renal injury.
Furthermore, it is important to determine the suitable
dosage of CDDP administration combined with radon
inhalation in a highly radiosensitive mouse strain.
Accordingly, the aims of the present study were toevaluate whether radon inhalation has different effects on

CDDP-induced renal injury caused by oxidative stress in
two different mouse strains differing in radiosensitivity,
and to determine the effective dosage of CDDP whenradon inhalation is used. Clarification of these points
should be beneficial for the appropriate use of CDDP insmall animals in research and veterinary practice.

MATERIALS AND METHODS
Animals
Eight-week-old male C57BL/6J mice, weighing
22.5–26.0 g, and 8-week-old male BALB/c mice, weighing
23.3–27.0 g, were purchased from Japan Charles River
Laboratories Inc. (Yokohama, Japan) and housed in clear
plastic cages with paper-chipping in a temperature (23.2± 2.4°C) and light cycle (12 h of light and 12 h of dark)controlled room, and maintained on a standard laboratory
diet with free access to water. Ethical approval for all
protocols and experiments was obtained from the Animal
Care and Use Committee of Okayama University.
Experimental procedure
The evaluation of clinical signs, and measurement ofurine specific gravity and body weight in each mouse wasconducted before radon inhalation, at the time of CDDP
administration, and at 72 h after CDDP administration.
In Experiment 1, C57BL/6J mice and BALB/c mice
were divided into the following groups with seven mice
per cage: control group, 1000 Bq/m3 radon inhalation only
group, 2000 Bq/m3 radon inhalation only group, CDDP
(20 mg/kg) only group (CDDP 20, sham), CDDP (20mg/kg) administration after 1000 Bq/m3 radon inhalationgroup (1000 Bq/m3+CDDP 20), and CDDP (20 mg/kg)
administration after 2000 Bq/m3 radon inhalation group(2000 Bq/m3+CDDP 20).
In Experiment 2, BALB/c mice were divided into
the following groups with seven mice per cage: control
group, CDDP (15 mg/kg) only group (CDDP 15, sham),
CDDP (15 mg/kg) administration after 1000 Bq/m3 radon
inhalation group (1000 Bq/m3+CDDP 15), and CDDP (15mg/kg) administration after 2000 Bq/m3 radon inhalationgroup (2000 Bq/m3+CDDP 15).
After two days of acclimatization, the clinical signs
were evaluated by calculating the body condition scores(BCS) for nutritional assessment according to the criteria of
the World Small Animal Veterinary Association (Freeman
et al., 2011): 1, easily palpable ribs and lumbar vertebrae,
pelvic bones and all bones without detectable body fat
and muscles; 2, easily visible ribs, lumbar vertebrae,
pelvic bones, and all bones without palpable body fat andwith slight loss of muscle; 3, easily palpable ribs without
palpable fat, visible top of the vertebrae, prominent
pelvic bones with an obvious waist; 4, easily palpable

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Effect of Radon Inhalation on Cisplatin-Induced Renal Damage

ribs with slight body fat covering, and easily noticeable
waist that can be viewed from above; 5, palpable ribs
without excessive body fat, and an observable waist; 6,slight excessive body fat covering the ribs, discernible but
prominent waist; 7, non-palpable ribs with heavy fat cover,
obvious fat deposition over the lumbar area, and absent
or barely visible waist; 8, no palpable ribs due to heavy
fat cover, fat deposition around the lumbar area, and an
absent waist; 9, massive fat deposition over the thorax andspine, absent waist, and abdominal trunk with heavy fat
deposition on the neck and limbs. Scale and hair condition
scores were based on the system described by Miyagawa
et al. (2013): 0, no scales, 1, a few substantial or systemic
scales, 2, easily recognized scales systemically, and 3,
massive scales systemically; and 0, smooth and glossy
hairs, 1, partially dried hair, 2, systemically dried hair, and3, densely tangled and rough hairs, respectively (Fig. 1).
Urine samples were collected either by the urination at thetime of the retention of mice or by the bladder pressure of
the mice for measuring urine specific gravity. Body weights
in all mice were also recorded at the time of evaluation,
before radon inhalation and CDDP administration for the
CDDP groups, and at 72 h after CDDP administration.

1159

of the serum creatinine (Cre) level (as an indicator of renal
dysfunction) using the Quantichrom Creatinine Assay
Kit (Funakoshi Co., Ltd., Tokyo, Japan) according to the
manufacturer’s recommendation.

Radon inhalation
Mice were exposed to air only (sham) or radon at the
specified concentration for 24 h using a radon exposuresystem as previously described (Kataoka et al., 2012). The
concentration of radon in the mouse cage was supervisedusing a radon monitor (CMR-510, femo-TECH, Inc., OH,
USA) and adjusted to 1000 Bq/m3 or 2000 Bq/m3.
CDDP administration
Immediately after radon inhalation, the CDDP groups
were administered a single dose of CDDP (Wako Pure
Chemical Industry Co. Ltd., Osaka, Japan) at either 15 mg/
kg body weight or 20 mg/kg body weight intraperitoneally(i.p.). The dose was determined so as to be sufficiently highto induce severe renal damage in mice (Ueki et al., 2013).
After treatment, the mice were allowed to acclimate in thecages under a similar condition as described above for 72
h. Since the greatest severity of renal damage was detected
at 72 h following CDDP i.p. injection, all mice weresacrificed by deep diethyl ether anesthesia or inhalationof carbon dioxide gas at 72 h after CDDP administration(Ueki et al., 2013).
Biochemical assay
Blood samples were drawn by cardiac puncture at 72 h
after CDDP injection, and then centrifuged at 3,000 ×g for
5 min at 4°C. Sera were kept at −80°C until measurement

Fig. 1. Representative photos of hair condition and scalescore in mice. A, indicates the normal hair condition andscale score of control mice; B, represents each score of haircondition; C, each score of scale score.

The left and right kidneys were quickly and surgicallyexcised, and the specimens were measured and rinsed in
10 mM phosphate buffer solution (PBS, pH 7.4) and storedat −80°C until analysis of the SOD and Cat activities and
GSH protein levels. The left kidneys were homogenizedin 10 mM PBS (pH 7.4) on ice, and the homogenates
were centrifuged at 12,000 ×g for 45 min at 4°C. The
supernatants were used for the assay of SOD activity using
the nitroblue tetrazolium (NBT) reduction method with the
Wako SOD test (Wako Pure Chemical Industry Co. Ltd.,
Osaka, Japan) according to the manufacturer instructions(Baehner et al., 1975).

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K. Sasaoka et al.

Fig. 2. Effects of radon inhalation on the body condition score (BCS), scale score, and hair condition score in C57BL/6J andBALB/c mice with CDDP-induced renal injury. ||| P < 0.001 compares the effects of the radon inhalation group at 2000 Bq/m3 vs.
the sham group in the C57BL/6J strain. +++P < 0.001 indicates the comparison of the radon inhalation group at 2000 Bq/m3 vs.
the sham in the BALB/c strain. N.D. means “not detected”. Photographs of mice show the methods for measuring the BCS, scalescore, and hair condition score.

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Effect of Radon Inhalation on Cisplatin-Induced Renal Damage

In brief, inhibition of the reduction of NBT was
measured at 560 nm using a spectrophotometer; oneunit of enzyme activity was defined as 50% inhibition of
NBT reduction. Cat activity was measured as the rate ofhydrogen peroxide (H2O2) reduction at 37°C detected at
240 nm using a spectrophotometer (Aebi et al., 1976). The
assay mixture consisted of 50 μL of 1 M Tris–HCl buffer
containing 5 mM ethylenediaminetetraacetic acid (pH7.4), 900 μL of 10 mM H2O2, 30 μL of deionized water,and 20 μL of the supernatants of the kidneys. Cat activity
was calculated using a molar extinction coefficient of 7.1
× 10-3 M-1cm-1. The GSH content was measured using
the GSSG/GSH Quantification Kit (Dojindo MolecularTechnologies Inc., Kumamoto, Japan). In brief, the left
kidneys were homogenized with 5% 5-sulfosalicylic acidand centrifuged at 8,000 ×g for 10 min. The supernatants
were used for the assay based on the colorimetric reactionof GSH by detecting the absorption at 415 nm. Protein
concentrations were measured using the Bradford methodwith the protein Quantification Kit-Rapid (Dojindo
Molecular Technologies Inc., Kumamoto, Japan)(Bradford, 1976).

1161

compared with that of the CDDP 20 group (P < 0.001; Fig.
2). By contrast, in the BALB/c strain, the hair condition
score in the 2000 Bq/m3+CDDP 20 group was significantly
increased compared with that of the CDDP 20 group (P <0.001; Fig. 2). In addition, the hair condition score wassignificantly higher in the 2000 Bq/m3+CDDP 15 groupthan that of the CDDP 15 group (P < 0.001; Fig. 3).

Statistical analysis
Data are presented as the mean ± standard error of
the mean (SEM). The statistical significance of differences
was determined by t-tests, Mann-Whitney’s U test andKruskal-Wallis test or post-hoc Tukey’s tests or Dunnet
test for multiple comparisons where appropriate. P < 0.05
was considered statistically significant.

RESULTSClinical signs
In Experiment 1, the BCS tended to be reduced at
72 h after CDDP administration in all CDDP 20 groups
compared with the control group for both strains (Fig. 2).
Scales were recognized in the C57BL/6J strain and thescale score tended to be reduced in the 2000 Bq/m3+CDDP
20 group compared with that of the CDDP 20 group (Fig.
2). No scales were detected in the BALB/c mice (Fig. 2).
Similarly, in Experiment 2 using BALB/c mice only witha lower CDDP dose (15 kg/mg), the BCS tended to be
reduced at 72 h after CDDP administration in all groups
compared with that of the control group (Fig. 3). However,there was no difference in the overall BSC between the
CDDP 15 and CDDP 20 administration groups at 72 h after
CDDP administration; no scales were observed (Fig. 3).
In the C57BL/6J strain, the hair condition score of the2000 Bq/m3+CDDP 20 group was significantly reduced

Fig. 3. Effects of radon inhalation on the body condition
score (BCS) and hair condition score in BALB/c mice with
15 mg/kg CDDP-induced renal injury. ||| P < 0.001 indicatesthe comparison of the group treated with 15 mg/kg CDDP
after radon inhalation at 2000 Bq/m3 vs. the sham. N.D.
means “not detected.” Pictures of mice indicate the methodof measuring the BCS and hair condition score.

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K. Sasaoka et al.

Fig. 4. Effects of radon inhalation on the influence of 20 mg/kg CDDP administration in body weight, the rate of body weight
loss, absolute and relative kidney weight, and urine specific gravity in C57BL/6J and BALB/c mice with CDDP-induced renal
injury. ¶¶¶P < 0.001 compares the data of the treatment groups vs. the control measured at 72 h after CDDP administration in the
C57BL/6J strain; the sham group received 20 mg/kg CDDP only with air inhalation. †††P < 0.001 indicates the comparison of
the treatment groups vs. the control in the BALB/c strain at 72 h after CDDP administration. $P < 0.05, $$P < 0.01, $$$P < 0.001
indicate comparisons of the treatment group vs. the control in the C57BL/6J strain.# P < 0.05, ## P < 0.01, ### P < 0.001 indicate thetreatment groups vs. the control in the BALB/c strain. §P < 0.05 and §§ P < 0.01 compare data at 72 h after CDDP administration vs.
before radon inhalation in the C57BL/6J strain. * P < 0.05, ** P < 0.01, and *** P < 0.001 compare the data at 72 h after CDDP
administration in the treatment groups vs. before radon inhalation in the BALB/c strain. +P < 0.05, ++P < 0.01, and +++P < 0.001
indicate the CDDP and radon inhalation groups vs. the sham in the BALB/c strain. :P < 0.05 indicates the CDDP and 1000 Bq/m3
inhalation group vs. the sham in the BALB/c strain.

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Effect of Radon Inhalation on Cisplatin-Induced Renal Damage

1163

Fig. 5. Effects of radon inhalation on the 15 mg/kg CDDP-induced renal injury clinical signs, body weight, the rate of body weight
loss, absolute and relative kidney weight, and urine specific gravity in BALB/c mice. #P < 0.05, ##P < 0.01, and ###P < 0.001 indicate
the radon inhalation groups vs. the control. |P < 0.05 indicates the data at 72 h after CDDP administration with 2000 Bq/m3 radon
inhalation vs. the sham (15 mg/kg CDDP only with air inhalation). +P < 0.05 indicates CDDP administration after 2000 Bq/m3
radon inhalation vs. the sham.

Body weight, body weight loss, kidney weight, and urine
specific gravity
Body weight was decreased at 72 h after theadministration of 20 mg/kg CDDP in all groups comparedto the control in both strains (P < 0.001; Fig. 4). The rateof body weight loss of all groups administered CDDP waslower than that of the corresponding control group, withno difference from that of the respective sham group in
both strains (P < 0.001; Fig. 4). Body weight significantly
decreased in all BALB/c mice treated with 15 mg/kg
CDDP at 72 h after CDDP administration compared withthat of the control group (P < 0.001; Fig. 5). In addition,

the body weight of the 2000 Bq/m3+CDDP 15 group was
significantly decreased compared with that of the CDDP
15 group (P < 0.05; Fig. 5). The rate of body weight loss of
all CDDP 15-administered groups was lower than that of
the control group (P < 0.001; Fig. 5).
In C57BL/6J mice, the right kidney weight wassignificantly reduced for all CDDP 20-administeredgroups compared with the control group (P < 0.05; Fig. 4).
In BALB/c mice, the left kidney weight of the CDDP 20
group was significantly decreased compared with that of
the control group (P < 0.05; Fig. 4). The left kidney weightof the 1000 Bq/m3+CDDP 20 group was significantly

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K. Sasaoka et al.

increased compared that of the CDDP 20 group (P <0.05; Fig. 4). In C57BL/6J mice, the relative weight of
the left kidney was significantly increased in all groups
administered CDDP 20 compared with that of the control
group (CDDP 20, P < 0.01; 1000 Bq/m3+CDDP 20, P <0.001; and 2000 Bq/m3+CDDP20, P < 0.01; Fig. 4). In
BALB/c mice, the relative weights of both kidneys in the1000 Bq/m3+CDDP 20 and 2000 Bq/m3+CDDP 20 groups
were significantly increased compared with those of thecontrol group (left and right kidneys 1000 Bq/m3+CDDP
20, P < 0.01; right kidney 2000 Bq/m3+CDDP 20, P <0.001; and left kidney 2000 Bq/m3+CDDP 20, P < 0.01;
Fig. 4). The relative weights of the left kidneys of the 1000
Bq/m3+CDDP 20 and 2000 Bq/m3+CDDP 20 groups werealso significantly higher than that of the CDDP 20 group(1000 Bq/m3+CDDP 20, P < 0.001; 2000 Bq/m3+CDDP
20, P < 0.01; Fig. 4). As shown in Figure 5, kidney weight
was also significantly reduced in the left kidneys of the
CDDP 15 and 1000 Bq/m3+CDDP 15 groups comparedwith that of the control group (both P < 0.05). Moreover,
the absolute and relative weights of both kidneys in the2000 Bq/m3+CDDP 15 group were increased comparedwith those of the CDDP 15 group (P < 0.05; Fig. 5) andcontrol group (P < 0.01; Fig. 5).
The urine specific gravities of the CDDP 20 and 2000
Bq/m3+CDDP 20 groups were significantly lower than thatmeasured before radon inhalation in C57BL/6J mice (P <0.01 and P < 0.05, respectively; Fig. 4). In BALB/c mice,the urine specific gravities of all groups administered
CDDP at 20 mg/kg were significantly decreased comparedwith that of the control group (P < 0.001; Fig. 4), and weresignificantly lower at 72 h after CDDP administration
than that measured before radon inhalation (CDDP 20, P< 0.05; 1000 Bq/m3+CDDP 20, P < 0.01; and 2000 Bq/
m3, P < 0.001; Fig. 4). The urine specific gravity of the1000 Bq/m3+CDDP 20 group was significantly decreased
compared to that of the CDDP 20 group at 72 h after CDDP
administration (P < 0.05; Fig. 4). Urine specific gravitiesof all groups treated with CDDP 15 were lower than thatof the control group (P < 0.001; Fig. 5), and the urine
specific gravity of the 2000 Bq/m3+CDDP 15 group was
also significantly lower than that of the CDDP 15 group (P< 0.05; Fig. 5).
Index of renal injury
To estimate the ability of radon inhalation to mitigate
the renal injury induced by CDDP, Cre levels were
determined as an index of renal injury. In C57BL/6J mice,
there were no significant differences in Cre levels among
any of the CDDP-treated mice. In BALB/c mice, the Crelevels of all CDDP-administered groups were significantly
higher than that of control mice (CDDP 20 P < 0.05; 1000
Bq/m3+CDDP 20, P < 0.05, and 2000 Bq/m3, P < 0.01; Fig.

6A), with no difference among the three CDDP-treatedgroups (Fig. 6A). The Cre levels of the CDDP 15 and 2000
Bq/m3+CDDP 15 groups tended to be increased comparedwith those of the controls (Fig. 7A). However, there wasa decrease in Cre of approximately 70% in the 1000 Bq/
m3+CDDP 15 group compared with that of the CDDP 15
group (Fig. 7A).
Effect of radon inhalation on renal antioxidant-related
substances
To assess the antioxidative effect of radon inhalation
against CDDP-induced renal injury, the antioxidant-related
substances SOD, Cat, and GSH were analyzed.
The SOD activity of the CDDP 20 group was
significantly decreased compared with that of the control
group in C57BL/6J mice (P < 0.05; Fig. 6B). In BALB/cmice, the SOD activity of the 2000 Bq/m3+CDDP 20 groupwas significantly lower than that of the control group (P <0.01; Fig. 6B), whereas the SOD activity of the 1000 Bq/
m3+CDDP 20 group was significantly increased comparedwith that of the CDDP 20 group (P < 0.001; Fig. 6B).
Similarly, the SOD activity of the 2000 Bq/m3 +CDDP 15
group was significantly decreased compared with that of
the control mice (P < 0.01; Fig. 7B), whereas the activities
of both the 1000 Bq/m3+CDDP 15 and 2000 Bq/m3+CDDP
15 groups were significantly increased compared with thatof the CDDP 15 group (both P < 0.05; Fig. 7B).
The Cat activities of all CDDP administration groups
were significantly lower than that of control mice (P <0.005; Fig. 6B), and there were no significant differencesamong the CDDP administration groups in C57BL/6J
mice. However, in BALB/c mice, Cat activities weresignificantly decreased in the 1000 Bq/m3+CDDP 20 and2000 Bq/m3+CDDP 20 groups compared with that of
the control group (P < 0.05 and P < 0.01, respectively)(Fig. 6B) and CDDP 20 group (P < 0.05 and P < 0.001,
respectively) (Fig. 6B). Similarly, the Cat activity of the2000 Bq/m3 +CDDP 15 group was significantly lower thanthat of the control group (P < 0.01; Fig. 7B) and CDDP 15
group (P < 0.001) (Fig. 7B).
In C57BL/6J mice, the GSH activity of the 2000 Bq/
m3+CDDP 20 group was 30% higher than that of the CDDP
20 group. However, in BALB/c mice, the GSH activities
were significantly decreased in all CDDP-administeredgroups compared with that of the control group (P < 0.01;
Fig. 6B), and the 2000 Bq/m3+CDDP 20 group showed a
26% decrease in GSH activity compared with that of the
CDDP 20 group (Fig. 6B). In addition, the GSH activity of
the 2000 Bq/m3 +CDDP 15 group showed a 52% decrease
compared with that of CDDP15 group (Fig. 7B).

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Effect of Radon Inhalation on Cisplatin-Induced Renal Damage

1165

Fig. 6. Effects of radon inhalation on the influence of 20 mg/kg CDDP administration on A) the serum creatinine level, andB) antioxidant-related substances in C57BL/6J and BALB/c mice with CDDP-induced renal injury. $P < 0.05 and $$$P < 0.001
indicate the treatment groups vs. the control in the C57BL/6J strain. The sham group represents the group administered with 20
mg/kg CDDP only with air inhalation. #P < 0.05, ## P < 0.01, and ### P < 0.001 indicate the treatment groups vs. the control in the
BALB/c strain. +P < 0.05 and +++P < 0.001 indicate the radon inhalation groups vs. the sham in the BALB/c strain.

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K. Sasaoka et al.

DISCUSSION

Fig. 7. Effects of radon inhalation on the changes inA) serum creatinine level and B) antioxidant-related
substances caused by 15 mg/kg CDDP in BALB/c micewith CDDP-induced renal injury. ##P < 0.01 indicates thetreatment groups vs. the control. The sham group wastreated with 15 mg/kg CDDP only with air inhalation. |P <0.05 and ||| P < 0.001 indicate the radon inhalation groups
vs. the sham.

Radon inhalation has been suggested to enhance theantioxidant functions of various organs such as the brain,
lung, heart, liver, and kidney (Kataoka et al., 2011), andROS-related diseases of these organs have been shown
to be inhibited via the suppression of ROS activity.
Furthermore, radon inhalation was found to effectively
improve clinical signs in a study with a renal kidney failure
animal model (Kataoka et al., 2012). Although CDDP isan effective chemotherapeutic agent, various side effects
have been confirmed, including nephrotoxicity as a major
adverse effect. The overarching aim of the present study
was to evaluate the different effects of radon inhalation
on mediating this CDDP-induced renal damage in two
different radiosensitivity mouse strains and to determine
an appropriate CDDP dose in combination with radon
inhalation in the highly radiosensitive mouse strain.
Our results revealed no notable differences between
strains with respect to BCS, body weight, and the rate ofbody weight loss upon 20 mg/kg CDDP administration
with or without radon inhalation at two concentrations.
Body weight loss is a common outcome of cancer therapy,
and therefore nutrition management is an importantconsideration (Nakahara et al., 2012). Accordingly, both
body weight loss and the rate of this loss are importantfactors for accurately assessing the effect of CDDP.
Body weight loss arises from the gastrointestinal toxicity
in response to CDDP treatment, and the consequent
reduction of food intake leads to a poor nutritional status(Noori and Mahboob, 2010; Rana et al., 2016). The BCS
reflects the nutritious condition of animals (Freeman et al.,2011). Therefore, it is considered that the poor nutrition
condition observed in C57BL/6J and BALB/c mice wascaused by the gastrointestinal toxicity from 20 mg/kg
CDDP administration. However, radon inhalation could
not protect against this gastrointestinal toxicity. With
respect to dose, gastrointestinal toxicity was detected withthe administration of both 15 mg/kg and 20 mg/kg CDDP
in combination with radon inhalation at 2000 Bq/m3 given
the reductions in the BCS, body weight, and rate of bodyweight loss, indicating that even a relatively high radon
concentration (2000 Bq/m3) cannot suppress the reductionof food intake due to the gastrointestinal toxicity induced by
CDDP administration at both doses evaluated in this study.
Poor hair quality with an increase of scales iscommonly observed in animals suffering from renal injury(Canon, 2016). Therefore, we also evaluated the haircondition as well as scale scores as clinical signs of renal
injury induced by CDDP to evaluate the effects of radon
inhalation. The results showed that both the hair conditionand scale scores were improved in mice administered with
20 mg/kg CDDP after 2000 Bq/m3 radon inhalation in the

1167

Effect of Radon Inhalation on Cisplatin-Induced Renal Damage

C57BL/6J strain, suggesting that this radon concentration
may mitigate the CDDP-induced renal injury in this strain.
By contrast, the hair condition further deteriorated in
BALB/c mice that received 20 mg/kg CDDP after 2000
Bq/m3 radon inhalation, suggesting that a relatively highradon concentration may exacerbate the CDDP-induced
renal damage, which is a mark of the high radiosensitivityof this strain. This same effect was noted with 15 mg/
kg CDDP administration, indicating that the extent ofrenal damage induced by CDDP administration may
be aggravated by the combination with a high radon
concentration regardless of the CDDP dose.
CDDP-treated animals also show polyuria, indicatinga reduction in the urine specific gravity because of a
deficiency of urine concentration (Ecelbarger et al.,
2001; Nematbakhsh et al., 2013). Polyuria was confirmed
following 20 mg/kg CDDP administration after inhaling
2000 Bq/m3 radon in C57BL/6J mice, and all BALB/cmice administered either 20 mg/kg or 15 mg/kg CDDP
after radon inhalation showed significant reductions ofurine specific gravity, indicating that radon inhalation
does not mitigate the renal damage induced by CDDP.
Specifically, there was a significant reduction of urine
specific gravity with 15 mg/kg CDDP administration after
2000 Bq/m3 radon inhalation and with 20 mg/kg CDDP
after both 1000 Bq/m3 and 2000 Bq/m3 radon inhalation.
These findings suggest that the combination of 15 mg/kg
CDDP administration and 2000 Bq/m3 radon inhalationand the combination of 20 mg/kg CDDP administrationand 1000 Bq/m3 and 2000 Bq/m3 radon inhalation does notprovoke the polyuria in CDDP-induced renal damage.
The severity of renal damage can be indicatedby a larger kidney weight (Nematbakhsh et al., 2013;
Tokunaga et al., 1996). This enlargement of the kidney
is not due to a relative effect linked to overall weightloss but rather results from edema caused by necrosis in
the tubular cells (Rana et al., 2016). Therefore, both theabsolute and relative kidney weights should be considered
for assessments of the ability of radon inhalation to
improve the CDDP-induced renal injury. The present
study showed that absolute and relative kidney weights
increased in the C57BL/6J mice administered 20 mg/kg
CDDP after radon inhalation, indicating that the edemaof the tubular cells in these kidneys was indeed induced
by CDDP and could not be mitigated by radon inhalation.
Similarly, the BALB/c mice showed significant increases
in both the absolute and relative kidney weights with 20
mg/kg CDDP administration after radon inhalation at
both concentrations, suggesting that radon inhalation
might actually worsen the necrosis of tubular cells in
the kidneys in this strain. The kidney is considered to
be a relatively weakly radiosensitive organ based on the1167

different radiosensitivities in various tissues and organs.
Furthermore, radon is a highly lipo-soluble substance(Yamato et al., 2013); therefore, it would be expectedthat radon inhalation would have minimal effects on renal
injury in these strains since the kidney has less fat thanthe other organs. The kidney weights of BALB/c mice also
increased with the combination of 15 mg/kg CDDP and2000 Bq/m3 radon, as well as 20 mg/kg CDDP and both1000 Bq/m3 and 2000 Bq/m3 radon inhalation, suggestingthat these combinations could further exacerbate the
CDDP-induced renal injury.
Cre is filtered out of the body by the kidneys, andthe level of Cre increases when the filtration function of
the kidney is damaged (Prabhu et al., 2013). Likewise, theserum Cre level is elevated with increasing histologicaldamage to the tubular cells (Palipoch and Punsawasd2013). We did not detect an effect of radon inhalation ateither concentration on the Cre level in C57BL/6J mice,indicating that radon inhalation cannot inhibit the effectsof CDDP on renal damage in this strain due to its low
radiosensitivity. However, in the BALB/c strain, the Crelevels actually increased with 20 mg/kg CDDP after radon
inhalation at both concentrations, suggesting that the highradiosensitivity of this strain resulted in exacerbation of
the CDDP-induced renal damage by radon inhalation inradon inhalation. Evaluation of the influence of CDDP
dose showed that 1000 Bq/m3 radon inhalation reduced the
Cre level with administration of 15 mg/kg CDDP, but notsignificantly, indicating that radon inhalation may increase
the glomerular filtration rate so that the renal injury is
mitigated by a relatively low radon concentration (1000
Bq/m3).
Given this decrease in the Cre level, we further
examined antioxidant activity in the C57BL/6J andBALB/c strains and at different CDDP administration
doses in the BALB/c strain. SOD converts O2- to hydrogen
peroxidase (H2O2), whereas Cat and GSH transform H2O2
to water (H2O) and then to oxygen (O2) (Sasaoka et al.,2016). In addition, O2- is generated by consuming oxygenin the mitochondria (Kruidering et al., 1997); indeed,O2- is mainly generated from the respiratory chain in the
mitochondria (Kruidering et al., 1997; Pabla and Dong,
2008). It has also been suggested that CDDP decreases
the activity of antioxidants after accumulating in the
mitochondria (Noori and Mahboob, 2010). In the present
study, the GSH content tended to increase in the C57BL/6J
strain after 2000 Bq/m3 radon inhalation. This suggeststhat GSH plays an important role for the mitigation of the
CDDP-induced renal injury in C57BL/6J mice. However,since this difference was not statistically significant, theeffect of radon inhalation in this strain may be small. By
contrast, in the BALB/c strain, although the SOD activity

K. Sasaoka et al.

1168

showed an increase in the GSH level following 1000 Bq/
m3 radon inhalation, it was reduced following 2000 Bq/
m3 radon inhalation with 20 mg/kg CDDP administration.
The radon concentration employed in this study wasdetermined according to the reference of the radon therapy
used at Misasa Medical Center, Okayama University, andbased on our previous study examining the change in
SOD activity in the kidneys of BALB/c mice (Kataoka
et al., 2011). In this previous study, the SOD activity inthe kidney sharply increased at 24 h, decreased at 72 h,
and then gradually increased with 1000 Bq/m3 radon
inhalation. However, the SOD activity did not increaseat 24 h and remained constant with 2000 Bq/m3 radon
inhalation. Therefore, the low SOD activity observed with
2000 Bq/m3 radon inhalation in the present study likely
reflects these dynamics. Consequently, the inhalation of2000 Bq/m3 radon may not contribute to the mitigation of
CDDP-induced renal injury in this strain. In addition, Cat
activity and the GSH contents were significantly reduced
following 1000 Bq/m3 and 2000 Bq/m3 radon inhalation.
As mentioned above, antioxidant activity was enhanced atabout 24 h and the effect was reduced by radon inhalation
at 72 h (Kataoka et al., 2011). Therefore, these reductionsof Cat activity and GSH content with 20 mg/kg CDDP
administration might be due to this sequential change
in these antioxidants. Moreover, our results showed that
SOD activity was significantly high with the combinationof 15 mg/kg CDDP and 1000 Bq/m3 radon inhalation,suggesting that the function of the antioxidant SOD wasenhanced by the radon inhalation. This finding is in linewith the results of our previous study demonstrating thechange of SOD activity in the kidneys of BALB/c mice
(Kataoka et al., 2011). Therefore, SOD activity may
contribute to the mitigation of 15 mg/kg CDDP-induced
renal injury conferred by 1000 Bq/m3 radon inhalation.
Furthermore, according to the analysis of antioxidant
activity in paratuberculosis positive cattle, there were no
variation of SOD and GSH-PX activities in their sera. Sinceradon inhalation enhances the antioxidative function, theelevation of antioxidant activity for the biological defense
system against the infection might be observed in thesecattle after radon exposure (Cenesiz et al., 2016).
Although we used healthy mice for evaluating theeffects of radon inhalation against the CDDP induced renal
damage model in this study, the further study are needed toassess the effects of radon inhalation with the combinationof various CDDP dose and radon concentration in thecancer mouse model.

CONCLUSION
In conclusion, evaluation of the differences in clinicalsigns, indexes of renal injury, and antioxidant activities

suggests strain-specific effects of radon inhalation on
CDDP-induced renal damage, with clear differences
detected between C57BL/6J and BALB/c mice. These
differences are assumed to result from intrinsic variations in
the radiosensitivities of these strains. C57BL/6J mice haverelatively low radiosensitivity characteristics, indicatingthat the effects of radon inhalation on CDDP-induced
renal damage would be minimal, whereas BALB/c mice
have relatively high radiosensitivity, and exhibited highsensitivity to radon inhalation, which may be associatedwith radiation-induced DNA damage to further exacerbate
the CDDP-induced renal damage. Moreover, comparison of
the CDDP administration dose in BALB/c mice suggested
that the combination of 15 mg/kg CDDP and 1000 Bq/m3
radon inhalation is likely to protect against CDDP-induced
renal damage to some extent via increasing the SOD
content. Based on these results, radon inhalation appears
to be a suitable candidate for further studies of mitigating
CDDP-induced renal injury in veterinary practice.

ACKNOWLEDGMENTS
The authors thank the staff of the Departments ofAnimal Resources and Radiation Research, Shikata
Laboratory, Advanced Science Research Center, Okayama
University for their technical support in this work.
Statement of conflict of interest
Authors have declared no conflict of interest.

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