Chemico-Biological Interactions 212 (2014) 65–71

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

NAD+ administration decreases doxorubicin-induced liver damage of

mice by enhancing antioxidation capacity and decreasing DNA damage
Ban Wang a,1, Yingxin Ma a,1, Xiaoni Kong a, Xianting Ding a, Hongchen Gu a, Tianqing Chu b,⇑,
Weihai Ying a,⇑
a
Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China
b
Respiratory Department, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, PR China

a r t i c l e i n f o a b s t r a c t

Article history: One of the major obstacles for cancer treatment is the toxic side effects of anti-cancer drugs. Doxorubicin
Received 23 June 2013 (DOX) is one of the most widely used anti-cancer drugs, which produces significant toxic side effects on
Received in revised form 16 January 2014 the heart and such organs as the liver. Because NAD+ can decrease cellular or tissue damage under multi-
Accepted 24 January 2014
ple conditions, we hypothesized that NAD+ administration may decrease DOX-induced hepatotoxicity. In
Available online 1 February 2014
this study we tested this hypothesis by using a mouse model, showing that NAD+ administration can sig-
nificantly attenuate DOX-induced increase in serum glutamate oxaloacetate transaminase activity and
Keywords:
decrease in liver weight. The NAD+ administration also attenuated the DOX-induced increases in the lev-
Doxorubicin
Hepatotoxicity
els of double-strand DNA (dsDNA) damage, TUNEL signals, and active caspase-3. Furthermore, our data
NAD+ has suggested that the NAD+ administration could produce protective effects at least partially by restor-
Antioxidation ing the antioxidation capacity of the liver, because NAD+ administration can attenuate the decreases in
Apoptosis both the GSH levels and the glutathione reductase activity of the DOX-treated liver, which could play
a significant role in the DOX-induced hepatotoxicity. This finding has provided the first evidence indicat-
ing that NAD+ is capable of increasing the antioxidation capacity of tissues. Collectively, our study has
found that NAD+ can significantly decrease DOX-induced liver damage at least partially by enhancing
antioxidation capacity and decreasing dsDNA damage. Because it can also selectively decrease tumor cell
survival, NAD+ may have significant merits over antioxidants for applying jointly with DOX to decrease
the toxic side effects of DOX.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction DOX interacts with DNA by intercalation and inhibition of macro-

Cancer is one of the leading causes of death in the world. One of
the major obstacles for establishing effective therapeutic strategies
for cancer is the toxic side effects of most anti-cancer drugs
[8,16,24,26], which can damage such organs and systems as the
immune system, liver and heart [13,14]. Thus, it is of crucial impor-
tance to find effective approaches to decrease the toxic side effects
of anti-cancer drugs. Doxorubicin (DOX) is one of the most widely
used anti-cancer drugs. The action mechanisms of DOX include:
⇑ Corresponding authors. Addresses: Med-X Research Institute and School of
Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road,
Shanghai 200030, PR China. Tel.: +86 21 6293 3075; fax: +86 21 6293 2302
(W. Ying), Respiratory Department, Shanghai Chest Hospital, Shanghai Jiao
Tong University, Shanghai 200030, PR China. Tel.: +86 21 6282 1990x3802;
fax: +86 21 3226 0856 (T. Chu).
E-mail addresses: weihaiy@sjtu.edu.cn (W. Ying), chutianqing@gmail.com
(T. Chu).
1
These authors contributed equally.
molecular biosynthesis, which inhibits the progression of the en-
zyme topoisomerase II, thus blocking the process of replication
[10,18]. The therapeutic efficacy of DOX have been severely limited
by the toxic side effects of the drug on heart as well as other organs
including the liver, brain and kidney [8,26]. Multiple studies have
reported that DOX can impair cardiomyocytes by generating oxida-
tive stress [9]. It has also been reported that such antioxidants as
Vitamin E and N-acetyl-cysteine (NAC) can decrease DOX-induced
liver damage [7,11,15]. It is of great significance to find new ap-
proaches that can decrease DOX-induced liver damage, so as to en-
hance the beneficial effects of DOX.
NAD+ is a fundamental molecule in cells, which plays important
roles in energy metabolism, mitochondrial functions and calcium
homeostasis [31]. Previous studies by our laboratory and other lab-
oratories have shown that NAD+ treatment can decrease oxidative
stress-induced cell death in vitro [1,2], and NAD+ administration
can also reduce both ischemic and traumatic brain damage
in vivo [29,32]. However, it remains unknown if NAD+ treatment

http://dx.doi.org/10.1016/j.cbi.2014.01.013
0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
66 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71
may directly increase antioxidation capacity of cells or tissues. In
current study, we used a mouse model to test our hypotheses that
NAD+ administration may decrease DOX-induced liver damage,
and that NAD+ administration can enhance the antioxidation
capacity of the DOX-exposed liver. Our results have provided evi-
dence supporting these hypotheses.
2. Materials and methods
2.1. Materials
The chemicals and antibodies used in this study were purchased
from Sigma Chemicals (St. Louis, MO, USA) except where noted.
2.2. Procedures of animal operation
Male ICR mice weighing 20–25 g were purchased from SLRC
Laboratory (Shanghai, China). All of the animal protocols were ap-
proved by the Animal Study Committee of the School of Biomedical
Engineering, Shanghai Jiao Tong University. To induce acute hepa-
tic injury, mice were injected intraperitoneally with 20 mg/kg
doxorubicin hydrochloride (DOX) dissolved in saline. For a subset
of mice, NAD+ at the doses of 100 or 200 mg/kg was also injected
intraperitoneally 1 h before the DOX administration. Animals were
sacrificed 48 h after the drug administration. The livers of the ani-
mals were removed and weighed, which were then stored at
80 °C until further analysis.
2.3. Assays of the activity of serum glutamate oxaloacetate
transaminase (SGOT)
The SGOT activities were measured by using a commercially
available kit (Jiancheng Bioengineering Institute, Nanjing, P.R. Chi-
na). The assay was performed using a plate reader according to the
manufacturer’s protocol.
2.4. GSH assay
The GSH level of the liver was measured by using a commer-
cially available kit (Jiancheng Bioengineering Institute, Nanjing,
P.R. China). The assay determines the reduction of 5,5-dithio-
bis(2-nitrobenzoic acid) (DTNB) by sulfhydryl groups to 2-nitro-
5-mercaptobenzoic acid, which is measured by determinations of
the optical absorbance of the samples at the wavelength of
540 nm under a plate reader.
2.5. Caspase-3 activity assay
The activity of caspase-3 was determined by using a caspase-3
activity kit (Beyotime Institute of Biotechnology, Haimen, P.R. Chi-
na) according to the manufacture’s protocol. The assay is based on
spectrophotometric detection of the chromophore, p-nitroanilide
(pNA), after its cleavage from the labeled substrate, acetyl-Asp-
Glu-Val-Asp p-nitroanilide (Ac-DEVE-pNA). Samples were mea-
sured with an ELISA reader at the absorbance of 405 nm. The pro-
tein concentrations were measured by BCA Protein Assay Kit
(Pierce Biotechnology, Rockford, IL, USA).
2.6. TUNEL staining
The TUNEL assay (ApopTagÒ Kit, CHEMICON, Catalog: S7101)
was used to assess apoptosis-like DNA fragmentation in situ. We
conducted the assay according to the manual provided with the
kit with minor modifications. Briefly, paraffin sections of the liver
were deparaffinized, and then freshly diluted proteinase K was
added. The endogenous peroxidase activity of the sections was
quenched by 3% hydrogen peroxide in PBS for 5 min at room tem-
perature. Then the sections were applied with equilibration buffer
and working-strength TdT enzyme, and incubated in a humidified
chamber at 37 °C for 1 h and incubated with anti-digoxigenin perox-
idase for 30 min at RT, which was detected by DAB. Sections were
counterstained in 0.5% (w/v) methyl green for 10 min at RT. After
washed with distilled water, the sections were dipped into 100%
N-butanol and dehydrated in xylene. After mounting, the sections
were viewed under a microscope. The slices of the brains obtained
24 h after middle cerebral artery occlusion were used as positive
controls. Negative control samples were prepared as other samples
except that the TdT enzyme was omitted in the procedures.
2.7. Immunostaining of activated capase-3
In brief, the paraffin sections were deparaffinized and then fixed
in 4% paraformaldehyde for 30 min, followed by three washes in
PBS. The slices were incubated in 10% goat serum for 1 h at room
temperature and then incubated with a rabbit anti-cleaved cas-
pase-3 antibody (Cell Signaling, Danvers, MA, USA) (1:300 dilution)
in PBS containing 1% goat serum overnight at 4 °C. After three
washes with PBS, the slices were incubated with Alexa Fluor 488
goat anti-rabbit secondary antibody (Molecular Probes, Eugene,
Oregon, USA) diluted at 1:300 containing 1% goat serum for 1 h
at RT. After the cells were washed three times with PBS, the cells
were counterstained with DAPI for 5 min at RT. Following washes
with PBS, the fluorescence images of the slices were photographed
under a Leica confocal fluorescence microscope.
2.8. Immunostaining of phosphohistone H2AX (c-H2AX)
In brief, the paraffin sections were deparaffinized and then fixed
in 4% paraformaldehyde for 30 min, followed by three washes in
PBS. The slices were incubated in 10% goat serum for 1 h at room
temperature and then incubated 2 lg/ml monoclonal anti-phospho-
histone H2AX (Millipore, Billerica, MA, USA) containing with 1% BSA
at 4 °C over night. After three washes with PBS, the slices were incu-
bated with Alexa Fluor 488 goat anti-rabbit secondary antibody or
Alexa Fluor 568 goat anti-mouse secondary antibody (Molecular
Probes, Eugene, Oregon, USA) diluted at 1:300 containing 1% goat
serum for 1 h at RT. After the cells were washed 3 times with PBS,
the cells were counterstained with DAPI for 5 min at RT. Following
washes with PBS, the fluorescence images of the slices were photo-
graphed under a Leica confocal fluorescence microscope.
2.9. NAD+ assay
NAD+ levels were measured by enzyme cycling method [4]. Tis-
sue was weighed and homogenized with 1.5 N perchloric acid
(40 lL/mg) kept at 0 °C for 20 min. The homogenate was centrifuged
for 10 min at 12,000 RPM and the supernatant was neutralized to
pH 7.2 with 3 N KOH and 1 M potassium phosphate buffer. The pro-
tein pellet was reconstituted by adding proper amount of 3 N KOH
and 1 M potassium phosphate buffer (to 500 lL total volume, pH
12) and the concentrations of protein were measured using Bradford
Protein Assay Kit assay (Beyotime Institute of Biotechnology, Hai-
men, P.R. China). After centrifugation at 12,000 RPM for 15 min,
the supernatants were mixed with a reaction medium containing
1.7 mg 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bro-
mide (MTT), 10.6 mg phenazine methosulfate, 1.3 mg alcohol dehy-
drogenase, 488.4 mg nicotinamide, and 2.4 mL ethanol in 37.6 mL
Gly–Gly buffer (65 mM, pH 7.4). The A560nm was determined imme-
diately and after 10 min, and the readings were calibrated with
NAD+ standards. Results were normalized to protein contents as
determined by Bradford Protein Assay.
 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71
2.10. Glutathione reductase (GR) assay
GR activity was determined by using a Glutathione Reductase
Assay kit (Jiancheng Bioengineering Institute, Nanjing, P.R. China)
according to the manufacture’s protocol. GR catalyzes the
NADPH-dependent reduction of oxidized glutathione (GSSG) to
reduced glutathione (GSH). The assay is based on spectrophoto-
metric detection of NADPH (kmax = 340 nm), which is oxidized by
GSSG.
2.11. Real-time PCR
Total RNA was isolated from the liver of mice by using TaKaRa
MiniBEST Universal RNA Extraction Kit (Takara Bio, Dalian, China).
500 ng total RNA was reverse-transcribed to cDNA using a Prime-
Script RT reagent kit (Takara Bio, Dalian, China) and RT-PCR was
performed as follows: 37 °C for 15 min, then 85 °C for 15 s. Quan-
titative real-time PCR was performed by using SYBR Premix Ex
Taq (Takara Bio, Dalian, China) and the following primers:GR
(sense 50 -GCCTTTACCCCGATGTATCACGCTGTG-30 and anti-sense
50 -TGTGAATGCCAACCACCTTTTCCTCTTTG-30 ); GAPDH (sense 50 -
AGGTCGGTGTGAACGGATTTG-30 and anti-sense 50 -TGTAGACCATG-
TAGTTGAGGTCA-30 ). PCRs were performed under the following
conditions: denaturing at 95 °C for 10 s, followed by 40 cycles of
95 °C for 5 s and 60 °C for 30 s. The data was analyzed by using
the comparative threshold cycle method, and results were
expressed as fold difference normalized to the level of GAPDH
mRNA.
2.12. Statistical analyses
All data are presented as mean ± SE. Data were assessed by
one-way ANOVA, followed by Student–Newman–Keuls post hoc
test. P values less than 0.05 were considered statistically
significant.
Fig. 1. Effects of NAD+ administration on DOX-induced hepatotoxicity in mice. The mice were injected by i.p. with 20 mg/kg DOX, and a subset of the mice were administered
(IP) with saline or 100 mg/kg or 200 mg/kg NAD+. Forty-eight hours after the DOX administration, the effects of NAD+ administration on DOX-induced hepatotoxicity were
assessed by measuring the activity of SGOT (A), the weight of the liver (B) and the weight of the body (C). N = 8–16 mice for each experimental condition. ⁄p < 0.05; ⁄⁄p < 0.01;
⁄⁄⁄
p < 0.001.
67
3. Results
3.1. Effects of NAD+ administration on DOX-induced increases in SGOT
activity and decreases in liver weight and body weight
We determined the effect of NAD+ administration on DOX-in-
duced hepatotoxicity of mice by measuring the activity of the he-
patic function-associated enzyme in the serum, as well as the
liver weight and the body weight (Fig. 1). We found that DOX in-
duced a significant increase in the activity of SGOT activity, which
was significantly attenuated by the NAD+ administration (Fig. 1A).
We also found that DOX administration led to significant decreases
in both the liver weight (Fig. 1B) and body weight of the mice
(Fig. 1C), which were also significantly attenuated by the NAD+
administration (Fig. 1B and C).
3.2. Effects of NAD+ administration on DOX-induced apoptotic changes
of the liver
We further conducted TUNEL assays to determine the effects of
administration of DOX and NAD+ on the apoptosis-like changes of
the liver. We found that DOX induced an increase in the TUNEL-posi-
tive signals in the liver, which was attenuated by NAD+ administration
(Fig. 2). We conducted caspase-3 activity assay on the liver, showing
that DOX induced a significant increase in the caspase-3 activity,
which was significantly attenuated by the NAD+ administration
(Fig. 3A). Our immunostaining assay of active caspase-3 also showed
that DOX induced an obvious increase in the levels of active caspase-3,
which was attenuated by the NAD+ administration (Fig. 3B).
3.3. Effects of NAD+ administration on DOX-induced dsDNA damage of
the liver
c-H2AX is a generally accepted marker of dsDNA damage [19].
To elucidate the effects of NAD+ and DOX on dsDNA damage in the
68 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71

Fig. 2. TUNEL staining showed that NAD+ administration significantly attenuated the DOX-induced apoptosis-like changes of the liver. The mice were intraperitoneally
injected with 20 mg/kg DOX, and a subset of the mice was administered intraperitoneally with saline or 100 mg/kg NAD+. Forty-eight hours after the DOX administration, the
effect of NAD+ administration on DOX-induced apoptosis-like-changes was assessed by TUNEL assay. N = 4–5 mice for each experimental condition. The length of the Scale
Bar denotes 50 lm.

Fig. 3. NAD+ administration significantly decreased DOX-induced increases in active caspase-3. (A) NAD+ administration significantly attenuated the DOX-induced increases
in caspase-3 activity. (B) Immunostaining assays showed that NAD+ administration attenuated DOX-induced increases in the active caspase-3. The mice were
intraperitoneally injected with 20 mg/kg DOX, and a subset of the mice was administered with saline or 100 mg/kg NAD+. The mice were sacrificed 2 days after the DOX
administration. Caspase-3 activity assays and immunostaining of active caspase-3 were conducted to determine the effects of NAD+ on the DOX-induced changes of the levels
of active caspase-3. N = 4–5 mice for each experimental condition. ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001. The length of the Scale Bar denotes 20 lm.

liver, we conducted immunostaining assay on c-H2AX. We found

that DOX induced a marked increase in c-H2AX in the liver, which
was decreased by the NAD+ administration (Fig. 4).

3.4. NAD+ administration significantly attenuated DOX-induced

decreases in the NAD+ levels in the liver

To investigate the mechanisms underlying the effects of NAD+

administration on DOX-produced damage of the liver, we deter-
mined the effects of administration of DOX and NAD+ on the
NAD+ levels in the liver. We found that DOX induced a significant
decrease in the NAD+ levels in the liver extracts, which was pre-
vented by the NAD+ administration (Fig. 5).

3.5. Effects of administration of NAD+ or NAC on DOX-induced

decreases in the GSH levels in the liver of mice

Our study has found that NAD+ administration can attenuate

the DOX-induced oxidative stress in the livers: DOX administration
led to a significant decrease in the GSH levels of the liver, which
was significantly attenuated by the NAD+ administration
(Fig. 6A). Because GR is the major enzyme that catalyzes the regen-
eration of GSH from GSSG [25], we tested our hypothesis that NAD+
prevents the DOX-induced decrease in GSH levels by preventing
DOX-induced decreases in GR activity. We found that DOX admin-
istration led to a significant decrease in the GR activity of the liver,
which was attenuated by the NAD+ administration (Fig. 6B). To
investigate the mechanisms underlying the effects of NAD+ admin-
istration on the GR activity, we conducted real-time PCR assays to
assess the effects of the NAD+ administration on the gene
Fig. 4. Immunostaining of c-H2AX shows that NAD+ administration attenuated
DOX-induced increases in the double-strand DNA (dsDNA) damage, as assessed by
c-H2AX immunostaining. The mice were intraperitoneally injected with 20 mg/kg
DOX, and a subset of the mice was administered with saline or 100 mg/kg NAD+.
The mice were sacrificed 2 days after the DOX administration. c-H2AX immuno-
staining was conducted to determine the effects of DOX on the levels of dsDNA
damage, and to determine the effects of NAD+ on the DOX-induced changes of
dsDNA damage. N = 4–5 mice for each experimental condition. The length of the
Scale Bar denotes 10 lm.
 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71 69

administration of 50 mg/kg NAC (Fig. 7A). The DOX-induced

increase in the SGOT activity was also significantly attenuated by
the NAC administration (Fig. 7B).

4. Discussion

The major findings of this study include: First, NAD+ adminis-

Fig. 5. NAD+ administration significantly attenuated DOX-induced decreases in the
NAD+ levels in the liver. The mice were intraperitoneally injected with 20 mg/kg
DOX, and a subset of the mice was administered with saline or 100 mg/kg NAD+.
Forty-eight hours after the DOX administration, the NAD+ levels in the homoge-
nates of the liver were assessed. DOX significantly decreased the NAD+ levels, which
were significantly attenuated by the NAD+ administration. N = 6 mice for each
experimental condition. Control NAD+ level is 244.2 + 19.7 lg/g protein. ⁄p < 0.05;
⁄⁄⁄
p < 0.001 (Student t-test).
expression of GR. We found that DOX treatment led to a significant
increase in the mRNA levels of GR, which was not affected by the
NAD+ administration (Supplemental Fig. 1).
We further determined the role of oxidative stress in the DOX-
induced liver damage, showing that DOX induced a significant de-
crease in the GSH levels in the liver, which was prevented by
tration can significantly attenuate DOX-induced increases in the
markers of liver injury; second, NAD+ administration can attenuate
DOX-induced apoptotic changes, including increased caspase-3
activity and increased TUNEL signals; third, NAD+ administration
can reduce DOX-induced dsDNA damage; and fourth, NAD+ admin-
istration can prevent DOX-induced decreases in the GSH levels in
the liver, which could at least partially account for its protective ef-
fects on DOX-induced liver injury.
DOX is one of the most widely used anti-cancer drugs. However,
this anti-cancer drug also produces significant toxic side effects by
damaging heart, liver, brain and kidney [8,26]. There are studies
suggesting that oxidative stress plays an important role in DOX-in-
duced damage of normal cells or tissues: DOX can impair normal
cardiomyocytes [9] and liver [20] by generating oxidative stress;
and such antioxidants as Vitamin E and NAC can decrease DOX-in-
duced liver damage [7,11,15]. It is of great clinical significance to
find new approaches that can attenuate DOX-induced liver
damage.
NAD+ is a fundamental molecule in cells, which plays important
roles in energy metabolism, mitochondrial functions and calcium
homeostasis [31]. Previous studies by our laboratory and other lab-
oratories have shown that NAD+ treatment can decrease oxidative
stress-induced cell death in vitro, and NAD+ administration can

Fig. 6. NAD+ administration significantly attenuated DOX-induced decreases in both the GSH levels and the glutathione reductase (GR) activity in the liver. The mice were
intraperitoneally injected 20 mg/kg DOX, and a subset of the mice was administered with saline or 100 mg/kg NAD+. Forty-eight hours after the DOX administration, both the
GSH levels (A) and GR activity (B) in the homogenates of liver were significantly decreased, which were significantly attenuated by the NAD+ administration. N = 5–8 mice for
each experimental condition. ⁄p < 0.05; ⁄⁄⁄p < 0.001.

Fig. 7. NAC administration decreased DOX-induced hepatotoxicity of mice. (A) NAC administration attenuated DOX-induced decreases in the GSH levels in the liver. The mice
were intraperitoneally injected 20 mg/kg DOX, and a subset of the mice was administered with saline or 50 mg/kg NAC. Forty-eight hours after the DOX administration, the
GSH levels in the liver were determined. (B) NAC administration attenuated DOX-induced increases in SGOT level. The mice were intraperitoneally injected 20 mg/kg DOX,
and a subset of the mice was administered with saline or 50 mg/kg NAC. Forty-eight hours after the DOX administration, the SGOT activity was determined. N = 5–8 mice for
each experimental condition. ⁄p < 0.05; ⁄⁄⁄p < 0.001.
70 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71
also attenuate both ischemic and traumatic brain damage in vivo
[29,31]. Because oxidative stress plays important roles in DOX-in-
duced liver damage, we hypothesized that NAD+ administration
may decrease the DOX-induced liver damage. Several lines of
evidence from our current study has supported the hypothesis:
First, NAD+ administration can attenuate several markers of DOX-
induced liver injury, including increases in SGOT activity, decreases
in the liver weight, and increases in both TUNEL signals and levels
of active caspase-3; second, NAD+ administration can reduce DOX-
induced dsDNA damage; and third, NAD+ administration can
attenuate DOX-induced decreases in the antioxidation capacity of
the liver.
Previous studies have indicated that oxidative stress, as indi-
cated by decreased GSH levels, plays a significant role in the
DOX-induced hepatotoxicity, since administration of such antioxi-
dants as NAC significantly attenuated the DOX-induced liver
damage [7,11,15]. In order to confirm that NAC is also effective
in decreasing DOX-induced liver toxicity in our experimental mod-
el, as well as to determine the extent of NAC-produced protection
on DOX-induced liver toxicity in our model, in current study we
determined the effects of NAC on DOX-induced liver injury in our
animal model. We found that NAC was also capable of significantly
decreasing DOX-induced injury of the liver in our model.
Our study has suggested that NAD+ produces its protective ef-
fects against DOX-induced liver damage at least partially by
enhancing the antioxidation capacity of the liver: We found that
NAD+ can significantly attenuate the decreases in the GSH levels
in the DOX-treated livers. Our study has also suggested that
NAD+ can significantly attenuate the decreases in the activity of
GR in the DOX-treated livers, which may at least partially account
for the NAD+-produced attenuation of DOX-induced GSH de-
creases. Because both previous reports and our current study have
indicated that GSH decreases play an important role in DOX-in-
duced liver toxicity, it appears that NAD+ administration produced
its beneficial effects at least partially by enhancing the antioxida-
tion capacity of the liver.
Previous studies have suggested that NAD+ decreased oxidative
stress-induced astrocyte and neuronal death by preventing the
pathological downstream events induced by oxidative stress, such
as mitochondrial permeability transition [1,2]. Our current study
has provided first evidence indicating that NAD+ can enhance the
antioxidation capacity of cells or tissues by increasing GSH levels.
Therefore, our current study has provided a novel mechanism
accounting for the protective effects of NAD+ on oxidative stress-
induced damage: NAD+ could decrease oxidative stress-induced in-
jury by directly decreasing oxidative stress, in addition to its re-
ported protective effects on the pathological downstream events
induced by oxidative stress.
We have found that the NAD+ administration can also prevent
DOX-induced decrease in GR activity. Because GR is a key enzyme
for maintaining GSH levels in cells, our study has suggested that
the NAD+ administration can enhance the GSH levels at least par-
tially by increasing GR activity. We have also conducted studies
on the effects of NAD+ administration on the gene expression of
GR by real-time PCR assay. We did not find any significant effects
of NAD+ administration on the gene expression of GR, thus arguing
against the possibility that the effects of NAD+ on GR activity re-
sults from its effects on gene expression. Future studies are war-
ranted to further investigate the mechanisms underlying the
effects of NAD+ administration on GR activity.
DNA damage belongs to the key damage produced by multiple
anti-cancer drugs [17]. Both single-strand DNA damage and dsDNA
damage can initiate cell death pathways [3,21,31]. It has been re-
ported that dsDNA damage can induce apoptosis by activating such
pathways as p53-dependent pathway, which can induce increased
Bax expression [3,22,30]. Our previous study has provided the first
evidence suggesting that NAD+ can decrease dsDNA damage under
certain pathological conditions: NAD+ administration can decrease
synchrotron radiation-induced dsDNA damage [23]. Our current
study has also shown that NAD+ administration can reduce DOX-
induced dsDNA damage of the liver. Collectively, both our previous
study and the current study have indicated that NAD+ may be a
promising drug for treating multiple diseases in which dsDNA
damage play significant pathological roles. Our current study
showed that DOX induced a marked increase in c-H2AX in both
intranuclear and extranuclear areas. Similar distributions of c-
H2AX have been observed by other laboratories in such organs
and tissues as the liver [5] and skin [27]. It is warranted to inves-
tigate the mechanisms underlying the extranuclear existence of
c-H2AX in the future.
The major apoptotic pathways include caspase-3-dependent
pathway and caspase-3-independent pathway. Various apoptotic
stimuli can activate caspase-3-dependent pathway, which is the
executioner of the apoptosis by activating such enzymes as caspase
activated deoxyribonuclease, leading to DNA fragmentation [28].
Previous cell culture studies have suggested that NAD+ treatment
can block oxidative stress-induced nuclear translocation of AIF
[2]. Our current study has provided first in vivo evidence suggest-
ing that NAD+ can prevent caspase-3-dependent apoptosis-like
changes of certain tissues.
We have found that DOX can significantly decrease the NAD+
levels in the liver extracts, which was significantly attenuated by
the NAD+ administration. This observation has suggested that
NAD+ has been uptaken and accumulated in the liver. This observa-
tion is also consistent with previous studies reporting that NAD+
administration can restore the NAD+ levels in the brain and de-
crease both ischemic brain injury and traumatic brain injury
[29,32].
Regarding the pathways by which NAD+ can enter cells, previ-
ous reports have indicated that NAD+ can enter cells though
P2X7 receptors in neurons [1] or through connexin 43 in fibro-
blasts [6]. In our current study, we did not conduct studies to elu-
cidate the exact pathways by which NAD+ enters the liver cells,
since these in vivo studies appear to be very time-consuming:
These studies would need use both P2X7 knockout mice and conn-
exin 43 knockout mice to solidly determine the pathways.
While it has been reported that NAD+ administration is benefi-
cial in animal models of brain ischemia, head trauma, and synchro-
tron radiation X-ray-induced tissue damage [23,29,32], our current
study is the first that indicates protective effects of NAD+ on the
toxic side effects of an anti-cancer drug. Therefore, our study has
further indicated major therapeutic potential of NAD+ for multiple
pathologies. Interestingly, out previous studies have indicated that
NAD+ can decrease tumor cell survival by such mechanisms as
increasing oxidative stress and inducing autophagy [12,33], in con-
trast to its beneficial effects on normal cells or tissue damage
[23,29,32]. These studies, together with our current finding, have
suggested that NAD+ may have significant merits over antioxidants
for applying jointly with DOX to decrease the toxic side effects of
DOX, since NAD+ may not only decrease tumor cell survival, but
also attenuate the toxic side effects of such anti-cancer drugs as
DOX on the liver.
Conflict of interest
There is no any competing interests.
Acknowledgment
This study was supported by a National Key Basic Research ‘973
Program’ Grant #2010CB834306 (to W.Y.), the Natural Science
 B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71
Foundation of Shanghai, China (Grant #12ZR1428800), Chinese
National Science Foundation Grants #81171098 and #81271305
(to W.Y.) and #81302004 (to T.C.), and Shanghai Jiao Tong Univer-
sity Grant for Interdisciplinary Research on Medicine and Engi-
neering (to W.Y. and T.C.).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cbi.2014.01.013.
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