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Chemico-Biological Interactions
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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.
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 added. The endogenous peroxidase activity of the sections was
current study, we used a mouse model to test our hypotheses that quenched by 3% hydrogen peroxide in PBS for 5 min at room tem-
NAD+ administration may decrease DOX-induced liver damage, perature. Then the sections were applied with equilibration buffer
and that NAD+ administration can enhance the antioxidation and working-strength TdT enzyme, and incubated in a humidified
capacity of the DOX-exposed liver. Our results have provided evi- chamber at 37 °C for 1 h and incubated with anti-digoxigenin perox-
dence supporting these hypotheses. 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
2. Materials and methods washed with distilled water, the sections were dipped into 100%
N-butanol and dehydrated in xylene. After mounting, the sections
2.1. Materials were viewed under a microscope. The slices of the brains obtained
24 h after middle cerebral artery occlusion were used as positive
The chemicals and antibodies used in this study were purchased controls. Negative control samples were prepared as other samples
from Sigma Chemicals (St. Louis, MO, USA) except where noted. except that the TdT enzyme was omitted in the procedures.
GR activity was determined by using a Glutathione Reductase 3.1. Effects of NAD+ administration on DOX-induced increases in SGOT
Assay kit (Jiancheng Bioengineering Institute, Nanjing, P.R. China) activity and decreases in liver weight and body weight
according to the manufacture’s protocol. GR catalyzes the
NADPH-dependent reduction of oxidized glutathione (GSSG) to We determined the effect of NAD+ administration on DOX-in-
reduced glutathione (GSH). The assay is based on spectrophoto- duced hepatotoxicity of mice by measuring the activity of the he-
metric detection of NADPH (kmax = 340 nm), which is oxidized by patic function-associated enzyme in the serum, as well as the
GSSG. liver weight and the body weight (Fig. 1). We found that DOX in-
duced a significant increase in the activity of SGOT activity, which
2.11. Real-time PCR was significantly attenuated by the NAD+ administration (Fig. 1A).
We also found that DOX administration led to significant decreases
Total RNA was isolated from the liver of mice by using TaKaRa in both the liver weight (Fig. 1B) and body weight of the mice
MiniBEST Universal RNA Extraction Kit (Takara Bio, Dalian, China). (Fig. 1C), which were also significantly attenuated by the NAD+
500 ng total RNA was reverse-transcribed to cDNA using a Prime- administration (Fig. 1B and C).
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- 3.2. Effects of NAD+ administration on DOX-induced apoptotic changes
titative real-time PCR was performed by using SYBR Premix Ex of the liver
Taq (Takara Bio, Dalian, China) and the following primers:GR
(sense 50 -GCCTTTACCCCGATGTATCACGCTGTG-30 and anti-sense We further conducted TUNEL assays to determine the effects of
50 -TGTGAATGCCAACCACCTTTTCCTCTTTG-30 ); GAPDH (sense 50 - administration of DOX and NAD+ on the apoptosis-like changes of
AGGTCGGTGTGAACGGATTTG-30 and anti-sense 50 -TGTAGACCATG- the liver. We found that DOX induced an increase in the TUNEL-posi-
TAGTTGAGGTCA-30 ). PCRs were performed under the following tive signals in the liver, which was attenuated by NAD+ administration
conditions: denaturing at 95 °C for 10 s, followed by 40 cycles of (Fig. 2). We conducted caspase-3 activity assay on the liver, showing
95 °C for 5 s and 60 °C for 30 s. The data was analyzed by using that DOX induced a significant increase in the caspase-3 activity,
the comparative threshold cycle method, and results were which was significantly attenuated by the NAD+ administration
expressed as fold difference normalized to the level of GAPDH (Fig. 3A). Our immunostaining assay of active caspase-3 also showed
mRNA. that DOX induced an obvious increase in the levels of active caspase-3,
which was attenuated by the NAD+ administration (Fig. 3B).
2.12. Statistical analyses
3.3. Effects of NAD+ administration on DOX-induced dsDNA damage of
All data are presented as mean ± SE. Data were assessed by the liver
one-way ANOVA, followed by Student–Newman–Keuls post hoc
test. P values less than 0.05 were considered statistically c-H2AX is a generally accepted marker of dsDNA damage [19].
significant. To elucidate the effects of NAD+ and DOX on dsDNA damage in the
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.
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.
4. Discussion
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 evidence suggesting that NAD+ can decrease dsDNA damage under
[29,31]. Because oxidative stress plays important roles in DOX-in- certain pathological conditions: NAD+ administration can decrease
duced liver damage, we hypothesized that NAD+ administration synchrotron radiation-induced dsDNA damage [23]. Our current
may decrease the DOX-induced liver damage. Several lines of study has also shown that NAD+ administration can reduce DOX-
evidence from our current study has supported the hypothesis: induced dsDNA damage of the liver. Collectively, both our previous
First, NAD+ administration can attenuate several markers of DOX- study and the current study have indicated that NAD+ may be a
induced liver injury, including increases in SGOT activity, decreases promising drug for treating multiple diseases in which dsDNA
in the liver weight, and increases in both TUNEL signals and levels damage play significant pathological roles. Our current study
of active caspase-3; second, NAD+ administration can reduce DOX- showed that DOX induced a marked increase in c-H2AX in both
induced dsDNA damage; and third, NAD+ administration can intranuclear and extranuclear areas. Similar distributions of c-
attenuate DOX-induced decreases in the antioxidation capacity of H2AX have been observed by other laboratories in such organs
the liver. and tissues as the liver [5] and skin [27]. It is warranted to inves-
Previous studies have indicated that oxidative stress, as indi- tigate the mechanisms underlying the extranuclear existence of
cated by decreased GSH levels, plays a significant role in the c-H2AX in the future.
DOX-induced hepatotoxicity, since administration of such antioxi- The major apoptotic pathways include caspase-3-dependent
dants as NAC significantly attenuated the DOX-induced liver pathway and caspase-3-independent pathway. Various apoptotic
damage [7,11,15]. In order to confirm that NAC is also effective stimuli can activate caspase-3-dependent pathway, which is the
in decreasing DOX-induced liver toxicity in our experimental mod- executioner of the apoptosis by activating such enzymes as caspase
el, as well as to determine the extent of NAC-produced protection activated deoxyribonuclease, leading to DNA fragmentation [28].
on DOX-induced liver toxicity in our model, in current study we Previous cell culture studies have suggested that NAD+ treatment
determined the effects of NAC on DOX-induced liver injury in our can block oxidative stress-induced nuclear translocation of AIF
animal model. We found that NAC was also capable of significantly [2]. Our current study has provided first in vivo evidence suggest-
decreasing DOX-induced injury of the liver in our model. ing that NAD+ can prevent caspase-3-dependent apoptosis-like
Our study has suggested that NAD+ produces its protective ef- changes of certain tissues.
fects against DOX-induced liver damage at least partially by We have found that DOX can significantly decrease the NAD+
enhancing the antioxidation capacity of the liver: We found that levels in the liver extracts, which was significantly attenuated by
NAD+ can significantly attenuate the decreases in the GSH levels the NAD+ administration. This observation has suggested that
in the DOX-treated livers. Our study has also suggested that NAD+ has been uptaken and accumulated in the liver. This observa-
NAD+ can significantly attenuate the decreases in the activity of tion is also consistent with previous studies reporting that NAD+
GR in the DOX-treated livers, which may at least partially account administration can restore the NAD+ levels in the brain and de-
for the NAD+-produced attenuation of DOX-induced GSH de- crease both ischemic brain injury and traumatic brain injury
creases. Because both previous reports and our current study have [29,32].
indicated that GSH decreases play an important role in DOX-in- Regarding the pathways by which NAD+ can enter cells, previ-
duced liver toxicity, it appears that NAD+ administration produced ous reports have indicated that NAD+ can enter cells though
its beneficial effects at least partially by enhancing the antioxida- P2X7 receptors in neurons [1] or through connexin 43 in fibro-
tion capacity of the liver. blasts [6]. In our current study, we did not conduct studies to elu-
Previous studies have suggested that NAD+ decreased oxidative cidate the exact pathways by which NAD+ enters the liver cells,
stress-induced astrocyte and neuronal death by preventing the since these in vivo studies appear to be very time-consuming:
pathological downstream events induced by oxidative stress, such These studies would need use both P2X7 knockout mice and conn-
as mitochondrial permeability transition [1,2]. Our current study exin 43 knockout mice to solidly determine the pathways.
has provided first evidence indicating that NAD+ can enhance the While it has been reported that NAD+ administration is benefi-
antioxidation capacity of cells or tissues by increasing GSH levels. cial in animal models of brain ischemia, head trauma, and synchro-
Therefore, our current study has provided a novel mechanism tron radiation X-ray-induced tissue damage [23,29,32], our current
accounting for the protective effects of NAD+ on oxidative stress- study is the first that indicates protective effects of NAD+ on the
induced damage: NAD+ could decrease oxidative stress-induced in- toxic side effects of an anti-cancer drug. Therefore, our study has
jury by directly decreasing oxidative stress, in addition to its re- further indicated major therapeutic potential of NAD+ for multiple
ported protective effects on the pathological downstream events pathologies. Interestingly, out previous studies have indicated that
induced by oxidative stress. NAD+ can decrease tumor cell survival by such mechanisms as
We have found that the NAD+ administration can also prevent increasing oxidative stress and inducing autophagy [12,33], in con-
DOX-induced decrease in GR activity. Because GR is a key enzyme trast to its beneficial effects on normal cells or tissue damage
for maintaining GSH levels in cells, our study has suggested that [23,29,32]. These studies, together with our current finding, have
the NAD+ administration can enhance the GSH levels at least par- suggested that NAD+ may have significant merits over antioxidants
tially by increasing GR activity. We have also conducted studies for applying jointly with DOX to decrease the toxic side effects of
on the effects of NAD+ administration on the gene expression of DOX, since NAD+ may not only decrease tumor cell survival, but
GR by real-time PCR assay. We did not find any significant effects also attenuate the toxic side effects of such anti-cancer drugs as
of NAD+ administration on the gene expression of GR, thus arguing DOX on the liver.
against the possibility that the effects of NAD+ on GR activity re-
sults from its effects on gene expression. Future studies are war-
Conflict of interest
ranted to further investigate the mechanisms underlying the
effects of NAD+ administration on GR activity.
There is no any competing interests.
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- Acknowledgment
ported that dsDNA damage can induce apoptosis by activating such
pathways as p53-dependent pathway, which can induce increased This study was supported by a National Key Basic Research ‘973
Bax expression [3,22,30]. Our previous study has provided the first Program’ Grant #2010CB834306 (to W.Y.), the Natural Science
B. Wang et al. / Chemico-Biological Interactions 212 (2014) 65–71 71
Foundation of Shanghai, China (Grant #12ZR1428800), Chinese [14] P.D. King, M.C. Perry, Hepatotoxicity of chemotherapy, Oncologist 6 (2001)
162–176.
National Science Foundation Grants #81171098 and #81271305
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(to W.Y.) and #81302004 (to T.C.), and Shanghai Jiao Tong Univer- acetylcysteine modulates doxorubicin-induced oxidative stress and
sity Grant for Interdisciplinary Research on Medicine and Engi- antioxidant vitamin concentrations in liver of rats, Cell Biochem. Funct. 28
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