marine drugs
ArticleProtective Effects of Hydrolyzed Nucleoproteins from
Salmon Milt against Ethanol-Induced Liver Injury
in Rats
Akiko Kojima-Yuasa 1, *, Mayu Goto 1 , Eri Yoshikawa 1 , Yuri Morita 1 , Hirotaka Sekiguchi 2 ,Keita Sutoh 2 , Koji Usumi 2 and Isao Matsui-Yuasa 1
1

2*

Department of Food and Human Health Sciences, Graduate School of Human Life Science,
Osaka City University, Osaka 558-8585, Japan; v-oooogue@ezweb.ne.jp (M.G.); eri92011@gmail.com (E.Y.);
morimaru.y@gmail.com (Y.M.); yuasa-i@hotmail.co.jp (I.M.-Y.)
Life Science Institute Co., Ltd., Tokyo 103-0012, Japan; sekiguchi@life-science.co.jp (H.S.);
sutoh@life-science.co.jp (K.S.); usumi@life-science.co.jp (K.U.)
Correspondence: kojima@life.osaka-cu.ac.jp; Tel.: +81-6-6605-2865

Academic Editor: Se-Kwon Kim
Received: 17 October 2016; Accepted: 15 December 2016; Published: 19 December 2016

Abstract: Dietary nucleotides play a role in maintaining the immune responses of both animals
and humans. Oral administration of nucleic acids from salmon milt have physiological functionsin the cellular metabolism, proliferation, differentiation, and apoptosis of human small intestinal
epithelial cells. In this study, we examined the effects of DNA-rich nucleic acids prepared from
salmon milt (DNSM) on the development of liver fibrosis in an in vivo ethanol-carbon tetrachloride
cirrhosis model. Plasma aspartate transaminase and alanine transaminase were significantly lessactive in the DNSM-treated group than in the ethanol plus carbon tetrachloride (CCl4 )-treated group.
Collagen accumulation in the liver and hepatic necrosis were observed histologically in ethanol plus
CCl4 -treated rats; however, DNSM-treatment fully protected rats against ethanol plus CCl4 -induced
liver fibrosis and necrosis. Furthermore, we examined whether DNSM had a preventive effect against
alcohol-induced liver injury by regulating the cytochrome p450 2E1 (CYP2E1)-mediated oxidative
stress pathway in an in vivo model. In this model, CYP2E1 activity in ethanol plus CCl4 -treatedrats increased significantly, but DNSM-treatment suppressed the enzyme’s activity and reduced
intracellular thiobarbituric acid reactive substances (TBARS) levels. Furthermore, the hepatocytes
treated with 100 mM ethanol induced an increase in cell death and were not restored to the control
levels when treated with DNSM, suggesting that digestive products of DNSM are effective for theprevention of alcohol-induced liver injury. Deoxyadenosine suppressed the ethanol-induced increasein cell death and increased the activity of alcohol dehydrogenase. These results suggest that DNSM
treatment represents a novel tool for the prevention of alcohol-induced liver injury.
Keywords: DNA-rich nucleic acid prepared from salmon milt (DNSM); in vivo ethanol-carbon
tetrachloride cirrhosis model; plasma aminotransferases (AST and ALT); collagen accumulation;
CYP2E1 activity; alcohol-induced liver injury; rats

1. Introduction
Alcoholic liver disease is a pathological process characterized by progressive liver damage that
leads to steatosis, steatohepatitis, fibrosis, and ultimately cirrhosis, which may further progress
to hepatocellular cancer [1–3]. Oxidative stress plays an important role in this process [4,5].
Alcohol-induced oxidative stress associated with ethanol metabolism in the liver plays a major role inethanol-induced liver injury. Alcohol dehydrogenase is the major enzyme responsible for oxidizing
ethanol to aldehyde in alcohol metabolism. Heavy consumption of ethanol induces cytochrome
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2 of 12p450
2E1(CYP2E1)
activity in hepatocytes. This enzyme complements the activity of a constitutively
expressed alcohol dehydrogenase in oxidizing ethanol to acetaldehyde [6]. However, CYP2E1 generates(CYP2E1) activity in hepatocytes. This enzyme complements the activity of a constitutively expressed
reactive oxygen species (ROS) quite efficiently, which appears to play a major role in ethanol-induced
alcohol dehydrogenase in oxidizing ethanol to acetaldehyde [6]. However, CYP2E1 generates
liver injury [7–10]. Therefore, possible strategies for preventing the production of these ROS may bereactive oxygen species (ROS) quite efficiently, which appears to play a major role in ethanol‐induced
effective
in attempts
minimize
the hepatotoxicity
ethanol inliver injury[7–10]. to
Therefore,possible
strategies for of
preventing
thehumans.
production of these ROS may be
Animalinmodelsof liverfibrosisthearehepatotoxicity
important forof
understanding
the underlying mechanisms of theeffective
attempts
to minimize
ethanol in humans.
treatments
usedtocombatthis
disease.
Currently,
twomodelshave
beendeveloped mechanisms
for administering
Animal models of liver fibrosis are important for understanding the underlyingofalcohol
to animals:usedthe Lieber-De
diet[11] and two
the Tsukamoto-French
model
the treatments
to combatCarli
this liquid
disease.
Currently,
models have been gastricdeveloped
for[12].
In theLieber-De Carliliquiddiet, ethanol
replaces the
carbohydrates
of aandnormaldiet. Tsukamoto andadministering
alcohol
to animals:
the Lieber‐DeCarliliquid diet [11]the Tsukamoto‐French
gastric
model [12].
Lieber‐De
Carli liquid
diet, ethanol
thecontinuously
carbohydratesadministeredof a normal to
Frenchdeveloped
an In
inthe
vivo
animal modelin whichenteral replaces
ethanol is
diet.
TsukamotoandFrenchdeveloped
an
in
vivo
animal
model
in
which
enteralethanol isthe animal via intragastric infusion. However, neither the Lieber De Caarli nor the Tsukamoto/Frenchcontinuously
administeredtotheanimalvia
intragastricinfusion.
However,neithertheLieber De
feeding protocol results in cirrhosis in rats. Tipoe et al. reported another model for administering
Caarlinor theand
Tsukamoto/French
feeding protocol
resultsthat
in cirrhosis
in rats.
Tipoe et al. reported
dietary
alcohol
fish oil (30% of calories)and showed
an increasein profibrogenic
mediators
anothermodel
foradministering
dietary
alcohol
and
fish
oil
(30%
of
calories)andshowedthatanet al.
was not associated with the presence of histological evidence of fibrosis [13]. Contrastingly, Siegers
increase in profibrogenic mediators was not associated with the presence of histological evidence ofdeveloped a model for the administration of low-dose carbon tetrachloride (CCl4 ) and a 5% ethanol
fibrosis [13]. Contrastingly, Siegers et al. developed a model for the administration of low‐dose
solution that produced histological changes in rats similar to those found in human alcoholic cirrhosis
carbon tetrachloride (CCl4) and a 5% ethanol solution that produced histological changes in rats
within four weeks [14]. We have also shown that hepatic histological changes occurred within four
similar to those found in human alcoholic cirrhosis within four weeks [14]. We have also shown thatweeksof theadministrationofoccurred
low-dose
CCl4 fourand weeks
a 5% ethanol
solution [15,16].
hepatic
histologicalchangeswithinof the administrationof low‐dose CCl4 andSalmonmilts
containmainlynucleic
acids,
protamine,andpolyamine.
These
components play
a 5% ethanol solution [15,16].
an important
role
in
the
diet.
Dietary
nucleicacids
areparticularly
importantforthe development
Salmon milts contain mainly nucleic acids, protamine, and polyamine. These components
play
andan
growthof tissues.
are nucleic
partly degradedby nucleases
in the intestine
and absorbed asimportantrole inNucleic
the diet.acids
Dietary
acids are particularly
important
for the development
nucleosides
andofnucleobases.
Dietary
nucleotides
play a rolein maintenanceof immune
and growth
tissues. Nucleic
acids arepartly degradedby nucleases
in the intestineand responses
absorbed inasanimals
nucleosides
nucleobases.
Dietary
nucleotides
playhavea rolealso
in maintenanceof immune
responses
bothandandhumans[17–19].
Severalresearchersreported thatorally administeredin bothanimals
humans[17–19].
have functions
also reported
thatasorally
administerednucleic
acids
fromandsalmonmilt
play aSeveral
role inresearchers
physiologicalsuchcellularmetabolism,
nucleicacids
fromsalmon
milt
play
a
role
in
physiological
functionssuchascellularproliferation, differentiation, and apoptosis in human small intestinal epithelial metabolism,
cells [17,20,21].
proliferation,differentiation,
and apoptosis
in humansmall intestinal
epithelial
cells inflammation[17,20,21].
Additionally,
Sakai
et al. have reported
that dietaryribonucleic
acid (RNA)suppresses
Additionally,
Sakai
et
al.
have
reportedthat
dietaryribonucleic
acid
(RNA)
suppresses
inflammationin adipose tissue and improves glucose intolerance in mice fed a high-fat diet [22].
in adipose tissue and improves glucose intolerance in mice fed a high‐fat diet [22].
In this study, we examined the effect of DNA-rich nucleic acids prepared from salmon milt
In this study, we examined the effect of DNA‐rich nucleic acids prepared from salmon milt
(DNSM) on the development of liver fibrosis in an in vivo ethanol-CCl4 -induced cirrhosis rat model(DNSM) on the development of liver fibrosis in an in vivo ethanol‐CCl4‐induced cirrhosis rat modeland in an in vitro alcohol-injury hepatocytes model, and we found that DNSM protected hepatocytes
and in an in vitro alcohol‐injury hepatocytes model, and we found that DNSM protected hepatocytes
againstethanol
inducedliver
againstethanol
inducedliverinjury.
injury.

2. Results
2. Results
Figure1 shows
changes
the experimental
experimentalperiod.
period.
The
bodyweights
Figure1 shows
changesininbody
bodyweightweight during
during theThebodyweightsof ofethanolplus
CCl-treated
rats
and
0.12%
DNSM
dietandethanolplus
CCl-treated
rats
tended
4 4‐treated rats and 0.12% DNSM diet‐ and ethanol plus CCl4‐treated4ethanol plus CClrats tended to be to
be lowerthan those
those rats
rats fed
fed thethe control
controldiet
dietororCCl
CCl4 4alone.
alone.However,
However,these
differenceswerelower thanthese
differenceswere
not not
statisticallysignificant.
statisticallysignificant.

4,
FigureChangesininbody
bodyweight.
weight. #:○: Control
diet
with
5%5%
ethanol
plusplus
CClCClFigure1. 1.
Changes
Controldiet,diet,●: :Control
Controldietwithethanol
4,ᇞ:Control
diet
with
CCl
4, ■: 0.12% DNSM diet with 5% ethanol plus CCl4.
4: Control diet with CCl4 , : 0.12% DNSM diet with 5% ethanol plus CCl4 .

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We examined the effect of nucleoprotein treatment on plasma aspartate
aspartate aminotransferaseaminotransferase (AST)(AST)
and alanine aminotransferaseaminotransferase (ALT) activities. As
shownin
Figure2,
inthe ethanol plus CCl44
As
shown
in
Figure2,in
We examined the effect of nucleoprotein treatment on plasma aspartate aminotransferase (AST)(0.1 mL/kg
of bodygroup, plasma
AST
ALT activities
increasedby 1.8‐andmL/kg
body weight)‐treatedweight)-treatedplasma
AST andandactivities
increased
1.8- CCland alanine ofaminotransferase(ALT)group,
activities.
As shown
in ALT
Figure2, in theethanolbyplus
4
3.5‐fold,
respectively, compared to the control group.
these same
enzymeswere
3.5-fold,group. However,
However,enzymes
weresignificantly
significantly(0.1 mL/kg of body weight)‐treated group, plasma
AST and these
ALT same
activities
increasedby 1.8‐ andless active in the DNSM‐treatedplus CCl
CCl4‐treated group.
DNSM-treatedgroup
comparedto theethanoltheseplus
3.5‐fold, respectively,
compared tothe control
group.
However,same4 -treatedenzymes were significantly
less active in the DNSM‐treated group compared to the ethanol plus CCl4‐treated group.

Figureof DNSMDNSM ononserum
serumAST
ASTandandALT
ALTactivity
activityinin
ethanolplus
CCl
4‐treatedrats.
EffectFigure 2.
2. The
The effecteffect ofethanol
plus
CCl
rats.
Effectof
4 -treated
of
DNSM
serum
AST
activity;
and
serum
ALT
activity.
Data
presented
as themean± S.E.
DNSM
onon(A)(A)
serum
AST
activity;
and(B)(B)
serum
ALT
activity.
Data
arearepresented
as themean± S.E.
ofFigure 2. The effect of DNSM on serum AST and ALT activity in ethanol plus CCl4‐treated rats. Effectofactivityof rats.
five rats.
Valueswithout
a commonare significantly
different0.01).
thetheactivityof five
Valueswithout
a commonletter areletter
significantly
different (p< 0.01). (p#: <Controlof DNSM on (A) serum AST activity; and (B) serum ALT activity. Data are presented as the mean ± S.E.
ethanolCCl4, ᇞ:Control
diet4 ,with
CCl4, DNSM■: 0.12%DNSM○:
Control
diet, ●:
Controldietwith 5%diet,: Control
dietwith 5%ethanolplus
CCl , plus
4: Control
dietwith CCl
: 0.12%dietwithof the activity of five rats. Values without a4 common letter are significantly
different (p < 0.01).
dietwith 5%plus
ethanol
CCl4.
5% ethanol
CCl4plus.
○: Control diet, ●: Control diet with 5% ethanol plus CCl4, ᇞ: Control diet with CCl4, ■: 0.12% DNSM
diet with 5% ethanol plus CCl4.

Histological analysis was performed by hematoxylin and eosin staining as well as elastic vanHistological analysis was performed by hematoxylin and eosin staining as well as elastic
Gieson (EVG) staining and Mason’s trichrome staining to assess liver damage (Figure 3). No histologicalvan Gieson(EVG)
staining
Mason’sbytrichrome
staining
to assessliverasdamage(Figurevan
3).
Histologicalanalysis
wasandperformed
hematoxylin
and eosin
staining
well as elastic
abnormalities were observed in the control rats or CCl4‐treated rats, but collagen accumulation in theNohistologicalabnormalities
weretrichrome
observedstaining
in the tocontrolCCl4 -treatedcollagen
Gieson(EVG) staining
and Mason’s
assessrats
liverordamage(Figure rats,3). Nobut
histologicalliver and hepatic necrosis were observed in ethanol plus CCl4‐treated rats. However, treatment withaccumulationin theobserved
liver andhepatic
necrosis
observed
inbut
ethanolplusaccumulation
CCl4 -treatedinrats.
abnormalities werein thecontrolrats orwere
CCl4‐treatedrats,
collagenthe
DNSM fully protected the rats from liver fibrosis and necrosis induced by ethanol plus CCl4.
However,treatmentwith were
DNSM
fully protected
rats
from
liver rats.
fibrosisand necrosis
inducedliver and hepatic
necrosis
observed
in ethanoltheplus
CCl4‐treated
However,treatment
withbyethanolplus
CCl4 . the rats from liver fibrosis and necrosis induced by ethanol plus CCl4.
DNSM
fully
protected

Figure 3. Cont.

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4 of 13
4 of 124 of 12Figure 3.
3. Theeffect
of
DNSM
on
the
changesin livermorphology.
Liversections
were
processedfor
FigureTheeffecteffectof
ofDNSM
DNSMononthechangeslivermorphology.
Liversections
were
processed
Figure3. Thethechangesin inlivermorphology.
Liversections
were
processed
for
(A)
EVG
staining;
and(B)
Masson’strichrome
staining.
(a)Controldiet;(b)Control
diet
with
5%
for (A)
EVG
staining;
Masson’strichrome
staining.
Control
diet;(b)(b)Control
Controldiet
dietwith
with5%
5%(A)
EVG
staining;
andand(B)(B)
Masson’strichrome
staining.
(a)(a)
Controldiet;ethanol plus
plus CCl
CCl4; (c) Control diet with CCl4; (d) 0.12% DNSM diet with 5% ethanol plus CCl4.
ethanol plus
CCl44; (c) Control diet with CCl44; (d) 0.12% DNSM diet with 5% ethanol plus CCl44. .

Ethanol‐induced oxidativestressstress fromCYP2E1
CYP2E1 appearsto
to playaamajor
major roleinin ethanol‐induced
Ethanol‐induced
Ethanol-induced oxidativeoxidative stress fromfrom CYP2E1 appears
appears to play
play a major rolerole in ethanol‐induced
ethanol-induced
liver
injury[7–10].
We
have
previously
demonstrated
that
Yerba
mate
extract
suppressed
the CTP2E1
liver
liver injury
injury [7–10].
[7–10].We
Wehavehavepreviously
previouslydemonstrated
demonstratedthat
thatYerba
Yerba mate
mate extract
extractsuppressed
suppressed thetheCTP2E1
CTP2E1
activity
inducedbyethanol
in
both
in
vitro
and
in
vivo
models[16].
Therefore,
weexamined
whether
activity
activity induced
induced byby ethanol
ethanol inin bothboth inin vitro
vitro andand ininvivovivomodels
models[16].
[16].Therefore,
Therefore,weweexaminedexaminedwhether
whether
DNSM treatment
treatment could
could also
also have
have aa preventive
preventive effecteffect against alcohol‐induced
alcohol‐induced liver
liver injury
injury byby
DNSM
DNSM treatment
could also
have a preventive
effect againstagainst
alcohol-induced liver injury
by regulating
regulating
theCYP2E1
enzyme
in
an
in
vivo
model.
In
ethanolplus
CCl
4‐treated rats, CYP2E1 activity
regulating
theCYP2E1in an
in vivoethanolplus CClrats,
4‐treated
rats,activity
CYP2E1increased
activity
the CYP2E1
enzymein enzyme
an in vivo
model.
In model.
ethanolIn
plus
CCl4 -treated
CYP2E1increased
2.1‐foldcomparedtothecontrolgroup.
However,
DNSM‐treatments
suppressed
CYP2E1increased
2.1‐fold comparedto thegroup.
controlHowever,group. However,
DNSM‐treatments
suppressed
2.1-fold comparedto the control
DNSM-treatments
suppressed
CYP2E1CYP2E1
activity
activity (Figure4).
4).We
Weexaminedexaminedthe
the effectof
of DNSMon
onthe
theincrease
increasein
inintracellular
intracellularlipid
lipidperoxidation
peroxidation
activity(Figure (Figure4). We examinedthe effect ofeffect
DNSMDNSM
on the increase
in intracellularlipid peroxidation
using
using thethe thiobarbituric
thiobarbituric acidacid reactive
reactive substances
substances (TBARS)(TBARS) assay.
assay. In
In ethanol
ethanol plus
plus CCl4‐treated
CCl4‐treated rats,
rats,
using
the thiobarbituric acid reactive substances (TBARS) assay. In ethanol plus CCl4-treated rats, hepatic
hepatic TBARSTBARS levels
levels were
were significantly
significantly increased.
increased. However,
However, DNSM‐treatment
DNSM‐treatment maintained
maintained thethehepatic
TBARS levels
were significantly
increased. However, DNSM-treatment
maintained the intracellular
intracellular
TBARS
levelsatthelowerlevelsofthecontrolrats(Figure5).
intracellularlevels levels
at the lower
levels ofratsthe(Figure
control5).
rats (Figure 5).
TBARS levelsTBARS
at the lowerof the control

Figure 4. The effect of DNSM on CYP2E1 activity in the livers of ethanol plus CCl4‐treated rats.
FigureFigure 4.
4. TheTheeffecteffect ofof DNSMDNSM on
on CYP2E1
CYP2E1 activity
activity inin thethe livers
livers ofof ethanol
ethanol plus
plus CCl
CCl44‐treated-treated rats.
rats.
CYP2E1
activity waswas determined
determined using
using thethe $-nitrophenol
ρ‐nitrophenol (PNP)(PNP) assay,
assay, asas described
described inin thethe Materials
Materials
CYP2E1 activity
activity
CYP2E1was determinedusing theρ‐nitrophenol (PNP)
assay, asdescribed in
the Materials
and Methods.
Methods. (C)Control
diet; (ET)(ET) Control
Control diet
diet with
with 5%5% ethanol
ethanol plus
plus CCl
CCl4;; (T)(T) Control
Control diet
diet withwithand(C)Control
Control diet;
diet;
and
Methods. (C)(ET) Control
diet with
5% ethanol
plus CCl44; (T) Control
diet with
0.12% DNSM
DNSM diet
diet with
with 5% ethanol plus CCl4. Data
Data areare presented as the mean±± S.E.
S.E. ofof
CCl4;; (DNSM)
CCl(DNSM)0.12%
0.12%
44; (DNSM)
DNSM dietwith 5% ethanol plus CCl44. Data
are presented as the mean
mean ± S.E. ofCClfiveanimals.
Valueswithoutacommonletteraresignificantly
different(p<0.01).
five animals.
animals. Values
Values without a common letter are significantly different (p < 0.01).
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5 of 13
5 of 12Figure5. effect
The effectof DNSM
onperoxidation
lipid peroxidationin theThe measurementof lipid
Figure5. Theof DNSMon lipidin the liver.
Theliver.
measurementof lipid peroxidation
peroxidation using a colorimetric reaction with thiobarbitric acid (TBA) was carried out according tousing a colorimetric reaction with thiobarbitric acid (TBA) was carried out according to the method
the method described by Ohkawa. The measured lipid peroxidation was expressed as
described by Ohkawa. The measured lipid peroxidation was expressed as malondialdehyde (MDA).
malondialdehyde (MDA). (C) Control diet; (ET) Control diet with 5% ethanol plus CCl4; (T) Control(C) Control diet; (ET) Control diet with 5% ethanol plus CCl4 ; (T) Control diet with CCl4 ; (DNSM) 0.12%
diet with CCl4; (DNSM) 0.12% DNSM diet with 5% ethanol plus CCl4. Each bar is the mean ± S.E. of
DNSM diet with 5% ethanol plus CCl4 . Each bar is the mean ± S.E. of five animals. Values without afive animals. Values without a common letter are significantly different (p < 0.01).
common letter are significantly different (p < 0.01).

Furthermore, we measured the weights of various organs after the experimental period.
Asshown in Table
1, there weredifferencesamongorgans
the weights
liver,
kidney, andperiod.
spleen As
in rats
Furthermore,
we measured
thenoweights
of various
afteroftheexperimental
shownof theHowever,
the amongmasses theof epididymal
andkidney,
visceraland
fat spleenof ethanol
plusofCClin Table
1, four
theregroups.
were nodifferences
weights of fat
liver,
in ratsthe4‐four
treatedrats andthe
DNSM
diet‐
ethanol plus
CCl4visceral‐treated rats
werelowerplus
thanCCl
those
rats fed thegroups.
However,massesofand
epididymal
fat andfat ofethanol
4 -treated rats andcontroldietorCCl
4 alone. These results suggest that loss of body weight in ethanol plus CCl4‐treated
DNSM diet- and ethanol plus CCl4 -treated rats were lower than those rats fed the control diet or CCl4
rats and DNSM diet‐ and ethanol plus CCl4‐treated rats may depend on the loss of fat and the loss
alone.
These results suggest that loss of body weight in ethanol plus CCl4 -treated rats and DNSM
may be induced by ethanol ingestion.

diet- and ethanol plus CCl4 -treated rats may depend on the loss of fat and the loss may be induced byethanol ingestion.
Table 1. Changes in organ weight of rats.
Weight(g) of rats.
Table 1. Changes Organin organweightLiver
KidneySpleenVisceral Fat Epididymal Fat
C
10.82 ± 0.38 1.60 ± 0.03 0.76Organ± 0.04 Weight
6.52 ±(g)0.557.49 ± 0.61
ET
10.54
±
0.46
1.58
±
0.05
0.75
±
0.03
4.54
±
0.48
4.74
± 0.37
GroupsLiver
KidneySpleen 6.22VisceralFat 6.02 ±Epididymal
FatT
12.35 ± 0.47 1.70 ± 0.08 0.72 ± 0.02
± 0.520.20
C DNSM 10.82± ±0.380.03 0.70
0.76± 0.04 4.79 6.52
7.49 ± 0.61
10.76
0.34 1.60
1.66 ±± 0.02± 0.03± 0.52± 0.55 5.44 ± 0.52
ET diet; (ET)10.54
± 0.46diet with1.58 5%± 0.050.03
4.54 ± 0.48
4.74 ± 0.37
(C) Control
Controlethanol 0.75
plus ±CCl
4; (T) Control diet with CCl4; (DNSM) 0.12%
T
12.35
±
0.47
1.70
±
0.08
0.72
±
0.02
6.22
±
0.52
6.02 ± 0.20
DNSM diet with 5% ethanol plus CCl4.
DNSM
10.76 ± 0.34
1.66 ± 0.02
0.70 ± 0.03
4.79 ± 0.52
5.44 ± 0.52
Groupselucidatewhetherprotective
effectplus
of DNSM
isControl
dependent
on digestion,
we0.12%
measured(C)To
Control
diet; (ET)Controlthe
dietwith 5% ethanol
CCl4 ; (T)diet with
CCl4 ; (DNSM)
DNSMthe
diet
with
5%
ethanolplus
CCl.
effect of DNSM treatment in 4an in vitro alcohol‐induced injury model in hepatocytes. We previously
demonstrated that a treatment of 100 mM ethanol for 24 h significantly decreased cell viability ofhepatocytes
control cells[15].ofHere,weismeasured
theon
celldigestion,
viability of
To elucidatecomparedwhether withthe protective
effectDNSM
dependent
wehepatocytes
measured thetreatedwith
100mM
ethanol
withorwithout
variousconcentrations
of
DNSM.
As
shown
in
Figure6A,
effect of DNSM treatment in an in vitro alcohol-induced injury model in hepatocytes. We
previously
DNSM did not prevent cell death. These results suggest that digestive products of DNSM are effective
demonstrated that a treatment of 100 mM ethanol for 24 h significantly decreased cell viability offor the prevention of alcohol‐induced liver injury. Therefore, the following experiments were carried
hepatocytes compared with control cells [15]. Here, we measured the cell viability of hepatocytes
out with the presence of deoxyadenosine. Adenosine which is the digestive product of RNA also wastreated with 100 mM ethanol with or without various concentrations of DNSM. As shown in Figure 6A,
measured. Treatments of deoxyadenosine and adenosine prevented cell death in the hepatocytes
DNSM
did with
not prevent
death.
These
resultssuggestthethatdigestive
products ofand
DNSM
are effective
treated100 mM cellethanol(Figure6B).
Furthermore,
effectof deoxyadenosine
adenosineonfor theof alcohol-induced
injury.
Therefore,
following
experiments examined
were carried
theprevention
activity of alcohol
dehydrogenaseliver(ADH)—amainpathwaytheof alcohol
metabolism—wasout with
deoxyadenosine.
is thedigestive productof RNA also
in thethecellspresence
incubatedoffor
4 h with 100 mM Adenosine
ethanol. The which
treatmentof deoxyadenosine
and adenosine
was increased
measured.
Treatmentsofdeoxyadenosine
andadenosine
prevented
cell
death
in
the
the activities of ADH compared with the cells treated with 100mM ethanol (Figurehepatocytes
7).

treated with 100 mM ethanol (Figure 6B). Furthermore, the effect of deoxyadenosine and adenosine on
the activity of alcohol dehydrogenase (ADH)—a main pathway of alcohol metabolism—was examinedin the cells incubated for 4 h with 100 mM ethanol. The treatment of deoxyadenosine and adenosine
increased the activities of ADH compared with the cells treated with 100 mM ethanol (Figure 7).

Mar. Drugs 2016, 14, 232
Mar. Drugs 2016, 14, 232
Mar. Drugs 2016, 14, 232

6 of 126 of 13
6 of 12Figure6.
The
effect
DNSMononethanol‐treatedethanol‐treated hepatocyte
hepatocyte
were
incubated
FigureThe
effectofofDNSM
DNSM
onethanol-treatedhepatocytecell
cellviability.
viability.Hepatocytes
6. Theeffectofcellviability.
Hepatocytes
were
incubated
with
100
mM
ethanol
withorwithout(A)
various
DNSM
concentrations;and(B)
deoxyadenosineor orwith 100 mM ethanol with or without (A) various DNSM concentrations; and (B) deoxyadenosine
adenosine
for
24
h.
Cell
viabilitywasmeasuredbytheNeutralRed
assay,
asdescribed
intheMaterials
adenosine
viability was measured by the Neutral Red assay,
assay, as described in the Materials
adenosine forfor 24 h. Cell viability
andMethods
section.Data
Dataarearepresented
presentedas
asthe
the meanmean ±±± S.E.
withouta aandMethods
section.
S.E.ofexperiments.Values
without
the
mean
S.E.
of threethreeexperiments.
Valuescommonlettersignificantly
different(p<0.01).
commonletterarearesignificantly
different(p<0.01).
significantly different (p <Figure 7. Effects of adenosine or deoxyadenosine on ADH activity in ethanol‐treated hepatocytes.
Hepatocytes
were incubated
4deoxyadenosine
h with 100 mM ethanol,
with or without
25 μM deoxyadenosine or
Figure 7.
7. Effects
adenosinefororon
FigureEffects ofof adenosine
or deoxyadenosine
on ADHADH activity
activity inin ethanol‐treatedethanol-treated hepatocytes.
hepatocytes.
adenosine.
ADH
activity
analysiswasperformed
asdescribed
intheMaterials
and deoxyadenosine
Methods section. or
Hepatocytes were
were incubatedincubated forfor 44 hh withwith 100
100 mMmM ethanol,
ethanol, with
with oror without
Hepatocytes
without 25
25 μMµM deoxyadenosineorData are presented
as themean ±was
S.E. performed as described in the Materials and Methods section.
adenosine.
ADH
activity
analysis
adenosine. ADH activity analysis was performed as described in the Materials and Methods section.
Data areare presented
presented asas thethe meanmean ±± S.E.
Data
S.E.

Mar. Drugs 2016, 14, 232

7 of 13

3. Discussion
This study has shown that DNSM protects against ethanol-induced liver injury. Here, we
demonstrated that the liver damage biomarkers, ALT and AST, were increased in a rat model
given ethanol plus CCl4 to induce liver damage, but this increase was reduced by nucleic acidsupplementation. Furthermore, the DNSM treatment did not affect cell viability in ethanol-treatedhepatocytes, suggesting the digestive products, which are created by the degradation of DNSM by
nucleases in the intestine, are effective against alcohol-induced liver injury.
Alcohol-induced liver injury is induced by heavy drinking and is accompanied by thedegeneration or necrosis of hepatocytes, which disrupts normal liver function via oxidative stress.
The CYP2E1 enzyme is one of the major producers of ethanol-induced ROS. Therefore, decreasing or
inhibiting CYP2E1 activity may be a feasible strategy for minimizing the hepatotoxicity of ethanol.
Recently, we demonstrated that Ecklonia cava polyphenol-treatment maintained CYP2E1 activity inethanol-treated hepatocytes below that of control cells [23]. We also reported that treatment withan extract of Yerba Mate tea suppressed ethanol-induced increases in CYP2E1 activity to the levelof the control cells in an in vitro, alcohol-induced hepatocyte model and an in vivo ethanol plus
CCl4 -induced liver-injury model [16].
In the present study, we have shown that DNSM treatment suppressed ethanol-induced increases
in CYP2E1 activity to the activity levels observed in the control rats and that the treatment of
deoxyadenosine increased the ADH activity compared with the cells treated with ethanol in an
in vitro alcohol injury hepatocyte model.
There are several reports that adenosine and deoxyadenosine has a protective effect against
various diseases. Modis et al. reported that adenosine and its metabolite, inosine, exerted
cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury [24]. Hasemi et al.
have shown that adenosine and deoxyadenosine induces apoptosis in human breast cancer cells
via the activation of the mitochondria/intrinsic apoptotic pathway [25]. Furthermore, Lee reported
that adenosine protected Sprague-Dawley rats from a high-fat diet and repeated acute restraint
stress-induced intestinal inflammation and altered expression of nutrient transporters [26].
In the present study, the precise mechanism of the DNSM protection against ethanol-induced
liver injuries in rats is not clear. However, there are two possible explanations for this protection.
The first is via the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. There are some reportsthat the cAMP/PKA signaling pathway regulates the activities of alcohol dehydrogenase andCYP2E1 in ethanol metabolism [27,28]. The administration of theophylline to rats, which inhibits
cAMP phosphodiesterase and thus increases endogenous cAMP levels, or the addition of dibutyrylcAMP to hepatocyte cultures, both increased ADH activity [27]. Contrastingly, cAMP-dependent
phosphorylation of CYP2E1 lead to a reduction in CYP2E1 activity [28]. We have shown that thetreatment of Ecklonia cava polyphenol with ethanol increased the activity of alcohol dehydrogenase
and inhibits CYP2E1 activity [23]. The changes in CYP2E1 and alcohol dehydrogenase activity were
suppressed by treatment with H89, an inhibitor of PKA, suggesting that Eclonia cava polyphenol has aprotective effect against ethanol-induced liver injury in a cAMP-dependent manner.
Charest et al. showed that adenosine and AMP increased cAMP concentration by interacting withthe adenosine receptor [29,30]. These results suggest that DNSM was digested by nucleases in theintestine and absorbed as nucleosides and nucleobases. Adenosine and AMP subsequently bind to the
adenosine receptor, which activates adenylate cyclase.
Another possibility is the involvement of the adenosine monophosphate-activated protein kinase(AMPK) signaling pathway. AMPK is a sensor that regulates cellular metabolism and oxidative
stress [31,32]. Chronic alcohol consumption results in inhibition of the hepatic AMPK signaling
pathway by ethanol, which leads to steatosis [33]. Fat accumulation in hepatocytes leads to thedevelopment of fatty liver. With continued alcohol consumption, fatty liver may progress to hepatitis
and cirrhosis. Therefore, it is important to enhance AMPK pathway signaling to protect the liver
against diseases induced by ethanol. Shin et al. have reported that β-lapachone, a naturally occurringMar. Drugs 2016, 14, 232

8 of 13quinone, activated the AMPK pathway in ethanol-fed rats [34]. Wang et al. also indicated thatoligomeric proanthocyanidines, a class of flavonoid compounds, alleviated liver steatosis and damage
through AMPK activation against alcohol-induced liver steatosis and injury [35]. On the other hand,
Stenesen et al. reported that dietary adenine controlled adult lifespan via adenosine nucleotidebiosynthesis and AMPK activation [36]. Dietary adenine feeding increases the ratio of AMP:ATP and
ADP:ATP and activates AMPK. These results suggest that nucleoproteins activate the AMPK signaling
pathway in the liver.
It is important to know the precise mechanism of the DNSM protection against ethanol-induced
liver injuries in rats. Especially, the involvement of cAMP/PKA pathway or AMPK pathway in an
in vivo ethanol-carbon tetrachloride cirrhosis model need to be elucidated further.
4. Materials and Methods
4.1. Materials
DNA-rich nucleic acids from salmon milt (DNSM) were water-solubilized using nuclease andprotease. The nucleotide and amino acid composition of the DNSM is shown in Table 2. DNSM
were provided by Fordays Co., Ltd. (Tokyo, Japan) and L•S Corporation. Williams’ Medium E andβ-nicotinamide adenine dinucleotide hydrate were obtained from Sigma-Aldrich Co. (St. Louis,
MO, USA). Fetal bovine serum (FBS) was purchased from Nichirei Biosciences, Inc. (Tokyo, Japan).
The other chemicals used in this study were special-grade commercial products purchased from
WAKO Pure Chemical Co., Ltd. (Osaka, Japan).
Table 2. Composition of nucleotides and amino acids in DNSM.
Nucleotides *Amount (g/100 g)

50 -dCMP

50 -dAMP
50 -dTMP
50 -dGMP

6.01
9.15
9.26
6.93

Total

31.35

Amino Acids

Amount (g/100 g)

Arg
Lys
His
Phe
Tyr
Leu
Ile
Met
Val
Ala
Gly
Pro
Glu
Ser
Thr
Asp
Trp
Cys

17.80
2.66
0.65
0.89
0.88
1.95
1.25
0.60
2.12
1.95
4.11
2.62
3.48
2.49
1.27
2.24
0.20
0.25

Total

47.41

* The amounts of nucleotides were analyzed after treatment of nuclease P1 .

Mar. Drugs 2016, 14, 232

9 of 134.2. Animals
Male Wistar rats were purchased from Japan SLC Inc. (Shizuoka, Japan). The rats were housed at
a constant temperature and were allowed free access to water and standard rat chow (LaboMR stock,
Japan SLC, Inc. Shizuoka, Japan). All animal experiments followed our institution’s criteria for thecare and use of laboratory animals in research, which meet the guidelines for animal experimentation
at Osaka City University.
4.3. Animal Experiments
Male Wistar rats weighing 180–210 g were fed a standard laboratory diet and water ad libitum
until three days prior to the experiment. The rats were then fed a control diet for three days and divided
into four groups. Group 1 was the control; Group 2 was treated with ethanol and CCl4 ; Group 3 wastreated with CCl4 alone; Group 4 was treated with ethanol, CCl4 , and 0.12% DNSM. The compositionof each diet is presented in Table 3. CCl4 (0.1 mL/kg of body weight diluted with olive oil to 25%) was
administered by intraperitoneal injection twice a week (on Mondays and Thursdays), and 5% ethanol
was administered in the drinking water ad libitum. The rats were euthanized after three weeks.
Table 3. Composition of diets.
Components (g)Control

0.12% DNSM

Casein200
3397.486
132
100
70
50
35
10
2.5
0.014
0
1000

200
3
396.286
132
100
70
50
35
10
2.5
0.014
1.2
1000

L -CystineCornstarchα-Cornstarch
SucroseSoybean oil
Cellulose powder
Mineral mix (AIN-93G-MX) 1
Vitamin mix (AIN-93VX) 2
Choline hydrogen tartrate
t-Butylhydroquinone
DNSM
Total
1 Composition in g/kg diet:

Calcium Carbonate, 357; Potassium Phosphate, Monobasic, 196; Potassium
Citrate·H2 O, 70.78; Sodium Chloride, 74; Potassium Sulfate, 46.6; Magnesium Oxide, 24; Ferric Citrate,
6.06; Zinc Carbonate, 1.65; Manganese Carbonate, 0.63; Cupric Carbonate, 0.324; Potassium Iodate, 0.01;
Sodium Selenate, 0.01025; Chromium K Sulfate·12H2 O, 0.275; Ammonium Molybdate·4H2 O, 0.00795;
Sodium Silicate·9H2 O, 1.45; Lithium Chloride, 0.0174; Boric Acid, 0.0815; Sodium Fluoride, 0.0635; Nickel
Carbonate·4H2 O, 0.0306; Ammonium Vanadate, 0.0066; Sucrose, 221.0032; 2 Composition in g/kg diet:
Vitamin A Acetate (500,000 IU/g), 0.8; Vitamin D3 (400,000 IU/g), 0.25; Vitamin E Acetate (500 IU/g), 15;
Phylloquinone, 0.075; Biotin, 2; Cyanocobalamin, 2.5; Folic Acid, 0.2; Nicotinic Acid, 3; Calcium Pantothenate,
1.6; Pyridoxine-HCl, 0.7; Riboflavin, 0.6; Thiamin HCl, 0.6; Sucrose, 974.655.

4.4. Histological Analysis
Liver samples were collected from each rat, fixed in 10% buffered formalin fixative, and thendehydrated in a graded alcohol series. Following xylene treatment, the specimens were embedded inparaffin blocks and cut into 5-µm sections. Consecutive sections were stained with EVG and Masson’s
trichrome staining. The pathologist was blinded to the rats’ group assignments.
4.5. Liver Damage Biomarkers
The activity of plasma AST and ALT were estimated using a Transaminase CII-test kit(Wako, Japan).

Mar. Drugs 2016, 14, 232

10 of 134.6. CYP2E1 Activity Analysis
Livers were homogenized in nine volumes of tris HCl buffer (containing 0.25 M sucrose, pH 7.4)
using a Polytron 1600E (Central Science Trade Co., Inc., Tokyo, Japan). The homogenates were
centrifuged at 700× g for 10 min at 4 ◦ C. The supernatant was collected as an S9 fraction. The activity
of CYP2E1 was determined by the rate of hydroxylation of PNP measured at 546 nm [37]. The S9
fraction was added to 100 mM KH2 PO4 (containing 0.2 mM PNP and 2.0 mM NADPH, pH 6.8) andincubated in a 37 ◦ C water bath for 20 min. The reaction was stopped using 0.6 M perchloric acid(250 µL) and 10 M NaOH (75 µL) was added to the remaining supernatant. The results were expressedas the amount of p-nitrophenol pmols/min/mg protein formed and the determined concentration of
4-nitrocatechol (ε = 10. 28 mM−1 ·cm−1 ).
4.7. Measurement of Lipid Peroxidation
Lipid peroxidation was measured according to the method described by Ohkawa using acolorimetric reaction with thiobarbitric acid (TBA) and the measured lipid peroxidation was expressed
as malondialdehyde (MDA) [38]. The frozen liver samples were excised and homogenized in ninevolumes of ice-cold 1.15% KCl. Samples consisting of less than 0.2 mL of 10% (w/v) tissue homogenate
were then added to 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid solution adjusted
to pH 3.5 with NaOH, and 1.5 mL of a 0.8% aqueous solution of TBA. The mixture was brought to a
total volume of 4.0 mL using distilled water, mixed with 5.0 mL of a mixture of n-butanol and pyridine(15:1, v/v), and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer wascollected and its absorbance was measured at 532 nm. 1,1,3,3-Terramethoxypropane was used as anexternal standard. The level of lipid peroxidation was expressed in nmol of MDA.
4.8. Hepatocyte Preparation and Culture
Hepatocytes were isolated by collagenase perfusion following their removal from 10-week-old
male Wistar rats anesthetized with sodium pentobarbital [39]. The viability of the isolated hepatocytes
was greater than 90%, as determined by 0.2% trypan blue exclusion. The cells were plated on 35-mm
plastic dishes at a density of 2.5 × 105 cells/mL in 2 mL of Williams’ Medium E supplemented with
10% FBS. The cells were cultured in a humidified atmosphere (5% CO2 /95% air) at 37 ◦ C overnight.
After pre-incubation, the cells were cultured in 10% FBS containing fresh Williams’ Medium E withdifferent concentrations of ethanol, with or without DNSM, adenosine, or deoxyadenosine for 0–24 h.
4.9. Cell Viability Assay
The cell viability of the hepatocytes was measured by the Neutral Red assay, as previously
described [40]. Neutral Red stock solution (0.4% Neutral Red in water) was diluted 1:80 inphosphate-buffered saline (PBS). Hepatocytes were incubated with the Neutral Red solution for
2 h at 37 ◦ C to allow for the uptake of the lysosomal dye into viable cells. The Neutral Red solution
was then removed, and the cultures were washed rapidly (in under 2.5 min) with a mixture of 1%
formaldehyde-1% calcium chloride. A mixture of 1% acetic acid-50% ethanol was added to the cells atroom temperature for 30 min to extract the Neutral Red from the hepatocytes. The optical density ofeach sample was then measured at 540 nm with a spectrophotometer. Cell viability was estimated as apercentage of the value obtained for untreated controls.
4.10. Assay of ADH Activities
After incubation, the cells were washed twice and then dissolved with cold PBS. The debris
was obtained by centrifugation at 2600× g for 1 min at 4 ◦ C, and then buffer (50 mM HEPES pH 7.5,
0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol (DTT), 3 mM MgCl2 , 1 mM phenylmethylsulphonyl
fluoride) was added. After two freeze-thaw cycles using liquid nitrogen, the cells were sonicatedand centrifuged at 12,000× g for 20 min at 4 ◦ C. Finally, the supernatant was collected. ADH activity

Mar. Drugs 2016, 14, 232

11 of 13

was determined at 25 ◦ C in a 1.5 mL volume (50 mM HEPES pH 8.0, 10 mM MgCl2 , 1 mM DTT,
300 µM NAD+ ) in the presence or absence of ethanol (50 µL). The reaction was started by adding
ethanol, and the absorbance at 340 nm was followed with a spectrophotometer. The linear initial
increase in absorbance was used to determine specific enzyme activities with an absorption coefficientof 6.2 mM·cm−1 .
4.11. Statistical Analysis
Statistical comparisons were performed between groups using one-way analysis of variance andpost hoc multiple comparisons using Tukey’s test. A p-value less than 0.05 was considered significant.
5. Conclusions
In conclusion, we found that DNSM had protective effects against ethanol-induced liver injury.
Although its precise mechanisms need to be elucidated further, DNSM may represent a novel tool for
preventing alcohol-induced liver injury.
Acknowledgments: This study was partially supported by a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (24500987).
Author Contributions: A.K-Y. and I.M-Y. contributed to the concept of the study and the manuscript preparation.
A.K-Y., I.M-Y., H.S., K.S. and K.U. wrote the paper. A.K-Y., M.G., E.Y., Y.M., H.S. and K.S. performed experiments
and analyzed data.
Conflicts of Interest: The authors declare no conflict of interest.

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