Original Article

Yonago Acta medica 2016;59:37–43

Preventive Effect of Geraniol on Diethylnitrosamine-Induced 				Hepatocarcinogenesis in Rats
Shintaro Sawada, Jun-ichi Okano, Ryu Imamoto, Yuki Yasunaka, Ryo Abe, Masahiko Koda, Yoshikazu Murawaki
and Hajime Isomoto
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504, Japan

ABSTRACT

diethylenetriamine pentaacetic acid (Gd-EOB) MRI.
Therapeutic options for HCC including radiofrequency
ablation (RFA), transcatheter arterial chemoembolization (TACE), surgical resection and molecular-targeted
therapy (Sorafenib), have also increased. Despite theseadvances in diagnosis and treatment, the prognosis ofpatients with HCC remains unsatisfactory.2 In order toimprove the outcomes of patients with HCC, novel chemopreventive and therapeutic compounds are urgently
required.
Geraniol is a natural acyclic monoterpene containedin the essential oils of many aromatic plants such as
geranium and lemon.3 It is widely used as an aromaticcomponent in many cosmetic products. In addition to
its aromatic properties, geraniol has been reported todemonstrate anti-tumor activity in a variety of cancer
cells including lung,4 pancreatic,5 colon6 and liver.7 The
mechanisms of anti-cancer effects of geraniol include
inhibition of angiogenesis3 and the mevalonate pathway,4
modulation of the Ras-extracellular signal-regulated
kinase (ERK) signaling pathway8 and inhibition of NF9
KB, then leading to the modulation of cellular proliferation and apoptosis. However, little is known about geraniol inhibiting hepatocarcinogenesis in vivo.
Diethylnitrosamine (DEN) is an established hepatocarcinogen in rats.10 DEN has been reported to induce
hepatocarcinogenesis by alteration of the DNA structure,formation of alkyl DNA adducts and induction of chromosomal aberrations and micronuclei in the liver.11 In
the present study, we evaluated anti-hepatocarcinogenic
effects of geraniol in DEN-induced hepatocarninogenic
rats.

Background

Geraniol is a plant-derived phytochemical possessing anti-cancer action. The anti-carcinogenic
effect of geraniol was investigated in the diethylnitrosamine (DEN)-induced hepatocarcinogenic rat model.
Methods Male Wistar rats were intraperitoneally injected with 300 μL of phosphate-buffered saline (PBS)(G1; n = 4) or DEN (100 mg/kg body weight) dissolved
in PBS (G2; n = 8) every 2 weeks on experimental
weeks 2, 4 and 6. The rats were treated with a low concentration (0.07%) of geraniol (G3; n = 9) and high concentration (0.35%) of geraniol (G4; n = 7) for 12 weeks.
To evaluate the effects of geraniol on the DEN-induced
hepatocarcinogenesis, we compared the relative liver
weight, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase(ALP) levels and expression levels of proliferating cell
nuclear antigen (PCNA) and glutathione S transferase-P(GST-P) by immunohistochemical analyses among eachgroup.
Results Relative liver weight was significantly higher
in G2 than in G1 (P < 0.01). Both serum AST and ALTlevels were significantly higher in G2 than in G3 and in
G4 (P < 0.05). Serum ALP levels did not show a significant difference among each group. Percentages of both
PCNA- and GST-P- positive area were significantly decreased in G3 and in G4 compared to in G2 (P < 0.001,
respectively), suggesting anti-hepatocarcinogenic effects
of geraniol.
Conclusion Geraniol is a promising compound usefulfor suppression of hepatocellular carcinoma. The mechanisms of action are required to be clarified in the future
intensive study.
Key words

diethylnitrosamine; geraniol; hepatocellu-

lar carcinoma

Corresponding author: Shintaro Sawada, MD
memento_mori_0524@yahoo.co.jp
Received 2015 December 7
Accepted 2015 December 18
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DEN, diethylnitrosamine; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; PBS, phosphate-buffered saline; PCNA, proliferating cell
nuclear antigen

Hepatocellular carcinoma (HCC) is one of the mostcommon malignancies in Japan, responsible for overthirty thousand deaths per year.1 Diagnostic imaging for
HCC in its early stage has progressed including contrastenhanced ultrasound and gadolinium ethoxybenzyl
37

S. Sawada et al.

MATERIALS AND METHODS

body weight) dissolved in PBS every 2 weeks on experimental weeks 2, 4 and 6. In order to examine the anticancer effects of geraniol on the liver, the rats in group 3
and 4 were fed with 0.07% and 0.35% geraniol in liquid
form, respectively during the experimental period (Fig.
1).
One week subsequent to the final treatments, the animals were sacrificed under anesthesia using pentobarbital. Blood samples were collected via cardiac punctureand serum samples were stored at –30 ˚C until analysis.
Immediately following the excision of the livers, thelivers were weighed and portions of the samples werestored in 10% neutral buffered formalin for histologicalexamination.

Chemicals

Geraniol and PBS were purchased from Cosmo Bio (Tokyo, Japan). DEN was purchased from Sigma Aldrich(St. Louis, MO). Pentobarbital was obtained from Dainippon Sumitomo Pharma (Osaka, Japan). Antibodies
against proliferating cell nuclear antigen (PCNA) andglutathione S‑transferase (GST)-P were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA) and Assay
Designs (Ann Arbor, MI), respectively.
Animals, treatments and tissue collection

Male Wistar rats weighing 200 g or less were purchasedfrom Japan SLC. (Hamamatsu, Japan). The rats wererandomized, divided into four groups and were housed
two per cage with rice husks for bedding in an air‑ventilated room under a 12‑h light/dark cycle. In the animalroom, the temperature (22 ˚C) and humidity (55%) were
kept constant. The animals were allowed free access tofood and tap water ad libitum during the experiment.
All animals received humane care and protocols wereapproved by the Animal Ethics Committee of Tottori
University (approval number 13-Y-35).
As a control group, animals in group 1 (G1) wereintraperitoneally injected with 300 μL of PBS (n = 4)
every 2 weeks on experimental weeks 2, 4 and 6. Inorder to generate a hepatocarcinogenic model, animals
in groups 2 (G2, n = 8), 3 (G3, n = 9) and 4 (G4, n = 7)
were intraperitoneally injected with DEN (100 mg/kg

Measurement of serum transaminase and alkaline
phosphatase levels

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)levels were measured at SRL. (Tokyo, Japan).
Histology and immunohistochemistry

The rat liver tissues embedded in paraffin were stainedwith hematoxylin and eosin. For immunohistochemistry with the PCNA and GST-P antibodies, Histofine
Simple Stain Rat MAX PO was employed (Nichirei
Biosciences, Tokyo, Japan). Briefly, after routine dewaxing with xylene and hydration through a graded ethanol
series, the sections were incubated with 1.5% hydrogen
peroxide solution for 15 min at room temperature toquench endogenous peroxidase activity. The sections
were washed in PBS, blocked with 1.5% serum solution

sacrifice	
PBS	

G1	

1	

2	

3	

4	

5	

6	

7	

8	

9	

10	
 11	
 12	

weeks	

normal diet/day	

G2	

1	

2	

3	

4	

5	

6	

7	

8	

9	

Rela&ve	
  liver	
  weight	
  (%)	

100 mg/kg DEN	
10	
 11	
 12	

normal diet/day	

100 mg/kg DEN	

G3	

1	

2	

3	

4	

5	

6	

7	

8	

9	

10	
 11	
 12	

0.07％ geraniol in diet/day	

100 mg/kg DEN	

G4	

1	

2	

3	

4	

5	

6	

7	

8	

9	

10	
 11	
 12	

0.35％ geraniol in diet/day	Fig. 1. Experimental schedules.
Figure 1	
Animals in group 1 (G1) were intraperitoneally injected with 300
μL of PBS (n =4) once every 2 weeks on experimental weeks 2, 4
and 6. Animals in groups 2 (G2, n = 8), 3 (G3, n = 9) and 4 (G4, n= 7) were intraperitoneally injected with DEN (100 mg/kg bodyweight) dissolved in PBS once every 2 weeks on experimental
weeks 2, 4 and 6. The animals in group 3 and 4 were treated with
0.07% and 0.35% geraniol in liquid form, respectively, during 12
weeks. DEN, diethylnitrosamine; PBS, phosphate-buffered saline.

**	

**	

**	

3.8	
3.6	
3.4	
3.2	
3.0	

G1	

G2	

G3	

G4	

2	
 among the groups (G1,
Fig. 2. The relative liver weightFigurecompared
G2, G3 and G4) demonstrated that the relative liver weight of the
DEN groups (G2, G3 and G4) was significantly higher than thatof the control group (G1). G1; PBS, G2; DEN, G3; DEN+0.07%
geraniol, G4; DEN+0.35% geraniol. **P < 0.01. DEN, diethylnitrosamine; PBS, phosphate-buffered saline.

38Geraniol on diethylnitrosamine-induced hepatocarcinogenesis

A

*

450	

B

*

ALT	
  (IU/L)	

AST	
  （IU/L）	

400	
350	
300	
250	
200	

G1	

G2	

G3	

*

130	
120	
110	
100	
90	
80	
70	
60	
50	

G1	

G4	

Figure 3A	

G3	

G4	

G3	

G4	

Figure 3B	

C

and incubated with primary antibodies overnight at 4
°C. After rinsing with PBS, the sections were incubatedwith biotinylated secondary antibody for 30 min at room
temperature and horse HRP-conjugated ABC solution(Vector Laboratories, Burlingame, CA) was applied for
30 min at room temperature. Peroxidase activity wasdeveloped with DAB solution (Vector Laboratories).
Counterstaining was performed with hematoxylin. The
PCNA labeling indices were represented as the percentage of positively stained nuclei by counting 1000 cells ina field at x200 magnification. The GST-P-positive area
was measured on images captured by a Charge CoupledDevice (CCD) camera on a Windows computer.

G2	

*

1200	

ALP	
  (IU/L)	

1100	
1000	
900	
800	
700	
600	
500	

G1	

G2	

Figure 3C	
Fig. 3. Serum AST (A), ALT (B)and ALP (C) levels comparedamong the groups (G1, G2, G3 and G4) demonstrated that serumtransaminase levels were significantly higher in the DEN administration group G2 than in the geraniol treatment groups G3 and G4(P < 0.05) and that serum ALP levels did not show any significant
differences among the DEN groups G2, G3 and G4. *P < 0.05.
ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST,
aspartate aminotransferase; DEN, diethylnitrosamine.

Statistical analysis

Values are expressed as mean ± SD. Means were compared using the Student t-test. Statistical significancewas inferred at P < 0.05.
RESULTS
Relative liver weight and serum liver enzyme levels

The relative liver weight (liver weight/body weight) was
significantly higher in the DEN groups (G2, G3 and G4)
than in the control group (G1) (Fig. 2, P<0.01), presumably due to the development of liver tumors. Among the
DEN groups G2, G3 and G4, the relative liver weight
did not show a significant difference. Both serum transaminases (AST and ALT) were significantly higher inthe DEN administration group G2 than in the geraniol
treatment groups G3 and G4 (P < 0.05, Figs. 3A and B).
Serum ALP levels did not show significant differencesamong the DEN groups G2, G3 and G4 (Fig. 3C). These
data suggest that both low (0.07%) and high (0.35%)
concentrations of geraniol attenuated the liver injury andhepatic tumor formation.

Macroscopic and histological examinations

Macroscopic and microscopic features of the liver were
evaluated. As expected, in the liver of control rats (G1),
no tumors were observed (Figs. 4A and B). Multiple
white tumor formation was macroscopically observed in
the DEN groups G2, G3 and G4 (Fig. 4A, arrows).
In the histological analysis, the white nodules induced by DEN were hyperplastic nodules (Fig. 4B).
Expression levels of PCNA and GST-P

PCNA is an essential regulator of the cell cycle and its
expression is a useful tool for the study of cell proliferation.12 Among the glutathione S-transferases (GSTs), a
family of detoxification enzymes catalyzing the conjuga39

S. Sawada et al.

A

B

G1	

G2	

G3	

G4	

G1	

Figure 4A	

G3	

G2	

G4	

Figure 4B	

Fig. 4. Representative macroscopic (A) and microscopic (B) features of the liver in the four groups (G1, G2, G3 and G4) demonstrated thatno tumors were observed in the liver of control rats (G1) and that multiple white tumor formation was macroscopically observed in the
DEN groups G2, G3 and G4. In the histological analysis, the white nodules induced by DEN were hyperplastic nodules (hematoxylin andeosin staining, Bars express 100 µm). Arrows indicate the representative nodules. DEN, diethylnitrosamine.

40

Geraniol on diethylnitrosamine-induced hepatocarcinogenesis

5	
4	
3	
2	
1	
0	

G2	

G3	
***	

G4	

G1	

G1	

G2	

G2	

G3	

***	

GST-­‐P	
  (%)	

PCNA	  (%)	

G1	

G3	

G4	

60	
50	
40	
30	
20	
10	
0	

***	

G1	

G2	

G4	

***	

G3	

G4	

Figure 5	
Fig. 5. Expression levels of PCNA examined by immunohistochemical analysis in the four groups (G1, G2, G3 and G4) demonstrated that no positive cells were observed in the control livers
and that administration of DEN induced the nuclear expression of
PCNA (G2), which was significantly attenuated by the treatment
with both low (0.07%, G3) and high (0.35%, G4) concentrationsof geraniol. ***P < 0.001. Bars express 200 µm. Arrows indicate
representative PCNA-positive nuclei. DEN, diethylnitrosamine．
PCNA, proliferating cell nuclear antigen.

Figure 6	
Fig. 6. Expression levels of GST-P examined by immunohistochemical analysis in the four groups (G1, G2, G3 and G4) demonstrated that no positive area was observed in the control livers
and that administration of DEN induced GST-P-positive area(G2), which was significantly inhibited by the treatment with bothlow (0.07%, G3) and high (0.35%, G4) concentrations of geraniol,
revealing no significant difference between G3 and G4. ***P <0.001. Bars express 200 µm. Arrows indicate representative GSTP-positive area. DEN, diethylnitrosamine; GST-P, glutathione S
transferase-P.

tion of glutathione with a large number of carcinogens,placental GST (GST-P) is specifically expressed during
rat hepatocarcinogenesis.13
In order to confirm the anti-tumor effects of geraniol, we evaluated the expression levels of PCNA and
GST-P by immunohistochemical analyses. Immunostaining with the PCNA (Fig. 5) and GST-P (Fig. 6)
antibodies revealed that no positive cells or areas wereobserved in the control livers (Figs. 5 and 6, G1). Administration of DEN induced the nuclear expression of
PCNA (Fig. 5, G2). Treatment with both low (0.07%)
and high (0.35%) concentrations of geraniol significantly
attenuated the percentages of PCNA-positive cells in G3
and G4 compared to G2 (Fig. 5, P < 0.001).
Immunostaining with the GST-P antibody revealed
mostly similar results as PCNA. Administration of DEN
induced the appearance of a GST-P-positive area (Fig.
6, G2). The GST-P-positive area was significantly decreased in G3 and G4 compared to G2 (Fig. 6, P < 0.001),
although there was no significant difference between G3
and G4. These results indicate that geraniol could be apotent compound useful for the prevention of HCC.

DISCUSSION
HCC is the third-leading cause of death from cancer
and the fifth most prevalent malignancy worldwide.14,15
Since most HCC is complicated with diseased liver including chronic hepatitis and liver cirrhosis secondary
to infection with hepatitis B virus (HBV), hepatitis C
virus (HCV) and alcohol, regular surveillance of thesepatients who are at high risk of HCC by serum tumor
markers and imaging has been conducted in Japan.16
However, the prognosis of patients with HCC remains
poor because HCC is often diagnosed at advanced
stages due to the inefficiency of ultrasonography instrument operators, dropout among the patients targeted by
the screening program17 and the presence of a number ofundiagnosed carriers of HBV and HCV18 which are difficult to include in the screening program. Thus, novel
preventive and therapeutic approaches for these patientsare urgently required.
Geraniol is an isoprenic derivative contained in theessential oils of many aromatic plants such as geranium
and lemon.3 Beside its cosmetic use, geraniol has been
demonstrated to have some bioactive properties including anti-inflammation,19 anti-angiogenesis3 and antitumor effects.5 Anti-tumor effects of geraniol have been
41

S. Sawada et al.

demonstrated on a variety of cells including lung,4 pancreatic,5 colon6 and liver.7 In the liver, anti-tumor effects
of geraniol have been demonstrated in culture cells7, 20
and rats.21–23
Thus geraniol is a promising candidate for the prevention and therapy of HCC. However, the number ofstudies investigating the anti-tumor effects of geraniol
on hepatocarcinogenesis is quite limited. Therefore, wesought to further examine the anti-hepatocarcinogenic
effects of geraniol employing the DEN-induced hepatocarcinogenic rat model which we have previously
established.24–26 In the present study, we have demonstrated that oral treatment by geraniol of the DENinduced hepatocarcinogenic rats significantly decreased
serum transaminase levels and tumor formation of theliver based on the immunohistochemical analyses by
the PCNA and GST-P antibodies. We determined theconcentrations of geraniol based on a previous report.22
However, appropriate concentrations of geraniol in humans need to be evaluated in future studies.
In the current study, we did not explore the mechanisms of antitumor action by geraniol. Although information available is limited, induction of apoptosis wasinvolved in the anti-proliferative effects of geraniol inrats.21, 22 In general, the proposed mechanisms by whichmiscellaneous plant-derived chemicals exhibit antitumor effects on the liver include cell cycle arrest, induction of apoptosis and inhibition of angiogenesis.27 It
is conceivable that geraniol, a plant-derived compound,also modulated these cellular mechanisms. It is essential to investigate the modulation of signaling pathways
which are relevant to the cell cycle, apoptosis and angiogenesis in DEN-administered rats after treating themwith geraniol in future studies.

24875281.
5 Jin X, Sun J, Miao X, Liu G, Zhong D. Inhibitory effect ofgeraniol in combination with gemcitabine on proliferationof BXPC-3 human pancreatic cancer cells. J Int Med Res.
2013;41:993-1001. PMID: 23801065.
6 Carnesecchi S, Bras-Gonçalves R, Bradaia A, Zeisel M,
Gossé F, Poupon MF, et al. Geraniol, a component of plant essential oils, modulates DNA synthesis and potentiates 5-fluorouracil efficacy on human colon tumor xenografts. Cancer
Lett. 2004;215:53-9. PMID: 15374632.
7 Crespo R, Montero Villegas S, Abba MC, de Bravo MG,
Polo MP. Transcriptional and posttranscriptional inhibition of
HMGCR and PC biosynthesis by geraniol in 2 Hep-G2 cellproliferation linked pathways. Biochem Cell Biol. 2013;91:1319. PMID: 23668785.
8 Chaudhary SC, Siddiqui MS, Athar M, Alam MS. Geraniol
inhibits murine skin tumorigenesis by modulating COX-2
expression, Ras-ERK1/2 signaling pathway and apoptosis. J
Appl Toxicol. 2013;33:828-37. PMID: 22760862.
9 Ahmad ST, Arjumand W, Seth A, Nafees S, Rashid S, Ali N,
et al. Preclinical renal cancer chemopreventive efficacy of geraniol by modulation of multiple molecular pathways. Toxicology. 2011;290:69-81. PMID: 21907755.
10 Hussein UK, Mahmoud HM, Farrag AG, Bishayee A. Chemoprevention of diethylnitrosamine-initiated and phenobarbital-promoted hepatocarcinogenesis in rats by sulfated polysaccharides and aqueous extract of ulva lactuca. Integr Cancer
Ther. 2015;14:525-45. PMID: 26130745.
11 Verna L, Whysner J, Williams GM. N-nitrosodiethylamine
mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation. Pharmacol
Ther. 1996;71:57-81. PMID: 8910949.
12 Alenzi FQ, El-Nashar EM, Al-Ghamdi SS, Abbas MY,
Hamad AM, El-Saeed OM, et al. Investigation of Bcl-2 and
PCNA in hepatocellular carcinoma: Relation to chronic HCV.
J Egypt Natl Canc Inst. 2010;22:87-94. PMID: 21503011.
13 Sakai M and Muramatsu M. Regulation of glutathione transferase P: a tumor marker of hepatocarcinogenesis. Biochem
Biophys Res Commun. 2007;357:575-8. PMID: 17434454.
14 El-Serag HB and Rudolph KL. Hepatocellular carcinoma:
epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557-76. PMID: 17570226.
15 Farazi PA and DePinho RA. Hepatocellular carcinoma
pathogenesis: from genes to environment. Nat Rev Cancer.
2006;6:674-87. PMID: 16929323.
16 Makuuchi M, Kokudo N, Arii S, Futagawa S, Kaneko S,
Kawasaki S, et al. Development of evidence-based clinicalguidelines for the diagnosis and treatment of hepatocellular
carcinoma in Japan. Hepatol Res. 2008;38:37-51. PMID:
18039202.
17 Okano J, Sawada S, Imamoto R, Abe R, Fujise Y, Murawaki Y.
Challenges of diagnosing primary hepatocellular carcinoma. J
Tottori med Ass. 2014;42:10-6. Japanese.
18 Tanaka J, Koyama T, Mizui M, Uchida S, Katayama K, Matsuo J, et al. Total numbers of undiagnosed carriers of hepatitis
C and B viruses in Japan estimated by age- and area-specificprevalence on the national scale. Intervirology. 2011;54:18595. PMID: 21454956.
19 Khan AQ, Khan R, Qamar W, Lateef A, Rehman MU, Tahir M,
et al. Geraniol attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation inmouse skin: possible role of p38 MAP Kinase and NF-κB.
Exp Mol Pathol. 2013;94:419-29. PMID: 23399806.

The authors declare no conflict of interest.

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