Phytomedicine 10: 292299, 2003

Urban & Fischer Verlag

http://www.urbanfischer.de/journals/phytomed

Phytomedicine

Inhibition of DNA synthesis by Carvacrol in mouse

myoblast cells bearing a human N-RAS oncogene
H. Zeytinoglu1, Z. Incesu2, and K. H. C. Baser3
1

Department of Biology, Faculty of Sciences, Anadolu University, Eskisehir, Turkey

Department of Biochemistry, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey
3
Medicinal and Aromatic Plant and Drug Research Center (TBAM), Anadolu University, Eskisehir, Turkey
2

Summary
Monoterpenes are dietary components found in the essential oils of a wide variety of plants.
A number of these monoterpenes have antitumor activity. We have investigated the effects of carvacrol obtained by fractional distillation of Origanum onites L. essential oil, on DNA synthesis of
N-ras transformed myoblast cells, CO25. Incubation of the cells with different doses of carvacrol
prevented DNA synthesis in the growth medium and ras-activating medium, which contains dexamethasone. This result demonstrates that carvacrol inhibits growth of myoblast cells even after
activation of mutated N-ras oncogene, suggesting the possibility that carvacrol may find application in cancer therapy.
Key words: Carvacrol, inhibition of DNA synthesis, mouse myoblast cells, human N-RAS oncogene

j Introduction
Carvacrol, the predominant monoterpenic phenol
which occurs in many essential oils of the family Labiatae including Origanum, Satureja, Thymbra, Thymus
and Corydothymus species used through the ages as a
source of flavour in food (Krimer et al., 1995). Substantial antibacterial, antifungal and insecticidal effects
of carvacrol on different organisms have been described in several studies (Didry et al., 1994; Rice and
Coats, 1994; Thomson, 1996; Ultee et al., 1998). Some
other activities such as analgesic (Aydin et al., 1996)
and antioxidant (Aeschbach et al., 1994) activities also
have been reported. In specifically, Ultee et al., (1999)
showed the interaction of this compound with the cytoplasmic membrane by changing its permeability for
protons and potassium ions and inhibition of diarrheal
toxin production from B. cereus by carvacrol has been
found (Ultee and Smid, 2001).
Effects of several terpenes have been shown in various types of tumorigenesis. Farnesol from this group
0944-7113/03/10/04-292 $ 15.00/0

showed a selective inhibitory effect on the growth of

human neoplastic cell lines by preventing of phosphatidylcholine biosynthesis (Melnykovych et al.,
1992; Adany et al., 1994). Treatment of Hep-2 cells derived from a human larynx carcinoma induced the
apoptotic phenotype (Stammatia et al., 1999). Recently, inhibitory effects of carvacrol on DMBA induced
tumorigenesis in rats and on the growth of melanomas
in vitro were reported (Zeytinoglu et al., 1998; He
et al., 1997). A well-studied monoterpene, limonene,
was also shown to have chemopreventive and
chemotherapeutic activity against chemically induced
mammary, lung and stomach cancer in rodents
(Elegbede et al., 1984; Wattenberg and Coccia, 1991;
Gould et al., 1994; Gelb et al., 1995). Limonene and its
metabolites were shown to inhibit isoprenylations of
p21ras, product of ras proto-oncogenes, which are well
documented for their activity in carcinogenesis
(Zeytinoglu et al., 1993, 1995; Bos, 1989; Marshall,

Inhibition of DNA synthesis by Carvacrol in mouse myoblast cells

1991), by disrupting the biological activity in NIH3T3
cells (Crowell et al., 1991).
In our study, a mouse muscle cell line (CO25) bearing a glucocorticoid inducible mutated human N-ras
oncogene was used. The cell line when grown in a
weakly mitogenic medium (containing 10% horse
serum) proceeds to fuse after 4 days and forms
branched myotubes after 710 days. On the other hand,
the cells fail to differentiate when grown in the presence of dexamethasone (Gossett et al., 1988). Here, the
cytotoxicities of carvacrol and range of effective doses
on DNA synthesis were determined in CO25 cells before or after N-ras activation. Induction of ras protein
was analysed by Western blotting. We report that carvacrol has an inhibitory effect on the proliferation of
CO25 cells.

j Materials and Methods

Plant Extract

The plant extract examined in this study, carvacrol

(2-methyl-5- (1-methyl ethyl) phenol), was isolated
from stem distillated essential oil of Origanum onites
L. collected from West Anatolia (Fig. 1). A fractional
distillation was performed using a lab-size glass fractional distillation unit containing column packed with
S/S Knit Mesh packing material (2.8 cm 1.35 m). Reflux ratio was adjusted at 10/1 to 20/1 and the medium
pressure was 810 mm Hg. Carvacrol-rich fractions
were bulked to obtain carvacrol with 99% purity (GCMS) (Azcan et al., 2000).

Fig. 1. The structural formula of carvacrol (2-methyl5(1-methylethyl) phenol).

293

Cell Culture

Dr. I. Gibson (University of East Anglia, UK) kindly provided the mouse skeletal muscle cell line (CO25). Cells
were maintained in Dulbecco Modified Eagle Medium
(DMEM) (Sigma) supplemented with 10% (v/v) of
foetal calf serum (FCS) (Gibco), penicillin/streptomycin
at 100 units/ml and 1% L-glutamine as adherent monolayers. Cells were incubated at 37 C under 5% CO2/95%
air in a humidified atmosphere.
The CO25 cells were derived by transfection of the
parental C2 cells with a plasmid containing a mutational activated human N-ras gene (in codon 61) under
transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal
repeat (MMTV-LTR). Therefore, to induce transformation, 1 M dexamethasone (Dex) (Sigma) was added to
the cells grown in 10% horse serum (HS). To induce
differentiation, the cells at approximately 80% confluence were switched to DMEM containing 10% HS
with low mitogenic activity as a fusion-promoting
medium (Gosset et al., 1988; Gerhart et al., 2001).
Immunoblotting

Cells were washed twice with ice-cold PBS and lysed

in a buffer containing 1% NP-40, 50 mM Tris base, pH
8.0, 50 mM NaCl, 50 g/ml Leupeptin and 1 mM
phenylmethyl sulphonyl fluoride. Amount of protein in
each sample was estimated by the method of Bradford
(1976). Aliqouts of cell extracts containing 40 g of
protein were mixed with a sample buffer (0.4 M Tris,
0.4 M dithiothreitol, 8% sodium dodecyl sulfate, 40%
glycerol and 0.04% bromophenol blue, pH 6.8) and
then separated by a 12% SDS-PAGE according to
Zeytinoglu et al. (1995). The proteins were transferred
onto a nitrocellulose membrane in a buffer containing
0.025 M Tris, 0.192 M glycine, 20% methanol, pH 8.5.
The blot was incubated with a mouse polyclonal antibody to p21ras 142EO5 (gift from Dr. P. Hawley, UEA,
Norwich, UK) and then with a Rabbit anti-mouse Ig
(HRP) (Dako Ltd.). The immunoblot was visualized by
enhanced chemiluminescence detection system (Amersham).

Fig. 2. Expression of p21N-ras in CO25 myoblasts transfected a steroid-inducible N-ras oncogene was determined by Western
blot analysis as described in Material and Methods. Cells were grown in 10% HS with or without 1 M of Dex for 14 days.
Exposure time of ECL treated blot is 3 seconds.

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H. Zeytinoglu et al.

MTT Dye Reduction Assay

CO25 cells in exponential growth phase were harvested and the cell number was determined using a haemocytometer. Samples were resuspended in fresh medium
to give a density of 2.5 104/ml and then various concentrations of carvacrol dissolved in DMSO (1 g/ml,
5 g/ml, 10 g/ml, 50 g/ml, 100 g/ml, 150 g/ml)
were added to the cells. As a solvent control, 0.1%,
0.5%, 1%, 5%, 10% and 15% of DMSO were added to
the parallel cells. Aliquots (200 l) of the cell suspen-

sions were placed into each of 88 wells of a 96 well microtitre plate and incubated for periods of time. At the
end of the exposure time, 20 l of MTT dye solution
(5 mg/ml in sterile PBSA) was added to each well and
the plates were incubated for a further 2 h. The medium
was removed and the dye uptaken by cells was then
solubilised by addition of 200 l of DMSO to each
well. Absorbance at 540 nm was determined by use of a
Dynatech, MR5000 (Dynatech Lab, USA) plate reader
with a reference beam of 690 nm. Each dose of carvacrol was repeated 4 times per experiment. The results
of repeat wells within the same experiment were averaged.
Analysis of DNA Synthesis

Fig. 3. Toxicity of carvacrol for CO25 cells. Cells were seeded at 2.5 104/ml and incubated with 1, 5, 10, 50, 100 and
150 g/ml concentrations of carvacrol or with 1, 5, 10, 50,
100 and 150 l/ml of DMSO as solvent controls. At each time
point, MTT assay was performed for quantization of viable
cells. Results are the mean of quadruplicate wells. s Day 1,
n Day 2; h Day 3; m Control (DMSO).

Cell proliferation assay was performed in 96-well

plates (Falcon, Beckton Dickinson) and the BrdU colorimetric kit (Boehringer Mannheim) was used to determine the DNA synthesis by the method as given by
the manufacturer. CO25 cells cultured as detailed previously (Zeytinoglu et al., 1993) were detached with
0.25% trypsin/EDTA and 1 103 cells/ml were transferred into each well containing 10% FCS, 10% HS
and 10% HS plus 1 M Dex.
To investigate the effects of carvacrol on DNA synthesis, the cells were incubated with various concentrations (10 g/ml, 5 g/ml and 1 g/ml) of carvacrol for
various periods of time those were chosen according to
MTT assay as described above. After each day, the
cells were labeled with 10 l of BrdU solution at 37 C
for 2 hours and then fixed with the addition of fixdenat
solution for 30 min at room temperature. After removing the fixdenat solution, cells were treated with 100 l
of anti-BrdU working solution for 90 min at room temperature. Then the cells were washed three times with

Fig. 4. Measurement of BrdU incorporation

in CO25 cells grown in 10% foetal calf serum
(FCS) or in 10% horse serum (HS) as a differentiation medium or 10% HS with 1 M Dex
as the transformation medium, and labeled
with BrdU for 2 hr before each time point harvest. Each point represents triplicate determination and two independent experiments gave
identical results.

Inhibition of DNA synthesis by Carvacrol in mouse myoblast cells

295

PBSA and incubated with substrate solution until the

color is sufficient for photometric detection that was
predetermined. The absorbance of the samples was
measured in an ELISA reader (Organom, Technica) at
492 nm. DMSO as a solvent control was added to the
cells during the time course.
Protein Determination

The CO25 cells grown in 10% FCS or 10% HS with or

without 1 M Dex were incubated with various concentration of carvacrol or DMSO for periods of time.
After each day, the cells were lysed with 0.5 ml of a
lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl,
1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 1 g/ml
aprotinin, 1 g/ml leupeptin). The lysate was scraped
off the dish and centrifuged at 13,000 rpm for 10 min at
4 C. Protein determination was performed by Bradford assay (Bradford, 1976).
Statistical analysis

Results of the BrdU incorporation assay were analysed

using Student-t test. The level of significance has been
given in the text as a p value.

j Results
CO25 mouse myoblast cells were grown in 10% HS
with dexamethasone to activate N-ras gene or without
dexamethasone to allow differentiation, and then protein extracts prepared at various times were analyzed
for p21ras level by Western blotting. The antibody used
is a polyclonal anti-ras and recognize all Ras proteins.
Incubation of the blot with the antibody showed that a
21 kDa polypeptide was significantly expressed and increased in the presence of Dex over the 14 days period
compared to samples extracted the cells grown in the
absence of Dex (Fig. 2). This result indicates that mainly the level of human p21N-ras was increased, since very
weak bands have obtained in the absence of Dex at
longer exposure time (data not shown).
Cytotoxicity of the carvacrol used in the cell proliferation assay was determined with a MTT assay as described in Material and Methods. As shown in Fig. 3,
1 g/ml, 5 g/ml and 10 g/ml concentrations of carvacrol used in cell proliferation assay showed between
1025% toxicity after 3 days. However, 60 g/ml of
carvacrol showed approximately 50% toxicity upon the
incubation time. 100 g/ml and 150 g/ml doses of
carvacrol were found to be significantly toxic and IC50
value for CO25 myoblast cells was determined as
IC50 = 60g.
The effects of carvacrol on DNA synthesis in CO25
cells with or without N-ras activation were examined
using a specific cell proliferation kit as described in

Fig. 5. Measurement of BrdU incorporation. CO25 cells

were grown in 10% FCS for period of time either (a) treated
with 1, 5 and 10 l/ml DMSO, or (b) with 1, 5 and 10 g/ml
carvacrol (Car). (c) Total protein contents of cells treated
with 10 g/ml were determined by Bradford assay. Each
point represents triplicate determination and two independent
experiments gave identical results. Results of each concentration were compared with the solvent control by Student-t test.
*p < 0.001.

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H. Zeytinoglu et al.

Fig. 6. Measurement of BrdU incorporation. CO25 cells

were grown in 10% HS either in the presence of 1, 5 and
10 l/ml DMSO (a), or 1, 5 and 10 g/ml Car (b) for period
of time. (c) Total protein contents of cells treated with
10 g/ml Car were determined by Bradford assay. Each point
represents triplicate determination and two independent experiments gave identical results. Results of each concentration were compared with the solvent control by Student-t test.
*p < 0.001.

Fig. 7. Measurement of BrdU incorporation. CO25 cells

were grown in 10% HS + 1 M Dex either in the presence of
1, 5 and 10 l/ml DMSO (a), or 1, 5 and 10 g/ml Car (b) for
period of time. (c) Total protein contents of cells treated with
10 g/ml Car were determined by Bradford assay. Each point
represents triplicate determination and two independent experiments gave identical results. Results of each concentration were compared with the solvent control by Student-t test.
*p < 0.001.

Inhibition of DNA synthesis by Carvacrol in mouse myoblast cells

Material and Methods. The cells normally grew in the
medium containing 10% FCS until reaching contact inhibition and ceased proliferation, then committed to
differentiation in 10% HS after 4 days (Fig. 4), as documented before (Gosset et al., 1988; Zeytinoglu et al.,
1993). However cells became transformed and lost
contact inhibition after ras activation by the addition of
Dex to the differentiation medium.
Basically, the treatment of CO25 cells grown in either 10% FCS (Fig. 5b), 10% HS (Fig. 6b) or 10% HS
plus Dex (Fig. 7b) with carvacrol at concentrations
ranging from 15 g/ml had no effect on cell proliferation compared to the controls (Fig. 5a, 6a and 7a). In
contrast, substantial effects on cell proliferation were
obtained when the cells were treated with 10 g/ml of
carvacrol. Thus, as shown in Fig. 5b, there was a significant reduction (p < 0.001) in the level of DNA synthesis after 2 days, when compared with the solvent control. This meant that at concentration of 10 g/ml, the
DNA synthesis of these cells was reduced about 75%
as compared to DMSO treated control cells (Fig. 5a).
In the experiment shown there appears to be a similar
reduction (by 43%) of DNA synthesis of CO25 grown
in differentiation medium (10% HS) (Fig. 6b) as compared to controls (Fig. 6a).
DNA synthesis was also significantly declined
(p < 0.001) in the ras activated transformed cells in the
presence of 10 g/ml of carvacrol for 3 and 4 days after
an elevation on day 2 (Fig. 7b) as compared to the control cells (Fig. 7a). Unlike the other medium conditions, ras-dependent proliferation of the cells appeared
to be inhibited by 1 g/ml and 5 g/ml of carvacrol
treatment as well.
To test these inhibitory effects of 10 g/ml of carvacrol on DNA synthesis, the total protein levels of
cells grown in parallel plates were measured as described in Material and Methods. In contrast to the cells
grown in transformation medium, which showed significant increase in the level of total protein (Fig. 7c),
Fig.5c and 6c show that the level of total protein obtained from each culture dish was slightly elevated.

j Discussion
CO25 cells provide an excellent model for investigation of the mechanism of differentiation and transformation (Zeytinoglu et al., 1993, 1995). Here, we have
used a mouse myoblast cell line, CO25, bearing a mutated human N-ras oncogene, for the first time to test
the potency of therapeutical agent. As shown in Fig. 2,
ras proteins were detected at high level after addition of
Dex, which was used as an inducer of mutated human
N-ras oncogene.
More recently, Crowell (1999) reviewed prevention
and therapy of cancer by monoterpenes. The inhibition

297

of cell growth in melanoma by isoprenoids including

carvacrol, d-limonene, its metabolites and farnesol has
reported by several grubs (Crowell et al., 1991; Adany
et al., 1994; He et al., 1997). Dietary limonene also inhibits the development of ras oncogene-induced mammary carcinomas in rats (Gould et al., 1994; Gelb et al.,
1995).
In our study, the cytotoxicity of carvacrol and the
range of effective doses were determined and its effect
on DNA synthesis was assessed using cell proliferation
assay. In vitro cytotoxicity of carvacrol reported here is
IC50 = 60 g. However, this IC 50 value is not the same
as reported by a previous study (Stammatia et al.,
1999), it might be due to cell type and purity of carvacrol. For the inhibition of DNA synthesis, the maximal dose of carvacrol was chosen as 10 g/ml. Because, the significant effect of carvacrol on the inhibition of DNA synthesis was thought to shown in the
least toxic doses, since it is quite important that minimize the cytotoxicity of a predrug for future use.
Result here showed that 10 g/ml dose of carvacrol
is very effective for the inhibition of DNA synthesis in
CO25 cells. Our findings support the tumor growthsuppressive action of carvacrol as reported by
Zeytinoglu et al. (1998) and He et al. (1997). This effect of 10 g/ml of carvacrol also determined by MTT
is not due to cytotoxicity since there was no change in
total protein levels of these cells. The inhibition of
DNA synthesis during growth phases especially transformation process is very important. However it seems
that this inhibitory effect might be specific for only
transformation phases or in other words for ras activated transformation since the doses of 1 g/ml and
5 g/ml of carvacrol was found to be effective on DNA
synthesis.
Here, especially 10 g/ml of carvacrol was shown to
affect the cells in growth or transformation medium.
On the other hand, the decrease in DNA synthesis in
the absence of ras activation seems to be not as a result
of carvacrol treatment, since the cells normally withdraw from the cell cycle and commit to differentiate
(Zeytinoglu et al., 1993).
DNA synthesis in the cells grown in the presence of
1 g/ml and 5 g/ml of carvacrol seems to be normal
for both growth and differentiation medium since it is
known that CO25 myoblasts cease proliferation when
they commit to differentiate after contact inhibition in
the growth medium. In contrast, after activation of
N-ras oncogene in the presence of Dex in differentiation medium, the cells enter transformation process;
therefore DNA synthesis is elevated (Zeytinoglu et al.,
1993). Our results demonstrate for the first time the inhibitory effect of carvacrol on DNA synthesis in N-ras
transformed cells. It is well documented that many
monoterpenes inhibit isoprenylation of proteins, which

298

H. Zeytinoglu et al.

occurs at the level of prenyl-protein transferase enzyme (Crowell et al., 1991; Elson, 1995; Gelb et al.,
1995). Prenylation of Ras enables it to associate with
plasma membrane, which is required for its oncogenic
activity (Bos, 1989; Marshall, 1991) and impairment of
the prenylation of Ras might account for the antitumor
activity of monoterpenes. Moreover, like ras proteins,
the prenylated proteins TC21, Rho and PRL-1/PTPCAAX tyrosine phosphatases can be oncogenic (Crowell et al., 1994), thus they may be important cellular targets of protein prenylation inhibitors such as monoterpenes.
Taken together, these data raise the possibility that
carvacrol and similar isoprenoids may find application
in cancer therapy by preventing prenylation of many
proteins including Ras. Therefore these results call for
further investigations of the suppression mechanism of
the DNA synthesis and ras expression.
Acknowledgments

We would like to thank Mrs. M. Irmak for technical assistance with ELISA and Department of Pharmacology, Faculty
of Medical, University of Osmangazi for providing us their
facilities. Anadolu University Research Fund grant 1996-15
and Medicinal and Aromatic Plant and Drug Research Center
(TBAM) supported part of the experimental work.

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j Address
H. Zeytinoglu, Anadolu University, Faculty of Sciences, Department of Biology, Yunusemre Campus,
26470 Eskisehir, Turkey
Tel.: ++90-222-3350580/ext. 5727;
Fax: ++90-222-3353616;
e-mail: hzeytino@anadolu.edu.tr