Mutation Research 585 (2005) 1–13

Cytotoxicity and gene induction by some essential oils in the

yeast Saccharomyces cerevisiae
F. Bakkali a,b , S. Averbeck b , D. Averbeck b,∗ , A. Zhiri c , M. Idaomar a
a Université Abdelmalek Essaadi, BCM, Département de Biologie, BP 2121 Tétouan, Morocco
b Institut Curie-Section de Recherche, UMR2027 CNRS/I.C., Bât. 110, Centre Universitaire d’Orsay, F-91405 Orsay Cedex, France
c S.A. PRANARÔM International, Ghislenghien, Belgium

Received 23 August 2004; received in revised form 25 January 2005; accepted 14 March 2005
Available online 21 June 2005

Abstract

In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications
we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba
alba, Cinnamomum camphora and Helichrysum italicum for genotoxic effects
using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells
being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order:
Origanum compactum > Coriandrum sativum > Artemisia herba alba > Cinnamomum camphora > Helichrysum italicum. In the
same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating
damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic
recombination were induced.
The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for
RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs
demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that
caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional
expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to
overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves
oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the
iron-chelating agent deferoxamine.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Essential oils; Origanum compactum; Coriandrum sativum; Artemisia herba alba; Cinnamomum camphora; Helichrysum italicum;
Saccharomyces cerevisiae; Cytotoxicity; Gene expression; Cytoplasmic petite mutations; RNR3; RAD51; Anti-radical effects

∗ Corresponding author. Tel.: +33 169867188; fax: +33 169869429.

E-mail addresses: fadil.bakkali@curie.u-psud.fr (F. Bakkali), dietrich.averbeck@curie.u-psud.fr (D. Averbeck), abzhiri@hotmail.com
(A. Zhiri), idaomar@hotmail.com (M. Idaomar).

1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2005.03.013
2 F. Bakkali et al. / Mutation Research 585 (2005) 1–13
1. Introduction
Essential oils (EOs) extracted from medicinal plants
are widely used in aromatherapy and in traditional
medicine, as well as in the food and perfume industry
and in agriculture. One can find them applied in AIDS
and cancer therapy, as condiments and as conserving
or flavoring agents in food and drinks, as insecticides
(aerosols) in agriculture, as perfumes in cosmetics, as
disinfectants in toothpaste and in household prepa-
rations. In this respect, their antiviral, antibacterial,
antifungal, antioxidant, cytotoxic and anticarcinogenic
properties are of special importance. In addition, they
can also influence the central nervous system affect-
ing humoral and cognitive functions and can exert
antispasmodic effects. EOs from five medicinal plants
are the focus of this paper: Origanum compactum
known for its antibacterial, antifungic, antimalaria,
anti-infectious and antihypotensive properties [1,2];
Coriandrum sativum known for its antiradical, antibac-
terial, antispasmodic and expectorant properties [3];
Artemisia herba alba known for its antibacterial, anti-
helminthic, antidiabetic and antihypotensive properties
[2]; Cinnamomum camphora
known for its anti-inflammatory and antiparasitic prop-
erties [4–6]; Helichrysum italicum
known for its antimutagenic effects [5,7]. EOs
can be classified as complex mixtures generally con-
taining more than 20 different compounds belonging to
different chemical classes. Indeed, the composition of
EOs depends on the plant species, developmental state,
environmental growth conditions (season, climate,
soil) and on the part of the plant used for extraction.
The genotoxicity of EOs has not yet been much
explored. Because of their complex chemical com-
position, the evaluation of their toxicity and possible
genotoxic effects is indeed a difficult matter. Since they
are usually employed as extracted complete entities of
oily consistency and not as compounds reconstituted
from pure ingredients, EOs have to be considered as
substances acting as a whole and should be tested as
such. Many known genotoxicity test systems, such as
those with bacteria and mammalian cells, are likely to
be affected by oily conditions because of insufficient
oxygen supply on the tester plates.
The present study was undertaken to estimate the
cytotoxic and possible genotoxic activities of five
EOs in a typical unicellular eukaryotic system, the
yeast Sacharomyces cerevisiae, which is frequently
employed in genotoxic risk assessment including stud-
ies at the mitochondrial and nuclear DNA level [8–11].
We investigate here the cytotoxic and genotoxic effects
in diploid yeast (D7) and the induction of two DNA
damage-responsive genes, the ribonucleotide reductase
gene RNR3 involved in the synthesis of DNA precur-
sors [12,13] and the DNA repair gene RAD51 involved
in the repair of DNA double-strand breaks by homolo-
gous recombination [14–16] in haploid Lac-Z fusion
strains after treatments with the five EOs extracted
from Origanum compactum, Coriandrum sativum,
Artemisia herba alba, Cinnamomum camphora
and Helichrysum italicum.
The RNR3 gene is known to be
induced in the presence of different types of DNA
damage including oxidative damage [12,13]. Also the
RAD51 gene is a DNA damage-inducible gene [14–16].
Using inhibitors of oxygen radical-mediated reac-
tions such as reduced glutathione (GSH) [17–20], cata-
lase [20] and deferoxamine (DFO) [17] we demonstrate
that the cytotoxic action of EOs from Origanum com-
pactum, Coriandrum sativum, Artemisia herba alba
and Cinnamomum camphora involves the production
of reactive oxygen species (ROS) and/or hydrogen per-
oxide.
2. Materials and methods
2.1. Essential oils (EOs) and reference compounds
We used five essential oils chemotyped by PRA-
NARÔM (B-7822 Ghislenghien, Belgium): Origanum
compactum of Moroco (extracted from flower head),
Coriandrum sativum of Russia (extracted from fruits),
Artemisia herba alba of Moroco (extracted from flower
head), Cinnamomum camphora
of Madagascar (extracted from leaves), Helichry-
sum italicum of France (extracted from flower head).
According to the chart of PRANARÔM, each EO was
extracted under the same conditions from the same part
of the plants, which were grown on the same soil, in
the same climate and season so that its composition was
quite reproducible. Throughout the paper we designate
the EOs by the name of the plant from which they were
extracted. Table 1 gives the list of main chemical con-
stituents of the EOs derived from the plant extracts as
determined by gas chromatographic analysis [6,7].
 F. Bakkali et al. / Mutation Research 585 (2005) 1–13 3

Table 1
Main ingredients of five essential oils (EOs) from Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora
and Helichrysum italicum
Ingredients Essential oils

Origanum Coriandrum Artemisia herba Cinnamomum Helichrysum

compactum (%) sativum (%) alba (%) camphora (%) italicum (%)
1,8-Cineole 3.26 50.20
Alpha curcumene 3.21
Alpha pinene 5.31 5.19
Alpha terpinene 2.59
Alpha terpineol 9.89
Alpha thuyone 36.67
Beta caryophyllene 2.85
Beta pinene 2.70
Beta thuyone 20.61
Camphene 3.01
Camphor 5.93 24.61
Carvacrol 30.53
Epi-gamma-eudesmol 3.25
Gamma terpinene 18.2 3.41
Geranyl acetate 4.43
Isoitalicene 2.98
Italicene 8.28
Italidione I, II, III 10.46
Limonene 2.29 3.84 4.75
Linalol 68.39
Nerol 2.61
Neryl acetate 33.90
Neryl propionate 6.47
p-Cymene 7.89 2.27
Sabinene 6.45
Terpinene-4-ol 2.65
Thymol 27.5
Viridiflorol 2.75
Not determined 10.44 7.97 11.84 16.33 24.09

In the study of gene induction, we included hydro-
gen peroxide (H2 O2 solution, 30%, w/w) (CAS 7722-
84-1, Sigma–Aldrich, USA) and methyl methanesul-
fonate (MMS) (CAS 66-27-3, Sigma–Aldrich, USA)
as DNA-damaging agents [21,22].
Antiradical effects were studied using inhibitors
of oxygen radical-mediated reactions such as defer-
oxamine mesylate salt (DFO) (CAS 138-14-7,
Sigma–Aldrich, USA) or reduced l-glutathione (GSH)
(CAS 70-18-8, Sigma–Aldrich, USA) and catalase
(product ref. C100, Sigma–Aldrich, USA).
2.2. Yeast strains
For gene expression analysis, we used the following
haploid strains of the yeast Saccharomyces cerevisiae:
DBY747 (MAT a RAD+ his3-11, leu2-3, 112 trpl-
289, ura3-52, carrying the RNR3–lacZ fusion) kindly
provided by Dr. Wei Xiao (Department of Microbi-
ology and Immunology, University of Saskatchewan,
Saskatoon, Canada, SK S7N 5E5) [12].
FF1082 (MATa RAD+ leu2, trpl, ura3, his7,
lysl, ura3+-51LACZ LEU2, carrying the RAD51–lacZ
fusion) kindly provided by Dr. Francis Fabre (DSV,
CEA, Fontenay aux Roses, France).
For the detection of cytotoxic effects, the induc-
tion of cytoplasmic petite mutations, ILV+ point muta-
tions, TRP+ mitotic gene convertants and mitotic
crossing-over involving the Ade2 gene, we used
the known diploid yeast tester strain D7 (a/␣,
Ade2-40/Ade2-119, Trp5- 12/Trp5-27, Ilv1-92/Ilv1-92)
[9].
4 F. Bakkali et al. / Mutation Research 585 (2005) 1–13
2.3. Growth media
The complete growth medium (YEPD) contained
2% d-glucose (Sigma–Aldrich), 1% bacto yeast extract
(Difco), 2% bacto peptone (Difco), 2.6% bacto agar
(Difco).
The minimum medium complemented or not with
the appropriate amino acids was composed of 2% d-
glucose, 3.2% bacto agar complemented with 0.67%
of bacto yeast nitrogen base without aminoacids and
5 mg adenine/L medium, 60 mg isoleucine/L for the
detection of TRP+ gene convertants (intragenic recom-
binants), 10 mg tryptophan/L for the detection of ILV+
revertants.
2.4. Treatments with essential oils (EOs) or with
H2 O2 or MMS as reference compounds
Cells of the strain D7 were grown in YEPD at
30 ◦ C up to exponential (2 × 107 cells/mL) or station-
ary phase of growth (2 × 108 cells/mL). Cells in expo-
nential phase or stationary phase were sedimented and
resuspended in distilled water. Generally, cell suspen-
sions of 2 × 107 cells/mL were treated with EOs or
the reference compounds at the desired concentrations
for a given time as indicated in the figure legends.
First, EOs were carefully diluted in absolute ethanol
(Merck) at different concentrations, and identical vol-
umes were added to the aqueous cell suspensions in
small flasks to give the chosen concentrations of EOs
at a given same final concentration of ethanol (1.25%).
Pipetman filter tips (ART) were used to avoid cross-
contamination between different EO preparations con-
taining some volatile molecules. The flasks containing
the cell suspensions with EOs were agitated at con-
trolled room temperature and at constant low speed
(200 rpm in a New Brunswick agitator) just sufficient to
keep the oil/ethanol mixture in the aqueous cell suspen-
sion well mixed. After 30 min incubation, cell aliquots
were diluted and spread onto YEPD solid medium and
selective minimal media to select for recombinants or
revertants. Colonies were counted after 6 days of post-
treatment incubation at 30 ◦ C.
2.5. Detection of cytotoxic and genotoxic effects
Cytotoxic and genotoxic effects after exposures to
different concentrations of EOs or of the reference
compounds H2 O2 or MMS at a given incubation time
or to a given concentration of EOs at different incu-
bation times, at room temperature, were determined
as previously described [8,23,24]. Clonogenic survival
was determined, and among the surviving colonies
cytoplasmic petite mutants were detected either by
replication on glycerol medium or by the triphenyl
tetrazolium chloride overlay technique [25]. Mitotic
intergenic recombinants involving the ade2 locus were
detected by accumulation of a red pigment (imidazole)
in purine metabolism on complete growth medium.
Mitotic gene convertants (TRP+ ) and gene revertants
(ILV+ ) were detected by their growth on the appro-
priate selective media. Experiments were performed at
least in triplicate with stationary phase cells and with
exponential phase cells.
2.6. Detection of reactive oxygen species using
inhibitors
The involvement of radical-mediated reactions in
the cytotoxic effects of four EOs (Origanum com-
pactum, Coriandrum sativum, Artemisia herba alba,
Cinnamomum camphora) was determined using l mM
DFO or 1 mM GSH during 1 h pre-incubation of cells
before treating with EOs for 30 min. One thousand units
per millilitre of catalase were added at the same time as
the EOs. For the control experiments with EOs alone
and the experiments with catalase, cells were also pre-
incubated for 1 h to obtain conditions comparable to
those used for the experiments with DFO or GSH. Yeast
cells (D7) were exposed in stationary growth phase to
Origanum compactum and Coriandrum sativum and
in exponential growth phase to Artemisia herba alba
and Cinnamomum camphora because these latter EOs
showed too little cytotoxicity to cells in the stationary
growth phase. Results presented are based on at least
three independent experiments.
2.7. Test of gene expression in RNR3–lacZ and
RAD51–lacZ fusion strains
Gene induction was analysed by determining
the ␤-galactosidase activity in the RNR3–lacZ and
RAD51–lacZ fusion strains using o-nitro-phenyl-␤-d-
galactopyranoside (ONPG) as a colorimetric substrate
and measuring spectrophotometrically the optical den-
sity at 420 nm [26,27].
 F. Bakkali et al. / Mutation Research 585 (2005) 1–13 5

Briefly, yeast cells were grown in YEPD up to expo-
nential phase (1.1–1.3 × 107 cells/mL), sedimented and
resuspended in distilled water. Aliquots were exposed
to the EOs or to the reference compounds at different
concentrations under the same conditions as above, for
30 min at room temperature. Cells were centrifuged,
resuspended in YEPD and then incubated for the indi-
cated times at 30 ◦ C. After this post-treatment incu-
bation, cells were collected by centrifugation, resus-
pended in a buffer with mercaptoethanol and per-
meabilized by chloroform and SDS. The activity of
␤-galactosidase was determined by means of ONPG
[12,28] and expressed as previously described [26,27].
The results reported are from at least three independent
experiments.
3. Results
3.1. Cytotoxic effects of EOs
growth phase at different EO concentrations for 30 min.
In most cases, shouldered survival curves are obtained.
Stationary phase cells are clearly more sensitive to
Origanum (LD50 = 0.45 ␮L/mL) and Coriandrum
(LD50 = 1.6 ␮L/mL) than to Artemisia, Cinnamomum
and Helichrysum (LD50 > 8 ␮L/mL). The cytotoxic
effectiveness decreases in the following order: Ori-
ganum > Coriandrum  Cinnamomum > Artemisia >
Helichrysum.
A different pattern is seen for cells treated
in the exponential phase of growth. Cells are
clearly more sensitive, especially to Artemisia and
Cinnamonum so that the cytotoxic effectiveness
decreases in a slightly different order: Origanum >
Coriandrum > Artemisia > Cinnamomum  Helichry-
sum than in stationary phase.
The results suggest that the cytotoxic effects of EOs
are facilitated by the less thicker cell wall at budding
sites of exponential phase cells and may be mediated
by effects on the cell membranes.

After treatment of diploid yeast cells (D7) with EOs
extracted from Origanum, Coriandrum, Artemisia,
Cinnamomum, Helichrysum, we observed an inhibi-
tion of cellular growth depending on the concentration
of the EOs. To quantify the cytotoxic effects involved,
we measured the clonogenic survival after treatment
of the cells with the five EOs. Fig. 1 shows the results
obtained for cells treated in exponential and stationary
3.2. Induction of cytoplasmic petite mutations
Yeast has the advantage that mitochondrial dam-
age can be easily assessed by detection of cytoplas-
mic petite mutations among the surviving cells. Fig. 2
shows the results obtained as a function of the EO con-
centration. The induction of cytoplasmic petite muta-
tions follows the same order as that for cytotoxicity:

Fig. 1. Survival curves obtained for diploid yeast cells (D7) in the stationary phase (a) and the exponential phase (b), after treatment with
five EOs from Origanum compactum (ORI) (, 䊉); Coriandrum sativum (COR) (
, ); Artemisia herba alba (ART) (♦, ); Cinnamomum
camphora (CIN) (Ravintsara aromatica) (, ); and Helichrysum italicum (HEL) (, ) at different concentrations. Data are presented with
standard deviations.
6 F. Bakkali et al. / Mutation Research 585 (2005) 1–13
Fig. 2. Induction of cytoplasmic petites in diploid yeast cells (D7)
in stationary (broken lines) and exponential phase (solid lines) after
treatment with five EOs from Origanum compactum (ORI) (, 䊉);
Coriandrum sativum (COR) (
, ); Artemisia herba alba (ART) (♦,
); Cinnamomum camphora (CIN)(, );
and Helichrysum italicum (HEL) (, ) at different concentrations.
Data are presented with standard deviations.
Origanum being the most active and Helichrysum being
nearly inactive. Also, cells treated in the exponential
phase are clearly more sensitive to the induction of
cytoplasmic petites than cells treated in the station-
ary phase of growth. The differential effect of the two
growth phases is slightly less pronounced in the case
of Coriandrum.
Also, when compared at equitoxic levels clearly
more cytoplasmic petite mutations are induced by EOs
such as Artemisia, Cinnamomum and Coriandrum in
exponential phase cells than in stationary phase cells
(data not shown). Most cytoplasmic petites form non-
sectored colonies suggesting that treatments with EOs
damage already the mitochondria of mother cells, and
the damage is directly transmitted to daughter cells.
3.3. Induction of point mutations and mitotic
recombination
Using the diploid yeast strain D7, several important
genotoxic endpoints such as induction of point muta-
tions and mitotic intragenic recombination (gene con-
version) and intergenic recombination (crossing-over)
can be detected simultaneously. Exposure to the five
EOs did not provide evidence for any induction of this
type of genetic events in cells in the stationary or expo-
nential phase of growth (data not shown). These types
of events were also not induced when cells were treated
at given concentrations for different incubation times,
at least for the three EOs tested (Origanum, Artemisia,
Cinnamomum) (data not shown). Genotoxicity (induc-
tion of mutations and mitotic recombinants) was also
not observed in cells that grew in liquid medium in
the presence of EOs from Origanum, Coriandrum,
Artemisia and were harvested at the end of growth (data
not shown). Also, there was no induction of CAN®
mutants after exposures of the haploid wild type strain
BY4741 to the EO of Origanum.
Taking MMS and H2 O2 as reference compounds
and in line with previous work [26–28], MMS effec-
tively induced point mutations and mitotic intra- and
intergenic recombination, but only a few cytoplas-
mic petites. H2 O2 induced strong lethality, just a few
nuclear genetic events and no cytoplasmic petite muta-
tions.
Thus, in contrast to MMS and H2 O2 , the five EOs
are unable to induce significant nuclear genetic events
in yeast. Induction of cytotoxicity appears to be pre-
dominant.
3.4. Modification of the expression of DNA
damage-responsive genes after treatments with
EOs
Changes in gene expression can be taken as a very
sensitive measure of genotoxic insult. We tested the
effects of EOs on the induction of two typical damage-
inducible genes RNR3 and RAD51 using appropriate
LacZ fusion strains of the yeast Saccharomyces cere-
visiae. Gene induction was monitored by measuring
␤-galactosidase activity.
We first studied the induction of the gene RNR3,
which encodes a subunit of ribonucleotide reductase,
an important enzyme involved in the synthesis of
precursors for DNA replication and repair [13].
Fig. 3a–e shows the results obtained for the kinetics of
induction of the RNR3 gene in the haploid RNR3–LacZ
fusion strain using different concentrations of EOs
from Origanum (a), Coriandrum (b), Artemisia (c),
Cinnamomum (d) and Helichrysum (e) giving rise to
comparable levels of cytotoxicity. With the exception
of Helichrysum, gene induction is starting 4 h after
treatment, and maximum gene induction occurs
approximately at the same time of post-treatment
 F. Bakkali et al. / Mutation Research 585 (2005) 1–13 7

Fig. 3. Induction of the RNR3 gene in the haploid RNR3–LacZ fusion yeast strain by five EOs. (a) Origanum compactum at different concentrations
giving rise to () 61.9 ± 2.7%; (
) 23.2 ± 3%; (♦) 2.3 ± 0.3% survival. (b) Coriandrum sativum at different concentrations giving rise to ()
75.1 ± 3.5%; (
) 54.5 ± 1.5%; (♦) 19.2 ± 8.2% survival. (c) Artemisia herba alba at different concentrations giving rise to () 79.1 ± 2.6%; (
)
47.3 ± 11.3%; (♦) 11.3 ± 4% survival. (d) Cinnamomum camphora at different concentrations giving rise to () 72.6 ± 3.6%; (
) 40 ± 9.5%;
(♦) 13.9 ± 3.6% survival. (e) Helichrysum italicum at different concentrations giving rise to () 65.6 ± 5.5%; (
) 39.5 ± 5.7%; (♦) 17.1 ± 2.4%
survival. Data are presented with standard deviations.

incubation, i.e. at 10–12 h after treatment in our
experimental conditions. In the case of Helichrysum,
the start of induction is clearly delayed up to 10 h
depending on survival levels. For all EOs, gene
induction is clearly concentration-dependent. At
comparable cytotoxic levels (Fig. 4a), the inducing
capacity is greatest for Cinnamomum and lowest
for Helichrysum and follows the order Cinnamo-
mum > Origanum > Coriandrum > Artemisia > Helich-
rysum.
For comparison, we also included in this study two
known DNA-damaging agents, the methylating agent
MMS and the oxidizing agent H2 O2 . Gene induction
was found to be concentration-dependent with a shift of
maximum induction from 5 to 10 h post-treatment incu-
bation for survival levels from approximately 70 down
to 6% (data not shown). At comparable cytotoxic lev-
els, gene induction occurs for both agents a few hours
earlier than for EOs (Fig. 4b). H2 O2 reaches induction
levels close to that of the most effective EOs (Cinnamo-
mum and Coriandrum). MMS, a DNA double-strand
break-inducing agent, is strikingly more effective (two
to three-fold). The comparable responses for some EOs
and H2 O2 suggest that in both cases oxidative radical-
mediated DNA lesions rather than DNA double-strand
breaks are initiating RNR3 gene induction.
In order to check whether the gene RAD51, a
DNA damage-inducible gene involved in double-strand
break repair and homologous recombination would
also respond to treatments by EOs, we tested its induc-
tion in a LacZ fusion strain after treatments with Orig-
anum and Cinnamomum (Fig. 5a and b). In contrast to
8 F. Bakkali et al. / Mutation Research 585 (2005) 1–13

Fig. 4. Induction of the RNR3 gene in the haploid RNR3–LacZ fusion strain by the five EOs from Origanum compactum (ORI), Coriandrum
sativum (COR), Artemisia herba alba (ART), Cinnamomum camphora (CIN) and Helichrysum italicum (HEL) at quasi-
equitoxic concentrations: ORI (23.2% survival), COR (19.2% survival), ART (11.3% survival), CIN (13.9% survival), HEL (17.1% survival)
(a); and by MMS (21.4% survival) and H2 O2 (14.9% survival) together with the five EOs for better comparison (b). Data are presented without
standard deviations for better clarity.

the results obtained with the RNR3–LacZ fusion strain,
after treatment with the two EOs the peaks of induc-
tion for RAD51 are shifted from 8 to 12 h dependent on
the survival level (from 64.8 down to 6.2%). The max-
ima of induction are clearly much lower for RAD51
(Fig. 5a and b) than for RNR3 (Fig. 3a and d) but still
significant for the two EOs used. At comparable sur-
vival levels (Fig. 5c) RAD51 induction by the two EOs
is approximately seven-fold lower than that caused by
MMS and H2 O2 .
As for RNR3 (Fig. 4b), RAD51 gene induction peaks
occur approximately at the same time (8–10 h after
treatment) for the two EOs tested, whereas the peaks
of induction are reached 2–3 h earlier after treatments
with MMS and H2 O2 (Fig. 5c). In contrast to the results
obtained on the induction of RNR3 (Fig. 4b), the max-
imal responses for RAD51 are comparable for MMS
and H2 O2 (Fig. 5c) suggesting that induction of these
two DNA damage-inducible genes involves different
signals for gene induction.
3.5. The effects of ROS inhibitors such as
deferoxamine (DFO), reduced glutathione GSH
and catalase
In order to get further insight into the possible
mechanisms involved in the cytotoxic and cytoplas-
mic effects of EOs we checked the possible contribu-
tion of radical-mediated reactions. For this, we used
three inhibiting agents: (1) the iron-chelating agent
DFO, an efficient inhibitor of Fenton reactions and
production of hydroxyl radicals; (2) GSH, an efficient
scavenger of ROS such as O2 •− ; and (3) catalase, a
hydrogen peroxide deactivating enzyme. The activity
of these inhibitors is partially overlapping. As shown
in Fig. 6, the inhibitors protect in a rather specific man-
ner against the cytotoxic effects of EOs (Origanum,
Coriandrum, Artemisia and Cinnamomum). The EO
from Helichrysum was not included because of its rel-
atively weak cytotoxicity. GSH and, to a lesser extent,
DFO protected against treatment with the EO from
Origanum, whereas catalase had no effect. DFO and
catalase protected efficiently against treatment with the
EO from Coriandrum, whereas GSH increased cyto-
toxicity. DFO protected against treatment with the EO
from Artemisia, whereas GSH and catalase were inef-
fective. Catalase efficiently protected against treatment
with the EO from Cinnamomum, and DFO was less
effective, whereas GSH increased cytotoxicity. The
results show that part of the oxygen radical-mediated
reactions produced by EO treatments can be inhib-
ited. Thus, depending on the EO used, reactive oxygen
species produced appear to contribute to the observed
cytotoxicity.
 F. Bakkali et al. / Mutation Research 585 (2005) 1–13 9

Fig. 5. Induction of the RAD51 gene in the haploid RAD51–LacZ fusion yeast strain by two EOs. (a) Origanum compactum at different
concentrations giving rise to () 64.8 ± 7.5%; (
) 26.3 ± 8.8%; (♦) 5.5 ± 2.3% survival. (b) Cinnamomum camphora
at different concentrations giving rise to () 61.0 ± 3.4%; (
) 18.9 ± 5.7%; (♦) 1.16 ± 0.44% survival. (c) Induction of the RAD51 gene by
Origanum compactum (ORI), Cinnamomum camphora (CIN) , MMS and H 2 O2 at quasi-equitoxic concentrations: ORI
(26.3% survival) (), CIN (18.9% survival) (
), MMS (24.2% survival) () and H2 O2 (21.3% survival) (). Data are presented with standard
deviations in (a) and (b) and without in (c) for better clarity.

4. Discussion All five EOs induced cytotoxic effects that were

The five EOs used in this study are complex mix-
tures extracted from Origanum compactum, Corian-
drum sativum, Artemisia herba alba, Cinnamomum
camphora and Helichrysum italicum, which are part
of traditional human pharmacopeia. EOs are usually
employed as whole entities. In the present study, we
attempted to get further insight into their possible cyto-
toxic and genotoxic effects in eukaryotic cells taking
the yeast Saccharomyces cerevisiae as a model system.
stronger in exponential than in stationary phase cells.
The more fragile cell wall structure of budding cells
in the exponential growth phase appears to facilitate
penetration of EOs. Treatment of yeast cells in the
exponential phase with the four most active EOs (Ori-
ganum compactum, Coriandrum sativum, Artemisia
herba alba, Cinnamomum camphora) gave rise to
shouldered survival curves. The steep final slopes of the
curves indicate strong cytotoxicity, possibly involving
damage to cellular membranes.
10 F. Bakkali et al. / Mutation Research 585 (2005) 1–13

Fig. 6. Modifications of cytotoxic effects of four EOs: (䊉) (a) Origanum compactum (ORI); (b) Coriandrum sativum (COR); (c) Artemisia
herba alba (ART); and (d) Cinnamomum camphora (CIN) in the presence of 1 mM reduced glutathione (GSH) (♦);
l mM deferoxamine (DFO) (
); or 1000 units/mL catalase (). Data are presented with standard deviations.

Treatment with EOs damages cell walls and mito-
chondria, which could also be observed by microscopic
analysis (data not shown).
The cytotoxicity of the five EOs was accompa-
nied by the induction of cytoplasmic petite mutations,
indicating mitochondrial damage and impairment of
oxidative metabolism [11]. Origanum was the most
active EO, Helichrysum was the least active. More
cytoplasmic petites were induced in exponential than
in stationary phase cells, probably also due to easier
penetration of EOs in exponential cells. The absence
of sectored colonies [11] suggested that EOs produce
dysfunction of mitochondria in the treated mother cells.
Damage to yeast mitochondria may, at least in part, be
linked to programmed cell death or apoptosis [29–31].
We found yeast cells lacking mitochondria to be more
sensitive to EOs than cells with mitochondria (data not
shown).
Absence of induction of genotoxic damage in
nuclear DNA, i.e. absence of the induction of point
mutations, mitotic gene conversion and mitotic inter-
genic recombination (crossing-over) in the diploid
 F. Bakkali et al. / Mutation Research 585 (2005) 1–13
tester strain D7 suggested that either the EOs do not
reach nuclear DNA and cause cell death by solely dam-
aging cellular membranes, or that they cause damage
to cellular organelles such as mitochondria, involving
oxidative stress that may lead to cytotoxicity.
In an attempt to better understand the cellular
responses after treatment with EOs we also studied
gene induction. In fact, not only bacteria but also
eukaryotic cells such as yeast and mammalian cells
respond sensitively to external stress from exposures to
various genotoxic agents by the transcription of a large
number of genes [32,33]. Many genes are inducible
by agents that induce damage to nuclear DNA. Using
RNR3- and RAD51–LacZ fusion strains of Saccha-
romyces cerevisiae we confirm here that the genes
RNR3 [12,13] and RAD51 [14–16] can be induced by
DNA-damaging agents such as MMS and H2 O2 pro-
ducing DNA double-strand breaks and oxidative dam-
age, respectively. Furthermore, we show that EOs are
also able to induce these two genes, which play impor-
tant roles in DNA metabolism and repair. Interestingly,
at comparable cytotoxic levels, RNR3 induction by
some EOs was close to that of H2 O2 although much
weaker than that of MMS, and in comparison to MMS
and H2 O2 , the peak of induction by EOs was delayed by
a few hours. These findings indicate that with EOs the
signal of induction may be comparable in amplitude but
may be produced rather delayed when compared with
that of H2 O2 . These results suggest that treatments with
EOs may trigger gene induction via a similar type of
damage. However, slightly different cellular compart-
ments may be affected. Oxidative damage produced
by treatments with H2 O2 can induce the RNR3 gene
[12] and can also cause cytotoxic and weak muta-
genic and recombinogenic effects in diploid yeast (D7)
[22,24].
Comparison of RAD51 induction by two EOs (Orig-
anum and Cinnamomum) with that produced by MMS
and H2 O2 revealed that the induction of the RAD51
gene by EOs was even weaker than that of the RNR3
gene and was also delayed when compared with that
produced by MMS and H2 O2 . However, in contrast to
RNR3 induction, the maximal RAD51 induction lev-
els for these latter agents were comparable indicating
that the pathways for gene induction differ for the two
genes and are not identical. From this, it appears that
MMS and H2 O2 induce the two genes more directly
and earlier than EOs.
11
Since treatments by EOs caused a variety of cellular
responses including cytotoxicity, induction of mito-
chondrial damage and gene induction, but no nuclear
genetic effects, we asked the question whether these
effects could be due to cellular stress responses induced
by EOs. Mitochondria are cellular organelles of energy
production, but at the same time they are important
sources of free radicals and oxidative stress including
ROS such as superoxide radicals (O2 •− ), hydroxyl rad-
icals (OH• ) and the termination product hydrogen per-
oxide (H2 O2 ) [34,35]. H2 O2 can be also produced by
oxidative enzymes in peroxisomes and easily permeate
membranes [33]. Exogenous stress disrupts the equilib-
rium between the production of such reactive species
and the cellular defense [34,35]. When mitochondria
are affected, loss of membrane potential, cytochrome
C release and cell death by apoptosis can occur [34].
Here, we tested the induction of oxidative stress by
EOs using suitable inhibitors such as GSH, DFO and
catalase. Reduced glutathione easily reacts with hydro-
gen peroxide and superoxide radicals (O2 •− ), leading
to less reactive oxidized glutathione or thiol radicals
[17–20]. Deferoxamine inhibits the Fenton reaction,
i.e. the production of hydroxyl radicals via the reac-
tion of Fe2+ iron with hydrogen superoxide [17], and
catalase inactivates hydrogen peroxide [17,20].
Indeed, we were able to demonstrate that ROS and
hydrogen peroxide are involved in the biological effects
of EOs. GSH and DFO, but not catalase, protected
against the EO of Origanum suggesting that this EO
produces hydroxyl radicals and superoxide anions but
not H2 O2 . DFO and catalase, but not GSH, protected
against the EO of Coriandrum suggesting that this
EO produces H2 O2 and hydroxyl radicals, but prob-
ably not much O2 •− . DFO protected against the EO of
Artemisia, but not GSH and catalase, suggesting that
this EO mainly produces hydroxyl radicals. Catalase
and DFO, but not GSH, protected against the EO of Cin-
namomum, suggesting that this EO produces mainly
H2 O2 . The fact that addition of GSH increased cytotox-
icity for Coriandrum and Cinnamomum may suggest
that GSH oxidized by H2 O2 reacted further in the pres-
ence of oxygen giving rise to reactive oxygen species
including sulphur peroxyl radicals, sulphinyl radicals
and O2 •− radicals [18]. Since the EOs and also the
inhibitors may differently affect various cellular com-
partments, these results are likely to reflect the different
interactions at various sites.
12 F. Bakkali et al. / Mutation Research 585 (2005) 1–13
Altogether, the production by EOs of highly dam-
aging agents such as OH• and O2 •− radicals and H2 O2
provides an explanation for the observed cytotoxic
effects, cytoplasmic petite induction and gene induc-
tion. In this respect, the quasi-simultaneous expression
of strong cytotoxic effects and mitochondrial damage
does not appear to be fortuitous. Exposure to EOs
strongly affects the cell wall and membranes and dam-
ages mitochondria. This may lead to mitochondrial
dysfunction and to a radical burst of reactive oxy-
gen species that triggers gene induction and apoptotic
cell death. Gene induction by EOs may be brought
about by the activation of mitogen-activated protein
kinases of signaling cascades involved in the activation
of c-Jun like transcription factors (Parl) [36]. In fact,
mitochondrial dysfunction is known to increase intra-
cellular concentrations of DNA-damaging species such
as superoxide and peroxide ions linked to apoptotic
death [34,35]. After treatment with EOs, apoptotic-like
cell killing appears to be predominant in yeast, and it
effectively avoids nuclear genetic events and genotoxi-
city. Thus, the beneficial effects of the five EOs for use
in humans are likely based on their capacity to induce
cytotoxicity via oxidative stress involving ROS and
H2 O2 . Because of the absence of nuclear mutagenic or
recombinogenic events in yeast after treatments with
EOs, the use of these EOs is unlikely to involve long-
term genotoxic risks.
Acknowledgements
This work was supported by the Institut Curie and
the CNRS. F.B. acknowledges a doctoral fellowship
from Agence Universitaire de la Francophonie (AUF).
We are also very much indebted to Dr. Wei Xiao from
the Department of Microbiology and Immunology,
University of Saskatchewan, Saskatoon, Canada, for
kindly sending us the RNR3–lacZ fusion tester strain.
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