THE EFFECT OF ESSENTIAL OIL OF BASIL ( OCIMUM BASILICUM L . ) ON UV-INDUCED MUTAGENESIS IN ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE

The antimutagenic potential of essential oil (EO) of basil (Ocimum basilicum L.�) and its major constituent linalool were studied with the E. coli K12 and �.� cerevisiae D7 assays.� In the E. coli assay�� ЕO and linalool inhibited UVinduced mutagenesis in a repair-proficient strain�� but had no effect on spontaneous mutagenesis in repair-proficient�� nucleotide excision repair-deficient�� and mismatch-deficient strains.� �y testing participation of different mechanisms involved in antimutagenesis�� it was concluded that the antimutagenic effect against UV-induced mutagenesis involved decrease of protein synthesis and cell proliferation which led to increased efficiency of nucleotide excision repair.� An antimutagenic effect of basil derivatives in S. cerevisiae was not detected.�


INTRODUCTION
A number of plant species contain biologically active compounds that promote health and provide promote health and provide protection from many chronic diseases.��ubstances �ubstances with beneficial health effects�� attributable to their beneficial health effects�� attributable to their antioxidative�� antimutagenic�� and anticarcinogenic properties�� can be found in fruits and vegetables�� as well as in medicinal and aromatic plants (K i t t s�� 1994).�It is assumed that their use in everyday life can be an effective way of preventing many genetic diseases�� including cancer (V e r h a g e n et al.�� 1997; K r i s -E t h e r t o n et al. �� 2002).�There are numerous ways in which mutagenesis can be reduced or prevented: mutagen scavenging�� interference by antimutagens with DNA repair or with mutagen metabolism�� and many others (D e F l o r a and F e r g u s o n �� 2005).�Many essential oils and their components exhibit antiviral�� antibacterial�� antioxidant�� and antimutagenic activities and are widely used in traditional medicine (T e p e et al.�� 2004; K n e ž e v i ć -V u k č e v i ć et al.�� 2005; M i t i ć -Ć u l a f i ć et al. �� 2005).��ased on reported data that basil ( �ased on reported data that basil (Ocimum basilicum L.�) and its main constituent�� the terpenoid alcohol linalool�� possess a strong antioxidative potential (C e l i k and Ö z k a y a�� 2002; J a v a n m a r d i et al. �� 2003)�� in our previous research we focused attention on the protective effect of essential oil of basil (EO) and linalool (69.�2 % in EO�� Table 2) against oxidative DNA damage and mutagenesis.�The mutagenic potential of EO and linalool was prescreened in the Salmonella/microsome mutagenicity assay (M a r o n and A m e s�� 1983)�� and no mutagenic effect of basil derivatives was detected in any tested strain (� t a j k o v i ć et al. ��� 2007).�Inhibitory influence of EO and linalool against t-�OOH-induced mutagenesis was shown in the E. coli K12 and WP2 reversion tests�� as well as against H 2 O 2 -induced oxidative DNA damage in the alkaline yeast comet assay (N i k o l i ć�� 2004; � t a n o j e v i ć et al. �� 2004�� 2006).�These findings suggest that the antimutagenic and antigenotoxic potential of EO and linalool can be attributed to their antioxidative properties.�However�� inhibition of t-�OOH-induced microsatellite instability in E. coli K12 by post-treatment with EO and linalool indicated involvement of other mechanisms (N i k o l i ć�� 2004).�

THE EFFECT OF ESSENTIAL OIL OF BASIL (OCIMUM BASILICUM L.) ON UV-INDUCED MUTAGENESIS IN ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE
The E. coli K12 assay is composed of four tests measuring different end-points at the DNA level (V u k o v i ć -G a č i ć et al. ��� 2006).�Antimutagenic influence against spontaneous and UV-induced mutagenesis is examined in the �Y252 repair proficient strain and its I�105 nucleotide excision repair deficient uvrA counterpart (Test A).� �pontaneous mutagenesis is also examined in the I�103 isogenic mismatch repair (MMR) deficient mutS strain (Test �).�All strains carry an ochre mutation (argE3) that can revert to prototrophy by base substitutions (T o o d et al.��� 1979).�The level of �O� induction�� corresponding to the induction of error-prone �O� repair (Q u i l l a r d and H o f n u n g�� 1993)�� is measured by monitoring the level of β-galactosidase in the I�111 repair-proficient strain lysogenized with non-inducible λ phage carrying the sfiA::lacZ fusion (Test C).�The given strain is also constitutive for alkaline phosphatase�� which is suitable for assessing the effect on overall protein synthesis (� e r i ć -� j e d o v �� 2003).�The effect on homologous recombination is measured using strains with two non-overlapping deletions in the duplicated lac op-eron�� in which intrachromosomal recombination results in the formation of Lac + recombinants (Test D).� �train GY8281 (recA + ) is recombination-proficient�� and an increased amount of activated RecA protein is formed only after DNA-damaging treatments.�On the contrary�� strain GY8252 (recA730) is partially recombination-deficient and has an increased level of activated RecA protein in the absence of DNAdamaging treatments.�It follows that strain GY8252 is constitutive for �O� induction (L a v e r y and K o w a l c z y k o w s k i �� 1992; E n n i s et al. ��� 1995).�To test the effect of basil derivatives in eukaryotic cells�� we used the S. cerevisiae D7 diploid strain (Z i m m e r m a n n et al.��� 1975)�� which permits simultaneous study of point mutations (ilv1-92→ Ilv + )�� mitotic crossing over (ade2→Ade + ) and mitotic gene conversion (trp5→Trp + ).�In our previous study�� this test successfully detected prokaryotic antimutagens and their effect on recombination (V u k o v i ć -G a č i ć et al. ��� 2001).� In this work we examined the potential of EO and linalool to modulate DNA repair and replication processes�� by studying their antimutagenic effect against spontaneous and UV-induced mutagenesis in E. coli K12 and S. cerevisiae D7 assays (V u k o v i ć -G a č i ć and � i m i ć�� 1993; � i m i ć et al. �� 1994�� 1997�� 1998; K n e ž e v i ć -V u k č e v i ć �� 1995; Z i m m e r m a n n et al., 1975).�We used UV-irradiation (254 nm) as a mutagen for several reasons: (i) it mainly induces base substitutions which can be detected in the assays; (ii) it shares cellular mechanisms of mutation avoidance (nucleotide excision and post-replication recombination repair) and mutation fixation (translesion error-prone replication) with many chemical mutagens and carcinogens; (iii) there is no chemical interaction between mutagen and antimutagen�� which is essential for detection of antimutagens with modulating effects on DNA replication and repair.�

Tester strains
The tester strains used in this study are listed in Table 1.� Preparation of essential oil of basil �asil (Ocimum basilicum L.�) was cultivated in the experimental field of the "Dr Josif Pančic" Institute for Medicinal Plant Research.�This field is located in Pančevo�� �erbia.�Essential oil was prepared according to Ph.� Jug.� IV�� by distillation of dried aerial parts (Basilicii herba) in a 2-m 3 steam distiller (Hromil) for 2 hours at a pressure of 3-4 bars and temperature of 135-145°C.�The composition of essential oil was determined using analytical GC/FID and GC/ M� techniques and the Wiley/N�� library of mass spectra .� The quality of essential oil meets standards Ph.� Jug.� IV and I�O 9909.�Essential oil was stored at 4°C.� �tock solutions of EO of basil and linalool (CA� No.� 78-70-6�� �igma-Aldrich�� �teinheim�� Germany) were freshly dissolved in 96% ethanol (1:9).�

Media and growth conditions
All bacterial strains were grown overnight at 37°C in L� medium (5 g NaCl�� 10 g bacto tryptone�� 5 g yeast extract�� 1000 ml distilled water).�S. cerevisiae D7 was grown in YPD medium (10 g yeast extract�� 20 g bacto peptone�� 20 g dextrose�� 1000 ml distilled water) at 30°C with aeration.�All media for the S. cerevisiae reversion assay were as described by Z i m m e r m a n n et al. (1975).�The semi-enriched minimal medium (�EM) for E. coli K12 reversion assays (Tests A and �) was minimal agar medium supplemented with 3 %

Ultraviolet irradiation
UV-irradiation was carried out with a germicidal lamp (from Camag) having maximum output at 254 nm (UV-C).�Dose rates were measured with the Latarjet dosimeter (L a t a r j e t et al., 1953).�Cell suspensions in 0.�01 M Mg�O 4 were irradiated in glass Petri dishes at a thickness of less than 1 mm.�Only in Test D were bacteria irradiated on plates.�Cell suspensions and plates were kept in the dark to prevent photoreactivation.�

Detection of antimutagenic potential against spontaneous and UV-induced mutagenesis (Tests A and B)
Overnight cultures of E. coli strains �Y252�� I�103 (mutS) and I�105 (uvrA) were washed by centrifugation and resuspended in 0.�01 M Mg�O 4 .�Cell suspensions of the �Y252 and I�105 strains were irradiated with UV-doses of 28 J/m 2 and 3 J/m 2 �� respectively.��amples (0.�1 ml) of unirradiated and UV-irradiated cells�� appropriately diluted for determination of cell survival and Arg + revertants�� were spread in duplicate onto 3% �EM plates with different concentrations of EO or linalool and incubated at 37°C for 48 h.� Ethanol was used as a negative control.�

Detection of effect on SOS induction and general protein synthesis (Test C)
The exponential culture of E. coli strain I�111 was washed by centrifugation�� resuspended in 0.�01 M Mg�O 4 ��and irradiated with 10 J/m 2 .�The cells were incubated for 20 minutes in L� medium with and without EO or linalool�� washed by centrifugation�� resuspended in minimal medium supplemented with 10% casamino acids�� and incubated for 20 minutes on ice.�Following incubation�� optical density OD 600 was measured�� the samples were diluted in appropriate buffer�� and cells were lyzed.�The mixture was incubated at 28°C for 5 minutes�� and the enzymatic reaction was started by adding appropriate substrates for enzymes (2-nitrophenyl-β-D-galactopyranoside CA� No.� 73660�� Fluka �igma-Aldrich�� �teinheim�� Germany; and p-nitrophenyl phosphate�� CA� No.� 104-0�� �igma-Aldrich�� �teinheim�� Germany).�The concentration of β-galactosidase and alkaline phosphatase was determined as described by Q u i l l a r d and H o f n u n g (1993).�

Detection of effect on intrachromosomal recombination (Test D)
Intrachromosomal recombination was measured in E. coli strains GY8281 (recA + ) and GY8252 (recA730) by monitoring Lac + recombinants on MacConkey lactose plates (K o n r a d�� 1977) with or without EO or linalool.��amples (0.�01 ml) of bacterial exponential cultures (3 x 10 8 cells/ml) were spread in the form of patches (2 x 2 cm) in triplicate on the same plate and irradiated with split UV-doses (5+5 J/m 2 for recA + ; 1+1 J/m 2 for recA730).�The first UV exposure was immediately after plating and the second after 3 h of incubation at 37°C.�The number of Lac + papillae was determined after incubation at 37°C for 48 h.� �train GY7066 (ΔrecA)�� which formed no papillae�� was used as a negative control in all experiments.�

Determination of bacterial growth rate
Overnight cultures of repair-proficient strain �Y252 and excision repair deficient mutant I�105 were diluted 50 fold in fresh L� medium�� with or without EO or linalool�� and incubated at 37°C with aeration.��amples were taken every 30 min and optical density OD 600 was measured using a �himadzu UV/VI�-120-02 spectrophotometer.�

S. cerevisiae D7 assay
The exponential culture containing about 3 x 10 7 cells/ml was washed by centrifugation and resuspended in sterile distilled water .�The cell suspension was irradiated with a UV-dose of 130 J/m 2 .��amples (0.�1 ml) of unirradiated and UV-irradiated cells�� appropriately diluted for determination of cell survival and Ade + recombinants�� and undiluted for determination of Ilv + and Trp + cells�� were spread in duplicate onto plates with different concentrations of EO or linalool.�Cell survival and Ade + recombinants were determined on YPD plates.�Ilv + revertants and Trp + convertants were scored on minimal plates supplemented with tryptophan or isoleucine�� respectively.�Plates were incubated at 30°C for 72 h.� Ethanol was used as a negative control.�

Statistical analysis
The �tudent's t-test was employed for statistical analysis.��ignificance was tested at the p<0.�05 level.�The results presented in figures and tables are expressed as the means obtained from three independent experiments�� with the standard error of the mean.�In all applied tests�� we calculated the percentage of inhibition of mutagenesis (% I) as described by W a l l et al.� (1988).�

Antimutagenic potential of EO and linalool against spontaneous and UV-induced mutagenesis in Escherichia coli
In order to investigate the possible role of DNA repair pathways in the antimutagenic effect of EO of basil and linalool�� we investigated their effect on spontaneous and UV-induced mutagenesis.��pontaneous mutagenesis was examined in repair-proficient�� nucleotide excision repair-deficient (uvrA)�� and MMR-deficient (mutS) strains.�The applied concentrations of both basil derivatives (up to 20 µl/plate) were not toxic and had no effect on spontaneous mutagenesis in any of the tested strains (data not shown).� The effect of basil derivatives on survival and UV-induced mutagenesis is shown in Fig. � 1.�In the range of applied concentrations�� both EO and linalool exhibited caused reduction of UV-induced mutagenesis in the repair-proficient strain (Figs.� 1a�� 1b).�Maximum reduction�� 32% for EO and 51% for linalool�� was achieved at a concentration of 20 µl/ plate�� with more than 80% surviving cells.�At all tested concentrations�� linalool exhibited a stronger antimutagenic effect in comparison with EO.�In the nucleotide excision repair-deficient strain�� neither derivative had any effect on UV-induced mutagenesis (Figs.� 1c�� d).�

Effects of EO and linalool on SOS induction in Escherichia coli
The effects of EO and linalool on the level of �O� induction�� corresponding to the induction of mutagenic �O� repair�� were monitored in repairproficient strain I�111.�In this strain�� expression of the lacZ gene�� coding for the enzyme β-galactosi-dase�� is placed under control of the sfiA gene.��ince sfiA is one of the �O� genes�� the level of β-galactosidase will reflect the level of �O� induction.�The non-specific effects of EO and linalool on general protein synthesis were determined by measuring the enzyme alkaline phosphatase�� which is constitutively expressed in this strain.� The applied concentration of EO and linalool (0.�7 μl/ml�� showing the highest antimutagenic response in the repair-proficient strain) decreased the amount of UV-induced β-galactosidase�� by 13 and 30%�� respectively (Fig. � 2).�However�� there was similar inhibition of alkaline phosphatase�� 10% for EO and 27% for linalool�� indicating that the obtained effect on β-galactosidase synthesis is non-specific.�

Effects of EO and Linalool on intrachromosomal recombination in Escherichia coli
The effects on homologous recombination were measured using strains with two non-overlapping deletions in the duplicated lac operon�� in which intrachromosomal recombination results in the formation of Lac + recombinants.�The strains carry different recA alleles and thus have different capacities for both recombination and �O� induction.�In the recA + strain�� with a functional recA gene�� there is significant inhibition of intrachromosomal recombination in the presence of EO�� both in unirradiated (37% and 24%) and UV-irradiated (29%) sample (Table 3).�In comparison with EO�� a weaker inhibitory effect is obtained with linalool.�In the recA730 strain�� which constitutively possesses a high level of activated RecA protein�� significant stimulation of recombination is detected in the presence of both basil derivatives.�The maximum stimulation of recombination by EO was 132% in unirradiated and 85% in UV-irradiated samples�� while linalool showed maximum stimulation of 177% in unirradiated and 48% in UV-irradiated samples.�The obtained results show that basil derivatives inhibit intrachromosomal recombination in the recA + strain�� but stimulate recombination in the recA730 mutant.�

Effect of EO and linalool on bacterial growth rate
To check whether the reduction of UV-induced mutagenesis results from selective inhibition of growth of the repair-proficient strain�� we compared the growth rates of repair-proficient �Y252 and excision repair-deficient I�105 cells in the presence of EO or linalool.�In both strains�� there was similar inhibition of cell growth during 300 min of incubation with EO or linalool (Fig. � 3).�

Effect of EO and linalool in the Saccharomyces cerevisiae D7 assay
Investigation of spontaneous and UV-induced mutagenesis in S. cerevisiae D7 showed no significant changes in the number of Ilv + revertants caused by EO or Linalool (Fig. � 4).�Although the applied concentrations (2.�5�� 5 and 7.�5 μl/ plate) were not toxic to S. cerevisiae D7 cells�� as determined by monitoring colony counts on YPD plates�� there was a dose dependant increase in the proportion of small colonies in both unirradiated and UV-irradiated samples�� indicating slower growth in the presence of basil deriv-Fig.2. Effects of basil derivatives on SOS induction (β-galactosidase) and protein synthesis (alkaline phosphatase) in repair proficient strain IB111 of E. coli K12 after UV-irradiation (10 J/m2) Fig. 3. Effects of EO and linalool on growth rate of E. coli K12 strains.EO 1 μl/ml for SY252, 0.5 μl/ml for IB105; linalool: 0.7 μl/ml for SY252, 0.3 μl/ml for IB105.
atives.�The number of pigmented twin-spot colonies�� resulting from UV-induced mitotic crossing over at the ade2 locus�� decreased 2-3 times relative to the control�� while mitotic gene conversion was not af-mitotic gene conversion was not affected (data not shown).�DI�CU��ION DNA repair is a dynamic process and can be modulated by many factors�� such as the rate of DNA replication and cell proliferation�� the level of expression of certain genes�� inactivation of certain repair enzymes�� etc.� Protective effect of antimutagens following DNA damage can be obtained mainly through increase in the fidelity of DNA replication�� stimulation of error-free repair of DNA damage�� and inhibition of error-prone repair systems (K a d a et al., 1985).�In the present work�� we examined the antimutagenic effects of basil derivatives (EO and linalool) against UV-induced mutations and participation of different mechanisms in antimutagenesis.� In the E. coli K12 assay system�� EO and linalool inhibited UV-induced mutagenesis in a repair-proficient strain�� but had no effect on spontaneous mutagenesis in repair-proficient�� nucleotide excision repair-deficient�� and MMR-deficient strains.�These results indicate that both basil derivatives have an antimutagenic potential that is independent of the MMR pathway and modulation of DNA replication.�Although EO and linalool had a similar inhibitory effect on the growth of repair proficient and nucleotide excision repair deficient cells (Fig.
� 3)�� an antimutagenic effect against UV-induced mutagen-esis is detected only in the repair-proficient strain (Fig.� 1).�We propose that by arresting bacterial growth and cell division�� basil derivatives increase the chances that DNA lesions will be repaired by nucleotide excision repair in an error free manner before the next division takes place.�Theresults obtained in other tests are consistent with this idea.�As clearly shown in Test C�� the reduction of �O� induction by basil derivatives is non-specific�� caused by general inhibition of protein synthesis (Fig.�2).�Increased recombination following DNA damage is also ruled out as a mechanism because both derivatives inhibited recombination in the UV-irradiated recA + strain (Test D�� Table3).�Our speculation that this effect is caused by inhibition of RecA protein amplification is supported by increased recombination in the recA730 mutant�� with high constitutive levels of RecA protein.�The observed reduction in the size of colonies and number of Ade + recombinants in S. cerevisiae D7 by basil derivatives could also be caused by inhibition of protein synthesis�� leading to reduced growth of the yeast population.�In addition�� published data suggest that essential oils in yeast induce damage to mitochondrial membranes resulting in liberation of RO��� oxidative stress�� and cell death (� a k k a l i etal., 2005).�Considering that liberation of RO� leads to increased incidence of oxidative DNA damage and that both UV photoproducts and oxidatively damaged DNA bases are removed by nucleotide excision repair (G e l l o n et al., 2001)�� saturation of the repair mechanism might account for the lack of antimutagenic effects of EO and linalool against

Table 2 .
Chemical composition of essential oil of basil (%).

Table 3 .
Effect of basil derivatives on intrachromosomal recombination in E. coli.