Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 296–305

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Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells a,∗ ˇ ˇ Hojnik Niderl c , Metka Filipiˇc a Bojana Zegura , David Dobnik b , Masa a b c

National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Veˇcna pot 111, 1000 Ljubljana, Slovenia National Institute of Biology, Department of Biotechnology and Systems Biology, Veˇcna pot 111, 1000 Ljubljana, Slovenia Vitiva d.d., Nova vas pri Markovcih 98, 2281 Markovci, Slovenia

a r t i c l e

i n f o

a b s t r a c t

Article history:

In the present study the chemopreventive effects of water soluble AquaROX® 15 and

Received 15 October 2010

oil soluble VivOX® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and

Received in revised form

2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse

13 June 2011

mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-

Accepted 16 June 2011

BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

Available online 23 June 2011

induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA dam-

Keywords:

age, whereas protection against indirect active genotoxic carcinogens was more efficient by

Antioxidants

VivOX.

Antigenotoxicity

© 2011 Elsevier B.V. All rights reserved.

Bacterial reverse mutation assay Comet assay HepG2 cells Rosmarinus officinalis L

1.

Introduction

Herbs and spices, which contain many phytochemicals with potential antioxidant capacity, have been used since ancient times for food flavouring. Many of these plant antioxidants are interesting in food industry to prolong the storage stability and the preservation of foods as well as in the pharmaceutical preparations in order to replace synthetic antioxidants (Ames, 1983; Baardseth, 1989). This has led to an increase in the use of natural antioxidants, especially those of plant origin, in

the form of chemically defined extracts. Most antioxidants isolated from higher plants are polyphenols and have been intensively studied during the last 20 years. The most commonly used herbs and spices as a source of antioxidants are Rosmarinus officinalis L., Salvia officinalis L. and Thymus vulgaris L. R. officinalis L., commonly referred as rosemary belongs to Laminaceae family of Mediterranean origin. It is an evergreen branched and bushy shrub and is a popular herb and spice. Rosemary is also used in the cosmetics and medicine. In folks medicine rosemary has been used as an analgesic,

Abbreviations: BaP, benzo(a)pyrene; IQ, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline; NQNO, 4-nitroquinoline-N-oxide; PhIP, 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; ROS, reactive oxygen species; t-BOOH, t-butyl hydroperoxide. ∗ Corresponding author. Tel.: +386 59232862; fax: +386 1 257 38 47. ˇ E-mail address: [email protected] (B. Zegura). 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.06.002

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anti-rheumatic, carminative, diuretic, expectorant, antiepileptic, for effects on human fertility and as hepatoprotectant (al-Sereiti et al., 1999). The extract of R. officinalis L. has been shown to have cancer preventive properties (Singletary and Nelshoppen, 1991; Huang et al., 1994; Singletary et al., 1996a) as well as anti-oxidative stress properties (Horváthová et al., 2010). Several studies showed that rosemary extract suppressed the binding of carcinogens to DNA and the formation of DNA adducts in several tissues (Singletary and Nelshoppen, 1991; Huang et al., 1994; Offord et al., 1995; Amagase et al., 1996; Singletary et al., 1996a). The most important constituents of rosemary are rosmarinic acid, carnosic acid, carnosol, rosmanol, flavonoids and other phenolic compounds (Aruoma et al., 1992; Haraguchi et al., 1995; Hernández-Hernández et al., 2009). Rosmarinic acid is an ester of caffeic acid and 3,4dihydroxyphenyllctic acid (Scarpati and Oriente, 1958). It has a number of interesting biological activities such as antidepresive (Takeda et al., 2002), hepato-protective (Osakabe et al., 2002), anti-inflammatory (Peng et al., 2007), antiangiogenic (Huang and Zheng, 2006), antitumor (Singletary and Nelshoppen, 1991; Huang et al., 1994; Singletary et al., 1996a), acts as photo-protective agent (Li et al., 2010) and has HIV-1-inhibiting properties (Aruoma et al., 1996). Recently, it has been shown that in peripheral polychromatic erythrocytes, rosmarinic acid reduced the formation of micronuclei induced by chemotherapeutic agent, doxorubicin (Furtado et al., 2008). The o-diphenolic diterpene carnosic acid and its oxidation product carnosol, possess anti-bacterial, anti-mutagenic, anti-inflammatory anti-proliferative, antitumorigenic and neuroprotective properties (Aruoma et al., 1992; Minnunni et al., 1992; Oluwatuyi et al., 2004; Izumi et al., 2007; Peng et al., 2007). Carnosic acid and carnosol are considered to account for more than 90% of the antioxidant properties of the rosemary extract; they are powerful inhibitors of lipid peroxidation and good scavengers of peroxyl radicals (Aruoma et al., 1992). Carnosol has been shown to interfere with tumor cell metastasis, chemotaxis, attachment and inhibition of invasion by targeting matalloproteinasemediated cellular events (Huang et al., 2005). The aim of the present study was to investigate the chemopreventive activity of two rosemary extracts, water soluble AquaROX® 15 and oil soluble VivOX® 40, on NQNO and IQ-induced mutations in the reverse mutation assays with Salmonella typhimurium TA98 and on DNA damage induced in human hepatoma HepG2 cells by direct (t-BOOH) and indirect (BaP, PhIP) mutagens using the comet assay.

2.

Materials and methods

2.1.

R. officinalis L. extracts and chemicals

Rosemary extracts AquaROX® 15 (301822) and VivOX® 40 (301803) were supplied by Vitiva d.d., Slovenia. The content of rosmarinic acid in AquaROX® 15 was 17% and the contents of 2 major rosemary antioxidative components in VivOX® 40, carnosic acid and carnosol, were 50.27 and 5.65%, respectively. All chemicals and solvents used for HPLC analysis were of analytical quality (p.a.).

2.2.

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HPLC analysis of R. officinalis L. extracts

Quantitative determination of the individual antioxidant components in the rosemary extracts AquaROX® 15 and VivOX® 40 were performed by Vitiva d.d. using high performance liquid chromatography (HPLC). The HPLC system for the determination of rosmarinic acid consisted of a SpectraSystem P1500 V3.02 pump (SpectraPhysics, USA), a UV/VIS detector (Spectra-Physics, USA), a PS 1000 V3.0 software (Spectra-Physics, USA) and a Rheodyne injector. A Kromasil 100 C18 (250 mm × 4.6 mm, 5 ␮m) column (Bia Separations, Slovenia) was used. The separation was isocratically undertaken with a mobile phase consisting of 0.03% (w/v) aqueous trifluoroacetic acid and acetonitrile (25:75, v/v) at a flow rate of 1.5 ml/min and detection wavelength 280 nm. The HPLC system and a column for determination of carnosic acid and carnosol was the same as for rosmarinic acid. The mobile phase was a mixture of acetonitrile and water (70:30, v/v), which contained 1.2 ml of phosphoric acid per liter of mobile phase. The mobile phase flow rate was 1.2 ml/min and the detection wavelength was 230 nm. The stock solutions of R. officinalis extracts AquaROX® 15 and VivOX® 40 were prepared in distilled water and 96% ethanol, respectively.

2.3.

Bacterial and human cell cultures

S. typhimurium TA98 was used for the mutagenicity and antimutagenicity testing and was obtained from Prof. B.N. Ames (Biochemistry Department, University of California, Berkeley). The working cultures were prepared from frozen permanents by overnight incubation (37 ◦ C) in nutrient broth N◦ 2. The human hepatoma cell line (HepG2) was a gift from Prof. F. Darroudi (Leiden University Medical Centre, Department of Toxicogenetics, Leiden, The Netherlands). The cells were grown in William’s medium E (Sigma, St. Louis, USA), supplemented with 15% foetal bovine serum (FBS), 2 mM lglutamine, and 100 IU penicillin/streptomycin at 37 ◦ C in 5% CO2 .

2.4.

Bacterial mutagenicity/antimutagenicity assay

The mutagenicity/antimutagenicity studies were performed with the Salmonella/microsomal reverse mutation assay according to Maron and Ames (1983). For the mutagenicity assay an overnight culture of S. typhimurium strain TA98 (100 ␮l), and AquaROX® 15/VivOX® 40 dilution (100 ␮l) were added to 2 ml of molten top agar (42 ◦ C), mixed and poured onto minimal agar plates. The final concentrations of AquaROX® 15 and VivOX® 40 were 0.05, 0.1 and 0.2 mg/plate. The experiment was performed in 3 replicates. For the antimutagenicity assay, the overnight bacterial culture (100 ␮l), 10 ng/plate IQ or 500 ng/plate NQNO, AquaROX® 15/VivOX® 40 dilution (100 ␮l) were added to the molten top agar, mixed and poured onto minimal agar plates. When tested the antimutagenic activity against IQ, 4% S9 mix (500 ␮l) was added to the mixture. The final concentrations of AquaROX® 15/VivOX® 40 extract were the same as in the mutagenicity assay. The number of His+ revertants was scored after incubation for 72 h at 37 ◦ C. The percentage of the inhibition

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of the mutagenic effect was calculated according to the equation: % inhibition = (1 − TM/M) × 100, where TM is the number of His+ revertants per plate in the presence of AquaROX® 15/VivOX® 40 in the combination with NQNO/IQ, and M is the number of His+ revertants per plate in the presence of NQNO/IQ alone. The antimutagenic effect was considered strong when inhibition of mutagenesis was higher than 40%, moderate when it was in the range between 25% and 40%, and weak or absent when the inhibitory effect was less than 25% (Ikken et al., 1999).

2.5.

Cytotoxicity assay in mammalian cells

The cytotoxicity of rosemary extracts was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay according to Mosmann (1983) with minor modifications (Zegura et al., 2003). HepG2 cells were seeded onto 96-well plates at a density of 5 × 103 cells/well and incubated overnight at 37 ◦ C to attach. The medium was then replaced with fresh complete medium containing 0.5, 5, 25, 50 and 100 ␮g/ml AquaROX® 15 and VivOX® 40 extract and incubated for 21 h (37 ◦ C/5% CO2 ). MTT (final concentration 0.5 mg/mL) was then added and the plates were incubated for additional 3 h. At the end of the incubation with MTT, the medium was removed, and the formazan crystals were dissolved in DMSO. The optical density (OD) was measured at 570 nm (reference filter 690 nm) using a microplate reading spectrofluorimeter (Tecan, Genios). Cell viability was determined by comparing the OD of the wells containing rosemary extract treated cells to that of the vehicle (0.1% ethanol) treated cells. 5 individual wells were measured per treatment point.

2.6. Genotoxicity/antigenotoxicity assays in mammalian cells

into fresh medium and the comet assay was performed as described below. The concentration of t-BOOH was 0.5 mM and was dissolved in PBS immediately before use. For all treatment conditions, three independent experiments were performed.

2.6.2. Protective effect of R. officinalis extracts against DNA damage induced by pro-carcinogens BaP and PhIP The cells were co-treated with BaP (40 ␮M) or PhIP (80 ␮M) together with different concentrations of AquaROX® 15 (0.05, 0.5, 5 in 50 ␮g/ml) or VivOX® 40 (0.05, 0.5 in 5 ␮g/ml) for 21 h at 37 ◦ C. After the treatments the cells were washed with PBS, trypsinized, centrifuged at 115 × g for 5 min and re-suspended into fresh medium and the comet assay was performed as described below.

2.7.

The assay was performed as described by Singh et al. (1988). 30 ␮l of cell suspension was mixed with 70 ␮l of 1% LMP (low melting point) agarose and added to fully frosted slides that had been covered with a layer of 1% NMP (normal melting point) agarose. The slides were lysed (2.5 M NaOH, 0.1 M EDTA, 0.01 M Tris and Triton X-100, pH 10) for 1 h at 4 ◦ C, transferred into electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH 13) for 20 min to allow DNA unwinding, and electrophoresed for 20 min at 25 V and 300 mA. Finally, the slides were neutralized with 0.4 M Tris buffer (pH 7.5), stained with ethidium bromide (5 ␮g/ml) and analyzed using fluorescence microscope (Nikon, Eclipse 800) and image analysis software (Comet IV, Perceptive Instruments). 50 nuclei were analyzed per experimental point in each of the 3 independent experiments, and the percentage of the fluorescence in the comet tail was scored as a reflection of DNA damage.

2.8. The genotoxicity and antigenotoxicity of AquaROX® 15 and VivOX® 40 were evaluated using the comet assay. The genotoxicity of the extracts was tested by the exposure of HepG2 to the highest non-cytotoxic concentration (50 ␮g/ml AquaROX® 15 and 5 ␮g/ml VivOX® 40) for 21 h.

2.6.1. Protective effect of R. officinalis extracts against t-BOOH induced oxidative DNA damage To determine the antigenotoxic potential of the AquaROX® 15 and VivOX® 40 against t-BOOH induced oxidative DNA damage three treatment schedules were applied: (1) the cells were pre-treated with AquaROX® 15 (0.05, 0.5, 5 in 50 ␮g/ml) or VivOX® 40 (0.05, 0.5 in 5 ␮g/ml) for 21 h at 37 ◦ C/5% CO2 , washed with PBS buffer and then exposed to t-butyl hydroperoxide (tBOOH) for 20 min at 4 ◦ C, (2) the cells were co-treated with t-BOOH together with different concentrations of AquaROX® 15 (0.05, 0.5, 5 in 50 ␮g/ml) or VivOX® 40 (0.05, 0.5 in 5 ␮g/ml) for 20 min at 4 ◦ C, and (3) the cells were pre-treated with AquaROX® 15 (0.05, 0.5, 5 in 50 ␮g/ml) or VivOX® 40 (0.05, 0.5 in 5 ␮g/ml) for 21 h at 37 ◦ C/5% CO2 , washed with PBS buffer and then exposed to t-BOOH together with AquaROX® 15 (0.05, 0.5, 5 in 50 ␮g/ml) or VivOX® 40 (0.05, 0.5 in 5 ␮g/ml) for 20 min at 4 ◦ C. After the treatments the cells were washed with PBS, trypsinized, centrifuged at 115 × g for 5 min and resuspended

Comet assay (single cell gel electrophoresis, SCGE)

Free-radical scavenging activity (DPPH assay)

The antioxidant activity of R. officinalis extracts was measured in terms of hydrogen-donating or radical-scavenging ability in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay (Blosi, 1958). The assay was performed as described by Plazar et al. (2007) with minor modifications. Reaction mixtures containing 0, 5, 10, 50 and 100 ␮g/ml AquaROX® 15/VivOX® 40 and 150 ␮M DPPH in ethanol were incubated at 37 ◦ C for 60 min in 96-well microtiter plates. The decrease of absorbance of the free radical DPPH was measured at 515 nm with a microplate spectrofluorimeter (Tecan Genios). Ascorbic acid was used as a positive control. The free radical scavenging activity was calculated as the percentage of DPPH radical that was scavenged, as follows: % radical scavenger activity = (1 − A1 /A0 ) × 100, where A0 was the absorbance of the control reaction (without R. officinalis extracts) and A1 the absorbance in the presence of R. officinalis extracts or ascorbic acid. 2 independent experiments with 5 replicates each were performed.

2.9.

Statistical analysis

The Student’s t-test was used for the statistical analysis of the data obtained in the bacterial mutagenicity and antimuta-

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Table 1 – The effect of rosemary extract AquaROX® 15 and VivOX® 40 on NQNO- and IQ-induced mutagenesis in Salmonella typhimurium TA98 strain. −S9 mix

Concentration (mg/plate)

−NQNO revertants/plate AquaROX® Vehicle 0 0.05 0.1 0.2 PC VivOX® Vehicle 0 0.05 0.1 0.2 PC

25.50 ± – 27.00 ± 31.33 ± 27.00 ± 412.33 ±

6.36

33.33 ± – 38.00 ± 40.00 ± 29.00 ± 412.33 ±

5.51

9.64 0.58 5.57 62.61

10.58 3.46 9.90 62.61

+S9 mix (4%)

+NQNO revertants/plate

−IQ revertants/plate

+IQ (10 ng/plate) revertants/plate 38 ± 561.67 ± 601.33 ± 489.67 ± 243.33 ± –

3 23.16 38.28 40.70 29.14*

± ± ± ± ±

3 23.16 71.60* 9.29* 12.17*

36.33 ± 401.67 ± 404.00 ± 387.67 ± 392.00 ± –

6.66 20.74 21.63 20.65 38.00

37.00 ± – 44.67 ± 33.67 ± 35.33 ± 225.67 ±

36.33 ± 401.67 ± 362.00 ± 277.00 ± 186.50 ± –

6.66 20.74 24.64* 9.54* 26.16*

38 ± – 44.00 ± 50.33 ± 46.00 ± 225.67 ±

5.29 4.16 40.70 5.51 38.79 67 11.36 3.51 2.67 38.79

38 561.67 404.67 302.33 174.00

The results are presented as the means ± SD. 4-Nitroquinoline-N-oxide (NQNO; 500 ng/plate) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ; 10 ng/plate) were used as the positive controls (PC) in the absence and presence of S9 metabolic activation, respectively. ∗ P < 0.05 (Student’s t-test).

genicity tests and the MTT cytotoxicity assay. The significance was tested at P < 0.05 level. For the results of the comet assay, one-way analysis of variance (non-parametric ANOVA, Kruskal–Wallis test) was used to analyze differences between the treatments within each experiment. Dunnet Post Test was used to compare median values of % of the fluorescence in the tail for all treatments; *P < 0.05; ** P < 0.01; ***P < 0.001 was considered as statistically significant.

3.

Results

3.1. Antimutagenicity of R. officinalis extracts in S. typhimurium TA98 None of the R. officinalis extracts was mutagenic in TA98 in the absence and presence of S9 metabolic activation (Table 1). The NQNO induced mutations were strongly suppressed by VivOX® 40 extract, whereas AquaROX® 15 extract was ineffective (Table 1 and Fig. 1A). The IQ induced mutations were suppressed by both extracts (Table 1 and Fig. 1B). VivOX® 40 extract exerted moderate to strong antimutagenic effect at all tested concentrations. AquaROX® 15 was ineffective at 2 lower concentrations, whereas at 0.2 mg/plate it exerted a strong antimutagenic effect.

3.2. Cytotoxicity and genotoxicity of R. officinalis extracts in HepG2 cells The effect of R. officinalis extracts on the viability of HepG2 cells was measured with the MTT assay. The incubation of cells with 0.5–100 ␮g/ml of AquaROX® 15 extract for 21 h had no effect on cell viability, while VivOX® 40 extract at concentrations 25 ␮g/ml and higher (25–100 ␮g/ml), significantly decreased cell viability (Fig. 2). Eventual genotoxic potential of

R. officinalis extracts was determined with the comet assay at the highest non-toxic concentration (50 ␮g/ml for AquaROX® 15 and 5 ␮g/ml for VivOX® 40). None of the extracts induced DNA damage at these concentrations (data not shown). Based on these results the concentrations 0.05, 0.5, 5 and 50 ␮g/ml for AquaROX® 15 and 0.05, 0.5 and 5 ␮g/ml for VivOX® 40 extract were used for the antigenotoxicity studies.

3.3. Protective effect of R. officinalis extracts against t-BOOH induced oxidative stress The antigenotoxic effects of AquaROX® 15 and VivOX® 40 extracts against t-BOOH induced DNA damage were assessed by the comet assay. The concentration of t-BOOH used for the induction of DNA damage in HepG2 cells was 0.5 mM. At these concentrations t-BOOH induced DNA damage without significant reduction of cell viability (data not shown). In HepG2 cells AquaROX® 15 extract significantly reduced t-BOOH induced DNA damage in the pre- (Fig. 3A) and pre/co- treatment (Fig. 3E) experiments, while in the co-treatment (Fig. 3C) experiment it only slightly, but not significantly, attenuated DNA damage. In pre-treated cells, AquaROX® 15 reduced DNA damage by 50% and 34% (median) at 50 and 5 ␮g/ml and in pre-/co-treated cells by 64% and 34%, respectively. Similarly, the VivOX® 40 extract, which was due to its cytotoxicity tested at lower concentrations, at 5 ␮g/ml, reduced DNA damage by 32% in pre- (Fig. 3B) and by 29% in pre-/co-treated cells (Fig. 3F).

3.4. Protective effect of R. officinalis extracts against DNA damage induced by pro-carcinogens BaP and PhIP The concentrations of BaP and PhIP used for the induction of DNA damage in HepG2 were 40 ␮M and 80 ␮M, respectively. At these concentrations BaP and PhIP induced DNA damage without significant reduction of cell viability (data not shown).

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Fig. 2 – The effect of rosemary extracts AquaROX® 15 and VivOX® 40 on viability of HepG2 cells. The viability was determined with the MTT assay after exposure of the cells to different concentrations of AquaROX® 15 and VivOX® 40 extracts (0.5, 5, 25, 50 in 100 ␮g/ml) for 21 h. *Significant difference between treated cells and control cells (Student’s t-test, P < 0.05).

ing activity comparable to that of the ascorbic acid, which is a strong antioxidant (Fig. 5). After a 60 min reaction at concentrations 50 and 100 ␮g/ml both extracts scavenged up to 60% DPPH radicals. At the lowest concentration (5 ␮g/ml) the scavenging of DPPH by AquaROX® 15 and VivOX® 40 was even more efficient than that of ascorbic acid.

4.

Fig. 1 – Antimutagenic effect of rosemary extract AquaROX® 15 and VivOX® 40 against NQNO (A) and IQ (B) induced mutations in S. typhimurium TA98 strain.

AquaROX® 15 extract significantly reduced BaP (Fig. 4A) and PhIP (Fig. 4B) induced DNA damage by 38 and 60%, respectively, only at the highest tested concentration (50 ␮g/ml). The VivOX® 40 extract showed the protective effect at lower applied concentrations; it significantly reduced BaP induced DNA damage by 22% and 28% (Fig. 4A), and PhIP induced DNA damage by 43% and 54% at 0.5 and 5 ␮g/ml, respectively (Fig. 4B).

3.5. The free-radical scavenging activity of R. officinalis extracts The free-radical scavenging activity of R. officinalis extracts was determined using the DPPH, a stable radical with a maximum absorption at 515 mm, which is readily reduced by an antioxidant. Both R. officinalis extracts showed DPPH scaveng-

Discussion

Phenolic compounds, which are widely distributed in plants, are considered to play an important role as dietary antioxidants in the prevention of oxidative damage in the living systems and can provide protection against cancer, coronary arteriosclerosis, and the ageing processes. In the present study, we showed the chemopreventive activity of R. officinalis extracts AquaROX® 15 containing 17% of rosmarinic acid, and VivOX® 40 containing 50.27% of carnosic acid and 5.65% of carnosol against NQNO- and IQ-induced mutations in S. typhimurium TA98 and DNA damage induced by oxidative agent t-BOOH and pro-mutagens BaP and PhIP in HepG2 cells. In the bacterial test system the VivOX® 40 extract was anti-mutagenic against direct acting mutagen NQNO and promutagen IQ, while AquaROX® 15 decreased only IQ-induced mutagenicity. Recently, it has been demonstrated that in S. typhimurium rosmarinic acid suppressed the mutagenicity of indirect acting mutagens sodium azide and N-methyl-N nitro-N-nitroguanidine (Vattem et al., 2006), whereas no data exist on the antimutagenic activity against pro-mutagens. Natural compounds exert their potential anticarcinogenic effects by multiple mechanisms. Beside the strong free radical activity phenolic compounds can act as antioxidants by chelating metal ions, preventing radical formation, and indirectly by modulating enzyme activities and altering expression levels of important enzymes, such as antioxidant and detoxifying enzymes (Ferguson, 2001; Ferguson et al.,

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Fig. 3 – Protective effect of AquaROX® 15 (0.05, 0.5, 5 and 50 ␮g/ml) and VivOX® 40 (0.05, 0.5 and 5 ␮g/ml) extract against t-BOOH induced DNA damage in HepG2 cells determined with the comet assay. The cells were either (i) pre-treated (white columns) with AquaROX® 15 (A) or VivOX® 40 (B) extract for 21 h, washed and then exposed to 0.5 mM t-BOOH, (ii) co-treated (light grey columns) with AquaROX® 15 (C) or VivOX® 40 (D) extract and 0.5 mM t-BOOH for 20 min or (iii) pre-treated (dark grey columns) with AquaROX® 15 (E) or VivOX® 40 (F) extract for 21 h, washed and then exposed to AquaROX® 15 or VivOX® 40, respectively, together with 0.5 mM t-BOOH for 20 min. Control cells were exposed to PBS for 20 min. The results are expressed as % tail DNA Fifty cells were analyzed per experimental point in each of the three independent experiments. Data are presented as quantile box plots with 95% confidence intervals. The edges of the box represent the 25th and 75th percentiles, the mean value is a solid line through the box. (*) Denotes a significant difference between the control and treated cells (Kruskal–Wallis test, P < 0.05).

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Fig. 5 – DPPH radical-scavenging activity of AquaROX® 15 extract, VivOX® 40 extract and ascorbic acid (AA). The direct interaction of AquaROX® 15, VivOX® 40 or AA with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured ex vivo in 96-well microtiter plates after 60 min, as described in Section 2. Data are presented as mean values of 5 replicates (±SD).

Fig. 4 – Protective effect of AquaROX® 15 (0.05, 0.5, 5 and 50 ␮g/ml) and VivOX® 40 (0.05, 0.5 and 5 ␮g/ml) extract against BaP (A) and PhIP (B) induced DNA damage in HepG2 cells determined with the comet assay. The cells (A) were either co-treated with AquaROX® 15 extract and 40 ␮M BaP (light grey columns) or co-treated with VivOX® 40 extract and 40 ␮M BaP (dark grey columns) or (B) were either co-treated with AquaROX® 15 extract and 80 ␮M PhIP (light grey columns) or co-treated with VivOX® 40 extract and 80 ␮M PhIP (dark grey columns) for 21 h. The results are expressed as % tail DNA Fifty cells were analyzed per experimental point in each of the three independent experiments. Data are presented as quantile box plots with 95% confidence intervals. The edges of the box represent the 25th and 75th percentiles, the mean value is a solid line through the box. (*) Denotes a significant difference between the control and treated cells (Kruskal–Wallis test, P < 0.05).

2004), which altogether may provide protection against cancer initiation. Since many carcinogens require metabolic activation, the use of cells that possess endogenous biotransforming activity has many advantages as exogenous activation mixtures prepared from rodent livers only partly reflects the bio-transformation of test compounds in the in vivo situation and the lack of detoxifying phase-II enzymes in conventional experimental models may lead to false positive results (Winter et al., 2008). The HepG2 cells, which express activity of several enzymes responsible for the activation of various xenobiotics, have been shown to be very promising for assessing the genotoxicity and antigenotoxicity (Knasmüller et al., 2004; Mersch-Sundermann et al., 2004; Plazar et al., 2007; Mitic-Culafic et al., 2009). Therefore, we further evaluated the antigenotoxic potential of rosemary extracts against oxidative DNA damage and DNA damage induced by pro-mutagens in metabolically competent HepG2 cells. The VivOX® 40 extract was toxic for HepG2 cells at concentrations 25 ␮g/ml and higher, whereas AquaROX® 15 extract at concentrations up to 100 ␮g/ml did not reduce cell viability. To investigate the protection of AquaROX® 15 and VivOX® 40 extracts against the cell injury initiated by acute oxidative stress we used t-BOOH, a short-chain analog of lipid hydroperoxide. We used 3 approaches to determine the mechanism of antigenotoxic potential of rosemary extracts against DNA damage induced by oxidants. In co-treatment procedure, where HepG2 cells were simultaneously exposed to rosemary extract and t-BOOH, extracts may protect cells against oxidant-induced DNA damage directly either by free radical scavenging activity or by decreasing free radical production through iron chelation (Anderson et al., 2000). In pre-treatment procedure, where cells were exposed to rosemary extracts for 21 h and then to t-BOOH for 20 min, AquaROX® 15 and VivOX® 40 extracts may indirectly act as antioxidants in cells by modulating the activity of antioxidant, detoxifying and repair enzymes as well as enzymes involved

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in the bioactivation of xenobiotics (Ferguson et al., 2004). The third approach pre-/co-treatment was the combination of two previously mentioned, in which the protective effect is due to the direct scavenging activity and induction of cellular protective mechanisms. We showed a very strong radical scavenging activity of VivOX® 40 and AquaROX® 15 extracts with the DPPH assay. The result is in line with the studies, which showed that the antioxidant activity of rosmarinic acid is mainly due to its redox properties, which play an important role in the adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Cervellati et al., 2002). Erkan et al. (2008) showed that the carnosic acid had higher antioxidant activity than rosmarinic acid and it has been suggested that carnosol and carnosic acid as powerful inhibitors of lipid peroxidation and good scavengers of peroxyl radicals and account for more than 90% of the antioxidant properties of rosemary extract (Aruoma et al., 1992). However, we did not observe significant decrease in t-BOOH induced DNA damage in the co-treatment procedure. This may be explained by possible poor permeation of AquaROX® 15 and VivOX® 40 extracts or their active components across the cell membrane during the relatively short time of exposure. Several studies showed that the biological antioxidant activity of phenolic compounds depends more on their polarity, hidrophobicity and bioavailability than on their intrinsic antiradical activity (Rice-Evans et al., 1996; Spencer et al., 2004; Lima et al., 2006). In the pre- and pre-/co-treatment experiments both rosemary extracts, at equal concentrations showed similar protective effects against t-BOOH induced DNA damage. The protective effect also did not significantly differ between pre- and pre-/co-treatment procedure, indicating that under applied experimental conditions induction of cellular defence mechanisms was involved in the protection of cells against oxidative damage induced by t-BOOH. Similarly to our results, pretreatment of human colon cancer (CaCo-2) and hamster lung (V79) cells with rosemary extract reduced the level of DNA strand breaks and oxidative DNA damage induced by H2 O2 and visible light-excited Methylene Blue (Slamenová et al., 2002). Recently, Horváthová et al. (2010) showed that in hepatocytes isolated from rats supplemented with rosemary oil for 14 days, decreased level of DNA damage induced by oxidative stress agents was detected compared to control rats. In HepG2 cells both extracts showed a significant protective effect against DNA damage induced by pro-carcinogens BaP and PhIP. Previously, it was observed that rosemary extract inhibited the genotoxic effects of the BaP in human bronchial epithelial cells (Offord et al., 1995) and aflatoxin B1 in human liver epithelial and human bronchial cells (Offord et al., 1997). In HepG2 cells rosmarinic acid showed a protective effect also against ochratoxin A and aflatoxin B1 induced cell death, oxidative stress and apoptosis (Renzulli et al., 2004). These results indicate that protective effects of the extracts against pro-carcinogens may be related to the modulation of drug metabolizing enzymes involved in carcinogen activation and detoxification. In human liver and bronchial cells carnosol and carnosic acid inhibited mRNA expression and activity of cytochrome P450 (CYP1A) and increased glutathione transferase (GST) and quinon reductase (QR) mRNA expression and activities (Offord et al., 1995, 1997). In rats rosemary

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extract after i.p. injection exhibited significantly higher activities of phase II enzymes: glutathione S-transferase (GST) and NAD(P)H quinone reductase (QR) in the liver (Singletary, 1996b). Debersac et al. (2001a) reported that after p.o. administration UDP-glucuronosyltransferases (UGT1A6 and UGT2B1) were increased, which is particularly important as UGTs are involved in the glucuronidation of the detoxification phase of a wide variety of compounds including carcinogens and their metabolites to unreactive derivatives (Mackenzie et al., 1993). Based on these results Singletary et al. (1996a) and Debersac et al. (2001b) proposed that carnosol, may have an important role in the antitumour activity of rosemary extracts. Taken together, the results of our study show that R. officinalis extracts AquaROX® 15, which contained rosmarinic acid (17%), and VivOX® 40, which contained carnosic acid (50.27%) and carnosol (5.65%), efficiently protected bacterial and human cells against genotoxicity of direct and indirect acting mutagens and ROS-inducing agent. In the bacterial test system the VivOX® 40 extract was anti-mutagenic against direct acting mutagen NQNO and pro-mutagen IQ, while AquaROX® 15 decreased only IQ-induced mutagenicity. In HepG2 cells both extracts protected cells against oxidative stress induced by t-BOOH, with AquaROX® 15 being slightly more effective, while the extracts protected the cells equally against indirect acting mutagens (BaP and PhIP). The underlying mechanisms of protection seem to involve the modulation of cellular antioxidant responses as well as the effects on the bioactivation and detoxification of xenobiotics. However, further studies should be performed to better understand the mechanisms and conditions underlying rosemary extracts chemopreventive activities. Due to their low cost and commercial availability rosemary extracts might be interesting candidates for the development of dietary or pharmaceutical cancer chemopreventive supplements.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgement This work was supported by grant J1-6712 (MF), J1-2054 (MF), and P1-0245 (TTL) provided by Slovenian Research Agency.

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