DOI:http://dx.doi.org/10.7314/APJCP.2012.13.10.4993 Effects of Vanillic Acid Against Mitomycin C-Induced Genomic Damage in Human Lymphocytes in Vitro RESEARCH ARTICLE Genotoxic and Anti-Genotoxic Effects of Vanillic Acid Against Mitomycin C-Induced Genomic Damage in Human Lymphocytes In Vitro Merve Guler Erdem1, Nilufer Cinkilic1*, Ozgur Vatan1, Dilek Yilmaz1, Deniz Bagdas2, Rahmi Bilaloglu1 Abstract Vanillic acid, a vegetable phenolic compound, is a strong antioxidant. The aim of the present study was to determine its effects on mitomycin C-induced DNA damage in human blood lymphocyte cultures in vitro, both alone and in combination with mitomycin C (MMC). The cytokinesis block micronucleus test and alkaline comet assay were used to determine genotoxic damage and anti-genotoxic effects of vanillic acid at the DNA and chromosome levels. MMC induced genotoxicity at a dose of 0.25 µg/ml. Vanillic acid (1 µg/ml) significantly reduced both the rates of DNA damaged cells and the frequency of micronucleated cells. A high dose of vanillic acid (2 µg/ml) itself had genotoxic effects on DNA. In addition, both test systems showed similar results when tested with the negative control, consisting of dimethyl sulfoxide (DMSO) in combination with vanillic acid (1 µg/ml)+MMC. In conclusion, vanillic acid could prevent oxidative damage to DNA and chromosomes when used at an appropriately low dose. Keywords: Vanillic acid - anti-genotoxic - mitomycin-C - genotoxicity - micronucleus - comet assay Asian Pacific J Cancer Prev, 13 (10), 4993-4998 Introduction a positive control for in vitro genotoxicity test systems (Pawar et al., 2009; Sontakke and Fulzele, 2009). The hydroxylated derivatives of benzoic and Our research aimed to examine vanillic acid from two cinnamic acids are known as phenolic acids. Vanillic acid perspectives. First, we determined the genotoxic effect (4-hydroxy-3-methoxybenzoic acid, Figure 1) is a dietary of vanillic acid in human lymphocytes. Experimental phenolic compound found in plants and fruits (Duke, genotoxicity tests, such as the cytokinesis block 1992) and an intermediate in the production of vanillin micronucleus (CBMN) and the alkaline comet assay, are from ferulic acid (Lesage-Meessen, 1996; Civolani, 2000). well-known models used to study the effects of different Additionally, vanillic acid is a metabolic product of caffeic physical and chemical agents on DNA. Second, the anti- acid that is found in the urine of humans after consuming genotoxic effect of vanillic acid on MMC-induced genetic coffee, chocolate or green tea (Falconnier et al., 1994) and damage was tested using both in vitro test systems. it can exhibit antioxidant, antimicrobial and antimalarial activity (Rechner et al., 2001; Yemis et al., 2011). It is a Materials and Methods key component of the vanilla plant and in recent years, it has been used as a preservative and antiseptic agent in Chemicals the food, pharmaceutical and cosmetic industries (Boyce The following chemicals were purchased from Sigma et al., 2003). Despite its wide application, the effects of Chemical Co. (St. Louis, MO, USA): vanillic acid, MMC, vanillic acid on human health are not yet fully understood, low melting agarose (LMA), dimethyl sulfoxide (DMSO), particularly from the genotoxicity perspective. Mitomycin triton X-100, cytochalasin-B, histopaque 1077, sodium C (MMC) is an antibiotic drug from the DNA alkylating agent group. It has potent DNA cross-linking activity and creates oxidative damage within the cell (Gresolia, 2002; Pawar et al., 2009). MMC induces clastogenesis and mutagenesis and inhibits DNA synthesis, therefore showing strong genotoxic effects (Sontakke and Fulzele, 2009). Due to these properties, MMC has been used as Figure 1. The Chemical Structure of Vanillic Acid 1Department of Biology, Faculty of Science and Arts, Cell Culture and Genetic Toxicology Laboratory, 2Faculty of Medicine, Uludag University, Experimental Animals Breeding and Research Center, Bursa, Turkey *For correspondence: aydemirn@uludag.edu.tr Asian Pacific Journal of Cancer Prevention, Vol 13, 2012 4993 Merve Guler Erdem et al sarcosinate, trypan blue, ethidium bromide, RPMI 1640 +4xN4)/N, where N1 to N4 represent the number of cells medium, penicillin-streptomycin, fetal calf serum (FCS), with one to four nuclei and N is the total number of cells phytohemagglutinin-A, L-glutamine, Dulbecco’s PBS and scored. Na2EDTA. Normal melting agarose (NMA) and Giemsa were obtained from E-Merck (Germany). All the other Alkaline comet assay chemicals used in the study were of analytical grade and The assay was performed essentially as described obtained from local commercial sources. by Singh et al. (1988). Briefly, blood samples were diluted 1:1 with phosphate-buffered saline (PBS) and Lymphocyte treatment with vanillic acid and MMC then layered onto the Ficoll-Histopaque using a 4:3 The vanillic acid was dissolved in 2% DMSO under ratio of (blood+PBS) to Histopaque. The samples were sterile conditions. Treatment with vanillic acid at doses centrifuged at 400 g for 35 min; then, the lymphocyte- higher than 2 µg /ml was shown to be highly cytotoxic by enriched layer was removed. The samples were washed trypan blue viability tests, so we used two non-toxic but twice more with PBS and centrifuged at 350 g for 10 effective doses of vanillic acid. The MMC was dissolved min; then, a final wash with RPMI-1640 medium was in sterile distilled water at a concentration of 0.25 µg /ml, performed. The number of viable cells was assessed by as suggested by published literature. All solutions were staining the cells with trypan blue and counting the cells by prepared immediately before performing the experiments a hemocytometer. Viable cells were suspended in RPMI- to prevent degradation. The cultured lymphocytes were 1640 media supplemented with 15% FCS, 200 mM of divided into six groups as follows. Group 1: Negative L-glutamine, penicillin (100 units/mL) and streptomycin (DMSO) control (2%). Group 2: Vanillic acid (1 µg/ml) (100 µg/mL). Phytohemagglutinin (0.2 mL) was added Group 3: Vanillic acid (2 µg/ml). Group 4: MMC (0.25 µg/ to cultured lymphocytes to initiate cell division. The ml). Group 5: MMC (0.25 µg/ml)+vanillic acid (1 µg/ml) cells were incubated at 37°C in a humidified incubator Group 6: MMC (0.25 µg/ml)+vanillic acid (2 µg/ml). maintained with 5% CO2. Roughened slides were cleaned with 100% methanol Blood samples and air-dried. Two solutions, 0.5% normal-melting Blood samples (a total volume of 6 ml) were taken agarose (NMA) and 0.5% low-melting agarose (LMA), with heparinized syringes from four healthy, non-smoking were prepared in Ca2+- and Mg2+-free PBS. NMA (0.1 ml) donors who were not exposed to radiation or drugs (two was used to create the first layer, upon which 1000 cells males and two females, ages 22-31). Informed consent was suspended in 75 µL LMA+10 µL PBS were used for the obtained from all participants; the study was performed second layer. To prevent any additional DNA damage, the in accordance with the Declaration of Helsinki and with remaining steps were conducted in the dark. To lyse the the approval of the local ethics committee. cells and denature the DNA, the slides were immersed in freshly prepared ice-cold lysis solution (1% sodium CBMN assay sarcosinate, 2.5 M of NaCl, 100 mM of Na2EDTA, 10 mM The presence of micronuclei (MN) in a binucleated of Tris–HClat a pH 10, 1% Triton X-100 and 10% DMSO). cell was assayed by blocking cytokinesis, as described by The slides were then incubated for 1 h at 4°C in the dark Fenech (1991). Duplicated cell cultures were prepared for and placed in an ice bath on a horizontal electrophoresis a MN test. Briefly, heparin-treated whole blood samples (1 unit. The electrophoresis unit was filled with fresh buffer ml) were added to a sterile culture tube containing RPMI (1 mM of Na2EDTA, 300 mM of NaOH; pH 13) to cover 1640 medium supplemented with 15% FCS, antibiotics the slides, and the slides were maintained in high-pH (100 units/ml of penicillin and 100 µg/ml of streptomycin), buffer for 20 min to denature the DNA and expose the 200 mMol L-glutamine and 2.5% phytohemagglutinin. alkali-labile sites. Electrophoresis was conducted for 20 The tubes were incubated at 37°C for 72h. Cytochalasin min at 25 V (300 mA). Following electrophoresis, the B was added, at 6 µg/ml, 44 h after the start of incubation. slides were washed gently in a neutralization buffer (0.4 M The MMC and vanillic acid treatments were performed Tris-HCl, pH 7.5) to remove the alkali and detergents, and 48 h after the beginning of the incubation period and then they were stained with 100 µl of ethidium bromide continued until the end of the culture. At the end of the (2 µg/ml). incubation, the cells were harvested by centrifugation. The cells were harvested, cast on a pre-cooled slide and Microscopic detection of comets stained with Giemsa. For each sample, two slides were prepared, and 200 cells from each slide were scored. Observations were made Slide scoring using 400× magnification on a fluorescence microscope One thousand binucleated cells from each donor’s equipped with a 530-nm excitation filter and a 590-nm blood sample were scored for the presence of micronuclei barrier filter. The genetic damage index (GDI) was visually (MN) under a light microscope at 400x magnification. determined based on the size and intensity of the comet The MN frequency was expressed as the number of MN tail. The tails were divided into five categories (0-4) as per 1000 binucleated cells scored. In addition, the nuclear follows: Class 0 (no damage), Class 1 (little damage division index (NDI) was taken into account by examining with a tail length that was shorter than the diameter of the number of nuclei in 2000 observed cells. NDI was the nucleus), Class 2 (medium damage with a tail length calculated according to the formula proposed by Eastmond one to two times the diameter of the nucleus), Class 3 and Tucker (1989) as follows. NDI= (1xN1+2xN2+3xN3 (significant damage with a tail length between two-and- 4994 Asian Pacific Journal of Cancer Prevention, Vol 13, 2012 DOI:http://dx.doi.org/10.7314/APJCP.2012.13.10.4993 Effects of Vanillic Acid Against Mitomycin C-Induced Genomic Damage in Human Lymphocytes in Vitro a-half and three times the diameter of the nucleus) and (p<0.005). However, MMC+vanillic acid (2 µg/ml) in Class 4 (significant damage with a tail longer than three combination significantly decreased the MN frequency times the diameter of the nucleus). These categories were when compared with MMC alone (p<0.05).Vanillic acid (1 based on those established by Collins (2004). We used this µg/ml) significantly decreased the MN frequency induced categorization to obtain a quantitative measurement of by MMC when compared to the MMC+DMSO control DNA damage based on a score average that was weighted levels (p<0.001). according to the number of cells with each grade of The average NDI values of all groups in this study damage.The formula for determining the DNA damage are shown in Figure 3. As shown in Table 1 and Figure was measured by the Genetic Damage Index (GDI)= 3, there were no differences between the NDI values of (Class 1+2xClass 2+3xClass 3+4xClass 4)/(Class 0+Class DMSO and vanillic acid (1 and 2 μg/ml). MMC induced 1+Class 2+Class 3+Class 4). DNA damage was expressed a significant decrease in NDI compared with the DMSO as the mean percentage of cells with medium, high and control (p<0.001). The NDI value of the low dose complete DNA damage and was calculated as the sum vanillic acid (1 µg/ml) and MMC combination was not of the cells categorized with Classes 2, 3 and 4 damage significantly different from that of the DMSO control (Palus et al., 2003). Percentage of damaged cells (% DC) (p>0.05). When compared with the MMC group, the = [Class 2+3+4/Sum of cells in all classes including 0 and low dose vanillic acid and MMC combination showed a 1] x 100. significant improvement in NDI values (p<0.001). The NDI values were significantly lower in the vanillic acid Statistical analysis (2 µg/ml)+MMC combination treatment when compared The IBM SPSS Statistics 20 program was used for with the MMC and also DMSO control group (p<0.001); statistical analyses. Statistical analysis was performed using one-way analysis of variance (ANOVA) and post- MN/1000  BNC   hoc Tukey tests. Values are represented as the means ± 40   S.D. for the samples in each group. P-values of<0.05 were 35   considered significant. 30   25   Results 20   15   Two different concentrations (1 and 2 μg/ml) of 10   vanillic acid and MMC (0.25 µg/ml) were evaluated 5   0   both in combination and alone in two different assays DMSO   1µg  /  µl  VA   2  µg  /  µl    VA   MMC   1µg  /  µl    VA    +   2µg  /  µl    VA+   MMC   MMC   (CBMN and alkaline comet) to determine the genotoxic Figure 2. MN Frequency of Vanillic Acid and Vanillic and cytotoxic effects of vanillic acid on human peripheral Acid+MMC in the CBMN Assay. VA, Vanillic acid; MMC, lymphocytes in vitro. DMSO (2%) was used as a negative Mitomycin-C. control. The frequency of vanillic acid-induced MN and NDI in human lymphocytes is summarized in Table 1. NDI   The MN frequency of all vanillic acid- and MMC- 1.65   treated groups is shown in Figure 2. Briefly, as shown 1.6   in Table 1 and Figure 2, the low dose of vanillic acid 1.55   (1 μg/ml) did not show any significant differences in 1.5   the MN frequency when compared with the DMSO negative control (p>0.05). On the other hand, the high 1.45   dose of vanillic acid (2 μg/ml) increased MN formation 1.4   significantly when compared with DMSO (p<0.05). 1.35   The MN frequency of the MMC-treated group was 1.3   significantly higher than that of the DMSO control DMSO   1µg  /  µl  VA   2  µg  /  µl    VA   MMC   1µg  /  µl    VA     2µg  /  µl    VA+  +  MMC   MMC   (p<0.001); in addition, the MMC+vanillic acid (2 µg/ Figure 3. The Average Values of Nuclear Division ml) combination treatment showed a significantly higher Index, (NDI) of Vanillic Acid and Vanillic Acid+MMC MN formation when compared with the DMSO control in the CBMN Assay.VA, Vanillic acid; MMC, Mitomycin-C Table 1. The Cyto-Genotoxicity of Vanillic Acid and Vanillic Acid+Mitomycin C on CBMN Assay in Human Blood Lymphocytes* Dose Groups MN/1000 No. of No. of No. of No. of Polynucleated No.of Binucleated Cells Mononucleate Cells Binucleate Cells Trinucleate Cells Cells (>4) NDI DMSO Control 14.2±2.75 1036.25±26.1 781.75±12.3 89.50±6.24 92.5±7.77 1.63±0.24 1µg/µl Vanillic acid 10.2±2.50 1007.50±34.4 801.00±19.0 86.50±5.07 105.0±11.1 1.64±0.31 2µg/µl Vanillic acid 20.0±2.16 1053.50±28.3 770.00±14.4 87.75±6.85 88.7±7.77 1.60±0.25 Mitomycin C 36.0±3.65 1316.75±36.1 554.25±22.9 60.50±7.05 68.5±7.23 1.44±0.29 1µg/µl Vanillic acid+Mitomycin C 16.2±2.06 1043.50±26.1 776.25±11.7 89.25±5.79 91.0±9.20 1.61±0.25 2µg/µl Vanillic acid+Mitomycin C 27.2±4.57 1194.50±33.9 648.75±17.5 74.75±8.77 82.0±8.29 1.52±0.29 *No. of Cells Scored=8000, MN, Micronuclei; NDI, Nuclear Divisio1n 0In0d.e0x; DMSO, Dimethyl sulphoxide Asian Pacific J6ou.3rnal of C1a0n.c1er Prev2e0nt.i3on, Vol 13, 2012 4995 12.8 10.3 75.0 25.0 30.0 56.3 46.8 75.051.1 51.7 50.0 54.2 31.3 30.0 25.0 38.0 31.3 31.3 30.0 33.1 23.7 25.0 27.6 0 0 Newly diagnosed without treatment Newly diagnosed with treatment Persistence or recurrence Remission None Chemotherapy Radiotherapy Concurrent chemoradiation Merve Guler Erdem et al nevertheless, the NDI value of the high dose vanillic acid when compared with DMSO treatment alone (p<0.05 and MMC combination was also higher than that of the and p<0.001, respectively). The MMC treatment alone MMC group (p<0.01). increased the percent of damaged cells over the DMSO To evaluate the comet assay, the concentration of treatment alone (p<0.001). The percent of damaged cells viable cells was assessed by a trypan blue dye exclusion was significantly higher in the combination of vanillic test (data not shown). As shown in Figure 4 and Table 2, acid (1 and 2 µg/ml) with MMC than in the DMSO a statistically significant decrease in the genetic damage control (p<0.01) and these combinations also significantly index (GDI) was observed in cells treated with vanillic attenuated the percent of damaged cells when compared acid (1 µg/ml) when compared with the DMSO control with the MMC treatment alone (p<0.001). (p<0.05). On the other hand, the GDI in the 2 µg/ml of vanillic acid treatment was significantly higher than in the Discussion DMSO control (p<0.001). The MMC treatment induced a significant increase in GDI when compared with the Initially, the genotoxic and cytotoxic effects of vanillic DMSO control (p<0.001).Vanillic acid (1 µg/ml)+MMC in acid, a dietary phenolic compound, were investigated in combination induced GDI at a level equal to the negative cultured human blood lymphocytes by both a CBMN and control (p>0.05) and this combination attenuated the an alkaline comet assay. A 1 µg/ml dose of vanillic acid GDI in comparison with MMC alone (p<0.001). The induced no significant cell damage, whereas a 2 µg/ml combination of 2 µg/ml of vanillic acid+MMC also dose of vanillic acid significantly increased the frequency showed a significantly lower GDI compared with the of micronuclei compared with the DMSO control in the MMC only treatment group (p<0.001). CBMN test. Neither dose of vanillic acid (1 or 2 µg/ As shown in Figure 5, the comet assay determined ml) displayed cytotoxic effects with regards to the NDI that while a 1 µg/ml dose of vanillic acid decreased the measurements from the CBMN test. In the comet assay, percent of damaged cells, the 2 µg/ml dose of vanillic a low dose of vanillic acid (1 µg/ml) decreased the GDI, acid significantly increased the percent of damaged cells but a 2 µg/ml dose of vanillic acid treatment elevated the GDI   GDI ratio compared with the DMSO control values. The 1.4   percentage of damaged cells, as measured by the comet 1.2   assay, decreased with the 1 µg/ml and increased with the 1   2 µg/ml dose of vanillic acid compared with the DMSO 0.8   control. Next, we examined the potential ameliorative 0.6   effects of vanillic acid on MMC-induced genotoxicity. 0.4   To determine these levels, the MMC-treated human 0.2   lymphocytes were examined. High MN frequency, low NDI, increased GDI and a higher degree of cell damage 0   DMSO   1µg  /  µl  VA   2  µg  /  µl  VA   MMC   1µg  /  µl    VA    +   2µg  /  µl    VA+   were found in the MMC-treated lymphocytes. These MMC   MMC   Figure 4. Genetic Damage Index (GDI) of Vanillic Acid results demonstrated the genotoxic effect of MMC in and Vanillic Acid+MMC in the Alkaline Comet Assay. vitro. MMC alone and in combination with vanillic acid V%A  ,D Vaamniallgiec da  cCide;l lMs  MC, Mitomycin-C treatment were used to evaluate the possible ameliorative 40   activity of vanillic acid on MMC-induced genotoxic 35   effects. As expected, both doses of vanillic acid reduced 30   the genotoxic and cytotoxic effects of MMC in human 25   lymphocytes for both cytogenetic endpoints. This effect 20   may arise from the antioxidant properties of vanillic 15   acid, which have been shown in recent studies (Tai et al., 10   2011; 2012). Possibly contributing to this activity is the 5   hydroxyl group, which places vanillic acid in the category 0   of phenolic antioxidants (Tai et al., 2012).It has been DMSO   1µg  /  µl  VA   2  µg  /  µl  VA   MMC   1µg  /  µl    VA    +   2µg  /  µl    VA+   MMC   MMC   reported that vanillic acid has both antimicrobial and anti- Figure 5. Damaged Cells (%) of Vanillic Acid and mutagenic activities and can exhibit a chemopreventive Vanillic Acid+MMC in the Alkaline Comet Assay. VA, effect in experimentally induced carcinogenesis in rats Vanillic acid; MMC, Mitomycin-C Table 2. DNA Damaging Effect of Vanillic Acid and Vanillic Acid+Mitomycin C on Alkaline Comet Assay in Human Lymphocytes* Dose Groups No. of Normal Class 1 Class 2 Class 3 Class 4 GDI % of Cells (Class 0) Damaged Cells DMSO Control 84.7±3.09 6.00±1.82 4.50±1.29 2.25±0.96 2.50±1.73 0.32±0.08 9.25±2.63 1µg/µl Vanillic acid 89.2±2.50 4.00±1.41 3.50±1.00 1.75±0.50 1.50±0.60 0.22±0.04 6.75±1.26 2 µg/µl Vanillic acid 77.5±2.38 6.50±1.73 9.00±1.63 3.00±0.82 3.25±1.26 0.49±0.08 16.0±3.37 Mitomycin C 60.5±2.64 4.25±1.50 7.75±2.22 8.25±2.36 19.3±1.89 1.21±0.05 35.3±1.71 1µg/µl Vanillic acid+Mitomycin C 81.7±1.26 4.00±2.16 7.75±1.50 3.50±1.29 3.00±0.00 0.41±0.03 14.2±1.89 2µg/µl Vanillic acid+Mitomycin C 72.5±2.08 6.00±1.41 6.50±2.64 5.75±1.26 9.25±2.50 0.73±0.09 21.5±1.91 *Number of Cells Scored=400, GDI, Genetic Damage Index; DMSO, Dimethyl sulphoxide. 4996 Asian Pacific Journal of Cancer Prevention, Vol 13, 2012 DOI:http://dx.doi.org/10.7314/APJCP.2012.13.10.4993 Effects of Vanillic Acid Against Mitomycin C-Induced Genomic Damage in Human Lymphocytes in Vitro (Tsuda et al., 1994; Rajaand, 2010). Except for these few in all parameters with a higher dose of vanillic acid, both reports, there is limited number of studies investigating doses of vanillic acid showed protective effects on the the genotoxic/anti-genotoxic properties of vanillic acid genotoxicity of the antineoplastic drug MMC. in vitro. However, the anti-mutagenicity, antioxidant We can conclude that the dietary, phenolic compound activity and anti-carcinogenicity of vanillin are well vanillic acid shows an anti-genotoxic effect on MMC- studied in the literature (Tai et al., 2011; Kumar et al., induced genetic damage in healthy human lymphocytes,as 2012). Vanillic acid is an oxidized form of vanillin and measured by the CBMN and alkaline comet assays. exhibits more free radical scavenging activity than vanillin Moreover, vanillic acid at higher doses may exert in (Sasaki et al., 1990). In our study, high doses of vanillic vitro genotoxic effects. Despite the anti-oxidant and anti- acid (2 µg/ml) combined with MMC did not result in a genotoxic capabilities of vanillic acid, care should be taken greater anti-mutagenic effect than that of the low dose with regard to its concentration in the daily diet. More vanillic acid+MMC combination. This might be due to the intensive research, both in vitro and in vivo, is required to genotoxicity of a high dose of vanillic acid. In the current clarify the genotoxic and anti-genotoxic effects of vanillic study, 1 µg/ml of vanillic acid did not show any cytotoxic acid. or genotoxic effects in cultured human blood lymphocytes. However, although a high dose of vanillic acid did not Acknowledgements develop cytotoxic effects, it did create genotoxicity in human lymphocytes. This result is compatible with other This research was funded by Uludag University, reports that show pro-oxidant and oxidative properties of Scientific Research Funds with Grant Number F (U)- some antioxidant phenolics (Childs et al., 2001; Sakihama 2009/39. et al., 2002; Kessler et al., 2003). It could be suggested that flavonoids should not be considered pure antioxidants References because under certain reaction conditions, they can also display pro-oxidant activity. This unexpected behavior Boyce MC, Haddad PR, Sostaric T (2003). Determination of of vanillic acid might be explained by its pro-oxidant flavour components in natural vanilla extracts and synthetic abilities. flavourings by mixed micellar electrokinetic capillary MMC is a chemotherapeutic agent that is still used chromatography. Analytica Chimica Acta, 485, 179-86. in the treatment of different cancers in some countries Childs A, Jacobs C, Kaminski T (2001). Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress Sontakke and Fulzele (2009). MMC is a highly in humans after an acute muscle injury induced by eccentric genotoxic agent that induces chromosomal aberrations exercise. Free Radic Biol Med, 31, 745-53. and sister chromatid exchanges in cultured human blood Civolani C, Barghini P, Roncetti AR, Ruzzi M, Schiesser, A lymphocytes (Krishnaja and Sharma, 2008), increases (2000). Bioconversion of ferulic acid into vanillic acid MN frequency (Fauth et al., 2000) and induces higher by means of a vanillate-negative mutant of Pseudomonas MN formation in human liver fibroblast cultures (Nesti fluorescens strain BF13. Applied and Environmental et al., 2000). In our study, MMC generated a high MN Microbiology, 66, 2311-7. and a low NDI in the CBMN assay and a high GDI and Collins AR (2004). The comet assay for DNA damage and repair: an increased percentage of damaged cells in the alkaline principles, applications, and limitations. Mol Biotechnol, comet assay. These findings agree with other studies 26, 249-61. that showed mutagenic and clastogenic activity for Duke JA (1992). Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton. CRC MMC (Fauth et al., 2000; Krishnaja and Sharma, 2008; Press FL USA, vol?, 654. Sontakke and Fulzele, 2009). It has been reported that in Eastmond DA, Tucker JD (1989). Identification of aneuploidy- vivo vanillin treatment decreased the MMC-induced MN inducing agents using cytokinesis-blocked human ratio in the bone marrow of mice (Inouye et al., 1988). lymphocytes and an antikinetochore antibody. Environ Additional researchdemonstrated that vanillin reduced the Mol Mutagen, 13, 34-43. DNA damage induced by H O Falconnier B, Lapierre C, Lesage-Meessen L, et al (1994). 2 2 in V79 cells in vitro (Tamai et al., 1992). In addition, the anti-mutagenic activity of Vanillin as a product of ferulic acid biotransformation vanillin was shown by an in vitro Ames test with the by the white-rot fungus Pycnoporus cinnabarinus I-937: TA104 strain of salmonella (Shaughnesy et al., 2001). Identification of metabolic pathways. J Biotechnology, 37, 123-32. Previous reports suggested that despite the reduced DNA Fauth E, Scherthan H, Zankl H (2000). Chromosome painting damage in vitro (Tamai et al., 1992), vanillin increased reveals specific patterns of chromosome occurrence in MMC the toxicity induced by N-methyl-N-nitroguanidine, MMC and diethyl stilbestrol-induced micronuclei. Mutagen, 15, and H2O2 in human-hamster hybrid A1 cells (Gustafson et 459-67. al., 2000). The results of our study revealed that the higher Fenech M (1991). Optimisation of micronucleus assays for dose of vanillic acid in combination with MMC showed a biological dosimetry. In ‘New Horizons in Biological higher MN frequency, GDI, proportion of damaged cells Dosimetry’ Eds Gledhill, BL, Mauro F. Wiley-Liss, ?, 373-86. and lower NDI than the lower dose combination treatment. Gresolia CK (2002). A comparison between mouse and fish These findings show that a higher dose of vanillic acid may micro- nucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat Res, 518, 145-50. exert DNA-damaging activity in human lymphocytes. We Gustafson DL, Franz HR, Ueno AM, et al (2000). Vanillin (3- did not observe any signs of synergistic effects between methoxy-4-hydroxybenzaldehyde) inhibits mutation induced MMC and the higher dose of vanillic acid. Given the by hydrogen peroxide, N-methyl- N-nitrosoguanidine and significant decrease in MMC-induced genotoxicity levels mitomycin C but not 137Cs γ-radiation at the CD59 locus in Asian Pacific Journal of Cancer Prevention, Vol 13, 2012 4997 Merve Guler Erdem et al human-hamster hybrid AL cells. Mutagenesis, 15, 207-13. occurring antioxidants on initiation of hepatocarcinogenesis Inouye T, Sasaki YF, Imanishi H, et al (1988). Suppression of by 2-amino-3-methylimidazo (4, 5) quinoline in the rat. Jpn mitomycin C-induced micronuclei in mouse bone marrow J Cancer Res, 85, 1214-9. cells by post-treatment with vanillin. Mutation Res/ Yemis GP, Pagotto F, Bach S, Delaquis P (2011). Effect of Fundamental and Molecular Mechanisms of Mutagenesis, vanillin, ethyl vanillin, and vanillic acid on the growth and 202, 93-5. heat resistance of Cronobacter species. J Food Prot, 74, Kessler M, Ubeaud G, Jung L (2003). Anti- and pro-oxidant 2062-9. activity of rutin and quercetin derivatives. J Pharmacy and Pharmacology, 55, 131-42. Krishnaja AP, Sharma NK (2008). Variability in cytogenetic adaptive response of cultured human lymphocytes to mitomycin C, bleomycin, quinacrinedihydrochloride, Co60 γ-rays and hyperthermia. Mutagenesis, 23, 77-86. Kumar R, Sharma PK, Mishra PS (2012). A review on the Vanillin derivatives showing various biological activities. Int J PharmTech Res, 4, 266-79. Lesage-Meessen L, Delattre M, Haon M, et al (1996). A two- step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. J Biotechnology, 50, 107-13. Nesti C, Trippi F, Scarpato R, Migliore L, Turchi G (2000). Cytokinesis block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and Mitomycin C. Mutagenesis, 15, 143-7. Palus J, Rydzynski Kl, Dziubaltowska E, et al (2003). Genotoxic effects of occupational exposure to lead and cadmium. Mutat Res, 540, 19-28. Pawar AA, Vikram A, Tripathi DN, et al (2009). Modulation of mitomycin C-induced genotoxicity by acetyl- and thio- analogues of salicylic acid. In vivo, 23, 303-7. Raja B, Mol SD (2010).The protective role of vanillic acid against acetaminophen induced hepatotoxicity in rats. J Pharmacy Res, 3, 1480-4. Rechner AR, Jeremy P, Spencer E, et al (2001). Novel biomarkers of the metabolism of caffeic acid derivatives in vivo. Free Radical Biol & Med, 30, 1213-22. Sakihama Y, Cohen MF, Grace SC, Yamasaki H (2002). Plant phenolic antioxidant and prooxidant activities: phenolics- induced oxidative damage mediated by metals in plants. Toxicology, 177, 67-80. Sasaki YF, Ohta T, Imanishi H, et al (1990). Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by x-rats in mice. Mutat Res, 243, 299-302. Shaughnessy DT, Setzer RW, DeMarini DM (2001). The antimutagenic effect of vanillin and cinnamaldehyde on spontaneous mutation in Salmonella TA104 is due to a reduction in mutations at GC but not AT sites. Mutat Res, 480-481, 55-69. Singh NP, McCoy MT, Tice RR, Schneider EL (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res, 175, 184-91. Sontakke YA, Fulzele RR (2009). Cytogenetic study on genotoxicity of antitumor-antibiotic Mitomycin C. Biomedical Res, 20, 40-4. Tai A, Sawano T, Yazama F, Ito H (2011). Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochim Biophys Acta, 1810, 170-7. Tai A, Sawano T, Ito H (2012). Antioxidative properties of vanillic acid esters in multiple antioxidant assays. Biosci Biotechnol Biochem, 76, 314-8. Tamai K, Tezuka H, Kuroda Y (1992). Direct modifications by vanillin in cytotoxicity and genetic changes induced by EMS and H2O2 in cultured Chinese hamster cells. Mutat Res, 268, 231-7. Tsuda H, Uehara N, Iwahori Y, et al (1994). Chemopreventive effects of beta-carotene, alpha-tocopherol and five naturally 4998 Asian Pacific Journal of Cancer Prevention, Vol 13, 2012