2001; Ferguson and Philpot, 2008; Ferguson et al., 2004) Thisis feasible owing to the fact that majority of the knownchemopreventive agents are phytochemicals present in our
838 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
Table 1 – DPPH free radical scavenging by PEL and CYN.
Anthocyanidin Concentration(mg/10 ml)
% Quenchingof free radicals
PEL 0.125 27.71 ± 1.25*
0.250 35.36 ± 1.49*
0.500 43.00 ± 1.54*
1.000 62.73 ± 2.85*
CYN 0.125 30.95 ± 1.47*
0.250 44.80 ± 1.59*
0.500 58.04 ± 2.23*
1.000 79.37 ± 1.30*
Values are mean ± SD.
2.3. Pre-treatment
∗ Significant against other three concentrations of the same antho-cyanidin (P < 0.05).
commonly consumed vegetables, fruits, cereals, spices, cocoa,tea and coffee (Surh, 2003; Wattenberg, 1985, 1992). World-wide research in the area of cancer chemoprevention for over35 years resulted in the identification of several groups ofphytochemicals which reduce the harmful effects of environ-mental toxicants by modulating important pathways leadingto mutagenesis and carcinogenesis (Kaefer and Milner, 2008;Mehta et al., 2010; Surh, 2003). The increasing importanceof anthocyanins as a group of phytochemicals with chemo-preventive effects has become evident from a series ofpublications which appeared during the preceeding decade(Tsuda, 2012; Wang and Stoner, 2008). Recent epidemiologi-cal surveys and human intervention studies have highlightedthe beneficial health effects associated with anthocyanin richfruits (Duthie, 2007).
Anthocyanins are one of the largest and probably the mostimportant group of water-soluble natural pigments (Cookeet al., 2005; Wang and Stoner, 2008). Till date, more than 635anthocyanins have been identified in nature (He and Guisti,2010). There are six anthocyanidins (aglycone form of antho-cyanins) commonly found in higher plants: cyanidin (50%),pelargonidin (12%), peonidin (12%), delphinidin (12%), petu-nidin (7%) and malvidin (7%) (Cooke et al., 2005; He andGuisti, 2010). These polyphenols are responsible for the blue,purple and red color of many fruits and vegetables (He andGuisti, 2010). Findings from several studies suggest that antho-cyanins possess anti-inflammatory, anticarcinogenic, as wellas preventive effects on cardiovascular diseases, obesity, anddiabetes (Cooke et al., 2005; He and Guisti, 2010; Tsuda, 2012;Wang and Stoner, 2008). Health benefits of anthocyanins havebeen attributed to their antioxidant activity/free radical scav-enging property (He and Guisti, 2010).
We initiated the present work to answer the questionwhether or not anthocyanidins can play a role in inhibiting thein vivo genotoxic stress induced by environmental toxicants.Information from in vitro studies suggests that anthocyani-dins can inhibit the genotoxic effects induced by diverse DNAdamaging agents (Abraham et al., 2007, 2012). These findingsprompted us to assess the possible protective effects of PELand CYN against in vivo genotoxicity of the environmentalcarcinogens diepoxybutane (DEB) and urethane (URE). In addi-tion we included experiments to evaluate the antinitrosating
effects of these phytochemicals.
DEB is the most toxic metabolite of 1,3-butadiene (BD)which is widely used in the manufacture of rubber, plastics
a r m a c o l o g y 3 7 ( 2 0 1 4 ) 837–843
and polymers. It has been classified by National ToxicologyProgram as a known human carcinogen. It is largely pro-duced during the processing of petroleum. BD is also foundin automobile exhaust and cigarette smoke. DEB is a bifunc-tional alkylating agent, which exhibits both inter-strand aswell as intra-strand DNA cross-linking ability (Goggin et al.,2009, 2011).
URE is a multisite carcinogen and it is capable of induc-ing tumors in various organs (Hernandez and Forkert, 2007;Kemper et al., 1995). It is a naturally occurring substance,which is formed during many fermentation processes. Humanexposure to URE was more during the early part of the lastcentury, when it was used for treatment of chronic leukemia,multiple myeloma and varicose veins (Hoffler et al., 2005). Atpresent exposure to URE can occur primarily through inges-tion of yeast breads, alcoholic beverages and use of fumigants,pesticides, cosmetics and textiles (Hoffler et al., 2005). URE hasbeen classified by International Agency for Research on Cancer(IARC) as a group 2A carcinogen that is “probably carcinogenicto humans.” (Hernandez and Forkert, 2007)
Cytotoxic, genotoxic and carcinogenic N-nitroso com-pounds can be formed endogenously when nitrite reacts withsecondary amines or N-substituted amides under acidic con-ditions in the stomach (Mirvish, 1975, 1994). This endogenousformation of harmful N-nitrosamines/N-nitrosamides can beinhibited by phytochemicals (d’Ischia et al., 2011; Mirvish,1994). Our recent work demonstrated that ascorbic acid (AA) incombination with polyphenols completely inhibited the geno-toxic damage in mice, induced by a model nitrosation reactionmixture (Abraham and Khandelwal, 2013). Hence we includedexperiments to investigate the antinitrosating effects of PEL,CYN and the combination of PEL and AA.
2. Material and methods
2.1. Chemicals
Cyanidin (CAS no. 528-58-5), pelargonidin (CAS no. 134-04-3),diepoxybutane (CAS no. 1464-53-5), urethane (CAS no. 51-79-6), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid andnew born calf serum were purchased from Sigma-Aldrich Pvt.Ltd., (India). Methyl urea (MU) and sodium nitrite (SN) wereobtained from Merck (India). All the other reagents and sol-vents used for the experiments were of analytic grade fromQualigens Chemicals Pvt. Ltd., India.
2.2. Animals
The experiments were carried out with 12–14-week-old maleSwiss albino mice weighing 30–34 g, in accordance with theguidelines of CPCSEA, India. These animals were bred andmaintained at 25 ± 2 ◦C on the standard mouse diet and waterad libitum. Approval for the work was obtained from the Insti-tutional Animal Ethics Committee (IAEC-JNU).
PEL and CYN were diluted in double distilled water and admin-istered to the experimental animals by gavage (10 ml/kg b.w.)
p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 837–843 839
3atd
2
Tbc(
2m
T(ewiar
2
MtTaacsiAs2fattwmo
2
Tsaa
2
Dtpg(rc
Fig. 1 – Dose response with three doses of DEB (10, 20 and40 mg/kg b.w.). Shown are means ± SD from four mice.These values are significant when compared to the negativecontrol (* P < 0.05). The PCEs/NCEs ratio is as follows:
test dose (2.5 mg/kg) was not significantly different from thatof the highest dose (20 mg/kg b.w.). Pretreatment with threedoses of CYN (1, 2 and 4 mg/kg b.w.) resulted in significant
Fig. 2 – DEB (40 mg/kg b.w.) induced micronucleusformation and its reduction by co-treatment with fourdoses of PEL. Shown are means ± SD from four mice. Thereduction in DEB genotoxicity by PEL pre-treatment (2.5, 5,
*
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
0 min before injecting the genotoxic carcinogens. Controlnimals received the same volume of distilled water. Eachreatment group consisted of four mice. The test doses wereecided based on preliminary experiments.
.4. Genotoxin treatment
he animals received DEB (40 mg/kg b.w.) or URE (800 mg/kg.w.) intraperitoneally, 30 min. after pretreatment with antho-yanidins. The control animals received the same volume10 ml/kg b.w.) of distilled water.
.5. Administration of the nitrosation reactionixture, PEL, CYN and combination of PEL with AA
he nitrosation reaction mixture consisting of methyl ureaMU) 300 mg/kg b.w. + sodium nitrite (SN) 15 mg/kg b.w. andach test anthocyanidin or the combination of anthocyanidinith AA was dissolved in distilled water just before the admin-
stration by gavage (10 ml/kg b.w.) as described by Abrahamnd Khandelwal (2013). The experimental animals were sac-ificed after 24 h for sampling the bone marrow cells.
.6. Micronucleus assay for evaluating genotoxicity
icronucleus test was carried out according to Schmid (1975)o evaluate the genotoxic effects in the mouse bone marrow.he experimental animals were sacrificed after 27 h treatmentnd bone marrow cells from both the femurs were flushed into
centrifuge tube containing 1 ml new born calf serum. Thisell suspension was centrifuged at 2000 rpm for 5 min. Theupernatant was discarded and the pellet was resuspendedn a drop of serum before being used for preparing slides.ir-dried slides were stained with May-Grünwald and Giemsatain. Four mice were used for each experimental point and000 polychromatic erythrocytes (PCEs)/animal were scoredrom a single slide to determine the frequency of micronucle-ted polychromatic erythrocytes (Mn PCEs). Special care wasaken to ensure that the doses selected for the combinationreatment did not lead to suppression of cell proliferation. Thisas done by monitoring the ratio of PCEs to NCEs (normochro-atic erythrocytes). All the slides were scored by the same
bserver.
.7. DPPH assay for radical scavenging activity
he DPPH assay was performed for assessing the free radicalcavenging activity of PEL and CYN as described by Abrahamnd Khandelwal (2013). The IC50 value was obtained for eachnthocyanidin.
.8. Statistical analysis
ata are expressed as mean ± standard deviation. Student’s-test was performed using Graph pad software for com-aring the incidence of bone marrow micronuclei of two
roups. The comparison was between the positive controlDEB/URE/nitrosation reaction mixture) and the group whicheceived the anthocyanidin. Results were considered statisti-ally significant at P < 0.05.
control, 1.26; DEB (10), 1.10; DEB (20), 1.07; DEB (40), 1.08.
3. Results
Data presented in Fig. 1 show results obtained from the mousebone marrow micronucleus test to evaluate genotoxicity ofDEB. Three doses of DEB were selected to evaluate the geno-toxic effect. DEB induced a dose-related genotoxic effect. Allthe three test doses show a significant increase in the inci-dence of MnPCEs. On the basis of this finding we selected thedose of 40 mg/kg b.w. for further investigations.
Fig. 2 shows the antigenotoxic effect of four doses of PEL(2.5–20 mg/kg b.w.) against DEB. Significant reductions wereobserved in the genotoxicity of DEB, irrespective of the doseadministered. However the antigenotoxic effect of the lowest
10 and 20 mg/kg b.w.) is significant ( P < 0.05). ThePCEs/NCEs ratio is as follows: control, 1.25; PEL (2.5), 1.23;DEB, 1.08; DEB + PEL (2.5), 1.21; DEB + PEL (5), 1.23; DEB + PEL(10), 1.21; DEB + PEL (20), 1.20.
840 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 7 ( 2 0 1 4 ) 837–843
Fig. 3 – Inhibitory effect of 3 doses of CYN (1, 2 and 4 mg/kgb.w.) on micronuclei induced by DEB (40 mg/kg b.w.) Thesevalues are means ± SD from four mice. * Significantlydifferent from DEB treatment (* P < 0.05). The PCEs/NCEsratio is as follows: control, 1.27; CYN (1), 1.24; DEB, 1.10;
Fig. 5 – URE (800 mg/kg b.w.) induced micronucleusformation and its reduction by co-treatment with 3 doses ofCYN. These values are means ± SD from four mice. *
Significantly different from URE treatment (* P < 0.05). ThePCEs/NCEs ratio is as follows:control, 1.25; CYN (1), 1.22; URE, 1.11; URE + CYN (1), 1.23;
DEB + CYN (1), 1.25; DEB + CYN (2), 1.23; DEB + CYN (4), 1.26.
reductions in genotoxicity of DEB (Fig. 3). From these resultsthere is no indication of significant dose-dependent reductionin genotoxicity. When compared to the negative control, therewas no significant increase in genotoxicity with the highesttest dose of either PEL (20 mg/kg b.w.) or CYN alone (4 mg/kgb.w.) (Figs. 2 and 3).
Results of the experiment to assess antigenotoxic effectsof three doses of PEL against URE have been shown in Fig. 4.
Significant dose dependent reductions in the frequencies ofMnPCEs have been observed. Fig. 5 illustrates the effect ofthree doses of CYN on the genotoxicity of URE. All the three
Fig. 4 – Protective effects of three doses of PEL (5, 10 and20 mg/kg b.w.) against the genotoxicity induced by URE(800 mg/kg b.w.). These values are means ± SD from fourmice. * Significantly different from URE treatment (* P < 0.05).The PCEs/NCEs ratio is as follows: control, 1.27; PEL (5),1.25; URE, 1.12; URE + PEL (5), 1.24; URE + PEL (10), 1.24;URE + PEL (20), 1.25.
URE + CYN(2), 1.21; URE + CYN (4), 1.24.
doses (1, 2 and 4 mg/kg b.w) have significantly reduced thegenotoxicity. These results indicate that the lowest test dose(1 mg/kg) has the maximum antigenotoxic effect.
Different doses of PEL were tested for inhibitory effects onendogenous nitrosation. Fig. 6 shows that two doses of PEL(5 mg/kg and 10 mg/kg) significantly reduced the MnPCE fre-quency. Fig. 7 shows the inhibitory effect of CYN (1, 2 and4 mg/kg) on induction of micronuclei by endogenous nitrosa-tion. Significant reductions in the genotoxicity were observedfollowing pre-treatment with all the three doses of CYN. Thereis no significant dose-related difference in the antigenotoxiceffect. Fig. 8 illustrates the result of an experiment to assesswhether the combination of AA and PEL is effective in inhibit-ing the genotoxic effects of endogenous nitrosation. Thecombination significantly reduced the incidence of Mn PCEs.Furthermore, the combination of PEL with AA is more effectivein reducing the genotoxic damage when compared with eitherPEL or AA alone. When the DPPH assay for free radical scaveng-ing was performed (Table.1), the IC50 values obtained for PELand CYN were 0.66 mg/10 ml and 0.405 mg/10 ml, respectively.
4. Discussion
The main objective of our present study was to focus onthe possible role of PEL and CYN as dietary agents forreducing genotoxic stress induced by carcinogenic environ-mental toxicants. Our results suggest that PEL and CYN, two
anthocyanidins found in many commonly consumed fruitsand vegetables have a chemoprotective effect against in vivogenotoxic stress induced by the environmental carcinogensDEB and URE. DEB is the ultimate carcinogenic form of the
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 7 ( 2 0 1 4 ) 837–843 841
Fig. 6 – Antigenotoxic effect of three doses of PEL (2.5, 5 and10 mg/kg b.w.) against the nitrosation inducedmicronucleus formation. These values are means ± SD fromfour mice. * Significantly different from MU + SN treatment (*
P < 0.05). The PCEs/NCEs ratio is as follows: control, 1.24;PEL (2.5), 1.22; MU + SN, 1.12; MU + SN + PEL (2.5), 1.23;M
eitOtef
FanST1(
Fig. 8 – Inhibitory effect of PEL (2.5 mg/kg), ascorbic acid(AA, 25 mg/kg) and their combination on micronucleiinduced by endogenous nitrosation. These values aremeans ± SD from four mice. Significantly different fromMU + SN treatment (* P < 0.05). The PCEs/NCEs ratio is asfollows: control, 1.27; PEL, 1.24; AA, 1.25; MU + SN, 1.08;MU + SN + PEL, 1.23; MU + SN + AA, 1.24; MU + SN + AA + PEL,
U + SN + PEL (5), 1.21; MU + SN + PEL (10), 1.23.
nvironmental pollutant 1,3-butadiene and hence our resultsndicate the protective effects of PEL and CYN againsthis directly acting genotoxin (Goggin et al., 2009, 2011).n the other hand URE (ethyl carbamate) is not geno-
oxic/carcinogenic per se. It is metabolized to vinyl carbamatepoxide, which is considered as the ultimate carcinogenicorm (Kemper et al., 1995). Apart from this, the present
ig. 7 – Protective effects of CYN (1, 2 and 4 mg/kg b.w.)gainst the genotoxicity induced by endogenousitrosation. These values are means ± SD from four mice. *
ignificantly different from MU + SN treatment (* P < 0.05).he PCEs/NCEs ratio is as follows: control, 1.26; CYN (1),.24; MU + SN, 1.15; MU + SN + CYN (1), 1.23; MU + SN + CYN
2), 1.21; MU + SN + CYN (4), 1.25.
1.24.
investigation has demonstrated the ‘antinitrosating’ effects ofthese two anthocyanidins against a model nitrosation reactionbetween methyl urea and sodium nitrite leading to formationof the genotoxic carcinogen methyl nitrosourea. Furthermore,from this work there is no indication of a genotoxic effectwhen the test doses of PEL and CYN are administered alone.
An interesting observation that has emerged from ourstudy is that a wide range of doses have shown this inhibitoryeffect on genotoxicity induced by DEB and URE. Low doses ofCYN (1 mg/kg) and PEL (2.5 mg/kg) had a significant inhibitoryeffect on DEB and URE. However CYN was more effective inreducing the genotoxic damage caused by DEB and URE whencompared to PEL. This could be possibly due to their structure-function properties. The six commonly found anthocyandinsvary in their free radical scavenging activity based on theirstructure. The antioxidant activity of anthocyanins are mainlyattributed to the presence of hydroxyl groups in position 3 ofring C and also in the 3′, 4′ and 5′ positions in ring B of themolecules (Wang and Stoner, 2008).
In this study, we observed that oral co-administration ofPEL/CYN with the model nitrosation reaction mixture leadsto a significant reduction in genotoxicity when comparedto animals which received N-nitroso compound precursorswithout anthocyanidins. Formation of genotoxic/carcinogenicN-nitroso compound from precursors under acidic con-ditions in the stomach is an extracellular process. Theresults we obtained suggest that PEL and CYN are inhibit-ing this process. Our recent work using ascorbic acid andpolyphenols demonstrated an antigenotoxic effect only whenthe antinitrosating agents are co-administered with the
precursors MU and SN (Abraham and Khandelwal, 2013).An inhibitory effect was not observed against the pre-formed N-nitroso compound N-nitroso–N-methylurea, which
842 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
indicates that formation of the harmful compound is inhib-ited (Abraham and Khandelwal, 2013). Phytochemicals areknown to scavenge nitrite and reduce its availability to reactwith methylurea (d’Ischia et al., 2011; Kono et al., 1995;Panzella et al., 2005). The results of our DPPH assay havedemonstrated the radical scavenging efficacy of PEL andCYN.
Many in vitro and in vivo investigations have been carriedout to elucidate the mechanisms associated with the chemo-preventive effects of anthocyanins (Duthie, 2007; Tsuda, 2012;Wang and Stoner, 2008). The findings from these studieshave clearly demonstrated the antioxidant/free radical scav-enging activity of anthocyanins (Rahman et al., 2006; Wangand Stoner, 2008). The antioxidant capacity of anthocyanin-treated cells are enhanced by activation of glutathione-relatedenzymes like glutathione reductase, glutathione peroxidaseand glutathione S-transferase (Wang and Stoner, 2008). Vinylcarbamate epoxide has been identified as the metaboloiteof URE ultimately responsible for its genotoxic/carcinogeniceffects (Kemper et al., 1995). The harmful effect of thismetabolite is reduced by glutathione S-transferase catalyzedconjugation with glutathione (Kemper et al., 1995). SimilarlyDEB is known to form glutathione conjugates mediated byglutathione S-transferase (Spanò et al., 1998). These are someof the likely mechanisms which can lead to detoxification ofDEB and URE. Furthermore, anthocyanins are known to exertpro-apoptotic effects and anti-inflammatory effects which areassociated with cancer chemoprevention.
There are many reports from in vitro studies which showthat CYN can inhibit DNA damage induced by ethyl methanesulfonate, hydrogen peroxide, mitomycin C, doxorubicin andcamptothecin in certain malignant cells (Esselen et al., 2011;Fimognari et al., 2004). The antimutagenic effect of CYN hasbeen observed in the Ames test (Gasiorowski et al., 1997). CYNwas more effective than antioxidants like kaempferol, sily-marin, vitamin C, vitamin E and its water soluble analoguetrolox when the same concentration (50 �M) was tested forprotective effects against H2O2 induced DNA strand breaks inhuman lymphocytes ex vivo (Duthie, 2007). Similarly, in vitroantigenotoxic effects of PEL have been observed against 4-nitroquinoline 1-oxide, mitomycin C, patulin and DEB in HL-60cells (Abraham et al., 2007, 2012). In addition to this, in vivostudies with CYN have shown protective effects against ochra-toxin induced DNA damage in rat kidney, liver and brain (DiGiacomo et al., 2007). Recently, Esselen et al. (2013) demon-strated the protective effect of CYN against irinotecan inducedDNA strand breaks in the colon of Wistar rats. An in vivostudy conducted by Roy et al. (2008) demonstrated that PELreduces oxidative stress in diabetic rats. In the rat model ofhemi-parkinsonism, orally administered PEL has exerted dosedependent neuroprotection (Roghani et al., 2010). Our presentfindings on the inhibitory effects of PEL and CYN against DEB,URE and endogenous nitrosation have highlighted the rolethese anthocyanidins can play as in vivo protective agentsagainst genotoxic stress.
Human intervention studies by Chung et al. (2002)
demonstrated the role of strawberry consumption inreducing endogenous formation of the carcinogen N-nitrosodimethylamine in healthy volunteers who receivednitrate with an amine-rich diet. Apart from PEL, strawberry
a r m a c o l o g y 3 7 ( 2 0 1 4 ) 837–843
is having a relatively high content of AA which is a wellknown inhibitor of nitrosation (Chung et al., 2002). Further-more, our recent findings show that combinations of AAand polyphenols are more effective inhibitors of endogenousnitrosation (Abraham and Khandelwal, 2013). These obser-vations prompted us to assess the antinitrosating effects ofthe naturally occurring combination of PEL and AA. From thepresent results, there is evidence for an enhanced inhibitionof endogenous nitrosation following administration of PEL incombination with AA.
Ideal chemopreventive agents are those with no toxiceffects, high efficacy, capability of oral administration, lowcost and known mechanisms of action (Morse and Stoner,1993). The findings from our present work and the publishedreports on the antigenotoxic/anticarcinogenic effects of PELand CYN suggest that these anthocyanins can be consideredas ideal chemopreventive agents in view of the absence oftoxic effects, efficacy to inhibit genotoxic/carcinogenic effectsof structurally diverse groups of environmental toxicants,easy availability through fruits and vegetables and the knownmechanisms of action.
5. Conclusion
In conclusion, the findings from our study have highlightedthe important role anthocyanidins PEL and CYN can play inreducing genotoxicity induced by environmental toxicants.Thus intake of a diet rich in anthocyanin containing fruits andvegetables can be an effective strategy for chemoprevention ofgenotoxic stress.
Conflict of interest
No conflict of interest.
Acknowledgement
This work was supported by the University Grants Com-mission (NETWORKING) and Department of Science andTechnology (PURSE). N.K. thanks the University Grants com-mission for the Meritorious Research Fellowship.
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