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Effect of natural phenolic acids on DNA oxidation in vitro
Food and Chemical Toxicology 39 (2001) 1205–1210 www.elsevier.com/locate/foodchemtox
Research Section
Effect of natural phenolic acids on DNA oxidation in vitro M. Lodovici*, F. Guglielmi, M. Meoni, P. Dolara Department of Pharmacology, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy Accepted 1 June 2001
Abstract We examined the antioxidant activity of the following natural phenolic compounds present in food: 3-OH-benzoic acid (3-OHBA); 4-OH-benzoic acid (4-OH-BA); 2,3-dihydroxybenzoic acid (2,3-diOH-BA); 3,4-dihydroxybenzoic acid (3,4-diOH-BA or protocatechuic acid); ferulic acid; caffeic acid; and 2-coumaric, 3-coumaric and 4-coumaric acids. We measured the inhibitory effect of these compounds on iron-dependent oxidative DNA damage in vitro [incubating herring sperm DNA with Fe(III)/GSH] or using cumene hydroperoxide (CumOOH) as a free-radical generating system; we also studied the interaction of these phenols with Fe(II) or Fe(III) spectrophotometrically. Among the tested compounds, 2,3-diOH-BA, 3,4-diOH-BA and caffeic acid interacted with Fe(II) and showed a potent inhibitory effect on iron-induced oxidative DNA damage. CumOOH-induced DNA oxidation was not modified by these compounds. On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids did not interact with iron but protected against oxidative DNA damage induced by Fe(III)/GSH and by CumOOH, indicating a direct free-radical scavenging activity of these compounds in both systems. The IC50 S.E.M. of the three coumaric acids against CumOOH-induced DNA oxidation was 44.2 2.0, 54.7 2.0 and 33.1 1.0 mm, respectively. On the contrary, 3-OH-BA and 4-OH-BA did not have scavenging activity and 3-OH-BA actually enhanced oxidative DNA damage. In conclusion, some natural phenolic acids, commonly present in food, have interesting protective activity against DNA oxidation in vitro and deserve further consideration as effective antioxidants in vivo. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Oxidative DNA damage; Reactive oxygen species; Iron; Cumene hydroperoxide; Phenolic acids
1. Introduction Numerous epidemiological studies have provided evidence that individuals consuming diets rich in fruit and vegetables have a lower risk of cardiovascular disease (Hertog et al., 1993, 1995), stroke (Keli et al., 1996) and cancer (Block, 1992). These protective effects have been attributed to various components, such as carotenoids, vitamin C and phenolic acids. Many studies have also focused on the biological activities of flavonoids, a group of plant polyphenolic compounds, which are potent antioxidants and free-radical scavengers (RiceEvans et al., 1995, 1996; Plumb et al., 1997; Ng et al., 2000). However, some of the polyphenolic compounds
Abbreviations: CumOOH, cumene hydroperoxide; 3-OH-BA, 3hydroxybenzoic acid; 4-OH-BA, 4-hydroxybenzoic acid; 2,3-diOHBA, 2,3-dihydroxybenzoic acid; 3,4-diOH-BA, 3,4-dihydroxybenzoic acid; 8-OhdG, 8-OH-20 -deoxyguanosine. * Corresponding author. Tel.: +39-055-427-1321; fax+39-055427-1280. E-mail address: [email protected]fi.it (M. Lodovici).
can act as auto-oxidants at physiological pH (Hodnick et al., 1986) or as pro-oxidants, producing toxic oxygen radicals. Sugihara et al. (1999), using a lipid peroxidation assay, reported that some flavonoids have a prooxidant activity in the presence of high concentrations of iron ions, although they act as antioxidants at lower iron concentration. Moreover, Smith et al. (1992), demonstrated that some synthetic and natural phenolic compounds utilised in food preservation, protect lipid against oxidation but can accelerate oxidative damage to carbohydrate or DNA. In recent studies we demonstrated that polyphenolic compounds extracted from red wine and from black tea have a protective effect on oxidative induced DNA damage in rat liver and intestine (Casalini et al., 1999; Giovannelli et al., 2000; Lodovici et al., 2000) and can protect against azoxymethane-induced colon carcinogenesis in the rat (Caderni et al., 2000). Phenolic acids (essentially hydroxybenzoic and hydroxycinnamic acid) are widely distributed in plants and can be present in considerable amounts in the human diet. The intake of hydroxybenzoic and hydroxycinnamic
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acid has been estimated to be around 11 mg/day and 211 mg/day, respectively, and the intake of caffeic acid, a hydroxycinnamic acid derivative, has been estimated in the order of 206 mg/day in subjects drinking coffee (Radtke et al., 1998). A protective effect of caffeic acid and its related catechols against hydroxyl radical formation in vitro has been reported (Iwahashi et al., 1990; Scott et al., 1993; Nardini et al., 1995; Abu-Amsha et al., 1996; Masella et al., 1999; Silva et al., 2000) and also protocatechuic acid has been well studied in this respect (Laranjinha et al., 1994; Ueda et al., 1996; Masella et al., 1999). On the contrary, data regarding the activity of hydroxybenzoic and coumaric acids are limited in the literature. On this basis, since DNA is one of the main molecular targets of the toxic effect of free radicals formed in cells, we investigated the effect of some phenolic acids on oxidative DNA damage and their interaction with iron in vitro.
2.5. Spectrophotometric measurements
2. Materials and methods
3. Results
2.1. Chemicals
3.1. Oxidative DNA damage
Herring sperm DNA, GSH, ferric chloride, ferrous sulphate, cumene hydroperoxide (CumOOH), 3-OHBA, 4-OH-BA, 3,4-diOH-BA, 2,3-diOH-BA, caffeic acid, ferulic acid, 2-coumaric acid, 3-coumaric acid and 4-coumaric acid were obtained from Sigma-Aldrich (Milan, Italy); bovine liver catalase was obtained by Roche Molecular Biochemicals (Milan, Italy).
To study the antioxidant effects in vitro of phenolic acids, we induced oxidative stress with Fe(III)/GSH, which produces H2O2 forming hydroxyl radical through the Fenton reaction; moreover, ferrous iron can also react with oxygen, forming perferryl ion species (FeO+ 2 and FeO3+) more reactive than the hydroxyl radical itself (Guengerich et al., 1997). In Fig. 1 we report the effect of hydroxybenzoic acid derivatives and the effect of hydroxycinnamic acid derivatives on iron-dependent oxidative DNA damage. In our experimental conditions oxidative DNA damage in the presence of Fe(III)/GSH, measured as 8-OHdG/dG, was increased about 30-fold when compared to control levels. We previously demonstrated that the addition of desferoxamine, an iron chelator, blocked the oxidation damage induced by Fe(III)/GSH (Lodovici et al., 2001). Similarly, when iron-induced oxidation damage was assayed in the presence of 2 mm EDTA, the chelation of iron prevented the production of H2O2 and oxidation damage (Table 1). Moreover, the addition of catalase (40 and 80 mg/ml), an enzyme capable of removing H2O2 derived from Fe(III)/GSH in the presence of O2 (Park and Floyd, 1994) reduced 8-OHdG levels dosedependently (Table 1). On the contrary, no inhibitory effect on oxidative damage induced by CumOOH was seen adding catalase at the same concentrations (data not shown), probably for the lack of involvement of H2O2 in CumOOH-induced DNA oxidation (Hix et al., 2000). In the presence of 3-OH-BA, oxidative DNA damage was increased. On the contrary, DNA oxidation was
2.2. Oxidative DNA damage induced by GSH/Fe(III) Herring sperm DNA (0.7 mg/ml, final concentration) was incubated in air for 2 h at 37 C in the dark in the presence of 3 mm FeCl3, 15 mm GSH and 4 mm HEPES, pH 7, according to Park and Floyd (1994). 2.3. Oxidative DNA damage induced by CumOOH Herring sperm DNA (0.7 mg/ml) was incubated in air for 2 h at 37 C in the dark with the test substances in the presence of 20 mm CumOOH in 4 mm HEPES, pH 7. 2.4. Measurement of 8-OHdG levels in DNA After incubation with Fe(III)/GSH or CumOOH, DNA was precipitated by ethanol in the presence of 20% 10 m ammonium acetate, washed with 70% ethanol, dried, dissolved in 20 mm acetate buffer pH 5.2, denatured at 90 C for 3 min and digested to a nucleoside pool; 8-OH20 -deoxyguanosine (8-OHdG) was measured with HPLC coupled with electrochemical detection, following established methods (Lodovici et al., 2000).
The absorption spectra of phenolic acids alone or in the presence of Fe(II) or Fe(III) were recorded from 190 to 700 nm on a Perkin-Elmer scanning spectrophotometer at 25 C. The cuvettes were closed with a cap during measurements. The final concentration of individual phenolic acids in 40 mm HEPES pH 7.1 were 15 mm; in the appropriate samples 3 mm FeSO4 or 3 mm FeCl3 were present; incubation buffer was used as reference. 2.6. Statistics Data were analysed with the Statgraphic Statistical Package, Statistical Graphic Corporation (Rockville, MD, USA) using one-way ANOVA analysis. IC50 values were obtained interpolating experimental data with a linear regression model.
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significantly reduced by 2,3-diOH-BA and 3,4-diOHBA (Fig. 1, panel A). Also hydroxycinnamic acid derivatives, except ferulic acid, significantly reduced irondependent oxidative DNA damage (Fig. 1, panel B). The highest inhibition of oxidative DNA damage was obtained in the presence of 3-coumaric and 4-coumaric acids. Fig. 2 shows the effect of the same compounds on oxidative DNA damage induced by CumOOH, which generates alkyl radicals directly (Hix et al., 2000). The ratio 8-OHdG/dG in DNA incubated with 20 mm CumOOH increased about 2.5-fold compared to control
levels (Fig. 2, panel A). The incubation with hydroxybenzoic acid derivatives did not modify 8-OHdG levels, but 2,3-diOH-BA and 3,4-diOH-BA induced a weak, non-statistically significant, increase in oxidative damage. As shown in panel B of Fig. 2, all analysed coumaric acids had a protective effect against oxidative DNA damage induced by CumOOH, with about a 40% reduction of DNA oxidation. Ferulic acid and caffeic acid, on the contrary, did not modify 8-OHdG levels.
Table 1 Effect of EDTA and catalase on DNA oxidation in vitro induced by Fe(III)/GSH
We determined the inhibition induced by 2-coumaric, 3-coumaric and 4-coumaric acids in the CumOOHinduced oxidative DNA damage assay, by estimating
3.2. IC50 values
Levels of 8-OHdG/dG105
Control EDTA (2 mm) Catalase (40 mg/ml) Catalase (80 mg/ml)
DNA
DNA +3 mm Fe(III)+GSH
5.00.3 – – –
121.811.7 7.00.4** 85.3 7.3* 60.5 7.2**
The values are means of five determinations S.E.M. *P< 0.05 and **P< 0.01 relative to control.
Table 2 IC50 values for hydroxycinnamic acids on CumOOH-induced oxidative DNA damage Phenolic compound
IC50 (mm)
2-Coumaric acid 3-Coumaric acid 4-Coumaric acid
44.2 2.0 54.7 2.0 33.1 1.0
The values are meansS.E.M. calculated with regression analysis using three different concentrations of the phenolic acids.
Fig. 1. Effect of hydroxybenzoic acid derivatives (a) and hydroxycinnamic acid derivatives (b) on 8-OHdG formation induced by Fe(III)/GSH in herring sperm DNA. Control values were obtained in the absence of Fe(III)/GSH. Each value is the mean of 10 determinationsS.E.M. *, ** P<0.05 or P <0.01 relative to control.
Fig. 2. Effect of (a) hydroxybenzoic acid derivatives and (b) hydroxycinnamic acid derivatives on 8-OHdG formation induced by CumOOH in herring sperm DNA. Control values were obtained in the absence of CumOOH. Each value is the mean of 10 determinationsS.E.M. ** P<0.01 relative to controls.
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Fig. 3. The UV/visible spectra of (a) caffeic acid, (b) 2,3-diOH-BA and (c) 3,4-diOH-BA in the presence of 3 mm FeSO4 (1) or 3 mm FeCl3 (2) or in the absence of iron (3). Table 3 Interaction with iron and inhibitory effect on the Fe(III)/GSH and CumOOH-induced oxidative DNA damage of the tested compounds Phenolic compound
Fe(II) interaction
Fe(III) interaction
Inhibition on Fe/GSH induced-DNA damage
Inhibition on CumOOH induced-DNA damage
3-OH-BA 4-OH-BA 2,3-diOH-BA 3,4-diOH-BA Caffeic acid Ferulic acid 2-Coumaric acid 3-Coumaric acid 4-Coumaric acid
+ + +
+ + + + + +
+ + +
+ Interaction with iron or inhibition of DNA damage. No interaction with iron or inhibition of DNA damage.
the IC50 values (the concentration of each compound inhibiting oxidative damage by 50%). As reported in Table 2, 4-coumaric acid was the most active, although the IC50 of the three compounds was similar. 3.3. Interaction with iron We investigated whether the analysed phenolic acids might interact with iron under our experimental conditions of induced oxidative DNA damage in vitro. We found that the absorption spectra of 2,3-diOH-BA, 3,4diOH-BA and caffeic acid were modified in the presence of Fe(II) (Fig. 3). Moreover, when EDTA was added to
the solutions containing iron and caffeic acid, 2,3-diOHBA or 3,4-diOH-BA, UV/visible spectra were identical to those of the phenolic acids alone (data not shown), confirming their interaction with ferrous ions. On the contrary, no interaction was observed between iron (II or III) and the other phenolic compounds tested at the concentration used in in vitro assay (Table 3).
4. Discussion The data in this paper indicate that two hydroxybenzoic acid derivatives, 2,3-diOH-BA and 3,4-diOH-BA,
M. Lodovici et al. / Food and Chemical Toxicology 39 (2001) 1205–1210
are potent inhibitors of iron-induced oxidative DNA damage in vitro, an effect described also in other reports about the antioxidant properties of these two molecules (Ueda et al., 1996; Exner et al., 2000). The protective effect of 2,3-diOH-BA and 3,4-diOH-BA should be attributed to their capability to bind iron, as suggested by our spectrophotometric measurements and by literature data (Peterson et al., 1976; Jacobs et al., 1977). In fact, 2,3-diOH-BA and 3,4-diOH-BA were devoid of activity on DNA damage induced by hydroxyl radical formation in the CumOOH assay. In the presence of 3-OH-BA and 4-OH-BA, CumOOH-induced DNA damage was unaffected, but ironinduced oxidative DNA damage tended to be higher compared to the control levels in accord with Montgomery et al. (1995), who previously observed that 4OH-BA increased the production of hydroxyl radicals in vivo in the rat cortex. Among the hydroxycinnamic acid derivatives, caffeic acid had an effect similar to 2,3-diOH-BA and 3,4diOH-BA, exclusively inhibiting iron-induced oxidation damage. This result is in agreement with the data of Nardini et al. (1995), who demonstrated the formation of transitional metal ion–caffeic acid complexes. Ferulic acid was totally devoid of activity in our experiments, although its antioxidant property has been reported (Scott et al., 1993; Aruoma, 1999). On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids were good inhibitors both of iron-catalysed and CumOOH-induced DNA oxidation, but they had no interaction with iron, suggesting a direct scavenging activity against oxygen radical species. The IC50 values of the three coumaric acids indicated that the inhibitory effect on oxidative DNA damage is induced at low concentration. In conclusion, our results demonstrate that 2,3-diOHBA and 3,4-diOH-BA, possessing two ortho-hydroxyl groups, exert their antioxidant activity essentially by interaction with Fe(II) and that caffeic acid, one of the most common components of dietary phenols, exerts antioxidant activity with a similar mechanism. On the contrary, coumaric acids are radical scavengers and have no affinity for iron at the concentrations used in our assay. We also observed that 3-OH-BA and 4-OHBA do not have scavenging activity, 3-OH-BA actually enhancing oxidative DNA damage. In conclusion, our data indicate that some of these dietary phenols protect at low concentration in vitro against DNA oxidation induced by iron and/or CumOOH, two methods capable of determining antioxidant and/or scavenger activities of natural molecules. Further studies will be planned to explore the protective effect of these compounds on oxidative damage induced by other oxygen radical species, in particular peroxynitrite (ONOO/ ONOOH), an important mediator of oxidative reactions, implicated in various diseases (Jourd’heuil et al., 2001).
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Acknowledgements This study was supported by grants from the European Community POLYBIND Program (QLRT-199900505) and by funds of MURST.
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Jourd’heuil D., Jourd’heuil F., Kutchukian P.S., Musah R.A., Wink D.A., Grisham M.B., 2001. Reaction of superoxide and nitric oxide with peroxynitrite. Implication for peroxynitrite-mediated oxidation reactions in vivo. Journal of Biological Chemistry. In press. Keli, S.O., Hertog, M.G., Feskens, E.J., Krombout, D., 1996. Dietary Flavonoids, antioxidant vitamins and incidence of stroke: the Zutphen study. Archives of Internal Medicine 156, 637–647. Laranjinha, J.A., Almeida, L.M., Madeira, V.M., 1994. Reactivity of dietary phenolic acids with peroxyl radicals: antioxidant activity upon low density lipoprotein peroxidation. Biochemical Pharmacology 48, 487–494. Lodovici, M., Casalini, C., De Filippo, C., Copeland, E., Xu, X., Clifford, M., Dolara, P., 2000. Inhibition of 1,2-dimethylhydrazineinduced oxidative DNA damage in rat colon mucosa by black tea complex polyphenols. Food and Chemical Toxicology 38, 1085– 1088. Lodovici M., Guglielmi F., Casalini C., Meoni M., Cheynier V., Dolara P., 2001. Antioxidant and radical scavenging properties in vitro of polyphenolic extracts from red wine. European Journal of Nutrition. In press. Masella, R., Cantafora, A., Modesti, D., Cardilli, A., Gennaro, L., Bocca, A., Coni, E., 1999. Antioxidant activity of 3,4-DHPEA-EA and protocatechuic acid: a comparative assessment with other olive oil biophenols. Redox Report 4, 113–121. Montgomery, J., Ste-Marie, L., Boismenu, D., Vachon, L., 1995. Hydroxylation of aromatic compounds as indices of hydroxyl radical production: a cautionary note revisited. Free Radicals in Biology and Medicine 19, 927–933. Nardini, M., D’Aquino, M., Tomassi, G., Gentili, V., Di Felice, M., Scaccini, C., 1995. Inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivatives. Free Radicals in Biology and Medicine 19, 541–552. Ng, T.B., Liu, F., Wang, Z.T., 2000. Antioxidative activity of natural products from plants. Life Sciences 66, 709–723. Park, J., Floyd, R.A., 1994. Generation of strand breaks and formation of 8-hydroxy-20 -deoxyguanosine in DNA by a thiol/Fe3+/O2-
catalyzed oxidation system. Archives of Biochemistry and Biophysics 312, 285–291. Peterson, C.M., Graziano, J.H., Grady, R.W., Jones, R.L., Vlassara, H.V., Canale, V.C., Miller, D.R., Cerami, A., 1976. Chelation studies with 2,3-dihydroxybenzoic acid in patients with beta-tb major. British Journal of Hematology 1, 127–150. Plumb, G.W., Price, K.R., Rhodes, M.J.C., Williamson, G., 1997. Antioxidant properties of the major polyphenolic compounds in broccoli. Free Radical Research 27, 429–435. Radtke, J., Linseisen, J., Wolfram, G., 1998. Phenolic acid intake of adults in Bavarian subgroup of the national food consumption survey. Zeitschrift fu¨r Ernahrungswissenschaft 37, 190–197. Rice-Evans, C.A., Miller, N.J., Bolwell, N.J., Bramley, P.M., Pridham, J.B., 1995. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radical Research 22, 375–383. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure-antioxidant activity relationship of flavonoids and phenolic acids. Free Radicals in Biology and Medicine 20, 933–956. Scott, B.C., Butler, J., Halliwell, B., Arouma, O.I., 1993. Evaluation of the antioxidant actions of ferulic acid and catechins. Free Radical Research Communications 19, 241–253. Silva, F.A., Borges, F., Guimaraes, C., Lima, J.L., Maros, C., Reis, S., 2000. Phenolic acids and derivatives: studies on the relationship among structure, radical scavenging activity, and physicochemical parameters. Journal of Agricultural and Food Chemistry 48, 2122– 2126. Smith, C., Halliwell, B., Aruoma, O.I., 1992. Protection by albumin against the pro-oxidant actions of phenolic dietary components. Food and Chemical Toxicology 30, 483–489. Sugihara, N., Arakawa, T., Ohnishi, M., Furuno, K., 1999. Anti-and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid. Free Radicals in Biology and Medicine 27, 1313–1323. Ueda, J., Saito, N., Shimazu, Y., Ozawa, T., 1996. A comparison of scavenging abilities of antioxidant against hydroxyl radicals. Archives of Biochemistry and Biophysics 333, 377–383.
Research Section
Effect of natural phenolic acids on DNA oxidation in vitro M. Lodovici*, F. Guglielmi, M. Meoni, P. Dolara Department of Pharmacology, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy Accepted 1 June 2001
Abstract We examined the antioxidant activity of the following natural phenolic compounds present in food: 3-OH-benzoic acid (3-OHBA); 4-OH-benzoic acid (4-OH-BA); 2,3-dihydroxybenzoic acid (2,3-diOH-BA); 3,4-dihydroxybenzoic acid (3,4-diOH-BA or protocatechuic acid); ferulic acid; caffeic acid; and 2-coumaric, 3-coumaric and 4-coumaric acids. We measured the inhibitory effect of these compounds on iron-dependent oxidative DNA damage in vitro [incubating herring sperm DNA with Fe(III)/GSH] or using cumene hydroperoxide (CumOOH) as a free-radical generating system; we also studied the interaction of these phenols with Fe(II) or Fe(III) spectrophotometrically. Among the tested compounds, 2,3-diOH-BA, 3,4-diOH-BA and caffeic acid interacted with Fe(II) and showed a potent inhibitory effect on iron-induced oxidative DNA damage. CumOOH-induced DNA oxidation was not modified by these compounds. On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids did not interact with iron but protected against oxidative DNA damage induced by Fe(III)/GSH and by CumOOH, indicating a direct free-radical scavenging activity of these compounds in both systems. The IC50 S.E.M. of the three coumaric acids against CumOOH-induced DNA oxidation was 44.2 2.0, 54.7 2.0 and 33.1 1.0 mm, respectively. On the contrary, 3-OH-BA and 4-OH-BA did not have scavenging activity and 3-OH-BA actually enhanced oxidative DNA damage. In conclusion, some natural phenolic acids, commonly present in food, have interesting protective activity against DNA oxidation in vitro and deserve further consideration as effective antioxidants in vivo. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Oxidative DNA damage; Reactive oxygen species; Iron; Cumene hydroperoxide; Phenolic acids
1. Introduction Numerous epidemiological studies have provided evidence that individuals consuming diets rich in fruit and vegetables have a lower risk of cardiovascular disease (Hertog et al., 1993, 1995), stroke (Keli et al., 1996) and cancer (Block, 1992). These protective effects have been attributed to various components, such as carotenoids, vitamin C and phenolic acids. Many studies have also focused on the biological activities of flavonoids, a group of plant polyphenolic compounds, which are potent antioxidants and free-radical scavengers (RiceEvans et al., 1995, 1996; Plumb et al., 1997; Ng et al., 2000). However, some of the polyphenolic compounds
Abbreviations: CumOOH, cumene hydroperoxide; 3-OH-BA, 3hydroxybenzoic acid; 4-OH-BA, 4-hydroxybenzoic acid; 2,3-diOHBA, 2,3-dihydroxybenzoic acid; 3,4-diOH-BA, 3,4-dihydroxybenzoic acid; 8-OhdG, 8-OH-20 -deoxyguanosine. * Corresponding author. Tel.: +39-055-427-1321; fax+39-055427-1280. E-mail address: [email protected]fi.it (M. Lodovici).
can act as auto-oxidants at physiological pH (Hodnick et al., 1986) or as pro-oxidants, producing toxic oxygen radicals. Sugihara et al. (1999), using a lipid peroxidation assay, reported that some flavonoids have a prooxidant activity in the presence of high concentrations of iron ions, although they act as antioxidants at lower iron concentration. Moreover, Smith et al. (1992), demonstrated that some synthetic and natural phenolic compounds utilised in food preservation, protect lipid against oxidation but can accelerate oxidative damage to carbohydrate or DNA. In recent studies we demonstrated that polyphenolic compounds extracted from red wine and from black tea have a protective effect on oxidative induced DNA damage in rat liver and intestine (Casalini et al., 1999; Giovannelli et al., 2000; Lodovici et al., 2000) and can protect against azoxymethane-induced colon carcinogenesis in the rat (Caderni et al., 2000). Phenolic acids (essentially hydroxybenzoic and hydroxycinnamic acid) are widely distributed in plants and can be present in considerable amounts in the human diet. The intake of hydroxybenzoic and hydroxycinnamic
0278-6915/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(01)00067-9
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acid has been estimated to be around 11 mg/day and 211 mg/day, respectively, and the intake of caffeic acid, a hydroxycinnamic acid derivative, has been estimated in the order of 206 mg/day in subjects drinking coffee (Radtke et al., 1998). A protective effect of caffeic acid and its related catechols against hydroxyl radical formation in vitro has been reported (Iwahashi et al., 1990; Scott et al., 1993; Nardini et al., 1995; Abu-Amsha et al., 1996; Masella et al., 1999; Silva et al., 2000) and also protocatechuic acid has been well studied in this respect (Laranjinha et al., 1994; Ueda et al., 1996; Masella et al., 1999). On the contrary, data regarding the activity of hydroxybenzoic and coumaric acids are limited in the literature. On this basis, since DNA is one of the main molecular targets of the toxic effect of free radicals formed in cells, we investigated the effect of some phenolic acids on oxidative DNA damage and their interaction with iron in vitro.
2.5. Spectrophotometric measurements
2. Materials and methods
3. Results
2.1. Chemicals
3.1. Oxidative DNA damage
Herring sperm DNA, GSH, ferric chloride, ferrous sulphate, cumene hydroperoxide (CumOOH), 3-OHBA, 4-OH-BA, 3,4-diOH-BA, 2,3-diOH-BA, caffeic acid, ferulic acid, 2-coumaric acid, 3-coumaric acid and 4-coumaric acid were obtained from Sigma-Aldrich (Milan, Italy); bovine liver catalase was obtained by Roche Molecular Biochemicals (Milan, Italy).
To study the antioxidant effects in vitro of phenolic acids, we induced oxidative stress with Fe(III)/GSH, which produces H2O2 forming hydroxyl radical through the Fenton reaction; moreover, ferrous iron can also react with oxygen, forming perferryl ion species (FeO+ 2 and FeO3+) more reactive than the hydroxyl radical itself (Guengerich et al., 1997). In Fig. 1 we report the effect of hydroxybenzoic acid derivatives and the effect of hydroxycinnamic acid derivatives on iron-dependent oxidative DNA damage. In our experimental conditions oxidative DNA damage in the presence of Fe(III)/GSH, measured as 8-OHdG/dG, was increased about 30-fold when compared to control levels. We previously demonstrated that the addition of desferoxamine, an iron chelator, blocked the oxidation damage induced by Fe(III)/GSH (Lodovici et al., 2001). Similarly, when iron-induced oxidation damage was assayed in the presence of 2 mm EDTA, the chelation of iron prevented the production of H2O2 and oxidation damage (Table 1). Moreover, the addition of catalase (40 and 80 mg/ml), an enzyme capable of removing H2O2 derived from Fe(III)/GSH in the presence of O2 (Park and Floyd, 1994) reduced 8-OHdG levels dosedependently (Table 1). On the contrary, no inhibitory effect on oxidative damage induced by CumOOH was seen adding catalase at the same concentrations (data not shown), probably for the lack of involvement of H2O2 in CumOOH-induced DNA oxidation (Hix et al., 2000). In the presence of 3-OH-BA, oxidative DNA damage was increased. On the contrary, DNA oxidation was
2.2. Oxidative DNA damage induced by GSH/Fe(III) Herring sperm DNA (0.7 mg/ml, final concentration) was incubated in air for 2 h at 37 C in the dark in the presence of 3 mm FeCl3, 15 mm GSH and 4 mm HEPES, pH 7, according to Park and Floyd (1994). 2.3. Oxidative DNA damage induced by CumOOH Herring sperm DNA (0.7 mg/ml) was incubated in air for 2 h at 37 C in the dark with the test substances in the presence of 20 mm CumOOH in 4 mm HEPES, pH 7. 2.4. Measurement of 8-OHdG levels in DNA After incubation with Fe(III)/GSH or CumOOH, DNA was precipitated by ethanol in the presence of 20% 10 m ammonium acetate, washed with 70% ethanol, dried, dissolved in 20 mm acetate buffer pH 5.2, denatured at 90 C for 3 min and digested to a nucleoside pool; 8-OH20 -deoxyguanosine (8-OHdG) was measured with HPLC coupled with electrochemical detection, following established methods (Lodovici et al., 2000).
The absorption spectra of phenolic acids alone or in the presence of Fe(II) or Fe(III) were recorded from 190 to 700 nm on a Perkin-Elmer scanning spectrophotometer at 25 C. The cuvettes were closed with a cap during measurements. The final concentration of individual phenolic acids in 40 mm HEPES pH 7.1 were 15 mm; in the appropriate samples 3 mm FeSO4 or 3 mm FeCl3 were present; incubation buffer was used as reference. 2.6. Statistics Data were analysed with the Statgraphic Statistical Package, Statistical Graphic Corporation (Rockville, MD, USA) using one-way ANOVA analysis. IC50 values were obtained interpolating experimental data with a linear regression model.
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significantly reduced by 2,3-diOH-BA and 3,4-diOHBA (Fig. 1, panel A). Also hydroxycinnamic acid derivatives, except ferulic acid, significantly reduced irondependent oxidative DNA damage (Fig. 1, panel B). The highest inhibition of oxidative DNA damage was obtained in the presence of 3-coumaric and 4-coumaric acids. Fig. 2 shows the effect of the same compounds on oxidative DNA damage induced by CumOOH, which generates alkyl radicals directly (Hix et al., 2000). The ratio 8-OHdG/dG in DNA incubated with 20 mm CumOOH increased about 2.5-fold compared to control
levels (Fig. 2, panel A). The incubation with hydroxybenzoic acid derivatives did not modify 8-OHdG levels, but 2,3-diOH-BA and 3,4-diOH-BA induced a weak, non-statistically significant, increase in oxidative damage. As shown in panel B of Fig. 2, all analysed coumaric acids had a protective effect against oxidative DNA damage induced by CumOOH, with about a 40% reduction of DNA oxidation. Ferulic acid and caffeic acid, on the contrary, did not modify 8-OHdG levels.
Table 1 Effect of EDTA and catalase on DNA oxidation in vitro induced by Fe(III)/GSH
We determined the inhibition induced by 2-coumaric, 3-coumaric and 4-coumaric acids in the CumOOHinduced oxidative DNA damage assay, by estimating
3.2. IC50 values
Levels of 8-OHdG/dG105
Control EDTA (2 mm) Catalase (40 mg/ml) Catalase (80 mg/ml)
DNA
DNA +3 mm Fe(III)+GSH
5.00.3 – – –
121.811.7 7.00.4** 85.3 7.3* 60.5 7.2**
The values are means of five determinations S.E.M. *P< 0.05 and **P< 0.01 relative to control.
Table 2 IC50 values for hydroxycinnamic acids on CumOOH-induced oxidative DNA damage Phenolic compound
IC50 (mm)
2-Coumaric acid 3-Coumaric acid 4-Coumaric acid
44.2 2.0 54.7 2.0 33.1 1.0
The values are meansS.E.M. calculated with regression analysis using three different concentrations of the phenolic acids.
Fig. 1. Effect of hydroxybenzoic acid derivatives (a) and hydroxycinnamic acid derivatives (b) on 8-OHdG formation induced by Fe(III)/GSH in herring sperm DNA. Control values were obtained in the absence of Fe(III)/GSH. Each value is the mean of 10 determinationsS.E.M. *, ** P<0.05 or P <0.01 relative to control.
Fig. 2. Effect of (a) hydroxybenzoic acid derivatives and (b) hydroxycinnamic acid derivatives on 8-OHdG formation induced by CumOOH in herring sperm DNA. Control values were obtained in the absence of CumOOH. Each value is the mean of 10 determinationsS.E.M. ** P<0.01 relative to controls.
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Fig. 3. The UV/visible spectra of (a) caffeic acid, (b) 2,3-diOH-BA and (c) 3,4-diOH-BA in the presence of 3 mm FeSO4 (1) or 3 mm FeCl3 (2) or in the absence of iron (3). Table 3 Interaction with iron and inhibitory effect on the Fe(III)/GSH and CumOOH-induced oxidative DNA damage of the tested compounds Phenolic compound
Fe(II) interaction
Fe(III) interaction
Inhibition on Fe/GSH induced-DNA damage
Inhibition on CumOOH induced-DNA damage
3-OH-BA 4-OH-BA 2,3-diOH-BA 3,4-diOH-BA Caffeic acid Ferulic acid 2-Coumaric acid 3-Coumaric acid 4-Coumaric acid
+ + +
+ + + + + +
+ + +
+ Interaction with iron or inhibition of DNA damage. No interaction with iron or inhibition of DNA damage.
the IC50 values (the concentration of each compound inhibiting oxidative damage by 50%). As reported in Table 2, 4-coumaric acid was the most active, although the IC50 of the three compounds was similar. 3.3. Interaction with iron We investigated whether the analysed phenolic acids might interact with iron under our experimental conditions of induced oxidative DNA damage in vitro. We found that the absorption spectra of 2,3-diOH-BA, 3,4diOH-BA and caffeic acid were modified in the presence of Fe(II) (Fig. 3). Moreover, when EDTA was added to
the solutions containing iron and caffeic acid, 2,3-diOHBA or 3,4-diOH-BA, UV/visible spectra were identical to those of the phenolic acids alone (data not shown), confirming their interaction with ferrous ions. On the contrary, no interaction was observed between iron (II or III) and the other phenolic compounds tested at the concentration used in in vitro assay (Table 3).
4. Discussion The data in this paper indicate that two hydroxybenzoic acid derivatives, 2,3-diOH-BA and 3,4-diOH-BA,
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are potent inhibitors of iron-induced oxidative DNA damage in vitro, an effect described also in other reports about the antioxidant properties of these two molecules (Ueda et al., 1996; Exner et al., 2000). The protective effect of 2,3-diOH-BA and 3,4-diOH-BA should be attributed to their capability to bind iron, as suggested by our spectrophotometric measurements and by literature data (Peterson et al., 1976; Jacobs et al., 1977). In fact, 2,3-diOH-BA and 3,4-diOH-BA were devoid of activity on DNA damage induced by hydroxyl radical formation in the CumOOH assay. In the presence of 3-OH-BA and 4-OH-BA, CumOOH-induced DNA damage was unaffected, but ironinduced oxidative DNA damage tended to be higher compared to the control levels in accord with Montgomery et al. (1995), who previously observed that 4OH-BA increased the production of hydroxyl radicals in vivo in the rat cortex. Among the hydroxycinnamic acid derivatives, caffeic acid had an effect similar to 2,3-diOH-BA and 3,4diOH-BA, exclusively inhibiting iron-induced oxidation damage. This result is in agreement with the data of Nardini et al. (1995), who demonstrated the formation of transitional metal ion–caffeic acid complexes. Ferulic acid was totally devoid of activity in our experiments, although its antioxidant property has been reported (Scott et al., 1993; Aruoma, 1999). On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids were good inhibitors both of iron-catalysed and CumOOH-induced DNA oxidation, but they had no interaction with iron, suggesting a direct scavenging activity against oxygen radical species. The IC50 values of the three coumaric acids indicated that the inhibitory effect on oxidative DNA damage is induced at low concentration. In conclusion, our results demonstrate that 2,3-diOHBA and 3,4-diOH-BA, possessing two ortho-hydroxyl groups, exert their antioxidant activity essentially by interaction with Fe(II) and that caffeic acid, one of the most common components of dietary phenols, exerts antioxidant activity with a similar mechanism. On the contrary, coumaric acids are radical scavengers and have no affinity for iron at the concentrations used in our assay. We also observed that 3-OH-BA and 4-OHBA do not have scavenging activity, 3-OH-BA actually enhancing oxidative DNA damage. In conclusion, our data indicate that some of these dietary phenols protect at low concentration in vitro against DNA oxidation induced by iron and/or CumOOH, two methods capable of determining antioxidant and/or scavenger activities of natural molecules. Further studies will be planned to explore the protective effect of these compounds on oxidative damage induced by other oxygen radical species, in particular peroxynitrite (ONOO/ ONOOH), an important mediator of oxidative reactions, implicated in various diseases (Jourd’heuil et al., 2001).
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Acknowledgements This study was supported by grants from the European Community POLYBIND Program (QLRT-199900505) and by funds of MURST.
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