Introduction

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Chlorogenic acid (CGA)¹, caffeic acid (CA), and dihydrocaffeic acid (DHCA) are nonflavonoid catecholic compounds, which are present in many plants (Fig. 1). These chemicals are present in the diet as part of fruits, tea, coffee, and wine (Buren et al., 1973; Challis and Bartlett, 1975). There is growing interest in the multiple biological and pharmacological properties of nonflavonoid catecholic compounds, such as CGA, CA, and DHCA (dihydroxycinnamic acids) (Laranjinha et al., 1994). It has been reported that these catecholic acids have anti-inflammatory, antimutagenic, and anticarcinogenic activities (Challis and Bartlett, 1975; Koshihara et al., 1984; Tanaka et al., 1993a,b). The focus of much of the current research is on their cancer chemoprevention and antioxidant properties (Challis and Bartlett, 1975; Koshihara et al., 1984; Tanaka et al., 1993a,b; Laranjinha et al., 1995; Nardini et al., 1995). However, there is little information available in the literature on the enzymatic oxidation of CGA, CA, and DHCA.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structures of dietary dihydroxycinnamic acids.

An example of enzyme-directed antimelanoma therapy includes the bioactivation of a phenolic or a catecholic agent to an o-quinone by tyrosinase-containing tumor cells (Riley et al., 1997). An agent that can undergo tyrosinase-mediated oxidation to generate a cytotoxic reactive intermediate species, such as o-quinone, but is not metabolically activated by P450-rich-containing tissues, such as liver or kidneys, would be an ideal candidate for an antimelanoma therapy trail. In search of such a candidate we have investigated the enzymatic oxidation of dihydroxycinnamic acids by three different metabolizing systems, i.e., horseradish peroxidase type I (HRP)/H₂O₂ and tyrosinase/O₂ or rat liver microsomal P450/NADPH systems.

We previously used tandem mass spectrometry, HPLC, and UV spectroscopy to show that quercetin and other flavonoid compounds formed glutathione conjugates by HRP/H₂O₂ and tyrosinase/O₂ (Galati et al., 1999, 2001). In this article, we have used similar techniques to show that CA, DHCA, and CGA underwent an enzymatic oxidation by HRP/H₂O₂ or tyrosinase/O₂ to form a transient o-quinone intermediate that formed a conjugate with glutathione or underwent hydroxylation by H₂O. Further oxidation of this hydroxylated adduct by O₂ formed a p-quinone.

Glutathione conjugate formation also occurred when these dihydroxycinnamic acids were metabolized by an NADPH/rat liver microsomes system. Conjugate formation was prevented by benzylimidazole, a cytochrome P450 inhibitor. Dicumarol (an NADPH/oxidoreductase inhibitor) increased hepatocyte cytotoxicity induced by these dihydroxycinnamic acids, whereas benzylimidazole prevented cytotoxicity. This suggests that dihydroxycinnamic acids were metabolically activated by cytochrome P450 to form cytotoxic-reactive quinoid intermediates.

	Materials and Methods

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Chemicals. CGA, DHCA, CA, mushroom tyrosinase, horseradish peroxidase, hydrogen peroxide, magnesium chloride, potassium borohydride, ethyl acetate, NADP⁺, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, glucose, glucose oxidase, cumene hydroperoxide, sodium azide, sodium periodate, benzylimidazole, glutathione (reduced), Tris, diethylenetriaminepentaacetic acid (DETAPAC), potassium phosphate monobasic, potassium phosphate dibasic, 5,5'-dithio-bis(2-nitrobenzoic acid), dimethyl sulfoxide, trichloroacetic acid, and 3-(N-morpholino)propanesulfonic acid were obtained from Sigma-Aldrich (Oakville, ON, Canada). The stock solutions of dihydroxycinnamic acids were prepared in dimethyl sulfoxide. Other chemicals were prepared in Millipore filtered water (Millipore Corporation, Bedford, MA) or buffer. The stock solution of DTNB was dissolved in Tris/HCl buffer (0.1 M, pH 8.9, containing 1 mM DETAPAC). All concentrations shown were final concentrations.

Preparation of 4-Propylcatechol. 4-Propylcatechol was prepared according to the method described by Bolton et al. (1994) and Iverson et al. (1995).

Oxidation Studies by UV-VIS Spectroscopy. The spectra of solutions containing 50 to 100 µM CGA, DHCA, and CA were recorded in the absence and presence of glutathione before or after the addition of HRP (0.1 or 3 µM)/H₂O₂ (50 µM) or tyrosinase (25 units/ml) in a potassium phosphate buffer (50 mM, pH 6.0, containing 1 mM DETAPAC). An Amersham Pharmacia Biotech Ultraspec 1000 (Piscataway, NJ) with the proprietary Swift 1000 software was used to record data.

o-Quinone Stability Kinetics. Sodium periodate (250 µM) was added to a solution of CA, DHCA, or CGA (250 µM) in 3 ml of sodium phosphate buffer at various pH (4.0, 5.0, 6.0, or 7.4). The absorbance was monitored at 420 nm over a period of 20 min.

Dihydroxycinnamic Acids Oxidation Kinetics. The rate of oxygen consumption was measured for auto-oxidation in a sealed chamber containing dihydroxycinnamic acids (500 mM) in phosphate buffer (0.1 M, pH 7.4, containing 1 mM DETAPAC) by a Clark-type O₂ electrode. The initial oxygen concentration was determined as 224 nmol/ml at standard temperature and pressure (Umbreit et al., 1964).

Distribution Coefficient Value. Phosphate buffer (50 mM, pH 4.0, containing 1 mM DETAPAC) was prepared and presaturated with 1-octanol overnight. Aliquots of 5 to 10 ml of 1-octanol (which was mutually presaturated with phosphate buffer) were added to the phosphate buffer containing CGA, CA, or DHCA (100 µM). The octanol was added in time intervals of 30 min during which the solution was constantly stirred. The absorbance of the aqueous solution was monitored at both 300 and 325 nm for CGA and CA, and 280 nm for DHCA before and after each octanol addition. All solutions were prepared and used at room temperature.

Glutathione Depletion Assay. HRP (0.1 µM) and H₂O₂ (50 µM) were added to a mixture containing dihydroxycinnamic acids (50 µM) and glutathione (200 µM) in 1 ml of sodium phosphate buffer (0.1 M, pH 4.0, containing 1 mM DETAPAC). The mixture was preincubated for 30 min from which 250 µl was added to 25 µl of 30% w/v trichloroacetic acid, vortexed, and left for 5 min. An aliquot of 100 µl of the supernatant was added to a solution containing 25 µl of 2 mg/ml DTNB (prepared in Tris/HCl buffer, 0.1 M, pH 8.9) and 875 µl of Tris/HCl buffer (0.1 M, pH 8.9) and vortexed. The absorbances of the solutions were monitored at 412 nm for CA and DHCA and at 460 nm for CGA. The glutathione depletion assay for CGA, DHCA, and CA was also carried out with tyrosinase (20 units/ml) at pH 4.0.

Mass Spectrometry Analyses. The reaction mixtures contained CGA, CA, or DHCA (1 mM) and GSH (4 mM) in 1 ml of Millipore filtered water to which was added HRP (3 µM)/H₂O₂ (2 mM) or tyrosinase (25 units/ml). The reactions were incubated for 5 min at room temperature prior to direct injection into a mass spectrometer (PE Sciex III, Biomolecular Mass; PE Sciex, Toronto, ON, Canada). Mass spectrometry analysis was also performed when GSH was added to the reaction mixture 5 min after HRP/H₂O₂ or tyrosinase addition.

Glutathione Conjugate Formation by Rat Hepatocyte Microsomes. Adult male Sprague-Dawley rats, 250 to 300 g, were obtained from Charles River Canada Laboratories (Montreal, QC, Canada), fed ad libitum, and allowed to acclimatize for 1 week on clay chip bedding. The animals were anesthetized by sodium pentobarbital (60 mg/kg of body weight). Livers were removed under sterile potassium buffer/KCl solution (1.18%, w/v, 4°C) as previously described (Anari et al., 1997a). Hepatic microsomes were prepared as described by Dallner (1978).

Isolated Rat Hepatocyte Cytotoxicity Studies. Hepatocytes (10 ml) (10⁶ cells/ml) were preincubated at 37°C under an atmosphere of 95% O₂ and 5% CO₂ in Krebs-Henseleit buffer, pH 7.4, with CA (5 mM), 4-propylcatechol (0.3 mM), dihydrocaffeic acid (5 mM), and chlorogenic acid (5 mM). Where shown the following inhibitors were used: 100 µM benzylimidazole (a P450 inhibitor; Quan et al., 1992), 20 µM dicumarol (an NADPH/quinone oxidoreductase inhibitor; Preusch et al., 1991), 4 mM sodium azide (a catalase inhibitor), and 200 µM bromoheptane (a GSH-depleting agent; Khan and O'Brien, 1991). Catalytic agents used included hydrogen peroxide (10 mM glucose/1 unit/ml glucose oxidase) or cumene hydroperoxide (130 µM), or tyrosinase (100 units/ml) in the presence or absence of benzylimidazole (100 µM). Cell viability at 60, 120, and 180 min of incubation was determined by trypan blue uptake. None of the inhibitors or catalytic agents affected the viability of control hepatocytes at the concentrations used. Three separate experiments were carried out. Values shown are means ± S.E.

HPLC Analysis of GSH and GSSG Contents of Isolated Rat Hepatocytes. A modified method reported by Reed et al. (1980) was used for the HPLC analysis of GSH and GSSG. An 800-µl aliquot of the isolated rat hepatocytes reaction mixture was added to 200 µl of 25% w/v metaphosphoric acid in a glass tube, vortexed, left for 30 min at room temperature, and centrifuged. A 500-µl aliquot of the supernatant and 50 µl of 15 mg/ml freshly prepared iodoacetic acid in water were cotransferred to a glass tube containing sodium bicarbonate (~100-200 mg), vortexed, left up to 1 h or overnight at room temperature in a dark room. To this was then added 500 µl of 1.5% w/v 2,4-dinitro-fluorobenzene (prepared in ethanol), which was vortexed and left to stand at room temperature in a dark room for a period of 4 to 6 h for immediate HPLC processes.

	Results

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Oxidation Studies by UV-VIS Spectroscopy. There were varying rates of oxidation for dihydroxycinnamic acids by HRP/H₂O₂ or tyrosinase/O₂-oxidizing systems (Table 1). DHCA was found to possess the lowest rate of oxidation by HRP/H₂O₂ but had the fastest rate of oxidation by tyrosinase and oxygen. CGA and CA, on the other hand, were more rapidly oxidized by HRP/H₂O₂ than tyrosinase/O₂.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Dihydrocaffeic acid metabolism by HRP/H2O2 or tyrosinase/O2.

o-Quinone Stability Kinetics. The stability of the o-quinones formed was found to be related to the pH and followed first order kinetics. The t_1/2 stability of the o-quinones formed by each compound decreased with an increase in pH. As shown in Table 1, the t_1/2 of o-quinone stability in a decreasing order was CA > DHCA > CGA. The CA o-quinone formed was slightly more stable than the DHCA o-quinone and was almost twice as stable as the CGA o-quinone at pH 7.4.

Dihydroxycinnamic Acids Oxidation Kinetics. As shown in Table 1, the rate of oxidation of dihydroxycinnamic acids by HRP/H₂O₂ was CA, CGA DHCA. Negligible oxidation (<1 nmol/ml/min) occurred by H₂O₂ in the absence of HRP. The rate of oxidation of dihydroxycinnamic acids by tyrosinase/O₂ in the presence of GSH was DHCA > CGA > CA. Negligible oxidation (<1 nmol/ml/min) occurred in the absence of tyrosinase.

Distribution Coefficient Value. The distribution coefficient measurements for CA, CGA, and DHCA were carried out at pH 4.0 (Table 3). The distribution coefficient value for CGA (50 µM) at pH 4.0 in a mutually presaturated phosphate buffer and 1-octanol solvent system was 1.1 ± 0.1, which means that CGA distributes equally between water and 1-octanol at pH 4.0. The distribution coefficient was higher for DHCA at pH 4.0, which was 3.0 ± 0.4. DHCA, therefore, partitions more into the organic phase than the aqueous phase at pH 4.0. CA with a distribution coefficient value of 14.0 ± 0.1 at pH 4.0, demonstrated a higher organic phase solubility than DHCA or CGA. The distribution coefficients of CA and CGA at pH 7.4 were previously published by Choudhury et al. (1999) as 0.04 and 0.02, respectively. These low distribution coefficients were expected to be due to the ionization of the carboxylic acid moieties at a higher pH.

Glutathione Depletion Assay. In the HRP/H₂O₂ oxidation system, CGA, CA, and DHCA depleted 1.6, 1.8, and 1.9 M equivalents of GSH, respectively, which suggests that 60, 80, and 90% of their mono-glutathione conjugates underwent a second glutathione conjugation, respectively, catalyzed by peroxidase. However, CGA, CA, and DHCA depleted only 1.1, 1.0, and 0.8 M equivalents of GSH in the tyrosinase/O₂ oxidation system, respectively, indicating that DHCA was the least suitable substrate for tyrosinase in comparison with CA and CGA. Unlike HRP/H₂O₂ oxidizing system, the mono-glutathione conjugate of CGA, CA, and DHCA were not substrates for the oxidative enzyme tyrosinase. Negligible GSH depletion (<0.1 M equivalent) occurred in the absence of the enzymes.

Mass Spectrometry Analyses. The glutathione conjugate metabolites were identified by direct injection of the samples into a mass spectrometer (Table 4). Both mono- and bi-glutathione conjugates were identified for CA, DHCA, and CGA when glutathione was present before HRP/H₂O₂ addition.

Glutathione Conjugate Formation by Rat Hepatocyte Microsomes. As shown in Table 3, the amount of GSH depleted as a result of dihydroxycinnamic acid oxidation catalyzed by rat liver microsomes/NADPH was determined to be 0.2, 0.5, and 0.6 equivalents of GSH per mole of CGA, CA, and DHCA, respectively. This suggests that only 20, 50, and 60% of the corresponding compounds in the reaction mixture underwent glutathione conjugation. The CGA-, CA-, and DHCA-induced GSH depletion by the microsomes/NADPH metabolic system was largely inhibited by benzylimidazole (a cytochrome P450 inhibitor) (data not shown). Negligible GSH depletion (<0.1 M equivalent) occurred in the absence of NADP⁺.

Cytotoxicity in Isolated Rat Hepatocytes. As shown in Table 3, the LD₅₀ (2 h) concentrations, determined by trypan blue exclusion technique as a measure of hepatocyte cell membrane intactness and viability, for CA, CGA, and DHCA were 7, 23, and 6 mM, respectively. The cytotoxicity of all three compounds was dose-dependent with a ranking order of DHCA, CA > CGA.

Hepatocyte GSH Levels. As shown in Table 5, hepatocyte GSH was depleted by the hydroxycinnamic acids with the following order of effectiveness: 4-propylcatechol CGA > CA > DHCA. The GSH levels of NQO-inactivated hepatocytes were also much more readily depleted by DHCA or 4-propylcatechol, which suggests that DHCA or 4-propylcatechol cytotoxicity was caused by a quinoid metabolite that was reductively detoxified be NQO. The cytochrome P450 inhibitor benzylimidazole prevented CA-induced hepatocyte GSH depletion. Similar results were also obtained for CGA, DHCA, or 4-propylcatechol (results not shown) and suggest that these catechols are metabolically activated by cytochrome P450.

	Discussion

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Using mass spectrometry analyses, we have shown for the first time that mono- and bi-glutathione conjugates were formed when GSH was present during the peroxidase/H₂O₂-catalyzed oxidation of CA, DHCA, or CGA. Hydroxylated adducts were formed in the absence of GSH or if GSH was added later. This hydroxylation reaction competed with GSH conjugate formation. Metabolism of the dihydroxycinnamic acids by tyrosinase/O₂/GSH, however, resulted in the formation of hydroxylated adducts and mono-glutathione conjugates only. Previously, Ploemen et al. (1993) also suggested a glutathione conjugate of CA was formed with the tyrosinase system but did not provide mass spectrometry evidence.

The bi-glutathione conjugates formed with HRP/H₂O₂ and not with tyrosinase/O₂ may be related to differences in the catalytic activity of the enzymes HRP/H₂O₂ and tyrosinase/O₂ as well as the differences in the oxidation mechanisms of the two enzymes. Peroxidase catalyzes a one-electron oxidation of catechols (Nakamura et al., 1985, 1989; Garcia-Moreno et al., 1999; Espin et al., 2000), whereas tyrosinase oxidizes catechols through a two-electron oxidation mechanism, thus bypassing the short-lived semiquinone intermediate (Passi and Nazzaro-Porro, 1981; Espin et al., 2000).

Although the mass spectrometry analyses do not show the pattern of glutathione conjugation, it is easy to predict the pattern for the formation of a mono-glutathione conjugate of dihydroxycinnamic acids. C-2 and C-5 of the aromatic ring of CA, CGA, or DHCA o-quinones are almost equally electrophilic reactive centers because they are adjacent to the carbonyl groups of the o-quinone formed, whereas the C-6 is the least electrophilic center (Fig. 3). However, what distinguishes the three electrophilic centers is the sterical hindrance in an increasing order of C-5 < C-6 < C-2. We have recently reported ¹H NMR data on the mixture of three possible isomers of mono-glutathione conjugate of catechin even though the isolation and purification of each mono-glutathione conjugate of catechin was not possible (Moridani et al., 2001). Using the integration of the protons, chemical shifts, and coupling constants for ¹H NMR on protons of the B-ring of catechin glutathione conjugate metabolite, we found that the sterical hindrance was playing a major role as a distinguishing factor for glutathione to attack C_6' electrophilic center more readily than C-2', despite the fact that C-2' is more electrophilic than the C-6' center in catechin. The C-5' center is the most electrophilic and the least sterically hindered center and therefore it does not come as a surprise that its glutathione conjugate derivative M1 was formed 1.5- and 3-fold more than the other two metabolites M2 and M3 (Fig. 3). Therefore, we have concluded that we could have a similar pattern for the mono-glutathione conjugates of CA, CGA, or DHCA except that C-2 on dihydroxycinnamic acids is less crowded than C-2' on catechin. This might lead to the different amounts of M2 and M3 formation found for dihydroxycinnamic acids.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Pattern for the mono-glutathione conjugate formation of dihydrocaffeic acid and catechin.

Spectral evidence of a transient o-quinone intermediate for CA, CGA, and DHCA was obtained at pH 4 by using HRP/H₂O₂-metabolizing system. These intermediate products reacted rapidly with glutathione or underwent addition by H₂O. Further oxidation of this hydroxylated adduct by O₂ formed a p-quinone. The immediate product formed during enzymatic oxidation had a peak at 250 nm and shoulder at 260 nm, which could be attributed to the formation of the o-quinone and the p-quinone, respectively. Evidence for this finding came from the addition of excess potassium borohydride that caused the disappearance of 250- and 260-nm peaks, o-quinone and p-quinone, respectively, and the reformation of CGA or CA or DHCA. The 250- and ~420-nm peaks observed for CGA, CA, and DHCA were tentatively postulated as an o-quinone product because the starting material (i.e., a catechol structure) was formed upon the addition of borohydride. The red shift observed in the UV spectra of CA, CGA, or DHCA after metabolism by HRP/H₂O₂ enzyme and reduction with borohydride could be attributed to the introduction of the third hydroxyl group to their aromatic rings. Similar results were found for CA, CGA, and DHCA with the tyrosinase/O₂ oxidation system. Previously, it was shown that the oxidation of 4-t-butylcatechol with HRP/H₂O₂ also formed the characteristic o-quinone peak at 420 nm (Garcia-Monreno et al., 1999). Oxidation of CGA by hypochlorite also led to the formation of peaks at 420 and 490 nm, which were suggested to be o-quinone or p-quinone, respectively (Kono et al., 1995).

Hydroxylation of catecholic compounds was previously reported as a result of p-coumaric oxidation to CA by spinach-beet phenolase (McIntyre and Vaughan, 1975). Upon potassium borohydride addition to the tyrosinase or peroxidase enzymatic oxidation products of CGA, CA, or DHCA, spectral evidence for the formation of hydroxylated products of CGA, CA, and DHCA was obtained. Mass spectrometry analyses confirmed this. Previously, it was suggested that water or H₂O₂ could add or hydroxylate, respectively, certain noncyclizable o-quinone (Napolitano et al., 1995; Jimenez and Garcia-Carmona, 1996). The conversion of the hydroxylated product of CGA, CA, and DHCA to the corresponding p-quinone probably resulted from auto-oxidation (Fig. 2) because the hydroxylated products are likely to have a higher oxidation potential than the hydroxycinnamic acids.

DHCA o-quinone can also undergo an intramolecular cyclization to form dihydroesculetin (Fig. 2) (Sugumaran et al., 1989). CA o-quinone, however, cannot undergo cyclization due to the presence of the trans double bound. Furthermore, CGA is also unable to cyclize due to the lack of nucleophilic carboxyl group on its side chain in a short vicinity to the o-quinone ring. However, esculetin, the cyclized product of CA, was found when rat livers were perfused with CA (Gumbinger et al., 1993). Under our experimental conditions, esculetin was not identified as a product of CA oxidation by tyrosinase although we have found that isolated rat hepatocytes could convert CA to DHCA (data not shown).

CA, CGA, or DHCA were also found to be substrates for the NADPH-supported microsomal cytochrome P450 system and resulted in glutathione conjugate formation. Because CA, CGA, or DHCA had a partition coefficient value of greater than one, we hypothesize that they can cross the isolated rat hepatocyte membrane and are metabolized by P450 to form a cytotoxic o-quinone. However, there is no obvious immediate correlation between LD₅₀ and log P, whereas there is a correlation between LD₅₀ and distribution coefficient values measured at pH 4 or calculated for pH 7.4; the higher the distribution coefficient the more toxic the compounds (Table 3). We also showed for the first time that CA, CGA, and DHCA were metabolically activated by cytochrome P450 peroxygenase activity (Anari et al., 1997a,b). Further evidence that the cytotoxic metabolite is probably an o-quinone is the marked increase in hepatocyte susceptibility to CA, CGA, and DHCA if NQO1 was inhibited. Most of the quinone substrates of NQO1 are p-quinones but two o-quinones, 9,10-phenanthrenequinone and cyclized dopamine o-quinone, have also been shown to be NQO1 substrates (O'Brien, 1991; Segura-Aguilar et al., 1992).

Tyrosinase is often abundant in melanoma and therefore can be considered as a useful enzyme for bioactivating CA, CGA, and DHCA if used for antimelanoma therapy. Enzyme-directed antimelanoma therapy includes the use of phenols or catechols that form o-quinones that are highly toxic to tyrosinase-containing melanoma tumor cells (Riley et al., 1997). Quinones are highly reactive species that can react with a variety of nucleophiles such as water, thiol-containing compounds, amino acids, and protein thiols, which are normally found in the cell. Glutathione is an important mechanism for cellular defense against reactive quinones (O'Brien, 1991). However, it needs to be shown that CGA, CA, and DHCA act as a substrate for melanoma tyrosinase and depletes GSH and protein thiols in the melanoma cells, resulting in cell death.