Modulation of cytochrome P450 and phase II enzymes by protocatechuic acid in mouse liver and kidney

Toxicology 216 (2005) 24–31

Modulation of cytochrome P450 and phase II enzymes by protocatechuic acid in mouse liver and kidney Violetta Krajka-Ku´zniak, Hanna Szaefer, Wanda Baer-Dubowska ∗ University of Medical Sciences, Department of Pharmaceutical Biochemistry, Grunwaldzka 6, 60-780 Pozna´n, Poland Received 24 June 2005; received in revised form 14 July 2005; accepted 14 July 2005 Available online 31 August 2005

Abstract Protocatechuic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. In this study the effect of protocatechuic acid on murine cytochrome P450 and phase II enzymes was evaluated. The activities of EROD, MROD, PROD, PNPH, GST, UDPGT and NQO1 were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with protocatechuic acid in the dose range of 80–800 mg/kg. At the highest doses, protocatechuic acid decreased the activities of EROD and MROD by ∼20–30% in mouse liver and kidney, while the activity of renal PNPH was reduced by 28%. Moreover, Western blot analysis with CYP1A1/1A2 and CYP2E1-specific antibodies showed the same effect on the levels of hepatic CYP1A1/1A2 and CYP2E1 proteins. This simple phenol affected also the phase II enzymes. The activity of GST was elevated in both tissues of the animals treated with protocatechuic acid at the dose of 80 mg/kg. The inhibition of hepatic NQO1 was the most striking effect. The effect was dose dependent and almost 70% inhibition was observed after treatment with protocatechuic acid at the dose of 800 mg/kg. In contrast to the liver, the renal NQO1 was not affected. These results indicate that protocatechuic acid, as other phenolic acids, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways, but this effect depends on tissue. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Protocatechuic acid; Cytochrome P450; Phase II enzymes; Mouse liver and kidney microsomes

1. Introduction Phenolic compounds are ubiquitous in edible vegetables, fruits and nuts and it is estimated that an average of 1–2 g/day of these components may be consumed in Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; DMBA, 7, 12-dimethylbenz[a]anthracene; EROD, ethoxyresorufin-O-deethylase; MROD, methoxyresorufin-O-demethylase; PROD, pentoxyresorufin-O-depentylase; PNPH, p-nitrophenol hydroxylase; GST, glutathione S-transferase; UDPGT, UDP-glucuronosyltransferase; NQO1, NAD(P)H:quinone oxidoreductase ∗ Corresponding author. Tel.: +48 61 8546621; fax: +48 61 8546609. E-mail address: [email protected] (W. Baer-Dubowska).

a human diet (Markham, 1989). Several phenolic compounds have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models (Yang et al., 1997; De Flora, 1998). Protocatechuic acid, a simple phenolic acid, is a constituent of apples, green and black tea, and herbal medicine (Hudson et al., 2000). A high level of this compound was found in the extract from the rind of Citrus reticulata Blanco (Ueno, 1993). This plant phenol is also an ingredient of popular beverages and a Chinese herbal medicine used to treat hypertension, pyrexia and liver damage (Tseng et al., 1998). Protocatechuic acid was indicated as an efficacious agent in inhibiting the carcinogenic action

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.07.013

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of diethylnitrosamine in the liver (Tanaka et al., 1993), 4-nitroquinoline-1-oxide in the oral cavity (Tanaka et al., 1994), azoxymethane in the colon (Kawamori et al., 1994), N-methyl-N-nitrosourea in glandular stomach tissue (Tanaka et al., 1995a), N-butyl-N-(4-hydroxybutyl) nitrosamine in the bladder (Hirose et al., 1995) and Nnitroso-bis(2-oxopropyl)amine in pancreas (Nakamura et al., 2000a,b). Ohnishi et al. (1997) demonstrated the inhibitory effect of dietary protocatechuic acid on 7,12dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch carcinogenesis. Moreover, a topical application of protocatechuic acid isolated from Hibiscus sabdariffa L. inhibited the 12-O-tetradecanoyphorbol ester-induced tumor promotion in mouse skin (Tseng et al., 1998). Our previous studies (Ignatowicz et al., 2003; Szaefer et al., 2004) showed that this compound reduces the formation of benzo[a]pyrene and DMBA–DNA adducts in vitro and in mouse epidermis in vivo. Its effect on the enzymatic systems involved in the activation and detoxification of these carcinogens was not univocal. In vitro protocatechuic acid was a potent inhibitor of methoxyresorufin O-demethylase (MROD)-CYP1A2, pentoxyresorufin O-depentylase (PROD)-CYP2B, ethoxyresorufin O-deethylase (EROD)-CYP1A1 (BaerDubowska et al., 1998). Single doses of protocatechuic acid administered by gavage to rats reduced (by 50%) the activities of hepatic EROD, MROD and PROD (Szaefer et al., 2003). This effect, however, was not observed after prolonged treatment (Krajka-Ku´zniak et al., 2004). In mouse epidermis this phenol increased the activity of AHH, marker of CYP1A1 (Szaefer et al., 2004). The similar discrepancies were observed in protocatechuic acid effect on phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase (NQO1) and UDP-glucuronosyltransferase (UDPGT). The above studies indicate that the experimental model, the method of animal treatment and the tissue specific response, affect to a great extent the change in the balance between potentially xenobiotic activating and detoxifying pathways by this plant phenol. The present study further characterizes the modulation of phases I and II enzymes in mouse liver and kidney after intraperitoneal treatment of mice with different doses of protocatechuic acid.

sorufin, pentoxyresorufin, resorufin, p-nitrophenol, glutathione, 1-chloro-2,4-dinitrobenzene (CDNB), 2,6dichlorophenoloindophenol, dicoumarol, dithiothreitol, NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase were purchased from Sigma (St. Louis, MO). Primary and secondary antibodies against CYP1A1/1A2, CYP2E1 and standard for CYP2E1 were supplied by Oxford Biomedical Research (Oxford, MI). Rainbow colored protein molecular weight markers was purchased from Amersham Pharmacia Biotechnology (UK). All the other compounds were readily available commercial products of the highest purity available.

2. Materials and methods

The ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase and pentoxyresorufin-O-depentylase activities were measured as described earlier (BaerDubowska et al., 1998; Burke et al., 1985). The pnitrophenol hydroxylase (PNPH) activity was determined according to Reinke and Moyer (1985) protocol.

2.1. Chemicals Protocatechuic acid (purity 97%) was obtained from Aldrich (Milwaukee, WI). Ethoxyresorufin, methoxyre-

2.2. Animals Female Swiss mice (7–9 week old, 25 g) were used in the all experiments. The animals were housed in polycarbonate cages containing hardwood chip bedding. Commercial mouse food and distilled water were available without restriction. The environment had a controlled light/dark cycle and temperature of 20–22 ◦ C. Fifteen mice were used for each experimental group. The animals were treated i.p. with 80, 200 or 400 mg/kg body weight of protocatechuic acid dissolved in olive oil for three consecutive days, or with a single dose of 800 mg/kg body weight. The control mice received vehicle only (0.4 ml of a olive oil). All the experiments were conducted according to the Regional Ethics Committee guidelines for animal experimentation. 2.3. Preparation of microsomes and cytosol Twenty-four hours after the last treatment, the mice were killed by cervical dislocation and livers and kidneys were removed. Both tissues were homogenized with 2 volumes of 0.25 M sucrose solution containing 0.05 M Tris (hydroxymethyl)aminomethane)–HCL buffer pH 7.5, 0.025 M KCl, and 0.003 M MgCl2 . Microsomal and cytosolic fractions were prepared by differential centrifugation as described previously (Gnojkowski et al., 1984). Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.4. Enzyme assays

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Table 1 The effect of protocatechuic acid on the activities of cytochrome P450-dependent enzymes in mouse liver Treatment

ERODa,b

MRODc

Control Protocatechuic acid (80 mg/kg) 3× Protocatechuic acid (200 mg/kg) 3× Protocatechuic acid (400 mg/kg) 3× Protocatechuic acid (800 mg/kg) 1×

66.07 ± 2.71 62.12 ± 1.86 (94)f 52.54 ± 2.93 (80)g 48.65 ± 2.95 (74)g 46.38 ± 2.44 (70)g

145.17 140.42 129.37 106.88 98.67

a b c d e f g

± ± ± ± ±

8.31 6.51 (97) 12.72 (89) 4.88 (74)g 1.50 (68)g

PRODd

PNPHe

42.77 ± 0.84 48.96 ± 5.45 (114) 51.16 ± 3.44 (120) 54.11 ± 2.05 (127)g 51.91 ± 1.75 (121)g

781.67 ± 50.56 731.81 ± 37.54 (94) 742.98 ± 39.45 (95) 694.56 ± 39.63 (89) 650.23 ± 25.01 (83)

Values are mean ± S.E.M. and represent analysis of three separate experiments (n = 5 mice per group). EROD pmol resorufin formed/min/mg. MROD pmol resorufin formed/min/mg. PROD pmol resorufin formed/min/mg. PNPH pmol p-nitrocatechol formed/min/mg. Percent of control. Significantly different from control by two-tailed Student’s t-test, P < 0.05.

The tissue glutathione S-transferase activity was determined by the method of Habig et al. (1974) using CDNB as substrate. The cytosolic NAD(P)H:quinone oxidoreductase activity was assayed according to the method of Benson et al. (1986). The microsomal UDP-glucuronosyltransferase activity was determined using p-nitrophenol as substrate as described by Jakoby (1981). 2.5. Immunodetection of CYP1A1/1A2 and CYP2E1 by Western blot

used. Densitometry was performed using BioRad GS 710 Image Densitometer (Bio Rad Laboratories, Hercules, CA, USA). 2.6. Statistical analysis The experimental groups were compared with their respective controls using ANOVA followed by the unpaired two-tailed Student’s t-test. The differences were considered significant at P ≤ 0.05. 3. Results

Microsomal proteins (50–100 ␮g) were separated on 10% SDS-polyacrylamide gels by the method of Laemmli (1970). The proteins were transferred to nitrocellulose membrane using method of Towbin et al. (1979). Membranes were incubated with a monoclonal mouse anti-rat CYP1A1/1A2 antibody or with a polyclonal goat anti-rabbit CYP2E1. As the secondary antibodies in the staining reaction the alkaline phosphataselabeled donkey anti-mouse or donkey anti-goat Ig were

3.1. Effects of protocatechuic acid on cytochrome P450 enzymes The effects of i.p. administered protocatechuic acid on cytochrome P450-dependent enzymes in mouse liver and kidney are summarized in Tables 1 and 2. After a single dose of 800 mg/kg treatment, this compound significantly inhibited the activities of EROD and MROD

Table 2 The effect of protocatechuic acid on the activities of cytochrome P450-dependent enzymes in mouse kidney Treatment

ERODa,b

MRODc

PRODd

PNPHe

Control Protocatechuic acid (80 mg/kg) 3× Protocatechuic acid (200 mg/kg) 3× Protocatechuic acid (400 mg/kg) 3× Protocatechuic acid (800 mg/kg) 1×

16.50 ± 0.62 15.68 ± 0.67 (95)f 14.43 ± 0.38 (87) 13.89 ± 0.86 (84)g 13.05 ± 0.35 (79)g

18.38 ± 0.79 21.82 ± 0.91 (119) 16.93 ± 0.74 (92) 14.73 ± 0.32 (80)g 15.05 ± 0.63 (82)g

16.37 ± 0.38 17.98 ± 5.78 (109) 15.04 ± 0.51 (92) 15.96 ± 0.44 (98) 16.43 ± 0.46 (100)

96.25 ± 5.35 91.8 ± 3.82 (95) 75.19 ± 4.04 (78) 69.54 ± 4.10 (72)g 69.32 ± 5.56 (72)g

a b c d e f g

Values are mean ± S.E.M. and represent analysis of three separate experiments (n = 5 mice per group). EROD pmol resorufin formed/min/mg. MROD pmol resorufin formed/min/mg. PROD pmol resorufin formed/min/mg. PNPH pmol p-nitrocatechol formed/min/mg. Percent of control. Significantly different from control by two-tailed Student’s t-test, P < 0.05.

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Fig. 1. Dose effect of protocatechuic acid on the expression of hepatic CYP1A1/1A2 in mouse. (A) Western blot analysis of liver microsomes that uses antibody against CYP1A1/1A2. (lane 1) Protein molecular weight markers; (lane 2) control; (lane 3) liver microsomes from mice treated with protocatechuic acid at the doses of 80 mg/kg; (lane 4) 200 mg/kg; (lane 5) 400 mg/kg; (lane 6) 800 mg/kg. Lanes 2–6 were applied with 50 ␮g microsomal proteins. Representative data are shown. (B) Data shown the percentage of CYP1A1/1A2 expression compared to control.

Fig. 2. Dose effect of protocatechuic acid on the expression of kidney CYP1A1/1A2 in mouse. (A) Western blot analysis of kidney microsomes that uses antibody against CYP1A1/1A2. (lane 1) Protein molecular weight markers; (lane 2) control; (lane 3) kidney microsomes from mice treated with protocatechuic acid at the doses of 80 mg/kg; (lane 4) 200 mg/kg (lane 4); (lane 5) 400 mg/kg; (lane 6) 800 mg/kg. Lanes 2–6 were applied with 100 ␮g microsomal proteins. Representative data are shown. (B) Data shown the percentage of CYP1A1/1A2 expression compared to control.

(∼20–30%) in the liver and kidney. A similar effect on hepatic and kidney EROD and MROD was observed at the dose of 400 mg/kg administered for three consecutive days. The same treatment regimen enhanced O-depentylation of pentoxyresorufin by 21–27%. In contrast to the liver, renal PROD was not affected. The hydroxylation of p-nitrophenol was not affected in liver microsomes. The highest doses of protocatechuic acid in the kidney inhibited PNPH by 28%. Western blot analysis of liver microsomes showed a decrease of the level of CYP1A1/1A2 in protocatechuic acid treated animals (Fig. 1) in comparison with control group. Densitometry of the bands in Fig. 1 indicated about 25% decrease in CYP1A1/1A2 in animals exposed to protocatechuic

acid at the dose of 800 mg/kg. In kidney expression of CYP1A1/1A2 was not statistically changed after treatment with protocatechuic acid (Fig. 2). Expression of hepatic CYP2E1 (Fig. 3) was slightly decreased (∼10–18%) but in renal microsomes CYP2E1 protein was not detected in our conditions. 3.2. Effects of protocatechuic acid on phase II enzyme activities In the second part of this study, the effect of protocatechuic acid on the phase II enzymes, GST, UDPGT and QR, was examined. These results are collected in Tables 3 and 4. Protocatechuic acid induced GST in

Table 3 The effect of protocatechuic acid on the activities of phase II enzymes in mouse liver Treatment

GSTa,b

UDPGTc

QRd

Control Protocatechuic acid (80 mg/kg) 3× Protocatechuic acid (200 mg/kg) 3× Protocatechuic acid (400 mg/kg) 3× Protocatechuic acid (800 mg/kg) 1×

1799.59 ± 37.78 2360.84 ± 181.18 (131)e,f 2078.62 ± 29.54 (116)f 1897.01 ± 18.07 (105) 1986.80 ± 29.34 (110)f

1.90 ± 0.19 2.09 ± 0.24 (110) 1.75 ± 0.11 (92) 1.63 ± 0.15 (86) 1.67 ± 0.13 (88)

31.49 23.96 14.40 10.19 8.50

a b c d e f

Values are mean ± S.E.M. and represent analysis of three separate experiments (n = 5 mice per group). GST nmol 1-chloro-2,4-dinitrobenzene conjugated/min/mg. UDPGT nmol 4-nitrophenyl-glucuronide formed/min/mg. QR nmol 2,6-dichloroindophenol reduced/min/mg. Percent of control. Significantly different from control by two-tailed Student’s t-test, P < 0.05.

± ± ± ± ±

1.54 2.31 (76)f 0.91 (46)f 0.19 (32)f 0.49 (27)f

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Table 4 The effect of protocatechuic acid on the activities of phase II enzymes in mouse kidney Treatment

GSTa,b

UDPGTc

QRd

Control Protocatechuic acid (80 mg/kg) 3× Protocatechuic acid (200 mg/kg) 3× Protocatechuic acid (400 mg/kg) 3× Protocatechuic acid (800 mg/kg) 1×

565.69 ± 19.53 813.50 ± 52.81 (143)e,f 727.27 ± 16.70 (129)f 580.41 ± 30.73 (103) 616.57 ± 21.17 (109)

0.29 ± 0.01 0.30 ± 0.07 (105) 0.26 ± 0.04 (91) 0.25 ± 0.01 (88) 0.24 ± 0.05 (85)

22.78 ± 0.58 23.08 ± 4.57 (101) 22.55 ± 0.77 (99) 26.48 ± 3.41 (116) 24.82 ± 1.22 (108)

a b c d e f

Values are mean ± S.E.M. and represent analysis of three separate experiments (n = 5 mice per group). GST nmol 1-chloro-2,4-dinitrobenzene conjugated formed/min/mg. UDPGT nmol 4-nitrophenyl-glucuronide formed/min/mg. QR nmol 2,6-dichloroindophenol reduced/min/mg. Percent of control. Significantly different from control by two-tailed Student’s t-test, P < 0.05.

liver and in kidney. The highest increase (31–43%) of the enzyme activity was observed when this phenol was administered for three consecutive days at the dose of 80 mg/kg. UDPGT was not affected by protocatechuic acid in both tissues. The most striking result was the reduced activity of hepatic NQO1 which was observed as an effect of i.p. treatment with protocatechuic acid. This effect was dose dependent and an almost ∼70% inhibition occurred after treatment with protocatechuic acid for three days with the dose of 400 mg/kg or a single 800 mg/kg dose. The activity of NQO1 in kidney was not affected by protocatechuic acid.

Fig. 3. Dose effect of protocatechuic acid on the expression of hepatic CYP2E1 in mouse. (A) Western blot analysis of liver microsomes that uses antibody against CYP2E1. (lane 1) CYP2E1 standard from microsomes rat liver induced pyridine; (lane 2) control; (lane 3) liver microsomes from mice treated with protocatechuic acid at the doses of 80 mg/kg; (lane 4) 200 mg/kg; (lane 5) 400 mg/kg; (lane 6) 800 mg/kg. Lanes 2–6 were applied with 50 ␮g microsomal proteins. Representative data are shown. (B) Data shown the percentage of CYP2E1 expression compared to control.

4. Discussion Protocatechuic acid, the simple phenolic is one of the major benzoic derivatives from edible plants and fruits and shows the variety of biological activities including anti-carcinogenic potential. This compound is also an antioxidant, what very often is related to induction of cytochrome P450 and/or phase II enzymes. The focus of this study was to investigate the effect of i.p. treatment of mice with protocatechuic acid on these enzymatic systems in murine liver and kidney. The doses and treatment protocols used were comparable to those applied by Nakamura et al. (2000a,b) in mouse skin protocatechuic acid bioassays and d-limonene i.p. treatment of rats (Reicks and Crankshaw, 1993). Our results demonstrate that intraperitoneally administered protocatechuic acid can modulate the phases I and II enzyme activities in both tissues. The observed effects are tissue and dose dependent. Protocatechuic acid reduced the activity of EROD and MROD in liver by 30–32%. This finding is in agreement with the results of our previous in vitro and in vivo studies, showing protocatechuic acid as an inhibitor of hepatic EROD and MROD (Baer-Dubowska et al., 1998; Szaefer et al., 2003). Western blot analysis of mouse liver microsomes confirmed the inhibition of CYP1A1/1A2 by protocatechuic acid as indicated the densitometric analysis showing ∼25% reduction in comparison with control. We demonstrated, however, that the activity of PROD in liver was increased by 21–27% after i.p. treatment with protocatechuic acid. The results of our current studies are in contrast to those observed in vitro (Baer-Dubowska et al., 1998), in which inhibition of PROD by protocatechuic acid was observed. These findings indicate that the results of in vitro studies are not always in agreement with the data obtained in vivo. Similar conclusions

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were drawn by Nims et al. (1997), who showed that many in vitro inhibitors of CYP2B were inducers of this P450 subfamily in vivo. This observation might be important since it has been reported that there is a good association between the ability of chemicals to induce the CYP2B1/2 and their potential to promote hepatocarcinogenesis. Therefore, strong liver P450 inducers, such as phenobarbital, which cause marked increase in CYP2B1/2 are generally considered to be liver tumor promoters (Vang, 2005). In our study protocatechuic acid decreased the PNPH activity, the marker of CYP2E1, in both tested organs. However, the observed changes were statistically significant in kidney of animals treated with protocatechuic acid in the highest doses (400 and 800 mg/kg). Moreover, Western blots demonstrated that protocatechuic acid administration resulted in a slight decrease in the expression of hepatic CYP2E1, but CYP2E1 protein, in kidney was not detected in our conditions. An interestingly similar effect was described by Hur et al. (2003) in rat treated with protocatechuic acid in combination with bromobenzene. They observed the reduced activity of aniline hydroxylase, a marker of CYP2E1. The P450 2E1 in rodents and humans is the principle enzyme involved in the bioactivation of a variety of low molecular weight suspect carcinogens, such as benzene, styrene and vinyl chloride, halogenated hydrocarbons as well as nitrosamines, such as N-nitrosodimethylamine (Yang et al., 1992). The inhibition of CYP2E1 is expected to block both the toxicity and the carcinogenicity of these compounds. Naturally occurring organosulfur compounds like diallyl sulfide are competitive and mechanism-based inhibitors of CYP2E1 and potent anticarcinogens (Yang et al., 1994; Premdas et al., 2000). Thus, the inhibition of CYP2E1 observed in our study might be important for anticarcinogenic activity of protocatechuic acid. Another interesting finding of the current study was the way in which protocatechuic acid affected the phase II enzymes. The lowest dose of protocatechuic acid increased the activity of GST in liver and kidney by 31% and 43%, respectively. The same effect was observed in our previous studies, but in the rat liver only (KrajkaKu´zniak et al., 2004). Additional studies using HPLC analysis demonstrated that GST induction was primarily caused by significant increase of GST class mu (Krajka-Ku´zniak et al., 2000). The induction of GST was described by Kampa et al. (2003) with caffeic acid representing the same chemical class of compounds as protocatechuic acid. Protocatechuic acid did not affect UDPGT in both tissues. The most marked effect was found for NQO1. An about 70% decrease of hepatic enzyme activity was observed in mice after treatment

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with protocatechuic acid at the highest doses. However, in contrast to the liver results, in renal NQO1 activity no differences were found between treated and control group of mice. Our current results are in contrast to those observed in rat liver (Krajka-Ku´zniak et al., 2004), where NQO1 activity was increased after protocatechuic acid treatment. These differences in activity of NQO1 between mice and rats can be partly explained by the different treatment regimen and species properties. The tissue-specific effects found in this study may reflect the differences in NQO1 activity in hepatic and extrahepatic tissues (Hietanen et al., 1987), but also the disposition of this phenol. Such dramatic effect in liver may suggest a suicide mechanism of the enzyme inhibition. More studies under way are needed in order to explain the detailed mechanism of this inhibition. NQO1 catalyzes two-electron transfer from both reduced pyridine nucleotides to some redox azo dyes and quinones (Smitskamp-Wilms et al., 1996; Begleiter et al., 1997). In this way, quinones are metabolized bypassing the formation of semiquine, and subsequently, of superoxide anion radicals (Winski et al., 1998). Thus, NQO1 is generally assumed to possess the important protective properties, both by detoxifying some carcinogenic compounds as well as by preventing the generation of oxygen radicals. However, this enzyme may provide not only a cellular detoxifying system, but also, with some substrates, an activating mechanism. These include important mutagens and carcinogens, such as heterocyclic amines (De Flora et al., 1988; Kitamura et al., 1999). NQO1 catalyzes the conversion of 4-nitroquinoline N-oxide to 4-hydroxyamino-quinoline N-oxide, an obligatory proximate metabolite (Nagao and Sugimura, 1976). Thus, on one hand the inhibition of NQO1 in concert with the inhibition of CYP2E1 might contribute to the anticarcinogenic activity of protocatechuic acid towards amine derivatives in experimental models, at least, in certain tissues (Tanaka et al., 1993, 1995b; Mukhtar et al., 1988). Conversely, it may accelerate the formation of the reactive oxygen species through semiquinones. Moreover, protocatechuic acid itself may be metabolized via the tyrosinase bioactivating pathway to reactive quinone intermediates (Nakamura et al., 2001). The antimutagenic and anticarcinogenic activity of this phenolic is thought to be mainly the result of scavenging the ultimate carcinogenic metabolites (Newmark, 1987). In this regard, our previous studies showed the reduction of the benzo[a]pyrene and DMBA–DNA adducts formation in the mouse epidermis in vivo and in vitro system by protocatechuic acid (Ignatowicz et al., 2003; Szaefer et al., 2004). The results of our present study indicate that this phenolic acid, besides scavenging active metabo-

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