Mutagenesis, Vol. 15, No. 3, 223-228,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Protection against Trp-P-2 mutagenicity by purpurin: mechanism of in vitro antimutagenesis
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima naka 1-1-1, Okayama 700 8530, Japan
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Abstract |
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Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a natural pigment isolated from madder root (Rubia tinctorum) which inhibits the mutagenicity of a number of heterocyclic amines in the Ames mutagenicity test. Two effects were observed in the presence of purpurin. The rate of degradation of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NHOH)] at neutral pH was increased. The major product of this purpurin-dependent degradation was identified as the parent amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Secondly, the rate of Trp-P-2 N-hydroxylation, the major route of bioactivation, by PCB-treated rat hepatic microsomes was markedly decreased. Cytochrome P450-dependent O-dealkylation of methoxy-, ethoxy- and pentoxyresorufin by these microsomes was also significantly inhibited by purpurin. The nature of this inhibition was competitive. Spectrophotometric investigations suggest no direct interaction between Trp-P-2 and purpurin. Furthermore, no evidence for Trp-P-2 binding was observed with carminic acid, a structural analog of purpurin, when it was immobilized on
-aminohexyl agarose. Therefore, in vitro the proposed mechanism by which purpurin protects against heterocyclic amine-induced mutagenesis involves competitive inhibition of cytochrome P450-dependent bioactivation and accelerated degradation of the N-hydroxylamine to the parent amine. |
Introduction |
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The cooking of meat and fish forms bacterial mutagens (Sugimura, 1982
), which have been identified as rodent carcinogens (Sugimura, 1985) and are suspected human carcinogens (Weisburger, 1993; Adamson et al., 1995; Schut and Snyderwine, 1999). These mutagens have been identified as heterocyclic amines (reviewed in Eisenbrand and Tang, 1993; Skog, 1993). Heterocyclic amines undergo bioactivation catalyzed mainly by the cytochromes P450 (CYP) (Guengerich et al., 1995, and references therein) but also by prostaglandin H synthase (Petry et al., 1986), an uncharacterized NADH-dependant microsomal oxygenase (Marczylo and Ioannides, 1999) and NADPH cytochrome P450 reductase (CYP reductase) (Maeda et al., 1995). However, the major route of bioactivation is via N-hydroxylation by the hepatic CYP1A isoforms, particularly CYP1A2.
Exposure to these environmental carcinogens is largely unavoidable (Sugimura and Sato, 1983). Therefore interest has recently focused upon chemoprevention. Epidemiological studies have implicated many dietary factors as possible chemopreventive agents. There are various protective mechanisms for these agents. Many act by modulating the metabolism of procarcinogens to decrease formation of the ultimate carcinogens. In contrast, chlorophyllin acts as a chemopreventive agent by complex formation with aromatic carcinogens, possibly preventing their absorption from the gut (Arimoto et al., 1993).
Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) (Figure 1A) is a pigment isolated from madder root (Rubia tinctorum). It is non-mutagenic in the majority of Ames tester strains in the presence and absence of S9 (Westendorf et al., 1990; Kawasaki et al., 1992) and only weakly mutagenic in TA1537 (Westendorf et al., 1990). We have previously investigated the effect of this pigment on heterocyclic amine-induced mutagenicity in the Ames test (Marczylo et al., 1999) and found it to be an efficient inhibitor of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NHOH)]-induced mutations comparable with the recognized chemopreventive agents chlorophyllin and (–)epigallocatechin gallate (Marczylo et al., 1999). In both the presence and absence of rat hepatic S9, incubation is required for purpurin to be antimutagenic (Marczylo et al., 1999). A decrease in Trp-P-2(NHOH) mutagenicity in the absence of S9 implies that this decrease is a result of some direct interaction between the mutagen and purpurin. The mutagenic response to 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) of Salmonella typhimurium TA1538ARO, a strain expressing recombinant human CYP1A2, CYP reductase and bacterial O-acetyltransferase, was also markedly decreased after preincubation of the bacterial culture with purpurin (Marczylo et al., 1999). This decrease in mutagenic response was accompanied by a decrease in expression of CYP1A2 and CYP reductase (Marczylo et al., 1999), implying that purpurin inhibits the bioactivation of Trp-P-2 by CYP1A2. Unlike other N-hydroxylated heterocyclic amines, acetylation of Trp-P-2(NHOH) does not increase the mutagenicity (Nagao et al., 1983; Saito et al., 1983a; Wild et al., 1995; Marczylo et al., 1999). Purpurin inhibits Trp-P-2(NHOH) mutagenicity to the same extent in TA98 and O-acetyltransferase-deficient TA98-1,8-DNP6 (Marczylo et al., 1999). Therefore inhibition of O-acetyltransferase is probably not involved in the antimutagenic mechanism of purpurin.
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Here we have investigated the mechanism of antimutagenesis in vitro, using Trp-P-2 and Trp-P-2(NHOH). The effect of purpurin upon the rate of Trp-P-2(NHOH) degradation, the N-hydroxylation of Trp-P-2 by hepatic microsomes and the dealkylation of alkoxyresorufins by CYP-dependent O-dealkylases have been studied. We have also explored possible complex formation between Trp-P-2 and purpurin.
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Materials and methods |
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Chemicals
Reagent grade (86% pure as determined by HPLC) purpurin and Trp-P-2 were purchased from Wako Pure Chemicals (Osaka, Japan). Ethoxyresorufin, methoxyresorufin, pentoxyresorufin, resorufin, cytochrome c, cysteamine, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid (CPTA) and
-aminohexyl agarose were obtained from Sigma-Aldrich (Tokyo, Japan). Pure purpurin and carminic acid (100% pure as determined by HPLC) were kind gifts from The San-Ei Gen Foundation for Food Chemical Research (Osaka, Japan). NADPH and PCB-54 (a commercial mixture of polychlorinated biphenyls) were purchased from Oriental Yeast Co. (Tokyo, Japan) and Tokyo Kasei Chemical Co. (Tokyo, Japan), respectively. Trp-P-2(NHOH), 3-nitro-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NO2)] and 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NO)] were synthesized from Trp-P-2 according to the literature (Saito et al., 1983b).
Animals and treatment
Adult male rats were pretreated with a single dose of PCB-54 (500 mg/kg i.p.) and hepatic post-mitochondrial fraction (S9) prepared as described previously (Ames et al., 1975) and stored at –80°C. Microsomal fractions were prepared on the day of use from the stored S9 by centrifugation at 109 000 g for 60 min.
Spectrophotometric analysis of the degradation of Trp-P-2(NHOH)
Trp-P-2(NHOH) (10 nmol) was added to 1 ml of 0.1 M potassium phosphate buffer, pH 7.4, in a quartz cuvette containing either 40 nmol purpurin dissolved in DMSO or DMSO only. The absorbance was measured immediately between 200 and 500 nm and at regular time intervals thereafter. The absorbances of Trp-P-2(NHOH) at 258 nm were recorded.
HPLC
In a final volume of 1.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, 6 nmol Trp-P-2(NHOH) and either 60 nmol purpurin dissolved in DMSO or solvent only were incubated in a shaking water bath at 37°C. At time intervals 20 µl aliquots were removed and analyzed. Resolution of Trp-P-2 and Trp-P-2(NHOH) was achieved using an inertsil ODS column (4.6 mm i.d.x250 mm) purchased from G.L. Sciences Inc. (Tokyo, Japan) at 25°C with an isocratic solvent system consisting of 15 mM ammonium dihydrogen phosphate, pH 5, acetonitrile (80:20) at a flow rate of 1 ml/min in a Waters 600 model HPLC. Eluent was monitored at 258 nm using a Waters model 481 LC spectrophotometer. Incubations were repeated under reducing conditions by the incorporation of 3 mM cysteamine.
For metabolism studies, 50 µl of PCB-induced rat liver microsomes (25% w/v), 25 nmol Trp-P-2 and 12.5 µmol NADPH in the presence of either 25 nmol purpurin or solvent alone were made up to a final volume of 0.5 ml with 0.1 M potassium phosphate buffer, pH 7.4, and incubated at 37°C. Aliquots (10 µl) were taken at regular time intervals. Protein was precipitated with an equal volume of ice-cold acetonitrile and the supernatant analyzed, after centrifugation at 4°C, as described above.
Assays for cytochrome P450 and NADPH cytochrome P450 reductase
The O-dealkylation of alkoxyresorufins was employed as a measure of CYP isoform activities. The formation of resorufin was followed fluorometrically as described previously (Burke and Mayer, 1974; Lubet et al., 1985). NADPH cytochrome P450 reductase was assayed by measuring the reduction of cytochrome c spectrophotometrically as described previously by Phillips and Langdon (1962). Direct reduction of 30 µM cytochrome c by 0–60 µM purpurin was also investigated in the absence of microsomal preparation or NADPH.
Spectrophotometric investigation of the interaction of Trp-P-2 with purpurin
A possible direct chemical interaction between Trp-P-2 and purpurin was investigated using mixtures of 5 mM Trp-P-2 and 0, 2.5, 5 or 10 mM purpurin in 0.1 M potassium phosphate buffer, pH 7.4. The solutions were mixed and incubated at room temperature for 30 min before the absorbance was measured between 350 and 650 nm.
Synthesis of -aminohexyl agarose-bound carminic acid and interaction with Trp-P-2
-Aminohexyl agarose slurry (2 ml), containing 5 µmol diaminohexane immobilized on 4% beaded agarose, was washed three times with dioxane solution (50%) then resuspended in 8 ml of 50% dioxane. Carminic acid (25 mg) was added followed by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (84 mg). The mixture was incubated at room temperature in the dark for 24 h with gentle shaking. After incubation the agarose was washed successively with dioxane solution (50%), dioxane, distilled water and ethanol. The residual ethanol was evaporated by spreading out the solid on a sheet of filter paper. The amount of bound carminic acid was estimated by complex formation with CPTA as follows. A preweighed amount of carminic acid-adducted -aminohexyl agarose was incubated, with constant mixing, at room temperature with 1 ml CPTA solution (100 nmol/ml) and the decrease in absorbance at 630 nm due to complex formation was determined. The decrease in absorbance observed with underivatized -aminohexyl agarose was subtracted before estimating the concentration of CPTA removed per mg of material from the standard curve.
To investigate the binding of Trp-P-2 to carminic acid, preweighed amounts of -aminohexyl agarose and carminic acid-adducted -aminohexyl agarose were incubated in Trp-P-2 solutions for 30 min with vigorous shaking at room temperature. The reaction was stopped by centrifugation and the supernatant collected and analyzed for Trp-P-2 content by UV spectrophotometry. The pellet was washed with distilled water then incubated with 2 ml of 1 M acetic acid for 30 min with vigorous shaking to liberate any bound Trp-P-2. The Trp-P-2 was then separated from the medium by centrifugation and the supernatant analyzed for the presence of Trp-P-2 by spectrophotometry.
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Results |
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Effect upon Trp-P-2(NHOH) degradation
Trp-P-2(NHOH) is unstable at neutral pH and its degradation can be followed at 258 nm. In the absence of purpurin this absorbance decreased by 47% over 60 min (Figure 2
). In the presence of a 4-fold excess of purpurin this rate of degradation was consistently greater. After 60 min, the absorbance of Trp-P-2(NHOH) had decreased by 68%. When this experiment was performed with equimolar (40 nmol) concentrations of Trp-P-2(NHOH) and purpurin, a similar, but less marked, effect was observed. After 90 min, Trp-P-2(NHOH) was degraded by 50 and 42%, respectively, in the presence and absence of purpurin (data not shown).
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) and absence (
) of purpurin. This experiment was repeated on three occasions with similar results.Degradation of Trp-P-2(NHOH) to Trp-P-2 in the presence of purpurin
The degradation of Trp-P-2(NHOH) was followed using HPLC. After incubation in the presence of purpurin three peaks were observed with retention times of 11, 14 and 17 min, with the peak at 14 min representing unmodified Trp-P-2(NHOH). Both the degradation of Trp-P-2(NHOH) and appearance of the two products were time dependent (Figure 3
). The major product (17 min) was identified as Trp-P-2 by co-elution with authentic standards on HPLC and by determination of the UV spectrum. A minor peak was also found, with a retention time of 11 min, which co-eluted with authentic Trp-P-2(NO2) and Trp-P-2(NO). When degradation of Trp-P-2(NHOH) was compared in the presence and absence of purpurin, marked differences were observed. As expected, the rate of degradation of the N-hydroxylamine was greater in the presence of purpurin (Figure 4A). Furthermore, no amine was formed in the absence of purpurin (Figure 4B). When Trp-P-2(NHOH) and purpurin were incubated together at 37°C for 20 min in buffer containing a 3 mM final concentration of cysteamine no significant degradation of Trp-P-2(NHOH) or formation of Trp-P-2 were observed for the first 20 min (data not shown). This suggests that formation of Trp-P-2 is a redox process (Wakata et al., 1985; Hiramoto et al., 1988).
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) in the presence of purpurin. These are typical results of HPLC analyses from several experiments.
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Inhibition of microsomal N-hydroxylation by purpurin
Trp-P-2(NHOH) was formed after incubation of 25 nmol Trp-P-2 with PCB-induced microsomes and NADPH in both the presence and absence of 25 nmol purpurin (Figure 5
). However, the rate of formation of Trp-P-2(NHOH) was significantly lower in the presence of purpurin (Figure 5).
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Effect of purpurin upon cytochrome P450-dependent dealkylation
Purpurin dose-dependently inhibited CYP-dependent O-dealkylation of methoxy-, ethoxy- and pentoxyresorufin (Figure 6A
). This inhibition was confirmed using pure purpurin (Figure 6B). Purpurin had no direct effect upon the fluorescence of resorufin at the concentrations employed (data not shown).
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When ethoxy- and methoxyresorufin O-dealkylases were investigated at increasing substrate concentrations, the inhibition by purpurin was not removed by increasing the substrate concentrations. However, analyses of these data by Lineweaver–Burke plots suggests that purpurin is a competitive inhibitor of the enzymes responsible (Figure 7
).
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Reduction of cytochrome c by purpurin
Initially purpurin appeared to modestly increase the activity of CYP reductase as determined by the reduction of cytochrome c (increased from 3.56 to 5.51 nmol/min in the presence of 166 µM purpurin). However, the addition of purpurin to oxidized cytochrome c in the absence of microsomal fraction demonstrated low level but dose-dependent `reductase-like' activity (Figure 8
).
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Direct interaction of Trp-P-2 with purpurin
Addition of increasing concentrations of purpurin to Trp-P-2 was not responsible for a significant change in the Trp-P-2 absorbance spectrum (data not shown). No new peaks or major shifts in peak wavelength were observed.
Synthesis of carminic acid–aminohexyl agarose and absence of binding with Trp-P-2
Carminic acid, an analog of purpurin (Figure 1B
) bearing carboxylic acid substituents, is expected to form amide linkages with an alkylamine upon condensation. Indeed, a stable, highly colored product was obtained by the reaction of -aminohexyl agarose with carminic acid in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. Complex formation between carminic acid and CPTA has been demonstrated previously (Hayatsu, 1992). Consequently, the amount of adducted carminic acid was estimated by complex formation with CPTA and calculated to be 5 nmol/mg. This is an estimate assuming 100% complex formation between CPTA and bound carminic acid. Incubation of 10 nmol Trp-P-2 with 5 or 50 mg of this medium, containing ~25 or 250 nmol carminic acid, for 30 min with vigorous shaking at room temperature did not decrease the amount of Trp-P-2 in solution as determined by absorbance at 260 nm. Furthermore, incubation of the washed material in 1 M acetic acid for 30 min with vigorous shaking did not liberate any mutagen (data not shown).
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Discussion |
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Purpurin is one of several hydroxyanthraquinone constituents found in madder root (Kawasaki et al., 1992
), extracts of which are used both as herbal remedies for bladder and kidney stones (Gebhardt, 1979) and as food colorings. Previously, we have demonstrated that purpurin is protective against a number of food-derived heterocyclic amines in bacterial mutagenicity assays (Marczylo et al., 1999). Here we demonstrate that the antimutagenesis is based upon inhibition of CYP-dependent N-hydroxylation (Figures 5 and 6) and direct chemical reduction of the N-hydroxylamine mutagen back to the non-directly mutagenic amine (Figure 4). No evidence could be found for chlorophyllin-like complex formation between purpurin and Trp-P-2.
At neutral pH, N-hydroxylated heterocyclic amines are unstable and degrade to nitroso derivatives (Arimoto-Kobayashi et al., 1998). An increase in the rate of this degradation has been demonstrated previously with hemin and myoglobin (Arimoto et al., 1987; Arimoto and Hayatsu, 1989), chlorophyllin (Arimoto-Kobayashi et al., 1998) and Monascus pigments (Izawa et al., 1997). Purpurin is also responsible for an increased rate of Trp-P-2(NHOH) degradation (Figure 2) and here we demonstrate that this increase is a result of reduction of the N-hydroxylamine to the amine (Figures 3 and 4). This effect is protective because the amine is not directly mutagenic. However, the presence of drug metabolizing enzymes such as CYP1A2 can reverse this reaction, leading to regeneration of the proximate carcinogen. In agreement with this hypothesis, we have demonstrated previously that the antimutagenic effect of purpurin is less marked in the presence of S9 mix (Marczylo et al., 1999). Purpurin is antimutagenic against Trp-P-2 in Salmonella typhimurium TA1538ARO, a strain expressing human CYP1A2, human CYP reductase and bacterial O-acetyltransferase, both in the presence of Trp-P-2 and in a two-stage test where bacteria were harvested and washed after incubation with purpurin before they were used in Ames tests with Trp-P-2 (Marczylo et al., 1999). These results imply that purpurin also inhibits CYP. To investigate this hypothesis further, we looked at the inhibition of Trp-P-2 N-hydroxylation, a reaction mainly performed by CYP1A isoforms (Guengerich et al., 1995), and the dealkylation of ethoxy-, methoxy- and pentoxyresorufin, marker substrates for CYP1A1, CYP1A2 and CYP2B1, respectively (Burke et al., 1994). In each case purpurin was an effective inhibitor of CYP-dependent metabolism (Figures 5 and 6). Therefore, the antimutagenic potential of purpurin is a consequence of inhibition of bioactivation by CYP in addition to degradation of the active metabolite. Modification of CYP has been implicated previously in the mechanism of action of chemopreventive agents (Alldrick et al., 1989; Guengerich and Kim, 1990; Maltzman et al., 1991; Yun et al., 1995; Hecht, 1997; Manson et al., 1998). This mechanism of action has also met with considerable skepticism (Paolini et al., 1995, 1997, 1998) because of the essential endogenous metabolic processes performed by CYP (e.g. in steroid and fatty acid metabolisms). Therefore, we further investigated the mechanism of inhibition. Lineweaver–Burke plots were generated for ethoxy- and methoxyresorufin O-dealkylases in the presence (0.5 and 0.25 mM) and absence of purpurin (Figure 7). The y-axis intercepts are comparable in the presence and absence of purpurin, demonstrating that 1/Vmax is unchanged. Therefore, the mechanism of inhibition is competitive, i.e. purpurin competes with substrate for the active site of CYP. Previously we have demonstrated that purpurin is an inhibitor of human CYP reductase when it is expressed in Salmonella typhimurium TA1538ARO (Marczylo et al., 1999). When we tried to measure this inhibition using PCB-induced rat hepatic microsomes, we found that the rate of reduction of cytochrome c was increased from 3.56 to 5.51 nmol/min. Further investigation in the absence of microsomal protein showed that this increase was due to direct reduction of cytochrome c by purpurin (Figure 8). This was not observed in experiments involving TA1538ARO because the majority of the purpurin had been removed when the bacteria were washed after preincubation with purpurin. Therefore it is possible that purpurin further interferes with CYP-dependent metabolism by blocking the transfer of electrons to CYP.
If inhibition of CYP reductase is the underlying mechanism of purpurin action, two aspects need to be considered. First, this mechanism would inhibit all CYP isoforms, including those concerned with endogenous metabolism. Consequently, any consideration of future use in chemoprevention would require caution due to the risk of side-effects. Second, direct reduction of cytochrome c may be indicative of a free radical generating compound. This latter possibility may explain the tumorigenicity of purpurin in the rat (Mori et al., 1996). When male F344 rats were fed purpurin at 1% of diet for 520 days an increased incidence of `progressive chronic nephropathies', hyperplasia of pelvic epithelium and urinary bladder tumors (papilloma and carcinoma) were observed (Mori et al., 1996). However, the insolubility of purpurin in aqueous and especially acidic conditions coupled with the weakly acidic urine in this species were responsible for crystallization in the target tissues. There was a good correlation between incidences of crystallization and tumorigenesis (Mori et al., 1996). Furthermore we have shown that purpurin is non-mutagenic, and therefore not bioactivated by CYP reductase, in Salmonella strains expressing human CYP reductase (TA1538R), human CYP reductase and human CYP1A2 (TA1538AR) and human CYP1A2, CYP reductase and bacterial O-acetyltransferase (TA1538ARO) (Marczylo et al., 1999). In addition, purpurin is not mutagenic in TA102, a strain sensitive to free radicals, either with or without S9 (Westendorf et al.,1990).
Chlorophyllin acts as a chemopreventive agent by complex formation with aromatic mutagens (Arimoto-Kobayashi et al., 1997). In the presence of chlorophyllin the rate of Trp-P-2(NHOH) degradation is also increased, an observation similar to that observed here with purpurin (Figure 2). Therefore, the possibility of complex formation between purpurin and Trp-P-2 was investigated. Purpurin has no suitable functional groups which can be utilized for the attachment to solid media. Therefore, we used an analog of purpurin for these investigations. Carminic acid (Figure 1B) was immobilized on -aminohexyl agarose, giving a stable highly colored medium. Incubation of this medium in a solution of Trp-P-2 was neither responsible for any decrease in Trp-P-2 concentration nor could any Trp-P-2 be recovered by treatment of the washed medium with acid (data not shown). Consequently, it can be concluded that no complex formation occurs between Trp-P-2 and carminic acid and it is implied that no stable complex formation is involved in the antimutagenicity of purpurin. Further evidence against complex formation comes from the UV spectrum of Trp-P-2 in the presence of purpurin, where no significant decrease in absorbance at 500 nm was observed.
Purpurin is an effective antimutagenic agent against Trp-P-2 in in vitro mutagenicity assays because it reduces the N-hydroxylamine to the non-mutagenic amine and competitively inhibits bioactivation by CYP1A. Non-specific inhibition of CYPs may be a result of CYP reductase inhibition. The consequences of CYP inhibition related to essential metabolic processes suggest that future consideration of purpurin as a chemopreventive agent will require careful monitoring of side-effects relating to steroid metabolism and other CYP-dependent processes.
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Acknowledgments |
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The authors would like to express their thanks to the Japanese Society for the Promotion of Science (JSPS) for their generous stipend to one of them (T.M) and to The Ministry of Education, Science, Sports and Culture, Japan, for funding the research through a Grant-in-Aid for JSPS Fellows (97439) and for Scientific Research on Priority Areas (A) (09253104). This work was supported by a Grant-in-Aid for Cancer Research (8-5) from the Ministry of Health and Welfare, Japan. We thank the San-Ei Gen Foundation for Food Chemical Research, Osaka, for funding and gifts of reagents.
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Notes |
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* To whom correspondence should be addressed. Present address: Edward Llwyd Building, University of Wales, Aberystwyth SY23 3DA, UK; Tel: +44 1970 621515; Fax: +44 1970 622350; Email: tom{at}aber.ac.uk
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Received on September 27, 1999; accepted on December 22, 1999.
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