Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

Cynthia Agus1,2, Kenneth F. Ilett2,4, Fred F. Kadlubar3 and Rodney F. Minchin1,2,5

1 Laboratory for Cancer Medicine, Royal Perth Hospital, Perth 6000, Australia,
2 Department of Pharmacology, University of Western Australia, Nedlands 6907, Australia,
3 National Center for Toxicological Research, Jefferson, AR 72079, USA and
4 Clinical Pharmacology and Toxicology Laboratory, Western Australian Centre for Pathology and Medical Research, Nedlands 6009, Australia


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [3H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 ± 0.3 and 0.9 ± 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and ~50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein. The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Abbreviations: DMSO, dimethyl sulphoxide; DTT, dithiothreitol; GSH, reduced glutathione; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; N-OH-IQ, N-hydroxy-IQ; N-OH-PhIP, N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine; PKC, protein kinase C; THF, tetrahydrofuran.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many mutagenic and carcinogenic heterocyclic amines are formed during cooking of protein-rich foods. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is amongst the most mutagenic of these compounds. In rodents IQ is capable of inducing tumours in tissues such as liver, small and large intestine, lung, pancreas and mammary gland (14), while in non-human primates it is a potent hepatocarcinogen (57). In addition to tumour formation, prolonged exposure to IQ results in cardiovascular changes in non-human primates (8,9) and in rodents (10). Human dietary exposure to heterocyclic amines ranges from 0.1 to 10 µg/person/day (1113) and the presence of these compounds and their metabolites has been detected in human urine, indicating that they are absorbed from the diet (14).

Like most chemical carcinogens, IQ requires metabolic activation to exert its genotoxic effects. Initially, IQ is metabolized by CYP1A2 (15) to N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ), which has mild alkylating activity for cellular macromolecules. However, subsequent esterification by phase II enzymes (O-acetyltransferase and sulphotransferase) generates highly reactive species capable of forming adducts with DNA and cellular proteins (16,17). The O-acetoxy and O-sulphate esters are hypothesized to rearrange rapidly to form a highly reactive nitrenium ion intermediate (1820) that is capable of attacking nucleophilic centres on DNA and, in particular, C8 and N2 of guanine.

DNA adducts are found in a variety of rodent and non-human primate tissues following single or multiple doses of IQ (6,9,21,22). Whether the reactive intermediates are formed in the liver and transported to extrahepatic tissues or whether they are formed in the latter has not been established. Moreover, the relative contributions of the different phase II pathways has not been determined. However, high adduct levels have been found in tissues, such as the pancreas and heart, where the levels of acetyltransferases and sulphotransferases are low. These data suggest that an alternative activation pathway may be important in some tissues. Previous studies have reported that some heterocyclic amines could be activated by ATP (16,17,23). Many of the individual enzymes involved in metabolic activation and detoxification of carcinogens are genetically variant or highly inducible. Therefore, understanding the various activation pathways that can participate in heterocyclic amine carcinogenesis is important for determining risk factors that may contribute to human cancers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
[5-3H]IQ (sp. act. 62.3 mCi/mmol) and [G-3H]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([3H]PhIP) (sp. act. 100 mCi/mmol) were purchased from Chemsyn Science Laboratories (Lenexa, KS). Rottlerin, staurosporine, tyrphostin A25 and wortmannin were purchased from Calbiochem (La Jolla, CA). Genistein, daidzein, calf thymus DNA, L-ascorbic acid, ATP, dithiothreitol (DTT), DNase I from bovine pancreas and 3'-phosphoadenosine 5'-phosphosulphate (PAPS) were obtained from Sigma Chemical Co. (St Louis, MO). Acetyl coenzyme A (AcCoA) and reduced glutathione (GSH) were obtained from Boehringer Mannheim (Castle Hill, NSW, Australia) while [{gamma}-32P]ATP (4000 Ci/mmol) and [{alpha}-32P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech (Castle Hill, NSW, Australia). Phenol was purchased from BDH Chemicals (Poole, UK). C18 Sep-Pak® Plus cartridges were purchased from the Waters Division of Millipore (Milford, MA) while Bio-Gel® P-6 DG desalting gel was from Bio-Rad (Hercules, CA). All other chemicals were of analytical or HPLC grade.

Synthesis of N-OH-IQ
[3H]N-OH-IQ was synthesized by conversion of [3H]IQ to [3H]2-nitro-3-methylimidazo[4,5-f]quinoline ([3H]NO2-IQ) followed by reduction to [3H]N-OH-IQ. Briefly, 45 µCi [3H]IQ was dissolved in 140 µl dimethylformamide and 150 µl glacial acetic acid was added. This mixture was then added dropwise to 200 µl of 6.8 M sodium nitrite and reacted at room temperature for 30 min. The reaction mixture was diluted with an equal volume of water and loaded onto a Waters C18 Sep-Pak® Plus cartridge pre-equilibrated with methanol followed by water. The column was washed with 5 ml of water and then eluted with 0.5 ml of ethanol and evaporated to dryness under argon (recovery of [3H]NO2-IQ was 50–60%). The [3H] NO2-IQ was dissolved in 100 µl of 1 M ascorbic acid in dimethyl sulphoxide (DMSO), 20 µl of 0.3 M copper sulphate was added and the reaction allowed to proceed for 8 min at room temperature. After the addition of 1 ml of 0.1 mM EDTA, the mixture was loaded onto a Waters C18 Sep-Pak® Plus cartridge, washed with 5 ml of water and eluted with 0.5 ml of DMSO:ethanol (4:1). Typical yields for [3H]N-OH-IQ were ~50–60%. The purity of the [3H]N-OH-IQ was determined by HPLC using a method modified from Snyderwine et al. (24). The system used a C8 column (Ultrasphere, 5 µm, 250x4.6 mm; Beckman) and a mobile phase (5% ammonium acetate, pH 5.85, methanol, 70:30) pumped at 1.2 ml/min. Eluting compounds were detected by their UV absorbance at 254 nm. Retention times for N-OH-IQ and IQ were ~11.2 and 14.8 min, respectively. Typical purity for the [3H]N-OH-IQ was >95%.

Synthesis of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP)
Synthesis of [3H]N-OH-PhIP was similar to that of [3H]N-OH IQ. Conversion of [3H]PhIP to 3H-labelled 2-nitro-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine ([3H]NO2-PhIP) was as described above, followed by reduction to [3H]N-OH-PhIP. Typical yields for this reaction were ~35%. Approximately 800 µCi of [3H]NO2-PhIP was dissolved in 0.5 ml of tetrahydrofuran (THF) and placed in an ice–salt bath for 5 min followed by addition of 1.5 mg palladium on charcoal. Hydrazine (15 µl) was dissolved in 0.25 ml of THF, which was then added dropwise to the reaction mixture. This was allowed to react in the ice bath for 15 min and stopped by the addition of 20 ml of 0.1 mM EDTA. It was then loaded onto a Waters C18 Sep-Pak® Plus cartridge pre-equilibrated with methanol and then water, washed with 20 ml of 0.1 mM EDTA and eluted in 2 ml DMSO:ethanol (4:1). The yield for this reaction was ~86% and a purity of >95% was determined by HPLC.

Preparation of rat tissue cytosols
Male Fischer 344 rats were obtained from the Animal Resources Centre (Murdoch, Western Australia). Animals were killed and tissues (liver, heart, kidney, lung, colon, duodenum and pancreas) removed and washed with ice-cold homogenization buffer containing 20 mM Tris–HCl buffer, pH 7.4, 1 mM EDTA, 1 mM DTT and 1.15% KCl. Differential centrifugation in 0.34 M sucrose was used to isolate nuclei (700 g for 10 min) and mitochondria (5000 g for 10 min) as described previously (25). Cytosolic and microsomal fractions from each tissue were prepared by differential centrifugation. Where indicated, cytosolic fractions were desalted on a Bio-Gel® P6-DG desalting gel. Protein concentrations were determined by the Bradford method (26) using bovine serum albumin as the standard.

DNA binding assays
ATP-dependent activation of [3H]N-OH-IQ was investigated using a DNA binding assay. The reaction mixture (total volume 0.5 ml) consisted of 1 mg desalted cytosolic protein, 0.5 mg DNA, 7.3 µM [3H]N-OH-IQ, 1 mM ATP, 3 mM magnesium acetate and 1 mM DTT in 50 mM N,N,-bis (2-hydroxyethyl)glycine buffer, pH 7.4. Reactions were started by addition of [3H]N-OH-IQ and incubated at 37°C typically for 7.5 min. Some reactions were incubated for up to 90 min. Incubations were terminated by the addition of 1.5 ml of reaction buffer and 3 ml of water-saturated n-butanol and vortexed vigorously. Modified DNA was then extracted as previously described (27). DNA was assayed fluorometrically (28) to determine recovery, followed by digestion with DNase I (0.2 mg/mg DNA) at 37°C for 30 min. Samples were then subjected to liquid scintillation spectroscopy to determine the extent of covalent binding.

HPLC analysis of the N-OH-IQ metabolite(s) formed in vitro
Reaction mixtures (see above) were subjected to HPLC analysis. The reactions were allowed to proceed at 37°C for 60 min unless otherwise specified. An aliquot (40 µl) of this was analysed by HPLC using the system described above. The eluate from the column was collected in 0.5 min fractions and radioactivity in each fraction was quantified by liquid scintillation spectroscopy. The recovery of radioactivity from the HPLC column was >95%.

Effect of kinase inhibitors on DNA binding and metabolite formation in vitro
A number of inhibitors were used to investigate whether the enzyme(s) responsible for ATP-dependent DNA binding and/or metabolite formation belonged to any of the typical kinase families. Genistein (a tyrosine kinase inhibitor), staurosporine (a broad spectrum kinase inhibitor), wortmannin (a phosphatidylinositol 3-kinase inhibitor), tyrphostin A25 (a tyrosine kinase inhibitor), rottlerin [a protein kinase C (PKC) inhibitor] and bisindolylmalemide (a PKC inhibitor) were used. DNA binding and metabolite formation in the absence and presence of these inhibitors were analysed as described above.


    Results
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 Materials and methods
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Tissue and subcellular distribution of ATP-dependent N-OH-IQ DNA binding
ATP-dependent DNA binding of N-OH-IQ in various subcellular fractions from liver showed that the majority of activity was present in the cytosol (Figure 1AGo). When this fraction was chromatographed on P6 gel (mol. wt cut-off 6000), activity increased 5-fold, indicating that rat liver cytosol contains low molecular weight inhibitors of N-OH-IQ covalent binding to DNA. Inactivation of the cytosol by heating at 95°C for 5 min abolished ATP-dependent DNA binding, suggesting that the reaction was enzymatic. Furthermore, the reaction displayed a pH dependency, with maximum DNA binding observed at pH 7 (data not shown). Neither IQ nor NO2-IQ could be activated by ATP to produce DNA-binding species. We also investigated ATP-dependent activation in other tissues (Figure 1BGo) and found significant activity in cytosols isolated from colon, pancreas and lung, although at levels much lower than that seen in liver.



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Fig. 1. Subcellular (A) and tissue (B) distribution of ATP-dependent DNA binding in the rat. Incubations were in the absence ({square}) and presence ({blacksquare}) of 1 mM ATP. For subcellular distribution, cytosolic fractions were not desalted prior to incubation. Data are presented as means ± SEM (n = 3). *Significantly higher than control incubations (P < 0.05).

 
Since previous studies have shown that reactive intermediates from the bioactivation of mutagens such as 2-aminofluorene (29), 1-nitropyrene (30) and N-methyl-4-aminoazobenzene (31) form conjugates with GSH, we investigated whether GSH might affect N-OH-IQ binding. Figure 2Go shows the GSH concentration-dependent inhibition of covalent binding, with an IC50 value between 1 and 3 mM, suggesting that GSH either conjugates with N-OH-IQ and/or its metabolic intermediates or that it reduces the reactive intermediate to a less reactive product.



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Fig. 2. Effect of GSH on ATP-dependent DNA binding in the absence ({circ}) and presence (•) of 1 mM ATP. Data are presented as means ± SEM (n = 3).

 
Kinetics for ATP-dependent N-OH-IQ activation
To further characterize ATP-dependent activation of N-OH-IQ, both time and concentration studies were performed. At a substrate concentration of 7.3 µM and a cofactor concentration of 1 mM, the reaction proceeded in a pseudo-linear manner for ~10 min and was essentially completed by 25 min (Figure 3AGo). A concentration–DNA binding curve showed saturation in the low micromolar range when the cofactor concentration was 1 mM (Figure 3BGo). From an Eadie–Hofstee plot of the data (Figure 3BGo, insert), the estimated maximum rate of reaction was 3.33 pmol/mg protein/min and the Km was 0.97 µM.



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Fig. 3. (A) ATP-dependent binding of 7.3 µM [3H]N-OH-IQ to DNA with respect to time. Incubations were carried out in the absence ({circ}) and presence (•) of 1 mM ATP. (B) Concentration–DNA binding curve in the presence of 1 mM ATP. From an Eadie–Hofstee plot of the data the estimated Vmax was 3.33 pmol/mg protein/min while the Km was 0.97 µM (insert). Data are presented as means ± SEM (n = 3).

 
Comparison with acetylation and sulphation
Recent studies suggest that acetylation is the major pathway for activation of fused 3-ring heterocyclic amines such as IQ, while sulphation is more important for 2-ring heterocyclic amines such as PhIP (32). We compared the ability of ATP to activate both N-OH-IQ and N-OH-PhIP under the same conditions (Table IGo). In addition, AcCoA and PAPS were used to investigate the activation of each substrate by acetylation and sulphation, respectively. Approximate physiological concentrations of cofactors were used: 1 mM ATP (33), 50 µM AcCoA (34) and 0.2 mM PAPS (35). Under these conditions, both N-OH-IQ and N-OH-PhIP were activated by all three cofactors, although N-OH-IQ resulted in consistently higher DNA binding. Moreover, binding in the absence of cofactors was greater with N-OH-IQ, indicating that it is more reactive than N-OH-PhIP.


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Table I. ATP-dependent DNA binding of N-OH-IQ and N-OH-PhIP
 
Effect of various kinase inhibitors on ATP-dependent DNA binding
The effect of a number of kinase inhibitors on N-OH-IQ activation was investigated. Genistein and tyrphostin A25, both tyrosine kinase inhibitors, inhibited ATP-dependent DNA binding by 86 and 98%, respectively (Table IIGo). Daidzein, an inactive analogue of genistein, inhibited DNA binding with similar potency to genistein. Rottlerin, a PKC inhibitor, also inhibited ATP-dependent DNA binding by 96%. In contrast, staurosporine, a kinase inhibitor with broad specificity, failed to inhibit DNA binding. Similar negative results were found with the phosphatidylinositol 3-kinase inhibitor wortmannin and the PKC inhibitor bisindolylmalemide. These results showed that the covalent binding of the N-OH-IQ intermediate(s) could be inhibited by a range of unrelated compounds, suggesting that the inhibition was not due to inactivation of a specific kinase, since staurosporine was ineffective while daidzein was as potent as genistein.


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Table II. Effect of kinase inhibitors on ATP-dependent DNA binding
 
Since the effect of the kinase inhibitors did not appear to be related to their kinase specificity, the ability of genistein, tyrphostin A25 and rottlerin to inhibit the AcCoA-dependent reaction was investigated. All three compounds were able to inhibit AcCoA-dependent covalent binding. However, the IC50 values were much higher in AcCoA-dependent incubations compared with those for ATP. For example, the IC50 for genistein in the presence of AcCoA was 36.8 µM, whereas it was only 2.6 µM in the presence of ATP (Table IIIGo).


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Table III. IC50 values of kinase inhibitors for the ATP- and AcCoA-dependent reactions
 
Formation of N-OH-IQ metabolite(s) in vitro
Reaction mixtures were analysed by HPLC for the presence of N-OH-IQ metabolite(s) formed during the incubation. In the presence of ATP, the major metabolite was IQ, although two stable metabolites (M1 and M2) were generated which were more polar than N-OH-IQ (Figure 4DGo). The retention times for M1, M2, N-OH-IQ and IQ were 3.0, 5.0, 11.2 and 14.8 min, respectively. N-OH-IQ was also evident in incubations without cofactor. Neither M1 nor M2 was formed when IQ or NO2-IQ was used as substrate (not shown). When AcCoA was used as cofactor, only M1 was formed (Figure 4EGo), suggesting that M2 is an ATP-specific metabolite. Incubations using heat-inactivated cytosol resulted in minimal amounts of M1 and M2 produced, suggesting that formation of the metabolites was enzymatic. Similarly, no metabolite was seen in the no cytosol incubations (data not shown).



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Fig. 4. HPLC profiles of [3H]IQ, [3H]N-OH-IQ and its metabolites (M1 and M2). The retention times for [3H]IQ and [3H]N-OH IQ (A and B) were 11.2 and 14.8 min, respectively. The retention times for M1 and M2 were 3.0 and 5.0 min, respectively (D). M1 was present in incubations without co-factor (C) as well as in AcCoA incubations (E), while M2 appears to be an ATP-specific metabolite. When the reaction mixture was evaporated to dryness and reconstituted in water, M1 could no longer be seen in both the ATP (F) and AcCoA incubations, suggesting that it was tritiated water.

 
To determine whether M2 was a phosphate conjugate, [{alpha}-32P]ATP or [{gamma}-32P]ATP was added to the incubation and the products extracted on a C18 Sep-Pak Plus cartridge. After removing unreacted [32P]ATP, the metabolite was eluted with ethanol. However, the radioactivity recovered was not different to that from incubations containing no enzyme. Moreover, HPLC analysis indicated that all of the recovered 32P co-chromatographed with the solvent front, indicating that no 32P was incorporated into M2. Interestingly, when the ATP-dependent reaction mixtures were evaporated under argon and reconstituted in water, M1 disappeared, suggesting that it was tritiated water (Figure 4FGo). This was also seen for AcCoA-dependent reactions (data not shown).

The time-dependent disappearance of N-OH-IQ and formation of the respective metabolites was determined over 60 min (Figure 5Go). The reaction was rapid, with all of the N-OH-IQ being consumed by the end of this time period, although residual amounts of the substrate could be seen in some incubations. The generation of M1 and M2 appeared to occur simultaneously. The amount of IQ, which is a degradation product of N-OH-IQ, increased with time, a phenomenon also seen in the absence of ATP.



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Fig. 5. Time course study for the formation of M1 (•), M2 ({blacksquare}) and IQ ({lozenge}) and the disappearance of N-OH-IQ ({circ}).

 
Effect of kinase inhibitors on ATP-dependent metabolite formation
The effect of various kinase inhibitors on the formation of M1 and M2 was also determined (Table IVGo). The same four compounds that inhibited DNA binding (genistein, daidzein, tyrphostin A25 and rottlerin) also inhibited M1 and M2 formation by up to 90%. In contrast, the broad spectrum kinase inhibitor staurosporine had no effect on either M1 or M2 formation. Neither wortmannin nor bisindolylmalemide had any effect on M1 formation. However, a 35% inhibition of M2 formation was seen with these compounds. The amount of IQ formed in the presence of these inhibitors did not change and no additional metabolites were seen.


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Table IV. Effect of kinase inhibitors on ATP-dependent formation of metabolites
 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies have shown that diet is a major factor in the occurrence of many types of cancers (36), in particular the consumption of cooked meats. Since cooked meats have been shown to contain a number of carcinogenic heterocyclic amines, knowledge of their carcinogenicity may be critical. In this study, activation of the N-hydroxylated food-derived carcinogen N-OH-IQ was investigated in the presence of ATP using a DNA binding assay as the end-point. The highest ATP-dependent DNA binding for the activation of N-OH-IQ was present in the rat liver cytosol. This is in agreement with previous studies where the liver had the highest level of DNA adducts (2123), as well as being a major site of tumour formation (3,5,7). The presence of significantly higher ATP-dependent activity in the colon, pancreas and lung indicates that metabolic activation could occur outside the liver. Moreover, this activity may be responsible for the formation of DNA adducts in these tissues.

GSH was able to inhibit ATP-dependent DNA binding in a dose-dependent manner. However, we could not confirm whether it was the removal of GSH that resulted in the 5-fold increase in activity seen in desalted cytosols. The thiol assay used to measure GSH levels also detected any free thiols present, hence there was little difference between desalted and non-desalted cytosols. However, we were able to show that GSH alone could be removed by the P6 gel.

Comparison of ATP-dependent DNA binding with other known pathways of activation, namely acetylation and sulphation, showed that ATP had a similar capacity for activation of N-OH-IQ. More importantly, we have shown that both N-OH-IQ and N-OH-PhIP could be activated by all three cofactors. However, regardless of the cofactor, N-OH-IQ activation was greater than that of N-OH-PhIP. This is in contrast to previous findings by Felton et al. (32), who reported that 2-ring heterocyclic amines such as PhIP relied on the sulphotransferase pathway for activation. The reactivity of N-OH-IQ found in this study was similar to a previous study by Sabbioni and Wild (20), where the overall adduct formation with IQ was higher than PhIP, possibly due to the high electrophilic reactivity of the IQ metabolite(s).

HPLC analysis of the reactions conducted in the presence of ATP showed two stable metabolites (M1 and M2) which were more polar than N-OH-IQ. The formation of M1 and M2 as well as the degradation product IQ was rapid and complete by ~20 min, which is similar to the kinetics seen for DNA binding. Furthermore, the formation of M1 and M2 appeared to occur simultaneously rather than in series, since neither disappeared over time. The lack of incorporation of 32P into M2 indicated that it was not a phosphate metabolite. The disappearance of M1 in reconstituted ATP- and AcCoA-dependent reactions suggested that it was tritiated water. Previous studies of IQ in rats have shown IQ-sulphamate (37) and IQ-N-glucuronide (38) as major metabolites. Since our system contains no source of UDP-glucuronic acid, it is unlikely that the metabolite(s) was a glucuronide. Similarly, it is unlikely that M2 was a sulphamate metabolite, since desalting of the cytosol would have removed any inorganic sulphate present in the liver cytosol. In addition, desalting had no effect on the amount of M2 produced. Identification of this metabolite is beyond the scope of the present study, but is currently being undertaken by LC-MS methods.

Since the inhibition of both DNA binding and metabolite formation by genistein, daidzein, tyrphostin A25 and rottlerin was not related to the target kinase, it appears that the inhibition did not occur at the enzyme level. Furthermore, it is unlikely that these compounds promoted the degradation of N-OH-IQ, since N-OH-IQ was present at the end of the incubation. An alternative mechanism by which these compounds may inhibit the DNA binding process is by scavenging the reactive intermediate(s). A common feature among the compounds that were inhibitory is the presence of hydroxyl groups, which have been shown to have an antioxidant activity through their reducing properties (39). Inhibition of heterocyclic amine-induced mutagenicity by flavones, flavonols and polyphenols, all of which possess hydroxyl groups, has been demonstrated in several studies (4044), where the inhibition occurred at the CYP 1A1/1A2 level (40,41). In this study we have shown that inhibition of mutagenicity may occur at the phase II stage of the activation pathway, possibly due to scavenging of the reactive intermediate(s).

Genistein, tyrphostin A25 and rottlerin also inhibited the AcCoA-dependent reactions, but with higher IC50 values compared with ATP. These results suggest that either the intermediate formed with AcCoA is not as reactive or that it is different. Genistein and daidzein are naturally occurring isoflavones found in high amounts in soy products (45). Consumption of soy products has been suggested to contribute to lower rates of breast, prostate and colon cancers in Asian countries (46). Since heterocyclic amines are capable of inducing these tumours in animal studies, the potential role of isoflavones as chemopreventive agents in vivo merits further investigation.

In summary, we have shown that activation of N-OH-IQ could be facilitated by ATP, as measured by DNA binding, and that N-OH-IQ appears to be more reactive than N-OH-PhIP. In addition, two stable, as yet inidentified, N-OH-IQ metabolites were generated during this reaction. The lack of specificity of the inhibition of both DNA binding and metabolite formation by kinase inhibitors suggest that they act as scavengers for the reactive intermediate(s) rather than by enzyme inhibition. Although carcinogenesis is a complex phenomenon, activation of heterocyclic amines by ATP may contribute to the overall carcinogenicity of these compounds in vivo.


    Notes
 
5 To whom correspondence should be addressed Email: rminchin{at}receptor.pharm.uwa.edu.au Back


    Acknowledgments
 
This research was supported by a grant from the Cancer Foundation of Western Australia and by the Elizabeth Stalker McEwan Trust.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 22, 1999; revised February 22, 2000; accepted February 24, 2000.