Pseudoenzymatic reduction of N-hydroxy-2-acetylaminofluorene to 2-acetylaminofluorene mediated by cytochrome P450

Shigeyuki Kitamura1, Koji Takekawa, Kazumi Sugihara, Kiyoshi Tatsumi and Shigeru Ohta

Institute of Pharmaceutical Science, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan


    Abstract
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N-hydroxy-2-acetylaminofluorene (N-OH-AAF) was reduced to 2-acetylaminofluorene by rat liver microsomes in the presence of both NAD(P)H and FAD under anaerobic conditions. The microsomal reduction proceeds as if it were an enzymatic reaction. However, when the microsomes were boiled, the activity was not abolished, but was enhanced. The activity was also observed with cytochrome P450 2B1 alone, without NADPH-cytochrome P450 reductase, in the presence of these cofactors. Hematin also exhibited a significant reducing activity in the presence of both a reduced pyridine nucleotide and FAD. The activities of microsomes, cytochrome P450 2B1 and hematin were also observed upon the addition of photochemically reduced FAD instead of both NAD(P)H and FAD. The microsomal reduction of N-OH-AAF appears to be a non-enzymatic reaction by the reduced flavin, catalyzed by the heme group of cytochrome P450.

Abbreviations: AAF, 2-acetylaminofluorene; N-OH-AAF, N-hydroxy-2-acetylaminofluorene.


    Introduction
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 Abstract
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2-Acetylaminofluorene (AAF) is a well-known carcinogen (1). N-hydroxylation of AAF is an essential first step in the metabolic activation of the carcinogen (24). The N-hydroxylated product, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), is further metabolized by such reactions as hydroxylation (5), reduction (614), deacetylation (911,15), conjugation (1) and isomerization (12). Among these reactions, the reduction of N-OH-AAF to the parent compound AAF is a detoxification step of the hydroxamic acid. Therefore, the balance of the N-hydroxylation of AAF and the reduction of N-OH-AAF is of importance in determining the carcinogenicity. These aspects of N-OH-AAF were reviewed in detail by Weisburger and Weisburger (16). Several workers (811) have demonstrated the reduction of N-OH-AAF by mammalian tissue homogenate or a 10 000 g supernatant fraction. A role of intestinal bacteria in the reduction of N-OH-AAF was also demonstrated by Weisburger et al. (68). Gutmann and Erickson (12) reported that N-OH-AAF was reduced to AAF by mammalian liver cytosol. We attempted to identify the liver cytosolic enzymes responsible for the reduction of N-OH-AAF. A new enzyme capable of reducing N-OH-AAF was purified from rabbit liver cytosol and was tentatively designated `N-hydroxy-2-acetylaminofluorene reductase' (17). Furthermore, we demonstrated that aldehyde oxidase (EC 1.2.3.1) is another liver cytosolic enzyme responsible for the reduction of N-OH-AAF (18). The present study provides evidence that N-OH-AAF is reduced non-enzymatically by the heme moiety of cytochrome P450 in liver microsomes in the presence of both a reduced pyridine nucleotide and a flavin.

AAF was purchased from Tokyo Chemical Industry (Tokyo, Japan). N-OH-AAF was synthesized according to the method of Cramer et al. (3). Reduced flavins were prepared photochemically by the method of Yubisui et al. (19). Livers from male Sprague–Dawley rats (130–150 g) were immediately perfused with 1.15% KCl and homogenized in 4 vol KCl solution using a Potter–Elvehjem homogenizer. The homogenate was centrifuged for 20 min at 9000 g and for 60 min at 105 000 g, sequentially. The microsomal fraction was washed by resuspension in the KCl solution and resedimentation. Cytochrome P450 2B1 was prepared from the rat liver microsomes by the method of Guengerich et al. (20). Conversion of cytochrome P450 to cytochrome P420 was performed by the method of Imai and Sato (21). Protein contents were determined by the method of Lowry et al. (22) with bovine serum albumin as the standard.

The incubation mixture consisted of 0.2 µmol N-OH-AAF, 1 µmol NADPH or NADH, 0.1 µmol FAD, 0.1 mmol sodium fluoride and a microsomal preparation equivalent to 100 mg wet wt liver in a final volume of 1 ml 0.1 M K,Na phosphate buffer (pH 7.4). The incubation was performed using a Thunberg tube under anaerobic conditions. The side arm contained NADPH or NADH and the body contained all other components. The tube was gassed for 3 min with nitrogen, evacuated with an aspirator for 10 min and again gassed with nitrogen. The reaction was started by mixing the components of the side arm and the body together, continued for 10 min at 37°C and stopped by adding 0.1 ml 2.5 N NaOH. The reaction mixture, following the addition of 20 µg phenacetin as an internal standard, was extracted once with 5 ml ether and the ether extract was then evaporated to dryness in vacuo. The residue was dissolved in 0.1 ml methanol and subjected to HPLC on an L-6000 chromatograph (Hitachi, Tokyo, Japan) fitted with a 130x4 mm Lichrosphere 100RP-18 column (Merck, Darmstadt, Germany). The mobile phases were acetonitrile/water (1:1 v/v) and the chromatograph was operated at a flow rate of 0.4 ml/min at a wavelength of 254 nm. The elution times of AAF and phenacetin were 4.2 and 8.0 min, respectively. AAF formed was determined from its peak area.

N-OH-AAF was reduced to AAF by liver microsomes from untreated rats in the presence of NADH or NADPH under both aerobic and anaerobic conditions. The reducing activity was stimulated in the presence of FAD, FMN or riboflavin under anaerobic conditions (Table IGo). Liver microsomes from phenobarbital- and 3-methylcholanthrene-treated rats also exhibited reducing activities toward N-OH-AAF 2- to 3-fold higher than that of untreated rats. These activities were sensitive to inhibition by carbon monoxide. When the liver microsomes of untreated rats were boiled, these activities were not abolished, but were enhanced in the presence of both NAD(P)H and FAD. The NAD(P)H-linked activities of the liver microsomes from phenobarbital- and 3-methylcholanthrene-treated rats were decreased by boiling of these microsomes. However, the stimulating effect of flavins was also observed in boiled liver microsomes from phenobarbital- and 3-methylchoranthrene-treated rats in the same way as with native microsomes. The activity of boiled microsomes was also markedly inhibited by carbon monoxide (Table IIGo).


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Table I. Reduction of N-hydroxy-2-acetylaminofluorene by rat liver microsomes
 

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Table II. Reduction of N-hydroxy-2-acetylaminofluorene by liver microsomes from untreated, phenobarbital-treated or 3-methylcholanthrene-treated rats
 
Rat liver microsomal cytochrome P450 2B1 alone exhibited a reducing activity in the presence of both NAD(P)H and FAD. When the cytochrome P450 was boiled, the activity was again not abolished, as described above (Table IIIGo). Cytochrome P420, an inactive form of cytochrome P450, also exhibited reducing activity (data not shown). Liver microsomes and cytochrome P450 2B1, as well as boiled microsomes and cytochrome P450 2B1, also exhibited reducing activity in the presence of the photochemically reduced forms of FAD, FMN or riboflavin instead of both a reduced pyridine nucleotide and a flavin (Table IVGo).


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Table III. Reduction of N-hydroxy-2-acetylaminofluorene by cytochrome P450 2B1 from rat liver
 

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Table IV. Reduction of N-hydroxy-2-acetylaminofluorene by rat liver microsomes and cytochrome P450 2B1 in the presence of reduced flavins
 
Hematin showed a significant reducing activity in the presence of both NADH or NADPH and FAD. Reduction of N-OH-AAF by hematin was also observed in the presence of reduced FAD (Table VGo). However, protoporphyrin, ferric chloride or ferrous chloride did not show activity, even in the presence of a reduced flavin (data not shown).


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Table V. Reduction of N-hydroxy-2-acetylaminofluorene by hematin
 
These results lead to the conclusion that N-OH-AAF is reduced to AAF non-enzymatically by the catalytic action of the heme group of cytochrome P450 in the presence of both FAD and a reduced pyridine nucleotide. However, a part of the NAD(P)H-dependent reducing activity of liver microsomes might be due to enzymatic reaction. Yamazoe et al. (14) reported that N-OH-AAF was reduced by rat liver microsomes supplemented with NADPH and suggested the involvement of the cytochrome P450 system. However, the reduction of N-OH-AAF by heat-treated microsomes was not examined. In the microsomal reduction, NADH was as effective as NADPH as an electron donor. This unusual requirement of a reduced pyridine nucleotide in the microsomal reaction also might support the non-enzymatic nature of the reduction of N-OH-AAF by liver microsomes.

The microsomal reduction of N-OH-AAF to AAF appears to proceed in two steps:



The first step involves the reduction of a flavin by a reduced pyridine nucleotide; in native microsomes, the flavin is mainly reduced by NADPH-cytochrome P450 reductase with NADPH or by NADH-cytochrome b5 reductase with NADH, as reported by Kato et al. (23). In boiled microsomes, the flavin appears to be reduced non-enzymatically by NADPH or NADH, as reported in Singer and Kearney (24). The second step is the non-enzymatic reduction of N-OH-AAF to AAF by the reduced flavin, catalyzed by the heme group of cytochrome P450.

As reported previously, aldehyde oxidase is also capable of reducing a variety of hydroxamic acids, including N-OH-AAF, in the presence of an electron donor of aldehyde oxidase such as N1-methylnicotinamide (18,25). In a previous study, moreover, a new enzyme responsible for the reduction of N-OH-AAF to AAF, which was named `N-hydroxy-2-acetylaminofluorene reductase', was purified from rabbit liver cytosol (16). The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol and either NADPH or NADH as an electron donor. Thus, the two liver cytosolic enzymes responsible for the reduction of the hydroxamic acid show a quite different mode of reduction from that of microsomal reduction. Weisburger et al. (6) isolated AAF and 7-, 5- and 3-hydroxylated AAF as reduced metabolites of N-OH-AAF from the urine of germ-free and conventional rats dosed with N-OH-AAF. Furthermore, when N-OH-AAF was administered to germ-free rats, the amount of reduced metabolites excreted in the urine was about a half of that in conventional rats. They suggested that the reducing enzyme systems in tissues of animals play as important a role as intestinal bacteria in the reduction of N-OH-AAF. The liver microsomal reducing activity was observed widely among animal species, including humans (data not shown), whereas considerable variability of the two cytosolic enzyme activities exists among species. Therefore, the microsomal reduction described here may play a central role in the reduction of N-OH-AAF in the liver.


    Notes
 
1 To whom correspondence should be addressed Email: kitamura{at}pharm.hiroshima-u.ac.jp Back


    References
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 Abstract
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Received July 22, 1998; revised October 1, 1998; accepted October 13, 1998.





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