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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Xenobiotica. 2018 Sep 28;49(7):791–802. doi: 10.1080/00498254.2018.1505064

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes

Haruna Nagayoshi 1, Norie Murayama 2, Kensaku Kakimoto 1, Shigeo Takenaka 3, Jun Katahira 4, Young-Ran Lim 5, Vitchan Kim 5, Donghak Kim 5, Hiroshi Yamazaki 2, Masayuki Komori 4, F Peter Guengerich 6, Tsutomu Shimada 4
PMCID: PMC6438780  NIHMSID: NIHMS1005798  PMID: 30048196

Abstract

1. The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes.

2. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2´-hydroxy-, 4´-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5.

3. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2´-hydroxylflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities.

4. Molecular docking analysis showed that the C2´-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2´ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively.

5. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

Keywords: Flavanone, Flavone, CYP2A6, Oxidation, Human, Liver microsomes, CYP2B6, CYP1A2

Introduction

Many types of plant flavonoids are found in the environment and these compounds show various biological properties, e.g. anti-allergic-, anti-inflammatory-, anti-oxidative, anti-microbial-, anti-tumorgenic-, and anti-mutagenic activities, thus affecting a number of diseases including cancer, heart disease, and bone loss in humans (Arct and Pytowska, 2008; Kale et al., 2008; Walle et al., 2007; Zhang et al., 2005). These biological activities have been shown to be related to the number and substitution positions of hydroxyl and/or methoxy groups in the flavonoid molecules (Hodek et al., 2002; Breinholt et al., 2001; Kim et al., 2005; Zhang et al., 2005; Wale, 2007; Walle and Walle, 2007; Walle et al., 2007; Shimada et al., 2010). A number of studies have been done to clarify how these flavonoids are metabolized by a variety of enzymes, particularly by P450s, in mammals as well as in plants (Akashi et al., 1998; 1999; Tanaka et al., 2010; Fliegmann et al., 2010; Tanaka and Brugliera, 2013; Du et al., 2010; Hodek et al., 2002; Kim et al., 2005; Zhang et al., 2005; Moon et al., 2006; Wale, 2007; Walle and Walle, 2007; Walle et al., 2007; NiKolic and van Breemen, 2004; Kagawa et al., 2004; Uno et al., 2013; 2015; Kakimoto et al., 2018).

We recently reported that racemic flavanone, consisting of its (2R)- and (2S)-enantiomers (Akashi et al., 1998; Si-Ahmed et al., 2010; Baranowska et al, 2016), is oxidized by human P450 enzymes to form at least eight mono-hydroxylated flavanones, three mono-hydroxylated flavones, and flavone (Figure 1) (Kakimoto et al., 2018). Among eight recombinant human P450 enzymes examined, CYP2A6 was the most active in oxidizing flavanone to form 2´- and 6-hydroxyflavanones and to form flavone. Flavone is also oxidized by CYP2A6 to form several as of yet uncharacterized mono-hydroxylated flavones—M1, M3, M4, M5, M6, and M7—as well as 6- and 5-hydroxyflavones and di-hydroxylated products M2 and M4. However, it is not known how flavanone and flavone are oxidized by human liver microsomes and which P450 enzymes contribute the most to the oxidation of these flavonoids in liver microsomes.

Figure 1.

Figure 1.

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LC-MS/MS analysis of oxidation of flavanone (A, B, and C) and flavone (D and E) by purified CYP2A6.1. Formation of mono-hydroxylated flavanones (from flavanone) was analyzed with m/z 239>137, 239>131, and 239>121 (A), formation of flavone (from flavanone) was analyzed with m/z 223>129 (B), and formation of mono-hydroxylated flavone (from flavanone) was analyzed with m/z 239>165, 239>137, and 239>121 (C). For the oxidation of flavone, formation of mono-hydroxylated flavones was analyzed with m/z 239>165, 239>137, and 239>121 (D), and formation of di-hydroxylated flavones was analyzed with 255>153 and 255>129 (E).

In this study, we examined oxidation of flavanone and flavone by liver microsomes of four human samples, one of which (sample HH2), was defective in coumarin 7-hydroxylation activity and was thus assigned as a poor metabolizer of CYP2A6. We also determined catalytic differences in the ability of CYP2A6.1 and 2A6.35 and CYP2A13.1 and 2A13.3 to oxidize flavanone and flavone; our previous studies have shown catalytic differences due to variations in humans (Shimada et al., 2018; Kakimoto et al., 2018). Other human P450 enzymes, including CYP1A2 and 2B6, were used to determine abilities to oxidize these flavonoids. Studies with chemical P450 inhibitors and antibodies against CYP2A6 were also done using human liver microsomes.

Materials and Methods

Chemicals

Flavanone [a racemic mixture of (2R)- and (2S)-enantiomers] (Akashi et al., 1998; Si-Ahmed et al., 2010; Baranowska et al, 2016), flavone, 5-, 6-, and 7-hydroxyflavone (5OHF, 6OHF, and 7OHF), 5,7- and 7,8-dihydroxyflavone (57diOHF and 78diOHF), 5-, 6- and 7-hydroxyflavanone (5OHFva, 6OHFva and 7OHFva), α-naphthoflavone (ANF), coumarin, and N,N´,N´´-triethylenethiophosphoramide (thio-TEPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Chemicals (Osaka, Japan). 2´-, 3´-, and 4´-Hydroxyflavanone (2´OHFva, 3´OHFva, and 4´OHFva) were purchased from Tokyo Kasei Co. (Tokyo). Other chemicals and reagents used in this study were obtained from the sources described previously or were of the highest quality commercially available (Shimada et al., 2018; Kakimoto et al., 2018).

Enzymes

Purified preparations of human CYP2A6.1 and 2A6.35 and CYP2A13.1 and 2A13.3 were used; the expression in Escherichia coli and purification of these P450 enzymes have been described previously (Han et al., 2012; Parikh et al., 1997; Shimada et al., 2009; 2013; Kim et al., 2018). Human CYP1A2, NADPH-P450 reductase, and cytochrome b5 (b5) were also purified from membranes of recombinant E. coli as described elsewhere (Parikh et al., 1997; Sandhu et al., 1993; 1994; Guengerich, 2015; Han et al., 2012; Shimada et al., 2013).

Recombinant CYP2B6 expressed in microsomes of Trichoplusia ni cells infected with baculovirus containing CYP2B6 and NADPH-P450 reductase cDNA inserts was obtained from GENTEST (Woburn, MA).

Liver microsomes of human samples HH2 (Cat No., 452002), HH47 (Cat No., 452047), HH54 (Cat No., 452054), and HG95 (Cat No., 452095) were obtained from GENTEST Corp (Woburn, MA). The data sheets provided by the manufacture indicated that these microsomes contained 0.19, 0.26, 0.35, and 0.25 nmol P450/mg protein, respectively. The oxidation activities towards typical P450 substrates such as (S)-mephenytoin, paclitaxel, diclofenac, bufuralol, chlorzoxazone, and testosterone were reported in these liver microsomes, except that HH2 had no detectable coumarin 7-hydroxylation activity and the data sheets indicated that this individual to be a poor metabolizer (PM) for CYP2A6 activity (Table 1).

Table 1.

Levels of P450, b5, and NADPH-P450 reductase and catalytic oxidation activities for typical P450 substrates in liver microsomes of humans used in this study (data from sheets provided by the manufacturer)

Marker enzyme and catalytic activity Human sample
HH2 HH47 HH54 HG95
P450 (nmol/mg) 0.19 0.26 0.35 0.25
b5 (nmol/mg) 0.64 0.55 0.31 0.28
NADPH-cytochrome c reductase (nmol/min/mg) 250 370 300 280
Phenacetin O-deethylation (nmol/mg/min) 0.51 0.36 0.44 0.26
Coumarin 7-hydroxylation (nmol/mg/min) PM* 0.51 2.5 0.18
S-Mephenytoin N-demethylation (nmol/min/mg) 0.010 0.0065 0.0010 0.0070
Paclitaxel 6a-hydroxylation (nmol/min/mg) 0.13 0.13 0.21 0.093
Dilofenac 4’-hydroxylation (nmol/mg/min) 3.3 2.8 1.8 1.9
S-Mephenytoin 4’-hydroxylation (nmol/min/mg) 0.020 0.0040 0.026 0.015
Bufuralol 1’-hydroxylation (nmol/min/mg) 0.0069 0.13 0.11 0.15
Chlorzoxazone 6-hydroxylaton (nmol/min/mg) 1.5 3.0 1.2 1.5
Testosterone 6β-hydroxylation (nmol/mg/min) 0.78 4.1 4.9 0.64
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*

PM. poor metabolizer for CYP2A6

Oxidation of Flavanone and Flavone by P450 Enzymes

Oxidative metabolism of flavanone and flavone by P450 enzymes was determined by the methods as described previously (Kakimoto et al., 2018). Briefly, liver microsomes (50 pmol of P450), baculosomal CYP2B6 (20 pmol of P450), or reconstituted monooxygenase systems consisting of each purified P450 (50 pmol), NADPH-P450 reductase (100 pmol), b5 (100 pmol, only in the cases of CYP2A6 and 2A13 enzymes) (Yamazaki et al., 2002), and L-α−1,2 dilaouryl-sn-glycero-3-phosphocholine (DLPC) (50 µg) were incubated with 60 µM flavanone or flavone at 37 °C for 20 min, following a pre-incubation period of 1 min before adding an NADPH-generating system (0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 unit of yeast glucose 6-phosphate dehydrogenase/ml). Each reaction was terminated by the addition of 0.5 ml of ice-cold CH3CN. Each mixture was mixed vigorously (with a vortex device) and centrifuged at 10,000 × g for 5 min. An aliquot of the upper CH3CN layer was injected directly into and analyzed with LC-MS/MS. Standard metabolites of flavanone and flavone were dissolved in ice-cold CH3CN and were analyzed as described above (internal standards were not used for quantification of the metabolites in this study).

LC-MS/MS analyses were performed using an HPLC system (ACQUITY UPLC I-Class system; Waters, Milford, MA) coupled to a tandem quadruple mass spectrometer (XevoTQ-S; Waters) by the methods described previously (Kakimoto et al., 2018). MS/MS analysis was performed in the positive electrospray ionization mode with a capillary voltage of 3000 V and cone voltage of 30 V as described previously (Kakimoto et al., 2018)..

Effects of CYP-specific inhibitors and anti-CYP2A6 IgG on flavanone and flavone oxidation by liver microsomes

The effects of ANF, coumarin, and thio-TEPA, which have been shown to be inhibitors of CYP1A2, 2A6, and 2B6, respectively (Shimada, 2006; 2017; Guengerich, 2015; Base et al., 2013; Rae et al., 2002; Walsky, 2007; Bae et al., 2013), on the oxidation of flavanone and flavone by human liver microsomes were studied. The substrate and microsomal P450 concentrations used were 60 µM and 0.2 µM, respectively, and varying concentrations of P450 inhibitors were added to the incubation mixtures.

The effects of anti-CYP2A6 IgG (Yun et al., 1991) on oxidation of flavanone and flavone by human liver microsomes were also studied using 60 µM substrate concentration, 0.2 µM liver microsomal P450, and 0.0125 mg-0.10 mg of anti-CYP2A6 IgG protein.

Other Assays

P450 and protein contents were determined by the methods described previously (Omura and Sato, 1964; Brown et al, 1989).

Docking Simulations of (2R)- and (2S)-Flavanone into CYP2A6 and 2A13

Crystal structures of CYP2A6 bound to 4,4´-dipyridyl disulfide (PBD 2FDY), an inhibitor of CYP2A6 (Fujita and Kamataki, 2001), and CYP2A13 bound to pilocarpine (PDB 3T3S) have been reported and were used in this study (Yano et al., 2006; DeVore et al., 2012a; 2012b; Kakimoto et al., 2018). Chemical structures of racemic- (CID 10251), 2R- (CID 689010), and 2S- (CID 439652) flavanone were taken from PubChem (an open chemistry database at the National Institutes of Health) and were optimized in MOE. Simulations were carried out by the methods described previously (Kakimoto et al., 2018), except that the Amber 10 force field described in the MOE software (ver. 2018.0101, Computing Group, Montreal, Canada) was used for analysis. Ligand-interaction energies (U values) were obtained by use of the program ASEdock in MOE.

Statistical analysis

Statistical analysis was examined using nonlinear regression analysis of hyperbolic plots using the program Kaleida-Graph (Synergy Software, Reading, PA, USA) or GraphPad Prism (GraphPad, La Jolla, CA, USA).

Results

LC-MS/MS Analysis of Oxidation of Flavanone and Flavone by CYP2A6.1

Oxidation of flavanone and flavone was studied in a standard reconstituted monooxygenase system containing purified CYP2A6.1, NADPH-P450 reductase, and b5, and the products formed were analyzed with LC-MS/MS as described in Materials and Methods (Figure 1). SRM analyses were conducted with the ion transitions 241>137, 241>131, and 241>121 for mono-hydroxylated flavanones, 223>129 for flavone, 225>103 for flavanone, 239>165, 239>137, and 239>121 for mono-hydroxyflavones and 255>153 and 255>129 for dihydroxyflavones (Figure 1).

Flavanone was oxidized by CYP2A6.1 to (at least) seven mono-hydroxylated products, e.g. 6OHFva, 2´-, 3´-, and 4´OHFva, and unidentified OHFvaM4, M5, and M6 [we have termed these products M1, M2, and M3, respectively, in our previous paper (Kakimoto et al., 2018)] (Figure 1A), to flavone (Figure 1B), and to four mono-hydroxylated flavones, e.g. 6OHF and OHFM6, M9, and M11 (Figure 1C). Flavone was oxidized by CYP2A6.1 to 6OHF and (at least) five unidentified mono-hydroxyflavones, e.g. OHFM6, OHFM7, OHFM9, OHFM11, and OHFM12 [we termed these products M3, M4, M5, M6, and M7, respectively, in our previous paper (Kakimoto et al., 2018)] (Figure 1D) and to di-hydroxyflavones, e.g. diOHFM1, diOHFM3, diOHFM4, and diOHFM5 (Figure 1E) as described recently (Kakimoto et al., 2018).

We compared mass fragmentation patterns of unidentified OHFvaM4, OHFvaM5, and OHFvaM6 with those of standard 6OHFva and 2´OHFva and found that OHFvaM4 and OHFvaM6 had fragmentation patterns, e.g. m/z 80.9, 102.9 (phenylacetylene ion), 130.9, and 136.9 (hydroxylated quinoid-type ion) similar to 6OHFva, the product oxidized on the A-ring of flavanone (Supplementary Figure 1A-1D) (Sasaki et al., 1966; Kagawa et al., 2004; Nikolic and van Breemen, 2004). On the other hand, OHFvaM5 showed similar fragmentation patterns, e.g. m/z 92.9 and 120.9, with 2´OHFva, the product oxidized on the B-ring (possibly hydroxylation at 5- or 6-position) (Supplementary Figure 1E and 1F) (Kakimoto et al., 2018).

We also examined the mass fragmentation patterns of unidentified products OHFM6, M7, M9, M11, and M12 derived from flavone and found that OHFM7 and M12 had patterns, e.g. m/z 80.9, 102.9, and128.9, and 136.9, similar to 6OHF oxidized on the A-ring of flavone (Supplementary Figure 2). The mass fragments of OHFM6, M9, and M11 were found to contain m/z 92.9 and 120.9, which are suggestive of oxidation on the B-ring of flavone, as described above.

Oxidation of Flavanone and Flavone by CYP2A6 and CYP2A13 Variants and by Human Liver Microsomes

Two CYP2A6.1 and 2A6.35 variants and two CYP2A13.1 and 2A13.3 variants were compared for their activities to oxidize flavanone to mono-hydroxylated products in reconstituted systems (Figure 2A-2D). Patterns of formation of mono-hydroxylated flavanones were found to be basically similar in CYP2A6.1 and 2A6.35, but the former enzyme was much more active than CYP2A6.35 (Figure 2A and 2B). Similarly, CYP2A13.1 had higher activities than CYP2A13.3 in the oxidation of flavanone (Figure 2C and 2D). Formation of 6OHFva and 2´OHFva was higher in CYP2A6 enzymes, while formation of OHFvaM4 and OHFvaM6 was high in CYP2A13 enzymes.

Figure 2.

Figure 2.

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Oxidation of flavanone by reconstituted monooxygenase system containing CYP2A6.1 (A), 2A6.35 (B), 2A13.1 (C), and 2A13.3 (D) and by liver microsomes of human sample HH2 (E), HH47 (F), HH54 (G), and HG95 (H). Major mono-hydroxylated metabolites of 6-, 2´-, and 4´- hydroxylated flavanone and uncharacterized mono-hydroxylated flavanone M4, M5, and M6 are shown in the figures by analyzing with m/z 241>137, m/z 241>131, and m/z 241>121.

Liver microsomes of four human samples were compared regarding flavanone oxidation to produce mono-hydroxylated products, where sample HH2 was defective in coumarin 7-hydroxylation activities (Table 1). Interestingly, sample HH2 microsomes formed a very small amount of 2´OHFva compared to other human samples, although the former sample produced significant amounts of 6OHFva, 4´OHFva, OHFvaM4, OHFvaM5, and OHFvaM6 (Figure 2E). The profiles of formation of mono-hydroxylated products catalyzed by the HH47, HH54, and HG95 samples were very similar to those catalyzed by purified CYP2A6 enzymes but not by CYP2A13 enzymes.

CYP2A6 was also found to oxidize flavone to various products at much higher rates than CYP2A13 enzymes and, again, wild-type CYP2A6 and 2A13 enzymes had higher activities in oxidizing flavone than the variant forms (Figure 3A-3D). Major products formed through metabolism of flavone by CYP2A6 enzymes were OHFM6, OHFM11, OHFM7, and OHFM9, and 6OHF was also detected as a minor product. Liver microsomes from HH47, HH54, and HG95 produced similar patterns of mono-hydroxylated products as CYP2A6.1, except that these liver microsomes formed a much higher amount of OHFM12 than CYP2A6 (Figure 3F, 3G, and 3H). As in the case of flavanone oxidation shown in Figure 1, HH2 sample exhibited very low activity in the formation of OHFvaM6, which was the most abundant product found with purified CYP2A6 and liver microsomes of other human samples (Figure 3E).

Figure 3.

Figure 3.

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Oxidation of flavone by reconstituted monooxygenase system containing CYP2A6.1 (A), 2A6.35 (B), 2A13.1 (C), and 2A13.3 (D) and by liver microsomes of human sample HH2 (E), HH47 (F), HH54 (G), and HG95 (H). 6-Hydroxyflavone and major uncharacterized mono-hydroxylated flavone products M6, M7, M9, M11, and M12 are shown in the figures by analyzing with m/z 239>165, m/z 239>137, and m/z 239>121.

Oxidation of Flavanone and Flavone by CYP1A2 and CYP2B6

These results suggest that CYP2A6 is a major enzyme involved in the oxidation of flavanone and flavone. However, other human P450 enzymes are involved in the oxidation process, because sample HH2 (defective in CYP2A6 activity) catalyzed the formation the of 6OHFva, 4´OHFva, OHFvaM4, OHFvaM5, and OHFvaM6 from flavanone and OHFM7, OHFM5, OHFM12, OHFM11, and 6OHF from flavone.

The oxidation of flavanone and flavone by purified CYP1A2 in reconstituted system and by CYP2B6 in T. ni microsomes was examined, and mono-hydroxylated flavanones and flavones were analyzed with LC-MS/MS (Figure 4). We did not examine reactions catalyzed by CYP2C9 and 3A4, two major P450s in human liver microsomes because our previous work showed only low activities of the purified preparations in the oxidation of these flavonoids (Kakimoto et al., 2018). CYP1A2 oxidized flavanone to form 4´OHFva, 6OHFva, OHFvaM5, and OHFvaM4 at high rates and 3’OHFva, 2’OHFva, and OHFvaM6 at significant levels (Figure 4A). CYP1A2 was also active in catalyzing flavone to form OHFM6, OHFM7, and OHFM11 and other products including OHFM5, OHFM9, OHFM12, and 6OHF (Figure 4C).

Figure 4.

Figure 4.

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Oxidation of flavanone (A and B) and flavone (C and D) by CYP1A2 in a reconstituted monooxygenase system (A and C) and by CYP2B6 in microsomes of T. ni cells (B and D). Formation of mono-hydroxylated flavanones on incubation of flavanone with CYP1A2 (A) and CYP2B6 (B) was analyzed with m/z 241>137, 241>131, and 241>121 and formation of mono-hydroxylated flavones on incubation of flavone with CYP1A2 (C) and CYP2B6 (D) was analyzed with m/z 239>165, 239>137, and 239>121.

CYP2B6 was found to show high oxidation of flavanone to form 6OHFva and was also active in producing 4´OHFva, 2´OHFva, 3´OHFva, and 2´OHFva (Figure 4B). CYP2B6 also catalyzed flavone to form OHFM6, at a high rate, and OHFM9 and 6OHF at significant levels but was not so active in producing OHFM7, OHFM11, and OHFM12 (Figure 4D).

Turnover Rates of Oxidation of Flavanone and Flavone by P450 Enzymes and by Human Liver Microsomes

We calculated turnover rates of oxidation of flavanone and flavone by P450 enzymes and by human liver microsomes, using available standards. The formation of flavone (from flavanone) was highest in CYP2A6.1, at a turnover rate of 0.68 (nmol product formed/min/nmol P450), followed by CYP2A13.1, 2A13.3, 2B6, and 2A6.35 (Figure 5A). Flavone formation was also detected in human liver microsomes at turnover rates ranging between 0.022 and 0.22 min-1. CYP2B6 had higher activity in forming 6OHFva than CYP2A6.1, while the latter enzyme catalyzed formation of 2´OHFva at much higher rates than CYP2B6 and other P450s (Figure 5B). Liver microsomes from sample HH2, defective in coumarin 7-hydroxylation, was very low in 2´OHFva formation; however, this sample catalyzed formation of 6OHF at rates similar to those by other three human samples (Figure 5C). CYP1A2 had much higher activities than other P450 enzymes in the formation of 3´OHFva and 4´OHFva from flavanone.

Figure 5.

Figure 5.

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Oxidation of flavanone by reconstituted P450 system, baculosomal CYP2B6 system, and human liver microsomes. Formation of flavone (A), 6OHFva (B), 2’OHFva (C), 3’OHFva (D), and 4’OHFva (E) was calculated using commercial standard materials. The results presented are means of three separate determinations ± S.D.

For the calculation of flavone oxidation by P450s and human liver microsomes, we had three standards available, namely 5OHF, 6OHF, and 7OHF. We did not detect any enzymatic formation of 7OHF in our assay system. The formation of 5OHF was detected in CYP2A6.1 and human liver microsomes at the rates of ~ 0.01 nmol/min/nmol P450 (Figure 6). CYP2A6.1 oxidized flavone to form 6OHF at 0.11 min−1, and the rate was the highest among other P450 enzymes determined, but liver microsomes catalyzed the formation of 6OHF at rates of 0.17–0.25 min−1 (Figure 6).

Figure 6.

Figure 6.

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Oxidation of flavone by reconstituted P450 system, a baculosomal CYP2B6 system, and human liver microsomes. Formation of 5OHF (A) and 6OHF (B) was calculated using commercial standard materials. The results presented are means of three separate determinations ± S.D.

Effects of ANF, Coumarin, and Thio-TEPA on Oxidation of Flavanone and Flavone by Human Liver Microsomes

The above results suggested that CYP1A2, 2A6, and 2B6 play important roles in the oxidation of flavanone and flavone by human liver microsomes. The effects of ANF, coumarin, and thio-TEPA, which have been shown to be inhibitors of CYP1A2, 2A6, and 2B6, respectively (Shimada, 2006; 2017; Guengerich, 2015; Base et al., 2013; Rae et al., 2002; Walsky, 2007; Bae eta l., 2013), were studied in human liver microsomes (Figure 7, 8, and 9).

Figure 7.

Figure 7.

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Effects of ANF (open circles), coumarin (closed circles), and thio-TEPA (closed triangles) on the oxidation of flavanone catalyzed by microsomes of a human liver sample HH54. The substrate and microsomal P450 concentrations used were 60 µM and 0.2 µM, respectively. Varying concentrations of P450 inhibitors were added to the incubation mixtures, and the products were measured. Control experiments in the absence of inhibitors were done in triplicate determinations and are represented as means ± S.D. Assays on the effects of inhibitors were done in single determination.

Figure 8.

Figure 8.

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Effects of ANF, coumarin, and thio-TEPA on chemical P450 inhibitors at 6 µM flavanone concentration in microsomes of human liver sample HH47. Experiments were done with triplicate experiments for controls (without chemicals, indicating “None”) and duplicate expereiments in the presence of inhibitors.

Figure 9.

Figure 9.

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Effects of ANF (open circle), coumarin (closed circle), and thio-TEPA (closed triangle) on the oxidation of flavone catalyzed by liver microsomes (human sample HH54). The substrate and microsomal P450 concentrations used were 60 µM and 0.2 µM, respectively. Other details are as in the legend to Fig. 5.

The effects of different concentrations of these three P450 inhibitors were studied in microsomes from human liver sample HH54 using a substrate concentration of 60 µM flavanone (Figure 7). The formation of flavone (from flavanone) was inhibited by both thio-TEPA and coumarin, and the formation of 6OHFva and OHFM6 was decreased with all three of the inhibitors used. As suggested by the above results, 2´OHFva formation was markedly decreased in the presence of a low concentration of coumarin (Figure 7E). The formation of 7OHFva, 3´OHFva, 4´OHFva OHFvaM5, and OHFvaM6 was inhibited by ANF (Figure 7).

We also examined the effects of these P450 inhibitors at a low flavanone concentration (6 µM) in human liver microsomes (Figure 8). The results strongly suggested that the formation of 2´OHFva was catalyzed by CYP2A6, the formation of flavone was both CYP2B6 and 2A6, and the formation of 6OHFva was by CYP2B6, 2A6, and 1A2. The contribution of several other products was dependent on CYP1A2 (Figure 8).

Similarly, the effects of these three inhibitors on flavone oxidation (at 60 µM substrate concentration) by liver sample HH54 microsomes were examined (Figure 9). OHFM6, OHFM9, and diOHFM4 were strongly inhibited by the low concentration of coumarin (Figure 9). Thio-TEPA was effective in inhibiting the formation of 6OHF, OHFM6, OHFM7, OHFM11, OHFM11 in liver microsomes. ANF inhibited the formation of OHFM11, diOHFM3, and diOHFM5 in liver microsomes (Figure 9).

Effects of Anti-CYP2A6 IgG on Oxidation of Flavanone and Flavone by Human Liver Microsomes

The effects of anti-CYP2A6 IgG on the oxidation of flavanone and flavone by microsomes from human liver sample HH54 were examined (Figure 10). It should be noted that anti-CYP2A6 IgG inhibits CYP2A13 as well as CYP2A6; however, roles of CYP2A13 can be ruled out because of its lower expression in human liver (Guengerich, 2015) and of its lower activities for the oxidation of flavanone and flavone by purified CYP2A13 described above. The formation of 2´OHFva from flavanone was markedly decreased with a low concentration of anti-CYP2A6 IgG, and the formation of 6OHFva, flavone, and OHFM6 was decreased by about 50%. Other products, e.g. 7OHFva, 3´OHFva, 4´OHFva, OHFvaM4-M6, were not attenuated by anti-CYP2A6 (Figure 10A-10E).

Figure 10.

Figure 10.

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Effects of anti-CYP2A6 IgG on oxidation of flavanone and flavone by human liver microsomes (sample HH54). Flavanone (A-E) and flavone (F-J) were incubated at 60 µM concentrations with human liver microsomes (50 pmol of P450) in 100 mM potassium phosphate buffer (pH 7.4) (in a total volume of 0.25 ml) in the absence (A and F) or presence [0.0125 mg (B and G), 0.025 mg (C and H), 0.05 mg (D and I), and 0.10 mg (E and J)] of anti-CYP2A6 IgG protein.

Flavone oxidation by liver microsomes was also inhibited by anti-CYP2A6 IgG (Figure 10F-10J). Formation of OHFM6 and OHFM9 was markedly inhibited by anti-CYP2A6, and the formation of diOHFM4, OHFM7, and OHFM11 was also affected (Figure 10).

Docking Simulations of Flavanone into CYP2A6 and 2A13

Molecular interactions of flavanone (Si-Ahmed et al., 2010) were investigated with CYP2A6 and CYP2A13 (Figure 11). The distances between the C2´ and C6 atoms of (2R)-flavanone and the iron center of CYP2A6 2FDY were calculated to be 3.80 and 10.02Å, respectively, and those of CYP2A13 3T3S iron were 3.75 and 10.30Å, respectively (Figures 11A and 11C). The distances between C2´ and C6 of (2S)-flavanone and iron center of CYP2A6 2FDY were calculated to be 6.91 and 5.42Å, respectively, and those of CYP2A13 3T3S iron were 7.53 and 3.90Å, respectively (Figures 11B and 11D). The ligand-interaction energy (U value) of interaction of (2S)-flavanone with active site of CYP2A6 was found to be –44.2, which was low compared with that of interaction of (2R)-flavanone with CYP2A6 (U = −2.79) (Figure 11A and 11B). The U value (−26.0) for the interaction of (2S)-flavanone with CYP2A13 was also lower than those of interaction of 2R-flavanone (−7.2) with CYP2A13 (figure 11C and 11D).

Figure 11.

Figure 11.

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Molecular docking of interaction of (2R)-flavanone (A and C) and (2S)-flavanone (B and D) with the iron center of CYP2A6.1 (A and B) and CYP2A13.1 (C and D). Experimental details are described in Materials and Methods.

Discussion

Our present results showed that CYP2A6.1 (“wild-type”) has unique characteristics in catalyzing the oxidation of flavanone and flavone to produce 2´OHFva and unidentified product OHFM6 as major products, respectively, in human liver microsomes. Formation of these products was very low in microsomes of human liver sample HH2, which was defective in CYP2A6-dependent coumarin 7-hydroxylation activities, and was strongly inhibited by both a CYP2A6-inhibitor coumarin and anti-CYP2A6 IgG when examined in microsomes of human liver samples HH47 and HH54, which had coumarin 7-hydroxylation activities. CYP2A6 was also suggested to be involved in the formation of flavone from flavanone and of OHFM9 and diOHFM4 from flavone; however, these reactions have been suggested to be catalyzed by other P450s, including CYP2B6 and 1A2 as well as CYP2A6. For example, CYP2A6-deficient sample HH2-catalyzed formation of flavone from flavanone at a rate of 0.15 nmol/min/nmol, which was comparable to those by other human samples (Fig. 5). Coumarin, thio-TEPA, and anti-CYP2A6 IgG inhibited this reaction in liver sample HH54 microsomes, which were active in coumarin 7-hydroxylation activity, suggesting possible roles of CYP2A6 and 2B6 in this reaction. Roles of CYP1A2 were supported in the experiments of inhibition with ANF in human liver microsomes, where formation of 7OHFva, 3´OHFva, 4´OHFva, OHFvaM4, OHFvaM5, and OHFvaM6 from flavanone and the formation of OHFM11, diOHFM3, and diOHFM5 from flavone appear to be catalyzed by CYP1A2 (Figure 7–9).

MS product ion spectra of unidentified products, e.g. OHFvaM4, OHFvaM5, and OHFvaM6, were analyzed and compared with those of standards 6OHFva and 2´OHFva (Supplementary Figure 1). On the basis of these results, OHFvaM4 and OHFvaM6 are suggested to be the products oxidized at A-ring of flavanone; the fragment patterns, e.g. m/z 80.9, 102.9 (phenylacetylene ion), 130.9, and 136.9 (hydroxylated quinoid-type ion) were similar to those of 6OHFva (Supplementary Figure 1A-1D) (Sasaki et al., 1966; Das et al., 1973; Kagawa et al., 2004; Nikolic and van Breemen, 2004). On the other hand, OHFvaM5 was suggested to be a product oxidized on the B-ring (of flavanone), having fragment ions, e.g. m/z 92.9 and 120.9 similar to those of 2´OHFva (Supplementary Figure 1E and 1F) (Kakimoto et al., 2018). Similarly, the unidentified flavone products OHFM7 and OHFM12 were found to have similar product ions, e.g. m/z 80.9, 102.9, and128.9, and 136.9, to those of 6OHF and to unidentified OHFM6, OHFM9, and OHFM11, containing m/z 92.9 and 120.9, suggestive of products oxidized on the B-ring of flavone (Supplementary Figure 2).

We used CYP2A6.1 and 2A6.35 variants and CYP2A13.1 and 2A13.3 variants to determine if there are differences in catalytic specificities in the oxidation of flavanone and flavone. The results showed that activities of CYP2A6.1 and 2A13.1 were always higher than those of the respective CYP2A6.35 and CYP2A13.3 variants, supporting previous results for lower activities of these variants with other substrates examined (Han et al., 2012; Shimada et al., 2018; Hosono et al., 2017; Kumondai et al., 2018; Kim et al., 2018). HPLC profiles of flavanone product formation in human liver microsomes were basically similar to those by CYP2A6.1, except when liver sample HH2 was used (Figures 2 and 3). CYP2A13.1, on the other hand, showed different patterns of oxidation of flavanone and flavone when compared human liver microsomes, consistent with the view that CYP2A13 is mainly expressed in extrahepatic organs (Su et al. 2000; Chiang et al., 2012; Shimada, 2015).

Commercial preparation of racemic flavanone contains (2R)- and (2S)-enantiomers (Si-Ahmed et al., 2010; Baranowska et al, 2016). Studies of the molecular interaction of these flavanone enantiomers with the active sites of CYP2A6.1 indicated that the C2´ atom of the (2R)-isomer was located nearer (distance 3.8Å) the iron center of CYP2A6 (2FDY) as compared with the (2S)-isomer (distance 6.9Å), while C6 of (2S)-flavanone was more closely located to the iron center of CYP2A6 than that of (2R)-isomer (Figure 11). It is not known at present whether these racemic enantiomers of flavanone can be oxidized at specific carbon atoms in the molecule. In this respect, it is interesting to note that there are differences in biotransformation reactions of S- and R-hesperetin enantiomers in vitro (Brand et al., 2010) and in stereospecific pharmacokinetics of hesperetin, naringenin, and eriodictyol in vivo (Yáñez et al. 2008). A similar tendency was also noted in the interaction of flavanone enantiomers with CYP2A13.1, indicating that CYP2A6 and 2A13 are basically similar in their interaction with flavanone enantiomers in molecular docking analysis, although the catalytic activities for flavanone oxidation were different for these enzymes. There are no optical isomers in flavone, and our docking studies showed that the interaction of flavone with CYP2A6 and 2A13 was found to be rather similar to those of interaction of (2R)-flavanone with the enzymes (Figure 11).

In conclusion, our present results showed that CYP2A6 has unique characteristics in oxidizing flavanone and flavone to form 2´OHFva and 6OHF, respectively, in human liver microsomes. This enzyme also catalyzed oxidation of flavanone and flavone at other carbon positions, however, and different human P450 enzymes, CYP1A2 and 2B6, were also suggested to play important roles in these flavanone and flavone oxidations in human liver microsomes.

Supplementary Material

Supplemental

Figure 12.

Figure 12.

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Summary of roles of human P450 enzymes in the oxidation of flavanone and flavone by liver microsomes.

Acknowledgements

This study was supported in part by JSPS KAKENHI [16K21710] (to H. N), [16K09119] (to K. K.), [15K07770] (to S. T.), [17K08630] (J. K.), [17K08426] (to N. M.), and [17K08425] (to H. Y.), National Research Foundation of Korea [NRF-2016R1D1A1B03932002] (to D. K.), and United States Public Health Service grant [R01 GM118122] (to F. P. G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abbreviations used:

P450 or CYP

cytochrome P450

b5

cytochrome b5

DLPC

L-α−1,2 dilaouryl-sn-glycero-3-phosphocholine

5OHFva, 6OHFva, 7OHFva, 2´OHFva, 3´OHFva, 4´OHFva

5-, 6-, 7-, 2´-, 3´-, and 4´-hydroxyflavanone, respectively

OHFva M1-M3

uncharacterized (mono-) hydroxyflavanones

5OHF, 6OHF, and 7OHF

5-, 6-, and 7-hydroxyflavones, respectively

OHFM1-M7

uncharacterized mono-hydroxyflavones M1-M7

ANF

α-naphthoflavone

Thio-TEPA

N,N´,N´´ -triethylenethiophosphoramide

Footnotes

Declaration of interest Statement

The authors declare no conflict of interest associated with this manuscript.

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