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. Author manuscript; available in PMC: 2023 Feb 3.
Published in final edited form as: Xenobiotica. 2022 Apr 13;52(2):134–145. doi: 10.1080/00498254.2022.2062486

Oxidation of 3′-methoxyflavone, 4′-methoxyflavone, and 3′,4′-dimethoxyflavone and their derivatives having with 5,7-dihydroxyl moieties by human cytochromes P450 1B1 and 2A13

Tsutomu Shimada a, Haruna Nagayoshi b, Norie Murayama c, Atsuki Sawai a, Vitchan Kim d, Donghak Kim d, Hiroshi Yamazaki c, F Peter Guengerich e, Shigeo Takenaka a
PMCID: PMC9896170  NIHMSID: NIHMS1866401  PMID: 35387543

Abstract

1. Oxidation of 3′-methoxyflavone, 4′-methoxyflavone, and 3′,4′-dimethoxyflavone and their derivatives containing 5,7-dihydroxyl groups by human cytochrome P450 (P450 or CYP) 1B1 and 2A13 was determined using LC-MS/MS systems.

2. 3′-Methoxyflavone and 4′-methoxyflavone were mainly O-demethylated to form 3′-hydroxyflavone and 4′-hydroxyflavone, respectively, and then 3′,4′-dihydroxyflavone at higher rates with CYP1B1 than with CYP2A13. 4′-Methoxy-5,7-dihydroxyflavone (acacetin) was found to be demethylated by CYP1B1 and 2A13 to form 4′,5,7-trihydroxyflavone (apigenin) at rates of 0.098−1 and 0.42 min−1, respectively. 3′-Methoxy-5,7-dihydroxyflavone was also demethylated by both P450s, with CYP2A13 being more active.

3. 3′,4′-Dimethoxyflavone was a good substrate for CYP1B1 but not for CYP2A13 and was found to be mainly O-demethylated to form 3′,4′-dihydroxyflavone (at a rate of 4.2 min−1) and also several ring-oxygenated products having m/z 299 fragments. Molecular docking analysis supported the proper orientation for formation of these products by CYP1B1.

4. Our present results showed that 3′- and 4′-methoxyflavone can be oxidized to their O-demethylated products and, to a lesser extent, to ring oxidation products by both P450s 1B1 and 2A13 and that 3′,4′-dimethoxyflavone is a good substrate for CYP1B1 in forming both O-demethylated and ring-oxidation products. Introduction of a 57diOHF moiety into these methoxylated flavonoids caused decreased in oxidation by CYP1B1 and 2A13.

Keywords: LC-MS/MS; 3′,4′-dimethoxyflavone; methoxylated flavones; oxidation; CYP1B1; CYP2A13

Introduction

Among the many structurally diverse plant flavonoids that have been examined, 5,7-dihydroxyflavone (57diOHF, chrysin), 4′5,7-trihydroxyflavone (4′57triOHF, apigenin), and 5,6,7-trihydroxyflavone (567triOHF, baicalein) have been reported to have significant anti-allergic, anti-inflammatory, anti-oxidative, anti-microbial, anti-tumorgenic, and anti-mutagenic properties in mammals, possibly in humans (Samarghandian et al., 2019; Farkhondeh et al., 2019; Li et al., 2012; Nabavi et al., 2015; Gao et al., 2016; Patel et al., 2007; Sung et al., 2016; Shukla and Gupta, 2010). Interestingly, recent studies have shown that among nine human cytochrome P450 (P450 or CYP) enzymes examined, the CYP1 family enzymes, including CYP1B1, catalyzed the oxidation of 57diOHF to form 567triOHF (Nagayoshi et al., 2019b; Williams et al., 2017), while CYP2A13 and 2A6 were the major enzymes involving in the formation of 4′57triOHF (Nagayoshi et al., 2019b).

Methoxy flavonoids, widely distributed in citrus fruits and other plants, have been shown to have altered biological activities after oxidation by P450 enzymes. For example, several methoxylated flavonoids—e.g., eupatorine, hesperidin, nobiletin, tamarixetin, tangeretin, and diosmetin—have been reported to be O-demethylated by human P450 enzymes, particularly CYP Family 1 enzymes, to active metabolites that inhibit proliferation of human cancer cells (Koirala et al., 2016; Wen et al., 2017; Androutsopoulos et al., 2008; 2009a; 2009b; 2009c; Surichan et al., 2012; 2018). 4′-Methoxyflavone (4′MeF) and 3′,4′-dimethoxyflavone (3′4′diMeF) have been reported to prevent decreases in cell viability of HeLa and SH-SY5Y cells caused by N-methyl-N′-nitro-N-nitrosoguanine, which induces parthanatos, a cell death signaling pathway in which excessive oxidative damage to DNA leads to over-activation of poly(ADP-ribose) polymerase (Fatokun et al., 2013; 2014). We have recently reported that human P450 enzymes, including CYP1B1 and 2A13, preferentially catalyze O-demethylation of 2′-, 3′-, and 4′-methoxyflavones (2′MeF, 3′MeF, and 4′MeF, respectively) to form mono-hydroxylated products and that these P450 enzymes also catalyze ring oxidations at much slower rates than the O-demethylation reactions (Nagayoshi et al., 2020; 2021).

In this study, we addressed the question of whether the presence of a 57diOHF moiety affects the P450-supported oxidation of 3′-methoxyflavone (3′MeF), 4′-methoxyflavone (4′MeF), and 3′,4′-dimethoxyflavone (3′4′diMeF) by comparisons with the 5,7-dihydroxy-substituted flavones (3′Me57diOHF, 4′Me57diOHF, and 3′4′diMe57diOHF). LC-MS/MS analysis was performed to determine product formation, and the products formed were analyzed by their fragment ion spectra. We used CYP1B1 and CYP2A13 (both wild type enzymes) in this study because these enzymes have different catalytic activities for 57diOHF and 2′-, 3′-, and 4′-MeF, as described earlier (Nagayoshi et al., 2019b).

Materials and methods

Chemicals

3′,4′-Dimethoxyflavone (3′4′diMeF), 3′,4′-dimethoxy-5,7-dihydroxyflavone (3′4′diMe57diOHF), 3′-methoxyflavone (3′MeF), 4′-methoxyflavone (4′MeF), 3′-methoxy-5,7-dihydroxyflavone (3′Me57diOHF), and 4′-methoxy-5,7-dihydroxyflavone (acacetin, 4′Me57diOHF) (Figure 1) were kindly donated by Dr. Maryam K. Foroozesh (Xavier University of Louisiana, New Orleans, LA, USA). 3′,4′-Dihydroxyflavone (3′4′diOHF), 5,7-dihydroxyflavone (chrysin, 57diOHF), 5,6,7-trihydroxyflavone (baicalein, 567triOHF), and 4′,5,7-trihydroxyflavone (apigenin, 4′57triOHF) were purchased from Tokyo Kasei Co. (Tokyo), Sigma-Aldrich (St. Louis, MO, USA), and Wako Pure Chemicals (Osaka, Japan). Other chemicals and reagents were obtained from sources described previously or were of the highest quality commercially available (Nagayoshi et al., 2020; 2021).

Figure 1.

Figure 1.

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Structures of flavonoids used in this study.

Enzymes

Purified preparations of wild type of human CYP1B1 and CYP2A13 expressed in Escherichia coli were obtained by the methods described previously (Parikh 1997; Han et al., 2012; Kim et al., 2018; Nagayoshi et al., 2020; 2021). NADPH-P450 reductase and cytochrome b5 (b5) were purified from membranes of recombinant E. coli by the methods described elsewhere (Parikh et al., 1997; Guengerich 2014).

Oxidation of flavonoids by recombinant human P450 enzymes

Oxidation of flavonoids by CYP1B1 and 2A13 was determined by the methods described previously (Nagayoshi et al., 2020; 2021). Briefly, reconstituted monooxygenase systems consisting of each purified P450 (50 pmol), NADPH-P450 reductase (100 pmol), b5 (100 pmol, in a case of CYP2A13), and L-α−1,2 dilaouryl-sn-glycero-3-phosphocholine (DLPC) (50 μg) were incubated (0.25 mL of total volume) with 60 μM concentrations of each flavonoid at 37 °C for 20 min unless specified, following a pre-incubation of 1 min before the addition of 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. The mixture was mixed vigorously (with a vortex device) and centrifuged at 10,000 × g for 5 min, and an aliquot of the upper CH3CN layer was injected and analyzed with LC-MS/MS as described (Nagayoshi et al., 2020; 2021). In our preliminary experiments, primary metabolite formation from 57diOHF or 3′MeF catalyzed CYP1B1 was linear within 30 min incubation under the present conditions.

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 as described previously (Nagayoshi et al., 2020; 2021). 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 (Nagayoshi et al., 2020; 2021).

Other assays

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

Docking simulations of 3′4′diMeF into CYP1B1 and 2A13

CYP1B1.3 variant (L432V) model was established by incorporating the mutation into CYP1B1.1 (3PMO) (Wang et al., 2011). The crystal structure of CYP2A13 (Protein Data Bank 3T3S) has been reported and was used in this study (DeVore and Scott, 2012). The chemical structures of flavonoids used in this study (Figure 1) were taken from PubChem (an open chemistry database at the National Institutes of Health) and were optimized in MOE software (ver. 2020.09, Computing Group, Montreal, Canada). Simulations were carried out in MOE by the methods described previously (Nagayoshi et al., 2020). We generated hundred solutions for each docking experiment and ranked according to the ligand-interaction energies (U values, kcal/mol) using the program of ASEdock in MOE. The lower the U value, the more effective in interacting chemicals with P450s.

Results

Comparison of oxidation of 57diOHF, 3′MeF, 4′MeF, 3′Me57diOHF, and 4′Me57diOHF by CYP1B1 and CYP2A13

We first examined oxidation of 57diOHF, 3′MeF, and 3′Me57diOHF by CYP1B1 and CYP2A13 and compared activities using LC-MS/MS (Figure 2). As has been reported previously (Nagayoshi et al., 2019b), CYP1B1 oxidized 57diOHF to form mainly 567triOHF, while CYP2A13 formed mainly 4’57triOHF (Figures 2A and 2B, respectively). Both CYP1B1 and 2A3 catalyzed the oxidation of 3′MeF to form 3′OHF and 3′4′diOHF, the former being faster than CYP2A13 (Figures 2B and 2G). Formation of m/z 269 products, suggested to arise from ring-oxidation products (vide infra), was also higher with CYP1B1 than CYP2A13, although the level was very low (showing peak intensities on the order of 2 × 105, Figures 2C and 2H) as compared with the formation of hydroxylated products (showing peak intensities on the order of 2 × 106, Figure 2B and 2G). When 3′Me57diOHF was used as a substrate, CYP2A13 was more active than CYP1B1 in forming an O-demethylated product of 3′57triOHF (not identified in this study, but having similar LC-MS behavior to 4′57triOHF, as described later). In addition, we found that both enzymes oxidized 3′Me57diOHF to form small amounts of a m/z 301 product suggested to be a ring oxidation product (vide infra, Figure 4J).

Figure 2.

Figure 2.

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LC-MS/MS analysis of oxidation of 57diOHF (A and F), 3′MeF (B, C, G, and H), and 3′Me57diOHF (D, E, I, and J) by CYP1B1 (A-E) and CYP2A13 (F-J). Detection of different products was by fragment ions (m/z) in the mass spectra, as indicated in the figures. In parts A, F, B, G, D, and I, the full-scale peak intensity was 2 × 106, while the scale for parts C, H, E, and J was 2 × 105, due to lower amounts of formation in the latter cases.

Figure 4.

Figure 4.

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Fragment ion spectra of various products of oxidation of 57diOHF (A and B), 3′MeF (C and D), 4′MeF (E), 3′Me57diOHF (F, G, and J), and 4′Me57diOHF (H and I). Spectra were obtained from the peaks of products shown in Figures 2 and 3. The suggested chemical structures and their fragments of these products are included in the figure.

Similarly, 4′MeF and 4′Me57diOHF were oxidized by CYP1B1 and CYP2A13 (Figure 3). 4′MeF was oxidized to form 4′OHF and 3′4′diOHF by CYP1B1 and 2A13, at a faster rate with CYP1B1 than CYP2A13 (Figures 3A and 3B). A very small amount of m/z 269 product was detected with CYP1B1 (Figure 3B). In contrast to 3′Me57diOHF (as a substrate), 4′Me57diOHF was not oxidized to a large extent to form 4′57triOHF (acacetin) with CYP1B1 or CYP2A13 (Figures 3C and 3G) and the m/z 269 product (Figures 3D and 3E).

Figure 3.

Figure 3.

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LC-MS/MS analysis of oxidation of 4′MeF (A, B, E, and F) and 4′Me57diOHF (C, D, G, and H) by CYP1B1 (A-D) and CYP2A13 (E-H). Detection of different products was by fragment ions (m/z) in the mass spectra, as indicated in the figures. In parts A, E, C, and G, the full-scale peak intensity was 2 × 106, while the scale in parts B, F, D, and H, the scale was 2 × 104, due to lower amounts of formation in the latter cases.

Fragment ion spectra of various oxidation products of 57diOHF, 3′MeF, 4′MeF, 3′Me57diOHF, and 4′Me57diOHF catalyzed by CYP1B1 and 2A13

The fragment ion spectra of various products of 57diOHF, 3′MeF, 3′Me57diOHF, 4′MeF, and 4′Me57diOHF incubated with CYP1B1 and 2A13 were determined (Figure 4) and their chemical structures and key fragments are shown. (Information about the spectra obtained either with CYP1B1 and 2A13 and from either Figure 2 or 3 is included in the right side of each of the figures.) The spectra of products of 567triOHF (Figure 4A), 4′57triOHF (Figure 4B), 3′4′diOHF (Figure 4C), 3′OHF (Figure 4D), and 4′OHF (Figure 4E), obtained either with CYP1B1 or 2A13, were identified using standard chemicals (results not shown). We obtained small amounts of m/z 269 materials on incubation of 3′MeF and 4′MeF with P450s and found that these products were oxidized on the A-ring of these flavonoids because of the presence of fragments of m/z 226 and 254 (Figure 4F) and of m/z 137, 226, and 253 (Figure 4I) on MS/MS (for the basis of assignment of flavone structures, see Nagayoshi et al. 2019b and also Lewars and March 2007, Burns et al. 2007, and van der Hooft et al. 2011). The spectrum of 3’57triOHF (Figure 4G) (from O-demethylation of 3′Me57diOHF) was found to be similar to that of 4’57triOHF (obtained from incubation of 57diOHF and of 4′Me57diOHF with P450s) (Figure 4B and 4H). Finally, we obtained an m/z 301 product on incubation of 3′Me57diOHF with P450s, and the spectra indicated that this was oxidized on the A-ring of this flavonoid because of the presence of fragments of m/z 258 and 286 (Figure 4J) (Nagayoshi et al. 2019b).

LC-MS/MS analysis of oxidation of 3′4′diMeF by CYP1B1 and 2A13

LC-MS/MS analysis showed that various products were formed when 3′4′diMeF was incubated with CYP1B1 (Figure 5A–5G) and CYP2A13 (Figure 5H–5N); the former enzyme was more active than the latter. The major products obtained were 3′4′diOHF (Figure 5A, m/z 255) and four m/z 299 products (Figures 5B and 5I) that were suggested to be ring oxidation products. These major products were formed with peak intensities on the order of 8 × 106 (Figures 5A, 5B, 5H, and 5I), while other minor products were on the order of 8 × 105 (Figures 5C–5G and 5J–5N).

Figure 5.

Figure 5.

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LC-MS/MS analysis of oxidation of 3′4′diMeF by CYP1B1 (A-G) and CYP2A13 (H-N). The products of 3′4′diOHF (A and H, as determined by m/z 255) and of the m/z 299 products (B and I). These products were found to be the major products and the peak intensity scale is 8 × 106 order, while for other products (m/z 269 (C and J), m/z 271 (D and K), m/z 285 (E and L), m/z 301 (F and M), and m/z 315 (G and N)) the full scale is 8 × 105.

We found various minor products having m/z 269, 271, 285, 301, and 315 peaks and, among these products determined, there were four m/z 285 products that are suggested to be oxidized on either the A, B, or C ring, based on the m/z 269 products (3′Me4′OHF or 4′Me3′OHF) (vide infra) (Figures 5C–5G) (Nagayoshi et al. 2019b).

Fragment ion spectra of various oxidation products of 3′4′diMeF formed by CYP1B1 and 2A13

The above oxidation products of 3′4′diMeF formed by CYP1B1 were characterized by fragment ion spectra using LC-MS/MS (Figure 6). As expected, the m/z 255 product was 3′4′diOHF (Figure 6A) and the m/z 269 products were either 3′Me4′OHF or 4′Me3′OHF, based on analysis of the spectra (Figure 6B). The m/z 271 product is suggested to be oxidized on the A-ring of 3′47diOHF (Figure 6C). We obtained four m/z 285 products, suggested to be ring-oxidized, either 3′Me4′OHF or 4′Me3′OHF (Figures 6D–6G). As described above, we obtained four m/z 299 products and these are suggested to be ring oxidation products based on the spectra (Figures 6H–6K). Finally, the m/z 301 and 315 products are suggested to have three hydroxyl group and one methoxy moiety and two hydroxyl group and two methoxy moieties, respectively (Figures 6L and 6M).

Figure 6.

Figure 6.

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Fragment ion spectra of products of oxidation of 3′4′diMeF by CYP1B1. The products with parent ions at m/z 255 (A, 3′4′diOHF), m/z 269 (B), m/z 271 (C), m/z 285a-285d) (D-G), m/z 299 (a-d) (H-K), m/z 301 (L), and m/z 315 (M)) are shown, along with structures of fragment ions.

LC-MS/MS analysis of oxidation products of 3′4′diMe57diOHF with CYP1B1 and 2A13

The oxidation products of 3′4′diMe57diOHF formed by CYP1B1 and CYP2A13 were examined by analysis with LC-MS/MS (Figure 7). In contrast to the case of 3′4′diMeF (Figure 5), the addition of a 57diOH moiety to 3′4′diMeF (i.e., 3′4′diMe57diOHF) caused decreased oxidation by CYP1B1 and CYP2A13 (Figures 7A and 7C). We detected only small amounts of products, suggested to be O-demethylated either at the 3′Me or 4′Me position, namely 3′Me4′57triOHF and 4′Me3′57triOHF, respectively (Figure 7D); only the fragment ion spectra of 3′Me4′57triOHF (Figure 7D) and 3′4′diMe57diOHF (Figure 7B) are shown in the figure.

Figure 7.

Figure 7.

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LC-MS/MS analysis of 3′4′diMe57diOHF oxidation products with CYP1B1 (A) and CY2A13 (C) and fragment ion spectra of 3′4′diME57diOHF (B, m/z 315) and its products (D, m/z 301). The products could be either 3′Me4’57triOHF or 4′Me3′57triOHF, and 3′Me4′57triOHF is shown as a model.

Rates of product formation from seven flavonoids by CYP1B1 and 2A13

Chemical standards of 4′OHF, 3′4′diOHF, 4′57triOHF, and 567triOHF were available and could be used to determine and compare rates of product formation catalyzed by CYP1B1 and CYP2A13 with three separate determinations (Table 1). CYP1B1 and CYP2A13 oxidized 57diOHF to form 567triOHF at rates of 2.2 min−1 and 0.041 min−1, respectively, while formation of 4’57triOHF was catalyzed by CYP2A13 at a rate of 0.79 min−1 (Table 1). When 3′MeF and 4′MeF were used as substrates, CYP1B1 produced 3′4′diOHF at rates of 2.6 and 3.2 min−1, respectively, and CYP2A13 at rates of 0.32 and 0.33 min−1, respectively (Table 1). 4′MeF was converted to 4′OHF by CYP1B1 and CYP2A13 at rates of 3.2 and 1.1 min−1, respectively. Formation of 4′57diOHF from 4′Me57diOHF was faster with CYP2A13 than CYP1B1. CYP1B1 and CYP2A13 oxidized 3′4′diMeF to form 3′4′diOHF at rates of 4.2 and 0.18 min−1, respectively.

Table 1.

Rates of oxidation of flavonoids by CYP1B1.1 and CYP2A13

Substrate Product Rate of product formation (nmol product /min/nmol P450)
CYP1B1 CYP2A13
57diOHF 4′57triOHF <0.001 0.79 ± 0.096
567triOHF 2.2 ± 0.40 0.041 ± 0.013
3′MeF 3′4′diOHF 2.6 ± 0.35 0.32 ± 0.081
4′MeF 4′OHF 3.2 ± 0.40 1.1 ± 0.20
3′4′diOHF 1.8 ± 0.35 0.33 ± 0.068
4′Me57diOHF 4′57diOHF 0.098 ± 0.010 0.42 ± 0.060
3′4′diMeF 3′4′diOHF 4.2 ± 0.31 0.18 ± 0.025
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Data are means ± S.D. of three separate determinations.

Molecular docking analysis of interaction of 3′4′diMeF with CYP1B1 and 2A13

Docking analysis suggested that there were two possible interactions between 3′4′diMeF and the active site of the CYP1B1 protein. First, both the 3′- and 4′- positions at B-ring were fit well in the active site of CYP1B1; the distances of these positions from the iron center of CYP1B1 were 5.13 and 4.90 Å, respectively, and the lowest ligand-interaction energy (U value) of this interaction was found to be –65.5, very low for possible oxidation reactions (Figure 8A). In addition, the 6- and 7-positions on the A-ring of 3′4′diMeF could interact with the active site of CYP1B1; the distances of these positions from the iron center of CYP1B1 were 4.28 and 4.51 Å, respectively, and the U value of interaction was –37.5, as the 51st of 100 docking solutions (Figure 8B). On the other hand, we found that U values for interaction of CYP2A13 with the B-ring and A-ring of 3′4′diMeF were 22.6 and 12.7 (results not shown), suggested to be unfavorable for oxidation reactions.

Figure 8.

Figure 8.

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Molecular docking analysis of two possible interaction of 3′4′diMeF with CYP1B1 in tow poses (A and B).

Discussion

We have previously shown that several flavonoids—including flavanone, flavone, 5-hydroxyflavone, 57diOHF, 2′MeF, 3′MeF, 4′MeF, and 2′-, 3′-, 4′-, and 6-hydroxyflavanones— are oxidized to various products by different forms of human P450 enzymes and that individual forms of P450 enzymes have different, but overlapping, substrate specificities in these oxidation reactions (Kakimoto et al., 2019; Nagayoshi et al., 2019a; 2019b; 2020; 2021; Shimada et al., 2021). For example, CYP Family 1 enzymes, including CYP1A1, 1A2, 1B1.1, and 1B1.3, are the major enzymes involved in 6-hydroxylation of 57diOHF to form 567triOHF, while CYP2A13 and 2A6 catalyze 4′-hydroxylation to form 4′57triOHF (Nagayoshi et al., 2019b). On the other hand, 3′MeF and 4′MeF are O-demethylated by these CYP1 and CYP2A13 enzymes to form 3′OHF and 4′OHF, respectively, and then 3′4′diOHF (Nagayoshi et al., 2020; 2021). Collectively, among several human P450 enzymes examined so far, CYP1B1 and 2A13 have been shown to be key enzymes in understanding the basis of oxidation of various flavonoids that contain hydroxyl- and/or O-methoxy-groups in the molecule (Nagayoshi et al., 2019b: 2020; 2021).

In this study, we examined the oxidation of 3′MeF, 4′MeF, and 3′4′diMeF by CYP1B1 and CYP2A13 when a 5,7diOHF group was substituted in these methoxylated flavonoids. As described above, 3′MeF and 4′MeF were O-demethylated by CYP1B1 and CYP2A13 to produce 3′OHF and 4′OHF and then 3′4′diOHF, with CYP1B1 being slightly more active than CYP2A13 (Figures 2 and 3). When 3′Me57diOHF and 4′Me57diOHF (acacetin) were used as substrates, we detected 3′57triOHF and 4′57triOHF, respectively, by CYP1B1 and CYP2A13 and found that CYP2A13 was more active than CYP1B1; the formation of 4′57triOHF by CYP1B1 and 2A13 was at rates of 0.098 min−1 and 0.42 min−1, respectively (Figures 2 and 3). CYP2A13 was active in converting 57diOHF to 4′57triOHF, but CYP1B1 had very low activity in this reaction (Figure 3). Although the rates of formation of 3′57triOHF from 3′Me57diOHF could not be determined in the absence of a standard chemical, the peak intensities seen with LC-MS/MS suggest that the rates are higher than those for formation of 4′57triOHF (Figure 2). It is not known whether 4′Me57diOHF (acacetin) and 3′Me57diOHF require metabolic activation by P450 enzymes to exert several biological activities (Kim et al., 2014; Bu et al., 2019; Chien et al., 2011).

CYP1B1 oxidized 3′4′diMeF more efficiently than CYP2A13 in forming various products, including 3′4′diOHF and at least four ring-oxidation products having a fragment ion at m/z 299. Among these m/z 299 products determined, three (299a, 299b, and 299d) are suggested to be products oxidized on the A ring, because of the presence of a fragment ion of m/z 137 (Nagayoshi et al., 2019b). Both 3′4′diOHF and the m/z 299 products are suggested to be the major ones formed on incubation of 3′4′diMeF with CYP1B1, because of the high peak intensities on the order of 2 × 106 (Figure 5), as compared with other products on the order of 2 × 105. The molecular docking analysis supported the conclusion that the 3′- and 4′-positions at B-ring and 6- and 7-positions at A-ring of 3′4′diMeF are close to the iron in the active site of CYP1B1, suggesting that 3′4′diMeF can be both O-demethylated and ring oxidized by CYP1B1.

It is not known whether or not these oxidative metabolites are more biologically active than the parent 3′4′diMeF. 3′4′diMeF has been reported to be an aryl hydrocarbon receptor antagonist in human breast cancer cells (Lee and Safe, 2000; Roblin et al., 2004), to cause inhibition of DNA binding by benzo[a]pyrene in human bronchial epithelial cells (Tsuji and Walle, 2006), and to prevent decreases in cell viability of HeLa and SH-SY5Y cells caused by N-methyl-N′-nitro-N-nitrosoguanine, which induces parthanatos (a form of programmed cell death) (Fatokun et al., 2013; 2014). 3′4′diOHF has also been reported to act as an antioxidant and antiapoptotic agent (Lee et al., 2011; Jomová et al., 2019) and to inhibit cell proliferation and in vitro angiogenesis in normal and tumor cells (Fotsis et al., 1997). Further work is needed to examine the roles that these oxidative products play in the biological activities of 3′4′diMeF.

A pathway for oxidation of 3′4′diMeF by CYP1B1 is suggested (Figure 9). 3′4′diMeF was mainly oxidized to form 3′4′diOHF and several m/z 299 products; the resulting 3′4′diOHF is further oxidized to tri-hydroxylated flavones (m/z 271) and the m/z 299 products to m/z 315 products. Small amounts of m/z 269 products, suggesting the formation of products that are demethylated at either the 3′- or 4′-position (3′Me4′OHF or 4′Me3′OHF), might be further oxidized to 3′4′diOHF (m/z 255), 3′MediOHF (m/z 285), and 4′MediOHF (m/z 285) products and then to the m/z 301 product (Figure 9).

Figure 9.

Figure 9.

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Proposed pathway for oxidation of 3′4′diMF by P450 enzymes. The protons are added to each compound to indicate the parent ion seen in the mass spectrometer (positive ion).

The introduction of a 57diOHF moiety into 3′4′diMeF, namely 3′4′diMe57diOHF, was found to decrease oxidation by CYP1B1 and 2A13; we detected only small amounts of the demethylated products (3′Me4′57triOHF and 4′Me3′57triOHF) with both P450 enzymes. It is interesting to note our previous results, in which we reported that 3′4′diMe57diOHF is more potent than 3′4′diMeF in inhibiting 7-ethoxyresorufin O-deethylation activities catalyzed by CYP1A1, CYP1A2, and CYP1B1.3 (a variant form of CYP1B1) (Shimada et al., 2010). In contrast, 57diOHF was reported to inhibit these P450 enzymes significantly and was oxidized by CYP1A1, CYP1A2, and CYP1B1 (wild type) to form 567triOHF at high rates (Shimada et al., 2010; Nagayoshi et al., 2019b; 2020; present results).

In conclusion, our present results showed that the O-demethylation reactions are likely to be faster than the ring oxidation reactions with methoxylated flavones (including 3′4′diMeF, as well as 3′MeF and 4′MeF), and that introduction of a 57diOHF moiety into 3′4′diMeF caused decreased oxidation by CYP1B1 and 2A13. Both 3′Me57diOHF and 4′Me57diOHF (acacetin) were easily demethylated by CYP1B1 and 2A13 to form 3′57triOHF and 4′57triOHF (apigenin) at rates of 0.098−1 and 0.42 min−1, respectively. Finally, 3′4′diMeF was found to be good substrate for the oxidation by CYP1B1, but not CYP2A13, and it was found that 3′47diMeF was mainly demethylated to form 3′4′diOHF and also ring-oxygenated to form several m/z 299 products. Our present results showing demethylation and oxidation of methoxylated flavonoids by P450 enzymes may be of use for understanding mechanisms by which these flavonoids play important roles in various biological activities in mammals including humans.

Acknowledgements

The authors thank Dr. Maryam Foroozesh for her supply of flavonoid derivatives used in this study.

Funding

This study was supported in part by JSPS KAKENHI [16K21710] (to H. N), [17K08426] (to N. M.), [17K08425] (to H. Y.), and [15K07770] (to S. T.), 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,

3′4′diMeF

3′,4′-dimethoxyflavone

3′4′diMe57diOHF

3′,4′-dimethoxy-5,7-dihydroxyflavone

3′MeF

3′-methoxyflavone

4′MeF

4′-methoxyflavone

3′Me57diOHF

3′-methoxy-5,7-dihydroxyflavone

4′Me57diOHF

4′-Me57dihydroxyflavone

57diOHF

5,7-dihydroxyflavone

3′4′diOHF

3′,4′-dihydroxyflavone

P450 or CYP

cytochrome P450

b 5

cytochrome b5

DLPC

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

Footnotes

Declaration of interest Statement

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

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