Metabolic activation and deactivation of dietary-derived coumarin mediated by cytochrome P450 enzymes in rat and human liver preparations

Abstract

Dietary-derived coumarin is of clinical interest for its potential hepatotoxicity in humans because such toxicity is especially evident in rats. In this study, the oxidative metabolism of coumarin to active coumarin 3,4-epoxide (as judged by the formation rates of o-hydroxyphenylacetic acid) and excretable 7-hydroxycoumarin was investigated in liver fractions from rats and humans. In rat liver microsomes, the formation rate of o-hydroxyphenylacetic acid (~6 pmol/min/mg microsomal protein) from coumarin at 10 μM was dependent on the presence of liver cytosolic fractions. Rat hepatocytes mediated similar formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin (~0.1 nmol/hr/10⁶ cells) at 0.20–20 μM coumarin. Human hepatocytes mediated the biotransformation of coumarin to o-hydroxyphenylacetic acid at roughly similar rates to those of rat hepatocytes. In contrast, the formation rates of 7-hydroxycoumarin by human hepatocytes were around 10-fold higher at ~1 nmol/hr/10⁶ cells. In the presence of human liver cytosolic fractions, the oxidative formation rate of o-hydroxyphenylacetic acid was relatively high in cytochrome P450 (P450) 1A2-rich human liver microsomes. The inhibitory effects of furafylline/α-naphthoflavone and 8-methoxypsoralen, P450 1A2 and 2A6 inhibitors, respectively, were seen on the rates of o-hydroxyphenylacetic and 7-hydroxylation formations, respectively, in pooled human liver microsomes. Human liver microsomes selectively inactivated for P450 1A2 and 2A6 showed low rates of o-hydroxyphenylacetic acid and 7-hydroxylation formation (~20–30% of control), respectively. Among the P450 isoforms tested, recombinant human P450 1A2 predominantly mediated o-hydroxyphenylacetic formation. These results suggested that the metabolic activation and deactivation of coumarin were mediated mainly by P450 1A2 and 2A6 enzymes, respectively. The metabolic oxidation of coumarin via 3,4-epoxidation forming o-hydroxyphenylacetic acid could inform individual human risk assessments of dietary-derived coumarin, for which hepatotoxicity is especially evident in rats.

INTRODUCTION

Coumarin (Fig. 1A) is a naturally occurring organic chemical found in food products and various plants such as cinnamon and flowering cherry (Lake, 1999; Abraham et al., 2010). It is estimated that coumarin is used in 90% of cosmetic and other fragranced products (Born et al., 2000). A risk assessment case study for coumarin in cosmetic products has been reported (Baltazar et al., 2020). Although it reportedly causes hepatotoxicity in rats via the reactive metabolite coumarin 3,4-epoxide (Rietjens et al., 2010; Vassallo et al., 2003), coumarin has been used as a medicine in humans, its tolerable daily intake being 0.1 mg/kg body weight (Abraham et al., 2010). Apparent species difference should be noted between the rapid coumarin 7-hyroxylation in humans (Peamkrasatam et al., 2006; Ujjin et al., 2002; Kiyotani et al., 2003) and the slow coumarin 7-hyroxylation seen in rats. Because the 7-hyroxylation of coumarin is slow in rats, the activation of coumarin to its reactive 3,4-epoxide assumes greater importance for an anticoagulant rodenticide.

Fig. 1

Metabolic pathways of coumarin (A) and a representative LC-UV chromatogram (B) for coumarin and its oxidative metabolites o-hydroxyphenylacetic acid and 7-hydroxycoumarin in pooled human liver microsomes and cytosolic fractions. In panel B, peaks 1, 2, 3, and 4 correspond to o-hydroxyphenylacetic acid, o-hydroxyphenylethanol, 7-hydroxycoumarin, and coumarin, respectively, in a reversed-phase analysis.

In in vitro experiments using human liver microsomes, coumarin was extensively eliminated via 7-hydroxylation mediated by cytochrome P450 (P450) 2A6 (Yamazaki et al., 1994), with a reported minor contribution of 3,4-epoxidation by P450 2E1 (Born et al., 2002). In contrast, in experiments with rat liver microsomes, coumarin elimination was slow and occurred mainly via 3,4-epoxidation forming o-hydroxyphenylacetic acid as the major metabolite (Born et al., 2000; Tanaka et al., 2017). We recently reported the pharmacokinetics of coumarin after virtual oral administrations were modeled in humans (Miura et al., 2020) based on the reported plasma concentrations in a rat study in which the main biotransformation of coumarin was to o-hydroxyphenylacetic acid (Tanaka et al., 2017). It has been reported that mouse and rat liver cytosolic fractions in in vitro experiments could mediate detoxication of coumarin 3,4-epoxide produced by liver microsomes by forming o-hydroxyphenylacetic acid (Vassallo et al., 2004). Although clinical data on hepatotoxicity in patients treated with coumarin as a medicinal drug and data on healthy subjects under the consumption of dietary foodstuff are available, there is no in-depth report on the in vitro metabolic profiles of coumarin in human liver fractions in comparison with those in rat liver fractions.

The aim of the present study is to focus on the roles for responsible oxidation enzymes for activation and deactivation of coumarin in humans. In the current study, we investigated coumarin oxidations via 3,4-epoxidation forming o-hydroxyphenylacetic acid and 7-hydroxycoumarin in liver preparations such as 9000 × g supernatant fractions (S9), liver microsomes, liver cytosolic fractions, and hepatocytes from rats and humans. We report herein activation and deactivation of coumarin mediated mainly by cytochrome P450 enzymes, especially in human liver preparations.

MATERIALS AND METHODS

Chemicals and enzymes

o-Hydroxyphenylacetic acid, o-hydroxyphenylethanol, 7-hydroxycoumarin glucuronide, and 7-hydroxycoumarin sulfate were purchased from Toronto Research Chemicals (Toronto, Canada). Other chemicals were obtained from the sources reported previously (Miura et al., 2020). Rat liver microsomes and liver cytosolic and S9 fractions were prepared from 7-week-old, male Sprague-Dawley rats (Miura et al., 2019); this study was approved by the Ethics Committee of Showa Pharmaceutical University. Individual and pooled human liver microsomes (HH2, low P450 2A6 activity; HH31, high P450 1A2 activity; and H150; pooled) and pooled liver S9 fractions were obtained from Corning Life Sciences (Woburn, MA, USA). Sets of control pooled human liver microsomes and liver microsomes selectively inactivated for P450 1A2, 2A6, and 3A4 (pretreated with mechanism-based inhibitors, Silensomes) were obtained from Biopredic International (Rennes, France). Human liver cytosolic and S9 fractions used were prepared previously (Okubo et al., 2013; Yamazaki et al., 2015). Recombinant human P450 1A2, 2A6, 2E1, and 3A4 were purchased from Corning Life Sciences. Cryopreserved rat and human hepatocytes, obtained from Biopredic International, were cultured under recommended medium conditions based on Williams E medium.

In vitro coumarin oxidation

In vitro oxidation rates of coumarin catalyzed by liver preparations and hepatocytes from rats or humans were measured using liquid chromatography (LC) with fluorescence and ultraviolet detectors (Yamazaki et al., 1999) with an analytical reversed-phase column. Briefly, coumarin (10 µM, unless otherwise specified) was incubated with liver microsomes (0.50 mg protein/mL) in the presence or absence of pooled liver cytosolic fractions (1.0 mg/mL) or liver S9 fractions (1.0 mg/mL) and an NADPH-generating system with or without uridine diphosphate glucuronic acid (UDP-GA, 2.0 mM) or 3’-phosphoadenosine-5’-phosphosulfate (0.20 mM) in a total of 0.50 mL of 100 mM potassium phosphate buffer (pH 7.4) at 37°C for 15 min. The effects of furafylline/α-naphthoflavone, 8-methoxypsoralen, 4-methylpyrazole, and ketoconazole (Sigma-Aldrich, Milwaukee, WI, USA), P450 1A2, 2A6, 2E1, 3A inhibitors (Yamazaki et al., 1992; Okubo et al., 2016), respectively, on coumarin oxidation activities were also investigated in human liver microsomes. Coumarin (0.20, 2.0, and 20 µM) was incubated in the culture medium for hepatocytes (5 × 10⁵ cells/well) at 37°C for 2 hr at 95% humidity and 5% CO₂ in a 0.50-mL reaction mixture. After test solutions were treated with an equal volume of acetonitrile, the aqueous supernatant was centrifuged at 2 × 10³ g for 10 min at 4°C and analyzed.

Determination of in vitro coumarin metabolites

LC conditions with an octadecylsilane (C₁₈) column (5 µm, 2.0 mm × 250 mm) were as follows: solvent A was acetonitrile and solvent B was 20 mM NaClO₄ (pH 2.5). The following gradient program was used, with a flow rate of 1.0 mL min^-1: 0–5 min, hold at 15% A; 5.1–11 min, linear gradient from 15% A to 32% A; 11.1–17 min, hold at 32% A; 17.1–18 min, linear gradient from 32% A to 15% A; and 18.1–25 min, hold at 15% A. The temperature of the column was maintained at 40°C. Samples (10 µL) were injected with an auto-sampler. The retention times of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin sulfate (with fluorescence detection), o-hydroxyphenylacetic acid, o-hydroxyphenylethanol, 7-hydroxycoumarin, and coumarin (with fluorescence and ultraviolet detection) were 8.5, 8.7, 13.8, 14.2, 15.0, and 19.0 min, respectively. Kinetic profiles for coumarin oxidation by recombinant human P450 enzymes were estimated by fitting the data to the Michaelis–Menten equation and mean differences were assessed using Prism (GraphPad Software, La Jolla, CA, USA).

RESULTS AND DISCUSSION

Figure 1A shows the reported metabolic pathways of coumarin and its metabolites (Born et al., 2000) as detected in plasma from rats after oral administration (Tanaka et al., 2017). Figure 1B illustrates a representative LC chromatogram obtained in this study for coumarin and its oxidative metabolites o-hydroxyphenylacetic acid and 7-hydroxycoumarin in pooled human liver microsomes in the presence of liver cytosolic fractions. In the current study, the formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin were investigated in rat and human liver preparations.

Coumarin oxidation rates by liver microsomes, liver S9 fractions, and hepatocytes from rats were investigated and are shown in Fig. 2. At a coumarin concentration of 10 μM in rat liver microsomes in the absence or presence of rat liver cytosolic fractions, the formation rates of 7-hydroxycoumarin were as low as ~0.2 pmol/min/mg microsomal protein (Fig. 2A). However, the formation rate of o-hydroxyphenylacetic acid, which was below the limit of detection in rat liver microsomes (< 0.01 pmol/min/mg microsomal protein) in the absence of rat liver cytosolic fractions, became as high as ~6 pmol/min/mg microsomal protein in the presence of liver cytosolic fractions (Fig. 2B). When liver S9 fractions were used as the enzyme source, the formation rates of 7-hydroxycoumarin and o-hydroxyphenylacetic acid were roughly similar (~4 pmol/min/mg S9 protein, Fig. 2C). Some of the 7-hydroxycoumarin formed by liver S9 fractions containing conjugating enzymes was in the form of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulfate. Rat hepatocytes mediated similar formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin (~0.1 nmol/hr/10⁶ cells) at 0.20, 2.0, and 20 μM coumarin (Fig. 2D).

Fig. 2

Coumarin oxidation rates by liver microsomes, liver S9 fractions, and hepatocytes from rats. The coumarin oxidation activities of rat liver preparations are shown at substrate concentrations of 10 μM in liver microsomes with or without rat liver cytosolic fractions and in liver S9 fractions fortified with UDP-GA and 3’-phosphoadenosine-5’-phosphosulfate (A-C) and at substrate concentrations of 0.20, 2.0, and 20 μM in rat hepatocytes (D). Data are mean values of triplicate determinations (and SD values for cell assays). Some 7-hydroxycoumarin formed in the presence of conjugating enzymes was transformed to 7-hydroxycoumarin glucuronide (light shaded) or 7-hydroxycoumarin sulfate (dark shaded).

In contrast, the more extensive coumarin oxidation activities of human liver microsomes are shown in Fig. 3A. Some 7-hydroxycoumarin formed extensively in the presence of conjugating enzymes and cofactors was converted to 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulfate (Fig. 3B). In the experiments with S9 fractions from rat and human livers, roughly similar rates of biotransformation of coumarin to o-hydroxyphenylacetic acid (~2 pmol/min/mg S9 protein) were evident. In rat liver S9 fractions, 7-hydroxylation activity was low, whereas human liver S9 fractions exhibited much higher 7-hydroxylation activities (~200 versus ~2 pmol/min/mg protein, Fig. 3C). At 0.20, 2.0, and 20 μM coumarin, human hepatocytes also mediated rapid formation rates of 7-hydroxycoumarin compared with rat hepatocytes (~1 versus ~0.1 nmol/hr/10⁶ cells). In contrast, the formation rates of o-hydroxyphenylacetic acid (Fig. 3D) by human and rat hepatocytes were similar at ~0.1 nmol/hr/10⁶ cells. The difference should be noted between the rapid oxidative elimination of coumarin via 7-hydroxylation in humans (Fig. 3), compared with rats (Fig. 2), in which the 7-hyroxylation of coumarin is slow in rats; however, the oxidation rates for the activation of coumarin to reactive 3,4-epoxide were similar in rat and human hepatocytes, as judged by the formation rates of o-hydroxyphenylacetic acid.

Fig. 3

Coumarin oxidation rates by liver microsomes, liver S9 fractions, and hepatocytes from humans. Coumarin oxidation activities of human liver preparations are shown at substrate concentrations of 10 μM in liver microsomes with or without human liver cytosolic fractions and in liver S9 fractions fortified with UDP-GA and 3’-phosphoadenosine-5’-phosphosulfate (A-C) and at substrate concentrations of 0.20, 2.0, and 20 μM in human hepatocytes (D). Data are mean values of triplicate determinations (and SD values for cell assays). Some 7-hydroxycoumarin formed in the presence of conjugating enzymes was transformed to 7-hydroxycoumarin glucuronide (light shaded) or 7-hydroxycoumarin sulfate (dark shaded).

The contributions of P450 enzymes to the formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin from coumarin were determined in individual and pooled human liver microsomes in the presence of human liver cytosolic fractions (Table 1). As expected, liver microsomes from a P450 2A6 poor metabolizer, HH2, showed low 7-hydroxylation activity toward coumarin; for which the formation ratio of 7-hydroxycoumarin to that of o-hydroxyphenylacetic acid was 9.5. The oxidative formation rate of o-hydroxyphenylacetic acid was high in liver microsomes from a P450 1A2 extensive metabolizer, HH31, for which the formation ratio of 7-hydroxycoumarin to o-hydroxyphenylacetic acid was 63. In contrast, a formation ratio of 7-hydroxycoumarin to o-hydroxyphenylacetic acid of 130 was seen in pooled human liver microsomes. Inhibitory effects of furafylline/α-naphthoflavone and 8-methoxypsoralen, P450 1A2 and 2A6 inhibitors, respectively, were seen on the rates of o-hydroxyphenylacetic acid formation and 7-hydroxylation. In separated experiments, liver microsomes selectively inactivated for 2A6 showed low activities of 7-hydroxylation, whereas those selectively inactivated for P450 1A2 showed low activities of o-hydroxyphenylacetic acid formation (~20% of control in each case) (Table 1).

Table 1. Oxidation rates of coumarin to its metabolites in individual and pooled human liver microsomes in the presence of liver cytosolic fractions.

Human liver microsomes	Inhibitor of P450 form	Product formation, pmol/min/mg microsomal protein		Formation ratio of 7-hydroxy-coumarin to that of o-hydroxy-phenylacetic acid
		7-Hydroxy-coumarin	o-Hydroxyphenyl-acetic acid
Experiment I
Individual liver microsomes HH2a		36	3.8	9.5
Individual liver microsomes HH31b		430	6.8	63
Pooled human liver microsomes H150		560 (100)	4.4 (100)	130
+ 10 µM furafylline	1A2	480 (86)	0.66 (15) *	730
+ 30 µM α-naphthoflavone	1A2	430 (77)	2.4 (55) *	180
+ 1.0 µM 8-methoxypsoralen	2A6	98 (18) *	3.2 (73)	31
+ 30 µM 4-methylpyrazole	2E1	550 (98)	2.6 (59) *	210
+ 1.0 µM ketoconazole	3A4	450 (80)	3.2 (73)	140
Experiment II
Non-inactivated control		470 (100)	2.3 (100)	200
Selectively inactivated by mechanism-based P450 1A2 inhibitor	1A2	360 (77)	0.80 (35) *	450
Selectively inactivated by mechanism-based P450 2A6 inhibitor	2A6	68 (15) *	1.7 (74)	40
Selectively inactivated by mechanism-based P450 3A4 inhibitor	3A4	490 (105)	3.0 (117)	160

Coumarin oxidation activities (at 10 μM) of human liver microsomes were determined in the presence of liver cytosolic fractions without UDP-GA or 3’-phosphoadenosine-5’-phosphosulfate as cofactors in triplicate determinations. ^a Low P450 2A6 activity. ^b High P450 1A2 activity. Numbers in parentheses are the percentage of the control, * p < 0.05.

Kinetic profiles for coumarin oxidation by recombinant human P450 enzymes were determined (Fig. 4). Recombinant human P450 1A2 and 2E1 were the dominant forms mediating o-hydroxyphenylacetic acid formation from coumarin among the P450 isoforms tested. Calculated K_m, V_max, and V_max/K_m values for o-hydroxyphenylacetic acid formation by recombinant human P450 1A2 and 2E1 were 2.1 ± 1.0 mM and 0.56 ± 0.30 mM; 11 ± 3 min^-1 and 2.1 ± 0.4 min^-1; and 5.2 min^-1/mM and 3.8 min^-1/mM, respectively, under the present conditions.

Fig. 4

Kinetic analyses of the formation of 7-hydroxycoumarin (A) and o-hydroxyphenylacetic acid (B) mediated by recombinant human P450 enzymes in the presence of human liver cytosolic fractions. Coumarin oxidations were determined for recombinant human P450 1A2 (open circles), 2E1 (triangles), 3A4 (squares), and 2A6 (closed circles) (5.0 nM) in the presence of human liver cytosolic fractions (1.0 mg/mL) without UDP-GA or 3’-phosphoadenosine-5’-phosphosulfate as cofactors.

In humans, coumarin was reportedly eliminated mainly via 7-hydroxylation by P450 2A6 (Yamazaki et al., 1994) with a minor contribution of 3,4-epoxidation by P450 2E1 (Born et al., 2002). In the current study, experiments with human liver preparations gave formation ratios of 7-hydroxycoumarin to o-hydroxyphenylacetic acid in the range 10–200. Variable ratios of urinary 7-hydroxycoumarin and o-hydroxyphenylacetic acid after oral administration with coumarin (2 mg) have been reported (Hadidi et al., 1998). Our findings suggested that metabolic activation and deactivation of coumarin was mediated mainly by P450 1A2 and 2A6 enzymes (Table 1). Although human P450 1A2 was the dominant form mediating o-hydroxyphenylacetic acid formation, a possible role of P450 2E1 in o-hydroxyphenylacetic acid formation in human liver microsomes could not be ruled out, because recombinant P450 2E1 had some activity toward o-hydroxyphenylacetic acid formation (Fig. 4B). However, it should be noted that the contribution of P450 2E1 is likely limited, as evidenced by the low suppressive effects of P450 2E1 inhibitor 4-methylpyrazole (Table 1), without any liver microsomes selectively inactivated by a P450 2E1 inhibitor commercially available. Further work following the current approach should clarify the metabolic balance and clearance rates of these activated/deactivated metabolites of food-derived coumarin, the production of which is mediated by P450 enzymes.

In conclusion, in the present study, the metabolic oxidations of coumarin to 7-hydroxycoumarin and its active 3,4-epoxide (as judged by the formation rates of o-hydroxyphenylacetic acid) were investigated. Under the current conditions, in humans, the metabolic ratios of coumarin to o-hydroxyphenylacetic acid, generated via 3,4-epoxidation, were two orders of magnitude less than those to 7-hydroxycoumarin. It is evident that the metabolic activation of coumarin to its active 3,4-epoxide by liver microsomal P450 1A2 does occur in humans in a similar manner to that in rats (Figs. 2D and 3D). Further studies will be of use to investigate whether P450 2A6 poor metabolizers and P450 1A2 ultrarapid metabolizers (such as smokers) are at risk from coumarin metabolite-induced hepatotoxicity. The current results suggested that the metabolic oxidation rates of coumarin via 3,4-epoxidation forming o-hydroxyphenylacetic acid mainly by includible liver microsomal P450 1A2 could inform individual human risk assessment of dietary-derived coumarin.

ACKNOWLEDGMENTS

The authors thank Drs. Takashi Yamada, Yusuke Kamiya, and Makiko Shimizu for their assistance and David Smallbones for copyediting a draft of this article. This work was supported in part by the Food Safety Commission of Japan (JPCAFSC20202006) and the METI Artificial Intelligence-based Substance Hazard Integrated Prediction System Project, Japan.

Conflict of interest

The authors declare that there is no conflict of interest.

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