Effect of Chrysin and Natural Coumarins on UGT1A1 and 1A6 Activities in Rat and Human Hepatocytes in Primary Culture

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Chemicals
• Coumarins and flavonoids
• Assay for the cytotoxicity
• Rat hepatocytes isolation and culture
• Human hepatocytes isolation and culture
• Hepatocyte treatment
• Hepatocyte microsome preparation
• Microsomal UGT1A6-dependent p-nitrophenol conjugation (PNP-UGT activity)
• Microsomal UGT1A1-dependent bilirubin conjugation (Bili-UGT activity)
• Protein content
• Statistical procedure
• Results
• Discussion
• References

Abstract

Flavonoids and coumarins are naturally occurring compounds that are widely distributed in vegetables and have a broad pharmacological activity. Inducibility of UDP-glucuronosyltransferases (UGTs) by xenobiotics is well documented and can be considered beneficial for health. In particular, UGT1A1-dependent bilirubin conjugation plays a critical role in the detoxification of neurotoxic bilirubin and phenobarbital-mediated UGT1A1 induction therapy is commonly used in the treatment of unconjugated hyperbilirubinemic diseases such as Crigler-Najjar type II disease. In the present study, the effects of the flavone chrysin and six natural coumarins isolated from various Rutaceous plants on UGT1A6-dependent p-nitrophenol and/or UGT1A1-dependent bilirubin glucuronoconjugation activities were evaluated in cultured rat and human hepatocytes and compared to those of the prototypical UGT1A inducers β-naphthoflavone, phenobarbital and clofibric acid. After 3 days of treatment at a concentration of 25 μM, the pyranocoumarins avicennin and cis-avicennol, and the furocoumarins bergapten and imperatorin, increased by 2-fold UGT1A1-dependent activity, equivalent to the increases obtained with chrysin at 25 μM, whereas in the presence of the simple coumarins such as coumarin or umbelliferone, UGT1A1-dependent activity was not modified. In terms of structural requirements for UGT1A1 induction, the present study suggests that the B-ring (phenyl) for chrysin and the furan or pyran rings for coumarins are essential for the biological activity.

Abbreviations

BCA:bicinchoninic acid

Bili:biliribin

BSA:bovine serum albumin

CLO:clofibric acid

FCS:foetal calf serum

HBSS:Hank’s buffered salt solution

HEPES:4-(2-hydroxyethyl)piperazine-1-ethane sulfonic acid

NF:β-naphthoflavone

PB:Phenobarbital

PBS:phosphate buffered saline

PNP:p-nitrophenol

UDPGA:Uridine 5′-diphosphoglucuronic acid

UGTs:UD-glucuronosyltransferases

Introduction

Uridine diphosphate glucuronosyltransferases (UGTs) [1] are microsomal phase II drug-metabolizing enzymes located in the endoplasmic reticulum of cells from a number of tissues. These enzymes catalyse glucuronidation, the major detoxification mechanism for a wide variety of drugs, environmental chemicals and numerous endogenous compounds. Two UGT families have been described: UGT1 isoforms that mainly catalyse the glucuronidation of bilirubin and exogenous agents (drugs, phenols, pesticides…), and UGT2 isoforms that mainly glucuronidate steroid hormones and bile acids. In humans, glucuronidation capacity is prominently present in the liver, with relatively high levels of most of the various UGT isoforms. Inducibility of these enzymes by various chemicals is well documented [2] and can be considered beneficial for health. Indeed, UGTs are implicated in the metabolism of carcinogens and play also a critical role in the detoxification of neurotoxic bilirubin. Various specific UGT inducers are currently known, including chemicals and drugs, but also food and dietary compounds. These molecules may contribute to a better detoxification of carcinogens and therefore act as cancer chemopreventive agents. For instance, induction of UGT1A6, the isoform involved in the detoxification of carcinogenic polycyclic hydrocarbons and arylamines, has been observed in livers from male F344 rats that consumed green tea [3] and from male Wistar rats supplemented with Brussels sprouts or other dietary compounds [4]. Walle et al. showed that dietary flavonoids, in particular chrysin, can induce UGT1A1 in the human gastrointestinal Caco-2 and hepatic HepG2 cell lines [5], [6]. In the inheritable unconjugated hyperbilirubinemic diseases, known as Gilbert's syndrome and Crigler-Najjar Type I and Type II syndromes, accumulation of bilirubin in the organism leads to jaundice. Crigler-Najjar type I disease, due to a complete lack of UGT1A1, can be fatal, whereas type II syndrome, due to a reduced activity of the enzyme, can frequently be treated by induction therapy with phenobarbital, a specific UGT1A1 inducer [1]. The side effects of phenobarbital, such as a strong sedation, justify the search of alternative drugs for induction therapy.

Coumarins, like flavonoids, constitute a broad class of polyphenolic compounds present in fruits, vegetables, spices and plant-derived beverages or products, and exhibit in general a wide range of pharmalogical properties such as antioxidant, anti-inflammatory, antimicrobial, cytotoxic, anticoagulant [7] or phototoxic activities [8]. But to our knowledge, coumarins have never been evaluated for their potential to modulate UGTs. In the present work, we examined the ability of a flavonoid, chrysin, and six natural coumarins, mainly isolated from different Rutaceous species, [9], [10], [11], [12] to modulate UGT1A6 and/or UGT1A1 activity, in cultured hepatocytes, from both rat and human origin.

Materials and Methods

Chemicals

β-Naphthoflavone (NF), phenobarbital (PB), clofibric acid (CLO), p-nitrophenol (PNP), bilirubin (Bili), uridine 5′-diphosphoglucuronic acid (UDPGA), bicinchoninic acid (BCA), collagenase type IV and Percoll were obtained from Sigma-Aldrich Co. (St Louis, MO, USA), BCA protein assay reagent from Pierce Co. (Oud Beijerland, The Netherlands). BSA, PBS, HBSS, FCS and William’s medium were purchased from Invitrogen Co. (Cergy Pontoise, France).

Coumarins and flavonoids

Coumarin (97 %) and chrysin (97 %) were purchased from Sigma-Aldrich. Umbelliferone, bergapten, imperatorin, avicennin and cis-avicennol were isolated from different Rutaceous species belonging to the genera Phebalium [9], [10], [11] and Philotheca [12] (Fig. [1]). All these compounds were identified by comparison of their spectral data with those in the literature and their purity checked with ¹H-NMR and GC-MS.

Assay for the cytotoxicity

Primary cultures of rat hepatocytes were plated in 96-multiwell culture plates. Twenty-four hours after plating, the medium was discarded and fresh medium containing 25 μM of the compound was added to the cultures. After 3 days, cellular viability was determined by measuring the reduction of the tetrazolium-blue compound MTT to a blue formazan product according to Carmichael et al. [13].

Rat hepatocytes isolation and culture

Rats were anesthetised with thiopental (Pentothal; Abbott; Rungis, France) administered intraperitoneally (100 mg/kg body). Hepatocytes were isolated by a two-step collagenase perfusion, as previously described by Binda et al. [14]. Cell viability, estimated by trypan blue exclusion, was 80 %. Hepatocytes were then plated onto 75 cm² flasks (Costar, Merck Eurolab; Fontenay-sous-bois, France) coated with home-prepared collagen at the density of 10 × 10⁶ cells/flask in William’s culture medium containing 10 % FCS, 2 mM glutamine, 50 μg/mL gentamycin, 4 μg/mL bovine insulin and 10^-5 M hydrocortisone. They were allowed to attach for 4 h at 37 °C in a humidified atmosphere of 95 % air/5 % CO₂. The medium was then changed to William’s medium culture without serum supplementation and was renewed every 24 h up to 3 - 5 days of culture.

Human hepatocytes isolation and culture

Human livers were obtained from 3 patients undergoing partial hepatectomy for primary or secondary tumours. All experimental procedures were done in compliance with French laws and regulations and were approved by the National Ethics Committee. The liver tissues were processed immediately after removal from the abdominal cavity and the margins of the tissues were selected to avoid tumour tissues. The hepatic tissue was then placed into ice-cold 25 mM HEPES buffer, pH 7.4, containing 0.8 mM Na₂HPO₄, 2.7 mM KCl, and 137 mM NaCl. Hepatocytes were isolated as previously described by David et al. [15]. After Percoll purification [16], cell viability was estimated by trypan blue dye exclusion. Human hepatocytes were plated onto 75 cm² collagen coated flasks at the density of 10 x 10⁶ cells/flask. After overnight culture, the medium was changed and replaced by serum-free medium. Medium was renewed every 24 h to 96 h of culture. The characteristics and viable hepatocyte yields of the patients are described in Table [1].

Hepatocyte treatment

The various inducers were added to the cell cultures: CLO, PB, NF were first dissolved in dimethyl sulfoxide (DMSO) and then diluted in serum-free culture medium to reach, respectively, 50 μM, 2mM and 250 μM, and a final concentration of 0.1 % DMSO. Control flasks received a medium containing 0.1 % DMSO only. The medium was renewed every 24 h for up to 3 (5 for kinetic assay) days of culture.

All coumarins and the flavonoid were dissolved in DMSO at final concentration 25 μM. At this concentration, the tested compounds did not show any cytotoxic effect on cell cultures (data not shown).

Hepatocyte microsome preparation

At the appropriate time points, hepatocytes were scraped in homogenisation buffer (50 mM, KCl 150 mM, EDTA 2 mM, pH 7.4), sonicated for 15 seconds and frozen at -80 °C. Microsomes were obtained by differential centrifugations from thawed homogenates and frozen at -80 °C as described by Richert et al. [17].

Microsomal UGT1A6-dependent p-nitrophenol conjugation (PNP-UGT activity)

PNP-UGT activity was determined according to the procedure described by Viollon-Abadie et al. [18]. Briefly, microsomes (0.25 mg of proteins/mL) were incubated with 100 μL of 100 mM Tris-HCl buffer (pH 7.8) containing PNP (1 mM), UDPGA (5 mM), for 1 h at 37 °C. The reaction was stopped by the addition of 1.9 mL NaOH (0.1 M) and PNP glucuronidation was quantified by measuring the decrease of absorbance at 405 nm. Activities were expressed as nmol PNP conjugated/min/mg cellular protein.

Microsomal UGT1A1-dependent bilirubin conjugation (Bili-UGT activity)

Bili-UGT activity was assayed by the method described by Heiwegh et al. [19] and modified by Viollon-Abadie et al. [18]. Briefly, microsomes (0.25 mg of proteins/mL) were incubated for 60 min at 37 °C with 100 μL of 400 mM Tris-maleate buffer (pH 7.8) containing bilirubin (0.125 mM), and UDPGA (5 mM). The reaction was stopped by addition of 250 μL glycine-HCl buffer (pH 2.7). Ethyl anthranilate diazo-reagent (250 μL) was added to the mixture for 30 min at room temperature and the reaction was stopped by addition of 250 μL of a freshly prepared ascorbic acid solution (10 %). The pigment formed was extracted with a mixture of pentan-2-one/n-butyl acetate (17 : 3, v/v). After freezing at -20 °C during 2 h, thawing, shaking and centrifuging 10 min at 250 × g, the absorbance of the organic phases was measured at 530 nm. Activities were expressed as pmol glucuronide formed/min/mg cellular protein.

Protein content

The protein content of cells was determined using the BCA determination kit [20] and BSA as standard.

Statistical procedure

The statistical significance of differences between control and treated groups was evaluated with Student's unpaired t-test.

Results

UGT1A1-dependent bilirubin (Bili-UGT) and UGT1A6-dependent p-nitrophenol (PNP-UGT) activities in rat hepatocytes in the presence or absence of specific inducers (Figs. [2] A and B): The evolution of UGT1A1 and UGT1A6 activities during the time of culture in rat hepatocytes monolayers was investigated by measuring respectively the glucuronoconjugaison of bilirubin (Bili-UGT) and p-nitrophenol (PNP-UGT) during 5 days, in the presence or absence of specific enzymatic inducers. Fig. [2] shows that Bili-UGT and PNP-UGT activities in rat hepatocytes were increased in the presence of specific inducers with a maximum at 72 h of treatment, as expected from previous work [21]. Bili-UGT activity was increased after CLO (250 μM) or PB (2 mM) treatment of rat hepatocytes (72 ± 13 and 43 ± 6 pmol/min/mg protein respectively versus 16 ± 1.6 pmol/min/mg protein in control), whereas the activity of PNP-UGT was increased after treatment with NF (50 μM), (49 ± 6 versus 20 ± 3 nmol/min/mg protein in control).

Effects of chrysin and coumarins on Bili-UGT and/or PNP-UGT activities in rat hepatocytes: Since maximal induction with the specific inducers was observed after 72 h of treatment, all the following assays were performed over a 3-day treatment period. Rat hepatocytes were first treated with chrysin (25 μM) and the effect on microsomal Bili-UGT activity was compared to that obtained with prototypical inducers. An increase in Bili-UGT activity was obtained after treatment with chrysin (48.5 ± 18.5 versus 21 ± 1.4 pmol/min/mg protein in control). This increase was equivalent to that observed with the specific inducer PB (36.5 ± 6.5 pmol/min/mg protein) whereas that observed in the presence of CLO was about two-fold stronger (72.0 ± 13 pmol/min/mg protein). In contrast, microsomal PNP-UGT activity was not affected by treatment with chrysin (18.5 ± 6.1 versus 17 ± 5.5 nmol/min/mg protein in control), while in the presence of the prototypical inducer NF, the activity was strongly increased (48.5 nmol/min/mg protein).

Since chrysin did not induce PNP-UGT activity, the effect of various coumarins (25 μM) was only tested on Bili-UGT activity as shown in Fig. [3]. An about 2-fold increase in activity compared to controls was observed after treatment of rat hepatocytes with chrysin, avicennin, cis-avicennol, bergapten and imperatorin, whereas in the presence of coumarin or umbelliferone, Bili-UGT activity was not modified.

Effects of coumarins on Bili-UGT activity in human hepatocytes (Fig. [4] ): Because of the scarcity of human liver availability, several human hepatocyte preparations were needed to test all compounds. As shown in Fig. [4], the microsomal activity of Bili-UGT was about 1.5- to 2.5-fold increased after a 3-day treatment period of hepatocytes from donor 1 with either chrysin, avicennin, cis-avicennol, bergapten or imperatorin and about 2- to 2.5-fold in hepatocytes from donor 2, while a 3-day treatment period with coumarin or umbelliferone did not affect Bili-UGT activity in human hepatocytes from either donor. For comparison, the effect of a 3-day treatment period of hepatocytes from donor 3 with PB (2 mM) are also given in Fig. [4], showing that Bili-UGT activity was about 2-fold increased.

Discussion

Over the last years, several groups have investigated the effect of natural or dietary compounds on UGTs. In particular, bioflavonoids have been described to modify phase I and phase II enzymes, which are key enzymes involved in the metabolism of xenobiotics. Among these compounds, the dietary flavonoid chrysin constitutes a reference compound and is extensively studied. Walle et al. demonstrated that chrysin strongly increased UGT1A1 mRNA and protein in Caco-2 and hepG2 cells [22], [23]. However, according to Smith et al. [24], immortalized cell lines may not be the best model for assessment of enzyme induction and human hepatocytes would be more representative of in vivo conditions. Therefore effects of prototypical inducers and chrysin on UGT1A in primary cultures of human hepatocytes have been recently explored [24], [25], [26], [27], but reports are often conflicting.

The first aim of the present study was to evaluate the effect of chrysin on UGT1A1- and UGT1A6-dependent activities in rat and in human hepatocytes in primary cultures.

Regarding the evolution of UGT1A1 and 1A6 activities in rat hepatocytes during time of culture (Figs. [2] A and B) in the presence of specific inducers, it appears that maximal induction was observed after 72 h of treatment. Therefore all the following assays were performed over a 3-day treatment period. We first found that after treatment of rat hepatocytes with chrysin (25 μM), the UGT1A1-dependent conjugation rate of bilirubin was 2.5-fold increased, while no such effect was observed on UGT1A6-dependent activities. The responses observed with the respective specific UGT1A1 inducers PB and CLO and UGT1A6 inducer NF confirm our previous observations that the response to prototypical UGT inducers in primary hepatocyte cultures from animal origin well mimicked the in vivo situation [18], [21]. This is, to our knowledge, the first report on the effect of chrysin on UGT1A-dependent activities in rat hepatocytes, strongly suggesting that chrysin is a UGT1A1, but not a UGT1A6 inducer in rats.

The second aim of the present study was to compare various natural coumarins, extracted from Rutaceous plants [9], [10], [11], [12] on UGT1A1-dependent bilirubin glucuronidation activity in cultured hepatocytes. We have chosen to measure Bili-UGT activity with these compounds, since chrysin did not induce PNP-UGT activity. The 3-day treatment period of rat hepatocytes with the pyranocoumarins avicennin and cis-avicennol, and the furocoumarins bergapten and imperatorin, induced a 2-fold increase in Bili-UGT, equivalent to the increase observed with chrysin, whereas in the presence of the simple coumarins, coumarin or umbelliferone, Bili-UGT activity was not modified. Bili-UGT activity was also obviously increased after treatment with the prototypical inducers CLO and PB. The effect of chrysin and coumarins was tested on human hepatocytes isolated from hepatic resections of two donors. Because of the scarcity of human liver availability, another donor was used for testing the PB effect The various compounds responded, qualitatively and quantitatively, similarly to rat hepatocytes: chrysin, furo- and pyranocoumarins led to an approximately 2-fold increase in Bili-UGT activity versus respective controls. It is noteworthy that, in spite of a difference of basal activity, which has been extensively reported [26], human hepatocytes of donors 1 and 2 responded similarly to the various compounds. Like in rat hepatocytes, simple coumarins did not affect Bili-UGT activity.

Since specific human anti-UGT antibodies are commercially available, while rat antibodies are not, Western blot analysis could be performed on microsomes prepared from human hepatocytes after the 3-day exposure to test compounds, but no evident UGT1A1 protein content increase, both for prototypical inducers and the compounds evaluated has been observed (data not shown). This is most probably related to the poor sensitivity of Western blots versus mRNA level determinations, since PB has been previously clearly found to induce UGT1A1 mRNAs [26], [28].

Taken together, the results from the present study suggest that increases of UGT1A1-dependent activity by coumarins are due to UGT1A1 induction.

In terms of structural requirements for UGT1A1 induction, by comparing the structure of the molecules tested, it clearly appears that the active compounds present an additional ring on the basal core benzopyrone, suggesting that the B-ring (phenyl) for chrysin and the furan or pyran rings for coumarins are essential for the biological activity.

In conclusion, coumarins, like flavonoids, may constitute a very interesting family of natural compounds able to induce UGT1A1 activity.

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Dr. Corinne Girard

Laboratoire de Pharmacognosie

EA 3921 ”Optimisation Métabolique et Cellulaire”

UFR des Sciences Médicales et Pharmaceutiques

Place Saint-Jacques

25030 Besançon cedex

France

Phone: +33-3-8166-5559

Fax: +33-3-8166-5568

Email: corinne.girard@univ-fcomte.fr

References

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  CrossrefPubMedSearch in Google Scholar
• 2 Tephly T R, Burchell B. UDP-glucuronosyltransferases: a family of detoxifying enzymes.  Trends Pharmacol Sci. 1990;  11 276-9.
  CrossrefPubMedSearch in Google Scholar
• 3 Embola C W, Sohn O S, Fiala E S, Weisburger J H. Induction of UDP-glucuronosyltransferase 1 (UDP-GT1) gene complex by green tea in male F344 rats.  Food Chem Toxicol. 2002;  40 841-4.
  PubMedSearch in Google Scholar
• 4 Van der Logt E MJ, Roelofs H MJ, Nagengast F M, Peters W HM. Induction of rat hepatic and intestinal UDP-glucuronyltransferases by naturally occuring dietary anticarcinogens.  Carcinogenesis. 2003;  24 1651-6.
  CrossrefPubMedSearch in Google Scholar
• 5 Walle U K, Walle T. Induction of human UDP-glucuronosyltransferase UGT1A1 by flavonoids - structural requirements.  Drug Metab Dispos. 2002;  30 564-9.
  CrossrefPubMedSearch in Google Scholar
• 6 Moon Y J, Wang X, Morris M E. Dietary flavonoids: effect on xenobiotic and carcinogen metabolism.  Toxicol In Vitro. 2006;  20 187-210.
  CrossrefPubMedSearch in Google Scholar
• 7 Rosselli S, Maggio A, Bellone G, Formisano C, Basile A, Cicala C. et al . Antibacterial and anticoagulant activities of coumarins isolated from the flowers of Magydaris tomentosa .  Planta Med. 2007;  73 103-9.
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  CrossrefPubMedSearch in Google Scholar
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Dr. Corinne Girard

Laboratoire de Pharmacognosie

EA 3921 ”Optimisation Métabolique et Cellulaire”

UFR des Sciences Médicales et Pharmaceutiques

Place Saint-Jacques

25030 Besançon cedex

France

Phone: +33-3-8166-5559

Fax: +33-3-8166-5568

Email: corinne.girard@univ-fcomte.fr