Evaluation of In Situ Generated Valproyl 1-O -Acyl Glucuronide...

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Evaluation of In Situ Generated Valproyl 1-O-β-Acyl Glucuronide in

Valproic Acid Toxicity in Sandwich-Cultured Rat Hepatocytes

Jayakumar Surendradoss, Thomas K. H. Chang, and Frank S. Abbott

Faculty of Pharmaceutical Sciences, The University of British Columbia,

Vancouver, British Columbia, Canada

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 21, 2014 as DOI: 10.1124/dmd.114.059352

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Running Title: Evaluating Toxicity of VPA Glucuronide in Rat Hepatocytes

Address correspondence to: Dr. Frank S. Abbott, Faculty of Pharmaceutical Sciences, The

University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada. Tel:

1-604-822-2566. Fax: 1-604-822-3035. E-mail: [email protected].

Number of text pages: 33

Number of tables: 3

Number of figures: 7

Number of references: 54

Number of words in the Abstract: 248

Number of words in the Introduction: 749

Number of words in the Discussion: 1494

ABBREVIATIONS: DFN, diclofenac; DFN-G, diclofenac 1-O-β-acyl glucuronide; DMSO,

dimethylsulfoxide; GC-MS, gas chromatography–mass spectrometry; GSH, glutathione; 4'-OH-

DFN, 4'-hydroxy diclofenac; 5-OH-DFN, 5-hydroxy diclofenac; LDH, lactate dehydrogenase;

UDP-GA, uridine 5'-diphospho-glucuronic acid; UGT, uridine 5'-diphospho-

glucuronosyltransferase; UHPLC-MS/MS, ultra-high performance liquid chromatography –

tandem mass spectrometry; VPA, valproic acid; VPA-G, valproyl 1-O-β-acyl glucuronide.


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ABSTRACT

Acyl glucuronides are reactive electrophilic metabolites implicated in the toxicity of carboxylic

acid drugs. Valproyl 1-O-β-acyl glucuronide (VPA-G), which is a major metabolite of valproic

acid (VPA), has been linked to the development of oxidative stress in VPA-treated rats.

However, relatively little is known about the toxicity of in situ generated VPA-G and its

contribution to VPA hepatotoxicity. Therefore, we investigated the effects of modulating the in

situ formation of VPA-G on LDH release (a marker of necrosis), BODIPY 558/568 C12

accumulation (a marker of steatosis), and cellular glutathione (GSH) content in VPA-treated

sandwich-cultured rat hepatocytes. VPA increased LDH release and BODIPY 558/568 C12

accumulation, whereas it had little or no effect on total GSH content. Among the various uridine

5'-diphospho-glucuronosyltransferase inducers evaluated, β-naphthoflavone produced the

greatest increase in VPA-G formation. This was accompanied by an attenuation of the increase

in BODIPY 558/568 C12 accumulation, but did not affect the change in LDH release or total

GSH content in VPA-treated hepatocytes. Inhibition of in situ formation of VPA-G by borneol

was not accompanied by substantive changes in the effects of VPA on any of the toxicity

markers. In a comparative study, in situ generated diclofenac glucuronide was not toxic to rat

hepatocytes, as assessed using the same chemical modulators, thereby demonstrating the utility

of the sandwich-cultured rat hepatocyte model. Overall, in situ generated VPA-G was not toxic

to sandwich-cultured rat hepatocytes, suggesting that VPA glucuronidation per se is not expected

to be a contributing mechanism of VPA hepatotoxicity.


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Introduction

Drug-induced hepatotoxicity is a common cause of acute liver failure (Tujios and

Fontana, 2011). Evidence for drug-induced hepatotoxicity leads to attrition during drug

development, refusal of drug approval, and black box warning or post-marketing withdrawal

(Regev, 2013). Drug-induced hepatotoxicity can be intrinsic, the occurrence of which is

relatively common, predictable, and dose-dependent (Russmann et al., 2009), or idiosyncratic,

which occurs in a rare, unpredictable, and often dose-independent fashion in a few susceptible

patients (Regev, 2013). Various mechanisms of drug-induced hepatotoxicity have been

proposed, including formation of reactive electrophilic metabolites (Srivastava et al., 2010;

Leung et al., 2012). One such class of reactive metabolites is the acyl glucuronides (Kalgutkar et

al., 2005).

Acyl glucuronides, which are enzymatic products formed by glucuronidation of

carboxylic acids (Stachulski et al., 2006), are capable of undergoing: 1) hydrolysis to parent

aglycone mediated by β-glucuronidases, non-specific esterases, hydroxyl ion, or serum albumin;

2) intra-molecular acyl migration to form positional isomers that are resistant to β-glucuronidase-

mediated hydrolysis; and 3) covalent binding to proteins via ‘transacylation’ or ‘glycation’

mechanisms (Regan et al., 2010). Acyl glucuronides, however, differ widely in their chemical

reactivity, which is attributed to the chemical structure of the ‘aglycone’ moiety (Stachulski et

al., 2006). Postulated mechanisms for the toxicity of acyl glucuronides include: 1) a direct

impairment of the function of a key protein that is covalently modified; 2) an indirect immune

reaction to the antigenic drug-protein adducts; and 3) formation of more reactive acyl-glutathione

thioester conjugates with intracellular glutathione (GSH), resulting in GSH depletion and

possibly covalent binding to proteins (Shipkova et al., 2003; Skonberg et al., 2008). Although


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acyl glucuronides are hypothesized to be involved in various toxicities, including hepatotoxicity,

of carboxylic acid-containing drugs (Regan et al., 2010; Boelsterli, 2011), it remains to be

established whether there is a causal role for these reactive species.

Valproic acid (VPA) is a commonly used antiepileptic drug that is effective against

various types of seizures and epileptic syndromes (Loscher, 2002). This drug undergoes

microsomal glucuronidation, mitochondrial β-oxidation, and microsomal cytochrome P450

(CYP)-mediated oxidation (Abbott and Anari, 1999). Glucuronidation is a major metabolic

pathway for VPA, and contributes to the biotransformation of about 30 to 50% of the

administered dose of VPA in humans (Silva et al., 2008). Valproyl 1-O- β-acyl glucuronide

(VPA-G) is one of the least reactive acyl glucuronides investigated to date (Stachulski et al.,

2006). Yet, it undergoes intra-molecular acyl migration to form positional isomers of VPA-G

(Dickinson et al., 1984), and appears to be responsible, at least partly, for the formation of VPA-

protein adducts in vitro in rat hepatocytes (Porubek et al., 1989). In rats (Tong et al., 2003) and

pediatric patients (Michoulas et al., 2006), VPA increases the in vivo levels of 15-F2t-isoprostane,

which is a marker of lipid peroxidation (Halliwell and Whiteman, 2004). The increase in plasma

and hepatic levels of 15-F2t-isoprostane in rats administered VPA was accompanied by an

increase in the levels of VPA-G (Tong et al., 2005b). In another in vivo study in rats, hepatic and

urinary concentrations of VPA-G did not correlate with serum levels of α-glutathione-S-

transferase (Lee et al., 2009), which is a marker of hepatotoxicity (Bailey et al., 2012).

The therapeutic use of VPA by humans is associated with a rare, but potentially fatal,

idiosyncratic hepatotoxicity (Nanau and Neuman, 2013). Although the mechanism of VPA

hepatotoxicity is not understood, VPA-G has been proposed to play a role (Tong et al., 2005b).

However, there is no direct evidence as to whether in situ generated VPA-G is hepatotoxic.


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Therefore, to increase our understanding of the toxicological significance of VPA-G, the present

study was conducted to determine whether in situ generated VPA-G is toxic and whether it

contributes to the toxicity of VPA in sandwich-cultured rat hepatocytes. It is now increasingly

recognized that the sandwich-cultured hepatocyte model is appropriate for studying hepatic

biotransformation and toxicity of drugs and other chemicals (Swift et al., 2010). Diclofenac is

another well-known carboxylic acid drug that is associated with idiosyncratic hepatotoxicity

(Tang, 2003) Although diclofenac undergoes glucuronidation to form an unstable acyl

glucuronide, the hepatotoxicity of this drug has been attributed to the cytochrome P450-mediated

oxidative metabolites of diclofenac (Tang, 2003). As a comparison, the present study also

determined the effect of modulating the in situ formation of a more reactive acyl glucuronide,

diclofenac 1-O-β-acyl glucuronide (DFN-G), on the release of lactate dehydrogenase (LDH) in

cultured hepatocytes treated with diclofenac. The findings of the present study provide insight to

the question as to whether VPA-G plays a role in VPA hepatotoxicity.


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Materials and Methods

Chemicals, Reagents, and Solvents. Sodium VPA (CAS # 1069-66-5), sodium diclofenac

(CAS # 15307-79-6), sodium phenobarbital (CAS # 57-30-7), β-naphthoflavone (CAS # 6051-

87-2), 3-methylcholanthrene (CAS # 56-49-5), L-sulforaphane (CAS # 142825-10-3), quercetin

(CAS # 117-39-5), dexamethasone (CAS # 50-02-2), clofibrate (CAS # 637-07-0), pregnenolone

16α-carbonitrile (PCN; CAS # 1434-54-4), trans-stilbene oxide (CAS # 1439-07-2), (-)-borneol

(CAS # 464-45-9), and dimethyl sulfoxide (DMSO; CAS # 67-68-5) were obtained from Sigma-

Aldrich (St. Louis, MO). DFN-G (CAS # 64118-81-6), 4'-hydroxy-diclofenac (4'-OH-DFN;

CAS # 64118-84-9), and 5-hydroxy diclofenac (5-OH-DFN; CAS # 69002-84-2) were purchased

from Toronto Research Chemicals, Inc. (North York, ON, Canada). The Lactate Dehydrogenase

(LDH) Cytotoxicity Detection Kit was obtained from Roche diagnostics (Indianapolis, IN) and

the Glutathione Assay Kit was from the Cayman Chemical Co. (Ann Arbor, MI). Williams’

medium E, liver perfusion medium, hepatocyte wash medium, heat-inactivated fetal bovine

serum, phosphate-buffered saline (pH 7.4), 10× Hank’s balanced salt solution, 10× Dulbecco’s

phosphate-buffered saline, penicillin-streptomycin, L-glutamine, and BODIPY 558/568 C12 were

obtained from Invitrogen (Burlington, ON, Canada). Matrigel basement membrane matrix was

obtained from BD Biosciences (Mississauga, ON, Canada). Percoll was purchased from GE

Healthcare (Baie d’Urfe, QC, Canada). Ammonium acetate, ethyl acetate, diethyl ether,

acetonitrile, methanol, n-hexanes, glacial acetic acid, and sodium hydroxide were obtained from

Fisher Scientific (Ottawa, ON, Canada).

Animals. Adult male Sprague-Dawley rats (175-200 g) were obtained from Charles

River Laboratories, Inc. (Senneville, QC, Canada), and were housed and cared for in our animal

care facility as described previously (Surendradoss et al., 2012). All animal experiments were


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approved by the University of British Columbia Animal Care Committee and were conducted in

accordance with the guidelines of the Canadian Council on Animal Care.

Isolation, Culture, and Treatment of Rat Hepatocytes. Rat hepatocytes were isolated

by a two-step collagenase perfusion method (Seglen, 1993), as described previously (Tong et al.,

2005a). Plating of hepatocytes (0.7 × 106 cells per well), preparing the sandwich-culture

configuration, and culturing of hepatocytes were performed as reported earlier (Surendradoss et

al., 2012). At 120 h after plating, sandwich-cultured rat hepatocytes were treated with VPA,

culture medium (vehicle for VPA), diclofenac, or DMSO (vehicle for diclofenac) for the next 24

h at the concentrations indicated in each figure legend. In other experiments, at 48 h after

plating, cultured hepatocytes were pretreated with an inducer of UGT or an inhibitor of

glucuronidation and then treated with VPA, diclofenac, or vehicle as described in each figure

legend.

Quantification of VPA-G Concentration. At the end of the drug treatment period,

culture supernatant was collected and hepatocytes were lysed with 2% Triton X-100 in

phosphate-buffered saline (PBS; pH 7.4) containing 20 mM EDTA. Each sample was

transferred into a microfuge tube and stored at -80oC until analysis. VPA-G concentrations in

culture supernatant and cell lysate were quantified using a validated ultra-high performance

liquid chromatography – tandem mass spectrometry (UHPLC-MS/MS) assay with [2H6]-VPA-G

as the internal standard (Surendradoss et al., 2013). The UHPLC-MS/MS system consisted of an

Agilent 1290 Infinity Binary Pump, a 1290 Infinity Autosampler, and a 1290 Infinity

Thermostatted Column Compartment (Agilent Technologies, Mississauga, Ontario, Canada),

which was connected to an AB Sciex QTRAP® 5500 hybrid linear ion-trap triple quadrupole


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mass spectrometer equipped with a Turbo Spray source (AB Sciex, Concord, Ontario, Canada).

The mass spectrometer was operated in negative ionization mode.

Quantification of DFN-G Concentration. As a precaution to maintain the stability of

DFN-G, the culture supernatant samples and the stock solutions of calibration standards were

thawed and maintained on wet ice during sample preparation, and were acidified with 2 M acetic

acid solution (4% v/v final concentration) to reduce intra-molecular acyl migration and/or

hydrolysis of DFN-G (Sparidans et al., 2008). To quantify DFN-G concentrations in culture

supernatant from diclofenac-treated hepatocytes, 10 µl of the sample and 10 µl of a 50 µg/ml

solution of [2H6]-VPA-G (internal standard) were added to 480 µl of assay diluent (85% of 2

mM ammonium acetate in water and 15% of 2 mM ammonium acetate in a 9:1 mixture of

acetonitrile and water), vortex-mixed for 10 s, and centrifuged at 10,600 × g for 5 min at 4oC. A

15 µl volume was injected onto the UHPLC-MS/MS system. The calibration curve of DFN-G

ranged from 2.1 to 2120 nM. The mobile phases, chromatographic gradient, and mass

spectrometric conditions were the same as those described previously for VPA-G assay

(Surendradoss et al., 2013). The declustering potential and collision energy settings were −60 V

and −12 V for DFN-G, and −40 V and −20 V for [2H6]-VPA-G, respectively. DFN-G was

analyzed using the total ion current of the multiple reaction monitoring transition m/z 470.2 →

192.8 (Koga et al., 2011), with the internal standard [2H6]-VPA-G transition pairs being m/z

325.1 → 149.3 and 325.1 → 174.9.

Lactate Dehydrogenase (LDH) Assay. LDH release was used as a marker of cell

necrosis (Jauregui et al., 1981). LDH activity in the culture supernatants and cell lysate samples

was determined using the Cytotoxicity Detection Kit (Roche Diagnostics, Indianapolis, IN)

(Surendradoss et al., 2012). LDH released into the culture supernatant was expressed as a


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percentage of the total cellular LDH activity (i.e. sum of the LDH activity in the culture

supernatant and cell lysate).

BODIPY 558/568 C12 Assay. Cellular accumulation of BODIPY 558/568 C12 was used

as an index of steatosis (Fujimura et al., 2009), and determined as described previously

(Surendradoss et al., 2012). Fluorescence was measured at a λex of 484 nm and λem of 618 nm in

a Biotek Synergy Mx microplate reader (Biotek Instruments, Winooski, VT). Each blank well

had culture medium containing BODIPY 558/568 C12 and a test compound, but without cells.

BODIPY 558/568 C12 accumulation was expressed as fold increase in fluorescence in drug-

treated wells over that in vehicle-treated control wells.

Total Glutathione (GSH) Assay. Cellular content of total GSH was quantified using the

Glutathione Assay Kit (Cayman Chemical Co.) as described previously (Surendradoss et al.,

2012). The rate of formation of the reaction product, 5-thio-2-nitrobenzoic acid, was determined

spectrophotometrically in a kinetic mode at a wavelength of 405 nm in a Labsystems Multiskan

Ascent® multiwell plate reader (Thermo Electron Corp., Burlington, ON, Canada). The blank

sample consisted of equal volumes of phosphate-buffered saline (pH 7.4; supplemented with 1

mM EDTA) and metaphosphoric acid, but without cell homogenate.

Quantification of Oxidative Metabolites of VPA. Concentrations of the oxidative

metabolites of VPA in culture supernatants from VPA-treated hepatocytes were quantified using

a gas chromatography–mass spectrometry (GC-MS) assay (Surendradoss et al., 2012).

Determination of 4'-OH-DFN and 5-OH-DFN. Concentrations of 4'-OH-DFN and 5-

OH-DFN in the culture supernatants of diclofenac-treated hepatocytes were determined semi-

quantitatively using a LC-MS/MS assay adapted from Sparidans et al. (2008). Briefly, 50 µl of

the culture supernatant sample was added to 150 µl of assay diluent (60% of solvent A, 8.5 mM


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ammonium acetate in water containing 0.0075% formic acid and 40% of solvent B, methanol),

vortex-mixed for 10 s, and centrifuged at 10600 × g for 5 min at 4oC. A 5 µl volume of the

supernatant was injected onto the UHPLC-MS/MS system. The calibration curves of 4'-OH-

DFN and 5-OH-DFN ranged from 0.03 to 16 µM. The mobile phases and the chromatographic

gradient conditions used in this assay were the same as those described previously (Sparidans et

al., 2008). Analysis was performed under multiple reaction monitoring mode on a QTRAP®

5500 linear ion trap mass spectrometer operated in positive electrospray ionization, using the

following instrument settings: curtain gas, 30 units; ion source gas 1, 60 units; ion source gas 2,

40 units; collision-activated dissociation gas level, high; ion source temperature, 400oC; ion

spray voltage, 5500 V; collision cell exit potential, 18 V; entrance potential, 10 V, and dwell

time, 150 ms. 4'-OH-DFN and 5-OH-DFN were analyzed using the sum of the total ion currents

of the multiple reaction monitoring transitions m/z 312.0 → 230.9, m/z 312.0 → 266.0, and m/z

312.0 → 294.0. Whereas the declustering potential was 66 V, the collision energy settings for

the three MRM transitions were 27, 19, and 15 V for the three transitions, respectively. Under

the assay conditions employed, the retention times of 4'-OH-DFN and 5-OH-DFN were 4.39 and

5.03 min, respectively.

Statistical Analysis. Data were analyzed by one-way or two-way analysis of variance as

appropriate and when there were significant differences, the data were further analyzed by the

Student-Newman-Keuls multiple comparison test (Sigmaplot for Windows, Version 11.0, Systat

Software, Inc., Chicago, IL). The level of statistical significance was set a priori at P < 0.05.


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Results

Concentration of In Situ Generated VPA-G in Culture Supernatant and Cell Lysate

of Sandwich-Cultured Rat Hepatocytes Treated with VPA. As assessed in hepatocytes

treated with VPA (1 mM), the concentration of VPA-G was 251 ± 12 and 6.8 ± 1.5 µM (mean ±

SEM; n = 4 rats per treatment group) in the culture supernatant and cell lysate, respectively. As

more than 97% of the in situ generated VPA-G was localized in the culture supernatant, VPA-G

concentration was quantified in culture supernatant in all the subsequent experiments, unless

indicated otherwise.

Time Course and Concentration-Response Relationship in the In Situ Formation of

VPA-G in Sandwich-Cultured Rat Hepatocytes Treated with VPA. As shown in Fig. 1A,

the in situ concentration of VPA-G continued to increase in the culture supernatants in a linear

fashion over the 1 to 24 h period. Concentration-response experiments indicated that the in situ

formation of VPA-G increased from 20 µM to 348 µM in response to increases in VPA

concentration from 0.03 mM to 3 mM, reached a peak at 3 mM to 10 mM VPA, and decreased at

≥ 20 mM VPA (Fig. 1B). Based on these results, VPA concentrations of 10 mM and 15 mM

were chosen to investigate the toxicity of in situ generated VPA-G, as these concentrations of

VPA resulted in maximal or near maximal formation of VPA-G (Fig. 1B) and elicited a

measurable response in the toxicity markers (Surendradoss et al., 2012).

Effect of Various Known UGT Inducers on In Situ Formation of VPA-G in

Sandwich-Cultured Rat Hepatocytes Treated with VPA. Except for rat UGT2B1 (Pritchard

et al., 1994), the identity of specific rat UGT enzymes catalyzing VPA glucuronidation is not

known. Therefore, prior to investigating the toxicity of the in situ generated VPA-G in

sandwich-cultured rat hepatocytes treated with VPA, initial experiments were performed to


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identify chemical modulators of VPA glucuronidation. Cultured hepatocytes were pretreated

with a known UGT enzyme inducer, such as β-naphthoflavone (Viollon-Abadie et al., 2000), L-

sulforaphane (Kohle and Bock, 2006), phenobarbital (Soars et al., 2004), 3-methylcholanthrene

(Jemnitz et al., 2000), quercetin (Soars et al., 2004), clofibrate (Jemnitz et al., 2000),

dexamethasone (Jemnitz et al., 2000), PCN (Shelby and Klaassen, 2006), or trans-stilbene oxide

(Shelby and Klaassen, 2006) once every 24 h for 72 h followed by VPA treatment for the next 24

h. As shown in Fig. 2A, β-naphthoflavone, L-sulforaphane, and phenobarbital increased the in

situ formation of VPA-G by 2.3-, 1.7-, and 1.9-fold, respectively, whereas none of the other

chemicals had an effect (data not shown). As a comparison, β-naphthoflavone, L-sulforaphane,

and phenobarbital increased the in situ formation of DFN-G by 3.4-, 4.6-, and 2.9-fold,

respectively (Fig. 2A). Among the UGT inducers investigated, β-naphthoflavone yielded the

greatest increase in in situ VPA-G formation (Fig. 2A). As evident from the concentration-

response data, pretreatment with β-naphthoflavone at 20 µM concentration produced the

maximal increase in in situ concentrations of VPA-G in culture supernatants and cell lysates of

VPA-treated cells (Table 1). Therefore, this concentration (20 µM) was used in subsequent

modulation experiments involving β-naphthoflavone.

Effect of Increasing In Situ VPA-G Formation by β-Naphthoflavone on Markers of

Toxicity in Sandwich-Cultured Rat Hepatocytes Treated with VPA. To investigate the

effects of increasing the in situ formation of VPA-G on VPA toxicity in sandwich-cultured rat

hepatocytes, we assessed LDH release (a marker of necrosis), BODIPY 558/568 C12

accumulation (marker of steatosis), and cellular content of total GSH, all of which are relevant to

VPA hepatotoxicity (Jurima-Romet et al., 1996; Silva et al., 2008). Cultured hepatocytes were

pretreated with β-naphthoflavone (20 µM) or DMSO (0.1% v/v; vehicle) once every 24 h for 72


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h followed by treatment with VPA (10 or 15 mM) or culture medium (vehicle) for the next 24 h.

At the end of the treatment period, LDH release, BODIPY 558/568 C12 accumulation, and

cellular concentration of total GSH were measured. As shown in Fig. 2B, β-naphthoflavone

alone did not affect LDH release, whereas 10 and 15 mM VPA increased it. β-Naphthoflavone

pretreatment did not further increase the LDH release by VPA (Fig. 2B). In a comparative

experiment, β-naphthoflavone increased DFN-G concentrations by 3.4-fold (Fig. 2A) and this

was accompanied by an attenuation of LDH release in diclofenac-treated hepatocytes (Fig. 2B).

Pretreatment with β-naphthoflavone alone did not affect BODIPY 558/568 C12 accumulation,

whereas 10 and 15 mM VPA increased BODIPY 558/568 C12 accumulation (Fig. 3A). This

increase in BODIPY 558/568 C12 accumulation by VPA was attenuated by β-naphthoflavone

pretreatment (Fig. 3A). In contrast to the increases observed for the LDH and BODIPY markers,

VPA (10 and 15 mM) had no effect on total GSH content (Fig. 3B). As shown in Fig. 3B, β-

naphthoflavone pretreatment alone led to an increase in the concentration of total GSH.

However, treatment with 15 mM VPA, but not 10 mM VPA, decreased the concentration of total

GSH (by ~ 25%) in the β-naphthoflavone-pretreated hepatocytes.

Effect of Borneol on In Situ Formation of VPA-G in Sandwich-Cultured Rat

Hepatocytes Treated with VPA. Another approach to investigate the toxicity of in situ formed

VPA-G is to determine the toxicological consequence of inhibiting its metabolic formation.

Initial experiments were performed to determine the effect of borneol, which is a known

inhibitor of glucuronidation (Watkins and Klaassen, 1983; Dong and Smith, 2009), on in situ

formation of VPA-G in sandwich-cultured rat hepatocytes treated with VPA. Cultured

hepatocytes were pretreated with borneol (0.25, 0.5, 0.75, or 1 mM) or DMSO (0.1% v/v;

vehicle) for 0.5 h followed by co-treatment with VPA (10 or 15 mM) or culture medium


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(vehicle) for the next 24 h. Borneol, over the concentration range of 0.25 to 1 mM, decreased

both extracellular and intracellular concentrations of VPA-G in VPA-treated hepatocytes (Table

1). As 1 mM borneol produced the greatest inhibition of in situ VPA-G formation (Table 1), this

concentration was selected for the subsequent experiments. As shown in Fig. 4A, whereas 1 mM

borneol decreased the extracellular concentration of VPA-G, the same concentration of borneol

did not affect the in situ formation of DFN-G in hepatocytes treated with diclofenac for 24 h

(Fig. 4A).

Effect of Decreasing In Situ VPA-G Formation by Borneol on Markers of Toxicity

in Sandwich-Cultured Rat Hepatocytes Treated with VPA. The next experiment was to

investigate the effects of decreasing in situ formation of VPA-G by borneol on markers of

toxicity in sandwich-cultured rat hepatocytes treated with VPA. Cultured hepatocytes were

pretreated with borneol (1 mM) or DMSO (0.1% v/v; vehicle) for 0.5 h, followed by a 24 h co-

treatment with VPA (10 or 15 mM) or culture medium (vehicle). As shown in Fig. 4B, borneol

alone did not affect LDH release, whereas 10 and 15 mM VPA treatment increased LDH release

by 2- and 4.5-fold, respectively. In hepatocytes treated with 10 or 15 mM VPA, co-treatment

with borneol (1 mM) decreased in situ concentration of VPA-G, but it had no effect on LDH

release (Fig. 4B). By comparison, borneol (1 mM) attenuated LDH release in hepatocytes

treated with 400 µM diclofenac (Fig. 4B), but this was not accompanied by a change in the in

situ concentration of DFN-G (Fig. 4A). As shown in Fig. 5A, treatment of cultured hepatocytes

with 10 and 15 mM VPA increased BODIPY 558/568 C12 accumulation by 1.9- and 2.5-fold,

respectively, and this increase was not affected by borneol. Treatment of cultured hepatocytes

with 15 mM VPA decreased the total GSH content by 35%, which was further decreased by


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borneol (Fig. 5B). In contrast, neither 10 mM VPA nor co-treatment with borneol affected total

GSH content (Fig. 5B).

Time Course of the Effect of Borneol on the In Situ Formation of VPA-G and LDH

Release in Sandwich-Cultured Rat Hepatocytes Treated with VPA. A time course

experiment was performed to further characterize the effect of borneol on in situ formation of

VPA-G and LDH release in VPA-treated hepatocytes. VPA increased VPA-G concentrations in

the culture supernatants over a 2 to 24 h period, and this increase was attenuated by borneol at 4,

8, and 24 h after VPA treatment (Fig. 6A). VPA increased LDH release, but this did not occur

until 24 h post-treatment, and the effect was further enhanced by borneol (Fig. 6B). By

comparison, diclofenac increased DFN-G concentrations over the 2 to 24 h time period (Fig.

6C), whereas borneol decreased the in situ formation of DFN-G only at 2 and 4 h after drug

treatment, but it had no effect at 8 h and in fact modestly increased DFN-G formation at 24 h

after drug treatment (Fig. 6C). Treatment with diclofenac resulted in an increase in LDH release

at 8 and 24 h post-treatment and this increase was attenuated by co-treatment with borneol (Fig.

6D).

Effect of β-Naphthoflavone and Borneol on In Situ Generated Oxidative Metabolites

of VPA in Sandwich-Cultured Rat Hepatocytes Treated with VPA. In addition to

glucuronidation, VPA is also biotransformed by mitochondrial β-oxidation and cytochrome

P450-mediated oxidation (Abbott and Anari, 1999). Therefore, we determined whether β-

naphthoflavone and borneol are capable of modulating the oxidative biotransformation of VPA,

as a means to gain insight into the observed lack of toxicity of VPA-G in cultured hepatocytes

treated with VPA (e.g., Fig. 2B and 4B). As shown in Table 2, β-naphthoflavone increased the

concentrations of 4-keto-VPA, 4-OH-VPA, and 3-OH-VPA (2- to 3-fold), but not the other


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oxidative metabolites of VPA in cultured hepatocytes treated with VPA. By comparison,

borneol decreased the concentrations of only 4-OH-VPA (60%) and 5-OH-VPA (40%) (Table

3).

Effect of β-Naphthoflavone and Borneol on In Situ Generated 4'-OH-DFN and 5-

OH-DFN in Sandwich-Cultured Rat Hepatocytes Treated with Diclofenac. Diclofenac

undergoes biotransformation not only by glucuronidation, but also by cytochrome P450-

mediated oxidation to produce 4'-OH-DFN and 5-OH-DFN, which undergo subsequent oxidation

to form highly electrophilic benzoquinone imine intermediates (Tang, 2003). Therefore, we

determined the effects of β-naphthoflavone and borneol on the in situ concentrations of 4'-OH-

DFN and 5-OH-DFN in diclofenac-treated hepatocytes in order to rationalize the attenuation in

LDH release by β-naphthoflavone (Fig. 2B) and borneol (Fig. 4B). Interestingly, β-

naphthoflavone pretreatment differentially altered the concentrations of the two hydroxy

metabolites of diclofenac; β-naphthoflavone increased the concentration of 4'-OH-DFN by over

2-fold, whereas it attenuated the concentration of 5-OH-DFN by 30% (Fig. 7). Borneol did not

affect the concentration of 4'-OH-DFN, but it decreased the concentration of 5-OH-DFN by 80%

(Fig. 7).


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Discussion

Acyl glucuronides have been implicated in the hepatotoxicity of carboxylic acid drugs;

however, there is no direct experimental evidence linking an acyl glucuronide to hepatotoxicity

(Stachulski et al., 2013). VPA-G, which is an acyl glucuronide (Dickinson et al., 1984), has

been linked to the formation of covalent VPA adducts with hepatocellular proteins (Porubek et

al., 1989) and the development of oxidative stress in rats by VPA (Tong et al., 2005b). Yet, the

hepatotoxic potential of VPA-G remains to be investigated. As shown in the present study, a

major finding is that in situ generated VPA-G did not appear to be toxic to sandwich-cultured rat

hepatocytes. This conclusion is based on the following experimental evidence: 1) an increase in

the in situ formation of VPA-G by β-naphthoflavone was not accompanied by an increase in

VPA toxicity (in fact, it led to an attenuation of BODIPY 558/568 C12 accumulation); and 2)

inhibition of in situ VPA-G formation by borneol did not result in an attenuation of VPA

toxicity. The lack of an effect of β-naphthoflavone on VPA toxicity cannot be due to the

increased cellular GSH content by β-naphthoflavone because depletion of GSH has been

reported as a consequence rather than a cause of VPA toxicity in cultured rat hepatocytes (Kiang

et al., 2011). In support of our finding that in situ generated VPA-G is non-toxic to rat

hepatocytes, previous studies have shown that VPA-G is one of the least reactive and the most

stable acyl glucuronides (Stachulski et al., 2006). Furthermore, as demonstrated under the cell

culture conditions employed in this study, acyl migration of VPA-G was not significant

(Surendradoss et al., 2013), suggesting that VPA-G did not form more reactive positional

isomers. Overall, VPA glucuronidation appears to be a typical detoxification pathway, as

determined in the current study in sandwich-cultured rat hepatocytes.


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Another novel finding of the present study was obtained from evaluating the effects of

various known inducers of UGTs on increasing in situ formation of VPA-G in sandwich-cultured

rat hepatocytes treated with VPA. Among the various inducers investigated in this study, only β-

naphthoflavone, phenobarbital, and L-sulforaphane, but not 3-methylcholanthrene, quercetin,

clofibrate, dexamethasone, PCN, or trans-stilbene oxide, increased in situ formation of VPA-G

in sandwich-cultured rat hepatocytes. The observed magnitude of the increase (~ two-fold) in

VPA glucuronidation by β-naphthoflavone, phenobarbital, and L-sulforaphane is consistent with

the notion that the inducibility of UGT enzymes is less than that of cytochrome P450 enzymes

(Soars et al., 2004). The increase in the extent of drug glucuronidation in response to

prototypical UGT inducers is usually ~ two-fold (Lin and Wong, 2002; Soars et al., 2004). As

shown in a previous ex vivo study (Shelby and Klaassen, 2006), β-naphthoflavone induces the

hepatic gene expression of rat UGT1A3, UGT1A6, and UGT1A7, whereas PB induces UGT2B1.

Therefore, these UGT enzymes are likely catalysts of VPA glucuronidation. In fact, rat

recombinant UGT2B1 has been shown to glucuronidate VPA (Pritchard et al., 1994).

As a comparison to the results obtained with VPA-G, the present study also investigated

the effects of modulating in situ formation of DFN-G on hepatocyte toxicity of diclofenac. The

increase in VPA-G formation by β-naphthoflavone did not affect LDH release in VPA-treated

sandwich-cultured rat hepatocytes, whereas the increase in DFN-G formation by β-

naphthoflavone resulted in an attenuation of LDH release in diclofenac-treated hepatocytes. The

reason for the differential effect of β-naphthoflavone on the toxicity of VPA and diclofenac is

not known, but it may involve distinct modulation of other biotransformation pathways of these

two drugs. Other than glucuronidation, pretreatment of rat hepatocytes with β-naphthoflavone

also increased cytochrome P450-mediated biotransformation of VPA, as shown in the present


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study. However, the in situ formation of these VPA oxidative metabolites has been reported not

to influence VPA toxicity in sandwich-cultured rat hepatocytes (Kiang et al., 2010; Surendradoss

et al., 2012). In the case of diclofenac, the hepatocyte toxicity of this drug has been attributed to

the formation of cytochrome P450-mediated oxidative metabolites of diclofenac (Kretz-Rommel

and Boelsterli, 1993). In the present study, β-naphthoflavone attenuated the hepatocyte toxicity

of diclofenac, even though it increased in situ formation of 4'-OH-DFN. The reason for the

attenuation of diclofenac toxicity is not known, but it may relate to the increase in cellular GSH

content by β-naphthoflavone. GSH is involved in the conjugation and detoxification of

cytochrome P450-mediated reactive metabolites of diclofenac (Tang et al., 1999). Overall, the

increase in the in situ formation of two acyl glucuronides (VPA-G and DFN-G), which differ

greatly (~ 150-fold) in chemical stability (Stachulski et al., 2006), did not enhance the hepatocyte

toxicity of the parent drugs VPA and diclofenac.

As shown in the present study, co-treatment of sandwich-cultured rat hepatocytes with

borneol and VPA decreased VPA-G formation over a period of 4 to 24 h after VPA treatment,

but this was not associated with a decrease in LDH release over the same time period. By

comparison, co-treatment of hepatocytes with borneol and diclofenac was seen to decrease DFN-

G levels for only up to 4 h after diclofenac treatment. The apparent recovery of the in situ levels

of DFN-G over the 4 to 24 h period was accompanied by a marked decrease in the extent of

LDH release in the 8 and 24 h time points after the co-treatment of hepatocytes with borneol and

diclofenac. A possible explanation for the observed decrease in LDH release by borneol in

diclofenac-treated hepatocytes is the substantive attenuation of the in situ levels of the oxidative

metabolite 5-OH-DFN. Previously, borneol was reported to increase rather than decrease LDH

release in diclofenac-treated rat hepatocytes (Kretz-Rommel and Boelsterli, 1993). The reason


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for the differences in the effect of borneol on LDH release in diclofenac-treated hepatocytes

shown in the present study and in the earlier study is not known; however, it may relate to the

differences in the duration of borneol treatment (24 h in the present study and 2 h in the 1993

study by Kretz-Rommel and Boelsterli).

Borneol inhibits the glucuronidation of drugs and other chemicals by depleting the

hepatocellular levels of the co-factor uridine 5'-diphospho-glucuronic acid (UDP-GA) and

inhibiting the catalytic activity of specific UGT enzymes that catalyze glucuronidation (Watkins

and Klaassen, 1982). As evident by our time course data, the inhibitory effect of borneol on in

situ VPA-G formation was apparent from 4 to 24 h after VPA treatment, whereas its inhibition of

in situ DFN-G formation no longer occurred at 8 h after diclofenac treatment. The differences in

the temporal profile on the effect of borneol on in situ formation of VPA-G and DFN-G suggest

that borneol does not inhibit the glucuronidation of VPA and diclofenac by the same mechanism.

Sandwich-cultured hepatocytes have been used to study the toxicity of acyl glucuronides

(Dong and Smith, 2009). However, a limitation of this model is the lack of enterohepatic

recycling. As evident from a preliminary experiment, hydrolysis of VPA-G was not apparent in

our sandwich-cultured rat hepatocyte model. Pretreatment or co-treatment with an inhibitor of

VPA-G hydrolysis (Suzuki et al., 2010), D-saccharolactone or a carbapenem (meropenem or

imipenem), did not enhance in situ concentrations of VPA-G, as assessed at 24 h after treatment

with VPA (data not shown). However, VPA-G is subject to efficient hydrolysis and

enterohepatic recycling in rats in vivo (Dickinson et al., 1979; Liu and Smith, 2006). Thus, the

conjugation-deconjugation cycling may increase the hepatocellular exposure to VPA-G in vivo.

It remains to be investigated whether such an increase in exposure to VPA-G in vivo contributes

to the hepatotoxicity of VPA.


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In conclusion, similar to the findings on DFN-G in the same cell culture model, in situ

generated VPA-G was not toxic to sandwich-cultured rat hepatocytes, as evident from the

experiments using chemical modulators of VPA glucuronidation. The findings of this study,

together with the available information regarding the chemical reactivity of VPA-G (Stachulski

et al., 2006), lead us to conclude that in situ generated VPA-G does not contribute to the

hepatocyte toxicity of VPA. As reported previously, the in situ formation of other reactive

metabolites of VPA, such as 4-ene-VPA (Kiang et al., 2010) and (E)-2,4-diene-VPA

(Surendradoss et al., 2012), also does not play a role in VPA toxicity. Valproyl-S-acyl CoA is

another metabolite of VPA formed by the β-oxidation pathway (Silva et al., 2008). It has been

proposed that bioactivation of carboxylic acids may also involve acyl-CoA thioesters and acyl-

glutathione thioesters, which are even more reactive than acyl glucuronides (Skonberg et al.,

2008). The toxicological significance of acyl-CoA thioester metabolites have been relatively

less studied in comparison to acyl glucuronides, although the intracellular localization of acyl-

CoA thioesters makes them more probable mediators of hepatotoxicity associated with

carboxylic acid drugs (Darnell and Weidolf, 2013). In a previous study, it was speculated that

the formation of valproyl-S-acyl CoA thioester may contribute to the hepatotoxicity of VPA

(Grillo et al., 2001). It would be of interest in the future to investigate the role of valproyl-S-

acyl-CoA and other downstream β-oxidation metabolites of VPA in the hepatotoxicity of VPA

using the sandwich-cultured hepatocyte model.


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Acknowledgment

The authors thank Andras Szeitz (Faculty of Pharmaceutical Sciences, The University of

British Columbia) for his technical assistance with the UHPLC-MS/MS assays for VPA-G and

DFN-G.

Authorship Contributions

Participated in research design: Surendradoss, Chang, Abbott.

Conducted experiments: Surendradoss

Performed data analysis: Surendradoss

Wrote or contributed to the writing of the manuscript: Surendradoss, Chang, Abbott.


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Footnotes

This work was supported by the Canadian Institutes of Health Research [Grant MOP-13744 to

F.S.A. and T.K.H.C.]. J.S. received a Four Year Doctoral Fellowship from The University of

British Columbia. T.K.H.C. received a Senior Scholar Award from the Michael Smith

Foundation for Health Research.

Address correspondence to: Dr. Frank S. Abbott, Faculty of Pharmaceutical Sciences, The

University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada. E-

mail: [email protected]


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Figure Legends

Fig. 1. Time course and concentration-response relationship in the in situ formation of VPA-G

in sandwich-cultured rat hepatocytes treated with VPA. (A) Hepatocytes were cultured for 120 h

and then treated with VPA (1 mM) or culture medium (vehicle). At 1, 2, 4, 8, 12, and 24 h after

drug treatment, an aliquot of culture supernatant was collected and VPA-G concentrations were

quantified by UHPLC-MS/MS. (B) Hepatocytes were cultured for 120 h and then treated with

VPA (0.03-100 mM) or culture medium (vehicle) for 24 h. Data are expressed as mean ± SEM

for three or four rats.

Fig. 2. Effect of UGT enzyme inducers on in situ formation of VPA-G and LDH release in

sandwich-cultured rat hepatocytes treated with VPA. (A) Hepatocytes were cultured for 48 h

and then pretreated with β-naphthoflavone (BNF; 20 µM), L-sulforaphane (L-SFN; 5 µM),

sodium phenobarbital (PB; 2 mM), or vehicle (0.1% DMSO for BNF and L-SFN; culture

medium for PB) once every 24 h for 72 h. Subsequently, the hepatocytes were treated with VPA

(10 mM), culture medium (vehicle for VPA), diclofenac (DFN; 400 µM), or DMSO (0.1% v/v;

vehicle for DFN) for the next 24 h. (B) Hepatocytes were pretreated with BNF and then treated

with VPA (10 or 15 mM), DFN (400 µM), or their respective vehicle as described above in (A).

Data are expressed as mean ± SEM for three rats. *Significantly different from the respective

vehicle-pretreated control group, P < 0.05. #Significantly different from the respective culture

medium-treated group, P < 0.05; †Significantly different from the DMSO + DMSO group, P <

0.05; ‡significantly different from the BNF + DMSO and the DMSO + DFN groups, P < 0.05.

VPA-G concentrations were 256 ± 45 and 213 ± 35 µM per 0.7 million cells in the culture


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medium- and DMSO-pretreated groups, respectively. DFN-G concentrations were 23 ± 2.0 and

20 ± 1.8 µM per 0.7 million cells in the culture medium- and DMSO-pretreated groups,

respectively.

Fig. 3. Effect of β-naphthoflavone on (A) BODIPY 558/568 C12 accumulation and (B) total

GSH content in sandwich-cultured rat hepatocytes treated with VPA. Hepatocytes were cultured

for 48 h and then pretreated with β-naphthoflavone (BNF; 20 µM) or DMSO (0.1% v/v; vehicle)

once every 24 h for 72 h. Subsequently, the hepatocytes were treated with VPA (10 or 15 mM)

or culture medium (vehicle) for the next 24 h. Data are expressed as mean ± SEM for three to

five rats. *Significantly different from the DMSO + culture medium group, P < 0.05;

#significantly different from the corresponding DMSO + culture medium or DMSO + VPA

groups; P < 0.05; ‡significantly different from the BNF + culture medium and DMSO + 15 mM

VPA groups, P < 0.05. The total GSH content was 7.4 ± 1.6 µM per 0.7 million cells in the

DMSO + culture medium group.

Fig. 4. Effect of borneol on in situ formation of VPA-G and LDH release in sandwich-cultured

rat hepatocytes treated with VPA. Hepatocytes were cultured for 120 h and then pretreated with

borneol (1 mM) or DMSO (0.1% v/v; vehicle) for 0.5 h. Subsequently, the hepatocytes were

treated with 10 mM VPA (A and B), 15 mM VPA (B), culture medium (vehicle for VPA; A and

B), 400 µM diclofenac (DFN; A and B), or DMSO (0.1% v/v; vehicle for DFN; A and B) for the

next 24 h in the presence of borneol (1 mM) or DMSO (0.1% v/v). Data are expressed as mean

± SEM for three rats. #Significantly different from the DMSO + VPA group, P < 0.05;

*significantly different from the DMSO + culture medium group or borneol + culture medium


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group, P < 0.05; †significantly different from the DMSO + DMSO group, P < 0.05;

‡significantly different from the borneol + DMSO and DMSO + DFN groups, P < 0.05. VPA-G

concentration was 283 ± 41 µM per 0.7 million cells in the DMSO + VPA group. DFN-G

concentration was 20 ± 1.8 µM per 0.7 million cells in the DMSO + DFN group.

Fig. 5. Effect of borneol on (A) BODIPY accumulation and (B) total GSH content in sandwich-

cultured rat hepatocytes treated with VPA. Hepatocytes were cultured for 120 h and then

pretreated with borneol (1 mM) or DMSO (0.1% v/v; vehicle) for 0.5 h. Subsequently, the

hepatocytes were treated with VPA (10 or 15 mM) or culture medium (vehicle) for 24 h in the

presence of borneol (1 mM) or DMSO (0.1% v/v). Data are expressed as mean ± SEM for three

or four rats. *Significantly different from the DMSO + culture medium group, P < 0.05;

#significantly different from the borneol + culture medium, P < 0.05; †significantly different

from the borneol + culture medium and DMSO + 15 mM VPA groups. The total GSH content

was 13 ± 1.1 µM per 0.7 million cells in the DMSO + culture medium group.

Fig. 6. Time course on the effect of borneol on (A) VPA-G concentration, (B) LDH release after

VPA treatment, (C) DFN-G concentration, and (D) LDH release following diclofenac treatment

in sandwich-cultured rat hepatocytes. Hepatocytes were cultured for 120 h and then pretreated

with borneol (1 mM) or DMSO (0.1% v/v; vehicle) for 0.5 h. Subsequently, the hepatocytes

were treated with VPA (15 mM), culture medium (vehicle for VPA), diclofenac (DFN; 400 µM),

or DMSO (0.1% v/v; vehicle for DFN) for the next 2, 4, 8, and 24 h in the presence of borneol (1

mM) or DMSO (0.1% v/v). Data are expressed as mean ± SEM for four rats. *Significantly

different from either DMSO + VPA (panel A) or DMSO + DFN (panel C) treatment group, P <


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0.05; †significantly different from either DMSO + culture medium (panel B) or DMSO + DMSO

treatment group (panel D), P < 0.05; ‡significantly different from either the borneol + culture

medium and DMSO + VPA treatment groups (panel B) or the borneol + DMSO and DMSO +

DFN treatment groups (panel D), P < 0.05.

Fig. 7. Effect of β-naphthoflavone and borneol on in situ formation of 4'-OH-DFN and 5-OH-

DFN in sandwich-cultured rat hepatocytes treated with diclofenac. At 48 h after plating,

hepatocytes were pretreated with β-naphthoflavone (BNF; 20 µM) or DMSO (vehicle; 0.1% v/v)

once every 24 h for 72 h, followed by treatment with diclofenac (400 µM) or 0.1% DMSO

(vehicle) for the next 24 h. In another experiment, hepatocytes were cultured for 120 h and

pretreated with borneol (1 mM) or DMSO (0.1% v/v; vehicle) for 0.5 h, followed by treatment

with diclofenac (400 µM) or 0.1% DMSO (vehicle) in the presence of borneol or DMSO for the

next 24 h. Data are expressed as mean ± SEM for three rats. *Significantly different from the

DMSO group, P < 0.05. 4'-OH-DFN and 5-OH-DFN concentrations were 1.0 ± 0.20 and 0.46 ±

0.20 µM per 0.7 million cells, respectively, in the DMSO group.


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TABLE 1

Concentration-response effect of β-naphthoflavone and borneol on in situ formation of VPA-G in

sandwich-cultured rat hepatocytes treated with VPA

Experiment 1. At 48 h after plating, hepatocytes were pretreated with β-naphthoflavone

(BNF; 5, 10, 15, or 20 µM) or 0.1% DMSO (vehicle) every 24 h for 72 h and then treated with

VPA (10 mM) or culture medium (vehicle) for the next 24 h. Experiment 2. At 120 h after

plating, hepatocytes were pretreated with borneol (0.25, 0.5, 0.75, or 1 mM) or 0.1% DMSO

(vehicle) for 0.5 h. Subsequently, hepatocytes were treated with VPA (10 mM) or culture

medium (vehicle) in the presence of borneol or DMSO at concentrations indicated above for 24

h. In both experiments, culture supernatants and cell lysates were collected at the end of the 24 h

treatment period and subjected to UHPLC-MS/MS assay for VPA-G concentrations. Data are

expressed as mean ± SEM for three or four rats.

Pretreatment / Treatment

In Situ Concentration of VPA-G

(fold-increase over control)

Culture Supernatant Cell Lysate

Experiment 1

DMSO 0.1% v/v 1.0 ± 0.0 1.0 ± 0.0

BNF 5 µM 1.7 ± 0.06* Not determined



BNF 20 µM 2.3 ± 0.20* 2.1 ± 0.09*


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Experiment 2

DMSO 0.1% v/v 1.0 ± 0.0 1.0 ± 0.0

Borneol 0.25 mM 0.90 ± 0.03* 0.85 ± 0.05*

Borneol 0.5 mM 0.83 ± 0.02* 0.78 ± 0.04*

Borneol 0.75 mM 0.72 ± 0.04* 0.72 ± 0.04*

Borneol 1 mM 0.64 ± 0.04* 0.65 ± 0.03*

*Significantly different from the DMSO vehicle control group, P < 0.05.


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TABLE 2

Effect of β-naphthoflavone on in situ formation of oxidative metabolites of VPA in sandwich-

cultured rat hepatocytes treated with VPA

At 48 h after plating, hepatocytes were pretreated with 20 µM β-naphthoflavone (BNF)

or 0.1% DMSO (vehicle) once every 24 h for 72 h and then treated with VPA (10 or 15 mM) or

culture medium (vehicle) for the next 24 h. Culture supernatants were collected at the end of the

24 h treatment period and subjected to GC-MS assay for the quantification of the concentrations

of oxidative metabolites of VPA. Data are expressed as mean ± SEM for three rats.

In situ Concentration of VPA metabolites (µM / 0.7 × 106 cells)

Metabolite 10 mM VPA 15 mM VPA

0.1% DMSO 20 μM BNF 0.1% DMSO 20 μM BNF

4-ene-VPA 0.06 ± 0.01 0.09 ± 0.01 0.05 ± 0.01 0.08 ± 0.01

4-keto-VPA 0.15 ± 0.01 0.50 ± 0.06* 0.22 ± 0.02 0.58 ± 0.08*

4-OH-VPA 2.5 ± 0.30 4.9 ± 0.54* 2.1 ± 0.25 5.1 ± 0.46*

5-OH-VPA 1.4 ± 0.27 1.2 ± 0.17* 1.3 ± 0.25 1.2 ± 0.24

(E)-2,4-diene-VPA None detected None detected None detected None detected

(E,Z)-2,3′-diene-VPA 0.16 ± 0.01 0.19 ± 0.01 0.15 ± 0.01 0.19 ± 0.02

(E,E)-2,3′-diene-VPA 1.7 ± 0.27 1.7 ± 0.21 1.7 ± 0.34 1.8 ± 0.34

3-ene-VPA 1.2 ± 0.20 1.3 ± 0.14 1.0 ± 0.15 1.1 ± 0.13


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(E)-2-ene-VPA 1.7 ± 0.13 1.5 ± 0.06 1.8 ± 0.21 1.8 ± 0.20

3-OH-VPA 5.7 ± 1.2 11 ± 4.2 5.5 ± 1.5 18 ± 5.0*

3-keto-VPA 2.0 ± 0.63 1.7 ± 0.32 2.1 ± 0.99 2.2 ± 0.79

*Significantly different from the DMSO-pretreated vehicle control group, P < 0.05.


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TABLE 3

Effect of borneol on in situ formation of oxidative metabolites of VPA in sandwich-cultured rat

hepatocytes treated with VPA

At 120 h after plating, hepatocytes were pretreated with 1 mM borneol or 0.1% DMSO

(vehicle) for 0.5 h. Subsequently, hepatocytes were treated with VPA (10 or 15 mM) or culture

medium (vehicle) in the presence of 1 mM borneol or 0.1% DMSO for 24 h. Culture

supernatants were collected at the end of the 24 h treatment period and subjected to GC-MS

assay for the quantification of the concentrations of oxidative metabolites of VPA. Data are

expressed as mean ± SEM for four rats.

In situ Concentration of VPA metabolites (µM / 0.7 × 106 cells)

Metabolite 10 mM VPA 15 mM VPA

0.1% DMSO 1 mM Borneol 0.1% DMSO 1 mM Borneol

4-ene-VPA 0.08 ± 0.01 0.05 ± 0.01* 0.04 ± 0.01 0.04 ± 0.01

4-keto-VPA 0.09 ± 0.01 0.09 ± 0.02 0.13 ± 0.01 0.16 ± 0.02

4-OH-VPA 3.4 ± 0.56 1.4 ± 0.45* 2.2 ± 0.22 1.9 ± 0.21

5-OH-VPA 1.6 ± 0.10 0.94 ± 0.05* 1.7 ± 0.23 1.3 ± 0.19

(E)-2,4-diene-VPA None detected None detected None detected None detected

(E,Z)-2,3′-diene-VPA 0.19 ± 0.02 0.19 ± 0.02 0.18 ± 0.01 0.20 ± 0.01

(E,E)-2,3′-diene-VPA 1.5 ± 0.09 1.4 ± 0.20 1.6 ± 0.22 1.5 ± 0.14


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3-ene-VPA 1.3 ± 0.13 1.2 ± 0.16 1.1 ± 0.13 1.0 ± 0.10

(E)-2-ene-VPA 1.8 ± 0.16 1.9 ± 0.20 2.2 ± 0.29 2.2 ± 0.23

3-OH-VPA 7.1 ± 0.36 6.2 ± 1.5 11 ± 3.1 7.2 ± 1.1

3-keto-VPA 1.4 ± 0.33 1.1 ± 0.35 2.8 ± 0.75 2.1 ± 0.97

*Significantly different from the DMSO-co-treated vehicle control group, P < 0.05.


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