Evaluation of in situ generated valproyl 1-O-ß-acyl glucuronide in valproic acid toxicity in sandwich-cultured rat hepatocytes.

Acyl glucuronides are reactive electrophilic metabolites implicated in the toxicity of carboxylic acid drugs. Valproyl 1-O-ß-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 lactate dehydrogenase (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, ß-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 for VPA hepatotoxicity.

A rapid and sensitive assay to quantify valproyl 1-O-acyl glucuronide in supernatants of sandwich-cultured rat hepatocytes using ultra-high performance liquid chromatography-tandem mass spectrometry.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of valproyl-1-O-acyl glucuronide (VPA-G) levels in hepatocyte culture medium. Chromatographic separation was achieved using a Waters Acquity UPLC(®) BEH C18 column (1.7µm, 2.1mm×50mm) with gradient elution and a total run time of 4min. [(2)H6]-VPA-G was used as internal standard (IS). Quantification was performed in the multiple reaction monitoring (MRM) mode using the total ion current of the MRM transition pairs m/z 319.1→142.7 and m/z 319.1→175.2 for VPA-G, and m/z 325.1→149.3 and m/z 325.1→174.9 for the IS under negative electrospray ionization mode. The assay was linear over the VPA-G concentrations of 0.5-500ng/mL, with a r(2) value of 0.995±0.002 (mean±SD). The intra- and inter-day accuracy (% deviation) ranged from -10.2% to 11.1%, whereas the intra- and inter-day precision (% RSD) were ≤7.43%. The method was applied successfully to the quantification of VPA-G levels in culture supernatants of sandwich-cultured rat hepatocytes treated with valproic acid (VPA). No significant difference in the levels of VPA-G over a culture period of 6 days was observed in an experiment that investigated the effect of the age of hepatocyte culture on the extent of VPA glucuronidation. The method presented here for the direct quantification of VPA-G is an improvement of existing methods in the literature and offers a shorter run time and greater sensitivity that enables the use of small volumes of sample. To the best of our knowledge, this is the first validated UHPLC-MS/MS method applied to the quantification of VPA-G in cell culture supernatants.

Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes.

Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by ß-oxidation. Another biotransformation pathway involves ß-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2',7'-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C12), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes.

Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity.

The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.

Role of oxidative metabolism in the effect of valproic acid on markers of cell viability, necrosis, and oxidative stress in sandwich-cultured rat hepatocytes.

Valproic acid (VPA) is a drug known for idiosyncratic hepatotoxicity and is associated with oxidative stress. It is metabolized extensively with at least one pathway leading to reactive metabolites. The primary aim of the present study was to determine whether oxidative metabolites of VPA generated in situ contribute to the toxicity of the parent drug in sandwich-cultured rat hepatocytes. Concentration-response experiments with VPA produced median effective concentration values (mean ± SEM) of 1.1 ± 0.4, 12.2 ± 1.4, and 12.3 ± 1.9mM in the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1; cell viability), lactate dehydrogenase (LDH; necrosis), and 2',7'-dichlorofluorescein (DCF; oxidative stress) assays, respectively. At equimolar concentrations, only the unsaturated metabolites of VPA gave responses comparable to VPA, with 2,4-diene-VPA calculated to be 3-, 6-, and 10-fold more potent than VPA in the WST-1, LDH, and DCF assays, respectively. In support of a role for reactive metabolites, 2-fluoro-2-propylpentanoic acid, which is relatively resistant to biotransformation to form a 2,4-diene metabolite, yielded little or no toxicity when compared with the nonhepatotoxic octanoic acid or the vehicle-treated control. By comparison, attenuating the in situ formation of 2-propylpent-4-enoic acid (4-ene-VPA), 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, and 5-hydroxy-2-propylpentanoic acid by an inhibitor of cytochrome P450 (1-aminobenzotriazole) did not alter the effects of VPA on the WST-1, LDH, or DCF assay. Overall, VPA toxicity in sandwich-cultured rat hepatocytes is independent of the in situ formation of cytochrome P450-dependent oxidative metabolites, including 4-ene-VPA. However, the data obtained from structural analogues of VPA suggest that biotransformation does appear to play a role in VPA toxicity in rat hepatocytes.

Effect of Ginkgo biloba extract on oxidative metabolism of valproic acid in hepatic microsomes from donors with the CYP2C9*1/*1 genotype.

We investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the oxidative metabolism of valproic acid (VPA) in hepatic microsomes from donors with the CYP2C9*1/*1 genotype. G. biloba extract decreased 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA formation with mean (+/- SE) IC50 values of 340 +/- 40 microg/mL, 370 +/- 100 microg/mL, 180 +/- 30 microg/mL, and 210 +/- 20 microg/mL, respectively. This was associated with inhibition of not only CYP2C9*1, but also CYP2A6 and CYP2B6. Bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were not responsible for the inhibition of VPA metabolism by the extract. When analyzed as the sum of the aglycone and total glycosides present in the extract, quercetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 76%, 51%, and 70%, respectively, kaempferol decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 65%, 46%, and 49%, respectively, and isorhamnetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 29%, 26%, and 31%, respectively. The 3 aglycones did not affect 3-OH-VPA formation. In summary, G. biloba extract decreased hepatic microsomal formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA, but the effect was not due to the terpene trilactones or flavonol glycosides investigated in our study.

Kinetics of valproic acid glucuronidation: evidence for in vivo autoactivation.

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (approximately 79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (V(max)(app)), the substrate concentration resulting in 50% of V(max)(app) (S(50)(app)), and Hill coefficient (n) of 2.10 +/- 0.75 micromol/min/kg, 117 +/- 56 microM, and 1.34 +/- 0.14, respectively. Comparable estimates of V(max)(app) (2.63 +/- 0.33 micromol/min/kg), S(50)(app) (118 +/- 53 microM), and n (2.06 +/- 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.

Oxidative stress as a mechanism of valproic acid-associated hepatotoxicity.

Valproic acid (2-n-propylpentanoic acid; VPA) has several therapeutic indications, but it is used primarily as an anticonvulsant. VPA is a relatively safe drug, but its use is associated with idiosyncratic hepatotoxicity, which in some cases may lead to fatality. The underlying mechanism responsible for the hepatotoxicity is still not well understood, but various hypotheses have been proposed, including oxidative stress. This article discusses the experimental evidence on the effect of VPA on the various indices of oxidative stress and on the potential role of oxidative stress in VPA-associated hepatotoxicity.

Oxidative stress in children receiving valproic acid.

OBJECTIVE: To determine whether valproic acid (VPA) influences urinary levels of 15-F2t -isoprostane (15-F2t -IsoP), a marker of oxidative stress, in children. STUDY DESIGN: Morning urine samples were collected from children with epilepsy receiving VPA (n = 25), carbamazepine (n = 16), or clobazam (n = 12) for > or = 4 weeks and from age-matched control subjects (n = 39). Urinary 15-F2t -IsoP levels were determined by enzyme-linked immunosorbent assay. RESULTS: The mean (+/- standard deviation) urine 15-F2t -IsoP levels (nmol/mmol Cr) were: valproic acid (0.36 +/- 0.15); carbamazepine (0.24 +/- 0.10); clobazam (0.23 +/- 0.10); control group (0.20 +/- 0.09). Patients treated with VPA had significantly elevated 15-F2t -IsoP levels when compared with the control, carbamazepine, and clobazam groups (P < .05). Multiple linear regression analysis demonstrated that younger patient age and exposure to second-hand smoke were significant predictors of elevated urine 15-F2t -IsoP levels within the control group (r2 = 0.261, P = .05 and P = .01, respectively). Subjects not exposed to second-hand smoke receiving valproic acid therapy had a significantly elevated mean urine 15-F2t -IsoP level compared to subjects not exposed to second-hand smoke in the carbamazepine, clobazam and control groups (P < .05). CONCLUSIONS: These data demonstrate that treatment of children with VPA is associated with higher urinary levels of 15-F2t -IsoP, a marker of oxidative stress.

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1-8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75-80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by approximately 50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.

Valproic acid glucuronidation is associated with increases in 15-F2t-isoprostane in rats.

Oxidative stress has been associated with valproic acid (VPA) treatment in rats and studies are ongoing to examine the relationship between VPA biotransformation and the increase in the lipid peroxidation biomarker 15-F2t-isoprostane (15-F2t-IsoP). This study investigated the effects of modulating VPA-1-O-acyl glucuronide (VPA-G) formation on 15-F2t-IsoP levels. Adult male Sprague-Dawley rats were pretreated with phenobarbital (PB; 80 mg/kg/day for 4 days), (-)-borneol (320 mg/kg), or a combination of both before VPA treatment (500 mg/kg). Liver VPA-G levels were determined by LC/MS and plasma and liver 15-F2t-IsoP levels were measured using an EIA method. PB, an inducer of VPA glucuronidation, elevated both liver VPA-G and plasma and liver 15-F2t-IsoP levels in VPA-treated rats. (-)-Borneol, an inhibitor of glucuronidation, significantly reduced the levels of liver VPA-G and decreased plasma and liver 15-F2t-IsoP levels in both the VPA and the PB + VPA groups. (-)-Borneol and PB alone did not elevate 15-F2t-IsoP levels compared to the vehicle control groups. The fluorinated analogue of VPA, alpha-fluoro-VPA, was a poor substrate for glucuronidation and did not elevate 15-F2t-IsoP levels. In summary, the VPA-induced formation of 15-F2t-IsoP is apparently associated with VPA glucuronidation.

Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes.

Oxidative stress has been associated with valproic acid (VPA) treatment, and mitochondrial dysfunction has been implicated in the pathogenesis of VPA-idiosyncratic hepatotoxicity. The present study investigated the effect of VPA and the role of GSH on oxidative stress, mitochondrial membrane potential, and toxicity in freshly isolated rat hepatocytes. Hepatocytes were isolated from Sprague-Dawley rats, and total levels of glutathione (GSH) reduced by pretreatment with a combination of L-buthionine sulfoximine (2 mM) and diethylmaleate (0.5 mM) prior to VPA (0-1000 microg/ml) treatment. Oxidative stress was determined by measuring the levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP) and 2',7'-dichlorofluorescein (DCF). Mitochondrial membrane potential (Deltapsi(m)) was determined by using the dual-fluorescent dye JC-1, and cell viability was evaluated by the water-soluble tetrazolium salt WST-1 assay. Exposure of rat hepatocytes to VPA (0-1000 mug/ml) resulted in a time- and dose-dependent increase in 15-F(2t)-IsoP and DCF fluorescence, and these levels were further elevated in GSH-reduced hepatocytes. In control hepatocytes, VPA had no effect on cell viability; however, significant cytotoxicity was observed in the glutathione-depleted hepatocytes treated with 1000 mug/ml VPA. The Deltapsi(m) was only reduced in glutathione-reduced hepatocytes at 500 and 1000 microg/ml VPA. Our novel findings indicate that acute treatment of freshly isolated rat hepatocytes with VPA resulted in oxidative stress, which occurred in the absence of cytotoxicity, and that glutathione confers protection to hepatocytes against mitochondrial damage by VPA.

Valproic acid I: time course of lipid peroxidation biomarkers, liver toxicity, and valproic acid metabolite levels in rats.

A single dose of valproic acid (VPA), which is a widely used antiepileptic drug, is associated with oxidative stress in rats, as recently demonstrated by elevated levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP). To determine whether there was a temporal relationship between VPA-associated oxidative stress and hepatotoxicity, adult male Sprague-Dawley rats were treated ip with VPA (500 mg/kg) or 0.9% saline (vehicle) once daily for 2, 4, 7, 10, or 14 days. Oxidative stress was assessed by determining plasma and liver levels of 15-F(2t)-IsoP, lipid hydroperoxides (LPO), and thiobarbituric acid reactive substances (TBARs). Plasma and liver 15-F(2t)-IsoP were elevated and reached a plateau after day 2 of VPA treatment compared to control. Liver LPO levels were not elevated until day 7 of treatment (1.8-fold versus control, p < 0.05). Liver and plasma TBARs were not increased until 14 days (2-fold vs. control, p < 0.05). Liver toxicity was evaluated based on serum levels of alpha-glutathione S-transferase (alpha-GST) and by histology. Serum alpha-GST levels were significantly elevated by day 4, which corresponded to hepatotoxicity as shown by the increasing incidence of inflammation of the liver capsule, necrosis, and steatosis throughout the study. The liver levels of beta-oxidation metabolites of VPA were decreased by day 14, while the levels of 4-ene-VPA and (E)-2,4-diene-VPA were not elevated throughout the study. Overall, these findings indicate that VPA treatment results in oxidative stress, as measured by levels of 15-F(2t)-IsoP, which precedes the onset of necrosis, steatosis, and elevated levels of serum alpha-GST.

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats.

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This study's objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.