Valproyl-dephosphoCoA: a novel metabolite of valproate formed in vitro in rat liver mitochondria.

The mitochondrial metabolism of valproic acid (VPA) was investigated in vitro to elucidate its beta-oxidation pathway since the characterization of VPA intermediates in the acyl-CoA thioester form, and not just in their free acid form, has not been fully achieved. Intact rat liver mitochondria were incubated with [4,5-3H2]VPA and [2-3H]VPA. The respective intermediates, valproyl-CoA, Delta2(E)-valproyl-CoA, 3-hydroxyvalproyl-CoA, and 3-oxovalproyl-CoA were analyzed by reverse phase high performance liquid chromatography (HPLC) with radioisotope and UV detection. An unknown metabolite, originating from both labeled substrates, was detected. It was identified as valproyl-dephosphoCoA (valproyl-dephCoA) by fast atom bombardment mass spectrometry (FAB-MS) analysis of the corresponding HPLC peak fraction. The FAB-MS spectrum of the authentic chemically synthesized valproyl-dephCoA proved to be consistent with that of the unknown compound. Valproyl-dephCoA is produced from valproyl-CoA in mitochondria, probably via a phosphatase-catalyzed reaction. This conversion was shown to be more dependent on the energy state involving [AXP] ([AXP] = [ATP] + [ADP] + [AMP]) and [phosphate] concentrations rather than the strict mitochondrial [ATP]/[ADP] ratio. The results indicate that higher concentrations of AXP and phosphate inhibit the dephosphorylation of valproyl-CoA. A complete understanding of the toxic significance of valproyl-dephCoA formation in vivo as a potential inhibitor of fatty acid beta-oxidation is important to clarify the pathogenesis of VPA-associated hepatotoxicity.     

Studies on the metabolism of tolmetin to the chemically reactive acyl-coenzyme A thioester intermediate in rats.

Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.     

Covalent binding of 2-phenylpropionyl-S-acyl-CoA thioester to tissue proteins in vitro.

In this study, we investigated the possible involvement of acyl-CoA, reactive intermediary metabolites of 2-arylpropionic acids (profens), in protein adduct formation in rat liver homogenate and in human serum albumin (HSA) in buffer. (RS)-[1-14C]-2-Phenylpropionic acid (14C-2-PPA, 1 mM) was incubated with rat liver homogenate (1.5 mg/ml) in the presence of cofactors of acyl-CoA formation (Mg2+, ATP, and CoA). Aliquots of the incubation mixture were analyzed for covalent binding and acyl-CoA formation over a 3-h period. High-performance liquid chromatographic analysis of the products from such incubations showed the presence of 2-phenylpropionyl-S-acyl-CoA (2-PPA-CoA), which was confirmed by coelution with authentic 2-PPA-CoA, as well as by mass spectrometry. In the same incubations, 2-PPA was shown to bind covalently to hepatic proteins in a time- and ATP-dependent fashion. Inhibition of 2-PPA-CoA formation by acyl-CoA synthetase inhibitors, such as palmitic acid, lauric acid, octanoic acid, and ibuprofen, markedly decreased the extent of covalent binding of 2-PPA to hepatic proteins. Results from these in vitro studies strongly suggest that acyl-CoA thioester derivatives are chemically reactive and are able to bind covalently to tissue proteins in vitro, and, therefore, may contribute significantly to covalent adduct formation of profen drugs in vivo.     

Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans.

The severe hepatotoxicity of valproic acid (VPA) is believed to be mediated through reactive metabolites. The formation of glutathione (GSH) and N-acetylcysteine (NAC) adducts of reactive intermediates derived from VPA and two of its metabolites, 2-propyl-4-pentenoic acid (4-ene-) and 2-propyl-2,4-pentadienoic acid [(E)-2,4-diene VPA], was investigated in the rat. Rats were dosed ip with 100 mg/kg of VPA, 4-ene-, or 2,4-diene-VPA, and methylated bile and urine extracts were analyzed by LC/MS/MS and GC/MS, respectively. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The NAC conjugate was also found to be a metabolite of VPA in patients. Both the GSH and NAC adducts were chemically synthesized and their structures established to be 5-(glutathion-S-yl)3-ene VPA and 5-(N-acetylcystein-S-yl)3-ene VPA by NMR and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo [about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr]. The characterization of the GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug.     

Acyl-coenzyme a formation of simvastatin in mouse liver preparations.

Formation of an acyl-CoA thioester has been proposed, but not directly demonstrated, to be a key step in mediating both lactonization and atypical beta-oxidation of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. Here, we describe studies to characterize formation of acyl-CoA thioesters in vitro in mouse liver preparations using the hydroxy acid form of simvastatin (SVA) as a model substrate. With an optimized chromatography method, three new products were detected in addition to the dehydration product (P1) and the lactone form of simvastatin, which have been characterized previously (Prueksaritanont et al., 2001). Based on high-pressure liquid chromatography analysis, UV spectroscopy, mass spectrometry, and NMR spectral characterization, two metabolites were identified as acyl-CoA thioester conjugates of SVA and P1, respectively, whereas the third metabolite (M1) was confirmed to be the L-beta-hydroxy isomer of simvastatin. M1 was probably formed by stereospecific hydration, a previously reported reaction, and subsequent lactonization of P1-S-acyl CoA. Among all the mouse liver subcellular fractions, microsomes exhibited the highest capacity to catalyze the CoASH-dependent metabolism of SVA, whereas such activity was totally absent in cytosol. Together, these results provide direct experimental evidence that SVA (and conceivably other statins as well) is able to form an acyl-CoA thioester, possibly by microsomal long-chain acyl-CoA synthetase(s), leading to formation of two parallel metabolic pathways, one resulting in the two diastereomers of statin lactones (simvastatin and M1) and the other to the beta-oxidation pathway of statin hydroxy acids.     

Panipenum, a carbapenem antibiotic, increases the level of hepatic UDP-glucuronic acid in rats.

To investigate the mechanism for the enhanced glucuronidation of valproic acid (VPA) by panipenem (PAPM), a carbapenem antibiotic, in rat liver, we carried out studies to investigate whether PAPM increases the activity of UDP-glucuronosyltransferase or the level of hepatic UDP-glucuronic acid (UDPGA) in rats. PAPM had no effect on the UDP-glucuronosyltransferase activity toward VPA both in vivo and in vitro. On the other hand, in vivo treatment with PAPM significantly increased the hepatic UDPGA level by about 1.7-fold (control: 434.5 +/- 65.5 nmol/g of liver; PAPM-treated: 755.2 +/- 92.3 nmol/g of liver). The in vitro formation of VPA glucuronide increased proportionally as a function of the UDPGA concentration up to 0.8 mM. Therefore, the increase in the level of hepatic UDPGA by PAPM is likely to be one of the causal factors for enhancing VPA glucuronidation in vivo.     

丙戊酸肝毒性高危因素易感性机制的实验研究

目的探讨丙戊酸(VPA)在不同年龄、以及与酶诱导剂联合用药时肝脏毒副作用差异及其机制。方法婴儿及成年W istar大鼠,以不同剂量VPA及苯巴比妥(PB)灌胃制作动物模型。差速离心法制备肝线粒体。测定血氨、L-肉碱、肝功酶谱、血液凝血因子、VPA与PB血药浓度、肝线粒体呼吸酶系,以及谷胱甘肽(GSH)、丙二醛(MDA)含量等。流式细胞仪、原位杂交法分别检测肝线粒体跨膜电位(MMP)及肝细胞色素P450(CYP450)还原酶mRNA的表达。O il-Red-O染色观察肝细胞脂肪变性。结果①无论婴儿鼠或成年鼠,即使较大剂量VPA/VPA+PB,对肝功酶谱ALT、AST均无明显影响,而凝血因子PT、TT、APTT、Fbg,以及血氨和L-肉碱含量均有明显异常,其中尤以婴儿鼠、以及与酶诱导剂联用时影响更为突出;②较大剂量VPA/VPA+PB使婴儿鼠肝线粒体细胞色素aa3含量分别下降58.80%和61.80%,成年鼠组分别下降37.55%和46.53%。与对照组比较,P均<0.01。检测肝线粒体呼吸链关键酶琥珀酸脱氢酶(SDH)活性表明,仅有较大剂量VPA/VPA+PB婴儿鼠SDH活性与对照组差异有显著性(P<0.01),分别降低44.8%和57.9%。婴儿和成年鼠细胞色素氧化酶(CCO)活性均明显降低(P<0.01),以婴儿鼠降低更明显。无论婴儿鼠或成年鼠组,较大剂量VPA/VPA+PB动物的肝线粒体GSH含量均低于同龄对照组(P<0.01),而线粒体MDA含量均增高(P<0.05),婴儿鼠更严重;③成年各组实验鼠之间肝线粒体MMP差异无显著性,但婴儿鼠中较大剂量VPA+PB组MMP下降21.47%,与对照组差异有显著性(P<0.05);④原位杂交检测显示较大剂量VPA引起婴儿鼠肝内CYP450还原酶mRNA平均光密度值(OD)增加(P<0.05),但对成年鼠无影响。合并使用PB时,则引起各年龄鼠该酶mRNA表达均增强;⑤组织学观察显示,婴儿鼠较大剂量VPA/VPA+PB组均出现门管区为主的肝细胞脂肪变性,脂肪细胞数分别高出成年鼠7.5和12倍,P<0.01。结论VPA对肝脏的毒副作用,主要表现为VPA对肝线粒体功能的影响,且在婴儿鼠年龄以及联合用药等高危因素存在时更为突出。另外,CYP450还原酶表达的明显增强也可能是高危因素时VPA肝毒性明显增强的重要机制之一。在VPA肝毒性高危因素存在时,尤其应警惕其肝毒性,然而,肝功酶谱并不是一种灵敏的用于预测VPA肝严重毒性发生的实验室指标。     

Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro.

Clofibric acid (p-chlorophenoxyisobutyric acid) is metabolized in vivo to a thioester-linked glutathione conjugate, S-(p-chlorophenoxyisobutyryl)glutathione (CA-SG). The formation of this metabolite is presumed to occur via transacylation reactions between glutathione (GSH) and reactive acyl-linked metabolite(s) of the drug. The present study examines the chemical reactivity of clofibryl-S-acyl-CoA (CA-SCoA), an acyl-CoA thioester intermediary metabolite of clofibric acid, with GSH to form the CA-SG in vitro. Incubations of CA-SCoA (1 mM) with GSH (5 mM) were carried out at pH 7.5 and 37 degrees C, with analysis of the formed reaction products by isocratic reverse-phase high-performance liquid chromatography (HPLC). Results showed a time-dependent and linear formation of CA-SG up to 4 h (50 microM CA-SG formed/h), and after a 1-day incubation, the reaction mixture contained 0.7 mM CA-SG. The identity of CA-SG was confirmed by analysis of HPLC-purified material by tandem mass spectrometry. The rate of CA-SG formation was found to be increased 3-fold in incubations containing rat liver glutathione S-transferases (4 mg/ml). Analysis of the chemical stability of CA-SCoA in buffer at 37 degrees C and varying pH showed the derivative to be stable under mildly acidic and basic aqueous conditions but to hydrolyze at pH values greater than 10 after a 1-day incubation (t(1/2) = approximately 1 day at pH 10.5). Results from these studies show that CA-SCoA is a reactive thioester derivative of clofibric acid and is able to acylate GSH and other thiol-containing nucleophiles in vitro and, therefore, may be able to acylate protein thiols in vivo, which could contribute to the toxic side effects of the drug.     

The relationship between glucuronide conjugate levels and hepatotoxicity after oral administration of valproic acid

The aim of this study was to investigate the relationship between hepatotoxicity, levels of glucuronide conjugates of valproic acid (VPA), and the toxic metabolites of VPA (4-ene VPA and 2,4-diene VPA). We also examined whether hepatotoxicity could be predicted by the urinary excretion levels of VPA and its toxic metabolites. VPA was administrated orally in rats in amounts ranging from 20 mg/kg to 500 mg/kg. Free and total (free plus glucuronide conjugated) VPA, 4-ene VPA, and 2,4-diene VPA were quantified in urine and liver using gas chromatography-mass spectrometry. Serum levels of aspartate aminotransferase, alanine aminotransferase, and α-glutathione S-transferase (α-GST) were also determined to measure the level of hepatotoxicity. The serum α-GST level increased slightly at the 20 mg/kg dose, and substantially increased at the 100 and 500 mg/kg dose; aspartate aminotransferase and alanine aminotransferase levels did not change with the administration of increasing doses of VPA. The liver concentration of free 4-ene VPA and the urinary excretion of total 4-ene VPA were the only measures that correlated with the increase in the serum α-GST level (p < 0.094 and p < 0.023 respectively). From these results, we conclude that hepatotoxicity of VPA correlates with liver concentration of 4-ene VPA and can be predicted by the urinary excretion of total 4-ene VPA.     

Valproic acid induced liver injury: An insight into molecular toxicological mechanism

Valproic acid (VPA) is an anti-seizure drug that causes idiosyncratic liver injury. 2-propyl-4-pentenoic acid (Δ⁴VPA), a metabolite of VPA, has been implicated in VPA-induced hepatotoxicity. This review summarizes the pathogenesis involved in VPA-induced liver injury. The VPA induce liver injury mainly by i) liberation of Δ⁴VPA metabolites; ii) decrease in glutathione stores and antioxidants, resulting in oxidative stress; iii) inhibition of fatty acid β-oxidation, inducing mitochondrial DNA depletion and hypermethylation; a decrease in proton leak; oxidative phosphorylation impairment and ATP synthesis decrease; iv) induction of fatty liver via inhibition of carnitine palmitoyltransferase I, enhancing nuclear receptor peroxisome proliferator-activated receptor-gamma and acyl-CoA thioesterase 1, and inducing long-chain fatty acid uptake and triglyceride synthesis. VPA administration aggravates liver injury in individuals with metabolic syndromes. Therapeutic drug monitoring, routine serum levels of transaminases, ammonia, and lipid parameters during VPA therapy may thus be beneficial in improving the safety profile or preventing the progression of DILI.     

Effect of valproic acid on zinc metabolism in the rat

Previous studies have suggested that the mechanism of valproic acid (VPA) hepatotoxicity may involve a drug-induced Zn deficiency. To test this hypothesis, the uptake of 65Zn or tissue Zn concentration was determined in plasma, liver, bone, kidney, and brain of adult male rats, administered parenteral VPA according to one of 3 schedules: 750 mg/kg; 500 mg/kg; and 100 mg/kg/day X 7 days. Histopathological changes in liver and weight loss were observed in rats 5 days after administration of VPA (750 mg/kg). The plasma Zn level in VPA toxic rats was significantly depressed compared to saline-injected controls, although the Zn content of liver and bone was unaffected. Furthermore, tissue uptake of 65Zn was not altered in rats 6 h after receiving VPA 500 mg/kg or after chronic administration at 100 mg/kg/day. On the basis of the present study, there is no evidence that Zn deficiency is induced by hepatotoxic doses of VPA in rats.     

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.     

Subchronic effects of valproic acid on gene expression profiles for lipid metabolism in mouse liver

Valproic acid (VPA) is used clinically to treat epilepsy, however it induces hepatotoxicity such as microvesicular steatosis. Acute hepatotoxicity of VPA has been well documented by biochemical studies and microarray analysis, but little is known about the chronic effects of VPA in the liver. In the present investigation, we profiled gene expression patterns in the mouse liver after subchronic treatment with VPA. VPA was administered orally at a dose of 100 mg/kg/day or 500 mg/kg/day to ICR mice, and the livers were obtained after 1, 2, or 4 weeks. The activities of serum liver enzymes did not change, whereas triglyceride concentration increased significantly. Microarray analysis revealed that 1325 genes of a set of 32,996 individual genes were VPA responsive when examined by two-way ANOVA (P<0.05) and fold change (>1.5). Consistent with our previous results obtained using an acute VPA exposure model (Lee et al., Toxicol Appl Pharmacol. 220:45-59, 2007), the most significantly over-represented biological terms for these genes included lipid, fatty acid, and steroid metabolism. Biological pathway analysis suggests that the genes responsible for increased biosynthesis of cholesterol and triglyceride, and for decreased fatty acid beta-oxidation contribute to the abnormalities in lipid metabolism induced by subchronic VPA treatment. A comparison of the VPA-responsive genes in the acute and subchronic models extracted 15 commonly altered genes, such as Cyp4a14 and Adpn, which may have predictive power to distinguish the mode of action of hepatotoxicants. Our data provide a better understanding of the molecular mechanisms of VPA-induced hepatotoxicity and useful information to predict steatogenic hepatotoxicity.     

A new approach on valproic acid induced hepatotoxicity: Involvement of lysosomal membrane leakiness and cellular proteolysis

Although valproic acid (VPA) a proven anticonvulsant agent thought to have relatively few side-effects VPA has been referred as the third most common xenobiotic suspected of causing death due to liver injury. In this study the cellular pathways involved in VPA hepatotoxicity were investigated in isolated rat hepatocytes. Accelerated cytotoxicity mechanism screening (ACMS) techniques using fluorescent probes including, ortho-phthalaldehyde, rhodamine 123 and acridine orange were applied for measurement of ROS formation, glutathione depletion, mitochondrial membrane potential and Lysosomal membrane damage, respectively. Our results showed that cytotoxic action of VPA is mediated by lysosomal membrane leakiness along with reactive oxygen species (ROS) formation and decline of mitochondrial membrane potential before cell lysis ensued. Incubation of hepatocytes with VPA also caused rapid hepatocyte glutathione (GSH) depletion which is another marker of cellular oxidative stress. Most of the VPA induced GSH depletion could be attributed to the expulsion of GSSG. Our results also showed that CYP2EI is involved in the mechanism of VPA cytotoxicity. We finally concluded that VPA hepatotoxicity is a result of metabolic activation by CYP2E1 and ROS formation, leading to lysosomal labialization, mitochondrial/lysosomal toxic cross-talk and finally general cellular proteolysis in the rat hepatocytes.     

Combined effects of a high-fat diet and chronic valproic acid treatment on hepatic steatosis and hepatotoxicity in rats

Aim： To investigate the potential interactive effects of a high-fat diet （HFD） and valproic acid （VPA） on hepatic steatosis and hepatotoxicity in rats. Methods： Male SD rats were orally administered VPA （100 or 500 mg.kgl.d1） combined with HFD or a standard diet for 8 weeks. Blood and liver samples were analyzed to determine lipid levels and hepatic function biomarkers using commercial kit assays. Low- molecular-weight compounds in serum, urine and bile samples were analyzed using a metabonomic approach based on GC/TOF-MS. Results： HFD alone induced extensive hepatocyte steatosis and edema in rats, while VPA alone did not cause significant liver lesions. VPA significantly aggravated HFD-induced accumulation of liver lipids, and caused additional spotty or piecemeal necrosis, accompanied by moderate infiltration of inflammatory cells in the liver. Metabonomic analysis of serum, urine and bile samples revealed that HFD significantly increased the levells of amino acids, free fatty acids （FFAs） and 3-hydroxy-butanoic acid, whereas VPA markedly decreased the levels of amino acids, FFAs and the intermediate products of the tricarboxylic acid cycle （TCA） compared with the control group. HFD aggravated VPA-induced inhibition on lipid and amino acid metabolism. Conclusion： HFD magnifies VPA-induced impairment of mitochondria113-oxidation of FFAs and TCA, thereby increases hepatic steatosis and hepatotoxicity. The results suggest the patients receiving VPA treatment should be advised to avoid eating HFD.     

Identification of N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, with DNA.

The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen

Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-S-ylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH-APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-450UT-A) catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P-450UT-A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P-450UT-F and the female specific isozyme P-450UT-I, produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by beta-naphthoflavone, cytochrome P-450 beta NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-450PB-B) a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450PB-B and P-450 beta NF-B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Valproic acid hepatotoxicity in human liver slices.

Precision cut human liver slices were incubated in organ culture with valproic acid (VPA) to identify patterns of sensitivity to VPA-induced hepatotoxicity. The slices were incubated in Krebs-HEPES buffer supplemented with 25mM glucose and 84 micrograms/ml gentamycin. At 2, 4, 6, 12, 18 and 24 hr slices were taken and analyzed for K+ retention, synthesis of protein and LDH leakage. All three of these viability indicators showed that certain human livers were more susceptible to VPA-induced hepatotoxicity than others. In the limited group of human livers investigated (n = 9) we found one to be particularly sensitive and two relatively insensitive to VPA toxicity. The remaining tissues were of intermediate sensitivity towards VPA. At this time there is no correlation between the human livers that were susceptible to VPA induced hepatotoxicity and age or sex. This study was designed to show that VPA does induce hepatotoxicity in vitro at therapeutically relevant concentrations. Future studies will show whether VPA hepatotoxicity correlates with VPA metabolism, nutritional status or concomitant therapy.     

Effect of aging on glucuronidation of valproic acid in human liver microsomes and the role of UDP-glucuronosyltransferase UGT1A4, UGT1A8, and UGT1A10

Valproic acid (VPA) is a widely used anticonvulsant that is also approved for mood disorders, bipolar depression, and migraine. In vivo, valproate is metabolized oxidatively by cytochromes P450 and beta-oxidation, as well as conjugatively via glucuronidation. The acyl glucuronide conjugate (valproate-glucuronide or VPAG) is the major urinary metabolite (30-50% of the dose). It has been hypothesized that glucuronidation of antiepileptic drugs is spared over age, despite a known decrease in liver mass. The formation rates of VPAG in a bank of elderly (65 years onward) human liver microsomes (HLMs) were measured by liquid chromatography/tandem mass spectrometry and compared with those in a younger (2-56 years) HLM bank. In vitro kinetic studies with recombinant UDP-glucuronosyltransferases (UGTs) were completed. A 5- to 8-fold variation for the formation of VPAG was observed within the microsomal bank obtained from elderly and younger donors. VPAG formation ranged from 6.0 to 53.4 nmol/min/mg protein at 1 mM substrate concentration (n=36). The average velocities at 0.25, 0.5, and 1 mM VPA were 7.0, 13.4, and 25.4 nmol/min/mg protein, respectively, in the elderly HLM bank. Rates of VPAG formation were not significantly different in the HLM bank obtained from younger subjects. Intrinsic clearances (V(max)/K(m)) for several cloned, expressed UGTs were determined. UGT1A4, UGT1A8, and UGT1A10 also were found to catalyze the formation of VPAG in vitro. This is the first reported activity of these UGTs toward VPA glucuronidation. UGT2B7 had the highest intrinsic clearance, whereas UGT1A1 demonstrated no activity. In conclusion, our investigation revealed no differences in VPAG formation in younger versus elderly HMLs and revealed three other UGTs that form VPAG in vitro.     

Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat

Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl-cysteine products rather than the S-acyl-NAC conjugate.