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Participation of lipid transport and fatty acid metabolism in valproate sodium-induced hepatotoxicity in HepG2 cells
Toxicology in Vitro 24 (2010) 1086–1091
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Participation of lipid transport and fatty acid metabolism in valproate sodium-induced hepatotoxicity in HepG2 cells Qiaoli Ji a, Xiaolian Shi a, Rong Lin a,*, Youhua Mao b, Xiaogang Zhai b, Qinqin Lin a, Jiye Zhang a a
Department of Pharmacology and Pharmacy, Key Laboratory of Environment and Genes Related to Diseases, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, PR China b Shaanxi’s Tiansen Drug Research and Development Limited Company, Xi’an, Shaanxi 710065, PR China
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Article history: Received 10 October 2009 Accepted 25 March 2010 Available online 3 April 2010 Keywords: Hepatotoxicity Valproate sodium VPA HepG2 Lipid transport Fatty acid metabolism
a b s t r a c t The hepatotoxicity induced by valproic acid (VPA) has been described in many clinical studies and the related mechanism has been partly elucidated. The objective of this study is to investigate the hepatotoxicity and its underlying mechanism of valproic acid on human hepatoma carcinoma cell line HepG2. The cell viability was evaluated by 3-(4,5-dimethyltyiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the medium were detected using spectrophotometry. The gene expressions of cytochrome P450 1 A1 (CYP1A1), ATP-binding cassette transporter G1 (ABCG1) and carnitine palmitoyltransferase 1 (CPT1A), related to lipid transport and fatty acid metabolism, were measured by quantitative real-time reverse transcriptase-PCR. Treatment with valproate sodium obviously decreased the viability of HepG2 cells, accompanied by the increased leakages of ALT, AST and LDH in a dose-dependent manner. Furthermore, the gene expressions of CYP1A1, ABCG1 and CPT1A were almost up-regulated in the treated groups. In conclusion, these data suggest that VPA-induced hepatotoxicity was critically enhanced with the elevation of valproate sodium, which may be correlated with up-regulated gene expressions of CYP1A1, ABCG1 and CPT1A. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Epilepsy is a prevalent and devastating disease which is characterized with mood swings. Drug therapy is still the basic means of treatment of epilepsy. As one of the most prescribed antiepileptic drugs, valproate sodium, an eight-carbon branched-chain saturated fatty acid sodium salt, has been employed clinically in the treatment of various forms of epilepsy for over three decades (McIntyre et al., 2003). Since it appeared in the market, valproate sodium has various side effects which have been reported during the clinical trials, such as nausea, vomiting, facial edema, hypoglycemia, and lethal hepatic injury (Powell-Jackson et al., 1984). Liver injury is considered the most serious side effect, and microvesicular steatosis, hepatocellular necrosis, cholestatic liver injury, and elevated levels of alanine aminotransferase and aspartate aminotransferase have been reported (Lewis et al., 1982; Lee et al., 2007). Abbreviations: ABC, ATP-binding cassette transporter; ALT, alanine aminotransferase; AST, aspartate transaminase; CPT1, carnitine palmitoyltransferase 1; CYP, cytochrome P450; HDL, high density lipoprotein; L-CFA, long-chain fatty acid; LDH, lactate dehydrogenase; RT-PCR, reverse transcriptional polymerase chain reaction; VPA, valproic acid. * Corresponding author. Tel.: +86 29 82657691; fax: +86 29 82657833. E-mail address: [email protected] (R. Lin). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.03.014
So far, a number of studies have been conducted following the interest in valproic acid (VPA)-induced liver injury and its potential mechanism. Hepatic steatosis has also been observed in the VPAtreated experimental animals such as rats and mice (Lewis et al., 1982; Roma-Giannikou et al., 1999). Despite the elusive underlying mechanism of VPA-induced hepatotoxicity, this viewpoint is to be generally acknowledged that VPA-induced hepatotoxicity is related with reactive VPA metabolites in vivo (Tong et al., 2005). More recently, a series of researches have obtained gene expression profiling for the VPA-induced murine fatty liver after VPA administration using the microarray analysis, and the gene expression profiling data showed significant changes in the gene expressions in the subchronic VPA treatment, which were consistent with the results in the acute VPA exposure model (Lee et al., 2007, 2008). Although these studies provided significant insight to the underlying mechanism of VPA-induced hepatotoxicity, contributions of individual gene still require further investigation. For instance, ATP-binding cassette transporter G1 (ABCG1), cytochrome P450 (CYP) and carnitine palmitoyltransferase 1 (CPT1), are related to lipid transport and fatty acid metabolism, respectively. In the present study, the hepatotoxicity of valproate sodium on human hepatoma cell line HepG2 was measured by detecting the cell viability and various biochemical endpoints (ALT, AST and
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LDH release). Furthermore, the potential mechanism of VPA-induced hepatotoxicity was approached in the expression of several genes, including CYP1A1, ABCG1 and CPT1A.
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Valproate sodium was obtained from Tiansen Bioengineering and Technology Limited Company of Shaanxi in China. New-born Calf Serum was purchased from Lanzhou Roya Bio-Technology Limited Company in China. Other reagents were of the highest grade commercially available. All those primers were purchased from ABI Company. Valproate sodium was dissolved in DMSO (Amersco) and prepared as a 0.1 M stock solution with culture medium containing no serum. The final concentration of DMSO is 0.5% and stock solution was diluted with culture medium without serum prior to use.
CO2. After 48 h, culture mediums in the wells were discarded before 0.5 ml cell culture medium containing the drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) were tested in five replicate wells. DMSO treated cells served as controls. After 24 h, the culture medium, containing sedimented cells, were collected and centrifuged at 1000g for 5 min at 4 °C. LDH leakages in culture medium have been determined by pyruvic acid method; the assay kit of LDH was commercially available and purchased from the Institute of Nanjing Jiancheng Biology Engineering (Nanjing, PR China). All operations followed the instruction of the kit. A 20 ll supernatant was mixed with 250 ll matrix buffer. Reaction was initiated by addition of 50 ll NAD at 37 °C, and 15 min later, 250 ll 2,4-dinitro-phenylhydrazine was added and warmed at 37 °C for 15 min. Then, 2.5 ml 0.4 M NaOH was added for color development. The absorbance was measured at 440 nm in an ultraviolet/visible scanning spectrophotometer and the activity of LDH calculated by formula applied by the assay kit.
2.2. Cell line and culture
2.5. Activity assessment of AST and ALT releasing in vitro
The HepG2 cells were obtained from the ATCC and cultured in 25 cm2 culture flasks according to the distributor’s recommendation in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated new-born calf serum, 1% sodium bicarbonate, 100 U/ml penicillin, 100 lg/ml streptomycin and no additional glutamine at 37 °C in humidified atmosphere of 5% CO2. The medium was changed every 3–4 days and cells were passaged when reaching 80–90% confluence. To subculture the cells, the culture medium was aspirated and cells were briefly washed with phosphate buffered saline (PBS) before trypsinized with 0.25% trypsin. Cells were resuspended and counted using a hemocytometer and a 0.4% Trypan Blue solution. Viability of the cells (P80%) was routinely checked.
The activity of ALT and AST were assayed as a marker of hepatotoxicity. Cell suspensions were seeded into a 24-well-plate and incubated in a 37 °C incubator with 5% CO2. After 48 h, culture mediums in the wells were discarded before 0.5 ml cell culture medium containing drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) were tested in five replicate wells. DMSO treated cells served as controls. After incubation for 24 h, the culture medium, containing sedimented cells, were collected and centrifuged at 1000g for 5 min at 4 °C. The activity of AST and ALT were detected by pyruvic acid method with CL-7150 automatic biochemistry analyzer made in Japan.
2. Materials and methods 2.1. Chemicals
2.3. MTT assay Cell suspensions were seeded into a 96-well-plate and incubated for 48 h. Cells were allowed to reach exponential growth and, then, the culture fluid in the wells were removed before 180 ll medium containing drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 1 and 3 mM) were tested in five replicate wells. DMSO treated cells served as controls. After 24 h, cells were incubated with 20 ll for 4 h, 5 mg/ml MTT 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), which was dissolved in PBS. Metabolically active cells cleaved the yellow tetrazolium salt to a purple formazan dye. A decrease of the number of living cells was correlated with the number of purple formazan crystals. Subsequently, discarded the clear supernant, kept the purple crystal granules and added 150 ll DMSO into the wells. Crystals were dissolved in lysis buffer (DMSO). The absorbance change of specimen was evaluated spectrophotometrically at 490 nm filter using a Multiskan Ascent microplate reader. The variation in metabolic activity shows a good correlation with the decline in cell growth. Viability results were expressed as percentages of solvent control group. Dose–effect curves for determining IC50 values were done by applying a graphical fitting method with Microsoft Excel 2003 (with ‘‘x = log” and ‘‘y = probit” scales). 2.4. Activity assessment of LDH releasing in vitro The activity of LDH releasing, as an index of hepatotoxicity, was also evaluated in the present study. Cell suspensions were seeded into a 24-well-plate and incubated in a 37 °C incubator with 5%
2.6. RNA isolation and gene expression analysis by a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay Cell suspensions were grown in a 12-well-plate at 5 105 and incubated in a 37 °C incubator with 5% CO2. After 48 h, culture mediums in the wells were discarded before culture medium containing the drugs were added into the wells. The concentrations of valproate sodium (25, 50, 100, 200 and 500 lM) were tested in four replicate wells. DMSO treated cells served as controls. After 24 h, cells treated with drugs or solvent were harvested by the trypsinization and total RNA was extracted with Qiagen M48 Kit according to the manufacturer’s instructions. The concentrations of total isolated RNA were precisely measured by spectrophotometry at 260 and 280 nm (Nanodrop ND-100 Spectrophotometer, Biolab). One gram of total RNA was then treated with RNase-free DNase I (Promega, Southampton, UK) at 37 °C for 15 min to remove any contaminating genomic DNA, followed by enzyme inactivation at 65 °C for 10 min prior to cDNA synthesis. cDNA was produced using SuperScript III First Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. The real-time PCR was performed using iQSupermix (BioRad) on an ABI 7900 Sequence Detector and analyzed using ABI Prism Sequence Detector Software v1.6.3 (PE Biosystems). Primer pairs used for the amplifying reaction of CYP1A1, ABCG1, CPT1A have been obtained from TaqMan Gene Expression Assays (Invitrogen). The change in gene expressions was determined by normalizing mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Melting curve analysis was performed to confirm production of a single product in each reaction. Gene expression levels were calculated from the threshold cycle values and expressed as the ratio to the GAPDH expression level.
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2.7. Data analysis Data are presented as mean ± standard deviations. Statistical analysis of the results from control and treatment groups were performed by one-way analysis of variance (ANOVA) test and the bivariate correlation was performed by Pearson’s correlation analysis using computerized statistical software (SPSS13.0). Two-tailed values of P < 0.05 were considered to be statistically significant. Each test must be duplicated and final data represent the mean of at least three experiments. 3. Results 3.1. Effect of valproate sodium on the viability of HepG2 cells Cell viability was observed in HepG2 cells after treatment with valproate sodium at tested concentrations of 0.1, 0.3, 1 and 3 mM for 24 h. As shown in Fig. 1, there was an obviously statistically significant difference between the treated groups and solvent control group (0.5% DMSO). With the increased concentration of valproate sodium, cell viability significantly decreased and the ratio of growth inhibition was gradually increasing in a dose-dependent manner. The value of IC50 was 20.72 mM. 3.2. Effect of valproate sodium on the activity of LDH in the culture media
Fig. 2. The effect of valproate sodium on the activity of LDH in the culture medium of HepG2 cells. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) for 24 h in vitro, the released LDH activity in the medium was assayed. Values are mean ± SD. The bars represent the mean values of five data points. *P < 0.05, **P < 0.01, compared with solvent control group.
of 0.1, 0.3, 0.5 and 1 mM. After 24 h, the leakage of ALT and AST markedly increased and the differences between the treated groups (0.3, 0.5 and 1 mM) and solvent control group had statistical significances. As described in Fig. 3A and B, treatment with val-
The measurement of the leakage of LDH in the culture medium was used to evaluate plasma membrane integrity. In this study, a dose-dependent response was observed when HepG2 cells were exposed to valproate sodium at tested concentrations of 0.1, 0.3, 0.5 and 1 mM. As shown in Fig. 2, significant differences in the leakage of LDH between the treated groups (0.3, 0.5 and 1 mM) and solvent control group were observed, and the activity of LDH further increased with the increased concentration of valproate sodium. 3.3. Effect of valproate sodium on the activity of ALT and AST in the culture medium To investigate the effects of VPA on the liver enzymes, HepG2 cells were treated with valproate sodium at tested concentrations
Fig. 1. The effect of valproate sodium on the cell viability. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 1 and 3 mM) for 24 h in vitro. Cell viability was measured by MTT assay. Data are expressed as mean ± SD. The bars represent the mean values at five data points from cultures. *P < 0.05, **P < 0.01, compared with solvent control group.
Fig. 3. The effect of valproate sodium on the activity of ALT and AST in the culture medium of HepG2 cells. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) for 24 h in vitro, the released ALT and AST activity in the medium was assayed: (A) the effect of valproate sodium on the activity of ALT; (B) the effect of valproate sodium on the activity of AST. Values are mean ± SD. The bars represent the mean values at five data points. *P < 0.05, **P < 0.01, compared with solvent control group.
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proate sodium caused a significant increase in the release of AST and ALT in all treated groups compared with solvent control group. 3.4. Effect of valproate sodium on the metabolism-related gene expression After treatment of HepG2 cells with valproate sodium (25, 50, 100, 200 and 500 lM) for 24 h, the gene expressions of CYP1A1,
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ABCG1, CPT1A and GAPDH were determined by real-time RTPCR. In this study, all the tested gene expressions remained at a low level at the low concentration of 25 and 50 lM. As shown in Fig. 4A, the amounts of CYP1A1 gene expression were about 9and 26-fold as many as solvent control group at the concentration of 200 and 500 lM. At the concentration of 200 and 500 lM, the amounts of ABCG1 gene expression were approximately 12- and 45-fold as many as solvent control group as described in Fig. 4B, respectively. However, the amount of CPT1A gene expression was just 1.7-fold at high concentration of 500 lM compared with solvent control group (Fig. 4C).
4. Discussion
Fig. 4. The effect of valproate sodium on gene expressions. The HepG2 cells were exposed with different concentrations of valproate sodium (25, 50, 100, 200 and 500 lV) for 24 h in vitro. The gene expressions were determined by real-time RTPCR: (A) the effect of valproate sodium on CYP1A1 gene expression; (B) the effect of valproate sodium on ABCG1 gene expression; (C) the effect of valproate sodium on CPT1A gene expression. Data are expressed as mean ± SD performed in triplicate. *P < 0.05, **P < 0.01, compared with solvent control group.
Drug-induced hepatotoxicity is an important healthcare issue with respect of serious morbidity and mortality (Kaplowitz, 2001). Therefore, it’s necessary to evaluate drug-induced hepatotoxicity for the design of safer therapeutic agents. This study focused on the VPA-induced hepatotoxicity and its potential molecular mechanism, which was performed upon the HepG2 cells treated with valproate sodium. Earlier studies have shown that VPA caused dose-dependent toxicity to rat hepatocytes and induced apoptosis in the rat hepatoma cell line, FaO (Kingsley et al., 1983; Phillips et al., 2003). Our present investigation confirmed that valproate sodium caused dose-dependent growth inhibition of HepG2 cells. LDH, a particularly important indicator of cell membrane integrity and existing in almost all histiocytes, and, ALT and AST, liver-specific enzymes, physiologically cannot be extracellular membrane leakage. The activities of ALT, AST and LDH are often used for evaluating the hepatotoxicity induced by chemicals or toxicants (Wang et al., 2007; Zhang et al., 2008). There was an obviously statistically significant difference at tested concentration up to 0.3 mM (**P < 0.01) in MTT experiment. So, we have chosen concentrations of valproate sodium from 0.1 to 1 mM for activity assessment of LDH, AST and ALT releasing. In the present study, the activities of enzymes obviously increased with the increase of drug concentration, which suggested that valproate sodium led to the damage of liver cell membrane and the leakage of intracellular enzymes. These data indicated that treatment with valproate sodium at tested concentrations resulted in damage to some cells and finally caused cell death. Many hypotheses, related with the mechanism of hepatotoxicity of VPA, have been raised and tested. It is widely accepted that VPA is extensively metabolized in the liver via glucuronic acid conjugation, mitochondrial b-oxidation and cytosolic (endoplasmic reticulum) x-oxidation to produce multiple metabolites, some of which are biologically active and involved in the VPA-induced hepatotoxicity. For instance, 4-ene-valproic (4-ene-VPA) is a more potent hepatotoxin than its parent, VPA (Tong et al., 2005; Fisher et al., 1998; Lheureux et al., 2005). Cells exposed to 4-ene-VPA experienced greater cytotoxicity than those treated with VPA, and CYP2E1 inducers enhanced toxicity in VPA-exposed HepG2 cells (Fisher et al., 1998). Hepatotoxicity after oral administration of VPA correlated with liver concentration of 4-ene-VPA and could be predicted by the urinary excretion of total 4-ene-VPA in rats (Lee et al., 2009). Multiple P450 isoforms in the CYP450 superfamily of enzymes play critical roles in the biotransformation of valproate sodium, including CYP2C9 and CYP2A6 in human liver, CYP2B4 in rat and rabbit liver, CYP4B1 in rabbit lung (Fisher et al., 1998). The formation of delta-VPA was catalyzed by CYP3A1 in rat liver microsomes, and the reaction was species selective in that neither human CYP3A4 nor rabbit CYP3A6 forms significant quantities of the metabolite (Neuman et al., 2001). In the present study, the expression level of CYP1A1, normally at a low level in extrahepatic tissues in humans but highly inducible in the liver
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and extrahepatic tissues, significantly increased following the increase of drug concentration. The increased gene expression of the drug-metabolizing enzyme would be profitable to chemical metabolism. Consequently, the level of reactive valproate sodium metabolite would be obviously increasing, which would enhance the VPA-induced hepatotoxicity. Subsequently, it might be possible that the hepatotoxicity of valproate sodium would enormously increase while the gene expression of CYP1A1 was up-regulated. The ATP-binding cassette transporters are involved in energydependent translocation of various substrates including sugars, lipids, peptides and xenobiotics across lipid membranes (Borst and Elferink, 2002). ABCB1, ABCC1 and ABCG2 are drug efflux transports, overexpression of which can decrease the intracellular drug concentrations (Ceckova et al., 2008; Kuo, 2007). Meanwhile, other studies in vitro have confirmed that up-regulating ABC1 mRNA was correlated with increased cholesterol efflux in a dose-dependent manner and ABCG1 mRNA levels were highly induced by lipid loading which stimulated reverse cholesterol transport in macrophages (Venkateswaran et al., 2000; Bortnick et al., 2000). Family member 1 of the G-subgroup of mammalian ABC transporters (ABCG1) have been proposed to function synergistically to promote cellular cholesterol efflux into lipid poor HDL, overexpression of which is protective against lipid accumulation (Oram and Vaughan, 2006; Engel et al., 2006; Vaughan and Oram, 2006). In this study, the gene expression of ABCG1 gradually increased with the increase of drug concentration. Then, a positive feedback mechanism, induced by nonphysiologically high concentrations of valproate sodium, which itself is a fatty acid, may be exerting and involved in the reinforcement of gene expression in the biosynthetic pathways. Thus, the gene expression of ABCG1 was highly regulated by overloading valproate sodium or its metabolites. The b-oxidation of long-chain fatty acids (LCFAs) in mitochondria is a major source of energy, and the carnitine palmitoyltransferase (CPT) enzyme system facilitates the transport of such fatty acids into the mitochondria (McGarry et al., 1977; McGarry and Brown, 1997). Carnitine palmitoyltransferase 1A (CPT1A), most abundant in liver and localized within the mitochondrial outer membrane, catalyzes the esterification of acylcoenzyme A to carnitine, which is the rate-limiting step of mitochondrial fatty acid oxidation and plays the major role in fatty acid oxidation (Fraser et al., 1997; Britton et al., 1995). A lot of studies are dedicated to getting more insight into the regulation of CPT1 gene expression in different experimental conditions, some of which found that expression of CPT1 gene is induced by LCFAs in the liver, which means that free LCFAs rather than acyl-CoA eaters are the signal metabolites responsible for the stimulation of CPT1 gene expression (Louet et al., 2001). In the present study, the expression of CPT1A gene gradually increased with the increase of drug concentration, which meant that VPA induced the gene expression of CPT1A. While the content of valproate sodium transported into the mitochondria was gradually increasing, the reactive metabolites of VPA were accumulating gravely in the body. Consequently, the VPA-induced hepatotoxicity would be more serious, forming a malignant circle. In summary, in our study the hepatotoxicity were evaluated and the expression of several genes in HepG2 cells treated with valproate sodium were identified. Our data suggest that the gene expressions were up-regulated in a dose-dependent manner, including CYP1A1, ABCG1 and CPT1A, which are important in fatty acid oxidation and lipid transport. Although the gene regulatory pathways in the valproate sodium-induced hepatotoxicity require further investigation, this study provides significant insight into the mechanisms underlying VPA-induced hepatotoxicity and further studies need to be undertaken to analyze the mechanism of gene expression and its pathway associated with hepatotoxicity.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Participation of lipid transport and fatty acid metabolism in valproate sodium-induced hepatotoxicity in HepG2 cells Qiaoli Ji a, Xiaolian Shi a, Rong Lin a,*, Youhua Mao b, Xiaogang Zhai b, Qinqin Lin a, Jiye Zhang a a
Department of Pharmacology and Pharmacy, Key Laboratory of Environment and Genes Related to Diseases, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, PR China b Shaanxi’s Tiansen Drug Research and Development Limited Company, Xi’an, Shaanxi 710065, PR China
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Article history: Received 10 October 2009 Accepted 25 March 2010 Available online 3 April 2010 Keywords: Hepatotoxicity Valproate sodium VPA HepG2 Lipid transport Fatty acid metabolism
a b s t r a c t The hepatotoxicity induced by valproic acid (VPA) has been described in many clinical studies and the related mechanism has been partly elucidated. The objective of this study is to investigate the hepatotoxicity and its underlying mechanism of valproic acid on human hepatoma carcinoma cell line HepG2. The cell viability was evaluated by 3-(4,5-dimethyltyiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the medium were detected using spectrophotometry. The gene expressions of cytochrome P450 1 A1 (CYP1A1), ATP-binding cassette transporter G1 (ABCG1) and carnitine palmitoyltransferase 1 (CPT1A), related to lipid transport and fatty acid metabolism, were measured by quantitative real-time reverse transcriptase-PCR. Treatment with valproate sodium obviously decreased the viability of HepG2 cells, accompanied by the increased leakages of ALT, AST and LDH in a dose-dependent manner. Furthermore, the gene expressions of CYP1A1, ABCG1 and CPT1A were almost up-regulated in the treated groups. In conclusion, these data suggest that VPA-induced hepatotoxicity was critically enhanced with the elevation of valproate sodium, which may be correlated with up-regulated gene expressions of CYP1A1, ABCG1 and CPT1A. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Epilepsy is a prevalent and devastating disease which is characterized with mood swings. Drug therapy is still the basic means of treatment of epilepsy. As one of the most prescribed antiepileptic drugs, valproate sodium, an eight-carbon branched-chain saturated fatty acid sodium salt, has been employed clinically in the treatment of various forms of epilepsy for over three decades (McIntyre et al., 2003). Since it appeared in the market, valproate sodium has various side effects which have been reported during the clinical trials, such as nausea, vomiting, facial edema, hypoglycemia, and lethal hepatic injury (Powell-Jackson et al., 1984). Liver injury is considered the most serious side effect, and microvesicular steatosis, hepatocellular necrosis, cholestatic liver injury, and elevated levels of alanine aminotransferase and aspartate aminotransferase have been reported (Lewis et al., 1982; Lee et al., 2007). Abbreviations: ABC, ATP-binding cassette transporter; ALT, alanine aminotransferase; AST, aspartate transaminase; CPT1, carnitine palmitoyltransferase 1; CYP, cytochrome P450; HDL, high density lipoprotein; L-CFA, long-chain fatty acid; LDH, lactate dehydrogenase; RT-PCR, reverse transcriptional polymerase chain reaction; VPA, valproic acid. * Corresponding author. Tel.: +86 29 82657691; fax: +86 29 82657833. E-mail address: [email protected] (R. Lin). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.03.014
So far, a number of studies have been conducted following the interest in valproic acid (VPA)-induced liver injury and its potential mechanism. Hepatic steatosis has also been observed in the VPAtreated experimental animals such as rats and mice (Lewis et al., 1982; Roma-Giannikou et al., 1999). Despite the elusive underlying mechanism of VPA-induced hepatotoxicity, this viewpoint is to be generally acknowledged that VPA-induced hepatotoxicity is related with reactive VPA metabolites in vivo (Tong et al., 2005). More recently, a series of researches have obtained gene expression profiling for the VPA-induced murine fatty liver after VPA administration using the microarray analysis, and the gene expression profiling data showed significant changes in the gene expressions in the subchronic VPA treatment, which were consistent with the results in the acute VPA exposure model (Lee et al., 2007, 2008). Although these studies provided significant insight to the underlying mechanism of VPA-induced hepatotoxicity, contributions of individual gene still require further investigation. For instance, ATP-binding cassette transporter G1 (ABCG1), cytochrome P450 (CYP) and carnitine palmitoyltransferase 1 (CPT1), are related to lipid transport and fatty acid metabolism, respectively. In the present study, the hepatotoxicity of valproate sodium on human hepatoma cell line HepG2 was measured by detecting the cell viability and various biochemical endpoints (ALT, AST and
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LDH release). Furthermore, the potential mechanism of VPA-induced hepatotoxicity was approached in the expression of several genes, including CYP1A1, ABCG1 and CPT1A.
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Valproate sodium was obtained from Tiansen Bioengineering and Technology Limited Company of Shaanxi in China. New-born Calf Serum was purchased from Lanzhou Roya Bio-Technology Limited Company in China. Other reagents were of the highest grade commercially available. All those primers were purchased from ABI Company. Valproate sodium was dissolved in DMSO (Amersco) and prepared as a 0.1 M stock solution with culture medium containing no serum. The final concentration of DMSO is 0.5% and stock solution was diluted with culture medium without serum prior to use.
CO2. After 48 h, culture mediums in the wells were discarded before 0.5 ml cell culture medium containing the drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) were tested in five replicate wells. DMSO treated cells served as controls. After 24 h, the culture medium, containing sedimented cells, were collected and centrifuged at 1000g for 5 min at 4 °C. LDH leakages in culture medium have been determined by pyruvic acid method; the assay kit of LDH was commercially available and purchased from the Institute of Nanjing Jiancheng Biology Engineering (Nanjing, PR China). All operations followed the instruction of the kit. A 20 ll supernatant was mixed with 250 ll matrix buffer. Reaction was initiated by addition of 50 ll NAD at 37 °C, and 15 min later, 250 ll 2,4-dinitro-phenylhydrazine was added and warmed at 37 °C for 15 min. Then, 2.5 ml 0.4 M NaOH was added for color development. The absorbance was measured at 440 nm in an ultraviolet/visible scanning spectrophotometer and the activity of LDH calculated by formula applied by the assay kit.
2.2. Cell line and culture
2.5. Activity assessment of AST and ALT releasing in vitro
The HepG2 cells were obtained from the ATCC and cultured in 25 cm2 culture flasks according to the distributor’s recommendation in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated new-born calf serum, 1% sodium bicarbonate, 100 U/ml penicillin, 100 lg/ml streptomycin and no additional glutamine at 37 °C in humidified atmosphere of 5% CO2. The medium was changed every 3–4 days and cells were passaged when reaching 80–90% confluence. To subculture the cells, the culture medium was aspirated and cells were briefly washed with phosphate buffered saline (PBS) before trypsinized with 0.25% trypsin. Cells were resuspended and counted using a hemocytometer and a 0.4% Trypan Blue solution. Viability of the cells (P80%) was routinely checked.
The activity of ALT and AST were assayed as a marker of hepatotoxicity. Cell suspensions were seeded into a 24-well-plate and incubated in a 37 °C incubator with 5% CO2. After 48 h, culture mediums in the wells were discarded before 0.5 ml cell culture medium containing drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) were tested in five replicate wells. DMSO treated cells served as controls. After incubation for 24 h, the culture medium, containing sedimented cells, were collected and centrifuged at 1000g for 5 min at 4 °C. The activity of AST and ALT were detected by pyruvic acid method with CL-7150 automatic biochemistry analyzer made in Japan.
2. Materials and methods 2.1. Chemicals
2.3. MTT assay Cell suspensions were seeded into a 96-well-plate and incubated for 48 h. Cells were allowed to reach exponential growth and, then, the culture fluid in the wells were removed before 180 ll medium containing drugs at different concentrations were added into the wells. The concentrations of valproate sodium (0.1, 0.3, 1 and 3 mM) were tested in five replicate wells. DMSO treated cells served as controls. After 24 h, cells were incubated with 20 ll for 4 h, 5 mg/ml MTT 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), which was dissolved in PBS. Metabolically active cells cleaved the yellow tetrazolium salt to a purple formazan dye. A decrease of the number of living cells was correlated with the number of purple formazan crystals. Subsequently, discarded the clear supernant, kept the purple crystal granules and added 150 ll DMSO into the wells. Crystals were dissolved in lysis buffer (DMSO). The absorbance change of specimen was evaluated spectrophotometrically at 490 nm filter using a Multiskan Ascent microplate reader. The variation in metabolic activity shows a good correlation with the decline in cell growth. Viability results were expressed as percentages of solvent control group. Dose–effect curves for determining IC50 values were done by applying a graphical fitting method with Microsoft Excel 2003 (with ‘‘x = log” and ‘‘y = probit” scales). 2.4. Activity assessment of LDH releasing in vitro The activity of LDH releasing, as an index of hepatotoxicity, was also evaluated in the present study. Cell suspensions were seeded into a 24-well-plate and incubated in a 37 °C incubator with 5%
2.6. RNA isolation and gene expression analysis by a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay Cell suspensions were grown in a 12-well-plate at 5 105 and incubated in a 37 °C incubator with 5% CO2. After 48 h, culture mediums in the wells were discarded before culture medium containing the drugs were added into the wells. The concentrations of valproate sodium (25, 50, 100, 200 and 500 lM) were tested in four replicate wells. DMSO treated cells served as controls. After 24 h, cells treated with drugs or solvent were harvested by the trypsinization and total RNA was extracted with Qiagen M48 Kit according to the manufacturer’s instructions. The concentrations of total isolated RNA were precisely measured by spectrophotometry at 260 and 280 nm (Nanodrop ND-100 Spectrophotometer, Biolab). One gram of total RNA was then treated with RNase-free DNase I (Promega, Southampton, UK) at 37 °C for 15 min to remove any contaminating genomic DNA, followed by enzyme inactivation at 65 °C for 10 min prior to cDNA synthesis. cDNA was produced using SuperScript III First Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. The real-time PCR was performed using iQSupermix (BioRad) on an ABI 7900 Sequence Detector and analyzed using ABI Prism Sequence Detector Software v1.6.3 (PE Biosystems). Primer pairs used for the amplifying reaction of CYP1A1, ABCG1, CPT1A have been obtained from TaqMan Gene Expression Assays (Invitrogen). The change in gene expressions was determined by normalizing mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Melting curve analysis was performed to confirm production of a single product in each reaction. Gene expression levels were calculated from the threshold cycle values and expressed as the ratio to the GAPDH expression level.
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2.7. Data analysis Data are presented as mean ± standard deviations. Statistical analysis of the results from control and treatment groups were performed by one-way analysis of variance (ANOVA) test and the bivariate correlation was performed by Pearson’s correlation analysis using computerized statistical software (SPSS13.0). Two-tailed values of P < 0.05 were considered to be statistically significant. Each test must be duplicated and final data represent the mean of at least three experiments. 3. Results 3.1. Effect of valproate sodium on the viability of HepG2 cells Cell viability was observed in HepG2 cells after treatment with valproate sodium at tested concentrations of 0.1, 0.3, 1 and 3 mM for 24 h. As shown in Fig. 1, there was an obviously statistically significant difference between the treated groups and solvent control group (0.5% DMSO). With the increased concentration of valproate sodium, cell viability significantly decreased and the ratio of growth inhibition was gradually increasing in a dose-dependent manner. The value of IC50 was 20.72 mM. 3.2. Effect of valproate sodium on the activity of LDH in the culture media
Fig. 2. The effect of valproate sodium on the activity of LDH in the culture medium of HepG2 cells. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) for 24 h in vitro, the released LDH activity in the medium was assayed. Values are mean ± SD. The bars represent the mean values of five data points. *P < 0.05, **P < 0.01, compared with solvent control group.
of 0.1, 0.3, 0.5 and 1 mM. After 24 h, the leakage of ALT and AST markedly increased and the differences between the treated groups (0.3, 0.5 and 1 mM) and solvent control group had statistical significances. As described in Fig. 3A and B, treatment with val-
The measurement of the leakage of LDH in the culture medium was used to evaluate plasma membrane integrity. In this study, a dose-dependent response was observed when HepG2 cells were exposed to valproate sodium at tested concentrations of 0.1, 0.3, 0.5 and 1 mM. As shown in Fig. 2, significant differences in the leakage of LDH between the treated groups (0.3, 0.5 and 1 mM) and solvent control group were observed, and the activity of LDH further increased with the increased concentration of valproate sodium. 3.3. Effect of valproate sodium on the activity of ALT and AST in the culture medium To investigate the effects of VPA on the liver enzymes, HepG2 cells were treated with valproate sodium at tested concentrations
Fig. 1. The effect of valproate sodium on the cell viability. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 1 and 3 mM) for 24 h in vitro. Cell viability was measured by MTT assay. Data are expressed as mean ± SD. The bars represent the mean values at five data points from cultures. *P < 0.05, **P < 0.01, compared with solvent control group.
Fig. 3. The effect of valproate sodium on the activity of ALT and AST in the culture medium of HepG2 cells. The HepG2 cells were exposed with different concentrations of valproate sodium (0.1, 0.3, 0.5 and 1 mM) for 24 h in vitro, the released ALT and AST activity in the medium was assayed: (A) the effect of valproate sodium on the activity of ALT; (B) the effect of valproate sodium on the activity of AST. Values are mean ± SD. The bars represent the mean values at five data points. *P < 0.05, **P < 0.01, compared with solvent control group.
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proate sodium caused a significant increase in the release of AST and ALT in all treated groups compared with solvent control group. 3.4. Effect of valproate sodium on the metabolism-related gene expression After treatment of HepG2 cells with valproate sodium (25, 50, 100, 200 and 500 lM) for 24 h, the gene expressions of CYP1A1,
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ABCG1, CPT1A and GAPDH were determined by real-time RTPCR. In this study, all the tested gene expressions remained at a low level at the low concentration of 25 and 50 lM. As shown in Fig. 4A, the amounts of CYP1A1 gene expression were about 9and 26-fold as many as solvent control group at the concentration of 200 and 500 lM. At the concentration of 200 and 500 lM, the amounts of ABCG1 gene expression were approximately 12- and 45-fold as many as solvent control group as described in Fig. 4B, respectively. However, the amount of CPT1A gene expression was just 1.7-fold at high concentration of 500 lM compared with solvent control group (Fig. 4C).
4. Discussion
Fig. 4. The effect of valproate sodium on gene expressions. The HepG2 cells were exposed with different concentrations of valproate sodium (25, 50, 100, 200 and 500 lV) for 24 h in vitro. The gene expressions were determined by real-time RTPCR: (A) the effect of valproate sodium on CYP1A1 gene expression; (B) the effect of valproate sodium on ABCG1 gene expression; (C) the effect of valproate sodium on CPT1A gene expression. Data are expressed as mean ± SD performed in triplicate. *P < 0.05, **P < 0.01, compared with solvent control group.
Drug-induced hepatotoxicity is an important healthcare issue with respect of serious morbidity and mortality (Kaplowitz, 2001). Therefore, it’s necessary to evaluate drug-induced hepatotoxicity for the design of safer therapeutic agents. This study focused on the VPA-induced hepatotoxicity and its potential molecular mechanism, which was performed upon the HepG2 cells treated with valproate sodium. Earlier studies have shown that VPA caused dose-dependent toxicity to rat hepatocytes and induced apoptosis in the rat hepatoma cell line, FaO (Kingsley et al., 1983; Phillips et al., 2003). Our present investigation confirmed that valproate sodium caused dose-dependent growth inhibition of HepG2 cells. LDH, a particularly important indicator of cell membrane integrity and existing in almost all histiocytes, and, ALT and AST, liver-specific enzymes, physiologically cannot be extracellular membrane leakage. The activities of ALT, AST and LDH are often used for evaluating the hepatotoxicity induced by chemicals or toxicants (Wang et al., 2007; Zhang et al., 2008). There was an obviously statistically significant difference at tested concentration up to 0.3 mM (**P < 0.01) in MTT experiment. So, we have chosen concentrations of valproate sodium from 0.1 to 1 mM for activity assessment of LDH, AST and ALT releasing. In the present study, the activities of enzymes obviously increased with the increase of drug concentration, which suggested that valproate sodium led to the damage of liver cell membrane and the leakage of intracellular enzymes. These data indicated that treatment with valproate sodium at tested concentrations resulted in damage to some cells and finally caused cell death. Many hypotheses, related with the mechanism of hepatotoxicity of VPA, have been raised and tested. It is widely accepted that VPA is extensively metabolized in the liver via glucuronic acid conjugation, mitochondrial b-oxidation and cytosolic (endoplasmic reticulum) x-oxidation to produce multiple metabolites, some of which are biologically active and involved in the VPA-induced hepatotoxicity. For instance, 4-ene-valproic (4-ene-VPA) is a more potent hepatotoxin than its parent, VPA (Tong et al., 2005; Fisher et al., 1998; Lheureux et al., 2005). Cells exposed to 4-ene-VPA experienced greater cytotoxicity than those treated with VPA, and CYP2E1 inducers enhanced toxicity in VPA-exposed HepG2 cells (Fisher et al., 1998). Hepatotoxicity after oral administration of VPA correlated with liver concentration of 4-ene-VPA and could be predicted by the urinary excretion of total 4-ene-VPA in rats (Lee et al., 2009). Multiple P450 isoforms in the CYP450 superfamily of enzymes play critical roles in the biotransformation of valproate sodium, including CYP2C9 and CYP2A6 in human liver, CYP2B4 in rat and rabbit liver, CYP4B1 in rabbit lung (Fisher et al., 1998). The formation of delta-VPA was catalyzed by CYP3A1 in rat liver microsomes, and the reaction was species selective in that neither human CYP3A4 nor rabbit CYP3A6 forms significant quantities of the metabolite (Neuman et al., 2001). In the present study, the expression level of CYP1A1, normally at a low level in extrahepatic tissues in humans but highly inducible in the liver
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and extrahepatic tissues, significantly increased following the increase of drug concentration. The increased gene expression of the drug-metabolizing enzyme would be profitable to chemical metabolism. Consequently, the level of reactive valproate sodium metabolite would be obviously increasing, which would enhance the VPA-induced hepatotoxicity. Subsequently, it might be possible that the hepatotoxicity of valproate sodium would enormously increase while the gene expression of CYP1A1 was up-regulated. The ATP-binding cassette transporters are involved in energydependent translocation of various substrates including sugars, lipids, peptides and xenobiotics across lipid membranes (Borst and Elferink, 2002). ABCB1, ABCC1 and ABCG2 are drug efflux transports, overexpression of which can decrease the intracellular drug concentrations (Ceckova et al., 2008; Kuo, 2007). Meanwhile, other studies in vitro have confirmed that up-regulating ABC1 mRNA was correlated with increased cholesterol efflux in a dose-dependent manner and ABCG1 mRNA levels were highly induced by lipid loading which stimulated reverse cholesterol transport in macrophages (Venkateswaran et al., 2000; Bortnick et al., 2000). Family member 1 of the G-subgroup of mammalian ABC transporters (ABCG1) have been proposed to function synergistically to promote cellular cholesterol efflux into lipid poor HDL, overexpression of which is protective against lipid accumulation (Oram and Vaughan, 2006; Engel et al., 2006; Vaughan and Oram, 2006). In this study, the gene expression of ABCG1 gradually increased with the increase of drug concentration. Then, a positive feedback mechanism, induced by nonphysiologically high concentrations of valproate sodium, which itself is a fatty acid, may be exerting and involved in the reinforcement of gene expression in the biosynthetic pathways. Thus, the gene expression of ABCG1 was highly regulated by overloading valproate sodium or its metabolites. The b-oxidation of long-chain fatty acids (LCFAs) in mitochondria is a major source of energy, and the carnitine palmitoyltransferase (CPT) enzyme system facilitates the transport of such fatty acids into the mitochondria (McGarry et al., 1977; McGarry and Brown, 1997). Carnitine palmitoyltransferase 1A (CPT1A), most abundant in liver and localized within the mitochondrial outer membrane, catalyzes the esterification of acylcoenzyme A to carnitine, which is the rate-limiting step of mitochondrial fatty acid oxidation and plays the major role in fatty acid oxidation (Fraser et al., 1997; Britton et al., 1995). A lot of studies are dedicated to getting more insight into the regulation of CPT1 gene expression in different experimental conditions, some of which found that expression of CPT1 gene is induced by LCFAs in the liver, which means that free LCFAs rather than acyl-CoA eaters are the signal metabolites responsible for the stimulation of CPT1 gene expression (Louet et al., 2001). In the present study, the expression of CPT1A gene gradually increased with the increase of drug concentration, which meant that VPA induced the gene expression of CPT1A. While the content of valproate sodium transported into the mitochondria was gradually increasing, the reactive metabolites of VPA were accumulating gravely in the body. Consequently, the VPA-induced hepatotoxicity would be more serious, forming a malignant circle. In summary, in our study the hepatotoxicity were evaluated and the expression of several genes in HepG2 cells treated with valproate sodium were identified. Our data suggest that the gene expressions were up-regulated in a dose-dependent manner, including CYP1A1, ABCG1 and CPT1A, which are important in fatty acid oxidation and lipid transport. Although the gene regulatory pathways in the valproate sodium-induced hepatotoxicity require further investigation, this study provides significant insight into the mechanisms underlying VPA-induced hepatotoxicity and further studies need to be undertaken to analyze the mechanism of gene expression and its pathway associated with hepatotoxicity.
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