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TOXICOLOGY
Clinical Pharmacology and Toxicology and Department of Research, University Hospital Basel, Basel, Switzerland (A.C.K., L.To., S.K.); Institute of Anatomy and Embryology (K.B.) and Institute of Pathology (L.Te.), University of Basel, Basel, Switzerland; and Institute of Clinical Pharmacology, University of Bern, Bern, Switzerland (H.S., J.R.)
Received for publication
September 4, 2007
Accepted
November 5, 2007.
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). A short time after the drug was introduced, cases of fulminant liver failure in patients treated with VPA were reported (Zafrani and Berthelot, 1982; Zimmerman and Ishak, 1982; Dreifuss et al., 1987; Krähenbühl et al., 1995); however, the underlying mechanism of VPA-induced hepatotoxicity is still not fully understood. One principal cause of liver failure that is associated with VPA therapy is most probably the inhibition of hepatic mitochondrial β-oxidation. Microvesicular steatosis of the liver, one of the most important histological findings in VPA-induced liver failure (Zafrani and Berthelot, 1982; Zimmerman and Ishak, 1982; Dreifuss et al., 1987; Krähenbühl et al., 1995), may be caused by impaired hepatic β-oxidation (Fromenty and Pessayre, 1995; Spaniol et al., 2001b). Different mechanisms have been proposed to explain inhibition of mitochondrial β-oxidation by VPA, among them microsomal production of toxic metabolites, e.g., 4-ene-VPA and 2,4-diene-VPA (Gram and Bentsen, 1985; Tennison et al., 1988; Ponchaut et al., 1992b; Ishikura et al., 1996), decreased activity of complex IV of the respiratory chain, and/or depletion of the hepatic pools of CoASH and/or carnitine (Ponchaut and Veitch, 1993; Krähenbühl et al., 1995). Pre-existing mitochondrial diseases, e.g., impaired β-oxidation or impaired function of the respiratory chain, have been proposed to represent risk factors for VPA-associated mitochondrial dysfunction and therefore for liver failure (Chabrol et al., 1994; Lam et al., 1997; Krähenbühl et al., 2000). Because carnitine is an essential cofactor for hepatic β-oxidation (Fromenty and Pessayre, 1995), systemic carnitine deficiency (OMIM 212140
[OMIM]
) is associated, among others, with microvesicular steatosis of the liver (Spaniol et al., 2001b). Therefore, we hypothesized that mice with decreased carnitine body stores are more susceptible to hepatic and possibly also skeletal muscle adverse effects associated with VPA treatment compared to control mice. To test this hypothesis, juvenile visceral steatosis (jvs) mice (formerly named C3H-H-2°) were used. The jvs mice, first described in 1988 by Koizumi et al. (1988), have a mutation in the gene coding for OCTN2 (Lu et al., 1998), leading to impaired renal absorption of carnitine and systemic carnitine deficiency. These mice are phenotypically characterized by liver steatosis and other features of carnitine deficiency, such as hyperammonemia, hypoglycemia, cardiac hypertrophy, mitochondrial abnormalities in the skeletal muscle, and progressive growth retardation (Horiuchi et al., 1993; Kaido et al., 1997). For the current study, heterozygous (jvs+/–) and not homozygous (jvs–/–) mice were used, because the carnitine tissue levels of jvs+/– mice are approximately half that of wild-type mice, and, in contrast to homozygous jvs–/– mice, jvs+/– mice can survive without carnitine replacement. An additional question that was addressed in the current study was the effect of VPA on carnitine homeostasis in an animal model with decreased carnitine stores such as jvs+/– mice. |
Materials and Methods |
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Animals. The jvs mice were obtained from Prof. Masahisa Horiuchi (University of Kagoshima, Kagoshima, Japan). The breeding pairs (wild-type and jvs+/– mice) and the offspring were supplemented with carnitine (1 g/250 ml of drinking water) before weaning to maintain an optimal survival rate. After weaning, the supplementation with carnitine was continued for the homozygous jvs–/– mice. For genotyping of littermates (wild-type, jvs+/–, and jvs–/– mice), DNA was extracted and purified from the mouse tails with a DNA-extraction kit (kit number 740952.250; Macherey-Nagel, Oensingen, Switzerland) and analyzed using a TaqMan allelic discrimination method, which combines polymerase chain reaction and mutation detection in a single step. Two allele-specific TaqMan probes were used, one for each allele (Applied Biosystems, Warrington, UK). Each probe consisted of an oligonucleotide with a 5' reporter dye (FAM for the detection of the wild-type L352 allele, and VIC for the detection of the mutant L352R allele) and a 3' quencher dye (TAMRA for both probes). The probes were as follows: FAM, 5'-atatggtcagcctgca-3' and VIC, 5'-tatggtccgcctgca-3' (boldface letters a and c give the position of the mutation). Identical primers (Microsynth, Balgach, Switzerland) were used for both alleles and designed as follows: forward primer, 5'-tccccatgcaagttaggagtgt-3', and reverse primer, 5'-tgctgctccagctctcttctg-3'. TaqMan analysis was performed using a 7900HT Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland), and identification of the mutation in OCTN2 was achieved using an allelic discrimination plot (Todesco et al., 2003). Cycling conditions were 10 min at 95°C for initial denaturation and activation of the DNA polymerase, followed by 40 cycles of 15 s at 95°C for denaturation, and 1 min at 60°C for combined annealing and primer extension. Fluorescence from the FAM reporter only reflected the presence of wild-type alleles, whereas fluorescence from the VIC reporter only indicated mutant alleles. In accordance, fluorescence from both reporters reflected the heterozygous population.
All experiments were reviewed and accepted by the Animal Ethics Committee of the canton of Basel-Stadt. Experiments were performed with animals 9 to 12 weeks old.
Study Design and VPA Administration. In this study, the following four groups of jvs mice were investigated: wild-type mice treated with VPA or 0.9% NaCl (vehicle), and heterozygous jvs+/– mice treated with VPA or 0.9% NaCl (n = 5 per group). VPA (0.1 mg/g b.wt./day) or vehicle was administered p.o. in a volume of 10 µl/g b.wt. once a day for 2 weeks. The used VPA dose was subtoxic, as established in earlier studies (Letteron et al., 1996). The mice were starved overnight before being used for the experiments. Urine of the mice was collected individually for 24 h, and a blood sample was obtained from the tail vein before the mice were killed by decapitation. Tissue samples were obtained from the liver and skeletal muscle (quadriceps femoris) for carnitine analysis. These samples were quickly frozen in liquid nitrogen and stored at –80°C until analysis. Additional liver samples were treated with 4% formaldehyde for histological analysis after staining with hematoxylin-eosin or with Sudan Black B. The remainder of the liver was quickly removed, put on ice, and used for the isolation of mitochondria.
Characterization of the Animals. The animals were characterized by their body and liver weights and the increased activity of aspartate aminotransferase, alkaline phosphatase, and creatine kinase in plasma. The enzyme activity was analyzed with commercially available kits on a MODULAR analyzer (Hoffmann-La Roche Diagnostics, Basel, Switzerland).
In Vivo Oxidation of Palmitate. To collect breath samples, mice were placed in a cylindrical vessel attached to a vacuum pump. [1-14C]palmitic acid (3 µCi/kg, 57.0 mCi/mmol) was diluted in thistle oil and administered i.p. at 0 min. To collect the 14CO2 resulting from the oxidation of [1-14C]palmitate, the exhaled air was pulled through successive solutions of ethanol (to dry the exhaled breath) and ethanolamine (4 M in ethanol) to trap exhaled 14CO2. The exhaled 14CO2 was quantified over 120 min by scintillation spectroscopy.
Isolation of Liver Mitochondria. The mitochondrial fraction of mouse livers was obtained by differential centrifugation according to the method of Hoppel et al. (1979). The mitochondrial protein content was determined using the biuret method with bovine serum albumin as a standard (Gornall et al., 1949).
Oxygen Consumption and in Vitro β-Oxidation of Intact Mitochondria. Oxygen consumption by freshly isolated liver mitochondria was measured in a chamber equipped with a Clark-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) at 30°C as described previously (Hoppel et al., 1979). The substrate concentrations were 20 mM for L-glutamate and succinate and 40 µM for palmitoyl-CoA. The incubation with palmitoyl-CoA also contained 2 mM L-carnitine and 5 mM L-malate.
In Vitro β-Oxidation of Intact Mitochondria. The β-oxidation of [1-14C]palmitic acid by liver mitochondria, which measures the formation of acid-soluble products from mitochondrial palmitate metabolism, was determined with freshly isolated liver mitochondria according to the method of Fréneaux et al. (1988), using some modifications as described by Spaniol et al. (2001a).
Determination of Carnitine in Plasma, Tissue, and Urine. The carnitine concentrations in plasma, liver, muscle, and urine were determined radioenzymatically as described by Brass and Hoppel (1978). Plasma and tissue samples were treated with perchloric acid (final concentration 3%), resulting in a supernatant and a pellet. Analysis of the supernatant yielded free carnitine and, after alkaline hydrolysis, total acid-soluble carnitine. The pellet yielded the long-chain acylcarnitines (acyl group chain length 
10 carbons) after alkaline hydrolysis. The short-chain acylcarnitine fraction (acyl group chain length <10 carbons) was calculated from the difference between total acid-soluble and free carnitine, and the sum of total acid-soluble and long-chain acylcarnitine represented total carnitine.
Histological Analysis of Liver Tissue. Pieces of the liver were fixed in 4% formaldehyde for histological analysis after staining with hematoxylin-eosin or immunohistochemistry for caspase-3.
For caspase-3 staining, paraffin sections were rehydrated and heated in EDTA buffer, pH 8.0 (100°C/5 min). Slides were then incubated in a quench solution (1.0 M sodium azide in a solution of 4:1 methanol and 30% hydrogen peroxide, v/v) and incubated with blocking solution (normal goat serum) for 30 min. Next, sections were incubated with caspase-3 antibody (cleaved caspase-3 antibody was obtained from Cell Signaling Technology, Beverly, MA) diluted 1:100 in phosphate-buffered saline, pH 7.1 to 7.3 (ChemMate Antibody Dilution Buffer; Ventana Medical Systems, Illkirch, France), for 1 h at room temperature. Negative controls were performed by omitting the primary antibody. After primary antibody incubation, slides were washed three times with Tris-buffered saline containing 0.05% Tween 20 and incubated for 30 min at room temperature with a mixture of biotinylated secondary antibodies in ChemMate Antibody Dilution Buffer. The slides were washed again and incubated for 30 min at room temperature with the avidin-biotin complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Staining was visualized by incubating the sections for 10 min in DAB solution (catalog number K3466; DakoCytomation, Baar, Switzerland). After incubation, the slides were rinsed in water, counter-stained with hematoxylin, dehydrated, and coverslipped.
The frozen liver tissue was cut into sections and stained with Sudan Black B to determine fat accumulation. The estimation of fat accumulation and the investigation of pathological changes in the liver were examined by light microscopy of the stained sections.
Statistical Analysis. All analyses were performed in duplicate. For each treatment group (n = 5 per group), the results are presented as the mean ± S.D. Significant differences between groups were determined by ANOVA with Bonferroni's multiple comparison post-hoc test. P values <0.05 were considered to be significant.
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Results |
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In Vivo Oxidation of Palmitate. VPA-treated jvs+/– mice showed a lower peak exhalation of 14CO2 (30 min after injection) compared with vehicle-treated jvs+/– mice (21% decrease) and to VPA-treated wild-type mice (20 min after injection, 23% decrease) (see Fig. 1 and Table 2 for quantitative results). In addition, the exhalation of 14CO2 over 2 h was significantly lower in VPA-treated jvs+/– mice. The decrease reached 23% compared to vehicle-treated jvs+/– mice and 20% compared to VPA-treated wild-type mice. On the other hand, there was no difference in these parameters between the vehicle-treated wild-type mice and the vehicle-treated jvs+/– mice, and no difference was found between the vehicle-treated mice and VPA-treated wild-type mice. These findings suggest that both carnitine deficiency and treatment with VPA are necessary to impair hepatic β-oxidation in vivo. Because tracer dilution could also be an explanation for these findings (increased hepatic content of nonesterified fatty acids as a consequence of impaired mitochondrial β-oxidation), histological sections of the livers were performed, and mitochondrial β-oxidation was studied in vitro.
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Histological Findings in the Liver. In support of the results obtained for in vivo β-oxidation, fat accumulation in the liver was lowest in vehicle-treated wild-type mice (Fig. 2A) and slightly higher in wild-type mice treated with VPA or in vehicle-treated jvs+/– mice (Fig. 2, B and C). The combination of carnitine deficiency (jvs+/– mice) and treatment with VPA was associated with the highest extent of fat accumulation (Fig. 2D). The accumulated fat was from the microvesicular type and compatible with impaired β-oxidation (Fromenty and Pessayre, 1995; Spaniol et al., 2003).
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Stains with hematoxylin-eosin confirmed the presence of microvesicular steatosis, predominantly in VPA-treated jvs+/– mice (Fig. 3A). In livers from VPA-treated jvs+/– mice, focal areas with hypereosinophilic, caspase-3-positive hepatocytes were detectable, suggesting cell death by apoptosis (Fig. 3B). In livers from vehicle- or VPA-treated wild-type mice or vehicle-treated jvs+/– mice, almost no caspase-3-positive cells were detectable. Similar histological findings have been reported in another study exploring the hepatic effects of VPA (Jezequel et al., 1984).
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), we examined the function of the respiratory chain in the presence of different substrates. The state 3 oxidation rate of L-glutamate was decreased by 32% in VPA-treated compared with vehicle-treated wild-type mice (Table 3). Likewise, a decrease of 58% was found in VPA-treated jvs+/– versus vehicle-treated jvs+/– mice. The state 3 oxidation rate of succinate was also significantly lower in VPA-treated jvs+/– mice compared with vehicle-treated heterozygous jvs+/– mice (decrease by 52%), whereas no significant decrease was found in VPA-treated wild-type compared with vehicle-treated wild-type mice. Palmitoyl-CoA state 3 oxidation rates were decreased by 43% in VPA-treated wild-type mice and by 73% in VPA-treated jvs+/– mice compared with their vehicle-treated controls.
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In Vitro β-Oxidation by Intact Liver Mitochondria. Because VPA has been shown to impair mitochondrial β-oxidation (Levy et al., 1990; Ponchaut et al., 1992b; Fromenty and Pessayre, 1995) and considering our results that were obtained in vivo (see Table 2), we investigated the effect of VPA on the metabolism of palmitate by isolated liver mitochondria. VPA treatment significantly decreased palmitate oxidation by 44% in wild-type and by 35% in jvs+/– mice compared with their vehicle-treated controls. For the interpretation of these results, it is important to take into account that the experiments were performed under saturating conditions regarding palmitate and in the presence of exogenous L-carnitine, excluding problems arising from tracer dilution with a high probability.
Plasma Carnitine Concentration. The free and total carnitine levels in plasma were significantly lower in wild-type mice treated with VPA compared with vehicle-treated wild-type mice (decrease by 58 and 24%, respectively) (Table 4). Similar results were obtained for VPA-treated jvs+/– mice, in which the decrease was 65% for free carnitine and 39% for total carnitine, respectively, versus vehicle-treated jvs+/– mice. A comparison of VPA-treated jvs+/– versus VPA-treated wild-type mice revealed a decrease of 40% in free carnitine and a decrease of 37% in total carnitine in jvs+/– mice. Treatment with VPA was associated with a decrease in the total carnitine concentration in both groups, namely by 24% in wild-type mice and by 39% in jvs+/– mice. It is interesting to note that the short-chain acylcarnitine/total acid-soluble carnitine ratio showed a 120% increase in VPA-treated wild-type mice and a 76% increase in VPA-treated heterozygous jvs+/– mice compared with their vehicle-treated controls.
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Urinary Excretion of Carnitine. Treatment with VPA over 2 weeks was associated with an increased excretion of free carnitine in wild-type mice and of free and total carnitine in jvs+/– mice (Table 5). In jvs+/– mice treated with VPA, the excretion of free carnitine was increased by 114% compared to vehicle-treated jvs+/– mice, whereas the increase in total carnitine excretion was 108%. Compared to vehicle-treated control mice, the renal clearance of free carnitine was approximately 6-fold higher in VPA-treated jvs+/– mice and approximately 3-fold higher in VPA-treated wild-type mice.
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Liver Carnitine Content. The hepatic-free carnitine content was 33% lower in vehicle-treated jvs+/– compared with vehicle-treated wild-type mice (Table 6). Although treatment with VPA did not significantly affect the free carnitine content in wild-type mice, VPA decreased the free carnitine content in jvs+/– mice by 53%. The short-chain-acylcarnitine content was increased by VPA treatment by a factor of two to three in both wild-type and jvs+/– mice compared with the respective vehicle-treated groups. In accordance, the short-chain-acylcarnitine/total acid-soluble carnitine ratio was increased in both VPA groups versus the vehicle-treated controls, reaching 157% in the wild-type and 195% in jvs+/– mice, respectively. The total carnitine content was decreased by 26% in vehicle-treated jvs+/– compared with wild-type mice. In both groups, treatment with VPA was not associated with significant changes in the total carnitine content.
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Skeletal Muscle Carnitine Content. Free carnitine levels were not different between vehicle-treated wild-type and jvs+/– mice (Table 7). Treatment with VPA decreased the free carnitine content by 31% in jvs+/– mice, but it had no significant effect in wild-type mice. The short-chain-acylcarnitine content and the short-chain-acylcarnitine/total acid-soluble carnitine ratio were not different between the vehicle-treated wild-type mice and jvs+/– mice and were not affected by VPA treatment. The total carnitine content was 16% lower in vehicle-treated jvs+/– mice compared with the respective wild-type mice. Treatment with VPA was associated with a 15% drop in the total carnitine content in jvs+/– mice, but it did not significantly affect the total carnitine content in wild-type mice.
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Discussion |
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In vivo determination of hepatic β-oxidation revealed a decrease in palmitate metabolism in VPA-treated jvs+/– mice, which was not the case for VPA-treated wild-type or vehicle-treated jvs+/– mice. In combination with the liver enzyme elevations, these findings suggest that VPA is more toxic in jvs+/– mice than in wild-type mice, supporting our initial hypothesis that a reduction in carnitine body stores deficiency is a risk factor for hepatotoxicity associated with VPA. VPA is metabolized primarily by conjugation with glucuronic acid or carnitine, and to a lesser extent by mitochondrial β-oxidation, microsomal 
-oxidation, and -1-oxidation (Zaccara et al., 1988). Microsomal VPA metabolism has been shown to be catalyzed by various cytochrome P450 isozymes, among them CYP2C9, CYP2A6, and CYP2B6 (Kiang et al., 2006). These oxidative pathways can yield potentially hepatotoxic products, e.g., pentanoate and propionate, as well as 4-ene-VPA and others. It is conceivable that a reduction in the hepatic availability of carnitine in jvs+/– mice can be associated with reduced conjugation of VPA, shifting more VPA into the oxidative pathways and possibly leading to hepatic toxicity. The observed decrease in the hepatic-free carnitine content in vehicle-treated jvs+/– versus wild-type mice and the even more pronounced decrease in the hepatic carnitine content of jvs+/– mice treated with VPA support such a mechanism. In addition, a reduction in the free carnitine pool is associated with similar changes in the CoASH pool (Ponchaut et al., 1992b; Krähenbühl et al., 1995), because these pools are connected with each other by the carnitine acyltransferases. A drop in cellular CoASH should impair enzymes and/or the metabolic pathways using CoASH, for instance pyruvate dehydrogenase and β-oxidation of fatty acids.
If the production and presence of toxic metabolites were only responsible for hepatic toxicity of VPA, this toxicity could be expected to decrease or even disappear in isolated mitochondria, due to loss of toxic metabolites during the isolation procedure (Spaniol et al., 2003). As shown in Table 2, this effect was clearly not the case in the current investigation, suggesting that mitochondrial changes on the gene expression and/or structural level are associated with VPA treatment. Earlier studies by Hayasaka et al. (1986) and Ponchaut et al. (1992a) have indeed demonstrated that long-term treatment with VPA is associated with changes in the composition of cytochrome c oxidase (complex IV), namely a loss cytochrome aa3. A reduced activity of complex IV associated with VPA treatment not only explains impaired oxidation of succinate and L-glutamate but also that of palmitate, as observed in this study (Table 3). On the other hand, the reduced activity of mitochondrial β-oxidation, which has been described in other studies assessing hepatic toxicity of VPA (Turnbull et al., 1983; Baldwin et al., 1996), can be explained most probably by interactions of toxic metabolites of VPA with enzymes involved in β-oxidation (Ito et al., 1990; Baldwin et al., 1996).
A comparison of palmitate oxidation in vivo and in vitro reveals that in vivo, β-oxidation was only impaired in VPA-treated jvs+/– mice, whereas in vitro, palmitate oxidation was also reduced in liver mitochondria from VPA-treated wild-type mice. As a possible explanation, it has to be taken into account that a tracer dose was only administered in vivo. Although tracer dilution may explain the observed decrease in 14CO2 exhalation by VPA-treated jvs+/– mice, it cannot explain the decrease in mitochondrial β-oxidation determined in vitro. With regard to the saturable nature of typical substrate concentration-activity curves, it is conceivable that differences in enzyme activity may be better detected at saturating versus at nonsaturating concentrations. Furthermore, under in vivo conditions, palmitate could have been metabolized to a minor part also by extrahepatic tissues, making small differences in hepatic activity of β-oxidation even more difficult to detect. As a result, it is possible that reduced in vivo β-oxidation of palmitate is primarily due to the reduction the hepatic carnitine stores, which was most accentuated in livers from VPA-treated jvs+/– mice (Table 6).
The fact that the skeletal muscle carnitine pools were much less affected by OCTN2 activity and by VPA administration than the hepatic carnitine pools may serve as one possible explanation for the reduced toxicity of VPA in the skeletal muscle. In addition, the cytochromes P450 involved in the metabolism of VPA have the highest expression in liver (Gonzalez, 1992) and not in the skeletal muscle. Nevertheless, VPA fat accumulation and morphological mitochondrial abnormalities have been described both in children and rats with long-term VPA treatment (Melegh and Trombitas, 1997).
Body carnitine homeostasis was affected by both activity of OCTN2 (jvs+/– versus wild-type mice) and treatment with VPA. As expected, jvs+/– mice had reduced plasma and liver carnitine pools compared to wild-type mice, demonstrating the importance of renal carnitine reabsorption associated with OCTN2. It is interesting to note that the effect of partial loss of OCTN2 activity was less accentuated for skeletal muscle than for liver, a finding that can at least partially be explained by the resistance of the plasmalemmal membrane for transport of carnitine (Rebouche and Engel, 1984). The effect of VPA treatment on the carnitine plasma and tissue stores was much more dramatic in JVS+/– mice than in wild-type mice, leading to additional and substantial losses in the plasma and tissue carnitine pools. As shown in Table 5, this is a consequence of a massive increase in the renal excretion of carnitine and acylcarnitines; in this case, it was most probably valproylcarnitine (Muro et al., 1995). Although precise data are lacking, the increase in renal carnitine excretion may be explained by the competition between valproylcarnitine and possible other acylcarnitines with carnitine for proximal tubular reabsorption by OCTN2 (Okamura et al., 2006).
In conclusion, hepatic toxicity of VPA is more pronounced in JVS+/– mice than in corresponding wild-type mice. Therefore, carnitine deficiency can be considered to be a risk factor for VPA-associated hepatotoxicity, showing the importance of a sufficient hepatic carnitine pool in patients treated with this drug.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: VPA, valproic acid; jvs, juvenile visceral steatosis; OCTN2, organic cation/carnitine transporter; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-N,N,N'N'-tetramethylrhodamine; CoA, coenzyme A.
Address correspondence to: Dr. Stephan Krähenbühl, Clinical Pharmacology and Toxicology, University Hospital, CH-4031 Basel, Switzerland. E-mail: kraehenbuehl{at}uhbs.ch
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