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Study of the underlying basis for valproate hepatotoxicity : drug metabolism and its interaction with homeostasis
| Summary: | Microvesicular steatosis is a chronic hepatocellular change potentially induced by valproic acid (VPA). This effect has often been associated with a disturbed fatty acid metabolism and an altered dynamic balance of the essential co-factor coenzyme A (CoA). Furthermore, the parent drug or its metabolites may interact with mitochondrial metabolism at several sites, thereby deranging cellular functions. The experimental work presented in this thesis aimed to 1.) elucidate the impact of valproate on CoA and carnitine homeostasis, 2.) shed new light on the mitochondrial oxidative biotransformation of the drug and 3.) understand the interactions of valproyl-CoA with different functions of mitochondria. Valproate-induced mitochondrial dysfunction is often related to a potential secondary carnitine deficiency. The study of valproate interference on mitochondrial metabolism and CoA homeostasis was first focused on the evaluation of carnitine status in animal tissues using electrospray tandem mass spectrometry, since both cofactors are strictly related. Our studies showed a significant dose-related increase of free carnitine and total acylcarnitine levels in the livers of rats treated with a single dose of sodium valproate. However, these levels appeared to recover after a subchronic treatment, except from specific acylcarnitines derived from branched-chain-2-ketoacids and straight medium-chain fatty acids that remain elevated. In addition, γ-butyrobetaine dioxygenase activity was induced and γ-butyrobetaine levels were increased in the liver. The inter-organ redistribution of carnitine and stimulation of endogenous carnitine biosynthesis seem to be crucial for the immediate rescue of hepatic free carnitine levels. Moreover, the disturbances in rat liver acylcarnitines were suggestive of an altered CoA metabolism. The hepatocellular levels of free and total CoA were quantified in the biological samples. A clear depletion of hepatic free CoA was observed at the onset of VPA treatment in rats. The levels of CoA normalized after prolonged treatment with VPA probably due to active adaptive cellular mechanisms to valproate-induced stress on mitochondrial metabolism. However, the decreased availability of this cofactor may contribute to the adverse effects associated with VPA, often observed at the start of VPA therapy or in cases of overdose. The structural similarity between the intermediates derived from the β-oxidation of VPA and from the oxidative metabolism of branched-chain amino acids (BCAAs) suggested that VPA might use key enzymes from the BCAAs catabolic pathways for its own metabolism. We have demonstrated that two enzymes from the isoleucine and leucine catabolism, namely short-branched chain acyl-CoA dehydrogenase (SBCAD) and isovaleryl-CoA dehydrogenase (IVD), are involved in the first step of the β-oxidation of VPA, the dehydrogenation of valproyl-CoA into Δ2-valproyl-CoA. We have further demonstrated that short-chain enoyl-CoA hydratase (ECHS1) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) are the unique enzymes hydrating Δ2-valproyl-CoA and oxidizing 3-hydroxyvalproyl-CoA, respectively. Moreover, the activity of IVD and SBCAD were inhibited in the presence of the tested VPA Summary xiv metabolites, valproyl-CoA and valproyl-dephosphoCoA. The drug metabolite 3-hydroxyvalproyl-CoA also inhibited the activity of MHBD. This result is in accordance with the increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid in patients under valproate therapy. It has been reported that patients treated with valproate reveal an increased excretion of 3-hydroxyisovaleric acid in urine, a secondary metabolite of the leucine oxidative pathway. This finding has not been explained and its biochemical basis remains elusive. Our results suggest that valproyl-CoA inhibits the activity of 3-methylcrotonyl-CoA carboxylase (3MCC) through a direct effect on the enzyme, or through the inhibition of biotinidase. This is in agreement with the significant increased levels of 3-hydroxyisovaleric acid observed in urine of patients treated with valproate as well as with the skin rash and hair loss, which are side effects often reported in VPA-treated patients. To elucidate the underlying mechanisms of VPA-induced interferences on oxidative phosphorylation (OXPHOS), the effects of VPA and specific metabolites were studied both on the mitochondrial respiration and on the formation of ATP. Our results show that oxygen consumption and ATP synthesis were inhibited by both valproate and Δ4-VPA, when 2-oxoglutarate or glutamate, were used as respiratory substrates. Accordingly, the results demonstrated a selective and mild inhibition of dihydrolipoyl dehydrogenase (DLDH) (E3 subunit) activity by valproyl-CoA, which suggest that valproate might limit energy production in mitochondria. In addition, the measurement of the GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL) activity revealed that valproyl-CoA was found to inhibit the activity of SUCL, in particular the ATP-specific enzyme. Since SUCL is a crucial enzyme in the Krebs cycle, interference with its activity might derange the cellular energy status. Furthermore, valproate might induce an imbalance of nucleotides in mitochondria explaining the potential valproate-induced liver failure in patients with deficiencies within the mitochondrial DNA replicase such as polymerase γ 1. The elucidation of the interference of valproate with mitochondrial metabolism promoted by the present studies, may explain the potencial idiosyncratic reaction reported in patients treated with the drug. The prescription of valproate should be carefully considered in patients with inborn errors of metabolism affecting not only fatty acid oxidation and urea cycle but also the oxidative pathways of branched-chain amino acids, the krebs cycle and the DNA replicase system. |
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| Main Authors: | Luís, Paula Maria Brazão Mendes, 1981- |
| Subject: | Teses de doutoramento - 2011 |
| Year: | 2011 |
| Country: | Portugal |
| Document type: | doctoral thesis |
| Access type: | open access |
| Associated institution: | Universidade de Lisboa |
| Language: | English |
| Origin: | Repositório da Universidade de Lisboa |
| Summary: | Microvesicular steatosis is a chronic hepatocellular change potentially induced by valproic acid (VPA). This effect has often been associated with a disturbed fatty acid metabolism and an altered dynamic balance of the essential co-factor coenzyme A (CoA). Furthermore, the parent drug or its metabolites may interact with mitochondrial metabolism at several sites, thereby deranging cellular functions. The experimental work presented in this thesis aimed to 1.) elucidate the impact of valproate on CoA and carnitine homeostasis, 2.) shed new light on the mitochondrial oxidative biotransformation of the drug and 3.) understand the interactions of valproyl-CoA with different functions of mitochondria. Valproate-induced mitochondrial dysfunction is often related to a potential secondary carnitine deficiency. The study of valproate interference on mitochondrial metabolism and CoA homeostasis was first focused on the evaluation of carnitine status in animal tissues using electrospray tandem mass spectrometry, since both cofactors are strictly related. Our studies showed a significant dose-related increase of free carnitine and total acylcarnitine levels in the livers of rats treated with a single dose of sodium valproate. However, these levels appeared to recover after a subchronic treatment, except from specific acylcarnitines derived from branched-chain-2-ketoacids and straight medium-chain fatty acids that remain elevated. In addition, γ-butyrobetaine dioxygenase activity was induced and γ-butyrobetaine levels were increased in the liver. The inter-organ redistribution of carnitine and stimulation of endogenous carnitine biosynthesis seem to be crucial for the immediate rescue of hepatic free carnitine levels. Moreover, the disturbances in rat liver acylcarnitines were suggestive of an altered CoA metabolism. The hepatocellular levels of free and total CoA were quantified in the biological samples. A clear depletion of hepatic free CoA was observed at the onset of VPA treatment in rats. The levels of CoA normalized after prolonged treatment with VPA probably due to active adaptive cellular mechanisms to valproate-induced stress on mitochondrial metabolism. However, the decreased availability of this cofactor may contribute to the adverse effects associated with VPA, often observed at the start of VPA therapy or in cases of overdose. The structural similarity between the intermediates derived from the β-oxidation of VPA and from the oxidative metabolism of branched-chain amino acids (BCAAs) suggested that VPA might use key enzymes from the BCAAs catabolic pathways for its own metabolism. We have demonstrated that two enzymes from the isoleucine and leucine catabolism, namely short-branched chain acyl-CoA dehydrogenase (SBCAD) and isovaleryl-CoA dehydrogenase (IVD), are involved in the first step of the β-oxidation of VPA, the dehydrogenation of valproyl-CoA into Δ2-valproyl-CoA. We have further demonstrated that short-chain enoyl-CoA hydratase (ECHS1) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) are the unique enzymes hydrating Δ2-valproyl-CoA and oxidizing 3-hydroxyvalproyl-CoA, respectively. Moreover, the activity of IVD and SBCAD were inhibited in the presence of the tested VPA Summary xiv metabolites, valproyl-CoA and valproyl-dephosphoCoA. The drug metabolite 3-hydroxyvalproyl-CoA also inhibited the activity of MHBD. This result is in accordance with the increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid in patients under valproate therapy. It has been reported that patients treated with valproate reveal an increased excretion of 3-hydroxyisovaleric acid in urine, a secondary metabolite of the leucine oxidative pathway. This finding has not been explained and its biochemical basis remains elusive. Our results suggest that valproyl-CoA inhibits the activity of 3-methylcrotonyl-CoA carboxylase (3MCC) through a direct effect on the enzyme, or through the inhibition of biotinidase. This is in agreement with the significant increased levels of 3-hydroxyisovaleric acid observed in urine of patients treated with valproate as well as with the skin rash and hair loss, which are side effects often reported in VPA-treated patients. To elucidate the underlying mechanisms of VPA-induced interferences on oxidative phosphorylation (OXPHOS), the effects of VPA and specific metabolites were studied both on the mitochondrial respiration and on the formation of ATP. Our results show that oxygen consumption and ATP synthesis were inhibited by both valproate and Δ4-VPA, when 2-oxoglutarate or glutamate, were used as respiratory substrates. Accordingly, the results demonstrated a selective and mild inhibition of dihydrolipoyl dehydrogenase (DLDH) (E3 subunit) activity by valproyl-CoA, which suggest that valproate might limit energy production in mitochondria. In addition, the measurement of the GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL) activity revealed that valproyl-CoA was found to inhibit the activity of SUCL, in particular the ATP-specific enzyme. Since SUCL is a crucial enzyme in the Krebs cycle, interference with its activity might derange the cellular energy status. Furthermore, valproate might induce an imbalance of nucleotides in mitochondria explaining the potential valproate-induced liver failure in patients with deficiencies within the mitochondrial DNA replicase such as polymerase γ 1. The elucidation of the interference of valproate with mitochondrial metabolism promoted by the present studies, may explain the potencial idiosyncratic reaction reported in patients treated with the drug. The prescription of valproate should be carefully considered in patients with inborn errors of metabolism affecting not only fatty acid oxidation and urea cycle but also the oxidative pathways of branched-chain amino acids, the krebs cycle and the DNA replicase system. |
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