FLUORINATED ANALOGS AS MECHANISTIC PROBES IN VALPROIC ACID HEPATOTOXICITY - HEPATIC MICROVESICULAR STEATOSIS AND GLUTATHIONE STATUS

It is postulated that the hepatotoxicity of valproic acid (VPA) result s from the mitochondrial beta-oxidation of its cytochrome P450 metabol ite, 2-propyl-4-pentenoic acid (4-ene VPA), to 2-propyl-(E)-2,4-pentad ienoic acid ((E)-2,4-diene VPA) which, in the CoA thioester form, eith er depletes GSH or produces a putative inhibitor of beta-oxidation enz ymes. In order to test this hypothesis, 2-fluoro-2-propyl-4-pentenoic acid (alpha-fluoro-4-ene VPA) which was expected to be inert to beta-o xidative metabolism was synthesized and its effect on rat liver studie d in comparison with that of 4-ene VPA. Similarly, the known hepatotox icant 4-pentenoic acid (4-PA) and 2,2-difluoro-4-pentenoic acid (F-2-4 -PA) were compared. Male Sprague-Dawley rats (150-180 g, 4 rats per gr oup) were dosed ip with 4-ene VPA (0.7 mmol/kg per day), 4-PA (1.0 mmo l/kg per day), or equivalent amounts of their alpha-fluorinated analog ues for 5 days. Both 4-ene VPA and 4-PA induced severe hepatic microve sicular steatosis (> 85% affected hepatocytes), and 4-ene VPA produced mitochondrial alterations. By contrast, alpha-fluoro-4-ene VPA and F- 2-4-PA were not observed to cause morphological changes in the liver. The major metabolite of 4-ene VPA in the rat urine and serum was the b eta-oxidation product (E)-2,4-diene VPA. The N-acetylcysteine (NAC) co njugate of (E)-2,4-diene VPA was also found in the urine. Neither (E)- 2,4-diene VPA nor the NAC conjugate could be detected in the rats admi nistered alpha-fluoro-4-ene VPA. In a second set of rats (3 rats per g roup), total liver GSH levels were determined to be depleted to 56% an d 72% of control following doses of 4-ene VPA (1.4 mmol/kg); and equiv alent alpha-fluoro-4-ene VPA, respectively. Mitochondrial GSH remained unchanged in the (alpha-fluoro-4-ene VPA treated group but was reduce d to 68% of control in the rats administered 4-ene VPA. These results strongly support the theory that hepatotoxicity of 4-ene VPA, and poss ibly VPA itself, is mediated largely through beta-oxidation of 4-ene V PA to reactive intermediates that are capable of depleting mitochondri al GSH.