Valproate-induced liver injury: modulation by the omega-3 fatty acid DHA proposes a novel anticonvulsant regimen

Abstract

Background: The polyunsaturated, ω-3 fatty acid, docosahexaenoic acid (DHA), claims diverse cytoprotective potentials, although via largely undefined triggers. Thus, we currently first tested the ability of DHA to ameliorate valproate (VPA)-evoked hepatotoxicity, to modulate its anticonvulsant effects, then sought the cellular and molecular basis of such actions. Lastly, we also verified whether DHA may kinetically alter plasma levels/clearance rate of VPA.

Methods and results: VPA (500 mg/kg orally for 14 days in rats) evoked prominent hepatotoxicity that appeared as a marked rise (2- to 4-fold) in serum hepatic enzymes (γ-glutamyl transferase [γ-GT], alanine aminotransferase [ALT], and alkaline phosphatase [ALP]), increased hepatic lipid peroxide (LPO) and tumor necrosis factor-alpha (TNFα) levels, as well as myeloperoxidase (MPO) activity (3- to 5-fold), lowering of serum albumin (40 %), and depletion of liver reduced-glutathione (GSH, 35 %). Likewise, histopathologic examination revealed hepatocellular degeneration, replacement by inflammatory cells, focal pericentral necrosis, and micro/macrovesicular steatosis. Concurrent treatment with DHA (250 mg/kg) markedly blunted the elevated levels of liver enzymes, lipid peroxides, TNFα, and MPO activity, while raising serum albumin and hepatic GSH levels. DHA also alleviated most of the cytologic insults linked to VPA. Besides, in a pentylenetetrazole (PTZ) mouse convulsion model, DHA (250 mg/kg) markedly increased the latency in convulsion evoked by VPA, beyond their individual responses. Lastly, pharmacokinetic studies revealed that joint DHA administration did not alter serum VPA concentrations.

Conclusions: DHA substantially ameliorated liver injury induced by VPA, while also markedly boosted its pharmacologic effects. DHA manipulated definite cellular machinery to curb liver oxidative stress and inflammation, without affecting VPA plasma levels. Collectively, these protective and synergy profiles for DHA propose a superior VPA-drug combination regimen.

Fig. 1

a–d Effect of VPA (500 mg/kg daily/2 weeks) with and without DHA (250 mg/kg/day) on serum hepatic enzyme and albumin levels. DHA was given orally 1 h after VPA, then blood was withdrawn from the orbital sinus for determination of enzymes (a–c; γ-GT, ALT, ALP, respectively) after 1 and 2 weeks, or albumin (d), after 2 weeks. Data represent the mean ± SEM of each group; n = 6–8. Symbols indicate significance against VPA-treated group (asterisks) and normal control group (dollar symbols), γ-GT γ-glutamyl transferase, ALT alanine aminotransferase, ALP alkaline phosphatase, DHA docosahexaenoic acid, VPA valproate

Fig. 2

a, b Effect of VPA (500 mg/kg daily/2 weeks) with and without DHA (250 mg/kg/day) on liver lipid peroxide (MDA) (a), and reduced glutathione (GSH) (b) levels. After 2 weeks of treatment, animals were sacrificed and a 10 % W/V liver homogenate was assayed for its content of MDA or GSH. Data represent the mean ± SEM of each group; n = 7. Symbols indicate significance against VPA-treated group (asterisks) and normal control group (dollar symbols), DHA docosahexaenoic acid, VPA valproate

Fig. 3

Effect of VPA (500 mg/kg daily/2 weeks) with and without DHA (250 mg/kg/day) on liver and serum levels of TNFα (a, b), and on liver activity of myeloperoxidase (MPO) (c). Blood was withdrawn for determination of TNFα after 1 and 2 weeks of treatments. Animals were sacrificed after 2 weeks and a 10 % W/V liver homogenate was assayed for both parameters. Data represent the mean ± SEM of each group; n = 8. Symbols indicate significance against VPA-treated group (asterisks) and normal control group (dollar symbols), DHA docosahexaenoic acid, TNFα tumor necrosis factor alpha, VPA valproate

Fig. 4

Necropsies of the liver of studied animals from each group to assess the pathologic changes. Photos 1, 2 are for the negative control group, showing average size/color of the liver with no detected histopathologic abnormalities. Photo 3: VPA control group showing grossly enlarged pale livers with multiple foci of focal lytic necrosis with replacement by inflammatory cells and hepatocyte degeneration. Also, combined macrovesicular and microvesicular steatosis occurring in the periportal zone were evident in four animals in this group (photo 4). DHA when combined with VPA showed only minimal small focal necrosis with no evidence of degeneration (photo 5). DHA docosahexaenoic acid, VPA valproate

Fig. 5

Individual and combined effects of VPA (175 mg/kg) and DHA (100–250 mg/kg) on onset of tonic convulsion (min) evoked by PTZ (85 mg/kg). PTZ was injected 30 min after VPA administration. The combination groups received DHA then VPA, respectively; at 30 min intervals, before PTZ was given. Data represent mean ± SEM of times recorded for each group (8 animals). Symbols indicate significance against VPA-treated group (asterisks) and normal control group (dollar symbols), DHA docosahexaenoic acid, PTZ pentylenetetrazole, VPA valproate