# Impact of phenytoin and valproic acid on cytotoxicity and inflammatory mediators in human mononuclear cells: with and without lipopolysaccharide stimulation

## TL;DR

The study compares how phenytoin and valproic acid affect human immune cells, finding that valproic acid at a specific dose reduces inflammation and cell damage, especially when exposed to bacterial signals.

## Contribution

The novel contribution is identifying valproic acid's dose-specific immunomodulatory effects in human mononuclear cells under inflammatory conditions.

## Key findings

- Valproic acid at 75 μM showed reduced cytotoxicity and inflammatory responses compared to phenytoin and higher VPA doses.
- LPS exposure increased cytotoxicity and altered inflammatory mediator levels in a dose-dependent manner.
- VPA at 75 μM reduced IL-18, IgA, IL-1β, and IL-6 levels, particularly under LPS challenge.

## Abstract

Valproic acid (VPA) is known for its broad-spectrum antiepileptic effects and is recommended for generalized epilepsy, in contrast to phenytoin, which has a more limited spectrum. This study investigated the cytotoxic and inflammatory responses to phenytoin and VPA in peripheral blood mononuclear cells (PBMCs), with and without bacterial lipopolysaccharide (LPS) stimulation.

PBMCs from healthy donors were divided into 12 groups: control (Ctrl), phenytoin (Phy), and four concentrations of VPA (Val-50, Val-75, Val-100, Val-200), with and without LPS. Assessments were conducted on days 1 and 3, including total, live, and dead cell counts, cell viability, and lactic acid dehydrogenase (LDH) cytotoxicity assays. Inflammatory mediators (IL-6, IL-1β) and immune markers (IL-18, IgA) were measured using enzyme-linked immunosorbent assay (ELISA) on day 3. Statistical analysis involved two-way ANOVA, Tukey’s HSD tests, and paired t-tests.

All treatment groups showed significant declines in cell counts and viability from day 1 to day 3, which were exacerbated by LPS. Val-50 + LPS maintained higher cell counts compared to Ctrl + LPS and Phy + LPS. Elevated LDH levels were primarily observed in the Val-100 and Val-200 groups, with and without LPS. In the absence of LPS, the Val-75 and Val-100 groups showed notable reductions in IL-18 and IgA levels, while all VPA treatments reduced IL-6 levels compared to controls. This effect was enhanced under LPS exposure, although IL-1β reductions in the Val-75, Val-100, and Val-200 groups were reversed in the presence of LPS. Val-75 demonstrated lower cytotoxic and inflammatory responses compared to Phy and higher VPA doses, showing moderate LDH increases and reduced IL-18, IgA, IL-1β, and IL-6 levels, particularly under LPS challenge.

Phenytoin and VPA induced significant cytotoxic and inflammatory responses, influenced by dosage and LPS exposure. Val-75 exhibited a dose-specific immunomodulatory effect, reducing both pro-inflammatory and immune markers.