Pyridoxine and valproic acid co-exposure induces ER-mediated calcium dysregulation and cardiac toxicity in zebrafish.

Abstract

Vitamin supplements are frequently utilized as adjunctive therapies in epilepsy care, yet the potential for toxic drug-nutrient interactions remains poorly defined. Using a pentylenetetrazol (PTZ) zebrafish seizure model, we conducted an exploratory screening of a multivitamin formulation containing vitamins B6, B9, D3, and E, together with CoQ10, in combination with common antiseizure medications (ASMs). This screening identified a severe, unexpected lethal interaction between pyridoxine (PN) and valproic acid (VPA). Co-exposure resulted in rapid, dose- and time-dependent mortality in both larval and adult zebrafish. Mechanistic investigations indicated that the calcium-dependent apoptosis, rather than necroptosis, contributes substantially to this toxicity. In vivo calcium imaging revealed marked cytosolic Ca2+ accumulation, which was alleviated by calcium chelation and by pharmacological inhibition of endoplasmic reticulum Ca2+ release through inositol 1,4,5-trisphosphate receptors and ryanodine receptors. PN/VPA co-exposure also caused pronounced cardiac dysfunction, characterized by reduced heart rate, ejection fraction, and cardiac output, together with apoptotic signals in cardiac tissue. Notably, no comparable cytotoxicity was observed in cultured zebrafish cardiomyocytes or mammalian cell lines, suggesting that organism-level or tissue-integrated factors may contribute to the observed toxicity. Supplemental pyridoxal 5'-phosphate (PLP) effectively rescued PN/VPA-induced mortality, supporting the possible involvement of altered vitamin B6/PLP-related cellular processes. Together, these results identify a previously unrecognized PN/VPA hazard and highlight the zebrafish as a sensitive in vivo platform for detecting complex drug-nutrient toxicities.