Developmental toxicity of valproic acid during embryonic chick vertebral chondrogenesis

Study Design. Valproic acid (2-n-propylpentanoic: acid, VPA), an anticonvulsant drug, was studied for its effects on cartilage matrix gene expression using dot blot hybridization with cDNA probes during early chondrogenesis in the developing lumbar spine. Objectives. To determine the noncytotoxic effects of VPA on vertebral chondrocytes at various stages of embryonic chick spine development. Summary of Background Data. Clinical and epidemiologic studies have indicated that maternal use of valproic acid during early pregnancy causes an increased risk for spina bifida. Methods. The sequence of chondrogenesis in the chick lumbar region was determined for stages HH23-32 and stage-correlated with matrix gene expression by dot blot hybridization analysis using cDNA probes for type II collagen, type IX collagen, and cartilage-specific aggrecan core protein. Results. The mesenchymal stage of lumbar chondrogenesis in the chick spine occurs from HH23-26, the pre-chondrogenic stage between HH27-28, and the chondrogenic period from HH29-32. Stabilization of the cartilage phenotype in the lumbar region is evident at HH-29. Type IX collagen and aggrecan core protein mRNA levels were significantly reduced (P < 0.01) after 48 hours of incubation of the lumbar spines from HH29-31 in the presence of 200 mu g/ml and 300 mu g/ml valproic acid. Lumbar spines cultured for an additional 24 hours after removal of valproic acid showed only an isolated instance of escape from the inhibitory action of 200 mu g/mL valproic acid for type IX collagen at HH31. Conclusions. Valproic acid significantly alters cartilage matrix gene expression during embryonic lumbar vertebral chondrogenesis. The alteration in gene expression for critical matrix proteins during vertebral chondrogenesis may be related to mechanisms underlying the failure of neural arch development in lumbar spina bifida.