Effects of ethanol on cartilage differentiation in vitro

Prenatal alcohol exposure induces diverse developmental anomalies that include skeletal defects of the limbs and face. However the mechanisms underlying its teratogenic actions remain unclear. The objective of my research was to test the hypothesis that ethanol exposure could selectively influence cartilage differentiation in embryonic chondroprogenitor cells. My results demonstrate that 1.0-2.0% ethanol treatment significantly elevates both cartilage matrix formation and the expression of cartilage-specific structural genes (type II collagen and aggrecan) in high-density micromass cultures of prechondrogenic mesenchyme isolated from limb buds and facial primordia of stage 23-25 chick embryos. Ethanol treatment stimulated cartilage differentiation even in low-density cultures of limb mesenchyme, and in maxillary and hyoid mesenchyme cultures, which undergo little or no chondrogenesis spontaneously. Ethanol's influence on cartilage differentiation appears to be exerted by the alcohol itself rather than a downstream metabolite since tertiary butanol, which is not metabolized to an aldehyde, was as effective as ethanol in promoting ' in vitro' chondrogenesis. The ability of alcohol to stimulate cartilage differentiation may be restricted to 'prechondrogenic' mesenchyme, since ethanol treatment failed to enhance chondrogenesis in cultures of phenotypically differentiated chondrocytes isolated from the 16-18 day chick embryo sternum. This suggested that ethanol might act at an early point in the chondrocyte differentiation pathway. Consistent with this hypothesis, I found that ethanol exposure rapidly elevated expression of transcripts for three chondrogenic regulatory genes, ' Sox-9, Ets-2', and BMPR-IB, that are expressed during the prechondrogenic cell aggregation events that precede overt cartilage tissue formation. Although it was recently suggested that alcohol-related birth defects might arise from inhibition of 'Msx-2' gene expression, levels of 'Msx-2 ' mRNA were unaffected by ethanol treatment in the limb mesenchyme cultures. My findings indicate that ethanol exposure has the capacity to directly influence embryonic cartilage differentiation and may provide insight into the molecular basis of alcohol's teratogenic effects on skeletal development ' in vivo'. (Portions of this work have been reported in Kulyk and Hoffman, 1996, 'Exp. Cell Res'. 223:290-300 and in Hoffman and Kulyk, 1999, 'Int. J. Dev. Biol'. 43:167-174.