Non-selective decrease of collagen synthesis by cultured fetal lung fibroblasts after non-lethal doses of ethanol

Abstract

Addition of ethanol to cultured fetal lung fibroblasts resulted in decreases of both collagen and noncollagen protein syntheses. The inhibitory effect of ethanol on protein synthesis was dependent on the concentration of ethanol and the number of treatments with ethanol. Significant inhibition of collagen and noncollagen protein synthesis was observed 3 hr after a single treatment in 0.25% (v/v) ethanol. The maximum inhibitory effect of ethanol on protein synthesis was observed at 6 hr after drug addition. Inhibition of protein synthesis was observed when either proline or glycine was used as the precursor amino acid. An inhibition of alcohol dehydrogenase did not block the ethanol-mediated inhibition of protein synthesis. Ethanol, added to cell cultures throughout the log phase, inhibited cell growth during the late log and stationary phases. Ethanol inhibition of collagen and noncollagen protein synthesis was reversed when the cell cultures were washed and suspended in fresh media for 24 hr. These inhibitory effects of ethanol on macromolecular syntheses were not engendered by killing of cells. The viability of the cells, as indicated by trypan blue exclusion, was not affected significantly at the concentrations of ethanol used. The inhibitory effect of ethanol on protein synthesis also did not originate from drug-mediated inhibition of precursor amino acid uptake. Polysomes isolated from ethanol-treated fibroblasts incorporated proline into protein at a rate which was reduced commensurate with cellular protein synthesis. The resultant inhibition by ethanol of protein synthesis was not attributable to a direct effect of drug on polysomes. Treatment of fetal lung fibroblasts with ethanol also caused a marked inhibition of radioactive thymidine and uridine incorporation, indicating a reduction of both total cellular DNA and RNA synthesis. Accordingly, the decrease of protein synthesis resulted from inhibition of RNA synthesis. Furthermore, messenger RNA synthesis may have decreased since polysomes isolated from ethanol-treated fibroblasts synthesized less protein in the wheat germ cell-free system. Unlike other biochemical variables that were inhibited by ethanol treatment, the level of prolyl hydroxylase activity was elevated significantly. The elevated level of prolyl hydroxylase activity, however, was related neither to the rate of collagen polypeptide synthesis nor to the degree of proline hydroxylation of cellular collagen. The data suggest that the growth-retarding effects of nonlethal doses of ethanol on fetal development may result from inhibition of macromolecular synthesis in fetal fibroblasts.