Butylated hydroxytoluene (BHT) is a pulmonary toxin and tumor promoter in mice presumably due to the formation of two quinone methides (QMs) that alkylate cellular nucleophiles. The activation of stress genes by these electrophilic metabolites was investigated with an assay system consisting of 14 recombinant cell lines derived from the human hepatoma line HepG2, each carrying a unique promoter or response element construct fused to the reporter gene for chloramphenicol acetyl transferase (CAT). The largest responses to QMs occurred in cells containing either the metallothionein IIA, glutathione S-transferase Ya, or 70 kDa heat shock protein promoter, or the xenobiotic response element. The other cell lines exhibited only small or no effects. These results are consistent with transcriptional activities reported for several other electrophiles known to undergo covalent interactions with proteins.

Chronic ethanol exposure has been associated with pleiotropic effects on cellular function in vivo and in vitro, including inhibition of growth. To date, it has been difficult to dissociate the primary effects of ethanol from the effects of ethanol metabolism, generation of acetaldehyde, and reducing equivalents. We have previously described the development of a Chinese hamster ovary cell line, A-10, which expresses a transfected murine-liver alcohol dehydrogenase. Cultures of these cells accumulate acetaldehyde due to the low level of aldehyde dehydrogenase. One noticeable effect of chronic acetaldehyde exposure, but not ethanol exposure, is the inhibition of cell growth. This study focuses on the mechanisms that underlie this growth inhibition. Our studies with the A-10 cell on the rates of [3H]thymidine incorporation and flow cytometry of asynchronous cultures indicated that acetaldehyde did not lead to arrest of the cell cycle in the G1 phase as has been found in other models of ethanol exposure. Rather, we observed a generalized delay in cell cycle progression. However, the slower cell cycle did not account exclusively for the slower rates of cell accumulation. Chronic exposure to acetaldehyde also increased the rate of cell death. The increased rate of cell death was both cumulative and dose-dependent. The dead cells accumulated in the medium and were apoptotic. Apoptosis was confirmed using morphological criteria and quantitation of DNA fragmentation. These data lend additional support to the idea that chronic acetaldehyde exposure can affect the mechanisms that regulate cell division and the apoptotic program.

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.

When propagated in cesium-rich medium, type 1 poliovirus incorporated enough cesium atoms to shift its buoyant density from 1.34 to an upper limit of about 1.40 g/ml. Studies with radioactive 137Cs indicated that this upper density limit corresponded to about 4200 cesium atoms per virion. The incorporated cesium was tightly bound, exchanging out of virions at a negligible rate at temperatures below 22°C. Cesium-loaded virions were normal with respect to specific infectivity, neutralizability by specific antisera, and electrophoretic profile of coat protein. Natural top component bound fewer than 20 cesium atoms per particle under the same conditions. These results demonstrate that the protein coat of poliovirus is impermeable to cesium ions. However, if the RNA of the virus is exposed to cesium ions while the virus is being assembled, the poliovirus binds approximately the same number of cesium ions as human rhinovirus. This similarity in cesium binding suggests that poliovirus and human rhinovirus may have the same fraction of their RNA neutralized by coat protein.