To determine the effects of repeated ethanol exposure on SH-SY5Y neuroblastoma cells, we used a 3-week chronic intermittent exposure to ethanol (CIE) paradigm. Cells were exposed to 40 mM ethanol (or control) for 4 h per day on 4 successive days, followed by 3 days without ethanol, for 3 weeks. The repeated exposures were akin to three weeks of binge drinking on 4 successive days with 3 abstinent days in between. The 40 mM ethanol dose is equivalent to a blood alcohol concentration of 0.18%, which can be surpassed in a bout of high intensity drinking. On the last week, after 3 days of exposure, half of the ethanol-treated cells were withdrawn for 24 h while the other half received the fourth 4 h exposure (Supplementary Figure 1), to determine how much recovery occurred in that time. The only ADH detectably expressed in these cells is ADH5, which has a very high Km for ethanol and is not likely to carry out significant metabolism; mRNA levels are the same under all 3 conditions and very similar to the levels seen in various brain regions in GTEx.
