Pyrazole

The reagents used were of the highest commercial quality available. Pyrazole, β-naphthoflavone (BNF), α-naphthoflavone (ANF), cadmium chloride, methanol, clotrimazole, orphenadrine, p-nitrophenol (PNP), chlorzoxazone, quinine, 7-methoxyresorufin (MRF), 7-hydroxycoumarin, 7-benzyloxyquinoline (BQ), 7-pentoxyresorufin (PRF), resorufin, phenobarbital, and sulfaphenazole were purchased from Sigma-Aldrich (St. Louis, MO). The P450 substrates and inhibitors, 3-cyano-7-ethoxycoumarin (CEC); 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 7-hydroxyquinoline, 3-cyano-7-hydroxycoumarin (CHC), and 3-[2-(diethylamino)ethyl]-7-hydroxy-4-methylcoumarin hydrochloride (AHMC) were purchased from BD Gentest (Woburn, MA).

Primary antibodies against CPR were obtained from Stressgen (Ann Arbor, MI). Anti-CYP2B4 and anti-CYP2E1 were purchased from Oxford Biomedical Research (Oxford, MI). The antibody for CYP1A2 was provided by Dr. Kristopher Krausz from NIH. Amplex Red cholesterol assay kit was purchased from Invitrogen (Eugene, OR). Bicinchoninic acid (BCA) protein assay kit and phenobarbital were purchased from Fisher Scientific (Rockford, IL). Methyl-β-cyclodextrin, Brij 98, pyrazole and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Mice were exposed to continuous ethanol vapor or air for 72 h, using a standardized method in the Finn and Crabbe laboratories that involved daily injections of the alcohol dehydrogenase inhibitor pyrazole (68.1 mg/kg; Sigma Chemical Company, St. Louis, MO) to help stabilize blood ethanol concentrations (BECs; see Finn and Crabbe, 1999 (link); Tanchuck et al., 2009 (link); Terdal and Crabbe, 1994 (link) for details). Controls also received daily pyrazole injections. Tail blood samples (20 μL) were collected from all the ethanol-exposed mice each day and at 72 h to determine BEC during and upon completion of the ethanol vapor exposure, and samples were analyzed by headspace gas chromatography (Finn et al., 2007 (link)). Ethanol vapor concentrations were measured hourly via an autosampler, and they were adjusted daily to achieve BECs in the range of 1.5 – 2.0 mg/mL. Briefly, ethanol vapor concentrations started at 7.0 mg ethanol/L air for the males and at 7.3 mg/L for the females. Ethanol vapor concentrations were increased by approximately 0.15 mg/L each day in the male and female WSP-1 mice, whereas they were increased approximately 0.5 mg/L in the male and female D2 mice. At 72 h, all animals were removed from the inhalation chambers, placed back into their home cages, and taken to a procedure room for behavioral testing.

Mice were subjected to chronic intermittent ethanol (CIE) exposure to induce chronic tolerance to ethanol-induced sedation and hypothermia, as well as to exacerbate withdrawal-induced hyperexcitability and make HICs more readily detectable (Becker et al., 1997 (link)) since C57Bl/6J mice show limited HICs (Metten and Crabbe, 1994 (link)). CIE was conducted in inhalation chambers (La Jolla Alcohol Research Inc., La Jolla, CA), as previously described (Gorini et al., 2013 (link); Kreifeldt et al., 2013 (link)). Before each 16-h vapor exposure period, mice received a loading dose of ethanol (1.5 g/kg, i.p.) and pyrazole (68.1 mg/kg, Sigma, St Louis, MO). Tail vein blood samples were collected periodically at the end of intoxication periods for BAL determination. Ethanol vapor concentration was adjusted so as to yield BALs approximating 200 mg/dl. Mice were exposed to a first series of 4 cycles of intoxication/withdrawal (16 h ethanol vapor/8 h air). The mice were then returned to their home cages for 4–6 weeks before being exposed to a second series of 3 cycles of intoxication/withdrawal. Approximately 26 h into withdrawal from the vapor chambers, mice were i.p. injected with 4 g/kg ethanol and were tested for sedation and hypothermia, and/or HIC.