330 Brain Research, 517 (1990) 330-332 Elsevier BRES 24092 Adaptation of in vitro rat brain protein synthesis to long-term ingestion of n-butanol R. Mufioz, J.M. Ferreras, R. Iglesias, M.J. Merino and T. Girb6s Departamento de Bioquimica, Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de Valladolid, Valladolid (Spain) (Accepted 30 January 1990) Key words: Protein synthesis; Adaptation; Brain; n-Butanol; Alkanol; Rat Long-term treatment of rats with n-butanol leads to a change in in vitro brain protein synthesis which increases the resistance of this process to either ethanol or isopropanol. The change seems to be related to ribosomal events since the synthesis of aminoacyl-tRNA was not affected in the same conditions. The ability to respond to long-time environmental changes by adaptation of physiological and metabolic processes is a crucial property for living systems. It has been known for many years that several lower and higher eukaryotes exhibit a phenomenon of adaptation to long-term contact with toxic drugs such as cyclohex- imide 2'3"6. Analysis of the biochemical changes induced by long-term exposure of Tetrahymena thermophila to this antibiotic, has indicated that the main factor respon- sible for the adaptation is a change in the machinery of protein synthesis 7. Since this antibiotic acts by specifically binding to ribosomal particles, we approached the ques- tion of whether the long-term exposure of a higher eukaryote, such as the rat, to a non-specific inhibitor like n-butanol may promote similar changes in the rat brain protein synthesis machinery equivalent to the adaptation observed in lower eukaryotes. Male Wistar albino rats (weighing 200-300 g) were fed ad libitum with the standard S-10 diet from Sanders (Madrid, Spain). Chronic exposure to n-butanol was carried out by giving the rats a solution containing the alcohol as the only available beverage. The schedule was: n-butanol 1% (v/v) for the first week; n-butanol 2% (v/v) for the second week and 4% (v/v) until their sacrifice. The net weight gain of n-butanol-treated animals, ex- tended for a period of 100 days, was 46% of that of control animals. The complete treatment took over four months and no rats died as a consequence of it. Under pentotal anaesthesia, the rats were killed, the brains rapidly removed and thereafter homogenized as described previously for rat liver 1°. The buffer solution used contained 100 mM KCi, 50 mM Tris-HCI (pH 7.6), 10 mM MgC12 and 5 mM dithiothreitol. The S-30 supernatant was obtained as follows: the homogenized tissue was centrifuged at 760 g for 5 min at 4 °C. The pellet was discarded and the supernatant was centrifuged again, this time at 31,000 g for 15 min at 4 °C. The clear supernatant was removed, avoiding contamination with the upper fat pad, and chromatographed through a Sephadex G-25 column (8 × 2.5 cm) equilibrated with the same solution as that used for disintegrating the tissue. The void volume containing UV-absorbing mate- rial was collected and stored in small fractions under liquid nitrogen until use. Polyuridylic acid-directed polyphenylalanine synthesis was carried out in 50/d reaction mixtures containing: 50 mM KC1, 100 mM NH4C1 , 20 mM Tris-HCl (pH 7.6), 5 mM dithiothreitol, 2 mM ATP, 1 mM GTP, 5 mM phosphoenolpyruvate, 2 ~g pyruvate kinase, 20 /~g polyuridylic acid, 52 nM L-[aH]phenylalanine (spec. act. 116.3 Ci/mmol), variable concentrations of MgCl 2 and alcohols as stated in the figures, and an optimized amount of the S-30 supernatant. Incubation was started with the S-30 supernatant and was continued for 60 min at 37 °C to ensure saturation of polymerization in both cell-free extracts; it was then stopped by adding 2 ml of cold 5% (v/v) trichloroacetic acid to each sample. The hot acid-insoluble radioactivity was determined as de- scribed previously4. Phenylalanine-tRNA synthesis was determined as the cold acid-insoluble radioactivity minus the hot acid-insoluble radioactivity. Protein content was analyzed by the method of Lowry 9. Fig. 1 shows the sensitivity of polyphenylalanine Correspondence: T. Girb6s, Departamento de Bioquimica, Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de Valladolid, 47005 Valladolid, Spain. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)