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MATERIALS AND METHODS Female ICR mice (Harlan, Houston, Tex.) weighing 18-23 g were ran- domly divided into 3 groups of 20 and given powdered feed (Purina Lab Chow 5002) supplemented with 0, 0.125, and 0.5% EQ HCI, respectively. Mice were killed by cervical dislocation, 4/group, after 2, 4, 6, and 10 wk of feeding; the liver, kidney, lung, and brain tissues were rapidly excised and weighed. Approximately 200 mg of each tissue sample with the ex- ception of the lung tissue was homogenized in 10 volumes (w/v) of ice- cold acetonitrile-water (7 : 3, v/v) with a Polytron (Brinckmann), then cen- trifuged for 10 min at 5000 x g. The clear supernatant was transferred into a screw-capped glass vial, centrifuged, then stored in a freezer (-20¡ãC) for 2-3 h or until the two layers separated. Due to the small size of the lung tissue, most of it was homogenized in 20 volumes of solvent. The clear upper layers, approximately 60% of the total volume, contained most (>90%) of the EQ; therefore, only the upper layers were analyzed by HPLC detected by the fluoromonitor. In order to avoid possible diur- nal fluctuations, mice were killed at 8:00-10:00. Approximately 200 mg of another liver sample from each mouse was homogenized in 10 volumes (w/v) of ice-cold 0.25 M perchloric acid and centrifuged for 10 min at 5000 x g for the determination of GSH level. Three groups of mice, 4/group, were killed after 14 wk of feeding, 1/group/d from each feeding group, and the mitochondria were isolated from the liver, saving 2 samples of approximately 200 mg each to deter- mine the EQ residue and GSH levels. The liver mitochondria were iso- lated in 2.5 M sucrose buffer by differential centrifugaron Gohnson and Lardy, 1967) and the protein concentration was determined by the biuret method (Gornall et al., 1949). |
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