5.0 statistical package (Abacus Concepts, Inc., CA, USA). Comparisons of the observed values for statistically significant differences were performed using the Student's t-test, with the aid of the StatView ver. To detect apoptotic cells in sections of fixed, paraffin-embedded tissue, the TUNEL method was applied using an ApopTag Peroxidase kit for in situ apoptosis detection according to the manufacturer's instructions (Intergen Co., NY, USA). Analysis of ~5,000 hepatocytes in each sample was performed, and BrdU labeling indices for rat liver were calculated as percent- ages of positively stained cells among ~1,000 hepatocytes. After deparaffinization, H 2 O 2 pre-treatment and microwaving, liver sections were treated sequentially with normal horse serum, anti-mouse BrdU antibody (Dako Japan Co., Ltd., Tokyo, Japan 1:1000) at 4 ̊C overnight, horse anti-mouse IgG (1:400) for 30 min and the avidin-biotin peroxidase complex reagent for 30 min. At autopsy, livers were excised and 3-μm slices were cut with a razor blade and fixed in 10% buffered formalin solution for subsequent immunohistochemical iden- tification of BrdU-positive cells. injection of BrdU (100 mg/ kg body weight). Twenty rats each were sacrificed for BrdU and apoptosis measurement in the former case 1 h after receiving an i.p. They were fed an MeIQx diet at doses of 0, 1, 10 and 100 ppm continuously for 2 weeks. Forty rats were used in the experiment for the immunohistochemical demonstration of BrdU labeling and apoptosis. Rat liver genomic DNA was prepared with a QIAamp DNA Mini kit according to the manufacturer's instructions (Qiagen, Hilden, Germany), and 1 μg of genomic DNA was amplified by high fidelity PCR using KOD-Plus-DNA polymerase. After 1-2 weeks of MeIQx administration, all rats in each group were sacrificed under anesthesia with diethyl ether, and the livers were quickly removed and frozen in liquid nitrogen for molecular assessment. (Tokyo, Japan), and the concentration in each diet was confirmed by HPLC.
The MeIQx diets were prepared by Oriental Yeast Co. They received MeIQx at doses of 0 (group 1, control), 0.001 (group 2), 0.01 (group 3), 0.1 (group 4), 1 (group 5), 10 (group 6) and 100 ppm (group 7) in a powdered basal diet for 1 or 2 weeks, continuously.
Seventy rats were employed in this experiment for measurement of H- ras mutation frequency. The animals were housed in an animal facility room maintained at a 12-h light/dark cycle at a constant temperature of 25☑ ̊C and a relative humidity of 55±5% and observed daily. A total of 110 male 21-day-old F344 rats were obtained from Charles River Japan, Inc. MeIQX (purity 99.9%) was purchased from the Nard Institute (Nishinomiya, Japan). The amounts of 33 P-labeled primers were measured using a bio-imaging analyzer (BAS 2500 Fuji Photo Film Co., Tokyo, Japan). Gels were run at 1,000 V constant voltage for 3.5 h. The reaction products were concentrated with a vacuum concentrator down to a 1/10 volume, mixed with 5 μl of deionized formamide containing 0.05% BPB and 0.05% XC, heated at 95 ̊C for 2 min, placed on ice and loaded on a 12% (W/V) denaturing polyacrylamide gel containing 7 M urea. TCEL reactions were performed in a GeneAmp 9600 thermal cycler with the following protocol: 50 cycles for 15 sec at 95 ̊C and 15 sec at 55 ̊C. Non-labeled dide- oxynucleotides were added to prevent a wrong addition of labeled dideoxynucleotides at the 3'-end of a detection primer. The PCR product (100 fmol) was used as a template for TCEL reactions in a total volume of 10 μl containing 26 mM Tris-HCl (pH 9.5), 6.51 mM MgCl 2, 2 pmol of the primer for mutation detec- tion (RHrasc12bA: 5'-GGGCACTCTTTCCCACGCCT-3'), 30 pmol ddCTP, 70 fmol ddATP, 70 fmol ddGTP, 70 fmol ddTTP, 30 fmol ddCTP or ddATP (1,500 Ci/ mmol) (Amersham Biosciences), 0.5 units Thermosequenase (Amersham Biosciences) and 10% DMSO. Thermosequenase Cycle End Labeling (TCEL). Biosciences Co., NJ, USA), incubated with 10 units exonu- clease I and 2 units shrimp alkaline phosphatase (Amersham Biosciences Co.) at 37 ̊C for 30 min, and then purified using a GFX PCR DNA Purification kit (Amersham Biosciences).
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