Baseline demographic data and medical comorbidities were collected on all subjects

mption is proportional to the GR activity and was measured at 340 nm. Catalase activity was determined in a reaction of H2O2 with ammonium-molibdate, which resulted in the formation of yellow complex, and its absorbance was measured at 405 nm. AchE activity was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19757875 measured spectrophotometrically at 412 nm, based on the formation of stable yellow complex in a reaction of DTNB with acetylcholine iodide degradation product. All enzyme activities are expressed as units per milligram of protein. Western blot analysis For Western blot analysis dorsolateral frontal cortex and hippocampus were dissected and homogenized in lysis buffer, protease inhibitor cocktail, 200 mM sodium orthovanadate and 1 M NaF ) on ice for 30 min, followed by centrifugation, and the supernatants were collected as the cell lysates. Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard. Equal amounts of protein from each sample were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis on 10% and 12% gels and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 hour in 5% nonfat dry milk in Tris-buffered saline/0.1% Tween 20. The following primary antibodies were used in this study: polyclonal goat anti-SOD1 and polyclonal goat anti-SOD2. After incubation with primary antibodies the membranes were incubated with the horseradish peroxidase labeled secondary anti-goat antibody in TBST for 1 hour at room temperature. Five HC-067047 manufacturer subsequent washes with 0.1% TBST were performed between each step. All membranes were stripped and re-probed with anti-actin antibodies to ensure that all wells were equally loaded. The signal was detected by enhanced chemiluminescence and subsequent exposure on an X-ray film. Western blots were scanned and densitometric analysis was performed using ImageQuant 5.2. Statistical analysis and drugs All results are expressed as meansSD. The significance of the difference was estimated by using analysis of variance with Fisher’s post hoc test, while the significance of the 4 / 14 Finasteride Has Regional Effects in the Brain Fig 1. The effect of finasteride and thioacetamide on plasma ammonia concentration. Daily doses of FIN and TAA were administered intraperitoneally PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755912 in three subsequent days and in FIN+TAA group FIN was administered 2 h before every dose of TAA. Blood samples were collected from the right side of the heart 24 h after administration of the last dose of TAA. The significance of the difference was estimated by ANOVA with Fisher’s post hoc test. doi:10.1371/journal.pone.0134434.g001 correlation between AchE activity and MDA level was determined by Pearson’s correlation test. The difference and correlation were considered significant if p<0.05. For statistical analysis computer software SPSS15.0 was used. TAA, FIN and 2-hydroxypropyl--cyclodextrin were products of Sigma-Aldrich Chemical Co., U.S.A. Results Blood ammonia concentration was significantly higher in TAA and FIN+TAA group by comparison with control. No significant difference in ammonia concentration was evident between TAA and FIN+TAA group. FIN alone also did not cause any change in ammonia concentration. MDA level was significantly higher in the cortex, the hippocampus and caudate nucleus in TAA vs. control group 24 h after the administration of the last dose of TAA. In contrast, TAA did not induce a significant change in MDA level in the thalamus when compared with control. While F