Lead acetate in drinking water is toxic to hippocampal tissue Measuring relative protein changes using tissue array detection

BACKGROUND： Lead can cause structural changes in the hippocampus, followed by damage to learning and memory functions, but its specific mechanisms are not yet clear. OBJECTIVE： To observe long-term toxicity of high-dose lead in drinking water on hippocampal tissue in rats, and analyze the potential association of oxidative damage, cell apoptosis, and pathology. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Center for Medical Experiment, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from May 2007 to October 2008. MATERIALS; Rabbit anti Bcl-2, Bax, and inducible nitric oxide synthase （iNOS） polyclonal antibodies were purchased from Santa Cruz Biotechnology, USA. An streptavidin-peroxidase immunohistochemistry kit and concentrated DAB kit were purchased from Beijing Zhongshan Biotechnology Company Limited, China. Crystal violet was purchased from Sigma, USA. METHODS： A total of 72 Wistar rats, aged 3 months, were randomly divided into control, low-, middle-, and high-dose lead groups, with 18 rats per group. Animal models were established through free drinking water contaminated by Pb2＋ for 1, 3, and 6 months, respectively. MAIN OUTCOME MEASURES： The general toxicity of lead was dynamically observed; the levels of Pb2＋ in the blood and brain tissue homogenete were detected using atomic absorption method; pathological changes were observed using hematoxylin-eosin staining and tigroid body staining; the protein expression levels of Bcl-2, Bax, and iNOS were dynamically observed using streptavidin-peroxidase immunohistochemistry of the hippocampus. RESULTS： Lead exposure reduced autonomic activities, produced a slumped appearance, slow responses, and lusterless fur, especially in the high-dose group. The amount of ingestion and hydroposia showed a decreasing trend, especially in middle- and high-dose groups. Lead levels in whole blood and brain homogenate were higher than controls （P 〈 0.01）. Lead caused degeneration of hippocampal neurons and pyknosis, with fewer tigroid bodies, especially in high-dose lead group. Bcl-2 expression decreased with increasing lead dose （P 〈 0.01）, whereas lead dose-dependently increased Bax levels （P 〈 0.01） and iNOS levels （P 〈 0.05）. CONCLUSION： High levels of Pb^2＋ may disrupt hippocampal structure by passing through the blood brain barrier. Oxidative damage and apoptosis may be a toxicity mechanism of Pb^2＋ on the hippocampus.

Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63is amplified in early carcinogenesis but down-regulated as disease progressed

AIM: By using comparative genomic hybridization, gain of 3q was found in 45-86% cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome 3q25.3-qter is the minimal common region with several oncogenes found within this region. However, amplification patterns of these genes in EC-SCC have never been reported. The possible association of copy number changes of these genes with pathologic characteristics is still not clear. METHODS: Real-time quantitative PCR (Q-PCR) was performed to analyze the copy number changes of 13 candidate genes within this region in 60 primary tumors of EC-SCC, and possible association of copy number changes with pathologic characteristics was analyzed by statistics. Immunohistochemistry (IHC) study was also performed on another set of 111 primary tumors of EC-SCC to verify the association between TP63 expression change and lymph node metastasis status. RESULTS: The average copy numbers (±SE) per haploid genome of individual genes in 60 samples were (from centromere to telomere): SSR3: 4.19 (±0.69); CCNL1: 5.24 (±0.67); SMC4L1: 2.01 (±0.16); EVI1: 2.02 (±0.12); hTERC. 5.28 (±0.54); SKIL 2.71 (±0.14); EIF5A2. 1.95 (±0.12); ECT2: 9.18 (±1.68); PIK3CA: 8.13 (±1.17); EIF4G1: 1.07 (±0.05); 557: 3.07 (±0.25); TP63: 2.51 (±0.22); TFRC. 2.42 (±0.19). Four clusters of amplification were found: SSR3 and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA at 3q26.31-q26.32; and 55T, TP63 and TFRC at 3q27.3-q29. Patients with lymph node metastasis had significantly lower copy number of TP63 in the primary tumor than those without lymph node metastasis. IHC study on tissue arrays also showed that patients with lymph node metastasis have significantly lower TP63 staining score in the primary tumor than those without lymph node metastasis. CONCLUSION: This study showed that different amplification patterns were seen among different genes within 3q25.3-qter in EC-SCC, and several novel candidate oncogenes (SSR3, SMC4L1, ECT2, and SST) were identified. TP63 is amplified in early stage of EC-SCC carcinogenesis but down-regulated in advanced stage of disease.

Proteome analysis and tissue array for profiling protein markers associated with type B thymoma subclassification

Background The prognostic relevance of World Health Organization （WHO） subtypes within type B thymomas is still controversial. Understanding of the molecular characteristics of the different histologic types of thymomas will provide meaningful information for diagnosis and therapeutic management in type B thymoma. Methods Proteins extracted from twelve type B thymoma tissue specimens （six type B1 and six type B2） were analyzed by two-dimensional electrophoresis （2-DE） coupled with MALDI-TOF-MS. Differentially expressed proteins were then assayed in sixty-nine type B thymoma tissues （including B1, B2 and B3） by tissue array analysis with immunohistochemistry staining. The relationship of their expression with clinicopathological parameters, such as tumor stage or WHO classification, was estimated by Spearman＇s Rank Correlation Test. Results Sixteen differentially expressed proteins between type B1 and B2 thymoma tissues were identified. The differential levels of ezrin and glutathione S-transferase pi （GSTP1） were validated using immunohistochemistry staining. A statistically significant difference was observed in the positive rate of ezrin expression between type B1 thymoma and type B3 thymoma （Z= -2.963, P 〈0.01）. Ezrin showed a tendency to be expressed in higher classification tumors from type B1 to B3. A statistical analysis demonstrated that type B2 and B3 tumors had significantly higher positive expression of GSTP1 than the B1 group （type B2 vs. BI： Z= -2.582, P 〈0.01; type B3 vs. BI： Z= -4.012, P 〈0.001）. The results also showed a strong correlation between GSTP1 and WHO type staging of B1 to B3 tumors （Spearman＇s correlation coefficient： 0.633, P 〈0.001）. Statistical analysis showed that there was close correlation between GSTP1 and ezrin expression with the clinical stage （Spearman＇s correlation coefficients, ezrin： 0.481, P 〈0.05; GSTPI： 0.484, P 〈0.01）. Conclusions Differentially expressed proteins between type B1 and B2 thymoma tissues were analyzed by comparative proteomic analysis. The techniques of proteomic analysis and tissue array provide a potential tool for screening of key molecules in type B thymoma histological sub-classifications. The statistical analysis of ezrin and GSTP1 expression by immunohistochemistry, especially GSTP1, may be a useful approach for type B thymoma classification.