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Objective: To screen the biomarkers in the exhaled breath of mice exposed to benzene by using exhaled breath online analysis system. Methods: Thirty 8-week-old male C57BL/6 mice were randomly divided into six groups (0, 3, 32, 324, 648, and 1 296 mg/m 3 ) and treated with benzene vapour for 28 days. At the end of the exposure, the peripheral blood cell counts and blood glutathione (GSH) were detected. The content of malondialdehyde (MDA) in HL60 cells treated by mice plasma was examined. Exhaled breath data from mice were collected by Secondary electrospray ionization source high resolution mass spectrometry (SESI-HRMS). Targeted analysis underlying benzene metabolites and oxidative stress metabolites was performed to screen the biomarkers in exhaled breath. Results: After benzene exposure, the number of peripheral blood cells was decreased in different degrees, particularly in the white blood cells (WBC) number. The WBC in 32 and 324 mg/m 3 groups was declined by 27.76% and 52.87%, respectively compared to that in control group ( P <0.05). Meanwhile, compared with the control group, the GSH content of peripheral blood cells from 324 mg/m 3 group decreased by 13.16% ( P <0.05). In addition, MDA content was increased by 18.11% in HL60 cells treated with plasma from 324 mg/m 3 group mice ( P <0.05). The phenol, hydroquinone/catechol, benzenetriol and trans , trans -Muconic acid ( t,t -MA) in the exhaled gas of mice could be used as biomarkers for benzene exposure ( R 2 >0.8, P <0.001). The peak intensity of five small molecular metabolites related to oxidative stress (ω-carboxylic fatty acid C 5 H 10 O 3 , ω-carboxylic fatty acid C 6 H 12 O 3 , glutamate, cysteine and MDA) increased with the increase of benzene concentration ( P <0.05), which was negatively correlated with WBC decline ( P <0.001), suggesting that these molecules mignt be used as biomarkers of benzene-induced toxicity. Conclusions: Phenol, hydroquinone/catechol, benzenetriol and trans , trans -Muconic acid ( t,t -MA) in exhaled breath of mice could be used as biomarkers for benzene exposure; ω-carboxylic fatty acid C 5 H 10 O 3 , ω-carboxylic fatty acid C 6 H 12 O 3 , glutamate, cysteine and MDA might be used as markers of benzene-induced toxicity.

Objective: To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study. Methods: Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD(50)) was calculated in linear regression. Results: Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD(50) of lead acetate was 2 025.0 μmol/L, the LD(50) of cadmium chloride was 36.6 μmol/L, and the LD(50) of sodium arsenite was 33.2 μmol/L. Conclusion: The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Objective: To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD). Methods: Wide type (WT) mice and CCM3(+)/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3(+/-) mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ(1-42) in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR. Results: The levels of blood lead WT (216.07±84.16) and CCM3(+/-) (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3(+/-) (11.79±8.20) μg/L, the difference was statistically significant ( t= 4.18, P= 0.006; t= 3.79, P= 0.016). The levels of brain lead WT (1.78±0.69) and CCM3(+/-) (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3(+/-) (0.97±0.64) μg/L, the difference was statistically significant ( t= 3.67, P= 0.018; t= 3.88, P= 0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ(1)-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3(+/-) mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3(+/-): 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3(+/-): 0.59±0.01) was decreased with statistical significance ( F =199.27, P< 0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3(+/-): 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3(+/-):2.12±0.18), the difference was statistically significant ( F= 288.29, P< 0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3(+)/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t= 26.90, P< 0.001). Conclusion: The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3(+/-) mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.

Objective: To evaluate the results of the active follow-up among registered cancer patients in 2002 - 2005 in urban areas of Beijing., Methods: A number of 63 997 cancer patients diagnosed during 2002 - 2005 were selected from the surveillance database of Beijing Office for Cancer Prevention and Control. By matching the identity information of the patients with the death surveillance database built by the vital statistic department in Beijing, 29 223 patients were confirmed to be alive.1149 cases were removed from the study due to lack of exact key variables, such as address and telephone numbers. 28 074 patients were, at last, included in the active follow-up study. The investigators and the inspectors, who accepted standard training program, investigated each patient's status of census register and survival condition by phone calling, household interview and visits at local police station or residential committee. The loss ratio of follow-up and the constituent ratio of the withdrawal reasons were calculated., Results: Among the 28 074 patients selected in active follow-up, 21 696 patients were followed successfully; 1453 of whom didn't have the census register of Beijing, which accounted for 6.70%. Out of the other 20 243 Beijing residents, 4715 patients (23.29%) were already dead and 84.22% (3971/4715) of them replenished the failure to report by passive follow-up. Among all the 4715 dead cases, 4405 (93.43%) patients were died from cancer. The follow-up study helped to replenish the vital statistics in different districts, the ratio ranged from 4.87% and 8.85%. 6378 patients were withdrawn from the study. The loss ratio was 22.72% (6378/28 074), and the total loss ratio was 12.03% ((6378 + 1149)/(63 997 - 1453)). Of these withdrawal cases, 3041 (47.68%) were lost to follow-up in that the investigators can't find the patients or the relatives of the patients according to the registered phone number or address information. The other reasons included: the patients removed to other areas (1199 cases, 18.80%), the patients and their family members were temporarily not at home (127 cases, 1.99%), the patients and their family members rejected to answer the interview (292 cases, 4.58%), and other reasons (1719 cases, 26.95%)., Conclusion: The method of active follow-up towards registered cancer patients can replenish the missing information which could not be collected from passive follow-up procedure; and therefore effectively improve the quality of data in cancer registration.

Objective: To explore potential epigenetic biomarkers for toxic effects, tumor-related chemical prevention and biological monitor by a genome-wide screening for differential DNA methylation during human cell malignant transformation in vitro., Methods: The two in vitro cell transformation models included B(a)P-induced human bronchial epithelial cell introduced by H-Ras (HBER) cell transformation and simian vacuolating virus 40 small T antigen induced (SV40 ST-induced) HBER cell transformation. Methylated genes were collected by methylated DNA immunoprecipitation and whole genome amplification (MeDIP-WGA) at three time points during cell transformation which represented different transformation stage. Then, CpG island microarray was used to screen differentially methylated genes. The mRNA levels of hypermethylated genes were also observed by RT-PCR., Results: The CpG island microarray showed that the number of hypermethylated genes in HBER, HBERNT, HBERT cells were 733, 661 and 738 respectively.83 genes were hypermethylated in pre-transformed cell and transformed cell. Moreover, 25 of 83 genes were also hypermethylated in SV40 ST-transformed cell (HBERST). We further confirmed that the mRNA expression of six of these 25 genes, namely family with sequence similarity 178, member A (FAM178A), retinoic acid receptor responder (tazarotene induced) (RARRES1), ubiquitin specific peptidase 28 (USP28), Scm-like with four mbt domains 2 (SFMBT2), family with sequence similarity 59, member A (FAM59A) and nuclear receptor subfamily 4, group A, member 3 (NR4A3) were suppressed during B(a)P-induced transformation., Conclusion: The abnormal hypermethylation of specific genes was a common event in the two kinds of human cell transformation models, which shed light on the study for chemical exposure monitor and tumor-related epigenetic biomarkers.

Objective: To describe the incidence trends and pathological characteristics of lung cancer in urban Beijing, China., Methods: A total of 32 845 medical records of the residents diagnosed as lung cancer in urban Beijing from 1998 to 2007 were retrieved through the cancer registry system of Beijing Cancer Registry. Crude incidence rate, age-specific incidence rate, adjusted incidence rate by world standardized population, annual percentage change (APC) and histological categorized incidence rate by world standardized population were calculated in order to compare the differences of the incidence trends in different time periods, or among different gender and age groups., Results: A total of 32 845 newly diagnosed lung cancer patients between 1998 and 2007 were included in our study. The crude incidence rate was 47.81/100 000 (32 845/68 704 429), increasing by 38.80% from 39.30/100 000 in 1998 to 54.55/100 000 in 2007 with APC at 3.35% in urban Beijing (Z = 9.984, P < 0.001). While it changed to 28.95/100 000 with an APC at 0.27% (Z = 0.846, P = 0.422) when adjusted by world standardized population. For male, the crude incidence rate was 58.28/100 000 (20 342/34 906 580, adjusted rate at 37.03/100 000, APC at 0.38%, Z = 1.008, P = 0.343); while for female, the crude incidence rate was 36.99/100 000 (12 503/33 797 849, adjusted rate at 21.48/100 000, APC at 0.14%, Z = 0.431, P = 0.678). 17 920 lung cancer patients being diagnosed according to histological evidence, accounted for 54.56%. The respective proportion of the patients with histological diagnosis was 43.14% (1095/2538) in 1998 and 65.55% (2641/4029) in 2007, with a 51.95% increase (χ(2) = 859.152, P < 0.001) in decade. In terms of subtypes of lung cancer, the proportion of squamous cell carcinoma decreased annually, from 30.41% (333/1095) in 1998 to 24.16% (638/2641) in 2007; while the proportion of adenocarcinoma increased from 42.83% (469/1095) to 46.80% (1236/2641). As a result, the squamous cell carcinoma to adenocarcinoma ratio declined from 0.71 (333/469) to 0.52 (638/1236) (χ(2) = 50.214, P < 0.001). For women, the ratio declined more significantly and the proportion of the squamous cell carcinoma and adenocarcinoma were 14.77% (925/6262) and 60.83% (3809/6262), respectively in the period between 1998 and 2007., Conclusion: No significant change was found in the incidence trend of lung cancer after the incidence rate adjusted by world standard population, but the proportion of the subtypes of lung cancer categorized by histological evaluation changed apparently.

Objective: To screen and identify differential serum proteins which might be involved in dermatitis medicamentosa-like of trichloroethylene (DMLT)., Methods: Three groups of sera were collected from population exposed to trichloroethylene (TCE) (group I), patients suffering from DMLT (group II), and the healed cases (group III). After removing albumin and IgG in the three pools of sera, a comparative proteomic analysis was carried out. The images were analyzed using ImageMaster Platinum 2D 5.0 to screen the differentially expressed proteins. The protein spots were then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides for further identification., Results: The depletion of albumin and IgG greatly increased the number of protein spots to 300 ± 12.Five differential spots were identified, which were complement component C4b, apolipoprotein A-I, apolipoprotein C-III apolipoprotein C-II and transthyretin. Compared with group I, the expression levels of complement component C4b in group III and apolipoprotein C-II in group II were up-regulated (1.352 88-fold, 1.512 14-fold, respectively); compared with group I, the expression levels of apolipoprotein A-I, apolipoprotein C-III and transthyretin in group II were down-regulated (1.601 17-fold, 1.034 49-fold, 1.313 35-fold, respectively)., Conclusion: The findings of this study show that most of the identified differential proteins are closely related to immunity and liver dysfunction, which provides some evidence on elucidating the mechanisms and screening of biomarkers of TCE intoxication.

Objective: To screen and identify differentially expressed proteins between adult female and male worms of Schistosoma japonicum(S.japonicum)., Methods: Two rabbits infected with the cercaria were perfused with saline in carotid, and approximately two hundred adult female and two hundred male worms of S.japonicum were collected. Approximately 300 microg soluble and hydrophobic proteins of adult female and male worms of S.japonicum were extracted and then the proteins were separated by two-dimensional gel electrophoresis respectively. The analysis using ImageMaster Platinum 2D 5.0 resulted in differentially expressed proteins between adult female and male worms, which were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides., Results: There were (255 +/- 10) and (224 +/- 12) spots detected for soluble proteins and (200 +/- 11) and (132 +/- 8) spots for hydrophobic proteins from adult female and male worms respectively. Six differential proteins were identified, five up-regulated proteins in female worms were thioredoxin, putative ferritin-1 heavy chain, chain B in solution structure of the human ubiquitin-conjugating-enzyme-like protein Mms2-Ubiquitin Complex, heat shock protein 10, cytoplasmic fatty acid binding protein variant H; while only one up-regulated proteins in male worms was identified as 48 kDa histamine receptor subunit peptide 4., Conclusion: Several differentially expressed proteins between female and male worms of S. japonicum were recognized through screening and identifying differential proteins between female and male worms of S.japonicum.