OBJECTIVE: Build and validate PBTK/TD with growth function of the Juvenile rat following the oral exposure to chlorpyrifos(CPF). METHODS: Model construction and validation: to determine the model structure and parameters;establish the growth function of body weight for rats,and write the differential equations as well as the program. Using experimental data to optimize the model parameters,make Sensitivity Analysis and model validation. Animal experiment: 21 days-old female SD rats were randomly divided into 1 control group and 3 treated groups (12.5,25.0 and 50.0 mg/kg) according to the weight. Serum and cerebral cortex were collected after single gavage at 1h,3 h,6 h,12 h and 24 h,then to determine the concentration of CPF and 3,5,6-trichloropyridinol(TCP) in serum,the activity of acetylcholinesterase (AChE) in cerebral cortex. RESULTS: Experimental data showed that the concentrations of serum CPF and TCP increased to the peak level at the 3rd hour,then the concentrations decreased. With time,the activity of AChE in serum and cerebral cortex decreased first and then rose slowly. The minimum activity of serum AChE and cortex AChE was observed at 6th hour and 12th hour,respectively. The model predictions is consistent with the experimental results,while there were an overvalued or undervalued problem when using the model to make extrapolation. CONCLUSION: The model has reasonable structure and parameters,and can be used for extrapolation,especially in prediction of the individual average level,but the model still need further improvement.

OBJECTIVE: To investigate the developmental toxicity and underlying mechanism of chlormequat chloride by utilizing embryonic stem cell test in vitro. METHODS: Mouse embryonic stem cells and 3T3 cells were treated with different doses of chlormequat chloride,the effects on ES and 3T3 cell proliferation and ES cell differentiation were determined. A cascade of genes related to normal cardiac development was examined by real time PCR. RESULTS: Chlormequat chloride (1 000 μg/mL) could evidently increase the viability of mouse fibroblasts 3T3 cells and mouse embryonic stem cells. Chlormequat chloride could influence embryonic stem cell cardiac differentiation in a U shape manner. Compared to the control group,only 69.4% beating EBs was observed at 250 μg/mL concentration. Chlormequat chloride was able to cause effect on gene expression during cardiogenesis,especially Nkx2.5 and α-MHC. CONCLUSION: Chlormequat chloride could not be classified according to the prediction model recommended by ECVAM. Chlormequat chloride at 250 μg/mL concentration could retard embryonic stem cell differentiation into myocardial cell without causing any morphological malformations of 3T3 and ES-D3. Chlormequat chloride could alter the expression of crucial genes during the early stages of cardiogenesis,and these effects might result in adverse developmental outcomes.

OBJECTIVE: To characterize the sequence variations of Epstein-Barr virus(EBV)-encoded latent membrane protein 1(LMP1) gene in extranodal NK/T cell lymphoma,nasal type (ENKTL) and healthy donors in Shandong province and to explore the association of LMP1 variants with ENKTL. METHODS: Nested-PCR and DNA sequencing were used to analyze the gene sequences of LMP1 in isolates from 47 ENKTL tissues and 70 throat washing samples of healthy controls in Shandong province. The sequences of LMP1 were compared with the prototype B95-8 strain and classified according to the signature changes and the phylogenetic tree. The distribution of LMP1 variants was compared between ENKTL and healthy controls. RESULTS: A total of 117 isolates from 47 ENKTL and 70 healthy controls were successfully sequenced and three main subtypes of LMP1 gene,China 1,China 2 and Med-,were identified. The frequencies of China 1,China 2 and Med- in ENKTL were 85.1%,8.5% and 4.3%,respectively,and those in healthy controls were 65.7%,5.7% and 21.4%,respectively. Another one(2.1%) ENKTL isolate and 5(7.2%) healthy controls isolates were recombinant strains of different subtypes. The distribution of LMP1 subtypes between ENKTL and healthy controls was significantly different (P=0.003). The frequencies of XhoI(-) (loss of an XhoI site in N-terminus of LMP1) and del-LMP1 (30 bp deletion in C-terminus of LMP1) in ENKTL were 95.7% and 85.1%,respectively,which were significantly higher than those in healthy controls (74.3% and 67.1%,P=0.003 and 0.029,respectively). CONCLUSION: China 1,Xho I(-) and del-LMP1 were the major LMP1 subtypes in ENKTL and may associate with the susceptibility of ENKTL.

OBJECTIVE: To explore the effects of benzo[a] pyrene and DDT exposure alone or in combination on ALT,AST and γ-GT in mice. METHODS: Fifty healthy male Kunming mice were divided into 10 groups at random: blank control group (normal feed);solvent control group(equal volume of oil);1,10 mg/(kg·d) B[a]P groups;0.6,6 mg/(kg·d) DDT groups;1 mg/(kg·d) B[a]P+0.6 mg/(kg·d) DDT group;1 mg/(kg·d) B[a]P+6 mg/(kg·d) DDT group;10 mg/(kg·d) B[a]P+0.6 mg/(kg·d) DDT group and 10 mg/(kg·d) B[a]P+6 mg/(kg·d) DDT group. Exposure groups were treated with oil contained B[a]P and DDT once a day. After 31 days of intraperitoneal injection,eyeballs were extracted and ALT,AST and γ-GT were examined in blood. HE slices were made to confirm the liver damage. Using two factors and three levels factorial ANOVA design for data analysis. RESULTS: The levels of ALT and AST in all B[a]P or DDT alone exposure groups were significantly higher than that of the control groups (F=41.308,P=0.000;F=20.083,P=0.000),showing dose-effect relationships in both. But no interaction was observed in co-exposure groups(P=0.258,P=0.264). No significant changes in γ-GT were observed in all B[a]P or DDT alone exposure groups. Neither was there any interactive effect in co-exposure groups(P=0.816). This was in accordance with the observation in HE slice: hydropic degeneration,endolysis and nuclear enlargement in liver cell. CONCLUSION: Under this experimental condition,increasing levels of ALT and AST could be detected in B[a]P or DDT separate exposure groups. But neither could affect γ-GT level. No interactive effects of ALT,AST and γ-GT could be induced in co-exposure groups.

OBJECTIVE: To understand the potential functions,signaling pathways and expression correlation of copy number variants related genes in esophageal adenocarcinoma. METHODS: From three references applying genome-wide association studies to analyze large scale clinical sample of esophageal adenocarcinoma,120 copy number variants related genes were collected for bioinformatics analysis,including gene function enrichment,construction of protein-protein interaction network,clustering of expression profile and expression correlation. RESULTS: The result of functional enrichment based on Gene Ontology showed that the most significant function of copy number variants related genes in esophageal adenocarcinoma is the “regulation of transcription”. Pathway enrichment result found the pathway involved largest number of genes is “Pathway in cancer”. Protein-protein interaction network for copy number variants related genes contained 3 356 nodes and 63 474 edges. The network characterized scale-free. Using a public esophageal adenocarcinoma expression profile,the clustering of these genes can almost distinguish esophageal adenocarcinoma and normal esophageal tissue. These genes also showed significant expression correlation in esophageal adenocarcinoma. CONCLUSION: The bioinformatics analyses of copy number variants related genes in esophageal adenocarcinoma provided for a solid foundation for future experimental validation of these genes in the development of esophageal adenocarcinoma.

OBJECTIVE: To explore the correlation between KCNQ4,GJB2 genetic polymorphisms and noise-induced hearing loss. METHODS: 1:1 matched case-control design was used in this study. The genetic polymorphisms of KCNQ4 rs34287852 and GJB2 rs3751385 were detected by polymerase chain reaction and direct sequencing in 103 pairs of hearing loss workers and normal workers with same noise exposure. The relationship between target SNPs and noise induced hearing loss (NIHL) were analyzed. RESULTS: There is no statistical difference of the frequency of T,G alleles and genotypes in KCNQ4 rs34287852 between cases and controls. The frequency of C allele and CC mutation genotype in GJB2 rs3751385 was significantly higher in case group than that in control group. Workers carried CC homozygous mutations were at the 2.78 fold risk of developing hearing loss than individuals with TT wild homozygous type when they exposed to the occupational noise. CONCLUSION: There was the correlation between GJB2 rs3751385 mutation and NIHL and the CC genotype might be susceptible genotype of NIHL.

OBJECTIVE: To establish a high-throughput screening method of cell transformation assay for carcinogens detection using Bhas 42 cell line,and evaluate the potential carcinogenic risk of genistein. METHODS: We established Bhas 42 cell transformation assay and compared the effects of traditional foci formation as well as H₂O₂ treatment methods on data analysis. In the traditional foci formation method,cells were fixed and dyed on day 21 after plating,whereas in the H₂O₂ treatment method,cells were treated with H₂O₂ for 24 h before dyeing and the transformation ratio measured at D(450) on day 20. Then the transformation potential of genistein on Bhas 42 cells was determined by the H₂O₂ treatment method,after a dose range-finding assay was performed to estimate the concentrations of genistein to be used based on the growth rates of cells. RESULTS: Apparent increase in the number of wells with transformed foci were noted in the positive control (3-MCA) in the initiation assay and the positive control (TPA) in the promotion assay,compared with the negative control in the traditional foci formation method(P<0.01). Meanwhile,apparent increase in the number of wells with transformed foci and D(450) values were noted in the positive control (3-MCA) in the initiation assay and the positive control (TPA) in the promotion assay,compared with the negative control in the H₂O₂ treatment method(P<0.01). The effects of genistein on Bhas 42 cell line was also examined. Although the D(450) values showed no difference between negative control and groups treated with genistein (0.03,0.1,0.3,1,3 μg/mL) in the initiation assay,a significant difference was noted in the promotion assay with the concentrations of genistein ranging from 0.03 to 3 μg/mL(P<0.01). CONCLUSION: This study has clearly demonstrated the H₂O₂ treatment method as a reliable improvement for Bhas 42 cell transformation assay. This cell transformation assay combined with initiation and promotion models is an effective and practical screening platform for non-genotoxiccarcinogens.,and it will be extremely helpful in the early stage of new drug development. Genistein could be a potential non-genotoxic carcinogen,however,this result needs to be further confirmed.

OBJECTIVE: To analyze the relationship between Toll-like receptor 9(TLR9) expression and clinical pathological staging in lung cancer and its impact on the results of postoperative radiotherapy in lung cancer. METHODS: The medical records of 63 patients who had received surgical treatment in our hospital in March 2007 to December 2011 were analyzed,TLR9 expression of cancer tissue was determined by SP immunohistochemistry methods. The relationship of TLR9 expression and clinical pathological staging in lung cancer was evaluated. We performed Kaplan-Meier survival analysis and multivariate Cox regression analysis on survival factors of 36 patients who received postoperative radiotherapy,and explored the significance of TLR9 expression in postoperative radiotherapy survivors. RESULTS: No TLR9 expression was found in normal pulmonary tissue,while TLR9 protein was localized in the cytoplasm of lung cancer cells. The positive rate of TLR9 protein was 68.3%(43/63). The TLR9 expression increased with the greater tumor size and lymph node metastasis of lung cancer (P<0.05). By analyzing the survival of 36 postoperative radiotherapy lung cancer patients,we found that TLR9 expression and tumor size of lung cancer significantly affected progression-free survival (PFS) and overall survival(OS) of these patients (P<0.05). Lymph node metastasis and histological type (squamous cell carcinoma or adenocarcinoma) of also affected OS and PFS of the patients (P<0.05). With Cox multivariate regression analysis,TLR9 expression,lymph node metastasis were independent prognostic factors for patient survival. CONCLUSION: Positive TLR9 expression increased in lung cancer. TLR9 expression and lymph node metastasis were independent risk factors on predicting patient survival. TLR9 could become a new molecular biological indicator to predict the survival of lung cancer patients after postoperative radiotherapy.

OBJECTIVE: To investigate the genotoxic effects of extractable organic matter (EOM) from coke oven emissions (COEs) and motor vehicle exhausts on human hepatoma HepG2 cells. METHODS: After treated with different concentrations of coke oven- and traffic-EOMs,the genotoxic damage in HepG2 cells were evaluated by comet assay and cytokinesis-block micronucleus cytome assay. RESULTS: Significant dose-dependent increases in Olive tail moment, tail DNA (%),and tail length were observed in both coke oven- and traffic-EOMs treated HepG2 cells,except for the Olive tail moment in 0.006 ng/L traffic-EOMs treated group. The micronuclei and nucleoplasmic bridges frequencies induced by 5.5 μg/L of coke oven EOM increased to a maximum of 3.6 and 2.5 folds,respectively. Coke oven EOM led to a significant increase in nuclear buds frequency that progressively increased to 2.3 fold of the control level after 24 h treatment. The traffic-EOM showed appreciable increases in micronuclei and nucleoplasmic bridges frequencies over the controls across a range of concentration (0.6-626.3 ng/L). However,compared with the control group,only 63 and 626.3 ng/L traffic-EOM led to significant increases in nuclear buds frequency in HepG2 cells. CONCLUSION: Both the coke oven- and traffic-EOMs could induce DNA strand breaks and genomic instability in the metabolically competent HepG2 cells. The comet assay could provide a sensitive method for rapid screening of the genotoxicity of low concentrations of environmental complex mixtures.

OBJECTIVE: This study attempted to establish the cytokinesis-block micronucleus cytomic test and to compare the susceptibilities of genotoxic biomarkers in a range of mammalian cell lines. The effects of TPL (triptolide) and DG(diammonium glycyrrhizinate) on CHL and MDCK were also investigated. METHODS: Method development: Cells were treated with mitomycin C in different concentrations for 6 h,followed by a CytoB (cytochalasins B) treatment covering around 1.5 cell doubling time. The cells were subsequently harvested,prepared on slides,stained and examined microscopically. The average CBPI (cytokinesis block proliferation index) and RI (replicative index) of each dose,and the Nec(necrosis rate permillage), Apop(apoptosis rate permillage),as well as the MN (micronucleus rate permillage), NBUD(nuclear bud rate permillage),NPB(nucleoplasmic bridge rate permillage) in binucleated cells were estimated. Method application: CHL was pretreated with DG for 24 h before treated with MMC for 6 h to evaluate the effect of DG on the genotoxicity induced by MMC. MDCK was treated with TPL (1 or 5 μg/mL) for 1 h as the potential protective effect of DG was also examined. RESULTS: Cytokinesis-block micronucleus cytomic test was successfully established in CHL,MDCK,L02 and Bhas 42 cell lines. The cell toxicity(CBPI,RI,Apop and Nec)- related indexes increased along with increasing MMC concentrations. The MN,NBUD and NPB also exhibited dose-dependent rising trend induced by MMC,however,the sensitivity for the above biomarkers varied for different cell lines. DG(10 and 100 μg/mL) significantly reduced MMC (0.2 μg/mL)-induced increment on MN,NBUD and NPB(P<0.05). TPL(5 μg/mL) treatment produced elevations of MN and NBUD in MDCK(P<0.05),which could be attenuated by DG (10 μg/mL) significantly(P<0.05). CONCLUSION: Cytokinesis-block micronucleus cytomic test made it possible to effectively evaluate multiple genetic toxicity indexes within a single slide. Our data clearly demonstrated that the genotoxic effects triggered by TPL were predominately caused by the DNA breakage or loss,while DG showed potent protective effects against the chromosome breakage in both CHL and MDCK cell lines and was able to reduce the toxicities induced by other agent.

OBJECTIVE: To study the inhibitory effect of sodium norcantharidin lipid microsphere injection on nude mice bearing human carcinoma A549. METHODS: Animal model of human lung cancer was established by inoculating human carcinoma A549 cells to BALB/c nude mice. 11 days after inoculation,mice were randomly divided into control group,positive control group,and other three groups with different doses of sodium norcantharidin lipid microsphere injection. The mice received physiological saline,2.5 mg/kg sodium norcantharidin injection,and 1.25,2.5 or 5 mg/kg sodium norcantharidin lipid microsphere injection at 1 d,4 d,8 d,11 d,15 d,18 d,and 22 d after grouping. The tumor volume (V_t),relative volume (V_RT),and the increment rate (T/C) were measured at 7 d,14 d,and 24 d after the first injection. The weight of tumors and the growth inhibition rate in vivo were measured at 24 d after the first injection. RESULTS: The tumor volume and the relative volume of all treated groups were much smaller than the group with negative control 7 days after the first injection,and the difference was significant (P<0.05). These effects showed drug dose-effect relationships (r_{7 d}=0.994,r_{14 d}=0.993,r_{24 d}=0.996). The tumor increment rate of all treated groups were lower than 25%,especially the medium dose and the high dose groups which dropped to below 20%. The average tumor weights of the treated groups were lower than the control group (P<0.01). The growth inhibition rates were more than 50%. CONCLUSION: Sodium norcantharidin lipid microsphere injection effectively inhibited the growth and reduced increment rate of human carcinoma A549 transplanted in nude mice.

OBJECTIVE: To explore the effect of transient receptor potential C class 1 (TRPC1) gene on spatial learning and memory of mice. METHODS: Using Morris water maze to perform a consecutive 5-days learning of spatial acquisition (spatial navigation test) on 4-month wild-type (S129) and TRPC1 gene knockout (TRPC1 KO) mice with S129 background (n=10) and record the four quadrants latency independently. Long-term memory on the seventh day after the end of the learning stage was measured,and some parameters that could reflect memory ability of the mice were analyzed as follows: the first time that mice reach the platform (probe time),swimming trajectories,platform crossing numbers,total distance,average speed,percentage of time in target quadrant and percentage of distance in target quadrant. Independent-sample t test was applied to analyze the behavioral differences between S129 and TRPC1 KO mice. RESULTS: During 5-days learning stage,wild-type mice and TRPC1 KO mice have no significant difference in latency. In the stage of long-term memory measurement,wild-type mice and TRPC1 KO mice displayed no significant difference in probe time,crossing number of platform,average swimming speed,total distance travelled,the percentage of time spent and the percentage of distance travelled in the correct quadrant. CONCLUSION: TRPC1 gene deficiency does not affect spatial learning and memory ability of 4-month-old mice.