Construction of an in vitro simulated one compartment extravascular administration model and its comparison with classic in vitro administration model in copper chloride induced HepG2 cell death.

In this study, we designed an in vitro administration device based on compartment model theory and utilized it to construct an in vitro simulated one compartment extravascular administration model of copper chloride. Within the Cmax range of 3.91-1000.00 µM, the measured concentration-time curves of the simulated one compartment extravascular administration model almost coincide with the corresponding theoretical curves. The measured values of toxicokinetic parameters, including ke, T1/2, ka, T1/2a, Tmax, Cmax, CL, and AUC0-∞ are close to the corresponding theoretical values. The fitting coefficients are >0.9990. In simulated one compartment extravascular administration and classic in vitro administration, copper chloride dose-dependently induced HepG2 cell death. When Cmax/administration concentration is equal, classic in vitro administration induces a higher cell death rate than simulated one compartment extravascular administration. However, there is no significant difference in inducing cell death between the two administration models when area under the curve is equal. In conclusion, the device designed in this study can be used to in vitro simulate one compartment extravascular administration, making in vitro toxicity testing more similar to in vivo scenarios. There are differences in copper chloride induced HepG2 cell death between simulated one compartment extravascular administration and classic in vitro administration.

Targeted metabolomics analysis identified the role of FOXA1 in the change in glutamate-glutamine metabolic pattern of BaP malignantly transformed 16HBE cells.

The carcinogenic mechanism of benzo[a]pyrene (BaP) is far from being elucidated. FOXA1 has been confirmed to play an oncogenic role in BaP-transformed cell THBEc1. To explore the changes in amino acid metabolic patterns, especially glutamate-glutamine (Glu-Gln) metabolic pattern caused by BaP-induced transformation and the possible role FOXA1 might play in it, we compared amino acid metabolic characteristics between THBEc1 cells and control 16HBE cells using a targeted metabolomics method and determined the effects of FOXA1 knockout on the amino acid metabolic pattern using FOXA1 knockout cell THBEc1-ΔFOXA1-c34. The amino acid metabolic patterns of THBEc1 and 16HBE cells were different, which was manifested by the differential consumption of 18 amino acids and the difference in the intracellular content of 21 amino acids. The consumption and intracellular content of Glu and Gln are different between the two types of cells, accompanied by upregulation of FOXA1, GLUL, SLC1A3, SLC1A4, SLC1A5 and SLC6A14, and downregulation of FOXA2 and GPT2 in THBEc1 cells. FOXA1 knockout changed the consumption of 19 amino acids and the intracellular content of 21 amino acids and reversed the metabolic pattern of Glu and the changes in FOXA2, GLUL, SLC1A3 and SLC6A14 in THBEc1 cells. Additionally, FOXA1 knockout inhibited cell proliferation and further increased the dependence of THBEc1 cells on Glu. In conclusion, FOXA1 knockout partially reversed the change in Glu-Gln metabolism caused by BaP-induced transformation by upregulating the expression of GLUL and SLC1A3. Our findings provide a clue for the possible role of FOXA1 in amino acid metabolism regulation.

Untargeted metabolomics and lipidomics analysis identified the role of FOXA1 in remodeling the metabolic pattern of BaP-transformed 16HBE cells.

Benzo[a]pyrene (BaP) is a strong carcinogen for lung cancer, and forkhead-box A1 (FOXA1) plays an oncogenic role in BaP-transformed cell THBEc1. To explore the remodeling of metabolic pattern caused by BaP-induced transformation and the possible role FOXA1 might play in it, we compared metabolic patterns between THBEc1 cells and control using untargeted metabolomics and lipidomics analysis, and determined the effects of FOXA1 knockout on the metabolic pattern of THBEc1 cells. Metabolomics and lipidomics identified a total of 15 and 46 differential metabolites and lipids between THBEc1 and 16HBE cells, respectively, and a total of 4 and 1 differential metabolites and lipids between FOXA1 knockout cell THBEc1-ΔFOXA1-c34 and control cell THBEc1-ctrl, respectively. Analysis results of metabolites and metabolic pathways indicated the metabolic pattern remodeling may be related to the alteration in glucose metabolism during BaP-induced transformation. Western blotting revealed the up-regulation of enolase-2 (ENO2), pyruvate carboxylase (PCB), aconitase-2 (ACO2) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Thr202/Tyr204), the down-regulation of succinate dehydrogenase complex subunit A (SDHA) and phosphoenolpyruvate carboxykinase 2 (PCK2) in THBEc1 cells. The detection results of metabolites related to glucose metabolism demonstrated the decreasing of lactic acid content in cells, lactic acid production in culture medium and citric acid content in mitochondria, and the increasing of ATP production of THBEc1 cells. FOXA1 knockout partially reversed the changes of ENO2, SDHA, PCK2 and p-ERK1/2 (Thr202/Tyr204) levels, lactic acid release, citric acid content in mitochondria of THBEc1 cells. In conclusion, FOXA1 knockout partially reversed the remodeling of glucose metabolism caused by BaP-induced malignant transformation. Our findings provide a clue for the possible role of FOXA1 in glucose metabolism regulation.

Label-free quantitative proteomic analysis identifies the oncogenic role of FOXA1 in BaP-transformed 16HBE cells.

Lung cancer has been the leading cause of cancer incidence and mortality in China for years. Benzo[a]pyrene (BaP) is a well-known carcinogen for lung cancer. To understand alternation of key proteins and their role in BaP-induced lung cancer, we compared proteome profiles between BaP-transformed 16HBE cell line T-16HBE-C1 (THBEc1) cells and control using label-free quantitative proteomic analysis. Forkhead box protein A1 (FOXA1) was selected and evaluated for its potential role in BaP-induced carcinogenesis in vitro and in vivo. Relationship between FOXA1 expression and survival of lung cancer patients were examined via The Cancer Genome Atlas (TCGA) database. A total of 183 differentially expressed proteins were identified, with 67 proteins including FOXA1 up-regulated and 116 proteins down-regulated in THBEc1 cells. Differentially expressed proteins mainly functioned in basic cellular metabolism, tumor related pathways and regulation of transcription factors. FOXA1 knockout inhibited colony formation and migration of THBEc1 cells in vitro. FOXA1 knockout inhibited tumor growth and metastasis in BALB/c-nude mice after subcutaneous and tail vein injection of FOXA1 knockout THBEc1 cells, respectively. FOXA1 mRNA expression was higher in tumor tissues for patients with lung squamous cell carcinoma, but not associated with survival of lung cancer patients. Our findings revealed oncogenic role of FOXA1 in BaP-induced lung cancer and improved understanding of mechanism in BaP-induced carcinogenesis.

MicroRNA expression profiling and the role of ALCAM modulating tumor growth and metastasis in benzo[a]pyrene-transformed 16HBE cells.

Benzo[a]pyrene (BaP) is a potent carcinogen and microRNAs (miRNAs) may play an important role in carcinogenesis. Activated leukocyte cell adhesion molecule (ALCAM) was up-regulated in BaP-transformed 16HBE cell line (THBEc1), and may be a key molecule for THBEc1 cells to gain and maintain the malignant phenotype. Here we screened the differentially expressed miRNAs which resulted in up-regulation of ALCAM in THBEc1 cells by comparing miRNA expression profiles between THBEc1 and 16HBE (HBE) cells. Results showed that a total of 555 miRNAs differentially expressed between THBEc1 and HBE cells, of which 351 miRNAs were down-regulated and 204 miRNAs were up-regulated in THBEc1 cells. MiR-152-3p, miR-142-5p and miR-211-5p down-regulated in THBEc1 cells were demonstrated to participate in the regulation of ALCAM. With THBEc1 as a tumor cell model, we determined the role of ALCAM in tumor growth and metastasis employing two ALCAM knockout THBEc1 cell lines via CRISPR/Cas9 technology. Results showed that ALCAM knockout inhibited colony formation and tumor growth, but enhanced cell migration and lung metastasis of THBEc1 cells. In conclusion, miR-152-3p/ALCAM, miR-142-5p/ALCAM and miR-211-5p/ALCAM axes may be involved in BaP-induced carcinogenesis. BaP might induce up-regulation of ALCAM via inhibiting miR-152-3p, miR-142-5p and miR-211-5p, which in turn allows ALCAM to exert its role promoting cell proliferation and tumor growth, and suppressing cell migration and metastasis.

Clusterin and neuropilin-2 as potential biomarkers of tumor progression in benzo[a]pyrene-transformed 16HBE cells xenografted nude mouse model.

Benzo[a]pyrene (BaP) is a ubiquitous environment contaminant and its exposure could increase incidence of human lung cancer. In order to confirm and compare potential biomarkers of BaP-induce carcinogenesis and tumor progression, time-dependent changes of clusterin (CLU) and neuropilin-2 (NRP2) levels were evaluated in sera of BaP-transformed 16HBE cell line T-16HBE-C1 cells xenografted nude mice. Performance of CLU and NRP2 on tissue classification and tumor progression forecast was also calculated. Levels of CLU and NRP2 were significant elevated in both culture supernatant of T-16HBE-C1 cells and sera of T-16HBE-C1 cells xenografted nude mice compared with control. CLU and NRP2 were both found positively stained in tumor tissue. CLU and NRP2 alone could well predicate tumor progression in nude mice and CLU appeared to be more sensitive than NRP2. When both of them combined, performance of predication would improve. In conclusion, CLU and NRP2 could serve as potential biomarkers of tumor progression in nude mice xenografted with T-16HBE-C1 cells.

PINK1/Parkin-mediated mitophagy play a protective role in manganese induced apoptosis in SH-SY5Y cells.

Manganese (Mn) as an environmental risk factor of Parkinson's disease (PD) is considered to cause manganism. Mitophagy is thought to play a key role in elimination the injured mitochondria. The goal of this paper was to explore whether the PINK1/Parkin-mediated mitophagy is activated and its role in Mn-induced mitochondrial dysfunction and cell death in SH-SY5Y cells. Here, we investigated effects of MnCl2 on ROS generation, mitochondrial membrane potential (MMP/ΔΨm) and apoptosis by FACS and examined PINK1/Parkin-mediated mitophagy by western-blotting and the co-localization of mitochondria and acidic lysosomes. Further, we explore the role of mitophagy in Mn-induced apoptosis by inhibition the mitophagy by knockdown Parkin level. Results show that MnCl2 dose-dependently caused ΔΨm decrease, ROS generation and apoptosis of dopaminergic SH-SY5Y cells. Moreover, Mn could induce mitophagy and PINK1/Parkin-mediated pathway was activated in SH-SY5Y cells. Transient transfection of Parkin siRNA knockdown the expressing level of parkin inhibited Mn-induced mitophagy and aggravated apoptosis of SH-SY5Y cells. In conclusion, our study demonstrated that Mn may induce PINK1/Parkin-mediated mitophagy, which may exert significant neuro-protective effect against Mn-induced dopaminergic neuronal cells apoptosis.

Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

ER stress and ER stress-mediated apoptosis are involved in manganese-induced neurotoxicity in the rat striatum in vivo.

Manganese (Mn) is an essential trace element found in many enzymes, however, excessive Mn-exposure can result in manganism which is similar to Parkinson's movement disorder. The mechanisms of manganism are not well-known. The present in vivo study was carried out to determine whether endoplasmic reticulum stress (ER stress) and ER stress-mediated apoptosis are involved in manganese-induced neurotoxicity. Sixty-four SD rats were randomly divided into four groups and were administered intraperitoneally with normal saline (NS, as control) or MnCl2 (7.5, 15 and 30 mg/kg body weight, respectively) for 4 weeks. We found that MnCl2 dose-dependently accumulate in striatal. HE staining and TUNEL assay results indicated that MnCl2 induced striatal neurocytes apoptosis in both male and female rats. The alterations of ultrastructures showed that MnCl2 resulted in chromatin condensation, mitochondria and ER tumefaction in rat striatal neurocytes. Furthermore, MnCl2 increased the expressions of p-IRE-1, ATF-6α, PERK, GRP78, Sigma-1R, CHOP, Bim, Bax, caspase-12 and caspase-3, and decreased the expression of Bcl-2 in rat striatal neurocytes. In conclusion, MnCl2 could induce ER stress and ER stress-mediated apoptosis in rat striatal neurocytes, which might be one of the important mechanisms of Mn-induced neurotoxicity.

The cell cycle distribution should be given more consideration in cell-based in vitro toxicological studies.

In this study, to discuss the importance of the cell cycle distribution in cell-based in vitro toxicity mechanism studies, diethyl sulfate (DES) was selected as a model chemical that induced the alteration of the cell cycle distribution in human bronchial epithelial cell line 16HBE 14o- (HBE) cells. Cells were treated with various concentrations of DES, cell proliferation and apoptosis were then determined. The results showed that DES concentration-dependently inhibited HBE cells proliferation and induced apoptosis. When cells were treated with 2.0 mM of DES for 20 or 28 h, significant S and G2/M phase accumulation was observed. Then, the relative cellular levels of Cdk4, p-Cdk2 (Thr160), Cyclins A and B1 in DES-treated HBE cells at 20 and 28 h were determined by two ways. The differences of the cell cycle distribution between DES and control groups were ignored in one way and eliminated by using flow cytometric cell sorting in the other. The results obtained by the two ways were quite different, which indicated that the cell cycle distribution might result in confounding if it was significantly different between the treated and control groups. Therefore, we propose that the cell cycle distribution should be given more consideration in cell-based in vitro toxicological studies.

Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells.

In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0mM) for 12, 24 or 36h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5-4.0mM) and then decreased (8.0mM). Down-regulation of p53 by RNA interference increased 4.0mM of DES-induced apoptosis but did not affect 2.0mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure.

In vitro malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene.

In this study, the human bronchial epithelial cells (16HBE) were treated five times with 10µM benzo(a)pyrene (BaP), followed by 20 passages culture, and the in vitro BaP-induced malignant transformation of 16HBE cells was established. Five colonies in soft agarose were then amplified and donated as T-16HBE-C1∼5 cells, respectively. T-16HBE-C1∼5 cells can form tumors subcutaneously in nude mice. Histopathological changes in the tumors indicated nests growth, high nuclear-cytoplasmic ratios, coarse and clumped chromatin, numerous and distinctly atypical mitoses, cell necrosis and surrounding normal adipose, muscle and connective tissue immersed. In addition, lung metastasis was observed in nude mice in T-16HBE-C1, 3 and 4 groups. In vitro cell migration assay results indicated that T-16HBE-C2∼5 cells showed much lower migration capabilities than 16HBE cells. Western blotting analysis showed that the expressions of p53 and p-Akt (Ser473) in T-16HBE-C1∼5 cells were significant higher than those in 16HBE cells. Our results demonstrated that BaP could induce the malignant transformation of 16HBE cells, and p53 and p-Akt (Ser473) might play crucial roles in BaP-induced carcinogenesis. The five monoclonal cell lines (T-16HBE-C1∼5) with different migration capabilities could be used as research models for further understanding the mechanisms of BaP-induced carcinogenesis and cell migration.

In vitro genotoxicity of danthron and its potential mechanism.

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100µg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.

Antigen-subtracted 2-DE/MS strategy, a novel proteomic analysis platform.

In the present study, we developed a novel proteomic research strategy named antigen-subtracted 2-DE/MS strategy and applied it to comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Following pre-purification by ammonium sulfate precipitation, rabbit antibodies against 16HBE G0/G1 cells were coupled with protein A/G PLUS-agarose under the maximal coupling rate of about 50%. The agarose-antibody complex was then used in immunoprecipitation known as antigen subtraction. When the mass ratio of antibody to the sample was 2.5-3:1, the subtraction rates for 16HBE and 16HBE-C G0/G1 cell proteins were 44 and 34%, respectively. Both subtracted and unsubtracted samples were then subjected to the 2-DE resolution. In 16HBE-subtracted 2-DE maps, 315 protein spots were subtracted and 49 new protein spots were detected, whereas in 16HBE-C-subtracted 2-DE maps, 287 protein spots were subtracted and 33 new protein spots were detected. Taken together, antigen subtraction results in 65 new protein spots being allowed to be detected, therefore, makes it possible to get more information of the samples. Finally, 4 protein spots only detected in 16HBE-C-subtracted 2-DE maps were analyzed by the Q-TOF MS/MS, and successfully identified as U6 snRNA-associated Sm-like protein LSm3, 60S acidic ribosomal protein P1, Peroxiredoxin-6 and 60S acidic ribosomal protein P2. These proteins are involved in carcinogenesis, oxidation stress and protein synthesis. In conclusion, the antigen-subtracted 2-DE/MS strategy could reduce the complexities of protein samples and therefore, improve the resolution for the sample analysis.

Diethyl sulfate induced Cdk2-dependent centrosome amplification in CHL cells.

The function of centrosome that serves as the main microtubule organizing center is essential to ensure the genomic integrity during the cell division cycle. Centrosome abnormalities are frequently observed in many tumors and cells exposed to genotoxic agents. Here, we investigated the centrosome abnormalities induced by diethyl sulfate (DES) in Chinese hamster lung (CHL) fibroblasts and the underlying molecular mechanisms. Our results showed that DES exposure at 0.3, 1 and 3mM for 48 h caused centrosome amplification in a dose dependent manner. This effect was associated with transient S and G2/M phase delay and up-regulating of Cdk2, Cyclin A expressions. Furthermore, inhibition of Cdk2 activities reversed the centrosome amplification induced by DES. These results reveal that centrosome is one of the key subcellular targets of DES. Centrosome abnormalities might be important mechanisms behind the aneuploidy induction and carcinogenicity of DES.

Comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed and normal human bronchial epithelial G0/G1 cells.

In the present study, we investigated the proteomic profiling of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE)-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Differential analysis of proteomic profiling indicated that 67 polypeptides were down-regulated and 77 polypeptides were up-regulated in 16HBE-C G0/G1 cells compared to 16HBE G0/G1 cells. Then 16 differentially expressed protein spots were analyzed with Q-TOF MS/MS. Of these spots, 3 down-regulated polypeptides were identified as sorcin, small ubiquitin-related modifier 2 precursor and eukaryotic translation initiation factor 5A-1, and 9 up-regulated polypeptides were identified as calmodulin, myosin light polypeptide 6, eukaryotic translation initiation factor 6, proliferating cell nuclear antigen (PCNA), tumor protein D52 (TPD52), superoxide dismutase [Cu-Zn], prohibitin, nuclear protein Hcc-1 and vimentin. These proteins are involved in cell proliferation, protein synthesis, signal transduction and carcinogenesis. Western blotting analysis verified the increased expression levels of PCNA and TPD52 in 16HBE-C G0/G1 cells. Based on the clues from proteomic analysis, the migration and invasion capabilities of 16HBE-C and 16HBE cells were tested. The results indicated that 16HBE-C cells showed much higher migration and invasion capabilities than 16HBE cells, and moreover, the suppression of TPD52 by RNAi resulted in significant decrease of migration and invasion capabilities of 16HBE-C cells. These results will be valuable for further investigating and understanding the mechanisms underlying BaP-induced carcinogenesis.

Cytotoxicity of polymerized resin cements on human dental pulp cells in vitro.

OBJECTIVES: This study was designed to evaluate the cytotoxicity of several resin-based cements (Panavia F, Super-Bond C&B, Chemiace II) after polymerization on cultured human dental pulp cells. METHODS: After polymerization, specimens from three resin-based cements were eluted with fresh Dulbecco's modified Eagle's medium (DMEM) without serum for 72 h, at 37 degrees C, using 0.4 g of each substance per milliliter of fresh medium. Elutes obtained during this step were passed through a 0.22-microm filter and diluted with the culture medium by a ratio of 75%, 50%, 25% (v/v). Cytotoxicity of elutes were evaluated by the relative growth rates (RGR) of pulp cells with a modified 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. The RGR of pulp cells were statistic analyzed by the one-way analysis of variance among the groups. RESULTS: The RGR of cells exposed to 100% concentration of elution of Panavia F, Super-Bond C&B, and Chemiace II were 74.42%, 85.54%, and 82.39%, respectively. The RGR increased along with the elution of cements diluted. There was significant difference between the Panavia F group and Super-Bond C&B group (p<0.01), but there was no significant difference in the cytotoxicity between Chemiace II and Super-Bond C&B. CONCLUSIONS: After polymerization, three resin-based cements (Panavia F, Super-Bond C&B, Chemiace II) induced slight cytotoxicity. The sensitivity of cytotoxicity to human pulp cells depended on the resin-based cements and the concentration of the elution. Super-Bond C&B is the least cytotoxic agent among the three resin-based cements.

Claspin is involved in S-phase checkpoint induced by benzo(a)pyrene in 16HBE cells.

Environmental carcinogen benzo(a)pyrene (BaP) can damage DNA by forming bulky adducts that are degraded further to DNA strand breaks, thus contributing to induce DNA damage checkpoint response. Claspin is a critical checkpoint protein in response to multiple forms of genotoxic stress including UV, IR and hydroxyurea (HU). In the present study we have investigated the role of human Claspin in the DNA damage checkpoint elicited by BaP in 16HBE cells. We observed that Claspin levels are increased in a time-dependent manner in response to S-phase arrest induced by BaP. In addition, the levels of phosphorylation of Chk1 on S345 were increased, but the levels of Cdc25A were decreased after treatment with BaP. Inhibition of Claspin expression (siRNA) attenuated the effect of BaP on S-phase arrest and abrogated the activation of Chk1 and degradation of Cdc25A in response to BaP. Taken together, these data imply that Claspin plays an important role in S-phase checkpoint induced by BaP, and it participates in the activation of Chk1 and Cdc25A in this checkpoint pathway.

Cyclin A is essential for the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells.

Benzo(a)pyrene (B(a)P) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. The p53 tumor suppressor as a cell cycle check-point determinant plays a critical role in cell proliferation. However, the mechanism of p53 that accounts for the remarkable toxicity of B(a)P remains elusive. Here we reported that exposure of B(a)P to A549 cells caused G1 to S and G2/M phase transition along with increased expression of p53, cyclin D1, Cdk2, Cdk4, p21 and decreased expression of cyclin E, but no change in cyclin A and p27 expression. Up-regulation of p53 expression via transfection caused G1 phase arrest with decreased expression of cyclin A, E, Cdk2 and Cdk4, and increased expression of p21, when the expression of cyclin D1 and p27 were not significant changed. While B(a)P exposure to A549 cells following p53 transfection, up-regulation of p53 significantly attenuated the B(a)P-induced enhancement of cell proliferation and cell arrest, with increased expression of cyclin D1, Cdk2 and Cdk4, and with declined expression of cyclin A, cyclin E and p21, and p27 were not significant changed. Compared to the untreated cells, cylin A expression reduced in p53-transfected cells and in the B(a)P-treated cells following p53 transfection, but showed no change in the only B(a)P-treated cells. These results indicated that cyclin A is regulated by p53, not by B(a)P, and it is essential in the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells, cyclin E and p21 also as downstream genes of p53 involved it, which is p27-independent.

TPX2 in malignantly transformed human bronchial epithelial cells by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide.

In order to elucidate the function of the targeting protein for Xenopus kinesin-like protein 2 (Xklp2) (TPX2) in the malignant transformation of human bronchial epithelial cells induced by anti-benzo[a]pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE), TPX2 was characterized in cells at both the gene and the protein levels. TPX2 was present at higher levels in 16HBE-C cells than in 16HBE cells as demonstrated by two-dimensional gel electrophoresis, immunocytochemistry, Western blot analysis and RT-PCR. TPX2 was also detected in lung squamous-cell carcinoma tissues by immunohistochemistry, but not in normal lung tissues. Depression of TPX2 by RNA interference in 16HBE-C cells led to a decrease in cell proliferation, S-phase cell cycle arrest and cell apoptosis. Abnormal TPX2 tyrosine phosphorylation was detected in 16HBE-C cells, and this could be inhibited, to different degrees, by tyrosine kinase inhibitors. Inhibiting tyrosine phosphorylation in 16HBE-C cells by three selected tyrosine protein kinase inhibitors, tyrphostin 47, AG112 and AG555, caused G(0)/G(1)-phase cell cycle arrest. Our results suggest that anti-BPDE can cause the over-expression of TPX2 and its aberrant tyrosine phosphorylation. Misregulation of TPX2 affects the cell cycle state, proliferation rates and apoptosis.