[Establishment of rat integrated discrete multiple organ cell culture (IdMOC) model].

OBJECTIVE: To establish the integrated discrete multiple organ cell culture (IdMOC) system. METHODS: Rat primary cell of hepatocyte, nephrocyte, cardiomyocytes, alveolar macrophage, dermal fibroblasts were isolated by collagenase digestion, separation of bronchial lavage, two-step digestion method and cultured respectively, with monolayer culture. To establish the integrated discrete multiple organ cell culture (IdMOC) system, glass slides of five different cells were used to the same dish with 10% FBS DMEM medium cultured 7d, using MTT comparison primary cells cultured alone and cocultured when growth. RESULTS: Established rat hepatocytes, renal cell, cardiomyocyte, alveolar macrophages, dermal fibroblasts separation method was stable, cell separation survival rate was about 90.0%. Hepatocytes separation survival rate 90.3% ,renal cell separation survival rate 91.9%, cardiomyocyte separation survival rate 93.0% and beating rate indifference curve among 3d-15d, alveolar macrophages cell separation survival rate 90.8%, dermal fibroblasts cell separation survival rate 92.7%. Five primary cells multiple organ cells coculture showed cocultured cell growth proliferation well, cultured alone and cocultured cells growth curve basic coincide. CONCLUSION: Established rat multiple organ cell co-culture is successful.

Rat ovarian follicle bioassay reveals adverse effects of cadmium chloride (CdCl2) exposure on follicle development and oocyte maturation.

Rat ovarian follicle culture, as a novel bioassay, is adopted in this study to explore the effects of cadmium chloride (CdCL(2)) on folliculogenesis and oocyte maturation in vitro; the feasibility for its application on detection of possible effects of chemicals on reproduction is discussed and evaluated as well. The results showed that follicle growth, differentiation, and steroidogenesis were significantly disturbed by ≥ 1.2 µg/mL CdCl(2). The germinal vesicle breakdown of oocyte was also disturbed dose-dependently after the culture follicles were exposed to ≥ 1.6 µg/mL CdCl(2). Exposure to CdCl(2) with concentrations of 1.6 µg/mL on day 2 had caused significant reduced (p < 0.05) survival rate and rate of antral follicles, and increased abnormal follicle rate significantly, compared to the group exposed on day 6. Rat preantral follicle culture is a potential tool to assess the hazards of chemical compounds on female fertility and can be used to elucidate their mechanisms of actions.

[Effect of DEHP and its metabolite MEHP on in vitro rat follicular development].

OBJECTIVE: To use rat preantral follicle culture as a novel bioassay in order to study the effects of di-2-ethylhexyl phttmlate (DEHP) and its metabolite mono-2-ethylhexyl phthalate (MEHP) on folliculogenesis in vitro. METHODS: The preantral follicles were mechanically dissected from ovaries and equally randomized to to 96-well plates. Six dose groups were setup to be acute exposed in 2.5, 5, 10, 20, 40 and 80 microg/ml MEHP concentration, 33.7, 67.5, 125, 250, 500 and 1000 nmol/L DEHP concentration on day 2,while another control group used solvent only, each group had 16 to 20 follicles. After subsequently cultured individually for 10 days, the follicles were induced to ovulate for 20h, follicle development and oocyte maturation were observed. RESULTS: Day 11 follicle survival rate (54.76% +/- 3.37%) had significantly dropped after exposure to >10 microg/ml MEHP concentration, comparing to the control group (91.57% +/- 1.32%), the difference is statistical significant (P < 0.05). There is correlation between dosage and follicle survival rate, with correlation coefficient R2 = 0.92. Abnormal follicle rate (45.24% +/- 3.37%) is remarkably higher, and the difference is statistically significant (P < 0.05) comparing to the control group. At the concentrations of >20 microg/ml MEHP, follicle growth and differentiation were dependently impaired:antral follicles (46.18% +/- 1.67%) had significantly reduced (P < 0.05) comparing to the control group (85.91% +/- 5.03%). DEHP showed no adverse effects upon follicle development, there were no significant defference between DMSO and control. CONCLUSION: At the dose of >10 microg/ml, MEHP could inhibit the in-vitro rat follicle development, DEHP seemed no adverse effects upon follicle development.

Evaluation of salidroside in vitro and in vivo genotoxicity.

It is reported that salidroside, the main component of a traditional Chinese medicine, Rhodiola rosea, has the efficacy of protecting Coxsackie virus impairment. As part of a safety evaluation on salidroside for use in the treatment of viral myocarditis, the present study evaluated potential genotoxicity of salidroside by using the standard battery of tests (i.e., bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay) recommended by the State Food and Drug Administration of China. The results showed that salidroside was not genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay conditions. The anticipated clinical dose seems to be smaller than the doses administered in the genotoxicity assays. With confirmation from further toxicity studies, salidroside would hopefully prove to be a safe anti-Coxsackie virus agent.

[Establishment of in-vitro mouse preantral follicle culture systems].

OBJECTIVE: To develop mouse preantral follicle culture for toxicology study. METHODS: The ovaries were from prepubertal mice (C57B1/6JxCBA/Ca) (at the ages of 12-14 days). The ovaries were from prepubertal mice (C57B1/6JxCBA/Ca) (at the ages of 12-14 days). The preantral follicles with a diameter around the range 100-130 microm were mechanically dissected and randomly allocated to 96-well plates. After subsequently cultured individually for 12 days, the follicles were induced ovulation. The in-vitro developmental characteristics of the follicles were observed including hormonogenesis, folliculogenesis and oogenesis 16h later after ovulation. In-vitro growth and maturation of oocytes (IVG) were compared with in-vivo growth and in-vitro maturation of oocytes (IVM) to assess the in-vitro assay. RESULTS: 89.74% (376/419) of follicles were visibly associated with theca cells and possessed a close follicle (granulosa) cell/oocyte apposition. At 12 days of culture, the average diameter of follicles were increased, the survival rate of follicles is 96.81% (364/376), 48.94% (184/376) of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity, and 56.38% (212/376) COCs were released by maturation induction in this study. From preantral follicle to oocyte maturation and the formation of first polar body, the morphological observation showed that developmental characteristics of mouse preantral follicles in-vitro were similar to those in-vivo. The patterns of hormone secretion observed in preantral follicle culture were similar to the characteristics of in-vivo follicle hormone secretion. The numbers of oocytes with germinal vesicle (GV) did not differ between IVG and IVM groups, 18.89% oocytes form the first polar body in IVG groups and 53.53% oocytes form the first polar body in IVM groups. No abnormal chromosome segregation found in this study. CONCLUSION: This study successfully developed in-vitro mouse pre-antral follicle culture system, which could be a efficient tool to study folliculogenesis physiology and toxicology system, which could be a efficient tool to study folliculogenesis physiology and toxicology.

[Detection of estrogenic effects of nonylphenol and bisphenol A in vitro reporter gene-based assays].

OBJECTIVE: To explore the estrogenic effects and disruptive mechanism of NP and BPA by reporter gene-based assays we developed. METHODS: pERE-Luc plamid was generated by inserting estrogen response element (ERE) fragment into MCS of pGL3-promoter vector. MCF7 cells were cotransfected with pERE-Luc and phRL-SV40 using Sofast transfection reagent. The cells then treated with 17beta-estradiol (E2), tamoxifen (Tam), nonylphenol(NP) and bisphenol A (BPA) and expression of the repoter gene in the cell lysates was assayed using Dual-Lucferase reporter assay system. RESULTS: The pERE-Luc plasmid was constructed. Luciferase activities of MCF7 cells transfected pERE-Luc showed dose-responed realitionship with E2. 1 x 10(-11) mol/L E2 could induce the expression of reporter gene and 1 x 10(-9) mol/L E2 resulted in the largest luciferase activity. E2 couldn't induce the luciferase activity without pERE-Luc. Tam is a complete antagonist, inhibited the E2-induced luciferase expression. NP induced the luciferase activity at concertrations > 1 x 10(-6) mol/L, BPA induced the luciferase activity at concertrations > 1 x 10(-6) mol/L. The estrogenic activity of NP was more than BPA. CONCLUSION: The assay we established is usful, NP and BPA showed estrogenic activities.

[Establishment of reporter gene-based assays for the identification of (anti) androgenic effects of chemicals in CHO cells].

OBJECTIVE: Develop reporter gene-based assays for the identification of (anti) androgenic effects of chemicals in CHO cells, explore the antiandrogenic effects of 2,4-DDT and methoxychlor. METHODS: Chinese Ovary cells were cotransfected with the human androgen receptor expression vector, mouse mammary tumour virus MMTV-luciferase vector and phRL-SV40 using the transfection reagent Sofast, test the luciferase activity. After treatment of the cells for 24 h with dihydrotestosterone (DHT), flutamide (FLU), validate the effectiveness and specificity, also test the effect of 2,4-DDT and methoxychlor (METH). RESULTS: DHT is a potent agonist, 1 x 10(-10) mol/L DHT could result in the enhancement of luciferase activity, FLU is a complete antagonist, that confirmed the effectiveness and specificity of test system. 2,4-DDT and METH are partial antagonists. 3 x 10(-7) mol/L and above 2,4-DDT and METH could inhibit the androgenic activity of DHT. CONCLUSION: The assay we developed is doful, 2,4-DDT and METH could antagonize the androgenic effects.

[Molecular mechanism of cleft palate induced by retinoic acid].

OBJECTIVE: To explore the molecular mechanism of cleft palate induced by chemicals. METHODS: Retinoic acid was used as a known teratogen to induce cleft palate in ICR mice and a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes that related to cleft palate of ICR mice. RESULTS: 14 reverse differently and 9 forward differentially expressed clones were obtained. Some clones were selected to be sequenced and aligned to GenBank. CONCLUSION: In this study, suppressed Gpc3 and Insulin-Induced protein 1 could affect growth of palate shelves and resulted in cleft palate by reducing the size of the palate shelves. Down-regulation of Ptprs interfered with a cell signal pathway and down-regulation of Tn C inhibited the cell de-adhesion and expression of Egfr, then suppressed Egfr prevented the normal expression of MMPs that influenced the medial edge epithelium disruption and caused cleft palate. Tn C could bind to Ptprs and Gpcs, and HSPGs were ligands for Ptrps. Up-regulate of Rps25 might play a role in cleft palate by excessively apoptosis.