Publications (12)17.43 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow® method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24 h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n = 200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n = 200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China.
- [Show abstract] [Hide abstract] ABSTRACT: Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.
- [Show abstract] [Hide abstract] ABSTRACT: Various biochemical and physiological stimuli may interfere with endoplasmic reticulum (ER) homeostasis, causing aggregation and accumulation of unfolded or malfolded proteins in the ER and resulting in ER stress (ERS), and the ER attenuates ERS through unfolded protein response (UPR). All-trans-retinoic acid (ATRA) can enhance or attenuate some ERS-induced physiological or pathological changes. Our previous studies showed that there was UPR in short forelimbs induced by ATRA. Many kinds of malformations induced by ATRA may correlate with cell death, and knowing that UPR is closely associated with cell death, so we speculate that the short forelimbs may caused by UPR-induced cell death. To test this hypothesis, the present study investigated the expression of UPR-related genes and proteins in the same short forelimb malformation model to determine whether ATRA-induced short forelimb malformation occurred through UPR-induced cell death. Subsequently, we further observed the differentiation and proliferation of chondrocytes and the expression of related genes and/or proteins. It was found that ATRA evoked UPR in this short forelimb model, thus activating the anti-cell death pathway and inhibiting the cell death-promoting pathway. Cell death was not evident in short forelimb, and ATRA inhibited the expression of Ccnb1 and Ccna1, thus retarding chondrocyte maturation. As a result, the number of immature chondrocytes in short forelimb was greater than the normal level. We therefore believe that ATRA induces short forelimb malformation most likely by inhibiting chondrocyte maturation rather than by evoking excess cell death.
- [Show abstract] [Hide abstract] ABSTRACT: Heat shock proteins (Hsps) are thought of as chaperones of morphologic development of cells and organisms and are believed to be closely related to normal and abnormal embryonic development. It was found, in the present study, that 19 Hsps of the three Hsp70, Hsp90, and Hsp110 families were expressed in embryonic forelimb tissue of normal mice, but in different patterns, showing a characteristic correlation with the developmental phases. The tendency that the expression of many genes changed with embryonic age, increasing in the all-trans retinoic acid (atRA)-induced limb malformation groups, was similar to that of the control group, but messenger RNA abundance of most of these genes was significantly different from that in the control group. HspA12B, HspA14, Trap1, and Hsp105 may be limb-development-related genes; Grp78 may play an important role in limb development.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Methods for knocking out genes and generating transgenic animals are available to study gene functions during embryogenesis. Recently, RNA interference (RNAi), a conserved eukaryotic mechanism in which double-stranded RNA induces sequence-specific degradation of homologous mRNAs, has become a powerful tool for investigating gene function in cells, whole animals, and embryos. Additionally, in vitro mouse limb bud culture is available to study limb development. By microinjection, we established a new method combining the use of RNAi strategies with in vitro mouse limb bud culture to investigate the functions of various genes during embryonic limb development. Using this new method, we found that HspB10 may play a role during skeletal development of limbs. It shows that the combined use of RNAi strategies (via microinjection) with in vitro mouse limb bud culture system may become a technique for investigating gene function in limb development.
- [Show abstract] [Hide abstract] ABSTRACT: 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. 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. 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. At the dose of >10 microg/ml, MEHP could inhibit the in-vitro rat follicle development, DEHP seemed no adverse effects upon follicle development.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Heat shock proteins (Hsps) are thought of as chaperones of morphologic development of cells and organisms, and believed to be closely related to normal and abnormal embryonic development. Previous studies only focused on a small number of HSPs and their relationships with embryogenesis, and roles of most HSPs in normal and abnormal embryonic development remain unclear. The present study detected expression of sHsps, Hsp40 and Hsp60 in normal and abnormal development of embryonic forelimbs in all-trans retinoic acid (atRA)-induced phocomelic, oligodactylic and atRA-induced abnormal limb bud development models in mice ex vivo. It was found that 17 Hsps of the three families were expressed in embryonic limb bud tissue of normal mice, though the expression patterns were different, showing a phase correlation with the development of embryo. There was a difference in the expression pattern and mRNA abundance of most genes between the atRA-indcued limb abnormal development model groups and the control group. Most members of the three families may play a stress-protective role in atRA-induced abnormal limb development, and abnormal expression of some genes (Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10 and Hsp40) may be related to atRA-induced phocomelic and other abnormalities.
- [Show abstract] [Hide abstract] ABSTRACT: To develop mouse preantral follicle culture for toxicology study. 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. 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. 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.
- [Show abstract] [Hide abstract] ABSTRACT: To provide the proof for the high throughput screening method in vitro by establishing the comet assay in vitro using HepG2 cells plated in 96-well plates. Plating HepG2 cells in 96-well plates, and then detecting the cytotoxicity using the neutral red method before comet assay. 8 genotoxicial agents could be detected using the method and the sensitivity was 100% , the ratio of the comet cells and the length of the tail of the comet were increased with the increasing dose when the cytotoxicity less than 50%. 11 non-genotoxicial agents could be detected within 12 non-genotoxicial agents and the specificity was 91%. (1) This method could detect the direct genotoxic agents and the indirect genotoxic agents at the absent of the S9. (2)4-5 agents could be detected in the same 96-well plate using the method. (3) Using the method, we could obtain an dose at which the agent could cause genotoxicity. (4) By plating cells in plates in vitro, increased the speed of the detection and reduced the consumption of the agents. (5) The method could be developed into the high throughput screening method in vitro.
- [Show abstract] [Hide abstract] ABSTRACT: To explore the molecular mechanism of cleft palate induced by chemicals. 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. 14 reverse differently and 9 forward differentially expressed clones were obtained. Some clones were selected to be sequenced and aligned to GenBank. 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.
Second Military Medical University, Shanghai
Shanghai, Shanghai Shi, China
- Faculty of Basic Medical Sciences