Xuying Wan

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.

Gene expression of sHsps, Hsp40 and Hsp60 families in normal and abnormal embryonic development of mouse forelimbs

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.

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 (CdCL2) 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 mg/mL CdCl2. The germinal vesicle breakdown of oocyte was also disturbed dose-dependently after the culture follicles were exposed to 1.6 mg/mL CdCl2. Exposure to CdCl2 with concentrations of 1.6 mg/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.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.

Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis

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.

Gene expression of Hsp70, Hsp90, and Hsp110 families in normal and abnormal embryonic development of mouse forelimbs.

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.

All-trans-retinoic acid induces short forelimb malformation during mouse embryo development by inhibiting chondrocyte maturation rather than by evoking excess cell death.

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.

Combined use of RNAi strategies with in vitro mouse limb bud culture.

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.