Interspecies Comparison of Control Data From Embryo-Fetal Development Studies in Sprague-Dawley Rats, New Zealand White Rabbits, and Göttingen Minipigs.

Species-dependent differences in relative incidence of spontaneous variations and malformations should be considered in the assessment of the translational value of reproductive and developmental safety assessments. The objective of this evaluation was to compare litter parameters and the frequency of external, visceral, and skeletal malformations and variations across species in the Sprague-Dawley rat, New Zealand White rabbit, and Göttingen minipig and to determine whether notable differences exist. Pregnant female rats (n = 824), rabbits (n = 540), and minipigs (n = 70) from vehicle control groups were included in the analysis, equating to 10,749 rat, 5,073 rabbit, and 378 pig fetuses collected at term by cesarean delivery. Preimplantation loss was more frequent than postimplantation loss in the rat and rabbit, whereas the opposite was observed in the minipig. Several external and visceral malformations and variations such as domed head, bent tail, abdominal edema, and anal atresia were observed in all 3 species. Visceral malformations of the heart and major blood vessels were remarkably more frequent in the minipig and rabbit, respectively; ventricular and atrium septum defects were observed in 1.9% and 2.1%, respectively, for the minipig fetuses, whereas they were observed in equal or less than 0.02% among the rat and rabbit fetuses evaluated in this study. Understanding species-dependent differences in spontaneous variations and malformations can be useful for the interpretation of embryo-fetal development study results. The current analysis identified relevant differences between commonly used species in reproductive toxicology with potential implications for data assessment.

Valproic Acid Induces the Hyperacetylation of P53, Expression of P53 Target Genes, and Markers of the Intrinsic Apoptotic Pathway in Midorganogenesis Murine Limbs.

In utero exposure to valproic acid (VPA), an anticonvulsant and histone deacetylase inhibitor (HDACi), increases the risk of congenital malformations. Although the mechanisms leading to the teratogenicity of VPA remain unsolved, several HDAC inhibitors increase cell death in cancer cell lines and embryonic tissues. Moreover, P53, the master regulator of apoptosis, is an established HDAC target. The purpose of this study was to investigate the effects of VPA on P53 signaling and markers of apoptosis during midorganogenesis in vitro limb development. Timed-pregnant CD1 mice (gestation day 12) were euthanized; embryonic forelimbs were excised and cultured in vitro for 3, 6, 12, or 24 hr in the presence or absence of VPA or valpromide (VPD), a non-HDACi analog of VPA. Quantitative RT-PCR and Western blots were used to assess the expression of candidate genes and proteins involved in P53 signaling and apoptosis. P53 hyperacetylation and a decrease (Survivin/Birc5 and Bcl2) or an increase (p21/Cdkn1a) in the expression of p53 target genes was observed only in VPA-exposed limbs. VPA exposure also triggered an increase in markers of apoptosis and DNA damage; the concentrations of cleaved caspase 9 and caspase 3, cleaved-poly (ADP-ribose) polymerase, and γ-H2AX were increased in VPA-exposed limbs. VPD treatment caused a small but significant increase in cleaved caspase 3. Thus, in vitro exposure to an HDACi such as VPA leads to P53 hyperacetylation, enhances the expression of P53 target genes, and triggers an increase in apoptosis that may contribute to teratogenicity.

The Murine Limb Bud in Culture as an In Vitro Teratogenicity Test System.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics the in vivo process, although at a slower rate, where growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided. The existence of transgenic mice with fluorescent markers for the different stages of endochondral ossification adds a further dimension to the technique by allowing striking time course observations of the developing limb. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signalling pathways.

The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis.

Histone deacetylases (HDACs) play a major role in chromatin remodeling, gene regulation, and cellular signaling. While the role of each class of HDAC during normal development is unclear, several HDAC inhibitors are embryotoxic; the mechanisms leading to the teratogenicity of HDAC inhibitors are not known. Here, we investigated the effects of class-specific HDAC inhibitors on the development of organogenesis-stage murine limbs. Timed-pregnant COL2A1-ECFP, COL10A1-mCherry, and COL1A1-YFP CD1 reporter mice were euthanized on gestation day 12; embryonic forelimbs were excised and cultured in vitro for 1, 3, and 6 days in the presence or absence of MS275 (a class I HDAC inhibitor), MC1568 (a class III HDAC inhibitor), Sirtinol (a class II HDAC inhibitor), or valproic acid, our positive control. Fluorescently tagged COL2A1, COL10A1, and COL1A1 served as markers of the differentiation of proliferative chondrocytes, hypertrophic chondrocytes, and osteoblasts, respectively. MS275 and valproic acid caused a reduction in expression of all three markers, suggesting effects on both chondrogenesis and osteogenesis. MC1568 had no effect on chondrocyte markers and mildly inhibited COL1A1 expression at 6 days. Sirtinol had no effect on COL2A1 expression or chondrocyte differentiation 1 day following exposure; however, it caused a drastic regression in limb cartilage and reduced the expression of all three differentiation markers to nearly undetectable levels at 6 days. MS275 and Sirtinol caused a 2.2- and 2.7-fold increase, respectively, in cleaved-caspase 3, a marker of apoptosis, suggesting embryotoxicity. These data demonstrate that inhibition of class I or III HDACs causes severe developmental toxicity and is highly teratogenic.

Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system.

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3-100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure.

Exposure to valproic acid inhibits chondrogenesis and osteogenesis in mid-organogenesis mouse limbs.

In utero exposure to valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, causes neural tube, heart, and limb defects. Valpromide (VPD), the amide derivative of VPA, does not inhibit HDAC activity and is a weak teratogen in vivo. The detailed mechanism of action of VPA as a teratogen is not known. The goal of this study was to test the hypothesis that VPA disrupts regulation of the expression of genes that are critical in chondrogenesis and osteogenesis during limb development. Murine gestation day-12 embryonic forelimbs were excised and exposed to VPA or VPD in a limb bud culture system. VPA caused a significant concentration- dependent increase in limb abnormalities, which was correlated with its HDAC inhibitory effect. The signaling of both Sox9 and Runx2, key regulators of chondrogenesis, was downregulated by VPA. In contrast, VPD had little effect on limb morphology and no significant effect on HDAC activity or the expression of marker genes. Thus, VPA exposure dysregulated the expression of target genes directly involved in chondrogenesis and osteogenesis in the developing limb. Disturbances in these signaling pathways are likely to be a consequence of HDAC inhibition because VPD did not affect their expressions.

The murine limb bud in culture as an in vitro teratogenicity test system.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context, since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics that in vivo, although the rate is slower. Growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signaling pathways. In this system, uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided.