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

Toxicological Sciences, Nov 2015

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.

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

TOXICOLOGICAL SCIENCES The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis France-He? le` ne Paradis 0 Barbara F. Hales 0 0 Department of Pharmacology and Therapeutics, McGill University , Montreal, QC , Canada H3G 1Y6 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 classspecific 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. VC The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: HDAC inhibitor; MS275; MC1568; Sirtinol; valproic acid; teratogen; limb development; chondrogenesis; osteogenesis; apoptosis - Histone deacetylases (HDACs) are enzymes that remove acetyl groups from the lysine residues of histone and nonhistone proteins, leading to effects on both gene expression and cellular signaling. HDACs are divided into four different classes; classes I, II, and IV comprise HDAC1?11, whereas SIRT1?7 are members of class III (Thiagalingam et al., 2003) . Inhibitors of class I and class II HDACs, such as valproic acid (VPA), an anticonvulsant, and vorinostat (SAHA), an anticancer agent used in the treatment of cutaneous T-cell lymphoma, are used as therapeutics (Duvic and Vu, 2007) . Several other HDAC inhibitors are currently under development and in clinical trials as anticancer agents (reviewed in West and Johnstone, 2014) . Studies suggest that HDAC class III inhibitors may have beneficial effects in the treatment of Parkinson?s disease (Outeiro et al., 2007; West and Johnstone, 2014) . However, some HDAC inhibitors are developmental toxicants in animal models, and the embryotoxicity of others is not known (Giavini and Menegola, 2014) . The specific mechanisms by which HDAC inhibitors cause malformations remain unresolved (Murko et al., 2013; Paradis and Hales, 2015) . As new therapeutic indications develop, an increasing number of women of childbearing age may be exposed to HDAC inhibitors. It is pivotal to decipher the specific roles of individual HDAC classes during development to predict the effects of their inhibition. The four classes of HDACs differ with respect to the homology of their catalytic domains, structures, and cofactor requirements (Blander and Guarente, 2004; Taunton et al., 1996) . HDAC1, 2, 3, and 8 are members of class I and homologous to the yeast RPD3 protein, whereas HDAC4, 5, 6, 7, 9, and 10 are class II HDACs, homologous to HDA1 (Yang and Seto, 2008). HDAC11 is the only member of class IV and has mixed properties, from classes I and II. While classes I, II, and IV are all Zndependent enzymes, class III HDACs are NAD?-dependent and are commonly referred to as Sirtuins (SIRT1?7), homologous to the yeast Sir2 (Thiagalingam et al., 2003) . The different HDACs have common targets, but they also have distinct high affinity substrates; HDAC1 and SIRT1 have a higher affinity for histones, whereas HDAC6 and SIRT2 target alpha-tubulin (Villalba and Alcain, 2012) . Mouse knockouts for HDAC1, 3, and 7 are embryonic lethal early during organogenesis; the knockouts of several others, such as HDAC2, SIRT1, and 6, are lethal perinatally, suggesting an important role for these enzymes during embryogenesis (Chang et al., 2006; Finkel et al., 2009; Montgomery et al., 2007, 2008) . However, little is known about the specific roles of HDAC classes during development. The limb has been used as a model system to study organogenesis for many years (Neubert and Barrach, 1977) . The vertebrate skeleton is formed through a process called endochondral ossification and requires the formation of a cartilage template that is subsequently replaced by bone matrix. The cartilage matrix is made of collagen type 2a1 (COL2A1) and is directly regulated by the transcription factor SOX9, expressed in proliferative chondrocytes (Bell et al., 1997) . These cells differentiate into hypertrophic chondrocytes, expressing the transcription factor RUNX2 and the structural protein collagen type 10a1 (COL10A1) (Ding et al., 2012) . Ultimately, these cells undergo cell death or differentiate into osteoblasts that secrete the bone matrix protein collagen type 1a1 (COL1A1) (Karsenty and Park, 1995) . In a previous study, our group has shown that VPA, a known human teratogen and inhibitor of class I and II HDACs, caused a decrease in Sox9, Col2a1, Runx2, and Col10a1 gene expression (Paradis and Hales, 2013) . Studies suggest that HDAC signaling intervenes in different steps of bone formation; HDAC1 modulates NKX3.2 and BMP signaling pathways, both known to play an important role during chondrogenesis (Cairns et al., 2012; Rigueur et al., 2015) . HDAC1, 3, 4, 5, 6, and 7 interact with RUNX2 (Bradley et al., 2011). HDAC4 knockout mice exhibit premature chondrocyte hypertrophy in the growth plate, while mice lacking HDAC2 exhibit a runted phenotype, suggesting congenital bone defects (Montgomery et al., 2007; Vega et al., 2004) . In this study, we investigated the effects of class-specific HDAC inhibitors on limb development in an in vitro limb bud culture system using class-specific HDAC inhibitors; MS275 (entinostat) inhibits HDAC1 and 3 (class I), MC1568 inhibits HDAC4 and 6 (class II), and Sirtinol inhibits SIRT1 and 2 (class III) (Beckers et al., 2007; Chiara et al., 2014; Mai et al., 2005) . VPA inhibits both class I and class II HDACs (Gottlicher et al., 2001) . We used triple transgenic mice expressing fluorescently tagged chondrogenic and osteogenic markers to readily assess the effects of our compounds on chondrogenesis and osteogenesis. MATERIALS AND METHODS Limb bud cultures and drug treatments. Col2a1-enhanced cyan fluorescent protein (Col2a1-ECFP), Col10a1-mCherry, and Col1a1-yellow fluorescent protein (Col1a1-YFP) CD1 reporter mice were a gift from David L. Butler (University of Cincinnati, Cincinnati, OH) and David Rowe (University of Connecticut Health Center, Farmington, CT) (Maye et al., 2011) . These mice express fluorescently tagged markers of cartilage and bone differentiation (Fig. 1). Mice were housed in the McIntyre Animal Resource Centre (McGill University, Montreal, QC, Canada), maintained on a 12-h light/ dark cycle, and allowed access to food and water ad libitum. The mice were mated overnight, and detection of a vaginal plug was considered gestation day (GD) 0. Timed-pregnant mice were euthanized by cervical dislocation on GD12, and their embryos were explanted. The embryonic forelimbs were cultured, as previously described (Paradis et al., 2012) . Briefly, limbs were excised in Hank?s balanced salt solution (HBSS), pooled, and cultured in vitro in 6 ml culture medium consisting of 75% BGJb medium (GIBCO BRL Products, Burlington, ON, Canada) and 25% salt solution supplemented with ascorbic acid (160 lg/ml) and gentamycin (1 ll/ml, GIBCO BRL Products). Each culture was gassed with 50% O2, 5% CO2, and 45% N2, and designated treatments were added; vehicle, sodium valproate (VPA, 3.6 mM, Sigma Chemical Co., St-Louis, MO, CAS number 1069-66-5) dissolved in distilled water, or MS275 (entinostat, 2.5 lM, SelleckChem, Houston, TX, CAS number 209783-80-2), MC1568 (2.5 lM, SelleckChem, CAS number 852475-26-4), or Sirtinol (50 lM, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, CAS number 410536-97-9) dissolved in dimethyl sulfoxide. Concentrations were chosen in accordance with previous studies and the manufacturers? IC50 recommendations (Poljak et al., 2014) . All animal studies complied with the guidelines established by the Canadian Council on Animal Care under McGill University protocol 1825. Limb morphology and differentiation Limbs (n ? 5 culture bottles, 5?6 limbs/bottle) were cultured for 6 days with a change of medium on day 3. On days 1, 3, and 6 of culture, pictures were taken using a Leica DFC450C digital camera (Leica Microsystems, Wetzlar, Germany) connected to a Leica M165 Fluorescent Stereo Microscope (Leica Microsystems). COL2A1-CFP-positive cartilage was quantified using a morphogenetic scoring system adapted from Neubert and Barrach and scores were normalized to control (Neubert and Barrach, 1977) . The proportions of limbs expressing the differentiation markers COL10A1-mCherry and COL1A1-YFP were quantified at 3 and 6 days. Primary cell cultures Embryonic forelimbs were collected on GD 12 and washed with low calcium HBSS (HBSS Ca/Mg free [Gibco, Life Technologies, Burlington, ON], 1 M HEPES pH 7.4, 0.15 M CaCl2). Limbs were cut and incubated in a collagenase solution (1.5 mg/ml collagenase type II, 300 mg/ml bovine serum albumin in low Ca HBSS) for 2 h at 36 C. Cells were sedimented from the suspension by centrifugation at 1000 g for 5 min and incubated with 2.5 g/ml Pancreatin for 15 min at room temperature. Cells were then washed with complete medium 199 (Medium 199, Gibco, Life Technologies, 26.2 mM NaHCO3, 25 mM HEPES pH7.4, 0.06 mg/ml gentamicin sulfate, 1% 100X Antibiotic-Antimycotic, 10% fetal bovine serum) and passed through a 40 lm filter cloth. Cells were counted with a hemocytometer, and approximately 1 106 cells were plated in a 12-well culture plate for 24 h. Cells were then treated with dimethyl sulfoxide (vehicle) or Sirtinol (50 lM, Santa Cruz Biotechnology). Cultures were stopped at designated times, and protein lysate was extracted. The experiment was repeated four times. Western blotting Whole cell lysates were obtained by sonication in lysis buffer containing protease inhibitors, consisting of 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5, 40 lg/ml bestatin, 0.2 M phenylmethylsulfonyl fluoride, 10 lg/ml leupeptin, and 6 lg/ml aprotinin. Total protein was extracted and quantified using spectrophotometric Bio-Rad protein assays (Bio-Rad Laboratories, Mississauga, ON, Canada). Proteins (15?30 lg per sample) were separated by SDS-PAGE acrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (BioSciences Inc., Baie d?Urf e?, QC, Canada). Precision standards (Bio-Rad Laboratories) were used as molecular weight markers. Membranes were blocked in 5% nonfat milk in TBS-T (137 mM NaC1, 20 mM Tris pH 7.4, 0.05% Tween 20) for 1 h at room temperature, probed overnight at 4 C with primary antibodies, washed, and incubated with the secondary antibody for 2 h at room temperature. Immunoblotting was done using polyclonal antibodies against histone 4 acetyl-lys 12 (H4K12Ac, EMD Millipore, Billerica, MA, 1:5000), alpha-tubulin acetyl-lys40 (Cell Signaling, Danvers, MA, 1:5000), cleaved-caspase 3 (Cell Signaling, 1:1000), HDAC1 (Cell Signaling, 1:2500), and beta-actin (Santa Cruz Biotechnology, Inc., 1:10 000). Monoclonal antibodies against SIRT1 (Abcam, Cambridge, MA, 1:2500) and HDAC6 (Abcam, 1:2500) were used. The secondary antibodies, conjugated to horseradish peroxidase (HRP), were anti-rabbit (GE Healthcare Limited, Baie d?Urf e?, QC, Canada, 1:5000), anti-mouse (GE Healthcare Limited, 1:5000), and anti-goat antibodies (Santa Cruz Biotechnology, Inc., 1:10 000). Western blots were visualized with the Enhanced Chemiluminescence Plus Kit (GE Healthcare Limited) and quantified by densitometry using a ImageJ imaging software (NIH, Bethesda, MD). Statistical analyses All data sets were analyzed statistically using SigmaPlot 11.0 (Systat Software, San Jose, CA). The Ranked Mann?Whitney U test and multiple comparisons were used for all data sets, with Bonferonni?s correction where multiple comparisons were done within a set. The minimum level of significance was P < .05. RESULTS HDAC inhibitory activity of MS275, MC1568, and Sirtinol in developing limbs To examine the HDAC inhibition activity of the class-specific HDAC inhibitors we chose, we used histone 4 acetylation as a marker of the activity of nuclear HDACs and tubulin acetylation as a marker of activity in the cytoplasm (Fig. 2). MS275, an inhibitor of HDAC1 and 3, induced a significant increase in H4k12Ac, whereas MC1568, an inhibitor of HDAC4 and 6, caused a hyperacetylation of tubulin. Surprisingly, Sirtinol, an inhibitor of SIRT1 and 2, did not cause an increase in acetylation of either target in cultured limb buds. We confirmed the expression of these targets in the developing limb by western blot (Supplementary Fig. 1). However, Sirtinol did induce tubulin hyperacetylation in limb primary cell cultures 3 h after exposure (Supplementary Fig. 2). The HDAC inhibition activity of VPA in the limb bud culture model was characterized in a previous study; VPA induced H4k12 hyperacetylation as early as 1 h following exposure (Paradis and Hales, 2015) . Class-specific HDAC inhibitors have distinct effects on chondrogenesis and osteogenesis To assess the effects of MS275 on chondrogenesis and osteogenesis, triple transgenic forelimbs were cultured in vitro in the presence of MS275 for 6 days. MS275 induced a decrease in CFP fluorescence (44% decrease in limb score) 1 d following exposure; changes were also observed at 3 and 6 days (76% and 66% decrease in score) (Fig. 3). Phalanges were often missing, and metacarpals were underdeveloped, short, and round. Hypertrophic chondrocytes and osteoblasts failed to differentiate, as shown by the complete absence of both mCherry and YFP fluorescence. In contrast, MC1568 treatment had no effects on chondrogenesis or on CFP-positive scores and only a mild nonsignificant effect on mCherry fluorescence at 3 days (Fig. 4). A decrease in COL1A1-YFP was observed at 6 days, suggesting that inhibition of HDAC class II affects osteoblast differentiation. Sirtinol exposure led to a phenotype distinct from that of either MS275 or MC1568 (Fig. 6). There were no changes in CFPpositive scores at 1 day and drastic decreases, to 42% and 91%, at 3 and 6 days, respectively, in Sirtinol-treated limbs. COL10A1mCherry and COL1A1-YFP fluorescence were also reduced at both 3 and 6 days. Moreover, qualitative analysis of the Sirtinoltreated limbs revealed radical effects on limb morphology, suggesting severe cytotoxicity (Fig 5, bright field). The effects of VPA on chondrogenesis and osteogenesis are shown in Figure 6. VPA treatment had a very similar effect to that of MS275; several digital condensations were missing, and a rapid decrease in CFP-positive score was observed at 1, 3, and 6 days following exposure (50%, 61%, and 60% decrease in limb score, respectively); the numbers of limbs exhibiting the differentiation markers COL10A1-mCherry and COL1A1-YFP were significantly reduced at both 3 and 6 days. MS275 and Sirtinol induce activation of caspase 3 Our next goal was to assess the extent of apoptosis in treated limbs using cleaved-caspase3 as a marker (Fig. 7). Both MS275 and Sirtinol significantly increased the cleavage of caspase 3 at 24 h following exposure, suggesting an increase in cellular death consistent with the limb phenotype we observed; in contrast, MC1568 did not increase cleaved caspase 3, suggesting that class II inhibition is not toxic to the cells. In a previous study using this model, VPA increased caspase 3 cleavage at 12 and 24 h following exposure (Paradis and Hales, 2015) . DISCUSSION Class-specific HDAC inhibitors had distinct effects on limb development. Inhibition of class I HDACs had highly detrimental effects on chondrogenesis and osteogenesis and was cytotoxic, whereas inhibition of class II HDACs had only mild effects on bone progenitor cell differentiation. Interestingly, inhibition of class III HDACs with Sirtinol had a delayed effect on chondrogenesis but was highly cytotoxic. This dramatic toxicity is interesting since the penetrance of Sirtinol in limb bud cultures is not clear. We found that treatment with Sirtinol induced the hyperacetylation of tubulin in cultures of primary limb cells but did not do this in cultures of the intact limbs. One possibility is that Sirtinol affects only a subset of limb cells, perhaps those on the surface of the organ. However, clear effects were observed on the limb morphology. A similar outcome was observed in a previous study investigating the effects of quantum dots in the limb bud culture system (Bigaeva et al., 2014); fluorescent quantum dots that were localized to the surface of the limb induced global limb malformations as well as the inhibition of chondrocyte and osteoblast differentiation. These observations suggest that the epidermal surface of the limb can affect the differentiation of the underlying mesenchyme. MS275 and Sirtinol both triggered programmed cell death in the developing limb. Teratogen-induced apoptosis in embryonic tissue has been associated with several birth defects. VPAinduced apoptosis in the somites of mouse embryos on gestational day 9 has been associated with an increase in neural tube defects (Di Renzo et al., 2010) . The severity of the malformations induced by treatment with hydroxyurea, an anticancer drug, was correlated with an increase in apoptosis and DNA fragmentation in specific tissues (Banh and Hales, 2013; Schlisser and Hales, 2013) . Thus, the enhanced apoptosis induced by HDAC class I and III inhibition in our limb model may also be observed in other malformation-sensitive tissues and organs during organogenesis. However, further studies are needed to investigate the molecular signalling pathways leading to this increase in cellular death. The VPA-induced rapid decrease in COL2A1 expression and decrease in mesenchymal condensation were remarkably similar to those observed with MS275, suggesting that these may be mediated through inhibition of HDAC class I, rather than class II. In a previous study, we showed that VPA exposure induced a rapid decrease in Sox9 and Runx2 gene expression; these effects were correlated with HDAC inhibition since valpromide, an analog of VPA lacking activity as an HDAC inhibitor, was inactive (Paradis and Hales, 2013) . Whether Sox9 and Runx2 signaling pathways are affected by our drug treatments remains to be investigated. The reduction in COL10A1 expression observed in MS-275 exposed limbs suggests that Runx2 signaling is affected by inhibition of class I HDACs. As mentioned previously, RUNX2 interacts with HDACs and becomes acetylated. However, its acetylation leads to an increase in its transcriptional activation that is inconsistent with the decrease in expression of the downstream targets that we observe in this study (Jeon et al., 2006) . The limb skeleton develops through the same process as other long bones, such as the ribs and vertebrae, via endochondral ossification. The flat bones of the body, including the bones of the skull, pelvis, scapula, and mandible, are formed by intramembranous ossification. Both processes require Runx2 and Col10a1 expression, suggesting that the formation of the entire skeleton may be affected by the inhibition of this pathway. MS275, MC1568, and Sirtinol target HDAC1/3, HDAC4/6, and SIRT1/2, respectively. HDAC1 knockout mice are embryonic lethal prior to limb bud formation (Montgomery et al., 2007) . In vitro, HDAC1 is involved in a number of pathways that regulate chondrogenesis, such as Nkx3.2 (Cairns et al., 2012) . HDAC3 knockout mice are runted, have fewer osteoblasts than their wild-type littermates, and exhibit a decrease in bone density (Razidlo et al., 2010) . These data are consistent with our results, suggesting that these members of the class I HDACs play pivotal roles during limb differentiation. The effects of MC1568 on limb development are inconsistent with the phenotype observed in HDAC4 knockout mice, which exhibit reduced cartilage formation, premature chondrocyte hypertrophy, and ossification (Vega et al., 2004) . MC1568 has been reported to inhibit matrix metalloproteinase 9 (MMP9) gene expression in extraembryonic tissue primary cell cultures (Poljak et al., 2014) . MMP9 is a matrix metalloproteinase that is essential for endochondral ossification; metalloproteinases degrade the cartilage extracellular matrix to create gaps where the osteoblasts will deposit the bone matrix (Ortega et al., 2003) . Thus, MC1568 may interfere with this degradation process in our model, thereby hindering osteoblast differentiation and Col1a1 expression. More studies are needed to assess whether the MC1568-triggered delay in ossification is transient or permanent. Our results are consistent with the phenotype observed in SIRT1 knockout mice; both the axial and appendicular skeletons of these mice exhibit abnormalities, although these are not as severe as the ones we observed in this study (Gabay et al., 2013) . This observation suggests that SIRT1 and SIRT2 may have complementary effects. Alternatively, Sirt1-/- mice may have compensatory mechanisms with the consequence that a pharmacologically induced transient inhibition of SIRT1 has a more detrimental effect than its deletion. This study provides valuable evidence for the diverse teratogenic effects of three class-specific HDAC inhibitors on mouse embryonic skeletal development. We showed that class I and III inhibitors are highly teratogenic and cause drastic structural malformations, whereas a class II inhibitor has only minor effects on ossification. Identifying the molecular pathways and targets leading to teratogenesis is pivotal to rapidly identify potential developmental toxicants and prevent their effects. ACKNOWLEDGMENTS We thank Dr. David L. Butler (University of Cincinnati, Cincinnati, OH) and Dr. David Rowe (University of Connecticut Health Center, Farmington, CT) for providing the Col2a1-ECFP, Col10a1-mCherry, and Col1a1-YFP reporter mice. FUNDING These studies were supported by grants from the Canadian Institutes of Health Research (CIHR, grant number: ?MOP86511?) awarded to B.F.H. and Fonds de recherche du Que? bec: Sant e? (FRQS) fellowship awarded to F.P. B.F.H. is a James McGill Professor. SUPPLEMENTARY DATA Supplementary data are available online at http://toxsci. oxfordjournals.org/. Banh , S. , and Hales , B. F. ( 2013 ). Hydroxyurea exposure triggers tissue-specific activation of p38 mitogen-activated protein kinase signaling and the DNA damage response in organogenesis-stage mouse embryos . Toxicol. 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Paradis, France-Hélène, Hales, Barbara F.. The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis, Toxicological Sciences, 2015, 220-228, DOI: 10.1093/toxsci/kfv174