ToxSci Advance Access originally published online on May 14, 2008
Toxicological Sciences 2008 104(2):397-404; doi:10.1093/toxsci/kfn094
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The Inhibition of Embryonic Histone Deacetylases as the Possible Mechanism Accounting for Axial Skeletal Malformations Induced by Sodium Salicylate
Department of Biology, University of Milan, 20133 Milan, Italy
1 To whom correspondence should be addressed at Department of Biology, University of Milan, via Celoria 26, 20133 Milan, Italy. Fax: +39-02-50314802. E-mail: elena.menegola{at}unimi.it.
Received April 21, 2008; accepted May 6, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In spite of the large use of salicylates, introduced into clinical practice more than 100 years ago, their anti-inflammatory and cancer preventive mechanisms are still under study. Teratogenic effects of salicylates have been reported in experimental animals since 1959 but the pathogenic pathways and the mechanisms of action were never described until now. The aim of this work is to verify if the inhibition of embryonic histone deacetylase (HDAC) enzymes and the consequent tissue hyperacetylation could be the mechanism responsible for axial skeletal defects described after the exposure of pregnant rodents to sodium salicylate (SAL). E8 pregnant CD-1 mice were intraperitoneally treated with SAL 0–150–300–450 mg/kg and sacrificed at 1, 3, 5 h after treatment or at term of gestation (E18). E8 embryos were processed for Western blotting and immunostaining analyses, while skeletons of E18 fetuses were double stained for bone and cartilage. A group of control E8 embryos were used to prepare embryonic nuclear extract for the HDAC enzyme assay. A significant SAL dose-related HDAC inhibition activity, compatible with a mixed-type partial inhibition mechanism, was detected. A clear dose-related hyperacetylation of histones was observed in embryos exposed in utero to SAL, with a peak at 3 h after treatment of dams. The most hyperacetylated organs were somites and the heart. Histone hyperacetylation is suggested to be the mechanism accounting for SAL-related axial skeletal and cardiovascular defects and is proposed as the mechanism responsible for other biological effects of salicylates.
Key Words: HDAC; hyperacetylation; histone; enzymatic activity; mouse; somite.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The inhibition of histone deacetylases (HDACs) has been recently proposed as a new mechanism of teratogenesis, correlated with axial skeletal malformations induced by a number of known HDAC inhibitors (HDACi) and by boric acid (BA) (Di Renzo et al., 2007a
, b; Menegola et al., 2005, 2006).
HDACs are nuclear and cytoplasmic enzymes, acting in prokaryotes and eukaryotes by removing acetyl groups from lysine residues of target proteins. In eukaryotes, nuclear substrates include histones. The most susceptible histones to epigenetical modifications by acetylation/deacetylation are H3 and H4 (Johnstone, 2002).
In general, histone deacetylation promotes a more condensed chromatin structure, allowing transcriptional repression, while histone acetylation increases gene activity (Mai et al., 2005).
The balance between deacetylation and acetylation, determined by the opposing activities of HDACs and histone acetyltransferases, controls cell proliferation, survival, and differentiation (Lehrmann et al., 2002).
HDACi are molecules with different structural characteristics. HDACi selectively alter gene expression in tumor cell lines by inducing specific acetylation of histones, transcription factors and other cell proteins directly or indirectly involved in transcription regulation (Bolden et al., 2006; Minucci and Pelicci, 2006). By increasing or decreasing the stability and function of these multiple target proteins, HDACi are known to induce cell growth arrest, cell death (apoptosis) and lineage differentiation (Rosato and Grant, 2005). For all these reasons, HDACi are considered promising molecules in the treatment of neoplastic diseases (Bolden et al., 2006; Minucci and Pelicci, 2006; Xu et al., 2007).
The ability of some HDACi (valproic acid [VPA], trichostatin A [TSA], apicidin, MS-275, sodium butyrate) to induce hyperacetylation on mouse embryo tissues has been recently reported and correlated to the teratogenic property of these molecules. In particular, hyperacetylation of target organs, such as the embryonic axial structures (somites), has been directly related to axial skeletal malformations (fusions of vertebrae and/or ribs, duplication of axial segments, homeotic transformations) in fetuses analyzed at term of gestation (Di Renzo et al., 2007a; Menegola et al., 2005, 2006). Moreover, on the basis of its inhibitory activity on HeLa cell and mouse embryonic HDACs, BA has been recently classified as a new HDACi. Significant HDAC inhibition activity, compatible with a mixed-type partial inhibition mechanism, has been observed at the developmental stage where BA also exhibited teratogenic effects. The direct correlation between embryonic hyperacetylation of axial tissues (somites) and axial malformations observed at term suggested HDAC inhibition as the mechanism explaining BA-related teratogenicity (Di Renzo et al., 2007b).
Similarly to VPA and BA, several embryotoxic agents, which include sodium salycilate (SAL), were reported to induce axial abnormalities when administered to pregnant rodents (Chernoff et al., 1987; Foulon et al., 1999; Kimmel and Wilson, 1973; Kimmel et al., 1971). In particular, supernumerary ribs (lumbar ribs) have been associated with the oral administration of SAL (300 mg/kg) to E9 pregnant rats and have been attributed to an anterior transformation of lumbar vertebrae identity into thoracic vertebrae identity, as suggested by Hox gene expression analysis (Wéry et al., 2005). Such a Hox gene expression shift has also been described in rats after BA exposure (Wéry et al., 2003).
The purpose of this study was to define if SAL-related axial skeletal defects could be explained by embryonic somite hyperacetylation, due to a specific HDAC enzymatic inhibition activity.
To answer these questions, we followed experimental procedures quite similar to those previously used for BA study (Di Renzo et al., 2007b). Teratogenic effects and embryonic hyperacetylation status were investigated on CD-1 mouse conceptuses exposed in utero at E8 to SAL 0–150–300–450 mg/kg dosed intraperitoneally. The enzymatic inhibitory profile was assessed on E8 embryo nuclear extracts and compared with the profile obtained with VPA.
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MATERIALS AND METHODS |
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Animals
CD-1 mice (Charles River Laboratories, Calco, Italy) were kept in standard conditions (light cycle 12 h, light from 7 to 19; humidity 55 ± 5%, T = 22 ± 2°C) at least 1 week before the initiation and during the study period. Virgin females were caged overnight with males of proven fertility; the presence of a vaginal plug the following morning (day 0 of gestation, E0) was considered as evidence of mating. Pregnant mice, randomly assigned to a treatment group, on day 8 (E8, embryonic stage 5–15 somites) received intraperitoneal injections (0.1 ml/10 g body weight) of 150, 300, 450 mg/kg SAL (sodium salicylate; Sigma, Milan, Italy), or of vehicle alone. Intraperitoneal injection was used in order to obtain a rapid concentration peak of the tested substance. This unusual route of exposure, in fact, allows a pharmacokinetic profile quite similar to the endovenous injection (not easy to perform in mice). Clinical signs were recorded after treatment and until sacrifice. Maternal body weight, food and water consumption were recorded at day 0 and daily from day 8 until sacrifice. Mice were killed after 1, 3, or 5 h from the treatment for Western blotting and immunohistochemical analysis. Some females treated with SAL were sacrificed at term of pregnancy (E18) for skeletal examination.
A group of untreated pregnant females were used to obtain E8 embryos for embryonic nuclear extract preparation.
Analysis at Term
Ten control females and 10 females for each SAL-treated group were sacrificed at term of gestation and the number of implants, live fetuses, dead fetuses, and resorptions were recorded in order to calculate the postimplantation loss index ([dead fetuses+ resorptions]/[total implantation sites] x 100). Live fetuses explanted from the uterus were externally examined, weighed and all processed for double staining for bone and cartilage, using the method previously described (Menegola et al., 2001). Placentas were weighed.
Analysis at Midgestation
Ten controls, 5–10 SAL-treated females for each sacrifice time were killed at midgestation. Embryos explanted from the uteri were randomly assigned to Western blot analysis or immunohistochemistry.
Western blotting.
Total cellular extracts were prepared from embryos explanted in saline. Samples were sonicated and centrifuged at 16,500 x g for 10 min at 4°C. Protein concentration was measured by the Bradford (1976) method. The supernatants were added to XT sample buffer (Bio Rad, Segrate, Italy) and heated at 100°C for 5 min for protein denaturation.
Equal amounts of protein (22.5 µg) were separated on 18% polyacrylamide gel (sodium dodecyl sulfate–polyacrylamide electrophoresis) and transferred to nitrocellulose membrane. Immunoblotting was performed using polyclonal antibody to histone H4 hyperacetylated (Upstate, Segrate, Italy) (dilution 1: 8000), to acetylated lysine (Cell Signalling Technology, Celbio SpA, Pero, Italy) (dilution 1:1000) or using monoclonal antibody to tubulin (Sigma) (dilution 1:200) to check protein loading. After incubation with secondary antibodies conjugated to alkaline phosphatase (anti-rabbit, 1: 2500 dilution; anti-mouse, 1:2500 dilution) (Sigma, Milan, Italy) protein expression was colorimetrically detected with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium (Sigma).
Quantitative analysis of signal was performed using the program Quantity One (Bio Rad). The integrated signal intensity obtained after immunodetection of hyperacetylated histone H4 was normalized on that obtained after immunodetection of tubulin in order to calculate the hyperacetylation index.
Immunohistochemistry.
After fixation with 3% paraformaldehyde 6 h at 4°C, control and SAL-exposed embryos were embedded in paraffin and sectioned (5 µm). After rehydration, sections were blocked for endogenous peroxidase activity in 0.3% H2O2 in distilled water and blocked in fetal calf serum. Immunolocalization was performed using the same antihyperacetylated histone H4 antibody also used for immunoblotting (overnight, diluted 1:8000). After incubation with secondary antibody anti-rabbit IgG peroxidase (Boehringer, Milan, Italy) (dilution 1:40) the staining was carried out with the substrate solution, diaminobenzidine (Sigma) and H2O2.
Isolation of Nuclear Extracts
Embryonal nuclear extracts were prepared from mouse embryos of 5–15 somites as previously described (Di Renzo et al., 2007b). Briefly, 200 embryos were collected into 1 ml of cold lysis buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 15mM MgCl2, 250mM sucrose, 0.5% NP-40, and 0.1mM ethylene-glycol-tetracetic acid), sonicated, diluted to 4 ml in the same buffer, and kept on ice for 15 min. The nuclei were collected by centrifugation through 16 ml of cold sucrose cushion (30% sucrose, 10mM Tris-HCl, pH 7.5, 10mM NaCl, and 3mM MgCl2) at 1300 x g for 10 min at 4°C, resuspended in 2 ml of cold 10mM Tris-HCl, pH 7.5, containing 10mM NaCl, and centrifuged at 1300 x g for 10 min at 4°C. The isolated nuclei were resuspended in 0.4 ml of extraction buffer (50mM hydroxyethylpiperazine ethane sulfonic acid, pH 7.5, containing 420mM NaCl, 0.5mM ethylenediaminetetraacetic acid, 0.1mM EGTA, and 10% glycerol) using a pellet pestle, incubated on ice for 30 min, and centrifuged at 10,000 x g for 10 min at 4°C. The supernatant containing the crude nuclear extracts was stored at –80°C until use. To determine protein concentration of the extracts, the bicinchoninic acid reagent assay (Micro BCA, Pierce, Rockford, IL) was used.
Enzymatic Activity Assay and Kinetic Measurements
HDAC activity was measured by using a fluorescence activity assay kit (Cayman Chemical, Ann Arbor, MI). The activity was assayed according to the manufacturer's instructions with at least three repeats. The concentration effect curve was obtained by incubating four micrograms of embryo nuclear extracts in presence of 100µM acetylated fluorogenic substrate and 0.1–1–10–100mM SAL or VPA. VPA was used as positive control, being a well known HDACi both in adult cells and in embryo nuclear extracts (Di Renzo et al., 2007b). These concentrations were selected on the basis of range finding tests in order to use concentration levels active for both VPA and SAL and on the basis of the pharmacokinetic evaluation of Higgs et al. (1987) showing a peak plasma concentration just over 1mM in male rats orally treated with 200 mg/kg salicylate. Kinetic analysis was performed by incubating four micrograms of embryo nuclear extracts with acetylated fluorogenic substrate (25–200µM) in 160 µl of assay buffer in the presence or absence of SAL (1–100mM). SAL was dissolved in water and neutralized before dilution with HDAC assay buffer (25mM Tris-HCl, pH 8.0, 137mM NaCl, 2.7mM KCl, and 1mM MgCl2). The deacetylated reaction was carried out at 37°C for 60 min, and stopped by the addition of 40 µl of HDAC developer containing 5µM TSA. After 15 min, fluorescence activity was measured with a Fluorocount reader (Packard BioScience, Walkham, MA) at 360 nm excitation and 465 nm emission. Experimental data were analyzed by a computer using a sigmoidal dose-response function or multiparameter, iterative, nonlinear regression program based on the Marquardt-Levenberg algorithm (Sigma Plot, Jandel, CA). For kinetic analyses, data obtained in the presence and in the absence of SAL were initially fitted to a simple Michaelis-Menten equation:
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Statistical Analysis
Data, given as mean ± SD, were analyzed for statistical significance by ANOVA followed by Tukey's test. For data collected at term of gestation, the litter was considered as the unit of comparison. For malformation analysis, fetuses were the experimental unit, and the percentage of malformed fetuses was analyzed using the Chi-square test. The level of significance was set at p < 0.05.
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RESULTS |
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Data at Term of Gestation
No clinical signs and other signs of maternal toxicity (as shown by body weight gain during gestation, food, and water consumption) were recorded in groups treated with SAL (data not shown). The means of implantation sites, dead and resorbed conceptuses, the postimplantation loss index, the means of fetal and placental weights in SAL-exposed groups did not differ from those of controls (data not shown). No external abnormalities were detected both in control and in SAL-treated groups. By contrast, a specific and dose-related effect was observed after skeletal examination in groups exposed to SAL. The abnormalities included homeotic respecifications of axial segments (transformation of body parts into the likeness of something else, as defined by Bateson, 1894
, including lumbar ribs) present also in controls but significantly increased in all dose groups, fusions of vertebrae and/or ribs and duplication of segments, both limited to SAL 300 and 450 mg/kg (Table 1, Fig. 1).
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Data at Midgestation
No abnormalities were observed after external morphological examination of embryos.
Western blotting.
Western blotting was performed on homogenates obtained from embryos explanted at 1, 3, 5 h after treatment. A clearly dose- and time-related signal for hyperacetylated histone H4 was observed in the protein extracts of the SAL-exposed embryos. Hyperacetylation was more evident in embryos exposed to SAL 450 mg/kg and explanted after 3 h from treatment (Figs. 2b and 2c). To verify the acetylation status of the entire proteome, Western blots using the antibody anti-acetylated lysine were performed on SAL 450 mg/kg embryos explanted at 3 h after treatment and revealed that only histones (H2, H3, H4) were acetylated (Fig. 2a).
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Immunohistochemistry.
Immunohistochemical analysis, using the antibody antihyperacetylated histone H4, was restricted to embryos explanted at 3 h after treatment. A weak signal, restricted to a small number of cells, was observed in control tissues, while a dose-related increased signal was observed in somites and the cardiac tissue of SAL-exposed embryos (Fig. 3).
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; mesenchyme, *; neural epithelium, °).Enzymatic Activity (HDAC) Assay
To test the effect of SAL and VPA on embryonic HDAC enzymes, we prepared nuclear extracts from E8 mouse embryos and performed an enzymatic inhibition assay. Fig. 4 shows that embryonic HDAC activity was significantly affected in a dose-dependent manner by SAL (IC50 = 23.9mM) and by VPA (IC50 = 1.21mM). We further investigated the SAL inhibition activity on a kinetic basis. Inhibition kinetics in the presence of SAL 10, 50, and 100mM as a function of substrate concentration was measured by using embryonic nuclear extracts. HDAC activity follows a Michaelis-Menten kinetics both in the presence and in the absence of SAL (Fig. 5a). The calculated Km and Vmax values are reported in Table 2. In the presence of SAL both Km and Vmax were affected: the Km value increased whereas the Vmax decreased suggesting that the aspirine metabolite behaves as a mixed-type inhibitor. This diagnosis was confirmed by plotting the data according to the Lineweaver-Burk equation: activities were interpolated by straight lines intersecting over the x axis in the second quadrant (Fig. 5b). The replotting of the slopes as a function of inhibitor concentration gives a straight line which intersects the x axis at the point -Ki. For SAL a Ki value of 8.6mM was calculated (Fig. 5b, inset).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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SAL, the natural deacetylated form of aspirin, is responsible for the anti-inflammatory action of this drug, introduced into clinical practice more than 100 years ago and until now one of the most widely used medicines. SAL is a phenolic compound also naturally present in fruits and plants, where it plays a central role in the development of local and systemic disease resistance to pathogenic infections (Dangl, 1998
; Dempsey and Klessig, 1994).
Salicylates have been recently evaluated for new multiple therapeutic activities, including range of antineoplastic properties of both synthetic salicylates and a vegetable-rich diet (Battezzati et al., 2006; Blacklock et al., 2001; Paterson and Lawrence, 2001; Paterson et al., 1988).
The antineoplastic effects of salicylates are still under investigation, and seem to be related to induction of apoptosis, inhibition of cell growth and suppression of angiogenesis (Borthwick et al., 2006; Cory and Cory, 2005; Katerinaki et al., 2006; Lee et al., 2003; Mahdi et al., 2006; Perugini et al., 2000). From a molecular point of view, the action of salicylates mainly involves inhibition of cyclooxygenase enzymes Cox-1 and Cox-2 that are responsible for the synthesis of prostaglandins and regulation of many homeostatic functions as well as inflammation (Vane, 1971). However, some lines of evidence suggest that aspirin and salicylate also act through Cox-independent mechanisms (Tegeder et al., 2001). Here, we supply the first evidence that inhibition of HDAC could be added to the mechanisms accounting the salicylate biological activities.
As far as teratological effects are concerned, salicylates have been tested since the 1950's, when Warkany and Takacs (1959) subcutaneously injected E9–E11 pregnant rats with 60–180 mg SAL and obtained axial defects, craniorachischisis, exencephaly, hydrocephaly, facial clefts, eye defects, and gastroschisis. About 20% of the fetuses with similar doses of SAL on days 9 and 10 of pregnancy also showed a variety of cardiovascular defects (transposition of the aorta, inter ventricular septal defects, dextrocardia) (Takacs and Warkany, 1968). Since then, numerous publications confirmed these results in rats and extended them to mice and monkeys, while no teratogenic effects were reported in rabbits (Cappon et al., 2003). SAL has been indeed identified as the causative agent in aspirin teratogenesis in rats (Kimmel et al., 1971). More recent in vitro studies on rat postimplantation whole embryo culture, described a direct embryotoxic effect (including central nervous system [CNS], limb, and axial defects) of SAL itself, rather than its generated metabolites (Ebron-McCoy et al., 1988; Greenaway et al., 1984).
The aim of the present work was to determine if SAL-induced axial defects were related to somite hyperacetylation and due to a specific HDAC enzymatic inhibition, similar to what has been previously described for a number of HDACi (VPA, TSA, apicidin, sodium butyrate, MS-275) and for BA (Di Renzo et al., 2007a, b; Menegola et al., 2005, 2006). The intraperitoneal treatment of E8 pregnant mice with SAL 150–300–450 mg/kg confirmed the previously described teratogenic effects of SAL at the axial skeleton level. No external abnormalities (including CNS abnormalities) were induced by SAL in all treated groups. The observed abnormalities as well as histone hyperacetylation of specific tissues appeared in a dose-related manner. Hyperacetylation peaked at 3 h after treatment, and has been related to the HDAC inhibition activity. The enzymatic assay, performed by using embryo nuclear extracts obtained from CD-1 embryos explanted at the same stage selected for the exposure to SAL, revealed that both VPA and SAL inhibited the embryonic HDAC activity. The comparison between the curves obtained after VPA or SAL incubation shows a greater embryonic HDAC inhibitory activity for VPA (the IC50 are 1.21 and 23.9mM for VPA and SAL, respectively). However, the kinetic analysis clearly revealed that SAL affected substrate affinity and maximal velocity, accordingly to the notion that SAL is a mixed-type HDACi. Even if HDAC inhibition observed by using embryo nuclear extracts appears significant only at high concentrations (10–100mM), the evidence that the effects are tissue-specific is consistent with the hypothesis that HDAC inhibition could be the direct mechanism for hyperacetylation of somites and, consequently, for axial malformations observed at term of gestation. After SAL exposure, in embryonic tissues, hyperacetylation was not restricted to somites, but also included the heart. Even if no visceral analysis was performed in the present study, cardiac hyperacetylation could be related to the SAL-induced cardiovascular defects reported in the literature (Takacs and Warkany, 1968). Further studies are, however, necessary to confirm or reject this hypothetical correlation. Neural tube defects have been described after subcutaneous SAL injection in rat in vivo models and in in vitro works (Ebron-McCoy et al., 1988; Greenaway et al., 1984; Warkany and Takacs, 1959) but were never observed both in the present study and by Foulon et al. (1999) and by Wéry et al. (2005) in rats treated by gavage. These data are in accordance with the observation that after immunohistochemistry histone hyperacetylation was never detected at the level of embryonic neuroepithelium.
This is the first evidence for embryonic HDAC inhibition by SAL, suggesting it as the molecular model for SAL-mediated specific hyperacetylation at the level of target tissues probably accounting for the observed axial teratogenic effects.
The identification of safe HDACi is one of the most urgent goals in the epigenetic treatment of neoplastic diseases. The anti-inflammatory properties of salicylates have been known for decades, and they are among the oldest and most widely used drugs in the world. The Greek physician Hippocrates wrote in the fifth century BC about a bitter powder extracted from willow bark that could ease aches and pains and reduce fevers. Their low toxicity in adults is well known and justifies their common use.
Recent evidence that SAL may reduce the risk of colorectal cancer, as observed in people taking aspirin or having vegetable-rich diet, suggests that this compound is active in the treatment of tumors (Paterson and Lawrence, 2001). Moreover, SAL-related inhibition of cell growth and angiogenesis, as well as induction of apoptosis, has been demonstrated in different experimental models (Borthwick et al., 2006; Cory and Cory, 2005; Katerinaki et al., 2006; Lee et al., 2003; Mahdi et al., 2006; Perugini et al., 2000). On the other hand, HDACi are known to induce cell growth arrest, cell death and lineage differentiation (Minucci and Pelicci, 2006; Rosato and Grant, 2003; Xu et al., 2007). The epigenetic activity of SAL, demonstrated in the present work in embryo models, could also help explain the described antitumoral properties of salicylates, even if its HDAC inhibitory activity has still to be established in adult and tumoral cells.
In conclusion, (1) teratogenic effects of SAL have been confirmed; (2) a relationship between HDAC inhibition, somitic histone hyperacetylation, skeletal defects has been suggested; (3) a probable relationship between HDAC inhibition, cardiac histone hyperacetylation, cardiovascular malformation has been proposed; (4) a possible molecular model for salicylate-mediated effects has been discussed.
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FUNDING |
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FIRST grant from the University of Milan.
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NOTES |
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2 These authors contributed equally to this work.
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ACKNOWLEDGMENTS |
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We would like to acknowledge Mr Calogero Bella for his skillful technical assistance.
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REFERENCES |
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