Key Event Title
|Level of Biological Organization|
Key Event Components
|enzyme inhibitor activity||histone deacetylase 1||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Histone deacetylase inhibition leading to testicular toxicity||MolecularInitiatingEvent|
|HDAC inhibition leads to impeded craniofacial development||MolecularInitiatingEvent|
|HDAC inhibition leads to neural tube defects||MolecularInitiatingEvent|
|Suberoylanilide hydroxamic acid|
|Adult, reproductively mature||Moderate|
Key Event Description
Site of action: The site of action for the molecular initiating event is a cell.
The nucleosome consists of core histones having classes of H2A, H2B, H3 and H4) [Damaskos, 2017]. DNA strand (about 200 bp) wound around the core histones, where histone deacetylase (HDAC) effects on the lysine residue of the histone to hydrolyze the acetyl residue [Damaskos, 2017]. Histone deacetylase inhibitor (HDI) inhibits HDAC and acetylate the histones and release the DNA strand to induce the binding of transcription factors [Taunton, 1996]. HDIs have potentials as anti-cancer pharmaceuticals since HDIs induce the transcriptional restoration of epigenetically silenced tumor suppressor genes by regulating acetylation of histones and non-histone proteins [Lee, 2016] [Minucci, 2006].
It is known that 18 HDAC isoforms are classified into four classes: class I HDACs (isoforms 1, 2, 3, 8), class II isoforms (4, 5, 6, 7, 9, 10) and class III HDACs (the sirtuins) and HDAC11 [Weichert, 2009, Barneda-Zahonero, 2012]. HDACs 1, 2 and 3 are ubiquitously expressed, whereas HDAC8 is predominantly expressed in cells with smooth muscle/myoepithelial differentiation [Weichert, 2009]. HDAC6 is not observed to express in lymphocytes, stromal cells and vascular endothelial cells [Weichert, 2009]. Class III HDACs sirtuins are widely expressed and localized in different cellular compartments [Barneda-Zahonero, 2012]. SirT1 is highly expressed in testis, thymus and multiple types of germ cells [Bell, 2014]. HDAC11 expression is enriched in kidney, brain, testis, heart and skeletal muscle [Barneda-Zahonero, 2012].
Description from EU-ToxRisk deliverable:
Eukaryotic histone deacetylases (HDACs) are grouped, according to phylogeny, into classes 1 through 4 (Gregoretti et al., 2004). The members of groups 1,2 and 4 are dependent on a zinc ion and a water molecule at their active sites, for their deacetylase function. The Sirtuins of class 3 depend on NAD+, and are considered impervious to most known HDAC inhibitors (Drummond et al., 2005).
Several structurally distinct groups of compounds have been found to inhibit HDACs of class 1, 2 and 4, among others short-chain fatty acids (e.g. butyrate and VPA), hydroxamic acids (e.g. TSA and SAHA) and epoxyketones (e.g. Trapoxin) (Drummond et al., 2005). The hydroxamic acids seem to exert their inhibitory action by mimicking the acetyl-lysine target of HDACs, chelating the zinc ion in the active site and displacing the water molecule (Finnin et al., 1999). Several recent high resolution crystal structures support this mode of inhibition (Decroos et al., 2015; Luckhurst et al., 2016). The mode of inhibition of epoxyketones seems to function the formation of a stable transition state analog with the zinc ion in the active site (Porter and Christianson, 2017). The inhibitory actions of the short-chain fatty acids are less well defined, but it has been speculated that VPA blocks access to the binding pocket (Göttlicher et al., 2001). It has been shown that VPA exert similar gene regulatory effects to TSA, on a panel of migration related transcripts in neural crest cells (Dreser et al., 2015) supporting a mode of action similar to hydroxamic acid type HDAC inhibitors.
How It Is Measured or Detected
The measurement of HDAC inhibition monitors the decrease in histone acetylation. The measurement methods include the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon, 2004]. The histones are isolated from pellets of cells treated with HDIs, followed by acid-urea-triton gel electrophoresis, western blotting, and immunohistochemistry [Richon, 2004]. Epigenetic modifications including the histone acetylation are measured using chromatin immunoprecipitation-microarray hybridization (ChIP-chip) [ENCODE Project Consortium, 2004, Ren, 2004]. ChIP detects physical interaction between transcription factors or cofactors and the chromosome [Johnson, 2007]. The HDAC activity is measured directly with ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) by calculating the ratio of deacetylated peptide and acetylated peptide [Zwick, 2016].
Description from EU-ToxRisk deliverable:
HDAC inhibition can be followed by several different approaches:
-Western blots applying antibodies targeting specific acetylated proteins.
-Commercial fluorimetric and colorimetric kits can be applied to assay HDAC activity from various biological extracts.
Domain of Applicability
The inhibition of HDAC by HDIs is well conserved between species from lower organism to mammals.
- HDIs reduced lethality in Drosophila model and the HDAC activity was inhibited with HDIs in rat PC12 cells [Steffan, 2001].
- HDIs inhibited restores the rate of resorption of subretinal blebs in hyper glycemia in brown Norway rat and HDAC activity was inhibited with HDIs in human ARPE19 cells [Desjardins, 2016].
- HDIs were approved as drugs for multiple myeloma and T-cell lymphoma by FDA [Ansari, 2016].
- HDIs inhibited cell growth in human non-small cell lung cancer cell lines [Miyanaga, 2008].
- HDAC acetylation level was increased by HDIs in MRL-lpr/lpr murine model of lupus splenocytes [Mishra, 2003].
- SAHA increased histone acetylation in brain and spleen of mice [Hockly, 2003].
- MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen, 2004].
- It is also reported that MAA inhibits HDAC activity in testis cytosolic and nuclear extract of juvenile rats (27 days old) [Wade, 2008].
- VPA and TSA inhibit HDAC enzymatic activity in mouse embryo and human HeLa cell nuclear extract [Di Renzo, 2007].
- HDAC inhibitors, phenylbutyrate (PB) (2 mM) and TSA (200 nM) acetylate histones H3 and H4 in synovial cells from rats with adjuvant arthritis [Chung, 2003].
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
HDIs are classified according to chemical nature and mode of mechanism: the short chain fatty acids (e.g., butyrate, valproate), hydroxamic acids (e.g., suberoylanilide hydroxamic acid or SAHA, Trichostatin A or TSA), cyclic tetrapeptides (e.g., FK-228), benzamides (e.g., N-acetyldinaline and MS-275) and epoxides (depeudecin, trapoxin A) [Richon, 2004, Ropero, 2007, Villar-Garea, 2004]. There is a report showing that TSA and butyrate competitively inhibits HDAC activity [Sekhavat, 2007]. HDIs inhibit preferentially HDACs with some selectiveness [Hu, 2003]. TSA inhibits HDAC1, HDAC3 and HDAC8, whereas MS-27-275 has inhibitory effect for HDAC1 and HDAC3 (IC50 value of ~0.2 mM and ~8 mM, respectively), but no effect for HDAC8 (IC50 value >10 mM) [Hu, 2003]. TSA inhibits HDAC1, 2, 3 of class I HDACs. HDAC 1, 4, 6 are related to tumor size [Damaskos, 2016]. MAA (2 or 5 mM) inhibited HDAC activity in dose-response manner in rat testis cytosolic and nuclear extracts [Wade, 2008].
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