Teratogenic Effects Mediated by Inhibition of Histone Deacetylases:  Evidence from Quantitative Structure Activity Relationships of 20 Valproic Acid Derivatives

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  • Teratogenic Effects Mediated by Inhibition of Histone Deacetylases:Evidence from Quantitative Structure Activity Relationships of 20

    Valproic Acid Derivatives

    Daniel Eikel, Alfonso Lampen, and Heinz Nau*,

    Department of Food Toxicology and Chemical AnalysissFood Toxicology, Center for SystemicNeuroscience HannoVer, Center for Food Science, UniVersity of Veterinary Medicine HannoVer, Foundation,Bischofsholer Damm 15, D-30173 HannoVer, Germany, and Department of Food Safety, Federal Institute for

    Risk Assessment (BfR), Thielallee 88-92, D-14195 Berlin, Germany

    ReceiVed August 12, 2005

    The widely used antiepileptic drug valproic acid (VPA), which is also used in migraine prophylaxisand the treatment of bipolar disorders, is also under trial as an anticancer agent. Despite its wide rangeof therapeutic applications, VPA also has two severe side effects: acute liver toxicity and teratogenicity.The mechanism of action for all these properties is unknown to date, but recently, it was shown thatVPA is able to inhibit the enzyme class of histone deacetylases (HDACs), proteins with a fundamentalimpact on gene expression and therefore possible molecular targets of VPA-induced signaling cascades.The purpose of this study was to determine if teratogenic side effects of VPA could be linked to itsHDAC inhibition ability by studying a large set of structurally diverse derivatives based on the VPAcore structure. We demonstrate that only VPA derivatives with a teratogenic potential in mice are ableto induce a hyperacetylation in core histone H4 in teratocarcinoma F9 cells. We also demonstrate thatthis marker of functional HDAC inhibition occurs almost immediately (15 min) after exposure of F9cells to VPA, whereas no influence on the HDAC protein levels (HDAC 2 and HDAC 3) could bedetected even after 24 h of treatment. Further measurement of the IC50(HDAC) values of VPA derivativesin a human HDAC enzyme test system revealed an activity range from 10 to 10 000 M; in somederivatives, HDAC inhibition ability was 40 times that of VPA. We also show a quantitative correlationbetween the IC50(HDAC) and the teratogenic potential of VPA derivatives, which clearly points towardHDACs as the formerly described teratogenic receptors of VPA-induced neural tube defects (NTDs).

    IntroductionValproic acid (VPA)1 is one of the antiepileptic drugs most

    frequently prescribed (1); it is also used clinically in a varietyof other pathologies including bipolar disorders (2) and migraineprophylaxis (3). Currently, VPA is in clinical trials and underinvestigation as an anticancer agent (4). In addition to its excitingbroad spectrum of properties, VPA is generally well-tolerated(5) but exhibits two rare but severe side effects: liver toxicity(6) and teratogenicity (7).

    Aside from malformations of the heart (8), the predominantVPA-induced teratogenic effects in humans are due to a failureof the neural tube to close (neural tube defects, NTDs) leadingto conditions such as spina-bifida-aperta, anencephaly, andexencephaly (9). These teratogenic effects can also be inducedin mice models by differential administration of VPA duringthe sensitive time of gestation and have been described as the

    NMRI-exencephaly-mouse model (10) and the NMRI-spina-bifida-aperta-mouse model (11).

    Our group has used these mouse models to establish astructure-activity relationship with a variety of derivatives basedon the VPA core structure, and important structural prerequisitesfor VPA-induced NTDs have been discovered (12). VPA itselfhas been shown to be teratogenic, and some of its plasmametabolites also exhibit teratogenic effects (13). Elongation ofone side chain and introduction of a triple bond in position C4in the second side chain resulted in VPA analogues withincreased induction of exencephaly in NMRI mice, while afurther branching of a side chain diminished the teratogeniceffects (14, 15). Derivatization of the carboxylic acid to thecorresponding ester, amides, or hydroxamic acids also decreasedor completely abolished the teratogenic potency (13, 16-18).In addition to these structural prerequisites, the most interestingand striking structural factor of VPA teratogenicity is the Rhydrogen atom at position C2. On one hand, teratogenic effectswere minimized or completely prevented in the NMRI-exen-cephaly-mouse model by substitution of the R hydrogen at C2with a methyl group (15), hydroxyl group (M. Radatz, unpub-lished results), or a fluorine atom (19) or by introduction of adouble bond between C2 and C3 (20). On the other hand,structure-activity relationship studies (SARs) have also dem-onstrated that there is differentiation between enantiomeric VPAanalogues if a chiral center is position C2 (21). These findingsultimately led to the theoretical prediction of a stereoselectivereceptor of VPA-induced teratogenic effects (22).

    * Corresponding author: Prof. Dr. Dr. h.c. Heinz Nau, University ofVeterinary Medicine Hannover, Foundation, Center for Systemic Neuro-science Hanover, Center for Food Science, Department of Food Toxicologyand Chemical AnalysissFood Toxicology, Bischofsholer Damm 15, 30173Hannover, Germany. E-mail, Heinz.Nau@tiho-hannover.de; tel., 0049-511-856-7600; fax, 0049-511-856-7680.

    University of Veterinary Medicine Hannover. Federal Institute for Risk Assessment (BfR).1 Abbreviations: HDAC(s), histonedeacetylase(s); VPA, valproic acid;

    NTD(s), neural tube defect(s); H4, core histone 4; AcH4, acetylated corehistone 4; NMRI, Naval Medical Research Institute; IC50(HDAC), substrateconcentration with half-maximum HDAC enzyme activity; TSA, tricho-statin A.

    272 Chem. Res. Toxicol. 2006, 19, 272-278

    10.1021/tx0502241 CCC: $33.50 2006 American Chemical SocietyPublished on Web 01/11/2006

  • Whereas the antiepileptic potential of VPA is much lesssensitive to changes in the chemical structure, even slightchanges in the molecular structure of VPA derivatives (e.g.,enantiomers) can completely prevent malformations in theNMRI-exencephaly-mouse model without substantially influ-encing the pharmacokinetic properties (23, 24). This phenom-enon represents a unique possibility for the studying of theunderlying molecular mechanisms of VPA-induced teratoge-nicity by investigating a proper set of structurally diversederivatives. Such a screening approach covering VPA deriva-tives with both much higher and much lower teratogenic potencyhas already been applied by our group to demonstrate theinvolvement of peroxisome proliferation-activated receptors(PPARs) in VPA-induced NTDs. While PPAR R and wereactivated nonstructurally, specifically the PPAR (PPAR )isoform was activated only by VPA analogues with highteratogenic potency (25, 26). Since it was not possible todemonstrate a direct binding of VPA by PPARs (27), it wassuggested that PPAR (PPAR ) is a molecular marker forVPA-induced NTDs rather than a molecular target of VPA (28).

    It was recently shown that the enzyme class of histonedeacetylases (HDACs) is inhibited by VPA, and it was proposedthat HDACs were a possible enzyme target structure of bothanticancer and teratogenic properties of VPA (29-32) due to

    the fundamental importance of HDACs in the chromatinremodeling of cells and therefore in gene expression andfunction of the cell collective. HDAC inhibition might thereforelead to cellular differentiation or apoptosis, both events thatcould ultimately also lead to embryonic malformations. Tri-chostatin A (TSA), a classical HDAC inhibitor, is also contro-versially discussed as a possible teratogen as it leads tomalformations similar to those of VPA if investigated in vitro(27, 33, 34) but not in vivo (35); this discrepancy might be dueto metabolism and possible detoxification of TSA in the mice(36). In addition, the well-known teratogen carbamazepine isalso proposed as an HDAC inhibitor (37, 38), which is furtherindication for HDACs as interesting molecular target structuresin the field of reproductive toxicology.

    In addition to functional inhibition of HDACs, it was alsoreported that HDAC inhibitors can alter the cellular protein levelof histone deacetylases, an effect that might also have an influ-ence on the sensitive balance of acetylation and deacetylationof core histones (39, 40).

    In this study, we used a structurally diverse set of 20 VPAderivatives (Figure 1, VPA and its derivatives, coded withRoman numerals) that had been extensively investigated forreproductive toxicity in the NMRI-exencephaly-mouse modelby our group. The analogues used here have both higher and

    Figure 1. Chemical structures of the 20 VPA derivatives investigated in this study (derivatives were numbered with Roman numerals, and theirrespective teratogenic potency are on the arbitrary scale from 0 (no detectable teratogenic potential) to +++++ (very high teratogenic potential)).

    HDAC Inhibition by Teratogenic VPA DeriVatiVes Chem. Res. Toxicol., Vol. 19, No. 2, 2006 273

  • lower teratogenic potency than VPA and represent the best-characterized set of test compounds for VPA-induced NTDsknown so far. The derivatives used in this study cover all ofthe known structural aspects of VPA-induced malformationssuch as carboxylic acid derivatization, side chain saturation, sidechain length, and especially the chirality at position C2.

    We show here that there is yet a quantitative correlationbetween functional HDAC inhibition and the teratogenic potencyof the corresponding VPA analogues, thus indicating HDACinhibition to be a crucial aspect of VPA-induced teratogenicitybut also demonstrating the possibility to utilize HDAC inhibitionas a prediction system for teratogenic side effects on an evenbroader selectivity.

    Experimental ProceduresMaterials and Valproic Acid Derivatives. All chemicals used

    were of analytical grade if not stated otherwise. Valproic acid (VPA)and trichostatin A (TSA) were obtained from Fluka-Sigma-AldrichGmbH (Germany); the valpromide (VPD) was a kind gift fromKatwijk Chemie (The Netherlands). Valproic acid derivatives weresynthesized as described in detail elsewhere (14-17, 19-21, 23,41). Standard GC-MS analysis showed that the chemical purityof the derivatives was >95%. The optical purity of chiralcompounds was measured after suitable derivatization with chiralreagents by standard GC-NPD analysis and found to be >95% ee(enantiomeric excess). All VPA derivatives used in the cell cultureassays were dissolved in dimethyl sulfoxide (DMSO) to give 1 Mstock solutions.

    Teratogenic Potency Measurement. The exencephaly rates usedas the model parameter for teratogenicity were derived fromprevious publications of our group. In these studies, the exencephalyrates had been measured in the NMRI-exencephaly-mouse model(7) at one or more dose levels (13-21). As a result of certaindifferences in the experimental procedures of these publications(e.g., pH of injected solutions, sc versus ip application, etc.), the

    exencephaly rates were grouped into an arbitrary range of terato-genic potency according to the decision criteria shown in Table 1in a range from 0 (no teratogenic potency detectable) to +++++(very high teratogenic potency). The resulting rating of the 20 VPAderivatives is given both in Figure 1 and Table 2.

    Cell Culture. The teratocarcinoma mouse cell line F9 (AmericanType Culture Collection, Rockville, MD) was cultured in HamsF-12/DMEM medium containing 2 mM L-glutamine, 10% (v/v)fetal bovine serum, 0.145 mM 2-mercaptoethanol, and 100 U/mLpenicillin/streptamycin (medium and supplements from Invitrogen,Germany). For the experimental setup, 106 cells were treated intriplicate in 6-well plates by incubation at 37 C in a humidatmosphere of air and 5% (v/v) CO2. After the indicated time,treated cells were scraped from the bottom of the wells, washedtwice with PBS, dissolved in 100 L of lysis buffer (62.5 mM Tris/HCl, pH 6.8, 2% (w/v) sodiumdodecyl sulfate, 1% (v/v) glycerin,2.5 M dithio-DL-threitol, 250 M phenylmethansulfonyl fluoride,0.05 g/mL bestatin, 2 g/mL aprotinin, and 0.05 g/mL leupeptin),and boiled immediately for 5 min at 90 C. Western blot analysisof acetylated histone 4 (AcH4), histone deacetylase 2 (HDAC 2),histone deacetylase 3 (HDAC 3), and -actin was generally madedirectly on 10 L of cell lysate, whereas the total cellular proteincontent was measured by the bicinichonic acid method (42) forcell samples treated longer than 6 h in order to compensateanalogue-dependent proliferation of cells.

    Western Blot Analysis of the Acetylated Core Histone H4. Atotal of 10 L of the cell lysate was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulosemembrane by semi-dry electroblotting. The blotted membrane waswashed with TBS buffer (2.4 g/L Tris/HCl and 8 g NaCl, pH 7.6)and blocked with TBS buffer containing 3% nonfat dry milk (TBS-M) for 1 h at room temperature. The nitrocellulose membrane(Amersham Bioscience, Germany) was incubated with a 1:2000dilution of anti-acetyl histone H4 antibody (Upstate/Biomol,Germany) in TBS-M at 4 C for 12 h. The membrane was washedonce with TBS buffer and incubated again with a 1:5000 dilutionof an anti-rabbit antibody (Amersham Bioscience ECL detectionkit) in TBS-M for 1.5 h at room temperature. Blots were washedthree times with TBS buffer, once with 0.05% (v/v) Tween 20 inTBS buffer, and again three more times with TBS buffer beforeantibodies were detected with the ECL detection kit (AmershamBioscience) according to the manufacturers instructions.

    Western Blot Analysis of HDAC 2, HDAC 3, and -Actin.Western blot analysis of HDAC 2, HDAC 3, and -actin wasperformed as described above but with the following changes: aftermeasurement of the protein content of the whole cell lysate, 10 gof proteins was separated by 8% SDS-polyacrylamide gel elec-

    Table 1. Decision Criteria for Teratogenic Potency Grading of VPADerivatives

    teratogenicpotency

    dose range(mmol/kg)

    exencephalyrate (%) description

    0 >3.0 0 no teratogenic potency detectable+ 2.0-3.0 1-5 low teratogenic potency++ 2.0-3.0 5-25 lower teratogenic potency than VPA+++ 2.0-3.0 25-60 equal teratogenic potency to VPA++++ 1.0-2.0 40-60 higher teratogenic potency than VPA+++++ 0.25-1.0 40-60 very high teratogenic potency

    Table 2. Summary of the Measured Properties of 20 Valproic Acid Derivatives (Teratogenic Potential, Hyperacetylation of Core Histone 4 inTreated F9 Cells, and Concentration of Half-Maximum Effect in the HDAC Enzyme Inhibition Assay) Sorted by HDAC Inhibition Potential

    VPA derivative teratogenic potential AcH4 (0 to ++) IC50(HDAC) ( SE (M)(()-2-heptyl-4-pentynoic acid (XVII) +++++ ++ 12 ( 2(()-2-hexyl-4-pentynoic acid (XVI) +++++ ++ 13 ( 2(()-2-propyl-octanoic acid (XI) ++++ ++ 25 ( 4(()-2-pentyl-4-pentynoic acid (XV) ++++ ++ 35 ( 10S-2-pentyl-4-pentynoic acid (XXI) +++++ ++ 48 ( 12(()-2-butyl-4-pentynoic acid (XIV) ++++ + 98 ( 18(()-2-propyl-heptanoic acid (X) +++ ++ 103 ( 21(()-2-propyl-hexanoic acid (IX) +++ + 144 ( 34valproic acid (I) +++ + 398 ( 50R-2-pentyl-4-pentynoic acid (XX) +++ + 869 ( 183(()-2-propyl-4-pentenoic acid (...

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