teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative...

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 000µM; 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).

Introduction

Valproic acid (VPA)1 is one of the antiepileptic drugs mostfrequently 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 theRhydrogen atom at position C2. On one hand, teratogenic effectswere minimized or completely prevented in the NMRI-exen-cephaly-mouse model by substitution of theR 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, [email protected]; 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 PPARR and γ wereactivated nonstructurally, specifically the PPARâ (PPAR δ)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 PPARâ (PPAR δ) 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, 2006273

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 Procedures

Materials and Valproic Acid Derivatives. All chemicals usedwere 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 Ham’sF-12/DMEM medium containing 2 mML-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 100µL of lysis buffer (62.5 mM Tris/HCl, pH 6.8, 2% (w/v) sodiumdodecyl sulfate, 1% (v/v) glycerin,2.5µM dithio-DL-threitol, 250µM phenylmethansulfonyl fluoride,0.05µg/mL bestatin, 2µg/mL aprotinin, and 0.05µg/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), andâ-actin was generally madedirectly on 10µL 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 µL 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 manufacturer’s instructions.

Western Blot Analysis of HDAC 2, HDAC 3, and â-Actin.Western blot analysis of HDAC 2, HDAC 3, andâ-actin wasperformed as described above but with the following changes: aftermeasurement of the protein content of the whole cell lysate, 10µgof 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 (µM)

(()-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(VI) ++ ++ 2620( 3170valproic hydroxamic acid(III) 0 + 5040( 67402-propyl-2-hydroxy-pentanoic acid(IV) + 0 5300( 365S-2-propyl-4-hexynoic acid(XIX) ++ + 5840( 19380R-2-propyl-4-hexynoic acid(XVIII) 0 0 7360( 10050valpromide(II) + 0 >10000(()-2-isobutyl-4-pentynoic acid(XII) + 0 >10000(()-2-propyl-4-hexynoic acid(XIII) + 0 >100002-propyl-2E-pentenoic acid(V) 0 0 >10000(()-2-isobutyl-4-pentenoic acid(VII) 0 0 >10000(()-2-ethyl-4-methyl-pentanoic acid(VIII) 0 0 >10000

274 Chem. Res. Toxicol., Vol. 19, No. 2, 2006 Eikel et al.

trophoresis. The blotted membranes were incubated either with theanti-HDAC 2 or anti-HDAC 3 antibody (Upstate/Biomol GmbH,Germany) in a dilution of 1:1000 or with the anti-â-actin antibody(Dunn Labortechnik GmbH, Germany) in a dilution of 1:2000 inTBS-M at 5°C overnight.

Human HDAC Enzyme Assay.HDAC activity was measuredby using an HDAC fluorescence activity assay kit (Biomol,Germany). Because of the pH dependency of the enzymatic testsystem, all compounds measured were first dissolved in water andneutralized before preparation of further dilution series with HDACassay buffer. The dose activity was assayed according to themanufacturer’s instructions with at least three repeats. In short, HeLanuclear extracts (1µL of between 6 and 9 mg/mL) were incubatedwith 500 µM acetylated Fluor-de-Lys substrate in 50µL of assaybuffer in the presence or absence of the respective valproic acidanalogue. The HDAC-inhibitor trichostatin A (TSA) at a concentra-tion of 5 µM served as positive control. The deacetylation reactionwas carried out at 37°C for 4 h and stopped by addition of 50µLof Fluor-de-Lys developer solution containing 2µM trichostatinA. After 15 min, fluorescence activity was measured with a Victor1420 fluorescence reader (Perkin-Elmar LAS GmbH, Germany) at355 nm excitation and 535 nm emission. The enzyme activity wascalculated relative to the measured fluorescence activity of fournegative controls (HDAC assay buffer only) on each 96-well plate.The IC50(HDAC) value was determined by computational fittingof at least six dose HDAC inhibition data to a mathematical enzymeinhibition function (Figure 2) with the pharmacodynamic moduleof the WinNonLin 4 software package (Pharsight Corporation,USA) to yield IC50 values inµmol/L with a standard error (SE)representing the goodness-of-fit between the computational modeland the experimental data.

ResultsTeratogenic Potency Grading of Valproic Acid Deriva-

tives. Data mining was conducted on previously publishedreproductive toxicity studies of our group. VPA-based sub-stances had been intensively characterized in the NMRI-exencephaly-mouse model, and animal experimental data wereobtained for all VPA derivatives used in this study. These werethen transformed into the arbitrary range of teratogenic potentialfrom 0 (no detectable teratogenic potency) to+++++ (veryhigh teratogenic potency) by means of the grading criteria givenabove. Teratogenic ratings for all VPA derivatives used in thisstudy are summarized in Figure 1 and Table 2.

Induction of Hyperacetylation of Core Histone H4 as aCellular Marker for Functional HDAC Inhibition. Terato-carcinoma F9 mouse cells were first treated for different periodsof time with valproic acid (I , +++) andS-2-pentyl-4-pentynoicacid (XXI , +++++) for characterization of the rate andintensity of the cellular response (Figure 3). Hyperacetylationof the core histone 4 (AcH4) served as the marker for inhibitionof enzymatic HDAC function and could be detected as soon as15 min (VPA at 0.25 mM), 2 h (S-2-pentyl-4-pentynoic acid at0.25 mM), or 60 min (Trichostatin A at 200 nM, data not shown)after treatment of cells. The intensity of hyperacetylation wasnot slowly increasing with time but appeared abruptly betweentwo time points without further increase at later time points.

Induction ofAcH4 was concentration-dependent as was shownfor both valproic acid (I ,+++) andS-2-pentyl-4-pentynoic acid

(XXI , +++++) after F9 treatment for 6 h with concentrationsraging from 0.05 to 3.00 mM (Figure 4). The grade of H4

acetylation was different from control samples at concentrationsas low as 500µM (VPA) and 50µM (S-2-pentyl-4-pentynoicacid) with respect to differences in band intensity of the H4

acetylation state of control samples. These differences representsthe basal acetylation of the core histone H4 due to normal cellactivity and are mainly due to slight differences in thedeveloping process of the Western blot film. A second bandsometimes occurring in the trichostatin A-treated positivecontrols is likely to be another hyperacetylated core histone;because of the kilodalton range of this band, it is likely to beAcH3.

All 20 VPA derivatives were screened for induction ofAcH4

in the F9 teratocarcinoma mouse cell line. There was a clearcorrelation between induction ofAcH4 and the teratogenicpotential of valproic acid analogues. The high sensitivity of thecellular test system is indicated by differentiation between VPAderivatives with only minor structural changes such as side chainelongation and further side chain branching (Figure 5 A). Whilechain elongation (I , IX , X, XI ) leads to consecutive higher levelsof acetylated H4, further branching at position C4 (VIII )completely averted this effect.

Figure 2. Mathematical enzyme inhibition function used for datafitting. E, enzymatic effect;C, concentration of test compound;Emax,maximal enzymatic effect with no test compound (negative control);E0, minimal enzymatic effect (positive control);δ, flexion of the sigmoidenzyme inhibition curve; and IC50, concentration of the test compoundwith half-maximum enzymatic effect.

Figure 3. Time-dependent hyperacetylation of core histone 4 after F9cell treatment with (A) 0.25 mM valproic acid (I , +++) and (B) 0.25mM S-2-pentyl-4-pentynoic acid (XXI , +++++). Positive control(Pos) represents F9 cell treatment for 6 h with 200 nM trichostatin A;negative control (Neg) represents 6 h treatment with 1% (v/v) DMSOin F9 cell medium.

Figure 4. Concentration-dependent hyperacetylation of core histone4 after F9 cell treatment for 6 h with (A) valproic acid (I , +++) and(B) S-2-pentyl-4-pentynoic acid (XXI , +++++). Positive control (Pos)represents F9 cell treatment with 200 nM trichostatin A; negative control(Neg) represents treatment with 1% (v/v) DMSO in F9 cell medium.

Figure 5. Hyperacetylation of core histone 4 after treatment of F9cells for 6 h with 1 mM each of VPA derivatives (A) side chainelongated and further branched derivatives and (B) analogues with thechiral center at C2. Positive control (Pos) represents F9 cell treatmentwith 200 nM trichostatin A; negative control (Neg) represents F9 celltreatment with 1% (v/v) DMSO in F9 cell medium; E represents anempty lane.


The high selectivity of the F9 cell system was demonstratedby its ability to distinguish between enantiomeric VPA deriva-tives with the chiral center at position C2 (Figure 5B). BothS-enantiomers studied here (XIX , XXI ) showed higher levelsof acetylated core histone 4 than the correspondingR-enanti-omers (XVIII , XX ).

All structural aspects of the induction of histone hyperacety-lation correlate qualitatively with the structural prerequisites forteratogenic effects of VPA derivatives known so far.

Influence of VPA Derivatives on the Cellular HDAC 2and HDAC 3 Levels.F9 mice cells were treated for 6, 12, and24 h with selected VPA derivatives with different grades ofteratogenic potential: high (XXI ), comparable to that of VPA(I , XX , XII ), and undetectable (III , V). The cellular HDAC 2and -3 levels were measured via Western blot analysis in directcomparison toâ-actin as control protein (Figure 6, data notshown for 6 and 12 h). The VPA derivatives had no influenceon cellular HDAC 2 and HDAC 3 levels after any of the threeincubation times (24, 12, and 6 h), although some VPAderivatives (I , XXI ) did influence the morphology and prolifera-tion of the F9 cells after 12 and 24 h treatment (data not shown;see ref25). These results suggest that a functional HDACinhibition and not HDAC protein degradation is correlated withthe teratogenic effects of VPA.

Measurement of the IC50(HDAC) Values of the 20 VPAAnalogues.The IC50(HDAC) values of the 20 VPA derivativeswere measured using an HDAC inhibition assay with humanHeLa cell nuclear extract as the scource of HDAC activity. Atypical set of computational fits of concentration effect curvesis shown in Figure 7. Side chain elongation of VPA derivativeswith a triple bond in position C4 resulted in decreasing IC50-(HDAC) values, while further branching of one side chain isdiminishing the HDAC inhibition effect.

The calculated IC50(HDAC) values range from 10 to 10 000µM, with some VPA derivatives exceeding the HDAC inhibitionpotential of VPA itself 40-fold. One can also detect a 20-foldincrease in IC50(HDAC) inhibition potential between VPAsteroisomers having a different teratogenic potential. This clearlyshows a stereoselective interaction with HDAC enzymes.

The resulting IC50(HDAC) values of all tested VPA deriva-tives are summarized in Table 2, and these measurementsconfirm quantitatively the aforementioned qualitative correlationof teratogenic potential and HDAC inhibition abilitiy of VPAderivatives.

Correlation of HDAC Inhibition Properties and Terato-genic Potential of VPA Derivatives.The correlation betweenthe IC50(HDAC) values and the graded teratogenic potential ofthe 20 VPA derivatives clearly indicates that these twocompound properties are not only related but also quantitativelyconnected with each other (Figure 8). The most potent HDACinhibitors, with IC50(HDAC) values between 10 and 50µM (XI ,

XV , XVI , XVII , XXI ), were also the VPA derivatives withthe highest teratogenic potential. This correlation strengthensthe hypothesis that VPA-induced NTDs are mediated by HDACinhibition and points toward HDAC inhibition as a potentialendpoint in screening systems of reproductive toxicity.

Discussion

Valproic acid (VPA) has recently been shown to bind andinhibit the enzyme class of histone deacetylases (HDACs),which are important regulators of the chromatin remodeling ofcells and therefore have a great impact on gene expression andcell function (29, 30). Inhibition of enzymatic HDAC functioncan lead to differentiation, apoptosis, or interruption of cellproliferation (43, 44), all cellular events that can possibly causeembryonic malformations. HDAC inhibition might therefore bepart of a molecular signaling cascade which results in VPA-induced neural tube defects (NTDs), and it can be hypothesizedthat HDACs are the theoretical “teratogenic receptors” for VPA-induced NTDs first described by our group (22). This hypothesishas been strengthened by further reports of chemicals with some

Figure 6. Protein levels of F9 whole cell lysate (10µg) of (A) â-actin(42 kDa), (B) HDAC 3 (48 kDa), and (C) HDAC 2 (55 kDa) after 24h treatment with 1 mM concentrations of VPA derivatives with noteratogenic potential (III , V), intermediate teratogenic potential (I , XII ,XX ), and very high teratogenic potential (XXI ). Negative control (Neg)represents treatment of F9 cells for 24 h with 1% (v/v) DMSO in F9cell medium.

Figure 7. Concentration effect curves fitted to the experimental HDACinhibition data of side chain elongated and further branched VPAderivatives with a triple bond in position C4 (XII -XVII ). All fitteddose-response curves are based on at least six concentrations with atleast three independent measurements of each concentration. Data pointsshown represent the mean of these measurements with error barsshowing the standard deviation of the mean (SD).

Figure 8. HDAC enzyme inhibition ability correlated with theteratogenic potency of the 20 investigated VPA derivatives. A linearregression and the 95% confidence interval visualize the quantitativerelationship of these two compound properties.


structural similarities to the VPA core structure that are alsonot HDAC inhibitors when being nonteratogenic (31, 32).However, these studies are mostly based on VPA-like structureswhich are less potent teratogens than VPA.

To prove the hypothesis that HDACs are the hypothesizedteratogenic receptors of VPA, we measured the HDAC inhibitionability of a structurally diverse set of 20 VPA derivatives withvarious structural modifications such as derivatization of thecarboxylic function, side chain elongation and further branching,introduction of double and triple bonds, and enantiomericderivatives with the chiral center at position C2. Thesecompounds had been extensively studied by our group in theNMRI-exencephaly-mouse model before and represent one ofthe best-characterized set of structurally diverse reproductivetoxicants so far. The VPA derivatives used in this study coverthe arbitrary range of teratogenic potency from undetectableteratogenic potency (0) to very high teratogenic potency(+++++). Therefore, this set is the first investigated set ofVPA derivatives that also considers structures with a muchhigher teratogenic potency than VPA itself. It also consists ofstereoisomers which could prove the enantioselective interactionwith HDAC enzymes and therefore represents a unique op-portunity for the study of qualitative and quantitative relation-ships between HDAC inhibition and teratogenic potency.

In fact, we found that VPA almost immediately (15 min)induced hyperacetylation of core histone 4 (AcH4) in F9 cells,which shows that HDAC inhibition is a very early cellular eventin the response to VPA. It is therefore reasonable to assumethat HDAC inhibition is not only part of, but might be the firststep in, a molecular signaling cascade leading toward neuraltube defects. The structurally different VPA derivativeS-2-pentyl-4-pentynoic acid (XXI ) leads toAcH4 only after 2 h ofcell treatment; this slower hyperacetylation rate might be dueto a slower passage of this congener through the cellularmembrane rather than to a delayed cellular response. Onlyteratogenic VPA derivatives inducedAcH4 in F9 cells, which isin accordance with previously published results (29-32).

VPA is reported not only to inhibit the function of HDACsbut also to induce a selective degradation of HDAC 2 proteinlevels both in vitro and in vivo (38). To date one, another HDACinhibitor (suberoyl anilide hydroxamic acid, SAHA) has alsobeen reported to decrease HDAC protein levels, but is selectivefor HDAC 3 (39). To determine whether degradation of HDACproteins could be involved in VPA-induced NTDs, we inves-tigated the cellular levels of both HDAC isoform 2 and 3 in F9cells after treatment with selected VPA derivatives covering thewhole teratogenic potential range from 0 to+++++.

Unlike the investigation mentioned above, we did not detectany degradation of either HDAC 2 or HDAC 3 after 6, 12, andeven 24 h of cell treatment. It is noteworthy that we were notable to reproduce the degradation of HDAC 2 after VPAtreatment in the ostensible same F9 cell culture used by Kraemeret al. (38). However, after 24 h of treatment, we already detectmajor changes in the cellular morphology of the F9 cells, andproliferation had already been strongly altered by both VPAandXXI , both effects described by our group before (25, 33).This VPA derivative, although possessing a much higherteratogenic potency than VPA, also did not induce HDAC 2degradation. It is also noteworthy that HDAC inhibition occursas soon as 15-60 min after cell exposure, whereas HDACdegradation did not occur up to 24 h of treatment.

Taking also into account that SAHA had been reported toinduce HDAC degradation of isoform 3 instead of isoform 2and with respect to the small data set, it seems likely that there

is no correlation between HDAC inhibition and HDAC degra-dation. In this regard, our results clearly demonstrate that thefunctional inhibition of HDACs is related to the VPA-inducedNTDs, and degradation of HDAC seems not to be linked tothis severe drug side effect.

The HDAC inhibition ability of the 20 VPA derivativesinvestigated here was measured quantitatively in a commercialHDAC inhibition assay with HeLa nuclear extracts as the sourceof HDAC activity. There was a wide range of activity at IC50-(HDAC) concentrations from 10 to 10 000µM with somederivatives exceeding the VPA HDAC inhibition potential 40-fold. Most interestingly, testing VPA stereoisomers, we detectedan up to 20-fold difference in HDAC inhibition potential, thus,demonstrating a stereoselective interaction with HDAC enzymes.

Here, too, the teratogenic potential of VPA derivativescorresponded to their IC50(HDAC) values. The correlationbetween the teratogenic potential of the investigated 20 VPAderivatives and their IC50(HDAC) is excellent when the terato-genic potential of the derivatives is equal or even higher thanthat of VPA, but the correlation was slightly smaller if theteratogenic potential of the VPA analogues was lower than thatof VPA. This also demonstrates that to disclose molecular targetsof VPA-induced NTDs it is important to investigate a properset of VPA derivatives consisting of analogues with both higherand lower teratogenic potential than VPA.

The correlation was surprisingly good, although no consid-eration was taken of differences in metabolism (activation ordeactivation) or pharmacokinetics in vivo. Our results clearlyindicate that HDAC inhibition is a molecular target for VPA-induced NTDs. We therefore conclude that HDACs are thetheoretically hypothesized “teratogenic receptors“ that cantrigger a VPA-induced molecular signaling cascade resultingin embryonic malformations.

Furthermore, there are strong indications that trichostatin A(TSA), a classical HDAC inhibitor, can also cause embryonicmalformations (33, 34), and there are controversial reports thatcarbamazepine, a known teratogenic drug, inhibits HDACactivity (36, 37). We can therefore assume that HDAC inhibitionnot only mediates VPA-induced NTDs but might also triggerother chemically induced embryonic malformations. In thisregard, our finding that the correlation between HDAC inhibitionand VPA-induced NTDs can be measured quantitatively dem-onstrates that HDAC inhibition might be a suitable molecularendpoint in screening systems of reproductive toxicology.

Ongoing studies by our group on both known HDACinhibitors as reproductive toxicants in the NMRI-execephaly-mouse model as well as screening of known teratogens forHDAC inhibition will reveal if HDACs can be successfullyutilized as endpoints in reproductive toxicity screenings.

Acknowledgment. We thank the Deutsche Forschungsge-meinschaft (DFG-NA 104/2-1), the European Research TrainingNetwork (RTN2-2001-00370), the European Commission (6thFramework Program: ReProTect), the Federal Ministry forEducation and Research (BBF) (Project 0313070D), and theAcademy for Animal Health (ATF) for generous financialsupport. We also thank Mrs. J. McAlister-Herman for valuablesuggestions and for the critical reading of this manuscript.

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