the teratogenic potencies of valproic acid derivatives and their effects on biological end-points...

The Teratogenic Potencies of Valproic Acid Derivatives and TheirEffects on Biological End-points are Related to Changes in Histone

Deacetylase and Erk1 ⁄2 ActivitiesKamil Gotfryd1, Maria Hansen1, Anna Kawa1, Ursula Ellerbeck2, Heinz Nau2, Vladimir Berezin1, Elisabeth Bock1 and Peter S. Walmod1

1Protein Laboratory, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen,Denmark, and 2Department of Food Toxicology, School of Veterinary Medicine, Hannover, Germany

(Received 7 October 2010; Accepted 11 March 2011)

Abstract: Valproic acid (VPA) is a known teratogen. In the present study, the effects of VPA and seven VPA derivatives withdifferent teratogenic potencies (isobutyl-, 5-methyl-, ethyl-, propyl-, butyl-, pentyl- and hexyl-4-yn-VPA) were investigated inL929 cells in vitro. Evaluated end-points included changes in cell proliferation, growth, cell cycle distribution, morphology,speed, glycogen synthase kinase-3b (GSK-3b) and Erk1 ⁄ 2 phosphorylation, and histone H3 acetylation. Changes in prolifera-tion, growth, speed, Erk1 ⁄ 2 and GSK-3b-Tyr216 phosphorylation, and H3 acetylation were significantly associated with theteratogenic potencies of the VPA derivatives. However, in contrast to changes in Erk1 ⁄ 2 phosphorylation and H3 acetylation,significant changes in GSK-3b phosphorylation could only be obtained in response to prolonged incubation at high drug con-centration. There was an association between changes in H3 acetylation and GSK-3b-Tyr216 phosphorylation, whereas noneof these end-points were associated with changes in Erk1 ⁄ 2 phosphorylation. These results suggest that the teratogenic poten-cies of VPA and VPA derivatives are related to effects on both Erk1 ⁄ 2 and histone deacetylase activities, whereas changes inGSK-3b activity are possibly a secondary effect.

Valproic acid (VPA) is a widely used antiepileptic drug [1]that is also used for the treatment of migraine, mood disor-ders and cancer [2,3]. Adverse effects associated with VPAtreatment include weight gain, hair loss and hepatotoxicity.Additionally, the drug is a teratogen that induces variousmalformations, including neural tube defects, in human foe-tuses exposed to the compound during early pregnancy [2].

In addition to effects related to its antiepileptic function,VPA affects many cellular and biochemical processes, includ-ing cell proliferation, morphology, differentiation, survival,apoptosis and the activity of Erk1 ⁄ 2, glycogen synthasekinase-3b (GSK-3b) and histone deacetylases (HDACs) [4,5].The teratogenic potencies of VPA and VPA derivatives havebeen correlated with drug-induced changes in the prolifera-tion [6–9], area and motility of L929 cells [10,11] and theaggregation of primary neurons in culture [12]. Moreover,the HDAC inhibitory activity of VPA and VPA derivativeshas been related to the teratogenic potencies of the com-pounds [13–15]. The HDAC inhibitory activity of VPA hasalso been proposed to account for the anticancer effects andother potentially beneficial effects of the drug [3,16].

In the present study, VPA and seven VPA derivatives withdifferent in vivo teratogenic potencies were tested in vitro inL929 cells for their effects on proliferation, growth, cell cycledistribution, area, speed, GSK-3b and Erk1 ⁄ 2 phosphoryla-

tion, and HDAC activity (reflected by histone H3 acetyla-tion).

Several of the estimated drug-induced changes in the cellu-lar and biochemical end-points correlated with the terato-genic potencies of the respective VPA derivatives. However,relative to changes in Erk1 ⁄ 2 phosphorylation and H3 acety-lation, significant changes in GSK-3b phosphorylationrequired longer exposure time and higher drug concentra-tion, suggesting that changes in GSK-3b, Erk1 ⁄ 2 andHDAC activities may all contribute to the teratogenic poten-cies of the compounds, but that changes in GSK-3b activitymay be a secondary effect to other changes induced by thedrugs.

Materials and Methods

VPA derivatives. Racemates of seven VPA derivatives (isobutyl-4-yn-VPA, 5-methyl-4-yn-VPA, ethyl-4-yn-VPA, propyl-4-yn-VPA, butyl-4-yn-VPA, pentyl-4-yn-VPA and hexyl-4-yn-VPA) were synthesizedas previously described [7,17–19]. Three molar stock solutions of thecompounds were prepared in dimethyl sulfoxide (DMSO), and allexperiments were performed in the presence of 0.1% (v ⁄ v) DMSOwith or without VPA derivatives. With the exception of the studiesof GSK-3b and Erk1 ⁄ 2 phosphorylation, and histone H3 acetyla-tion, all experiments were performed on coded compounds. Table 1shows the chemical structures and the previously determined in vivoteratogenic potencies of the respective compounds.

Cell culture. The mouse fibroblastoid cell line L929 (L-cells) wasobtained from the European Collection of Animal Cell Cultures.Cells were grown in Dulbecco’s modified Eagle medium supple-mented with 10% (v ⁄ v) heat-inactivated foetal calf serum, 2.5 lg ⁄ ml

Author for correspondence: Peter S. Walmod, Protein Laboratory,Panum Institute, Blegdamsvej 3C, Building 24.2.21, DK-2200 Copen-hagen N, Denmark (fax +45 3536 0116, e-mail [email protected]).

Basic & Clinical Pharmacology & Toxicology, 109, 164–174 Doi: 10.1111/j.1742-7843.2011.00702.x

� 2011 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2011 Nordic Pharmacological Society

fungizone, 100 U ⁄ ml penicillin and 100 lg ⁄ ml streptomycin (allfrom Invitrogen, Taastrup, Denmark). Cells were dislodged with0.5 mg ⁄ ml trypsin and 0.54 mM ethylenediaminetetraacetic acid inmodified Puck’s saline (Invitrogen).

Cell proliferation and growth. The effects of the compounds on cellproliferation were determined by measuring 5-bromo-2-deoxyuridine(BrdU) incorporation using the Biotrak ELISA system (GE Health-care, Hillerød, Denmark) as previously described [7]. Briefly, cellswere plated in 96-well Nunclon D MicroWell plates (Nunc, Roskilde,Denmark) at a density of 5–7 · 103 cells ⁄ well and grown in the pres-ence of 0, 0.3, 1.0, 3.0 and 10 mM of the test compounds and10 mM BrdU, with each treatment replicated in six wells ⁄ plate.Incorporated BrdU was visualized by horseradish peroxidase(HRP)-conjugated anti-BrdU antibodies and quantified with anELISA reader using a 450-nm long-pass filter.

To estimate cell growth, cells were plated at a density of7 · 103 cells ⁄ well in 96-well plates (Nunc) and grown in the absenceor presence of the test compounds, with each treatment replicated insix wells per plate. Cultures were rinsed twice in phosphate-bufferedsaline (PBS), fixed in 3.7% (v ⁄ v) formalin and 1% (v ⁄ v) methanol inPBS and stained with 0.2% (w ⁄ v) crystal violet in 0.1 M citric acid.Cell-bound crystal violet was solubilized with 1% (w ⁄ v) sodiumdodecyl sulphate (SDS) and quantified with an ELISA reader usinga 600-nm long-pass filter.

Flow cytometry. For analysis of DNA content, 5 · 105 L-cells wereplated in T-25 Nunclon D flasks (Nunc) and grown for 24 hr in nor-mal medium followed by 48 hr in the absence or presence of 3 mMof the test compounds. Subsequently, cells were trypsinized, stainedwith propidium iodide as previously described [20] and analysedusing a CellQuest apparatus (Becton Dickinson, Broendby, Den-mark). Data from 1 · 104 cells were collected for each analysis usingLysys II software (Becton Dickinson). The percentage distribution of

cells in the various phases of the cell cycle was calculated using MOD-

FIT LT software (Verity Software House, Topsham, ME, USA). Thepercentage of apoptotic cells was determined after excluding cellaggregates and cellular debris as the fraction of cells with subnormalDNA content plus the fraction of cells with a DNA content between2 and 4 n, with n the haploid number of chromosomes.

Determination of cell morphology and motility. Cells from subconflu-ent cultures were dislodged and plated in 6-well Nunclon D Multi-dishes (approximately 4 · 104 cells ⁄ well; 48 or 72 hr; Nunc) in theabsence or presence of 3 mM of the test compounds in a mediumcontaining 25 mM HEPES (Invitrogen).

Digital images for the determination of cellular morphology wereacquired using a Nikon Diaphot 300 inverted microscope with a 10·objective equipped with a black-and-white charge-coupled devicecamera (Burle, Lancaster, PA, USA). For each experimental condi-tion, 20 non-overlapping images were recorded. Subsequently, thecontours of the cells were obtained semi-automatically by means ofcomputerized thresholding and binary image transformation as pre-viously described [10,21].

Time-lapse video-recordings of live cells for the determination ofcell motility were performed essentially as previously described [22].Briefly, live cells from approximately 25 non-overlapping areas fromeach well of a multi-well dish were recorded for 60 min. at 10-min.intervals using a Nikon microscope workstation equipped with acomputerized movable stage and a CCD camera. The individual esti-mates of cell speed were based on the analyses of an average of 91cells.

The positions of the individual cells were determined by semi-automatic marking of nuclear centres. The cell positions fromconsecutive images were utilized to calculate the mean displacement(<d>) for each cell. Subsequently, the mean-cell speed (Ss =<d> ⁄ s) was determined using a 10-min. time interval of interest(ti) [11,23].

Table 1.Test compounds.

Compound Structure Teratogenicity % (concentration, mmol ⁄ kg)

VPA COOH 42 (3.00)

Isobutyl-4-yn-VPA COOH 0 (1.85)

5-methyl-4-yn-VPA COOH 0 (2.00)

Ethyl-4-yn-VPA COOH 0 (1.85)

Propyl-4-yn-VPA COOH 12 (1.25)1

Butyl-4-yn-VPA COOH 71 (1.25)1

Pentyl-4-yn-VPA COOH 60 (1.25)1

Hexyl-4-yn-VPA COOH 79 (1.25)1

The indicated teratogenicity of the individual compounds reflects the percentage of exencephaly observed in mice.1[7] Reproduced with permission.VPA, Valproic acid.

IN VITRO STUDIES OF THE EFFECTS OF VALPROIC ACID DERIVATIVES 165

� 2011 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2011 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 109, 164–174

Immunoblotting. Cells were plated in Nunclon D dishes (Nunc) at adensity of 1.3 · 104 cm)2 and grown for 24 hr in normal mediumfollowed by 24 hr in the absence or presence of test compounds. Sub-sequently, whole-cell lysates were prepared as previously described[24]. Proteins of whole-cell lysates were resolved by SDS–polyacryl-amide gel electrophoresis and transferred to Immobilon-P mem-branes (Millipore, Billerica, MA, USA). Subsequently, membraneswere blocked in PBS, 0.1% (v ⁄ v) Tween-20 and 5% (w ⁄ v) bovineserum albumin and incubated overnight with antibodies againstphospho-GSK-3b (Tyr216; Sigma-Aldrich, Broendby, Denmark),phospho-GSK-3b (Ser9), phospho-Erk1 ⁄ 2 (Thr202 ⁄ Tyr204) or acet-ylated histone H3 (Lys9 ⁄ Lys14; all from Cell Signaling, Danvers,MA, USA). After incubation with HRP-conjugated anti-rabbit anti-bodies (Cell Signaling), the blots were visualized by chemilumines-cence (SuperSignal West Dura Extended Duration Substrate; PierceBiotechnology, Rockford, IL, USA), and blot images were acquiredusing the GeneGnome chemiluminescence capture and analysis sys-tem (SynGene, Cambridge, UK). To verify equal protein loading,membranes were stripped (30 min. at 50�C) in 62.5 mM Tris–HCl(pH 6.8), 2% (w ⁄ v) SDS and 50 mM 1,4-dithio-L-threitol, blockedand reprobed with antibodies against GSK-3b (27C10), Erk1 ⁄ 2(both from Cell Signaling) or actin (Sigma-Aldrich) and visualizedby HRP-conjugated anti-rabbit antibodies (Cell Signaling) andchemiluminescence. The subsequent blot quantifications were per-formed using GeneTools software (SynGene).

Results

Effects of VPA derivatives on cell proliferation and growth.Valproic acid and VPA derivatives have previously been dem-onstrated to inhibit cellular proliferation in a manner thatcorrelates with the teratogenic potencies of the respectivecompounds [6–9]. In the present study, changes in prolifera-tion were estimated by BrdU incorporation. Table 2 showsthe IC50 values for the estimated inhibitions of proliferationafter 48-hr growth in the presence of the individual com-pounds. All seven VPA derivatives concentration-depen-dently inhibited proliferation. One of the derivatives, thenon-teratogenic ethyl-4-yn-VPA, did not exhibit an IC50

value below the highest concentration used (10 mM). Theremaining six compounds exhibited IC50 values between 0.4and 9.5 mM, and a significant inverse correlation was foundbetween the teratogenic potencies of the test compounds and

the corresponding IC50 values for proliferation (n = 6,R2 = 0.9331, p < 0.017).

The effects of the test compounds on the proliferation ofL-cells were also investigated by measuring cell growth(determined by crystal violet staining). Figure 1 shows theeffects of 72-hr exposure to 3 mM of the respective test com-pounds on cell growth. At this concentration, VPA and thefour teratogenic VPA derivatives had significant effects oncell growth, with the effect most pronounced for the strongteratogens butyl-, pentyl- and hexyl-4-yn-VPA. A significantlinear correlation was demonstrated between the teratogenicpotencies of the test compounds and their inhibition of cellgrowth (n = 28, R2 = 0.5783, p < 0.001). Therefore, an asso-ciation was observed between the teratogenicity of the VPAderivatives and their ability to inhibit the proliferation andgrowth of L929 cells.

Effects of VPA derivatives on cell cycle distribution.The inhibitory effect of VPA on cell proliferation has previ-ously been shown to result from cell cycle arrest in the G1phase [25]. To determine whether VPA derivatives have simi-lar effects, the cell cycle distribution of L-cells was investi-gated by flow cytometry after growth in the absence orpresence of the respective test compounds.

Figure 2 shows the average effects of VPA and the sevenderivatives on the relative distribution of cells in the G1, Sand G2 ⁄ M phases of the cell cycle, respectively. As expected,VPA significantly increased the percentage of cells in the G1phase, accompanied by a significant decrease in the percent-age of cells in the S phase. The teratogenic butyl-4-yn-VPAalso increased the percentage of cells in the G1 phase. In

Table 2.IC50 values for cell proliferation.

Compound IC50 (mM) IC50 € S.E.M. (mM)

VPA 1.9 (0.7–4.0)Isobutyl-4-yn-VPA 9.5 (8.8–9.9)5-methyl-4-yn-VPA 7.8 (5.6–>10)Ethyl-4-yn-VPA >10 –Propyl-4-yn-VPA 6.1 (4.8–7.6)Butyl-4-yn-VPA 2.5 (2.2–2.7)Pentyl-4-yn-VPA 1.8 (1.6–2.0)Hexyl-4-yn-VPA 0.4 (0.30–0.60)

IC50 values for proliferation determined from bromo-2-deoxyuridineincorporation. The values were determined by interpolation of aver-age values derived from two independent concentration-responseexperiments. Numbers in parentheses indicate interpolated values forIC50 € S.E.M.VPA, Valproic acid.

Fig. 1. Effects of Valproic acid (VPA) and VPA derivatives on cellgrowth. The bars indicate cell growth after exposure to the respectivetest compounds (3 mM, 72 hr). Open bars, non-teratogens; solidbars, teratogens. One-way anova F8,31 = 9.951, p < 0.0001, followedby the Tukey–Kramer multiple comparison test. *p < 0.05,***p < 0.001.

166 KAMIL GOTFRYD ET AL.


contrast, the teratogenic hexyl-4-yn-VPA significantly decre-ased the percentage of cells in the G1 phase and increased thepercentage of cells in the G2 ⁄ M phase. However, although twoof the teratogens, propyl- and pentyl-4-yn-VPA, showed thesame tendency as VPA and butyl-4-yn-VPA, five of the sevenVPA derivatives did not significantly change the cell cycle dis-tribution. These data demonstrate no consistent effect of VPAand VPA derivatives on cell cycle distribution.

Effects of VPA derivatives on cell morphology.Consistent with previous studies of other VPA derivatives,the VPA derivatives tested in the present study caused no sig-nificant alterations in the morphology of L-cells at concen-trations <1 mM or with exposure times <24 hr (data notshown). However, with longer exposure times and higherconcentrations, alterations in cell morphology were observed.None of the three non-teratogens induced significantchanges in the mean-cell area of the cultured cells. In con-trast, all four teratogens caused time-related changes in themean-cell area at this concentration (fig. 3). Notably, the

strong teratogen hexyl-4-yn-VPA significantly and dramati-cally decreased the cell area, whereas the remainingteratogens significantly increased the mean-cell area. Conse-quently, teratogenic VPA derivatives appear to induce largeralterations in the morphology of cultured L-cells than non-teratogenic VPA derivatives. However, the teratogens do notinduce a qualitatively consistent morphological response.

Effects of VPA derivatives on cell motility.In the present study, the cell motility of L-cells was estimatedas the mean-cell speed of individual cells. As shown in figs 4and 5 of the seven VPA derivatives significantly inhibited cellmigration. VPA and the four teratogenic VPA derivativesinhibited cell speed more strongly than the non-teratogens,and a significant linear correlation was found between theteratogenic potencies of the VPA derivatives and their effectson the mean-cell speed (n = 14, R2 = 0. 7868, p < 0.0001).

Effects of VPA derivatives on GSK-3b activity.The effects of VPA exposure on GSK-3b activity appear tobe cell type- or design dependent [26]. GSK-3b activity canbe regulated by direct phosphorylation at Ser9 (inhibitory)and Tyr216 (stimulatory) [27]. In the present study, GSK-3bactivity in L-cells was investigated by estimating VPA- andVPA derivative-induced changes in GSK-3b phosphorylationby immunoblotting. Figure 5 shows the relative changes inGSK-3b-Ser9 and GSK-3b-Tyr216 phosphorylation levels inresponse to VPA and VPA derivatives. A 48-hr exposure to3 mM VPA significantly increased GSK-3b-Ser9 phosphory-

Fig. 2. Effects of Valproic acid (VPA) and VPA derivatives on cellcycle distribution. The bars indicate the relative distribution of L-cells in the G1, S and G2 ⁄ M phases of the cell cycle after exposureto the respective test compounds (3 mM, 48 hr). Hatched bars,dimethyl sulfoxide; open bars, non-teratogens; solid bars, teratogens.One-way repeated-measures anova F8,27 = 14.54, p < 0.0001 (G1),F8,27 = 8.640, p < 0.0001 (S), F8,27 = 13.87, p < 0.0001 (G2 ⁄ M), fol-lowed by the Tukey–Kramer multiple comparison test. *p < 0.05,**p < 0.01,***p < 0.001.

Fig. 3. Effects of Valproic acid (VPA) and VPA derivatives on cellmorphology. The bars indicate the mean-cell area of L-cells exposedto the respective compounds (3 mM) for 48 hr (left bars) and 72 hr(right bars), respectively. The average mean-cell areas of controlcells (100%) were 630 and 598 lm2 after 48 and 72 hr, respectively.Open bars, non-teratogens; solid bars, teratogens. One-way repeated-measures anova F8,18 = 22.97, p < 0.0001 (48 hr), F8,18 = 16.19,p < 0.0001 (72 hr), followed by the Tukey–Kramer multiple compar-ison test. *p < 0.05, **p < 0.01,***p < 0.001.



lation levels and significantly decreased GSK-3b-Tyr216phosphorylation levels (thereby supposedly inhibiting GSK-3b activity). Figure 5A (left) shows representative immuno-blots of the levels of phosphorylated GSK-3b in response toVPA (3 mM, 48 hr). Figure 5A (right) shows the quantifiedchanges in GSK-3b phosphorylation levels from severalexperiments. At shorter incubation times (3 mM, 24 hr) orlower concentrations (1 mM, 48 hr), VPA had no significanteffect on GSK-3b-Ser9 and GSK-3b-Tyr216 phosphoryla-tion, respectively (data not shown).

Subsequently, the effects of VPA derivatives on GSK-3b-Ser9 and GSK-3b-Tyr216 phosphorylation levels were inves-tigated. Figure 5B shows representative immunoblots of thelevels of phosphorylated GSK-3b after treatment with therespective VPA derivatives (3 mM, 48 hr). Figure 5C and dshow quantifications of the relative changes in GSK-3b-Ser9and GSK-3b-Tyr216 phosphorylation levels, respectively.None of the VPA derivatives induced significant changes inGSK-3b-Ser9 phosphorylation when exposed to the com-pounds at 3 mM for 48 hr (fig. 5B,C) or at 1 mM for 24 hr(data not shown), respectively. In contrast, GSK-3b-Tyr216phosphorylation was significantly reduced in response toexposure to 3 mM hexyl-4-yn-VPA for 48 hr, and a signifi-cant linear correlation was found between the teratogenicpotencies of the VPA derivatives and their effects onGSK-3b-Tyr216 phosphorylation (n = 22, R2 = 0.3552, p <0.0034). In conclusion, VPA and teratogenic VPA derivativesappear to inhibit GSK-3b activity through several mecha-nisms in L-cells, but only after prolonged incubation and athigh concentration.

Effects of VPA derivatives on Erk1 ⁄ 2 activity.Valproic acid has recently been demonstrated to time- andconcentration-dependently inhibit the activity of the MAPkinases Erk1 ⁄ 2 in L-cells [24]. To determine whether theinhibition of Erk1 ⁄ 2 activity in L-cells is a general responseto exposure to VPA derivatives, changes in relative Erk1 ⁄ 2phosphorylation, which reflects Erk1 ⁄ 2 activity, were studiedafter exposure to the respective VPA derivatives. Theseexperiments (similar to the investigations of changes inGSK-3b phosphorylation levels) were performed at a con-centration of 1 mM because this VPA concentration wasfound to cause only a partial inhibition of Erk1 ⁄ 2 activity[24], thereby making the detection of compounds with bothmore and less potent inhibitory properties possible.

Figure 6A (left) shows representative immunoblots of thelevels of phosphorylated Erk1 ⁄ 2 in response to VPA treat-ment. Figure 6A (right) shows the quantified changes inErk1 ⁄ 2 phosphorylation levels from several experiments.Treatment with VPA (1 mM, 24 hr) was found to lead to asignificant 37% reduction in the degree of Erk1 ⁄ 2 phosphor-ylation, an inhibition comparable to what has recently beenreported for L929 cells [24]. Figure 6B shows representativeimmunoblots of the levels of phosphorylated Erk1 ⁄ 2 aftertreatment with the respective VPA derivatives (1 mM, 24 hr).Figure 6C shows the relative changes in Erk1 ⁄ 2 phosphory-lation levels from several experiments. The three non-terato-gens had no significant effects on Erk1 ⁄ 2 activity, whereasthe four teratogens significantly inhibited Erk1 ⁄ 2 activity.Furthermore, a significant linear correlation was foundbetween the teratogenic potencies of the VPA derivatives andtheir effects on Erk1 ⁄ 2 activity (n = 35, R2 = 0. 1249,p < 0.0373).

Effects of VPA derivatives on histone H3 acetylation.Valproic acid is a known HDAC inhibitor [28]. In the presentstudy, the HDAC activities of VPA derivatives were investi-gated by estimating histone H3 acetylation in L-cells byimmunoblotting. Estimations of changes in histone H3 acety-lation levels induced by VPA and the respective VPA deriva-tives were performed after 24-hr incubations with 1 mM ofthe test compounds, similar to the estimated changes inErk1 ⁄ 2 phosphorylation levels. Figure 7A (left) shows repre-sentative immunoblots of the levels of acetylated histone H3in response to VPA treatment. Figure 7A (right) shows thequantified changes in histone H3 acetylation levels from sev-eral experiments. Treatment with VPA (1 mM, 24 hr) wasnot found to have a significant effect on histone H3 acetyla-tion, which is in agreement with what has recently beenreported for L-cells incubated with 3 mM VPA for 48 hr [24].Figure 7B shows representative immunoblots of the levels ofacetylated histone H3 and loading control in response toincubation of L-cells in the presence of the respective VPAderivatives. Figure 7C shows the relative changes in histoneH3 acetylation levels from several experiments and showsthat none of the three non-teratogens had significant effectson histone H3 acetylation, whereas the three strongest terato-gens (i.e. butyl-, pentyl- and hexyl-4-yn-VPA) significantly

Fig. 4. Effects of Valproic acid (VPA) and VPA derivatives on mean-cell speed. The bars indicate the mean-cell speed of L-cells exposedto the respective compounds (3 mM, 48 hr). The average mean-cellspeed of control cells (100%) was 0.39 lm ⁄ min. Open bars, non-teratogens; solid bars, teratogens. One-way anova F8,13 = 16.37,p < 0.0001, followed by the Tukey–Kramer multiple comparisontest. *p < 0.05, **p < 0.01, ***p < 0.001.



increased the degree of histone H3 acetylation. Further-more, a significant linear correlation was found between theteratogenic potencies of the respective compounds and theireffects on histone H3 acetylation (n = 42, R2 = 0. 5080,p < 0.0001).

Correlation of end-point values.Of the various cellular and biochemical end-points esti-mated previously, several demonstrated a significant linearcorrelation with the teratogenic potencies of the seven VPA

derivatives. These end-points included cell growth, motilityand changes in Erk1 ⁄ 2 activity (as determined by Erk1 ⁄ 2phosphorylation), GSK-3b activity (as determined by GSK-3b-Tyr216 phosphorylation) and HDAC inhibition (asdetermined by histone H3 acetylation). These correlationsare summarized in fig. 8A. Moreover, drug-induced changesin the end-points for cell GSK-3b activity, growth andmotility correlated with the corresponding drug-inducedchanges in HDAC activities; changes in growth and motilitycorrelated with changes in Erk1 ⁄ 2 activity; and changes in

A

B

C D

Fig. 5. Effects of Valproic acid (VPA) and VPA derivatives on glycogen synthase kinase-3b (GSK-3b) phosphorylation. (A) Left: Representativeimmunoblots showing the effects of VPA (3 mM, 48 hr) on GSK-3b-Ser9 and GSK-3b-Tyr216 phosphorylation levels with total GSK-3b levelsas loading control. Right: Bar diagram showing the effects of VPA on GSK-3b-Ser9 and GSK-3b-Tyr216 phosphorylation levels normalized tothe corresponding untreated controls. Wilcoxon matched-pair signed-rank test, p < 0.0391 (Ser9); p < 0.0078 (Tyr216). (B–D) Effects of expo-sure to VPA derivatives (3 mM, 48 hr). Because of lack of compound, ethyl-4-yn-VPA was not included in these experiments. (B) Representativeimmunoblot showing the effects GSK-3b-Ser9 and GSK-3b-Tyr216 phosphorylation levels with total GSK-3b levels as loading control. (C) Bardiagram showing the effects of VPA derivatives on GSK-3b-Ser9 phosphorylation levels normalized to the corresponding untreated controls.One-way repeated-measures anova F6,19 = 0.8814, followed by the Tukey–Kramer multiple comparison test. p > 0.05. (D) Bar diagram show-ing the effects of VPA derivatives on GSK-3b-Tyr216 phosphorylation levels normalized to the corresponding untreated controls. One-wayrepeated-measures anova F6,19 = 2.797, p < 0.0402, followed by the Tukey–Kramer multiple comparison test. *p < 0.05, **p < 0.01.



growth were correlated to changes in GSK-3b activity(fig. 8B). In contrast, no significant correlation was foundbetween the drug-induced changes in Erk1 ⁄ 2 and HDAC

activities (n = 7, R2 = 0. 3702, p < 0.1471) or betweenchanges in GSK-3b and Erk1 ⁄ 2 activities (n = 7, R2 = 0.4893, p < 0.1219) (data not shown).

A

B

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Fig. 7. Effects of Valproic acid (VPA) and VPA derivatives on his-tone H3 acetylation. (A) Effects of exposure to VPA (1 mM, 24 hr).Left: Representative immunoblots showing histone H3 acetylationlevels with actin protein levels as loading controls. Right: Bar dia-gram showing the effect of VPA normalized to the correspondinguntreated controls. Wilcoxon matched-pair signed-rank test, p <0.6250, n = 9. (B, C) Effects of exposure to VPA derivatives (1 mM,24 hr). (B) Representative immunoblot showing the histone H3acetylation levels with actin protein levels as loading controls. (C)Bar diagram showing the effects of the VPA derivatives normalizedto the corresponding untreated controls. One-way repeated-measuresanova F7,40 = 5.844, p < 0.0001, followed by the Tukey–Kramermultiple comparison test. *p < 0.05, ***p < 0.001.

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B

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Fig. 6. Effects of Valproic acid (VPA) and VPA derivatives onErk1 ⁄ 2 phosphorylation. (A) Effects of exposure to VPA (1 mM,24 hr). Left: Representative immunoblots showing the effects of VPAon Erk1 ⁄ 2 phosphorylation levels with total Erk1 ⁄ 2 protein levels asloading control. Right: Bar diagram showing the effect of VPA onErk1 ⁄ 2 phosphorylation levels normalized to the correspondinguntreated controls. Wilcoxon matched-pair signed-rank test, p <0.0195, n = 9. * p < 0.05. (B, C) Effects of exposure to VPA deriva-tives (1 mM, 24 hr). (B) Representative immunoblot of Erk1 ⁄ 2 phos-phorylation levels with total Erk1 ⁄ 2 protein levels as loadingcontrols. (C) Bar graph showing the effects of the VPA derivativesnormalized to the corresponding untreated controls. One-wayrepeated-measures anova F7,32 = 5.473, p < 0.0005, followed by theTukey–Kramer multiple comparison test. *p < 0.05, **p < 0.01,***p < 0.001.



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Fig. 8. Linear regressions of selected end-points. (A) Associations between the in vivo teratogenicity of the respective test compounds and thecorresponding changes in cell growth (see fig. 1), mean-cell speed (see fig. 4), glycogen synthase kinase-3b (GSK-3b)-Tyr216 phosphorylation(see fig. 5D), Erk1 ⁄ 2 phosphorylation (see fig. 6C) and histone H3 acetylation (see fig. 7C). (B) Associations between changes in histone H3acetylation, GSK-3b-Tyr216 phosphorylation, and Erk1 ⁄ 2 phosphorylation and the corresponding changes in GSK-3b-Tyr216 phosphorylation(top row), mean-cell speed (middle row) or cell growth (bottom row). Solid lines indicate linear regressions with a 95% confidence interval(dashed curves). Symbols and error bars show mean € S.E.M. calculated on the basis of the number of independent experiments (see previousfigure legends for details).



Discussion

Effects of VPA derivatives on cellular end-points.The observed effects of VPA derivatives on the proliferationand growth of L-cells are consistent with previous studiesdemonstrating an association between the teratogenic poten-cies of VPA derivatives and their effects on cell cycle progres-sion [6–9]. The antiproliferative effects of VPA have beenshown to be caused by an arrest of cells in the G1 phase ofthe cell cycle. This was first observed in C6 glioma cells [25]and was later observed in a number of other cell types [e.g.29, 30]. However, in mesenchymal stem cells, VPA has beenreported to arrest cells in the G2 ⁄ M phase [31], suggestingthat the drug may have cell type–specific effects on cell cycledistribution. In the present study, we found that VPA arrestsL-cells in the G1 phase and, consistent with the results onproliferation and growth, the non-teratogen VPA derivativeshad no significant effects on cell cycle distribution, whereasmost of the teratogenic VPA derivatives did. However, wedid not observe consistent changes in the cell cycle distribu-tion in response to the respective test compounds.

The drug-induced changes in cell morphology demon-strated in the present study are consistent with previousobservations that showed a correlation between the terato-genic potencies of compounds and the relative morphologychange induced in L-cells [8,9]. However, a direct correlationbetween the teratogenic potencies of the compounds andtheir effects on the mean-cell area of L-cells, as previouslyreported [10], could not be demonstrated because of theopposite effects of the strongest teratogen tested, hexyl-4-yn-VPA.

The fact that hexyl-4-yn-VPA had opposite effects on cellcycle distribution and cell morphology compared with theother teratogens, causing cells to round up and becomearrested in the G2 ⁄ M phase, might suggest that the com-pound had toxic effects. However, the results obtained with aterminal deoxynucleotidyl transferase dUTP nick end label-ling (TUNEL) assay did not reveal a significant increase inapoptosis after hexyl-4-yn-VPA exposure at the tested con-centration (data not shown).

Valproic acid is known to have highly cell type–specificeffects on cell motility, migration and invasion [24,29,32].However, the results of the present study are consistent withprevious studies demonstrating that the motility of L-cells isinhibited by VPA and VPA derivatives in a manner that corre-lates with the teratogenic potencies of the compounds [11,22].

Effects of VPA derivatives on biochemical end-points.The effects of VPA on the activity of GSK-3b reported inthe literature are contradictory. Estimates of the direct orindirect modulation of GSK-3b activity by VPA have dem-onstrated either inhibition or no change in activity levels[e.g. 4, 33, 34]. Nevertheless, in vivo studies suggest that VPA,at least under certain conditions, directly or indirectlyreduces GSK-3b activity [35–39].

In the present study, we did not evaluate GSK-3b activitydirectly, but instead estimated changes in inhibitory Ser9 and

stimulatory Tyr216 phosphorylation. Interestingly, VPA(3 mM) led to significant changes in the levels of both phos-phorylations after 48 hr, but not after 24 hr, thereby suppos-edly inhibiting GSK-3b activity. Treatment with therespective VPA derivatives induced significant changes inonly GSK-3b-Tyr216 phosphorylation, and only at a highconcentration and after a long incubation time (3 mM,48 hr). This observation suggests that only high and pro-longed, but not brief, exposures to VPA or certain VPAderivatives are able to modulate GSK-3b activity.

Valproic acid is a known HDAC inhibitor. Consistent withprevious studies in other cell types, we found that VPA andVPA derivatives have HDAC inhibitory properties that cor-relate with their teratogenic potencies [13–15].

Valproic acid has been reported to time- and dose-depen-dently stimulate the activity of the MAP kinases Erk1 ⁄ 2 inmany cell types [e.g. 15, 40, 41]. However, in some cells,Erk1 ⁄ 2 activity is unaffected or even inhibited by VPA [24].VPA has recently been shown to time- and dose-dependentlyinhibit the activity of Erk1 ⁄ 2 in L-cells by affecting signallingthrough the Ras-MAPK pathway at the level of Raf [24].The results obtained in the present study suggest that VPAderivatives also inhibit Erk1 ⁄ 2 activity in L-cells and do soin a manner that correlates with the teratogenic potencies ofthe compounds.

Association between cellular and biochemical end-points.A recent study demonstrated that changes in HDAC orErk1 ⁄ 2 activities in response to VPA exposure did not cor-relate with the corresponding changes in cell proliferationor motility when comparing different cell types [24]. How-ever, the present study of a single cell line demonstrates thatVPA derivatives in L-cells inhibited cell growth and speedin a manner that correlated with their effects on bothErk1 ⁄ 2 and HDAC inhibition (fig. 8B), suggesting thatboth Erk1 ⁄ 2 and HDAC inhibition may represent biochem-ical changes responsible for the observed cellular responsesin L-cells.

The observed changes in Erk1 ⁄ 2 activity could be an indi-rect effect of HDAC inhibition because HDAC inhibitorshave been shown to inhibit Erk1 ⁄ 2 activity [e.g. 42, 43].However, in a study of human umbilical vein endothelialcells exposed to VPA or 2 VPA derivatives, no associationwas found between HDAC inhibition and changes in Erk1 ⁄ 2activity [15]. Moreover, in the present study, no significantcorrelation was found between the degrees of Erk1 ⁄ 2 andHDAC inhibition in L-cells. Thus, it is possible that VPAand VPA derivatives in part affect Erk1 ⁄ 2 and HDAC activi-ties through independent mechanisms.

Changes in GSK-3b activity correlated with changes inHDAC activity (fig. 8B), but not Erk1 ⁄ 2 activity. Moreover,as mentioned earlier, changes in GSK-3b activity in responseto treatment with VPA or VPA derivatives required highconcentration and prolonged exposure. These observationssuggest that the effects of the compounds on GSK-3b activ-ity are indirect and potentially a result of the HDAC activi-ties of the compounds.



The ability of HDAC inhibitors to affect cell cycle progres-sion is well described [44]. Similar to other HDAC inhibitors,VPA arrests cell cycle progression partially by up-regulatingp21 [45]. Additionally, HDAC inhibitors affect cell motilityin several ways. For example, in some cells, VPA can inhibitHDAC6 [46], the activity of which can modulate cell motility[47]. In a recent study, VPA enhanced stromal cell-derivedfactor-1a-mediated mesenchymal stem cell migration throughan HDAC-mediated increase in CXC chemokine receptor 4expression [48].

The ability of Erk1 ⁄ 2 to regulate cell proliferation andmotility is also well described [49,50], and the effects of VPAon Erk1 ⁄ 2 activity have been investigated in many studies[4]. Moreover, VPA partially modulates Erk1 ⁄ 2 activity inthe Ras-MAP kinase pathway at the level of Raf [15,24].

Administration of VPA for a single day during gestation issufficient to induce malformations in mice [51], emphasizingthe rapid teratogenic properties of the drug. Therefore, long-term changes in gene transcription that result from HDACinhibition or alterations in Erk1 ⁄ 2 activity do not explainthe teratogenic potential of VPA compounds, although long-term effects may contribute to the teratogenic potencies ofthe compounds. The inhibition of L-cell motility, one of thecellular end-points that correlate with the teratogenic poten-cies of VPA derivatives, has been observed as early as20 min. after VPA exposure [24].

Most of the assays performed in this study have been per-formed at a drug concentration of 3 mM. VPA at this con-centration has been reported not to affect the viability ofL929 cells [8,9], and the high concentration is therefore suit-able for obtaining good signal-to-noise relationships, henceimproving the likelihood of identifying significant relation-ships between the teratogenic potency and biological or bio-chemical processes. However, it should be kept in mind thatalthough a significant effect on a given biological or bio-chemical process is observed at a concentration of 3 mM,the same process may not be significantly affected at lower,therapeutically relevant concentrations.

In conclusion, the present study demonstrates that cellularresponses (such as changes in cell proliferation and motility)of L-cells to exposure to VPA or VPA derivatives correlatewith their effects on Erk1 ⁄ 2 and HDAC activities. Moreover,the changes in Erk1 ⁄ 2 and HDAC activities correlate withthe teratogenic potencies of VPA derivatives, suggesting thatthe effects of the drugs on both Erk1 ⁄ 2 and HDAC activitiesare responsible for the teratogenic properties of thecompounds.

AcknowledgementsWe thank Dr Eugene E. Lepekhin and Associate Professor

Carsten Røpke (formerly at the Department of MedicalAnatomy, Section A, Panum Institute, University of Copen-hagen, Denmark) for technical assistance with the cell cycleinvestigations. We gratefully acknowledge the financial sup-port of the European Union program on In vitro Develop-mental Toxicology (contract no. BIO2-CT-93-0471), theResearch Training Network ‘Nutritional and Environmental

Nuclear Receptor Modulators: Transcriptional Pathways toAbnormal Development and Cancer’ (no. RTN2-2001-00370, HPRN-CT-2002-00268), the Danish Biotechnologyprogram, The Agnes and Poul Friis Foundation, The DanishResearch School in Molecular Cancer Research, FabrikantEinar Willumsens Mindelegat, Fonden Victoria og HenryAndersens Legat, Th. Maigaards Eftf. Fru Lily BenthineLunds Fond af d. 1. juni 1978, Civilingeniør Bent Bøgh oghustru Inge Bøghs Fond, Else og Mogens Wedell-Wedells-borgs Fond, and Anders Hasselbalchs Fond til LeukæmiensBekæmpelse.

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