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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Jergil M, Kultima K, Gustafsson AL, Dencker L, Stigson M.

Valproic Acid-Induced Deregulation In Vitro of Genes Asso-ciated In Vivo with Neural tube Defects Toxicological Sciences. 2009; 108(1), 132-148

II Kultima K, Jergil M, Gustafsson AL, Salter H, Nau H, Dencker L, Stigson M. Early Transcriptional Responses in Mouse Em-bryos as a Basis for Selection of Molecular Markers Predictive of Valproic Acid Teratogenicity Submitted manuscript under revision

III Jergil M, Forsberg M, Salter H, Nau H, Dencker L, Stigson M. Short-Time Gene Expression Response to Valproic Acid and Valproic Acid Analogs in Mouse Embryonic Stem Cells. Manuscript

IV Jergil M, Strehl R, Salter H, Dencker L, Hyllner J, Stigson M. Exploring Transcriptional Response to Valproic Acid and Val-proic Acid Analogs in Human Embryonic Stem Cells. Manuscript

Reprints were made with permission from the publisher.

Contents

Introduction ..................................................................................................... 9 Perspective of developmental toxicology ................................................... 9 Alternative methods in developmental toxicology ................................... 10

Limb bud micromass test ..................................................................... 11 Rat whole embryo culture test ............................................................. 11 Embryonic stem cell test ...................................................................... 11

Pluripotent stem cells of human and mouse ............................................. 12 Embryonal carcinoma cells .................................................................. 13 Embryonic stem cells ........................................................................... 13 Epiblast stem cells ............................................................................... 16 Embryonic germ cells .......................................................................... 16 Induced pluripotent stem cells ............................................................. 17

Transcriptomics ........................................................................................ 17 Toxicogenomics ....................................................................................... 18 The teratogen valproic acid ...................................................................... 19

Aims of the studies........................................................................................ 21

Comments on Methods ................................................................................. 22 Experimental designs ............................................................................... 22 Animals .................................................................................................... 22 Stem cell culture ....................................................................................... 23

Results and discussion .................................................................................. 25 Potential of in vitro systems - detection of effects on gene regulation relevant for the in vivo phenotype after VPA exposure in P19 EC cells (paper I) .................................................................................................... 25 Gene deregulation in mouse embryos and search for markers of VPA teratogenicity through in vivo - in vitro comparison (paper II) ................ 28 Transcriptional responses in mouse ES cells (paper III) .......................... 30 Transcriptional responses in human ES cells (paper IV) ......................... 33

Concluding remarks and future perspective .................................................. 37

Acknowledgements ....................................................................................... 40

References ..................................................................................................... 42

Abbreviations

(S)-4-Pe-Pentyn (S)-4-pentyn-2-pentynoic acid 2-Et-4-Me-Penta 2-ethyl-4-methyl-pentanoic acid 3-Pr-Hepta 3-propyl heptanioc acid AP Alkaline phophatase ATRA All-trans retinoic acid BMP Bone morphogenetic protein cDNA Complementary deoxyribonucleic acid DAVID The Database for Annotation, Visualization and Inte-

grated Discovery DMSO Dimethylsulfoxide EC cell Embryonal carcinoma cell EG cell Embryonic germ cell EpiS cell Epiblast stem cell ES cell Embryonic stem cell EST Embryonic stem cell test GABA Gamma-aminobutyric acid GD Gestational day GO Gene Ontology HDAC Histone deacetylase hES cell Human embryonic stem cell IC50 50% inhibition of growth ICM Inner cell mass ID50 50% inhibition of differentiation iPS cell Induced pluripotent stem cell KEGG Kyoto encyclopedia of genes and genomes LIF Leukemia inhibitory factor Log2 Base two logarithm MEF Mouse embryonic fibroblast mES cell Mouse embryonic stem cell MM Limb bud micromass test mRNA Messenger ribonucleic acid NTD Neural tube defect PCR Polymerase chain reaction PG cell Primordial germ cell qPCR Semi-quantitiative real-time reverse transcription PCR

REACH Registration, Evaluation, Authorization and restriction ofChemicals

SSEA Stage specific embryonic antigen TSA Trichostatin A VPA Valproic acid WEC Whole embryo culture test

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Introduction

Toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals, and these procedures consume large resources and many laboratory animals. To lower the costs and animal consumption, the pharmaceutical industry pursue to implement in vitro embryotoxicity tests in high throughput screening for lead optimiza-tion (Genschow et al., 2004). In addition, due to the new European Union legislation on chemicals, called REACH (Registration, Evaluation, Authori-zation and restriction of Chemicals), in 2007 as many as 30,000 chemicals need to be toxicity evaluated (Hartung, 2009b). This necessitates new strate-gies and the development of new techniques (Hartung, 2009a; b).

In this thesis the concept of using short-term in vitro assays for develop-mental toxicity based on pluripotent stem cells has been investigated. The developmental toxicant valproic acid (VPA) has been used to explore the possible implementation of toxicogenomics and gene expression as markers of developmental toxicity in such assays.

Perspective of developmental toxicology In developmental toxicology the effects on developmental processes are usually examined from the stage of the zygote (the fertilized egg), through organogenesis and the fetal period to the term of pregnancy. Disturbances of development resulting in developmental defects can be divided into intrinsic and extrinsic causes. Intrinsic causes include various genetic alterations such as mutations and chromosomal aberrations, whereas extrinsic causes com-prise environmental factors such as infections, hyperthermia, nutritional deficiencies, chemicals and pharmaceuticals. It is estimated that 2-3% of all live born infants have a major congenital defect. Of these, about 5% are at-tributed to environmental insults, which in addition to chemicals and drugs include infections and diseases like diabetes (Bremer and Hartung, 2004; Fraser, 1977). However, these numbers do not include neurological or beha-vior problems or spontaneous abortions and still-birth, which greatly in-crease the number of adverse outcomes in human pregnancy (Hass, 2006).

In 1933, Hale showed that retinol deficiency during pregnancy induced malformations in piglets, this was one of the first developmental toxicology studies conducted using appropriate controls (Mark et al., 1999). Gregg was, in 1941, the first to show that maternal exposure can cause congenital mal-

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formations in humans when he reported a connection between maternal ru-bella virus infection and congenital cataract (Gregg and Banatvala, 2001). However, the most prominent and well-documented event, that brought im-mediate attention to modern studies of developmental toxicology, was the recognition of thalidomide as a human teratogen (Lenz, 1966; McBride, 1961). Thalidomide was initially marketed as a mild sedative and then sub-sequently used for morning sickness in pregnant women. In 1961 and 1962 its use in pregnancy was associated with an epidemic of malformed children. Between 7000 and 15000 children worldwide were born with severe mal-formations including phocomelia (absence of the long bones) before the removal of the drug from the international market, at the end of 1961, brought the epidemic to an end (Lenz, 1988). Thalidomide was not tested for teratogenicity before marketing, but acute toxicity testing in rats and mice had indicated it was not acutely toxic and it was thought to be safe for hu-man use. After the human catastrophe, thalidomide was shown to induce malformations in rabbit and non-human primates with a pattern of malfor-mations similar to that seen in humans. However, no malformations were seen in rodents (Wilson, 1973). This highlighted the complexity of extrapo-lating animal data to the human condition, and prompted new regulatory guidelines requiring the use of at least two species, whereof at least one has to be non rodent, in developmental toxicity assessment.

In Europe 2003, approximately 40,000 laboratory animals were used in developmental toxicity testing annually, and within the REACH initiative it is estimated that developmental toxicity will use about 20% (perhaps as many as 3.5 million in total) of the animals (Bremer and Hartung, 2004; Piersma, 2006). This emphasizes the urgent need for the development of alternative methods of assessing human developmental toxicity.

Alternative methods in developmental toxicology As an initiative from the European council 1991, ECVAM (European Center for Validation of Alternative Methods) was created to support the develop-ment and validation of methods that could reduce, refine or replace (known as “the 3R:s”) the use of laboratory animals (http://ecvam.jrc.it/, 2009). Current-ly, ECVAM has evaluated three in vitro tests designed to predict developmen-tal toxicity: the limb bud micromass test (MM), the rat whole embryo culture test (WEC) and the embryonic stem cell test (EST). These in vitro tests have been shown to be relatively good at predicting the embryotoxicity for a num-ber of substances classified in vivo as non-teratogenic or weak or strong tera-togens (Genschow et al., 2004; Genschow et al., 2002; Piersma et al., 2004; Spielmann et al., 2004). However, further development of these assays is needed before they can be implemented as a standard battery in developmental toxicity testing. Below follows a description of each of these tests.

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Limb bud micromass test In the MM, the inhibition capacity of chemicals on the differentiation of limb bud cells to chondrocytes is investigated. Cultures of dissociated cells from the limb buds of rat embryos at gestational day (GD) 14 (approx. 45 somites) are studied (Flint and Orton, 1984; Kistler, 1987). The cells are seeded at a high density (micromass) and cultured in the presence or absence of test chemicals for 5 days, and growth and differentiation of the cells into chondrocytes are then evaluated (Genschow et al., 2000). Alcian blue is used to specifically stain cells which have developed into chondrocytes and the cytotoxic effect is detected after which ID50 (50% inhibition of differentia-tion) and IC50 (50% inhibition of growth) values are calculated (Genschow et al., 2000). Although an in vitro technique, the MM is still dependent on ani-mals for the derivation of cells which makes it more of a refinement than a replacement of animal testing.

Rat whole embryo culture test In the WEC, embryos are explanted and cultured for a period during organo-genesis (New, 1978). More specifically, post implantation rat embryos at GD 10 (1-5 somite stage) are cultured for 48 hours in rat serum and exposed to test substances (Piersma et al., 1995). During this culture period, major steps in the organogenesis occur, such as neural tube closure, heart development, as well as development of the ear and eye, and limb bud formation. There-fore, WEC enables the detection of dysmorphogenesis of specific structures and general retardation of growth and development. Function and morpholo-gy are examined at the end of the incubation period, and the embryos are scored according to a criteria system adopted from Brown and Fabro (Brown and Fabro, 1981). The mean value of total morphological score of seven embryos per concentration are then calculated and compared to a control group (Genschow et al., 2000). In addition, the number of malformed or dead embryos is compared with the control group, to assess the no-effect concentration and maximal effect concentration as well as IC50 (Piersma et al., 2004). As with MM, WEC relay on live animals as the source of em-bryos, but since all embryos in a litter can be monitored separately a reduc-tion of the number of animals can be achieved.

Embryonic stem cell test The embryonic stem cell test (EST) has two principle procedures. The first takes advantage of the inherent ability of mouse embryonic stem (ES) cells to differentiate in vitro and measures the ability of the test chemical to inhi-bit the formation of cardiomyocytes. The second measures the viability of ES cells and fibroblast cells after exposure to test substances (Scholz et al.,

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1999a). The test begins when ES cells are grown to form so-called embryoid bodies (EBs), small aggregates of cells that induce differentiation, for three days with or without test chemicals. After this, the EBs are transferred to bacterial dishes, to avoid their adherence, for 2 days and thereafter they are plated in tissue culture plates for outgrowth. After 5 days on the plate, when the cells have been in the presence of the test chemicals for a total of 10 days, the presence of contracting areas of cardiomyocytes are evaluated and compared to the control cultures for calculation of the ID50 (Genschow et al., 2000; Scholz et al., 1999a). For viability measurements, ES cells and fibrob-last cells are cultured for 10 days in the presence or absence of the test sub-stance and a MTT-viability assay is performed. The IC50 values from ES and fibroblast cells are then used together with the ID50 value, and applied to a biostatistical prediction model to classify embryotoxic potential (Genschow et al., 2000; Genschow et al., 2002; Scholz et al., 1999b).

In an attempted to improve the EST there has been implementation of molecular markers and the use of different techniques such as flow cytome-try and gene expression. This is aimed at shortening the assay time and mak-ing it compliant with high throughput screening (Buesen et al., 2009; Seiler et al., 2004; van Dartel et al., 2009; zur Nieden et al., 2004). ES cells can also differentiate into the neural lineage, an approach also under develop-ment in test assay context (Stummann et al., 2007). Human ES cells poten-tially offer an in vitro system closer to the human embryo and would conse-quently be a better option in risk assessment. Work has been done to adopt the EST protocol also to human ES cells (Adler et al., 2008a; Adler et al., 2008b).

Pluripotent stem cells of human and mouse The most simplistic definition of a stem cell describes a cell that is able to self replicate (self-renew) over an indefinite time, and with the right condi-tions and given the right signals, is able to differentiate into specialized cell types. Most embryo-derived stem cells are considered pluripotent which means they have the ability to develop into all different cell types of an or-ganism, or at least to cells in all three germ layers (Solter, 2006). This can be contrasted to the totipotent state, stipulated only for the zygote and the cells of the first cell divisions, which in addition to all cell types in the body can contribute also to extra-embryonic cell types (Jaenisch and Young, 2008; Nagy et al., 1993). Below follows a schematic overview of a number of plu-ripotent stem cells that has been established and characterized in vitro.

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Embryonal carcinoma cells Teratocarcinomas are tumors that are typically found in the gonads and may be composed of a mixture of tissues such as hair, muscles, skin and even teeth. The teratocarcinoma may also contain a core of undifferentiated stem cells that can be isolated and further propagated in culture as embryonal carcinoma (EC) cells (Figure 1) (Andrews et al., 1984; Kahan and Ephrussi, 1970; Solter, 2006). In addition, grafting mouse embryos in an early stage to ectopic sites in adult mice may also generate teratocarcinomas from which EC cells can be established (McBurney, 1993; McBurney and Rogers, 1982; Smith, 2001). EC cells have the ability to divide repeatedly and are consi-dered pluripotent as they can be differentiated in vitro by extrinsic factors (e.g. retinoic acid) and form all three germ layers after injection subcuta-neously in mice (Jones-Villeneuve et al., 1982). Mouse EC cells can even make chimaeras, however the contribution of EC cells in the fetus is often low and their germline transmission capacity is disputed (Brinster, 1974; Rossant, 2008). Chimeras with EC cells readily develop tumors, derived from the EC cells, indicating that the tumor properties are still resident in the cells (Rossant, 2008; Rossant and McBurney, 1982).

Mouse EC cells express the surface antigen stage-specific embryonic an-tigen (SSEA) -1 and turn on SSEA-3 and SSEA-4 upon differentiation (Solter and Knowles, 1978). In contrast, human EC cells express SSEA-3 and SSEA-4 and turn on SSEA-1 upon differentiation. EC cells of both spe-cies have a high enzymatic activity of alkaline phosphatase (AP) and charac-teristically express the transcription factor OCT4 (Andrews et al., 2005).

Embryonic stem cells

Mouse After the establishment and characterization of EC cell lines, the next step was to establish pluripotent cells directly from the mouse embryo to culture in vitro for an extended time. In 1981, Evans and Kaufman were able to propagate cells from the inner cell mass of the blastocyst on a layer of mouse fibroblast cells and keep them undifferentiated for a prolonged period of time. In this way the cell type known as embryonic stem cell was established (Evans and Kaufman, 1981). Mouse ES (mES) cells have revolutionized life sciences through their use in transgenic and knockout mice, a technology that rendered Evans the 2007 Nobel Prize in Physiology or Medicine togeth-er with Mario R. Capecchi, and Oliver Smithies.

mES cells grow in tight, rounded 3D colonies (Figure 1) and divide ap-proximately every 12 hour. During self-renewal, most ES cells are only tran-sient in the G1 phase of the cell cycle and instead they spend most time are spent in the S phase (Niwa, 2007).

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Figure 1. Pluripotent stem cells. (A) Schematic overview of the blastocyst with the inner mass cells, from which ES cells are derived (B) mouse EC cells (C) mouse ES cells grown on feeder layer (D) human ES cells grown on feeder layer.

To continuously self-renew in vitro mES cells are dependent on either a feeder cell layer, consisting of embryonic fibroblast cells, or a serum-containing medium supplemented with leukemia inhibitory factor (LIF) (Smith et al., 1988). To promote pluripotency, LIF binds to the LIF-receptor (LIFR)-gp130 heterodimer receptor to mediate JAK-STAT3 activation. This induces STAT3 dimerization and phosphorylation with subsequent tranloca-lization to the nucleus where STAT3 functions as transcription factor (Niwa et al., 1998). STAT3 is reported to regulate itself in an autoregulatory loop where it also controls the expression of gp130 and LIFR (Davey et al., 2007). However, in serum-free conditions LIF alone is not sufficient to pre-vent differentiation, and the system requires bone morphogenetic protein (BMP) in the medium (Ying et al., 2003a). BMP4 is considered to inhibit differentiation of mES cells into the neuronal lineage through activation of Smad4 which in turn activates the inhibition of differentiation (Id) gene (Ying et al., 2003a). BMP seems to be the serum-derived factor that prohi-bits differentiation together with LIF.

In addition to the extrinsic factors, pluripotency of ES cells is believed to be maintained by a few essential transcription factors consisting of Oct4, Sox2 and Nanog (Chambers and Smith; Mitsui et al., 2003; Nichols et al., 1998). These three factors repress and activate transcription of genes in order to maintain pluripotency, often through their co-binding on transcription

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sites of target genes. It is suggested that it is mainly activation of genes that is essential for pluripotency (Jaenisch and Young, 2008; Sharov et al., 2008). However, Nanog may be more of a stabilizing factor which prohibits diffe-rentiation rather than being essential for the pluripotent state (Silva and Smith, 2008). Sox2 mediates its effect on pluripotency mainly through hete-rodimerization with Oct4 to regulate the levels of Oct4 (Masui et al., 2007), in fact all three of Oct4, Sox2 and Nanog seems to be autoregulated since they all bind to their own promoters (Loh et al., 2006). Also the Wnt-pathway is suggested to promote pluripotency since extrinsic GSK3 inhibi-tion sustains the expression of Oct4 and thus maintains pluripotency (Sato et al., 2004).

Characterized markers for the undifferentiated state of mES cells are the transcription factors mentioned above Oct4, Nanog, and Sox2, together with the cell surface antigen SSEA1, AP and a high activity of telomerase enzyme (Boiani and Scholer, 2005). The most profound marker of pluripotency is the ability of mES cells to generate chimeras and readily contribute to germ line when injected into recipient blastocysts (Nagy et al., 1993). In addition to the in vitro differentiation to cardiomycytes mentioned above, protocols are also available for the directed differentiation of mES cells into various cell types such as neuronal cells, pancreatic cells, or hepatic cells (Naujok et al., 2009; Shiraki et al., 2008; Ying et al., 2003b).

Human The first reports of established primate ES cells came in the middle of the 1990s, with successful derivations of ES cells from the common marmoset and the Rhesus monkey (Thomson et al., 1995; Thomson et al., 1996). Hu-man ES cells were derived from the inner cell mass for the first time 1998 by Thomson and co-workers (Thomson et al., 1998).

In contrast to mES cells, human ES (hES) cells do not rely on LIF to maintain pluripotency and self-renewal, instead they need bFGF and a feeder cell layer (Amit et al., 2000). However, a combination of activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Se-rum Replacer have been shown to support pluripotency (Vallier et al., 2005). This indicates that pluripotency in hES cells is promoted through the TGF�/Activin/Nodal signaling which involves SMAD2 and 3 signaling (Vallier et al., 2005; Yu and Thomson, 2008). As in mES cells the three transcription factors OCT4, SOX2 and NANOG have a central role in main-taining pluripotency in hES cells through activation and repression of several genes. Many of these target genes encode developmentally important tran-scription factors for lineage specificity and differentiation (Boyer et al., 2005).

Human ES cells grow in flattened two-dimensional (2D) compact colo-nies (Figure 1) with a cell cycle of approximately 25 h. They are characte-rized by the expression of the “trinity of pluripotency” OCT4, SOX2 and

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NANOG and of the cell-surface antigens SSEA3, SSEA4, TRA-1-60, and TRA-1-81 as well as enzymatic activities of TRA-2-54 (AP) and telomerase (Boiani and Scholer, 2005; Henderson et al., 2002). In addition, hES cells that are transplanted into severe combined immunodeficiency (SCID)-mice, form teratomas consisting of cells from all three germ layers (Thomson et al., 1998). As with mES cells, protocols for directed differentiation of hES cells in vitro into specialized cell types such as neural, cardiac, or hepatic cells are available (Agarwal et al., 2008; Hu et al., 2009; Shiraki et al., 2008; Synnergren et al., 2008).

Epiblast stem cells ES cells are derived from the pre-implantation blastocyst whereas epiblast stem (EpiS) cells are derived from the post-implantation embryos (Brons et al., 2007; Tesar et al., 2007). Mouse EpiS cells are not dependent of LIF or BMP as are mES cells, but dependent on FGF and activin A as are hES cells (Brons et al., 2007). They also resemble hES cells morphologically as they form flat compact colonies (Brons et al., 2007; Tesar et al., 2007). EpiS cells express the pluripotency markers Oct4, Sox2, and Nanog but not AP, and can form teratomas with cells from all germlayers, but cannot produce chi-meras (Brons et al., 2007). It is suggested that mouse EpiS cells have gene expression profiles and transcription factor networks similar to hES cells and are in a closer relationship to hES cells than mES cells (Brons et al., 2007; Rossant, 2008).

Embryonic germ cells Primordial germ (PG) cells are progenitors of the germ cell lineage and re-side in the post implantation embryo in a small population. From PG cells a type of ES-cell-like cells can be derived, called embryonic germ (EG) cells (Matsui et al., 1992; Resnick et al., 1992; Shamblott et al., 1998). For deriva-tion on fibroblast layers, LIF and bFGF are needed as well as forskolin, however, for maintenance of mouse EG cells FGF is dispensable (Durcova-Hills et al., 2006; Matsui et al., 1992). EG cells closely resemble mES cells in growth and both mouse EG and human EG cells express pluripotency markers AP and SSEA-1. Just like hES cells, human EG cells also express SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81. Human EG cells have the ability to differentiate in vitro and form cells from all three germ layers while the mouse counterpart may form chimeras when injected into blasto-cyst (Durcova-Hills et al., 2006; Matsui et al., 1992).

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Induced pluripotent stem cells Pioneering work by the group of Yamanaka in 2006 resulted in the devel-opment of a new type of pluripotent ES-cell-like cells designated induced pluripotent stem cells (iPS). The iPS cells were induced from differentiated adult mouse cells (fibroblasts) into pluripotent self renewing cells through ectopic transcription factors consisting of Oct4, Sox2, c-Myc, and Klf4 (Okita et al., 2007; Takahashi and Yamanaka, 2006). Recently, iPS cells have been established also from human primary fibroblasts which have gen-erated great expectations in the field of regenerative medicine (Huangfu et al., 2008; Takahashi et al., 2007). Like EG cells, iPS cells are pluripotent and resemble ES cells closely, both through colony formation and through the expressed of pluripotency markers. Mouse iPS cells, like mES cells, express SSEA-1, while the human counterpart expresses SSEA-3 and 4, TRA-1-60, and TRA-1-81, just like the hES cell. In addition, Nanog, Sox2, and Oct4 are maintained at a high level as well as AP (Okita et al., 2007; Takahashi et al., 2007; Takahashi and Yamanaka, 2006). Chimeras have been achieved with iPS (mouse) and even germline transmission which ultimately proves their pluripotency (Okita et al., 2007). It has been suggested that creation of iPS cells is likely to include a process of epigenetic conversion from the diffe-rentiated cell back into the pluripotent cell, involving activated Nanog, a balanced expression of the endogenous pluripotency factors, and minimized extrinsic disturbance (Silva and Smith, 2008).

Transcriptomics The publication of the structure of the DNA helix by Watson and Crick was the dawn of molecular biology (Watson and Crick, 1953). This was the be-ginning of understanding the processes by which genetic material is stored, copied, and transferred through generations, and how genes are sequenced, which culminated with the publication of the whole human genome in 2001 (Venter et al., 2001). Ribonucleic acid (RNA) in the form of messenger RNA (mRNA) is the product of gene expression and is transcribed from DNA, and further translated into proteins. The complete set of mRNA in a given cell or organism is named the transcriptome and represents the ongo-ing activity of gene expression.

Since the middle of the 1970s several techniques have evolved for quanti-fying mRNA and thus the activity of genes. Northern blot, a gel based tech-nique for assessing mRNA, was one of the first methods described (Alwine et al., 1977). The invention of the polymerase chain reaction (PCR) by Nobel laureate Kary Mullis, enabled the exponential amplification of specific DNA constructs using a set of primers through thermal cycling (Mullis et al., 1987). In combination with reverse transcriptase (an enzyme capable of turn-

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ing RNA into complementary DNA) PCR is a powerful technique to assess gene expression. Higuchi and co-workers greatly simplified PCR as a tool for assessing mRNA, when they introduced the concept of real-time quantit-ative reverse transcription PCR (qPCR) (Higuchi et al., 1992). In qPCR the level of amplified product is measured simultaneously throughout the whole procedure with a fluorescent dye.

PCR techniques have meant a big leap forward in reproducible gene ex-pression analysis, however, it measures only one transcript at a time and presupposes knowledge of which gene might be altered. Thus, PCR is not very useful in hypothesis generation or for investigating global gene expres-sion responses, gene expression microarrays on the other hand offers this potential. Microarrays contain many thousands of cDNA clones or oligonuc-leotides (fragments of genes) fixed in arrays on a solid surface, to which fluorescent labeled RNA can hybridize. The fluorescent signal emitted when analyzing the array with a laser scanner is proportional to the amount of RNA hybridized to a specific probe, and hence an estimation of the level of expression of specific genes.

Toxicogenomics The development of microarray techniques opened the possibility to assess gene expression after exposure to a toxicant in a global perspective and paved the way for toxicogenomics. Classical toxicological evaluations in animals rely on histological findings, pathology, and clinical chemistry. The toxicogenomics approach on the other hand aims to classify substances from the outcome of gene expression profiles that occur after an exposure (Nuwaysir et al., 1999). The principle is that certain toxicity outcomes are preceded by specific gene expression changes as a consequence of toxicant exposure (Maggioli et al., 2006; Suter et al., 2004). If this hypothesis holds true, gene expression profiling and toxicogenomics would generate mecha-nistic knowledge for toxicity as well as classification of a toxic exposure (Gant and Zhang, 2005). Since microarrays generate data from thousands of genes simultaneously, a hurdle is how to discriminate gene expression res-ponses that are induced by an exposure but are not relevant for the prediction of toxicity (Maggioli et al., 2006). Different techniques such as hierarchical clustering, K mean clustering, and multivariate analysis with principal com-ponent analysis, can be useful tools in assessing the global gene expression and the elucidation of relevant gene expression changes (Hayes and Bradfield, 2005). Also, additional gene attribute knowledge (such as Gene Ontology and pathways analysis) or phenotypic anchoring (combination of the expression data with specific conventional toxicological parameters) will enhance the interpretation of the data to discriminate for relevant gene ex-pression (Ashburner et al., 2000; Davis et al., 2009; Paules, 2003). In com-

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bination with profiles of gene expression, a smaller set of genes responding to a toxic exposure could be applied as biomarkers for a certain class of tox-icant.

The teratogen valproic acid Valproic acid (VPA) is a short chain fatty acid, mainly used as an antiepilep-tic drug, particularly in patients with absence seizures and myoclonic genera-lized tonic-clonic seizures (Loscher, 2002). It is also used in the management of mania associated with bipolar disorders and as a migraine prophylaxis (DeVane, 2003). Moreover, VPA has shown promising results as an anti-cancer agent (Blaheta et al., 2005; Duenas-Gonzalez et al., 2008). VPA was first synthesized in 1882, and used as an organic solvent for several years, and its anticonvulsant effect was revealed in the middle of the 1960s. The finding was serendipitously made when VPA was used as a vehicle for a number of compounds in a screen aimed at finding anti-seizure activity. VPA was marketed in France in 1967 and released for antiepileptic use on the US-market in 1978, and in Sweden 1980 (Peterson and Naunton, 2005; Pinkston and Walker, 1997; Sztajnkrycer, 2002).

The mode of action of VPA´s different applications is not definitely es-tablished. However, the antiepileptic action is suggested to relate to effects on the neurotransmitter gamma butyric acid (GABA) levels in specific re-gions in the brain as a result of inhibition of enzymes responsible for GABA metabolism (Johannessen, 2000; Sztajnkrycer, 2002).

VPA has relatively few side effects, but one major adverse effect is that when taken in pregnancy it increases the risk of birth defects. The probabili-ty of adverse effects of VPA in the human fetus was at first neglected, even though animal studies had indicated a risk, and the first indication of VPA induced teratogenicity in human came in a case report in 1980 (Dalens et al., 1980). The relationship was further established in a birth monitoring pro-gram where children born to mothers treated with VPA showed a higher incidence of neural tube defects (NTDs) (Robert and Guibaud, 1982). A number of other monitoring programs and publications have since then con-firmed that there is a distinct risk increase in birth defects in children whose mothers ingest VPA during pregnancy (Artama et al., 2005; Lindhout et al., 1992; Meador et al., 2008; Ornoy, 2009; Vajda et al., 2006; Wide et al., 2004). The most prominent teratogenic effect of VPA is NTDs, primarily spina bifida with a 1-2% incidence, that is a 10-20-fold increase in risk over non-exposed pregnancies (Ornoy, 2006). Cardiovascular and limb defects, skeletal malformations and urogenital malformations are also sometimes seen after VPA exposure (Holmes, 2002; Kozma, 2001; Wide et al., 2004). A pattern of facial dysmorphy together with other malformations in children exposed in utero to VPA has been described as the fetal valproate syndrome

20

(DiLiberti et al., 1984). VPA is also reported to induce neurodevelopmental effects such as autism and low IQ (Ornoy, 2006; Vinten et al., 2009). A dose-response relationship has been proposed for these developmental ef-fects, and a maternal daily dose of more than 1000 mg is suggested to ele-vate the risk for the child (Meador et al., 2006) but no dose can be consi-dered safe.

The teratogenic effects of VPA are seen also in other vertebrates includ-ing the Rhesus monkey (Hendrickx et al., 1988), rat (Menegola et al., 1996), rabbit (Petrere et al., 1986), zebrafish, and xenopus (Phiel et al., 2001). In mice, NTDs and a variety of other malformation such as skeletal malforma-tion, fused ribs and heart malformations, are seen after VPA exposure (Menegola et al., 1996; Nau, 1985). VPA given to pregnant mice on GD 8 induces exencephaly, thus representing a model of VPA-induced NTDs (Nau and Loscher, 1986).

The teratogenic mechanism of VPA is poorly understood, but it has re-cently been suggested to be dependent on inhibition of histone deacetylase (HDAC) enzymes of class I (Kultima et al., 2004; Menegola et al., 2005; Phiel et al., 2001).The HDAC inhibiting capability of VPA structure analogs have been correlated with increased potential of inducing NTDs (Eikel, 2005; Eikel et al., 2006b). HDACs mediate the removal of acetyl groups on lysines in the tail of core histones, proteins that make up the chromatin struc-ture, which is recognized as a more condensed chromatin. The inhibition of HDAC results in increased acetylation of histones with subsequent activation and repression of genes (Dokmanovic and Marks, 2005; Marks et al., 2004; Zupkovitz et al., 2006). The formation and closure of the neural tube is a highly organized procedure that has been closely investigated, a substantial number of genes are reported to be implicated in this procedure (Copp et al., 2003; Harris and Juriloff, 2007; Kibar et al., 2007; Sadler, 2005). The possi-bility that VPA may deregulate essential genes through HDAC inhibition and thus cause NTD is a plausible mechanism behind the teratogenicity, although a recent study has suggested that HDAC inhibition is not the cause of teratogenicity (Wise et al., 2007).

Other suggested mechanisms of VPA induced teratogenicity are reactive oxygen formation (Defoort et al., 2006), interactions with the actin cytoske-leton (Walmod et al., 1999), activation of the peroxisome proliferator-activated receptor (PPAR)-delta (Werling et al., 2001) and interactions with folic acid metabolism (Dawson et al., 2006).

21

Aims of the studies

Both the general public and industry desire to replace, or at the least reduce the number of, animals used in toxicology evaluations of pharmaceuticals and chemicals with in vitro assays. The work in this thesis is a continuation of in vitro studies and developmental toxicology, with VPA as a model sub-stance, previously initiated in the group. The overall objective has been to explore in vitro systems consisting of pluripotent stem cells of embryonic origin in short-time assays with applications to developmental toxicology. VPA, and in later studies some analogs of VPA, have been used in the stu-dies since this drug induces similar developmental effects (NTDs) in both human and mouse after exposure during the embryonic period, and thus may bridge between the species in inter-species comparisons.

Specific aims were:

• Study the effects of the antiepileptic and teratogenic drug VPA on gene expression, particularly of those genes known to be involved in formation and closure of the neural tube, using an in vitro model sys-tem of EC cells.

• Find potential transcriptional markers of VPA-induced teratogenicity useful in an in vitro assay through in vivo - in vitro comparison

• Explore mouse ES cells as an in vitro model system of developmen-

tal toxicity with comparisons with EC cells and mouse embryos.

• Find specific transcriptional markers of VPA effects in vitro using VPA analogs with varying potency of HDAC inhibition and terato-genic potential and test those in mouse ES cells.

• Explore the transcriptional response of VPA and VPA analogs in human ES cells.

• Compare the effects of VPA and VPA analogs on global gene regu-lation in mouse versus human ES cells.

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Comments on Methods

Experimental designs The ability of VPA to induce the NTDs spina bifida in human embryos and exencephaly in mouse embryos (when administered at GD 8) has been used as the central paradigm in the experimental design of the studies conducted in this thesis.

Exposures that induced only low cytotoxicity and small effects on cell proliferation over 24 h in mouse cells were used for the toxicogenomic eval-uations. An exception is the 10 mM VPA exposure in EC cells (paper I). For hES cells, visual evaluation of the cells was used to determine that there were no apparent cytotoxicity over 24 h for the exposure concentrations.

VPA analogs with different potential for HDAC inhibition and teratoge-nicity that, in addition to VPA, have been used in paper III and IV and are listed in Table 1.

Table 1. VPA and VPA analogs.

Animals NMRI mice (B&K Universal AB, Sollentuna, Sweden) were kept on a 12-h light cycle. Females were mated with males for 2 h at the end of the dark

Compound Abbrevation StructureTeratogenic

potencya

HDAC-inhibition

capabilityb

Valproic acid VPA +++c

+++b

3-Propyl-heptanoic acid 3-Pr-Hepta ++++c

-e

(S)-2-Pentyl-4-pentynoic acid (S)-2-Pe-4-Pentyn +++++c

+++++b

2-Ethyl-4-methyl- pentanoic

acid2-Et-4-Me-Penta -

d-b

aDecision critera for teratogenicity investigated in NMRI-exencephaly mouse model (Nau et al., 1986)bHyper acetylation of H4 and effect in HDAC enzyme inhibition assay (Eikel et al., 2006)c(Volland, 2005)d(Bojic, 1996)e(Eikel, 2005)

CH3CH3

COOH

CH3CH3

COOH CH3

CH3

COOH

CH3

CHCH3

COOH

23

period and were then checked for vaginal plugs. The midpoint of the mating period was taken as 0 days postcoitum. VPA (600 mg/kg) was administered intraperitoneally on GD 8.

Stem cell culture Three pluripotent stem cell lines derived from embryos have been used in this thesis; P19 mouse EC cells, R1 mouse ES cells, and SA002 human ES cells.

P19 mouse EC cells were cultured at 37 °C and 5% CO2 humidified air in Dulbecco’s Modified Eagle’s Medium (Swedish National Veterinary Insti-tute, Uppsala, Sweden), supplemented with 10% fetal bovine serum (Swe-dish National Veterinary Institute), 2 mM L-glutamine, 100 U/ml penicillin and 100 �g/ml streptavidin. Cells were split 1:10 every second to third day. For exposures the culture medium was exchanged for medium containing test substance.

R1 mouse ES cells were maintained on a layer of irradiated mouse embryo fibroblast (MEF) cells at 37°C and 5 % CO2 humidified air in complete ES cell medium containing D-MEM with glutamax (Invitrogen) supplemented with 10 % ES cell qualified FBS (Invitrogen) 0.1 mM non-essential amino acids, 0.1 mM �-mercaptoethanol, and 1000 U/mL of leukemia inhibitory factor (LIF) (Chemicon). Change of medium was performed every day and cells were passed through trypsination at least every second day. ES cells were feeder depleted and grown on gelatin coated dishes before and during exposure to minimize effects of feeder cells and their contribution to gene expression. When exposing the ES cells, the culture medium was exchanged for medium containing the test substance but no LIF.

hES cells of the line SA002 (Cellartis AB, Gothenburg, Sweden) were main-tained on a mitomycin-C inactivated mouse embryo fibroblast (MEF) feeder cell layer at 37°C in VitroHESTM medium (Vitrolife, Gothenburg, Sweden) supplemented with 4 ng/ml human recombinant bFGF (Invitrogen Co., Carlsbad, CA, USA). Cells were passaged in fresh medium every 4-5 day through mechanical dissociation using a “Stem cell tool” (Swemed Lab In-ternational AB, Billdal, Sweden). For exposure, a portion of the culture me-dium was withdrawn and mixed with test substance and subsequently put back to the cells. This procedure was done to provide the cells with condi-tioned medium.

24

Other methods used in this thesis are listed in Table 2 and the reader is re-ferred to the Materials and Methods section in respective paper for a com-prehensive description.

Table 2. Methods used in the studies in this thesis. Method used Paper (s) Cluster analysis II, III, IV Gene ontology analysis I, II, III, IV Microarray analysis one-channel (CodeLink Human Whole genome Bioar-rays)

IV

Microarray analysis one-channel (CodeLink Mouse CodeLink 20K Bioar-rays)

I, (II)

Microarray analysis one-channel (CodeLink Mouse Whole Genome Bioar-rays)

III

Microarray analysis Two-channel (Agilent Mouse 20k (development) Oligo) II Multivariate analysis I, III,IV Proliferation/viability assay (flow cytometry) III Proliferation/viability assay (trypan blue exclusion) I qPCR (SYBR Green I) I, II, IV qPCR (TaqMan Low density array) III RNA extraction (Qiagen) III, IV RNA extraction (TRIzol) I, II Western blot (Caspase detection) I Western blot (Histone acetylation detection) III

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Results and discussion

Potential of in vitro systems - detection of effects on gene regulation relevant for the in vivo phenotype after VPA exposure in P19 EC cells (paper I) In paper I, the effect of VPA exposure (1, 2.5 and 10 mM) for 1.5, 6 or 24 h on gene expression in the EC cell line P19 was investigated. Particular inter-est was given the effects on genes known to be involved in the formation and closure of the neural tube and genes presumed to be regulated through HDAC.

VPA induced a significant response in a substantial number of genes at any of the time points, 7.7% (F-test p-value <10-7) to 19.4% (F-test p-value <10-3) of the 15,308 probes in gene list after normalization and filtering (Figure 2). There was a significant VPA response across all times points for 133 (F-test p-value <10-7) to 421 (F-test p-value<10-3) of these probes. The highest per-centage of probes with a significant VPA-induced change was found at the 6-h time point. In addition to the F-test, genes having an average of |log2 fold change| >0.75 calculated over all arrays at any time point, or a consistent average response down (log2 fold change < -0.5) or up (log2 fold change >0.5) across two consecutive time points were regarded as deregulated. This gave an additional 776 probes. In total 3,748 probes were considered as po-tential VPA targets, which expands the number of deregulated genes found in previous microarray studies (Gurvich et al., 2005; Kultima et al., 2004; Massa et al., 2005; Okada et al., 2005).

Among the many probes showing signs of deregulated gene expression in response to VPA, a core subset was defined. It consisted of nearly 200 VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) (Supplemental Tables2 and 3, paper I) with high probability (F-test p-value <10-7) after both 1.5 h and 6 h VPA exposure. Of these there were 37 and 93 genes, up-and downregulated respectively, highly significant also at the 24-h time point. The shape of the dose-response curves for many of the genes in this core subset indicated that their expression may not only be deregulated within �6 h of VPA exposure, but also at relatively low con-centrations (�1 mM) of VPA.

26

Figure 2. Gene expression changes in P19 embryonal carcinoma cells after 1.5 h, 6 h, and 24h exposure to 1 mM and 2.5 mM VPA. (A-E) Venn diagrams showing the number of array probes showing a significant VPA response in the F test at p-value p <10-3 (A), p <10-4 (B), p <10-5 (C), p <10-6 (D), and p <10-7 (E), respectively. The total number of significant probes and their percentage among 15,308 array elements are given for each cut-off level. (F) Venn diagram showing a comparison between the number of probes showing a significant (p <10-3) VPA response in the F-test and the analysis based on average fold change (Mav) across all arrays per time point (see Materials and methods in paper I).

This finding indicated an apparently high sensitivity to VPA (low EC50) for these genes. Since no apparent adverse cellular effects were obsereved after a short exposure time (�6 h) and no cytotoxic effects �1 mM of VPA over 24 h, it is not likely that gene deregulation is induced secondary to cyto-toxicity. In fact cytotoxcity and inhibited cell proliferation may be the result of early changes in gene expression rather than the other way around. Many of the genes in the core subset encode proteins involved in apparently rele-vant biological functions, such as transcription, translation, signaling, vari-ous transport processes, cell cycle control, and apoptosis. Significantly enriched Gene ontology (GO) terms among the down-regulated genes were

27

transcriptional regulation and cell growth, and among the up-regulated genes it was apoptosis and response to unfolded protein.

As the major toxicological outcome of VPA exposure during early em-bryogenesis, is induction of NTDs, it was investigated to what extent genes known to be crucial for neural tube development were deregulated by VPA. Of the 15,308 array probes for which expression data were obtained, a total of 179 represented genes known from genetic mouse models to be NTD-related, compiled from recent literature listings (Copp et al., 2003; Harris and Juriloff, 2007) and Gene Ontology biological process terms related to the neural tube formation and closure. In total, 59 of these NTD-related genes showed evidence of altered mRNA levels in P19 cells after VPA ex-posure. A significant VPA response at any time point was found for 17 (F-test p-value <10-7) to 43 (F-test p-value <10-3) of these genes (Table 2, paper I). Among the down-regulated NTD genes was for example Folr1, which encodes the folate receptor 1 (also known as folate-binding protein 1, Folbp1) responsible for uptake of folate into the cells. Abolished expression of this gene is sufficient to produce an NTD in the mouse embryo, as indi-cated by the Folr1-/- phenotype (Piedrahita et al., 1999). Similar reasoning can be made for more of the NTD genes but through other mechanisms. Several deregulated NTD genes encode proteins that directly (at the level of DNA) or indirectly (signaling pathways) influence transcriptional regulation. In several cases they may act together (e.g., Arnt and Hif1a, Deaf1 and Lmo4, Sall1 and Sall4) or in the same pathway (e.g., Axin1 and Lrp6, Cyp26a1 and Aldh1a2), while others act on the same processes, such as the regulation of G1-S progression (e.g., Gtf2i, Sall4, Crebp). These findings indicate that developmentally important genes may be altered in an in vitro system of cultured EC cells in monolayer.

The effect on genes related to HDAC inhibition was also investigated to investigate the mechanisms by which VPA is proposed to alter gene regula-tion (Gottlicher, 2004; Phiel et al., 2001), and induce teratogenicity, includ-ing NTDs (Eikel et al., 2006b; Gurvich et al., 2005; Menegola et al., 2005). A data set generated with Hdac1-/- ES cells (Zupkovitz et al., 2006) was rea-nalyzed and a list of putative Hdac1 target genes was compiled in which an apparent VPA response of 40% (60 of 145 probes for which data were ob-tained) were found. Of these there were 31genes upregulated and 9 downre-gulated in the same direction as in the Hdac1-/- ES cells. Among the downre-gulated putative Hdac1 target genes were Lefty1 and the NTD-related gene Gja1. We also compared our list of deregulated genes with a set of 15 genes reported to be commonly deregulated in three human tumor cell lines by the three HDAC inhibitors trichostatin A (TSA), Suberoylanilide hydroxamic acid and MS-275, or reported to be consistently deregulated by HDAC inhi-bitors in gene expression studies (Butler et al., 2002; Glaser et al., 2003; Huang and Pardee, 2000). Of the 10 genes for which data was obtained, 7 showed an altered expression, all in the predicted direction. Interestingly,

28

only a few known or putative HDAC target genes were among the large number of significantly affected genes with an early (1.5 h) response to VPA. Early gene deregulation induced by VPA might thus not be predomi-nantly driven through HDAC inhibition, which has been suggested previous-ly. It is also interesting that a relatively high number of the putative HDAC deregulated genes showed an opposite direction to the expected.

Gene deregulation in mouse embryos and search for markers of VPA teratogenicity through in vivo - in vitro comparison (paper II) In paper II, the in vitro data from paper I (generated with Codelink arrays) was compared to mouse embryos exposed to VPA for 1.5, 3 or 6 h on GD 8, an exposure that induces high frequency of NTD (Nau and Loscher, 1986). This was done to anchor the previous in vitro data to the in vivo phenotype of induced NTD. The in vivo data was generated with an independent micro-array platform (Agilent arrays) since genes measured with different probes may be regarded as essentially confirmed. This will increase the probability of the results and make the comparison and selection of potential markers of VPA induced developmental toxicity more stringent.

Effects in embryo VPA induced significant (adjusted p-value <0.01) deregulation of a total of 342 probes at any time point in the embryos. A total of 85 probes were sig-nificant at all three time points (Table 1, paper II) and more probes were significant at 3 and 6 h compared to 1.5 h (226, 228, and 179 respectively).

Many genes that were deregulated by VPA in embryos belong to biological process GO terms that are compatible with adverse effects on morphogenetic processes, including neural tube closure. Other GO terms overrepresented for downregulated genes were growth, morphogenesis, organ morhpogenesis and tube development, and the more general term positive regulation of transcrip-tion (Table 2, paper II). For the upregulated genes, we found overrepresentation of cellular carbohydrate metabolism/catabolism, response to stress, cell ion homeostasis, and microtubular-based process. The genes Fdft1, Gja1, Gtf2i, Ptch1, Shroom3 and Zfp36l1 known from the compiled list of NTD genes were found to be significantly deregulated by VPA at one or more exposure times. Moreover, several genes known from the literature to be expressed in the de-veloping nervous system, and/or to produce defects in the nervous system (in-cluding NTD) when mutated (e.g., Marcksl1, Fst, Id3, Sox2, Wnt5b, Smad4, and Hap1) were also deregulated. The deregulation of other genes such as Jag1, Mycn and Tbx4 may be compatible with VPA exposure inducing various other types of malformations, such as axial skeletal defects, somite defects,

29

growth retardation, and heart malformations (Ehlers et al., 1992; Faiella et al., 2000; Menegola et al., 1996) depending in part on the stage of gestation.

Comparison of in vivo and in vitro data and selection of potential markers of VPA effects Gene deregulation induced by a teratogen may be caused by a range of dif-ferent mechanisms such as direct effects on nuclear receptors, through inter-ference with signaling pathways, or through global effects on regulatory proteins like HDAC. However, regardless of the mechanism of action, gene expression a short time after exposure might be used as end-points and markers in an in vitro assay for developmental toxicity. To find genes useful as in vitro markers of adverse effects seen in embryo, a gene should prefera-bly be deregulated similarly in vitro as in vivo, with a reasonably good re-sponse over time and/or dose and preferably belong to a relevant functional category for the in vivo phenotype.

To do the comparison between embryos and the previous data set on P19 cells an exon-exon mapping between the Agilent and CodeLink arrays was performed (see Materials and Methods paper II). The best agreement in VPA response for the 2389 probes, that were mapped between the platforms, was found for the 6-h exposure of P19 cells versus the 6-h exposure of embryos. A set of 163 genes were commonly upregulated (81) or downregulated (82) by VPA in embryos and P19 cells after 6 h of exposure (Supplemental Table 2, paper II) with |log2 fold change| >0.25 and 0.4 for Agilent and CodeLink, respectively. Some of these genes belong to developmentally relevant over-represented GO terms such as organ morphogenesis, tube development, posi-tive regulation of transcription, cell proliferation, as well as organelle organ-ization and biogenesis, including the term microtubule-based process.

Through the microarray comparison a number of genes emerged as candi-date in vitro molecular markers of VPA effects in the embryo. Some belonged to overrepresented developmentally relevant GO terms (Gja1 and Hap1), or were related to NTD (Gja1 and Sall2), or showed exceptionally strong deregu-lation (H1f0). Some other developmentally interesting genes which showed a response to VPA in P19 cells (paper I), were not represented in the Agilent array (Cyp26a1, Fgf15, and Lin7b), or showed only weak response in the mi-croarray results (Otx2 and Sall4). These five genes were, however, verified as putative VPA targets in embryos through qPCR, and thus emerged as possible markers (Table 4, paper II). In summary, a number of genes emerged as poten-tial markers of VPA effects to be used in an in vitro assay. Some were mecha-nistically plausible to induce NTDs, and others showed a particularly high de-gree of deregulation (in combination with a relevant biological function).

30

Transcriptional responses in mouse ES cells (paper III) Paper III investigated whether gene deregulation induced by the teratogenic features of VPA could be distinguished from possible compound related effect. This was done in the mouse ES cell line R1, using the two VPA ana-logs (S)-2-pentyl-4-pentynoic acid ((S)-2-Pe-4-Pentyn) (0.25 and 0.5 mM), a more potent teratogen than VPA (see Table 1 in the Comments on Methods section), and 2-ethyl-4-methyl-pentanoic acid (2-Et-4-Me-Penta) (1 mM) a potentially non-teratogenic analog, in addition to VPA (1 mM) (Bojic et al., 1996; Eikel, 2005; Eikel et al., 2006a). In addition to teratogenic properties, (S)-2-Pe-4-Pentyn (like VPA) is also a potent inhibitor of HDAC, while 2-Et-4-Me-Penta is not. Incubation over 6 h was used since this was indicated as a proper assay length for EC cells in paper II. A panel of genes as poten-tial markers of teratogenicity was defined based on the effects of the analogs. This panel was then tested against some other teratogenic substances with presumed different mechanism of action.

Figure 3. Overlapping gene expression profiles in mouse ES cell R1 exposed to VPA and VPA analogs for 6 h. Venn diagrams showing number of deregulated probes (p < 0.001) with fold change |log2| > 1.

VPA and (S)-2-Pe-4-Pentyn induced significant (p <0.001) more than two fold change (|log2| >1) deregulation of a large number of array probes as illustrated in the Venn diagrams in Figure 3. The non-teratogenic substance 2-Et-4-Me-Penta affected only a low number, and in general with only mod-est effects on the gene expression. Substance specific as well as overlapping

(S)-2-Pe-4-Pentyn (0.5 mM)

25

1

910

1493

391

8

1

VPA (1 mM)

2-Et-4-Me-Penta (1mM)


25

1

1566

837

614

7

2


VPA (1 mM)


VPA (1 mM)

836

682

885

26

337

115

277


31

expression profiles were seen, the teratogenic substances VPA and (S)-2-Pe-4-Pentyn (particularly at the higher concentration) shared a large set of dere-gulated probes. Interestingly, most of the few probes deregulated by 2-Et-4-Me-Penta were deregulated also by VPA and the teratogenic analog.

Many of the genes deregulated by VPA and (S)-2-Pe-4-Pentyn were in-cluded in overrepresented GO terms, such as morphogenesis in general and neural tube formation, neural tube closure, and neural tube development specifically. Since NTDs occur in mouse embryos after maternal administra-tion of both VPA and (S)-2-Pe-4-Pentyn this finding is interesting. They also affected genes among signaling pathways like the MAPK signaling pathway and TGF-beta signaling pathway, both important pathways in embryonic development and stem cell regulation.

When taking a global approach using multivariate analysis with principal component analysis, a relatively good separation between treatments was seen, as well as clustering within treatments. When teratogenic/HDAC po-tential was used as a variable, a minimal subset of 5 probes could effectively separate the data. Since as many as 2500 genes could be correlated to this separation, a strong influence of the teratogenic/HDAC inhibition potential as a driving force for gene deregulation is indicated.

A substantial overlap of genes deregulated by the teratogens in R1 cells and genes deregulated by VPA in P19 and embryos could be seen when comparing with the data generated in papers I and II. The response to VPA in R1 cells was relatively similar to that in P19 cells, 208 probes were simi-larly deregulated more than two fold after 6 h of 1 mM VPA exposure. However, differences in deregulated genes were also seen which may be caused by differences in signaling pathways supporting pluripotency in ES and EC cells. A certain degree of overlap of deregulated genes between em-bryos and ES cells was also seen, as with EC cells, when investigating mod-est deregulation. However, whether ES cells or EC cells are the most equiva-lent to embryos is uncertain from this data.

Based on the gene expression response to VPA and the analogs in mES cells and the previous data on VPA (paper I and II), a panel of genes was defined as potential markers of VPA teratogenicity and evaluated with Taq-Man low density arrays (LDA). To investigate the effect of HDAC inhibi-tion, we used an additional teratogenic VPA analog without HDAC inhibito-ry properties, 3-propyl heptanioc acid (3-Pr-Hepta) (Eikel, 2005; Volland, 2002), and two HDAC inhibitors consisting of trichostatin A (TSA) and butyrate (Di Renzo et al., 2007). 3-Pr-Hepta was used at a molar equivalent (1 mM) to VPA exposure. TSA and butyrate induced deregulation of the vast majority of the genes deregulated by VPA and (S)-2-Pe-4-Pentyn in the pan-el and would probably been classified as teratogenic in blinded test. Howev-er, 3-Pr-Hepta seriously affected only Ddit3 (up) (Figure 4).

32

Figure 4. Hierarchical cluster of TaqMan LDA results for test substance exposures in mES cells over 6 h. Brighter blue indicates downregulation and brighter red indi-cates upregulation.

This indicates that gene deregulation induced by VPA is strongly driven by the inhibition of HDAC. As an investigation of the potential prediction value of the gene panel on general teratogenicity it was tested against the terato-genic substances all-trans retinoic acid (ATRA), carbamazepine, thalido-mide, and lithium chloride (LiCl). There were few genes affected by these exposures with the exception of the upregulation of Cyp26a1 (downregu-lated by VPA) and Ddit3 and downregulation of Otx2 by ATRA. The results suggest that the other teratogens exert their effect through a mechanism other than HDAC inhibition, although carbamazepine has been reported as a HDAC inhibitor (Beutler et al., 2005). It might also be argued that the ab-

33

sence of response to thalidomide is expected since this substance shows a lack of teratogenic potential in rodents.

In summary, the results in paper III indicated that gene deregulation in-duced by teratogens can be evaluated and classified as teratogenic exposure (see butyrate and TSA) a short time after exposure in mouse ES cells. How-ever, a larger set of teratogens, from diverse classes, should preferably be evaluated. This would potentially enable the selection of a set of marker genes for a comprehensive prediction of general teratogenicity.

Transcriptional responses in human ES cells (paper IV) In paper IV the gene expression response to VPA and VPA analogs in

human ES cells was investigated. hES cells were exposed to 1 mM VPA, 2-Et-4-Me-Penta or 3-Pr-Hepta or to 0.25 or 0.5 mM (S)-2-Pe-4-Pentyn for 24 h. The substantially longer cell cycle of hES cells compared to mES cell was the reason for using 24 h rather than 6 h that was used in mES cells.

For VPA, 449 probes (about 2% of the probes with expression) showed a

significant deregulation (p <0.01 and |log2| >1) and (S)-2-Pe-4-Pentyn dere-gulated 262 and 474 probes (0.25 respectively 0.5 mM) (Figure 5). The non HDAC inhibiting analogs induced much more modest gene deregulations, 75 and 25 probes deregulated by 3-Pr-Hepta and 2-Et-4-Me-Penta respectively. VPA and (S)-2-Pe-4-Pentyn showed a substantial overlap in deregulated genes (Supplemental Table 2, paper IV) while only 10 probes were com-monly deregulated by all three teratogenic substances. This is in line with the results for mES cells (paper III), where almost none of the panel of genes deregulated by VPA showed the same response to the non HDAC inhibitor 3-Pr-Hepta. That HDAC inhibition may be the predominant factor for VPA gene deregulation was also indicated when multivariate analysis was applied to the data, 190 probes explained 80% of the separation (Figure 3, paper IV) when HDAC inhibition was used as variable.

Gene Ontology analysis of genes deregulated by VPA and (S)-2-Pe-4-Pentyn showed a number of overrepresented GO terms, many of which were related to neural processes, morphological processes and cellular processes such as cell proliferation, cell adhesion and cell-cell signaling. This indicates that genes relevant for the in vivo phenotype of VPA exposure may be dere-gulated in hES cells. An overlap of affected GO terms between all the tera-togenic substances was also observed (Figure 4, paperIV). Since 3-Pr-Hepta and VPA induce the same type of developmental toxicity in mice (Volland, 2002), this overlap of teratogenic properties may be caused through effects on the same signaling pathways or within the same groups of gene, instead of specifically the same individual genes.

34

Figure 5. Venn diagrams showing number of deregulated probes (p <0.01 and |log2| >1) after 24 h exposure with VPA and VPA analogs in hES cells.

The hES cell data was also compared to the mES cells data in paper III to

investigate similarities (and differences) in response to VPA and VPA ana-logs. HomoloGene was used to map genes between the two species and when using a criteria of |log2| >0.5 and p <0.05 in both human and mouse data, 1358 unique genes (a total of 1818 probes) were deregulated by VPA (Figure 6). However, only 332 unique genes (392 probes) were deregulated in the same direction. For (S)-2-Pe-4-Pentyn 325 unique genes were deregu-lated in the same direction in both species. VPA and (S)-2-Pe-4-Pentyn simi-larly deregulated 206 of the same genes in both species (Supplemental Table 3 in paper IV) indicating a set of genes that are potentially HDAC driven in both human and mouse, which may include genes potentially causing the teratogenic effects seen in both species. Using multivariate analysis, a set of 13 genes could separate the data according to HDAC inhibition. An associa-tion of 95 genes with those 13 genes also indicates a driving force of HDAC inhibition for gene deregulation in both human and mouse.


4

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Figure 6. Hierarchical cluster of genes matched be-tween human and mouse through homologene. In-cluded genes have a deregulation of |log2| >0.5 by 1 mM VPA in both mouse ES cells and human ES cells (though not necessarily the same direction). A co-deregulation as well as species specific deregulation of genes is visualized in the cluster. Bright blue indicates downregulation and bright red indicates upregulation, maximum visualized log2 fold change is sat to 2.5. _H = human and _M = mouse.

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36

The similarities in gene response to VPA and (S)-2-Pe-4-Pentyn are nota-ble. This may offer an inter-species bridge in the understanding of molecular events leading to effects on developmental processes in human and mouse embryos, seen e.g. as NTDs after VPA exposure. However, in addition to these similarities, the comparison revealed many genes deregulated in oppo-site directions. Different mechanism by which pluripotency is maintained may account for these differences. Since mouse ES cells are dependent on LIF and BMP whereas human ES cells depend on bFGF and activin for sup-port of pluripotency (Heins et al., 2004; Nagy et al., 1993), different signal-ing pathways are activated and may thus influence the response to teratogen exposures.

VPA, (S)-2-Pe-4-Pentyn and 3-Pr-Hepta all deregulated CYP26A1 in hES. This transcript was deregulated by VPA also in mouse embryos as well as in P19 cells. CYP26A1 encodes a retinoic acid degrading enzyme and is important for the spatial restriction of retinoic acid signaling during embryo-genesis. Exogenous retinoids induce serious defects in the developing human embryo (McCaffery et al., 2003), and Cyp26a1-/- mouse embryos show simi-lar defects as wild-type embryos exposed to excessive retinoic acid (Abu-Abed et al., 2001; Sakai et al., 2001). Downregulated transcription of the main retinoic acid metabolizing enzyme with subsequent altered metabolism of retinoic acid, may thus be one of several mechanisms behind the deve-lopmental effects caused by VPA in the human fetus (McCaffery and Simons, 2007).

37

Concluding remarks and future perspective

A total replacement of laboratory animals in toxicological evaluations is probably not realistic. However, the implementation of in vitro assays in the early phase of drug development might rationalize lead optimization. This will save animals and resources since fewer substances would reach full-scale animal risk assessment. This thesis has explored the early molecular action of teratogenic substances in in vitro systems consisting of pluripotent stem cells of embryonic origin. By using short-term assays, primary effects on the gene expression are investigated and it is plausible that these initial effects mediate the adverse effects seen in vivo. The use of ES and EC cells are favored in such toxicogenomics assays as these cells have a relatively open chromatin structure which enables easy access of gene expression modulators. Since pluripotent cells also are less restricted in their gene ex-pression compared to adult differentiated cells, a higher number of active and non-silenced genes are available as possible targets for these modulators. This is especially important in assays aiming to predict teratogenicity since the development of the embryo is a highly specialized process with a multi-tude of genes as potential targets of teratogen exposure.

The use of VPA as a model substance in these initial studies is motivated by its ability to induce NTDs in both humans and mice. This makes an inter-species comparison possible, and may expand the knowledge of mechanistic similarities in response to teratogens in these species.

The results in this thesis show that pluripotent stem cells may respond with altered gene expression to the action of teratogens early after exposure. For both VPA and its teratogenic analog (S)-2-Pe-4-Pentyn this deregulation was massive with a large number of altered genes. That developmentally important genes involved in the morphogenesis of the neural tube were dere-gulated by these exposures in mouse cells is promising. This shows that genes relevant for the in vivo phenotype may be affected in a monolayer of stem cells. It was also shown by the in vitro – in vivo comparison that similar gene deregulation may occur both in vivo and in vitro after exposure to tera-togens. Further, paper III showed that the teratogenic potential of a substance may be detected in an assay of early gene response. In a blinded test, both the teratogenic substances butyrate and TSA would have been classified as teratogens due to their gene expression profiles. However, the results also indicated that a strong gene expression deregulation mechanism, such as HDAC inhibition, could potentially overshadow mechanisms involving few-

38

er genes exerted by other classes of developmental toxicants. Hence, it seems most important to carefully choose which substances are used to de-fine a set of genes used in such an assay. The most straight forward approach to solve this issue may be the use of a large set of teratogens, preferably with diverse mechanisms of action, and profile these to gather a database of gene expression data. Included in such a set should also be non teratogens to be able to discriminate developmental toxicants from non-toxicants.

The results also suggest that it might be difficult to discriminate between gene responses relevant for the phenotype or toxic outcome and other, less harmful, gene expression changes by an exposure. This is a well known problem regarding microarray studies and the use of techniques as Gene Ontology, pathway analyses, clustering multivariate analysis and so on may be helpful in determining relevant deregulations.

The choice of pluripotent cell type for short-time assays and their poten-tial capacity to predict developmental toxicity is of great interest. All three cell lines used in these studies have their advantages. EC cells are easy to handle and culture, but their potential chromosomal aberrations and residual tumor properties may be a disadvantage. hES cells are in closer relation to the human fetus and thus a better choice from that aspect, however, to extra-polate to the in vivo situation is difficult as there is limited data available on teratogen exposure in humans. In this respect mES cells are better suited since there is much more teratology data available, however, it is still a mouse cell to be used for human risk assessment. So, even if valuable data can be generated on the in vivo – in vitro comparison in a short perspective by using mES cells, data on hES cells may be preferred in the long run. However, along with implementing hES cells in assays it might be argued that it is appropriate to investigate the full potential of mES cells, and use that knowledge to extrapolate results to the human counterpart. Pluripotent embryo-derived EpiS cells are under investigation in our laboratory as a potential cell system to use in assays, and since they are potentially more like hES cells in gene expression and pluripotency factors they may bridge the gap between hES and mES cells.

Future mechanistic investigations in the area of HDAC-inhibition may in-volve chromatin immunoprecipitation on microarrays (ChIP-on-chip) or ChIP-sequencing experiments to reveal explicit localization of hyper-acetylated histones on the chromatin. This data could then be correlated with gene expression profiles which would potentially provide more in depth insights in the mechanism by which VPA exerts its actions on gene expres-sion. Achieving this for both human and mouse ES cell could reveal distinct epigenetic mechanisms in the two species. The potential contribution of oth-er epigenetic mechanisms, such as DNA and histone methylation, in the action of VPA would be interesting as well.

39

For the NTD-induction aspect of VPA, directed studies of embryos for example with laser micro dissection of the developing neural structure would be preferred. This would enable assessment of gene expression in the specif-ic cells most probably affected by VPA.

40

Acknowledgements

This work was carried out at the Department of Pharmaceutical Biosciences, Division of Toxicology, Faculty of Pharmacy, Uppsala University.

This work was supported by the Swedish Board of Agriculture, the Swedish Animal Welfare Agency, Vinnova and the EU Project ReProTect (LSHB-CT-2004-503257). The Swedish Academy of Pharmaceutical Sciences and the foundation of Helge Ax:son Johnson are acknowledge for travelling grants. I express my sincere gratitude to all the people around me that have made this thesis possible. Especially I would like to mention:

My supervisor Lennart Dencker for giving me the opportunity to become a PhD student in your group. Thank you for the true enthusiasm you express and especially for the belief you have had in my projects. You have always a smiling face and are always ready to invite to a party! Michael Stigson, my co-supervisor, for your support and the substantial contributions that made this thesis possible, and thank you for interesting discussions about all that matters in science. Members of the Dencker team: Birger Scholz, for always having an idea about what to do next. Your com-mitment and dedication to science and all projects around you is truly amaz-ing. Mats Nilsson, for sharing cakes and stories and for being great compa-ny in and outside the sports field. Henrik Alm, for all these years we have wandered in the path of PhD studies side by side. Thank you also for great conference company and all nice parties. Kim Kultima, for your contribu-tions to the biostatistical matters in the projects. However, that you intro-duced me to hunting is probably worth more ;-). Present colleagues at the Toxicology department: Lena Norgren and Raili Engdahl the hub around which the whole toxicol-ogy department rotates. Without you this place would not be the same. Thank you for all help and assistance with my work. Helene Andersson my most recent roommate, for introducing me to French hiphop and for always

41

sharing a smile and a positive thinking. Oskar Karlsson, for very nice con-ference company and most interesting sports discussions. Björn Hellman and Eva Brittebo, for creating a nice working atmosphere at the department. Anna Karlsson, for bringing some vocals almost from my home ground to the department. Malin Andersson, for positivism and laughter in the corri-dors, and of course for bringing new spirit to the department. Jemal Dem-ma, for your hard work at the lab. Elena Piras, for your smile and for al-ways reminding me of the Mediterranean. Former colleagues: Anna Östergren, my first roommate, for sharing the room with me without complaining, not even ones. Per Stenqvist, also former roommate, we shared a golden moment but it lasted far too short. Maria Andersson, Esti-baliz Lopez and Camilla Svensson, for all fun times and nice parties. Magnus Jansson, all your support regarding computer issues and layouts are highly appreciated. Marianne Danersund, Agneta Hortlund, Agneta Bergström, for all help with administrative business. Collaborators and co-writers: Maud Forsberg, the stem cell guru, thank you for introducing me to the stem cell field and for all the knowledge and wisdom you have shared with me. Hugh Salter, for the multivariate business and comments on my manu-scripts. Robert Söderlund for the assistance you provided me with the mi-croarray analyses. The people at Cellartis, especially Raimund Strehl and Johan Hyllner, for always making me feel welcome when visiting Göte-borg, and thank you for the fun times in Toronto. Anne-Lee Gustafsson, Heike Hellmold, Ulf Andersson and Kenneth Stockling at AstraZeneca, for all help and comments concerning the projects and manuscripts. Bill Webster, your comments and thoughts about my text have meant a lot to me! All my friends outside BMC, for reminding me that there is a world outside this building. My parents Stellan and Kerstin and my brother Erik, for all the support and care all these years. And finally the most important person in my life, Anna, thank you for your never ending support and love, for your patience with me and for always encouraging me! Timbuktu sings ”Det kommer ordna sig, det gör det alltid, det kommer fixa sej till slut, det är det inget tvivel om”. And do you know what? It will!

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