Monitoring developmental toxicity in vitro, by using mouse and human embryonic stem cells

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M M on o n it i t or o ri in ng g d de ev ve el lo o p p me m en nt ta al l t t ox o xi ic ci it t y y i in n v vi it tr ro o, , b b y y u u s s i i n n g g m m o o u u s s e e a a n n d d h h u u m m a a n n

em e mb br ry yo o ni n ic c s st t em e m c ce el ll ls s

Di D i ss s se er rt ta at ti io on n

Z

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Tag der mündlichen Prüfung: 13. Mai 2005 1. Referent: Prof. Dr. Thomas Hartung 2. Referent: Prof. Dr. Albrecht Wendel

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M M ar a rc ch h 2 20 00 05 5

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Alla persona piu’

importante della mia vita

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A

A c c k k n n o o w w l l e e d d g g m m e e n n t t

The work presented in this thesis was carried out between March 2002 and December 2004 at ECVAM in the Reproductive toxicity research group, under the supervision of Dr. Susanne Bremer and Prof. Thomas Hartung.

First I would like to thank Dr. Susanne Bremer to give me the opportunity to start my work in ECVAM and especially for her friendship.

My thanks also go to Prof. Thomas Hartung to offer me the possibility to do the Ph.D at the University of Konstanz and to coach me in the last months.

Especially, I want to thank them both for their patients for each time that I came in their office saying “do you have 5 minutes for me?”.

There is a long list of people to who I can say “thank you” but I am sure that they know.

At the end, a big hug goes to Sarah because she was always next to me.

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Cristian Pellizzer P.h.D. Thesis University of Konstanz

Major parts of this thesis are:

- published publication:

• • MMoonniittoorriinngg ofof tteerraatotogegenniicc efefffeeccttss iinn vivittrroo bbyy ananaallyyssiinngg a a seselleecctteedd gegennee eexxprpreessssiioonn ppaatttteerrnn

CCrriissttiiaann PPeelllliizzzzeerr,, SSaarraahh AAddlleerr,, RRaaffffaaeellllaa CCororvvii,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr TTooxxiiccoollooggyy IInn VViittrroo 1188 ((33)),, 332255--333355,, 22000044

• • DDeetteeccttiioon n ofof ttiissssuuee ssppeecciiffiicc efefffeeccttss bby y MeMetthhootrtreexxatatee onon ddiiffffeerreenntitiaatitinngg m

moouusese eemmbbrryyoonniicc sstteemm cceellllss

CCrriissttiiaann PPeelllliizzzzeerr,, EEzziiaa BBeelllloo,, SSaarraahh AAddlleerr,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr BBiirrtthh ddeeffeeccttss rreesseeaarrcchh ((PPaarrtt BB)) 771 1 ((55)),, 333311--33441,1, 22000044

•

• DDeevveellooppmmeennttaall totoxxiciciittyy tetessttiinngg frfroomm ananiimmaall totowwaarrddss eemmbbryryononiicc ststeemm cecellllss

CCrriissttiiaann PPeelllliizzzzeerr,, SSuussaannnnee BBrreemmeerr aanndd TThhoommaass HHaarrttuunngg AALLTTEEXX,, iinn pprreessss

- submitted paper:

• • DDeevveellooppmmeenntt ofof aann iinn vivittrroo sysysstteemm foforr ththee dedetteeccttiioonn ofof cchheemmiiccaallss cacauusisinngg g

grroowwtthh rreettaardrdatatiioonn iinn tthhee ddeevveelloopipinngg eemmbbrryyoo

CCrriissttiiaann PPeelllliizzzzeerr,, EEzziiaa BBeelllloo,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr SSuubbmmiitttteedd ttoo TTooxxiiccoollooggy y LLeetttteerrss

- manuscript in preparation:

•

• CCaarrddiioommyyooccyytteess ddififffeerreenntitiaattiioonn frfroomm huhumamann emembbrryyononicic ststeemm cecellllss foforr ddeevveellooppmmeennttaall ttooxxiicciittyy ssttuudydy

CCrriissttiiaann PPeelllliizzzzeerr,, SSaarraahh AAddlleerr,, LLaarrss HHaarreenngg,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr MMaannususccrriipptt iinn pprereppaarraattiioon n

Significant contributions have been made to:

• • DDeevveellooppmmeenntt ofof aa tetessttiinngg ststrraatteeggyy foforr ddeetteeccttiinngg emembbrryyoottooxxicic hahazzaarrddss ofof c

chheemmiiccaallss iinn vviittrroo bbyy uussiinngg eemmbbrryyoonniicc sstteemm cceellll mmooddeellss

SSuussaannnnee BBrreemmeerr,, CCrriissttiiaann PPeelllliizzzzeerr,, SSaarraahh AAddlleerr,, MMaarrttiinn PPaappaarreellllaa aanndd JJaann ddee LLaannggee AATTLLAA 3300 SSuuppppll 22,, 110077--110099,, 2200002 2

•

• DDeetteeccttiioon n ooff didiffffeerreennttiiaatitioonn inindducuciinng g cchheemmiiccaallss byby ususiinngg ththee ggrreeeenn fflluuoorreesscceenntt pprrootteeiinn exexpprreessssiioon n inin gegenneettiiccaallllyy enenggiinneeeerriinngg t

teerraatotoccaarcrciinnoommaa cceellllss

SSaarraahh AAddlleerr,, MMaarrttiinn PPaappaarreellllaa,, CCrriissttiiaann PPeelllliizzzzeerr,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr AATTLLAA iinn pprreessss

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• • TThhee eeffffeeccttss ooff ssoolvlveennttss oon n eemmbbrryyoonniicc sstteemm cceellll ddiiffffeerreennttiiaatitioonn

SSaarraahh AAddlleerr,, CCrriissttiiaann PPeelllliizzzzeerr,, MMaarrttiinn PPaappaarreellllaa,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr SSuubbmmiitttteedd ttoo TTooxxiiccoollooggy y iinn VViittrroo

- Poster presented at the 11^th Congress on Alternatives to Animal testing, Linz, Austria (19-21 Sept. 2003).MMoonniittooririnngg ofof teterraattooggeneniicc efefffeeccttss inin vivittrroo bby yananaallyysisinngg a a sseelleecctteedd ggenenee eexxpprreessssiioonn ppaatttteerrnn

SuSussaannnnee BBrreemmeerr,, SSaarraahh AAddlleerr,, RRaaffffaaeellllaa CCoorrvvii,, TThhoommaass HHaarrttuunngg aanndd CCrriissttiiaann PPeelllliizzzzeerr

- Poster presented at the 11^th Congress on Alternatives to Animal testing, Linz, Austria (19-21 Sept. 2003).TThhee efefffeecctt ofof sosollvveennttss onon eemmbbrryyoonniicc ststeemm cecellll didiffffeerreennttiiaattiioonn

SaSarraahh AAddlleerr,, CCrriissttiiaann PPeelllliizzzzeerr,, MMaarrttiinn PPaappaarreellllaa,, TThhoommaass HHaarrttuunngg aanndd SSuussaannnnee BBrreemmeerr

- Poster presented at the fourth World Congress on Alternatives and Animals use in the life sciences, New Orleans Louisiana U.S.A. (11-15 Aug. 2002). DDeetteeccttiioon n ofof DiDiffffeerreennttiiaatitioonn inindduucciinngg ChCheemmiiccaallss bbyy uusisinngg ththee GGrreeeenn FFlluuoorreesscceenntt PrProotteeiinn ExExprpreessssiioonn iinn ggeenneettiiccaallllyy eenngiginneeeerreedd TTeerraattooccaarrcciinnoommaa CCeellllss

S

Saarraahh AAddlleerr,, MMaarrttiinn PPaappaarreellllaa,, CCrriissttiiaann PPeelllliizzzzeerr aanndd SSuussaannnnee BBrreemmeerr

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5-FU 5-Fluorouracil

6AN 6-Amminonicotinamide

ANF Atrial natriuretic factor

BA Boric acid

bFGF Basic fibroblast growth factor

CTRL Control

DMEM Dulbecco’s modified Eagles medium DMSO Dimethyl sulfoxide

EB Embryoid body

ECVAM European centre for the validation of alternative methods EINECS European inventory of existing commercial chemical substances EPA Environmental protection agency

ESAC ECVAM Scientific Advisory Committee

ES Embryonic stem cells

EST Embryonic stem cells test

EU European Union

FACS Fluorescence-activated cell sorting

FCS Fetal calf serum

FETAX Frog embryo teratogenesis assay GFP Green fluorescence protein GSK-3β Glycogen synthase kinase-3β

hEB Human embryoid body

HEPT Hamster egg penetration test hES Human embryonic stem cells HOS Hypoosmotic swelling test

IC50 Concentration that inhibited 50% of growth

ICCVAM Interagency coordinating committee for the validation of alternative methods IUGR Intrauterine growth retardation

LiCl Lithium chloride

LIF Cytokine leukemia inhibitor factor

MHC Myosin heavy chain

MLC-2v Myosin light chain-2

MM Micromass test

MTX Methotrexate

NOEL No observable effect level

OD Optical density

OECD Organisation for economic co-operation and development

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RA All-trans retinoic acid RARs Retinoic acid receptors

REACH Registration evaluation and authorisation of new existing chemicals RT-PCR Real time-PCR/semi-quantitative reverse transcriptase-PCR RXRs Retinoic 9-cis receptors

SAC Saccharin

SAR Structure activity relations SEM Standard error of the mean STDV Standard deviation

UhES Undifferentiated human embryonic stem cells α-MHC Alpha myosin heavy chain

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Page

1.Introduction ¹

1. A Birth defects 2

1. B Teratology 2

1. C Principles of teratogenesis 3

1. C. 1 Susceptible species 4

1. C. 2 Susceptible stages of development 5

1. C. 3 Mechanisms and pathogenesis 6

1. C. 4 Dose dependency 7

1. D Manifestation of deviant development 9

1. D. 1 Malformations 9

1. D. 2 Growth retardation 10

1. D. 3 Embryolethality 11

1. D. 4 Functional impairment 12

1. E European Chemicals Policy 16

1. F OECD guidelines for testing of chemicals 18

1. G Use of the animal models to assess human risk 19 1. H Alternative tests for developmental toxicity 20

1. H. 1 Embryonic Stem Cells Test 23

1. H. 2 Evaluation of teratogenic potential in vitro 25

1. I Human Embryonic Stem Cells 27

2. Aims of the study 32

3.A Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern

34

3. A. 1 Introduction 35

3. A. 2 Materials and Methods 37

3. A. 3 Results 40

3. A. 4 Discussion 46

3. A. 5 Acknowledgement 49

3.B Detection of tissue specific effects by Methotrexate on differentiating mouse embryonic stem cells

50

3. B. 1 Introduction 51

3. B. 2 Materials and Methods 52

3. B. 3 Results 57

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3. B. 4 Discussion 63

3. B. 5 Acknowledgement 67

3.C Cardiomyocytes differentiation from human embryonic stem cells for developmental toxicity study

68

3. C. 1 Introduction 69

3. C. 2 Materials and Methods 70

3. C. 3 Results 73

3. C. 4 Discussion 81

3. C. 5 Acknowledgement 85

3.D Development of an in vitro system for the detection of chemicals causing growth retardation in the developing embryo by using embryonic stem cells

86

3. D. 1 Introduction 87

3. D. 2 Materials and Methods 89

3. D. 3 Results 91

3. D. 4 Discussion 96

3. D. 5 Acknowledgement 99

4. Discussion 100

5. Summary 108

6. Zusammenfassung 7. References

111 115

8. Annex 129

Poster presented at the 11th Congress on Alternatives to Animal testing, Linz, Austria (19-21 Sept. 2003).

“Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern”

129

Poster presented at the 11th Congress on Alternatives to Animal testing, Linz, Austria (19-21 Sept. 2003).“The effect of solvents on embryonic stem cell differentiation”

130

Poster presented at the fourth World Congress on Alternatives and Animals use in the life sciences, New Orleans Louisiana U.S.A. (11-15 Aug. 2002).“Detection of Differentiation inducing Chemicals by using the Green Fluorescent Protein Expression in genetically engineered Teratocarcinoma Cells”

131

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List of Tables Page

Table 1. Suspected causes of birth defects in humans 2

Table 2. Critical period of organogenesis in various species 5 Table 3. Spontaneous malformations rates for various species of animals 10 Table 4. Physiological changes that may alter toxicokinetics during pregnancy 15 Table 5. Some tests proposed to reduce or replace animal experiments for developmental

toxicity studies 23

Table 6. Key genes used in this study, involved in cardiac differentiation 36 Table 7. Length of PCR products and sequence of genes primers used for semi-

quantitative RT-PCR 38

Table 8. Key genes used in this study, involved in cardiac and osteoblast differentiation 52 Table 9. Length of PCR products and sequence of gene primers used for semi-quantitative

RT-PCR. 56

Table 10. The PCR conditions used for semi-quantitative RT-PCR 56 Table 11. Key genes used in this study, involved in human cardiac differentiation 72 Table 12. Composition of different condition medium tested for hES cardiomyocytes

differentation 72

Table 13. hEBs size obtained using image analysis software 80

Table 14. Correlation between hEBs size and cells death 80

Table 15. Percentage of cardiac cells and cells like neurons in the surviving hEBs 81 Table 16. Relation between hEBs spontaneous beating cells like neurons and the hEBs size 81

Table 17. Chemicals tested in this study 88

Table 18. IC50 values (in M) resulting from the MTT tests and monitoring growth EBs 96

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List of Figures Page

Fig. 1. Human embryonic development showing sensitive periods 6 Fig. 2. Gene expression in EBs derived from mouse ES cells 28 Fig. 3. Time course of mRNA levels of cardiac genes during untreated ES differentiation 41 Fig. 4. Concentration response curve of toxicants to ES during 10 days of differentiation 42

Fig. 5. Morphological effects of RA and LiCl on EBs 43

Fig. 6. Percentage of embryoid bodies showing, at day 10, spontaneously beating

cardiomyocytes. 43

Fig. 7. Time course of mRNA genes specific for cardiac development 45 Fig. 8. Gel electrophoresis of semi-quantitative RT-PCR products 45 Fig. 9. Concentration response curve of MTX to ES during 10 days of differentiation 57 Fig. 10. Bone mineral deposition detected by Alizarin Red S stain 58

Fig. 11. Methotrexate effects on cardiac activity 59

Fig. 12. Effects of MTX on the time course of mRNA genes specific for cardiac

development 61

Fig. 13. Effects of MTX on the time course of mRNA genes specific for skeletal

development 61

Fig. 14. Gel electrophoresis of semi-quantitative RT-PCR products 62

Fig. 15. Effects of MTX on EB growth curves 63

Fig. 16. Stages in EB production and differentiation 73

Fig. 17. Gene expression on day 4 of hES cells differentiation 75 Fig. 18. Time course of Oct-4, hTERT and Brachyury mRNA genes during cardiac

specification

76 Fig. 19. Time course of GATA-4 and Nkx2.5 mRNA genes during cardiac specification 76

Fig. 20. Time course of specific cardiac genes 78

Fig. 21. Morphological evaluation of hEBs 80

Fig. 22. Linear correlation between hEB’s size and cell death 80

Fig. 23. Concentration response curve of toxicants 92

Fig. 24. EB growth monitored by image analysis 93

Fig. 25. Dose-response curves measuring MTT (ES/D3 and BALB/3T3) and EBs monitoring growth: A) BA; B) 5-FU; C) 6AN treatment 94 Fig. 26. Dose-response curves obtained from MTT (ES/D3 and BALB/3T3) and EBs

monitoring growth: A) MTX; B) SAC treatment 95

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1

1 1 . . I I N N T T R R O O D D U U C C T T I I O O N N

Developmental toxicology is an increasingly important area of toxicology. It is well known that a significant percentage of newborns have anatomical defects and that birth defects are a major cause of hospitalization of infants. In addition,

“spontaneous” abortion and perinatal death are common, and numerous individuals suffer from congenital functional deficits, such as mental retardation. Although considerable progress has been made in determining causes, the etiology of the majority of birth defects is unknown or only poorly established. It still has to be understood how toxicological mechanisms of congenital defects are working and what is the role of the genetic and environmental factors in triggering such mechanisms. Thus, it is certain that both mechanistic studies of known developmental toxicants and the toxicological assessment of pharmaceutical agents, food additives, pesticides, industrial chemicals, environmental pollutants, and the like to which pregnant women may be exposed will be of importance in the future.

Congenital malformations or birth defects are a major public health concern.

In U.S.A., birth defects occur in a frequency of 20 to 30:1000 live birth, and additional 60 to 70: 1000 are observed in the interval between birth and 1 year of age (Hook, 1981). Minor anomalies represent another 140:1000 (Hook, 1981), and minor to severe mental retardation accounts for 0.7-0.8% incidence (Rosenberg, 1984). Congenital malformations account for approximately 14% of all infant deaths (Warkany, 1957). Birth defects have been the leading cause of infant mortality for more than 20 years, with a rate of 173:100,000 live births in 1994 (Petrini et al., 1997).

The causes of birth defects are varied, but the etiology of most malformations is unknown (Table 1). An agent that harms a baby in one pregnancy may be harmless in others, produce different birth defects in still others, and have no effect in a subsequent pregnancy for the same woman (Ajl, 1982). Earlier, Wilson (1977) considered chemical agents putatively being responsible for 4-6% of birth defects. Nonetheless, it is a worthwhile goal to identify those potentially hazardous chemical agents in the environment and consumer products to which the gravid

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mother and her conceptus are exposed to minimize the risk of congenital malformations.

Because some birth defects would be preventable by simply avoiding known teratogens, the prevention of one or two anomalies in every thousand births would be a reputable accomplishment. The overhelming size of the “unknown” category in the etiology of birth defects is of concern, and it may reflect that chemical teratogen by specific agents is so difficult to establish. Indeed, physicians derive information on drug and chemical hazards to the human conceptus mainly from isolated case reports; only a few are based on epidemiological studies.

Table 1. Suspected causes of birth defects in humans

Source: Brent and Beckman, 1990.

11.. AA BBiirrtthh ddeeffeeccttss

Birth defects are those malformations observable at birth (congenital) or thereafter. Birth defect is usually used to describe structural or anatomical, but the term also includes physiological or functional and behavioral defects. Presumably, these include dysfuction, impairment, or deficit of any biological system (i.e., immunological, hormonal, biochemical, metabolic, and neurobehavioral).

1

1.. BB TTeerraattoollooggyy

The sciences of birth defects, “teratology”, or the process of induction of malformation, “teratogenesis”, stem from the Greek word root “teras”, meaning

Suspected cause % of total

Genetic

• Autosomal genetic diseases 15-20

• Cytogenetic 5

Enviromental

• Maternal conditions 4

• Maternal infections 3

• Mechanical problems (deformations) 1-2

• Chemicals/drugs/radiation/hyperthermia <1

Preconception exposures ?

Unknown (polygenic) 65

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malformation or monstrosity (i.e., the induction of “terata”). In this work

“teratogenicity” is meant as structural malformations in selected target tissue noticed at birth or in a defined postnatal period induced by chemicals. The other functional or behavioral effects are considered as another class of developmental toxicity.

1

1..CC PPrriinncciipplleess ooff tteerraattooggeenneessiiss

The determination of whether a given chemical has the potential or capability to induce congenital malformations in the human or other animal species is governed by essentially three fundamental established principles of teratogenesis. James G.

Wilson, beginning in 1959 and elaborated on later (Wilson, 1965), is largely credited with formulation of these basic concepts. Brent (1964) was an early contributor, and Johnson (1981, 1988) has added further, more recent enhancements of principles.

Karnofsky (1965), these principles can be illustrated by the axiom:

The principles of teratology, as stated by Wilson 1973, are the following:

• Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with adverse environmental factors (section 1.C.1).

• Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence (section 1.C.2).

• Teratogenic agents act in specific ways (mechanism) on developing cells and tissues to initiate sequences of abnormal developmental events (pathogenesis) (section 1.C.3).

• Manifestation of deviant development increase in frequency and degree as dosage increases from the no-effect to the totally lethal level (section 1.C.4).

• The results of adverse influences to developing tissues is death,

“A“A tteerraattooggeenniicc rreessppoonnssee ddeeppeennddss uuppoonn tthhee aaddmmiinniissttrraattiionon ooff aa ssppeecciiffiicc ttrreeaattmmeenntt ooff aa paparrttiiccuullarar ddoossee ttoo aa ggeenneettiiccaallllyy ssuusscceeppttiibbllee ssppeecciieses wwhheenn tthhee eemmbbrryyooss aarree iinn

a

a ssuusscceeppttiibbllee ssttaaggee ooff ddeevveellooppmmeenntt””

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malformation, growth retardation, and functional defect (section 1.D).

11..CC..11 SSuusscceeppttiibbllee ssppeecciieess

Not all species are equally susceptible or sensitive to teratogenic influence by a given chemical. Some species respond more readily than others. Some species differences are due to genetic factors. However, the latter variability of response is commonly observed even in inbred animals, and this cannot be solely due to genetic constitution. Metabolic differences and several auxilliary factors contribute in producing further variation.

As noted by Kalter (1968b), inter- and intraspecies variability may be manifested in several ways: an agent that is teratogenic in some species may have little or no teratogenic effect in others; a teratogen may produce similar defects in various species, but these will vary in frequency; a teratogen may induce certain abnormalities in one species that are entirely different from those induced in other species. Similarly, there are genetic differences within given strains or breeds of the same species that influence the teratogenic response. Both the nature and incidence of effects are modulated by the genetic constitution of mother and fetus. Such factors as maternal parity and weight, fetal weight, number of young, size and constitution of placenta, intrauterine associations, fetal weight, fetal and maternal production of hormones, and maternal utilization of vitamins and other essential nutrients, are all variables in strain susceptibility. These may be further modified by environmental factors, such as diet, season, temperature, etc. (Woollam and Millen, 1960; Kalter, 1965).

The reaction to a specific teratogen may also be due to differences in species- specific rate of metabolism, as well as to qualitative differences in metabolic pathways (Burns and Conney, 1964). In the human, for example, drugs are metabolized both in the liver and at extrahepatic sites, such as the adrenal gland, whereas extrahepatic activity is negligible or absent in fetal rats, guinea pigs, rabbits, and swine (Rane et al., 1973). Even when the two species metabolize a drug at the same rate, differences in the metabolic products may cause different teratogenic responses in the two species (Tuchman-Duplessis, 1970).

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Because substances cross the placental membrane by various mechanisms, some differences in species reactivity to teratogens may be due to accessibility of the drug to the embryo. Only drugs with a molecular weight of more than 1000 Da do not readily cross the placenta; those with molecular weights of less than 600 usually do.

Because most drugs have molecular weights in range of 250-400, there is usually no difficulty in transfer (Mirkin, 1973). Several different animal species have been used in teratological research in an attempt to determine the most satisfactory model for predicting the hazard to humans. It is universally recognized that teratogenesis studies should be conducted in mammals. Ideally, in the case of drugs, the species chosen should metabolize the administered drug in a manner similar to that of humans. No single species thus far evaluated, however, fulfills all criteria.

Therefore, usually a rodent and a non-rodent species are selected for regulatory safety testing.

Table 2. Critical period of organogenesis in various species

**Following fertilization

***Also may be given as days 35-70 after last menstrual period

Source: Schardein, 2000a.

11..CC..22 SSuusscceeppttiibbllee ssttaaggeess ooff ddeevveellooppmmeenntt

Another cardinal principle of teratogenesis is related to timing of treatment.

The organogenesis is the period when the embryological differentiation can be observed and the drugs or the chemicals administrated can exert their teratogenic effects. The severity and the nature of the effects are directly linked to the

Species Days of organogenesis**

Mouse 7-16 Hamster 7-14 Rat 9-17

Guinea pig 11-25

Armadillo 1-30 Ferret 12-28 Rabbit 7-20 Cat 14-26 Rhesus

monkey 20-45

Baboon 22-47 Dog 14-30 Sheep 14-36 Cow 8-25 Pig 12-34

Human 20-55 ***

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development stage of the insulted fetus. In thalidomide teratogenesis (Wilson, 1972) the importance of this principle, rather than dosage, was the decisive factor.

Organogenesis varies among the various species and is dependent on the length of gestation. In early gestation, during the predifferentation, the embryo is generally resistant to congenital malformations, while the death of the embryo or abortion can occur. Following differentiation, the conceptus becomes progressively less susceptible to teratogenic stimuli. Abnormalities in the central nervous system may occur later in gestation. During development it is possible to observe one or more periods of a maximum effect caused by teratogen insult. In mouse, the frequency of cleft plate inducted by 5-fluorouracil exposure was highest on days 10 to 13 (Dagg, 1960). In Table 2 the susceptible period of various species used in teratological studies are shown. The time of greatest teratogenic insult corresponds to the time at which particular organ is developing most rapidly. The temporal relation for a number of specific types of defects in the human is shown in Fig.1.

Fig. 1. Human embryonic development showing sensitive periods (darks area mark the most sensitive stage)

Source: Moore, 1988.

11..CC..33 MMeecchhaanniissmmss aanndd ppaatthhooggeenneessiiss

Many efforts have been spent in order to address specific biological events that might be responsible for the various abnormalities observed subsequent to teratogenic insult. The initial event(s) in all instances was defined as the mechanism.

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The nine proposed mechanisms were:

1. Mutation

2. Chromosomal nondisjunction 3. Interference with mitosis

4. Alteration of nucleic acid function 5. Lack of substrates and/or precursors 6. Lack of energy sources

7. Inhibition of enzymes

8. Altered membrane characteristics 9. Osmolar imbalance

Pathogenesis is second in the sequence of events leading to expression of a malformation and may be represented by one or more of the following: (1) cell death, (2) failure of cell interaction, (3) reduced biosynthesis, (4) impaired morphogenic movement, or (5) mechanical disruption of tissues (Wilson, 1977).

Current experimental data are most convincing in support of these pathogenic events. For example, investigators studying forelimb cartilage of osteochondrodysplastic rats (ocd/ocd) on days 16 to 21 of gestation examined the distribution of glycosaminoglycans, type II collagen, and fibronectin. It was concluded that chondrocytes from fetus affected by a lethal form of dwarfism might be defective in their release of extracellular matrix components. Failure to release specific type and/or amount of extracellular matrix constituents may be related to differentiation and/or cell death in these chondrocytes (Parker and Cheng, 1990).

11..CC..44 DDoossee ddeeppeennddeennccyy

Dose-effect relation also governs teratogenicity. Teratogenic induction is a threshold, not a stochastic phenomenon, which characterizes cancer and mutation induction (Brent, 1986). The dosage of a given teratogen properly applied lies within a narrow zone between that which will kill the fetus and that which has no discernible effect. It is probable that death and abnormal development are simply different degrees of reaction to the same stimuli, with the rate of mortality and the rate and severity of malformations increasing in roughly parallel fashion as dosage is

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increased, as a continuum. All embryos have threshold dosage, above which irreparable changes occur, as a malformation or in severe cases death. A correlation between embryolethality and teratogenicity does not always exist, nor are death and malformations necessarily correlated. Some normal or malformed or resorbed offspring are generally the result of an administration of a suitable dosage of teratogen, with or without maternal toxicity. When exposure increases above the no observable effect level (NOEL), the incidence and the severity of adverse developmental effects increase. If the dose of agent administrated is too high, all offspring may be dead or resorbed. If it is too low, there may be no effect on the fetus, and the agent give apparent negative result. Indeed, it is universally recognized that the test can be considered adequate until a dose low enough to permit survival of some normal offspring can be found (Wilson, 1968).

Dose-response curves occasionally show a plateau phase in relation to the malformation induced. The anticancer drug 5-chlorodeoxyuridine, administrated to pregnant mice at 200 mg/kg, resulted in a 40 % incidence of malformed digits of the hindfeet, but this frequency did not change significantly with doses two and three times higher (Nishimura, 1964).

It should also be considered that the embryo is usually more susceptible to chemicals than the adult. In fact, a teratogenic agent needs no to be toxic to the mother. Neuber et al. (1971) have pointed out that this is due to a particular vulnerability of certain embryonic cells that is not found in corresponding adult cells. Due to the lack in the embryo of some enzymes necessary for detoxification, the fetus may be exposed to greater concentrations of drugs (Tuchmann-Duplessis, 1970).

The drug treatment duration is another variable. Normally acute dosing schedules result in a greater teratogenic insult to the embryo than chronic dosing schedules.

For example, in rats treated with the anticancer drug dactinomcyn, malformations were induced in 28% of the survivors when a single teratogenic dose (200 µg/kg) was given on day 9, the day of greatest sensitivity, yet an even higher total dose (250 µg/kg) given as ten daily injections of 25 µg/kg on days 0-9 resulted in only 9%

malformed survivors (Wilson, 1966).

Finally a combination of drugs or chemicals may result in a teratogenic effect when either alone has little or no teratogenicity. For example, individual administration of

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cyclophosphamide and 5-fluorouracil to rats at doses of 10 mg/kg produced malformations in 26 and 10% in the offspring, respectively (Wilson, 1964).

Administrating both compounds resulted in 100% malformations. Synergism or potentiation is one type and the most important one for teratological considerations in human.

1

1..DD MMaanniiffeessttaattiioonn ooff ddeevviiaanntt ddeevveellooppmmeenntt

Malformations, growth retardation, embryolethality and functional impairment are the four principals manifestations of deviant or disruptied development.

1

1..DD..11 MMaallffoorrmmaattiioonnss

Malformations are the principal parameters assessed in the determination of potential teratogenicity of a drug or other chemical. Malformations are one indicator of developmental toxicity. Administration of a teratogenic agent may increase the frequency of a spontaneously occurring malformation, or it may induce malformations rarely seen spontaneously. At the beginning, the teratogenic effect probably occurs through apoptosis, or alteration in the rate of cell growth, but the final deformity represents not only the consequence of this direct injury, but also of secondary regenerative processes that follow it (Wilson, 1959; Haring and Lewis, 1961). Mutation, chromosomal abberation, mitotic interference, altered nucleic acid or energy sources, biosynthetic imbalance, enzyme inhibition and osmolar imbalance are possible mechanisms of the pathogenesis of malformations.

There are also some differences between malformations in various species. Whereas most types of malformations are observed in most or all species of animals, the individual malformations and their frequency of occurrence are somewhat species- dependent. For example, eye defects, exencephaly, polydactily, and cleft palate are seen quite commonly in mice (Flynn, 1968; Kalter, 1968a). Rabbits, however, have limb defects, umbilical hernias, and craniofacial defects more frequently than other types of malformations (Chai and Degehardt, 1962; Staples and Holtkamp, 1963).

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Induced malformations are usually bilateral in paired organs. This means simple involvement of both sides. For example, the limb defects in thalidomide disaster were always bilateral (Lenz, 1971).

Table 3. Spontaneous malformations rates for various species of animals

Source: Schardein, 2000b.

A teratogen might produce a single specific or a whole pattern or syndrome defects, but these would have some degree of uniformity from case to case, if chemically induced. Chemicals capable of inducing malformations also hold the potential for inducing other classes of developmental toxicity. One often-misunderstood aspect, in relation to developmental toxicity is that all teratogens are developmentally toxic, but not all agents that are developmental toxic are teratogenic.

11..DD..22 GGrroowwtthh rreettaarrddaattiioonn

The fetal size is another important parameter to be considered to detect potential Species Range (%) of malformations

reported

Mouse <1-18.6 Rat 0.02-1.9 Rabbit 0.7-14.1 Ferret <1

Guinea pig 0.02

Hamster 0.4 Dog 0.2-7.1 Cat 1.2 Sheep 1.8-9.1 Cow 0.2-3 Swine 0.6-9.8 Rhesus

monkey

0.1-7.1 Squirrel

monkey

1.3 Japanese

monkey

16.8

Cyno monkey 0.4-5.5

Baboon 0.5 Bonnet

monkey 0.3

Chimpanzee 0.6 Human 0.14-13.8

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teratogens. This parameter is an indicator of developmental toxicity. Reduction in size or growth retardation commonly occurs among fetus of dams given dosages that are toxic to the dam, to the offspring, or both. Numerous agents are known to cause intrauterine growth retardation (IUGR). In a large series of consecutive pregnancies in the human, the incidence of IUGR was 5.3%; perinatal mortality among these infants occurred three times more often than normal (Low and Galbraith, 1974). In another study (Callan and Witter, 1990), 86% of perinatal deaths were found in the IUGR group. Up to 20% of spontaneous abortions exhibit severe embryonic growth retardation.

Generally accepted definition for IUGR is: “Birth weight less than the tenth percentile for gestational age” (Lubchenco et al., 1963). An alternative definition is:

“Birth weight more than two standard deviations below the mean for gestational age, corresponding to approximately the third percentile of the intrauterine growth curves” (Gruenwald, 1966).

Growth inhibition during stage will produce an undersized fetus with fewer cells, but normal cell size, causing symmetric IUGR. Growth inhibition during stage II and III will cause a decrease of cell size and fetal weight with less effect on total cell number and fetal length and head circumference, causing asymmetric IUGR.

IUGR is not uncommon in infants with severe and multiple malformations (Warkany, 1971). In addition, overwhelming congenital infections, neonatal deaths, and long term neurological and intellectual deficits have been recorded more frequently than among infants with normal growth (Miller, 1981).

11..DD..33 EEmmbbrryyoolleetthhaalliittyy

Death of the offspring is another class of developmental toxicity. Embryonic and early fetal loss occur in approximately one out of every two pregnancies in the human (Shepard and Fantel, 1979). Fetal death is often associated with the occurrence of congenital malformations; the pattern may simply be a positive relation between several forms of toxicity to the embryo or fetus.

Intrauterine death is distributed along a dose-response curve (Wilson, 1980). It is manifested in a temporal relation and referred to as pre- or postimplantation loss.

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When there is undue toxicity early in pregnancy in animals, the embryo dies, is resorbed and only the presence of the site of implantation is visible; its counterpart in the human is miscarriage or death occuring later in pregnancy, the fetus can not be wholly resorbed and a stillborn or dead and often macerated fetus, is the result in both animals and humans.

Mortality may primarily be due to the direct action of chemicals on the conceptus or it may be secondary to maternal effects and some times it is impossible to distinguish between the two (Kalter, 1980). Some agents have non-specific effects on the dam and the embryo coincident with their effects on morphological development. If embryos do not recover, the outcome will then be concurrent induction of death and malformation, and the frequency of both will vary depending on the chemical. Other teratogens cause primarily morphological maldevelopment that may lead to embryonic death. In humans, several correlations between malformations and death have been noted. It appears that spontaneous abortion serves as a mean of selectively terminating abnormal conception; indeed, 95% of abnormal pregnancies are believed to terminate in this way (Haas and Schottenfeld, 1979).

11..DD..44 FFuunnccttiioonnaall iimmppaaiirrmmeenntt

In recent years, increasing attention has been placed on the more subtle, non- structural alterations produced by drugs and chemicals when given prenatally. Such parameters as motor ability, sociability, emotional, and learning capacity are examples. Evidence has accumulated indicating that if there is exposure to certain agents during critical periods in fetogenesis, specific types of behavioural alterations may result. Such alterations, or abnormalities, in behaviour arise as a result of drug- induced modification of development of specific neurotransmitter systems (Leonard, 1981). Some alterations occur after teratogenic doses; others occur after minimally teratogenic or even subteratogenic dose levels. Aspirin (Okamoto et al., 1986), ethoxyethanol (Nelson et al., 1981) and hydroxyurea (Vorhees et al., 1979) are agents that produce, in animal species, behavioural effects at low dose trigger.

A corrected terminology to describe this fact is “functional developmental toxicity”.

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This branch of toxicology studies the causes, mechanism and manifestations of alterations or delays in functional competence of the organism or organ system following exposure to an agent during periods of development pre- and/or postnatally.

Functional effects are not normally evaluated, because the standard protocol traditionally followed in drug and chemical teratology assessments does not include postnatal assessment. However, behavioral tests have become a common component of developmental toxicity assessment of pharmaceuticals in recent years. Good examples of behavioral effects attributed to teratogens are: mental retardation in children born of women exposed to anticonvulsants or alcohol and the abnormal reflexes observed in offspring of methyl mercury-exposed mothers. In addition to malformations and other classes of developmental toxicity, there are other usually, less subtle, reproductive parameters which are assessed in animals that may have relevance to the human species and are reflective of environmental hazard.

For this reason we can consider some other parameters to assess developmental toxicity:

• MaMallee--mmeeddiiaatteedd eeffffeeccttss: sperm cells collected from rabbits were suspended in a solution containing the drug colchicin (Chang, 1944). Of the 32 offspring produced, 3 were defective. In another study, an uncontrolled one with the classic teratogen thalidomide, male rabbits were treated before mating with untreated females; there were “gross malformations” in some offspring (Lutwak-Mann, 1964). These are some example in which the male seemingly mediated effects. The absence of extensive human evidence for this should be interpreted as a deficiency in research, rather than an absence of male-mediated adverse reproductive outcomes, and according to one review, some 194 chemical agents have been identified as possessing data relating to male-mediated adverse reproductive outcomes (Davis et al., 1992).

• DeDevveellooppmmeennttaall vavarriiaattiioonnss: another factor in assessing fetal development is related to minor aberrations in structure and variations in ossification that occur in fetal evaluation, including control animals. These occur more frequently than malformations, and represent delay in growth, minor

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changes in structure and form, or alteration in differentiation. The most common of these are supernumerary ribs, ossified cervical centra and unossified sternebrae in rats as well as supernumerary ribs, unossified sternebrae, and extra vertebrae in rabbits. Among the variations, extra and way ribs appear to be most consistently associated with maternal toxicity (Schardein, 1987). Variations are interpreted to be indicators of developmental toxicity when elicited in a dose-related manner at incidences significantly above control values.

• ThThee mmooddiiffyyiinngg iinnfflluueennccee ofof mmaatteerrnnaall ttooxxiicciittyy: maternal toxicity has long been recognized as an possible outcome in teratology assessment, the rationale of this statement being that toxicity in the mother is intentionally elicited in mandated testing requirements, to maximize detection of toxicity in the offspring. It is clear that toxicity to the embryo may be modified or influenced by toxicity to the mother, but it is not yet understood why, under what circumstances, or just how extensive the impairment of maternal homeostasis and resultant compromised health status must be affected to impair development. It has been suggested by Khera (1987) that some fetal malformations frequently occur in conjunction with maternal toxicity in animals, including various rib, vertebral, and sternebral defects. This conclusion has been subject to controversy, and is not supported by several experiments conducted to confirm or deny the association (Rosen et al., 1988; Chernoff et al., 1989). Distinction must be made whether teratogenicity and other developmental toxicity result from a primary action of a chemical on the conceptus, of whether such toxicity is secondary to, and coexistent with, maternal toxicity.

• PhPhaarrmmaaccookkiinneettiicc coconnssiiddeerraattiioonnss: pharmocokinetics studies address the factors that govern the time course of the concentrations of the biologically active forms of the agent relative to the incidence and magnitude of toxicological response (Gilette, 1987). There are four basic factors that determine the action of chemicals and drugs on tissue: absorption, distribution, biotransformation (metabolism), and elimination. Pregnancy itself alters pharmocokinetics through physiological changes that occur

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normally (Table 4). These changes are required for successful pregnancy and lactation. It is the pharmocokinetic properties of the chemicals or drugs that influence the amount of exposure to the fetus and, hence, the toxicological response to its disposition. Pharmocokinetic modelling in developmental toxicity studies is becoming very common.

Table 4. Physiological changes that may alter toxicokinetics during pregnancy

Source: Mattison et al., 1991.

Physiological parameters Change

Absorbtion

Gastric emptying Increased

Intestinal motility Decreased

Pulmunary function Increased

Cardiac output Increased

Blood flow to skin Increased

Distribution

Plasma volume Increased

Total body water Increased

Plasma proteins Decreased

Body fat Increased

Metabolism

Hepatic metabolism ±

Extrahepatic metabolism ±

Excretion

Renal blood flow Increased

Glomerular filtration rate Increased

Pulmunary function Increased