1. Developmental toxicology
Structural malformations
Growth retardation
Functional impairment
Death of the organism

2. Teratology Teratogen Any agent that causes a birth defect
1. the study of malformations or serious deviations from the normal type in organisms
2. the branch of science concerned with the production, development, anatomy, and classification of malformed fetuses.
Teratogen
Any agent that causes a birth defect
After Greek “monster creating”

3. Environmental conditions (1200)
Maternal nutritional deficiencies (1930)
Rubella virus infection (1941)
Thalidomide (1961)

4. (65%)
35-40 /4100 chemicals alter prenatal development, Table 10-1

5. Thalidomide

6. Thalidomide Thalidomide was released in 1956 as a mild sedative used to combat nausea in pregnant women. It was later (1961) withdrawn from the market once it was discovered thalidomide was a human teratogen. As little as one dose could cause a significant birth defect. Approximately 5,000-7,000 malformed infants were born to women who ingested thalidomide during pregnancy. Symptoms: malformed intestines, hearing defects, absent ears, and/or ocular and renal anomalies. However, the most striking phenotype is phocomelia: severe limb malformations in which the long bones of the limb are either greatly reduced in length or absent all together.

7. Teratogenic between 20-36 days after fertilization
Mechanisms –unknown?
Proposed mechanisms (more than 30)
Angiogenesis
Integrin regulation
Oxidative DNA damage
growth factor antagonism
Now approved for
Oral ulcer for AIDS, erythema nodosum leprosum
-new anticancer drug? Anti-angiogenesis

8. Diethylstilbesterol (DES)
DES was prescribed between 1940 and 1970 to prevent miscarriages in high risk pregnancies. This was accomplished by DES increasing estrogen and progesterone synthesis by the placenta. In the mid 1970 cases of vaginal adenocarcinoma in women ages were linked to fetal exposure through maternal DES ingestion early in the pregnancy. Approximately 1 in 1000 pregnancies were exposed, 75% of which resulted in female children with vaginal and cervical carcinomas as well as uterine anomalies. Male offspring had epididymal cyst, hypotrophic testes, decreased semen volume and poor semen quality.

9. Fetal Alchohol Syndrome (FAS) Fetal Alchohol Effects (FAE)
Alcohol (Ethanol)
Fetal Alchohol Syndrome (FAS)
Fetal Alchohol Effects (FAE)
                          
Cranial facial dysmorphism
Intrauterine and postnatal growth retadation
Retarded psychomotor and intellectual development
IQ 68

10. Tobacco
Nicotine restricts uterine blood vessels and restricts blood flow to the fetus resulting in chronic hypoxia and malnutrition leading to birth defects. On average, offspring of smoking women weigh g less at birth as compared to a non smoker’s child. There is a dose dependence in that the child weight decreases in proportion to number of cigarettes smoked by the mother. There is also a reduction in overall fetal length, reduced head circumference, intrauterine growth retardation as well as behavioral alterations after birth.

11. Possible outcomes of smoking during preganancy include:
spontaneous abortion
perinatal deaths
increase risk of sudden infant death syndrome
increased risk of learning, behavioral, and attention disorders.
Perinatal exposure to tobacco smoke can affect branching morphogenesis and maturation of the lung.
Smoking during pregnancy increases the risk for premature delivery, abruption placenta, placenta previa and perinatal mortality.

12. Cocaine
Cocaine is an anesthetic and vasoconstrictor. Cocaine is thought to induce birth defects by disrupting the vasculature in the placenta thereby inducing intrauterine hypoxia and malnutrition
These pregnancies are at risk for premature labor, spontaneous abortion, increased perinatal mortality and fetal death.
Exposed fetuses often have intrauterine growth retardation, microcephaly, altered presencephalic development, decreased birth weight, a neonatal neurologic syndrome of abnormal sleep, tremor, poor feeding, irritability, and occasional seizures.

13. Retinoic Acid
                                        

14. Retinoic acid
Retinoic acid is the active ingredient in “Accutane”, a drug used to treat severe acne. Since its introduction in September of 1982, an estimated 160,000 women of child bearing age have ingested the drug. Between 1982 and 1987, approximately malformed children, spontaneous abortions and elective abortions are due to Accutane exposure. Exposed children may have hydrocephaly, ear malformations, cardiovascular defects and decreased IQ. Accutane carries a pregnancy category X warning, meaning it is a known human teratogen.

15. Mechanism
A proposed mechanism is that biologically active retinoic acid binds retinoic acid receptors which in turn bind DNA enhancer elements such as the retinoic acid response elements. Several Hox genes (responsible for early patterning of the embryo) contain this enhancer element in their promotors. Therefore, Hox signaling may be altered due to increased retinoic acid concentrations resulting in multiple birth defects.

16. Retinoid Actions in vivo
Myeloid differentiation
Epithelial growth – keratinocytes
Embryo development
Anti-oxidants

17. Retinoid Therapies Use Drugs Psoriasis Tazartene (Zorac),
Etritinate (Tegison)
Acne
Adapalene (Differin),
Tretinoin (Renova),
Isotretinoin (Accutane)
Leukemia
Tretinoin/ATRA (Vesanoid)

18. Retinoid Excess in Embryogenesis
Retinoids are teratogens
Embryos exposed to excess RA develop posterior neural tube defects
Particularly affected are the retina, spinal chord and hind brain
Posteriorization of anterior structures
Marshall et al., FASEB J, 1996

19. RAR and RXR (Simple Version)
Nuclear Receptors (like ER, PPAR, VDR and others)
RXR/RAR Heterodimer is functional unit
Bind selectively to REs in genome
Act as transcription factors
Up-regulate or Repress the expression of particular genes

21. SIGMA-ALDRICH.com/rbi
Hormonal Targeting of Nuclear Complexes to Chromatin
SIGMA-ALDRICH.com/rbi

22. RAR and RXR types Gene Major Isoforms Endogenous Ligand RAR 1, 2
ATRA, 9-cisRA
RAR
1, 2, 3, 4
RAR
1, 2
RXR
9-cis RA
RXR
1, 2
RXR
Summarized from: Chambon, FASEB J., 1996

23. Valproic Acid
Valproic acid was released in 1967 in Europe and in 1978 in the United States to treat epilepsy. Approximately 11,500 epileptic women become pregnant each year, many of which use valproic acid. By 1980, publications began linking malformed children to in utero exposure to valproic acid (greater than 500 mg/day).

24. These children were born with lumbosacral spina bifida with menigomyelocele or menigocele, often accompanied by midfacial hypoplasia, deficient orbital ridge, prominent forehead, congenital heart disease and decreased postnatal growth. The proposed mechanism of action is that valproic acid influences folate metabolism, thereby altering the closure of the spinal column resulting in spina bifida.

25. Congenital Minamata Disease
Methylmercury was used in the past as a fungicide on wheat and grains. Cases have been documented in Iraq ( ), Sweden, Japan and New Mexico of birth defects due to maternal ingestion of bread made with contaminated grain. There have also been documented cases in Canada, New York and Sweden of paper mill contaminants polluting the water with inorganic mercury. This mercury is converted to the biologically active methylmercury by microbes that live on the bottom of the lakes. It is then concentrated in the flesh of fish. Here fetal damage may occur by maternal intake of fish and shellfish containing methylmercury. Exposure in utero may result in sensory and motor impairments, cerebral palsy, mental retardation and behavioral damage.

26. Between 1953 and 1965 there were over a hundred adult men and women developing symptoms of central nervous system disorders such as ataxia, alterations in gait, tremors, altered sight and sensation. In 1955 in the Minamata Bay area of Kyushu, Japan, there was a large influx of cases of severe neurological disorders in newborn children. There were cases of cerebral palsy, some children were diplegic and others were tetraplegic. They were all mentally handicapped. Some villages had 6-12% of their newborns affected. Together, these disorders are now known as Congenital Minamata Syndrome. In 1959, it was found that methylmercury was being dumped into the bay by a plant of the Chisso Corporation.

27. Children with Congential Minamata Syndrome seem to be normal at birth and begin to present symptoms at approximately six months of age. They have instability of the neck, convulsions, reduced IQ, microcephaly, malformed limbs, restricted growth and an altered cerebellum. In utero exposure to methylmercury induces general brain atrophy and hypoplasia.
                                                
                       

28. The bombing of the Japanese cities of Hiroshima and Nagasaki (in 1945) induced an increase in newborns with microcephaly and mental retardation. There was also a marked increase in fetal and neonatal death. Studies have since been able to link the incidence of microcephaly directly with the distance of the mother from the explosion of the bomb.
                                                       
                                        

29. The 6 Principles of Teratology
Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with environmental factors.
Susceptibility to teratogenic agents varies with the developmental stage at the time of exposure.
Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis).
The final manifestations of abnormal development are death, malformation, growth retardation, and functional disorder.
The access of adverse environmental influences to developing tissue depends on the nature of the influences (agent).
Manifestations of deviant development increase in degree as dosage increases from the no-effect to the totally lethal level.

30. Principle of developmental toxicology
1.  genetic influences-由於遺傳差異，引起個體對致畸作用的敏感度不同
2.  critical periods-不同時期對致畸作用的敏感度不同
3.  initiating mechanism-致畸原以特定的機轉對細胞組織作用引發一連串的不正常發育
4.  access to embryo and fetus-致畸原的特性決定其與胚胎接近的難易
5.  abnormal development-不正常的發育的結果→death, malformation, growth retardation, and functional disorder
6.  dose-response relationship-however,一般致畸原存在threshold level
Attribution of threshold
1. high restorative growth potential of mammalian embryo
2. cellular homeostatic mechanisms
3. maternal metabolic defenses

31. Critical periods of susceptibility and endpoints of toxicity
Gametogenesis and Fertilization
Mechanism unclear, may be related to
imprinting
Cytosine methylation and change in chromotin conformation
受精後6hr暴露ethylene oxide, ethylmethane sulfonate, ethylnitrosourea→malformed fetus

33. DNA Methylation Methyl groups may be attached
to cytosine (C5 position)
Methyltransferases
Methyl groups provide a tag Concentrated in CG-rich domains, often in promoter regions
Maintains a gene in inactive state rather than initiating gene repression – Example:
Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation
Implantation – a new wave of methylation occurs
Early Zygote – most methylation tags removed

34. DNA Methylation – Genomic Imprinting
Certain genes are active or inactive during early development
Depending on whether they are paternal or maternal genes
Eg – IGF-2 is only active in the gene from the male parent
The gene is imprinted according to parental origin
Mammalian genome has > 100 imprinted genes in clusters
Imprinted due to selective methylation of one of the alleles

35. 2.Preimplantation著床前期 (blastocyst)
囊胚形成，細胞分裂到1000個細胞，僅3個細胞將發育成胎兒，餘發育成胎盤等支持組織，在此期暴露，理論上不影響或稍微影響胎兒生長，不然就導致死胎。
DDT, nicotine, methylmethane→body and/or brain weight deficits and embryo lethality but not malformation
然而, Methylnitrosourea, cyproterone→malformation

37. 3. Implantation 著床 第6-13days
4. Gastrulation-三胚層形成, 第3週
在此期暴露有害物質將造成眼、腦及臉部的畸形

38. 5. Organogenesis 器官形成，第3-8週
為最容易受影響的時期，因為本期
Cell proliferation
Cell migration
Cell-cell interactions
Morphogenetic tissue remodeling

39. 6. Fetal period胎兒期 第8wk-birth
在此期暴露，影響生長和功能的成熟，需要在出生後仔細觀察才能察覺。如中樞神經的異常包括行為、智力、運動的缺失，生殖力降低，以及免疫系統、心臟、肺臟、腎臟功能受損等。
*若有構造的改變乃是破壞原本正常的構造稱為deformation，不同於前述malformation

41. Dose-response Patterns and the threshold concept

42. 6 Principles of Teratology
1.  genetic influences-由於遺傳差異，引起個體對致畸作用的敏感度不同
2.  critical periods-不同時期對致畸作用的敏感度不同
3.  initiating mechanism-致畸原以特定的機轉對細胞組織作用引發一連串的不正常發育
4.  access to embryo and fetus-致畸原的特性決定其與胚胎接近的難易
5.  abnormal development-不正常的發育的結果→death, malformation, growth retardation, and functional disorder
6.  dose-response relationship-however,一般致畸原存在threshold level

43. Mechanisms and pathologenesis of developmental toxicology
1.   mutation 突變
somatic mutation in the early embryo, ex.mutagen
2.   chromosomal abnormalities 染色體異常
ex. advanced maternal age, viral infection, irradiation, and chemical agents
3.   mitotic interference干擾細胞分裂
slow or arrest DNA synthesis (hydroxyurea or irradiation), interfere with spindle formation (colchicine, vincristine)
4.   interference with nucleic acid function干擾核酸的功能
including replication , transcription, translation ex. antibiotics and antineoplastic drugs
5.   nutritional deficiencies營養缺乏
ex. vitamins , minerals  

44. 6. deficient or alter energy supply 缺少或改變能量的供給
ex. inadequate glucose supply (hypoglycemia), interference with glycolysis (iodoacetate, 6-aminonicotinamide), inhibition of the citric acid cycle (riboflavin deficiency), blockage of the terminal electron transport (hypoxia, cyanide)
7.   changes in osmolarity滲透壓的改變
ex. hypoxia, trypan blue, hypertonic solutions, adrenal hormone→edema, hematoma, and blisters
8.   changes in cell membranes細胞膜的改變
ex. solvent, vitamin A
9.   enzyme inhibition酵素的抑制
抑制代謝酵素,DNA repairing, polymerase

45. Example of cyclophosphamide (CP)
A teratogenic chemotherapeutic agent
Damage to DNA inhibit cell cycle progression
cell cycle arrest too long apoptosis
Bind to protein
Single strand DNA break

46. CP induces DNA damage
(predominant occur in
S phase)
leading to
Cell cycle perturbation
Cell death
Sensitivity is determined
by cell cycle length and
cell predisposition to apoptosis

48. Cell death in the neural tube by CP
Sensitivity to CP-induced cell death
Neuroepithelium >heart
Cell cycle length
9.5 hr vs 13.4 hr (longer Go/G1)

52. Advances in the Molecular basis of dysmorphogenesis
1.Using either singly or double gene knockout
Retinoic acid receptor family (syndactyly)
2. Antisense oligonucleotide
Wnt-1, Wnt-3a (mid and hindbrain malformation)
3. Reporter transgenes
RA  activate hoxb-1-lacZ

53. Pharmacokinetics and metabolism in pregnancy
1.Changes in maternal physiology
hepatic metabolism, GI tract, cardiovascular system, excretory system, respiratory system
2.Overall decrease in hepatic xenobiotic transformation
3.Roles of placenta in influence embryonic exposure
help to regulate blood flow
-offer a transport barrier-pH gradient, weak acid rapidly transfer
-metabolize chemicals
2-acetylaminofluorene (proteratogen)
7-hydroxyl metabolites(proximate teratogen)
4.Maternal metabolism of xenobiotics
2-methoxyethanol methoxyacetic acid

54. Maternal factors affecting development
Genetics
high incidence of cleft lip/palate in white mother
Disease-chronic hypertension
diabetes
infection-cytomegalovirus, Taoxoplasma gondii
Hyperthermia-CNS malformation
Nutrition-folate neural tube defect
Stress-noise, restraint
Placenta toxicity -46 toxicants, Cd

55. placental toxicity
Metals, Cd, As, Hg, ethanol, cocaine, cigratte, sodium salicylate
Maternal injection vs fetal injection of Cd
Production of metallothionein
Interaction with Zn

56. Maternal toxicity- acetazolamide inhibits carbonic anhydrase
forelimb ectrodactyly
diflunsial results in anemia
skeleton defects in rabbits
phenytoin affects folate metabolism and heart rates
metallothionein synthesis inducer-urathane, mercaptopurine, valproic acid
Zn deficiency

58. Develpmental toxicity of endocrine-disrupting chemicals
Definition of endocrine-disrupting chemicals
“Exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones responsible for the maintenance of homeostasis and the regulation of developmental processes.”

59. Endocrine-disrupting chemicals
Four modes of action
1. Serving as steroid receptors ligands
2. Modifying steroid hormone metabolizing enzymes
3. Perturbing hypothalamic-pituitary release of trophic hormones
4. Uncharacterized proximate modes of action

60. Modern safety assessment
Regulatory guidelines for in vivo testing
Multigeneration tests
Children’s health and the food quality protection act
Alternative testing strategies
Epidemiology
Concordance of data
Elements of risk assessment
use-in pregnancy rating:A, B, C, D, X

64. Impact on screening and testing programs
Expansion of the periods of dosing from the end of
organogenesis to the end of pregnancy in order to include the urogenital differentiation
2. EDSTAC recommended a high through put screening (HTPS) cell-based, receptor-mediated gene transcription assay

65. Tier I screening battery for EDS
In vitro:Estrogen receptor binding or transcriptional activation
assay
Androgen receptor binding or transcriptional activation
Steroidogenesis assay using minced testis
In vivo:Rodent urotrophic assay,
A rodent 20-day pubertal female assay for thyroid function
A male rodent 5-7 day Hershberg assay,
A frog metamorphosis assay for thyroid effects
A fish partial life cycle test
T2T: more defined toxicological response would be characterized

66. Summary of in vivo regulatory protocol guidelines for evaluation of developmental toxicity

73. Alternative tests for developmental toxicity
Mouse ovarian tumor
Human embryonic palatal mesenchyme
Micromass culture
Mouse embryonic stem cell test
Chichen embryo neural retina cell culture
Drosophila
Hydro
FETAX (Venopus embryo)
Rodent whole embryo culture
Chernoff/Kavlock assay
Sensitivity(+)/Specificity (-)

74. Sonic Hedge-hog signal pathway
Cholesterol synthesis inhibitor
cyclopamine
jervine
Holoprosencephaly

76. Birth Defect Prevention Measures
Folate supplementation
Healthy lifestyle
Genetic counseling; diagnostic testing

77. Consequences of Folate Deficiency
Result of low dietary intake, genetic error of folate metabolism, lifestyle exposures
DNA Hypomethylation
Gene overexpression, uncontrolled cell growth, genomic instability
Hyperhomocysteinemia
Excessive accumulation of Hcy
Base Misincorporation
Decrease in thymine synthesis; replaced by uracil
DNA strands prone to nicks, breaks and vulnerable to mutagen insertion

78. How Do I Get Folate? Eat a healthy diet
Fruits, green leafy vegetables, beans, corn, peas, bananas,
orange juice
Eat fortified cereal and grain products
Total®, Special K®, Product 19®
Take a multivitamin
400µg of folic acid