Structural malformations Growth retardation Functional impairment Death of the organism

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

2. Teratology 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 It was later (1961) withdrawn from the market once it was discovered thalidomide was a human teratogen.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 between20-36 daysafter 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 16-20 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. 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 170-200 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 900-1300 malformed children, 700-1000 spontaneous abortions and 5000-7000 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

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

22. RAR and RXR types 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 withmenigomyelocele 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 (1971-1972), 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)