Developmental Toxicology

1
Developmental Toxicology
2
4 manifestations of developmental toxicity

• Structural malformations
• Growth retardation
• Functional impairment
• Death of the organism

3
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

4

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

5
Adverse Outcomes in Pregnancy

• 57 of pregnancies detected by hCG at 8-9 days
  after fertilization do not develop as clinically
  detectable pregnancies
• 15-20 of recognisable pregnancies end in
  spontaneous abortion 90 in the first trimester
• 2 of pregnancies end in miscarriage gt20 weeks.
• 2-3 of newborn have a major malformation severe
  enough to require hospitalisation.

6
Cause of human birth defects

• 15-25 genetic cause
• 4 maternal conditions
• 3 maternal infections
• 1-2 deformation (mechanical problem)
• lt1 chemical and environmental influences
• 65 unknown etiology

7
Chemical teratogenicity

• gt4100 chemicals have been tested
• 66 non-teragenic
• 7 teratogenic more than one species
• 18 teratogenicin most species tested
• 9 equivocal experimental results
• 50-60 chemicals or conditions alter prenatal
  development in humans (Table 10-1)

8
Pregnancy Risk Categories
From A Textbook of Psychopharmacology. Ed. by
Schatzberg and Nemeroff. American Psychiatric
Publishing Company, 2004.
9
Therapeutic Drugs Teratogenic to Humans

• Anticonvulsants
• Phenytoin, primidone, trimethadione, valproic
  acid, carbamazepine
• Anticancer agents
• Alkylating agents busulfan, cyclophosphamide,
  chlorambucil, mechlorethamine
• Antimetabolites-aminopterin, methotrexate,
  cytarabine
• Androgenic hormones-danazol
• Coumarin anticoagulants-warfarin
• Retinoids-accutane, isotretinoin, etretinate,
  acitretin
• Antihyperlipidemic agents-lovastatin,
  atorvastatin
• Other drugs-diethystilbestrol, thalidomide,
  penicillamine, lithium, fluconazole, misoprostol

10
Thalidomide
11
Thalidomide

• Susceptible period- 20-36 days after
  fertilization
• Proposed mechanisms (gt24)
• Embryonal DNA oxidation (PBN can prevent)
• Misregulation of the expression of genes
  critical for outgrowth of limb
• The inability of NF-kappaB, a redox-sensitive
  transcription factor, to bind to its DNA promoter
  results in the failure of limb cells to express
  fibroblast growth factor (FGF)-10 and twist in
  the limb progress zone mesenchyme, which in turn
  attenuates expression of FGF-8 in the apical
  ectodermal ridge.

12
Diethylstilbesterol (DES)

• DES was prescribed between 1940 and 1970 to
  prevent miscarriages in high risk pregnancies.
• DES increases 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.
• Female children - vaginal and cervical
  carcinomas, uterine anomalies.
• Male offspring - epididymal cyst, hypotrophic
  testes, decreased semen volume and poor semen
  quality.

13
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

14
(No Transcript)
15
Tobacco smoke

• Spontaneous abortions
• Perinatal deaths
• Lower birth weight
• Increased risk of
• Sudden infant death syndrome
• Behavioral attention disorders
• Orofacial cleft (particular xenobiotic gene
  polymorphisms)
• Gastroschisis (with variant alleles N053, ICAM1,
  NPPA)
• Branching morphogenesis and maturation of the
  lung
• Nicotine-related adverse nerodevelopmental
  outcomes

16
Cocaine

• At risk for premature labor, spontaneous
  abortion, increased perinatal mortality and fetal
  death.
• intrauterine growth retardation, microcephaly,
  altered presencephalic development, decreased
  birth weight, a neonatal neurologic syndrome of
  abnormal sleep, tremor, poor feeding,
  irritability, and occasional seizures.
• Genitaouinary tract malformation
• Impaired uditory process

17

Retinoic Acid
18
c 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.
19
Retinoids

• Malformations of the face, limbs, heart, CNS, and
  skeleton
• RXR a receptor
• Schizophrenia

20
Retinoid Therapies
Use Drugs
Psoriasis Tazartene (Zorac), Etritinate (Tegison)
Acne Adapalene (Differin), Tretinoin (Renova), Isotretinoin (Accutane)
Leukemia Tretinoin/ATRA (Vesanoid)
21
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

22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
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).
26
Valproic Acid

• spina bifida with menigomyelocele or menigocele
• The proposed mechanism of action is that valproic
  acid influences folate metabolism

27
Angiotensin Converting enzyme inhibitors and
angiotensin antagonists

• 2-3 trimester
• related reduced amniotic fluid volume and
  impaired fetal renal function
• Oligohydromnios
• Fetal growth retardation
• Pulmonary hypoplasia
• Renal failure
• Hypotension
• Death
• First trimester
• Congenital malformation

28

• Wilsons General Principles of Teratology (Table
  10-2)
• 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.

29

• Critical periods of susceptibility and endpoints
  of toxicity
• Gametogenesis and Fertilization
• Mechanism unclear, may be related to
• imprinting
• Cytosine methylation and change in chromatin
  conformation
• ???6hr??ethylene oxide, ethylmethane sulfonate,
  ethylnitrosourea?malformed fetus

30
(No Transcript)
31

• 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

32
DNA Methylation vs 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 gt 100 imprinted genes in
  clusters
• The majority of imprinted genes in mammals have
  been found to have roles in the control of
  embryonic growth and development, including
  development of the placenta
• Imprinted due to selective methylation of one of
  the alleles

33
2.Preimplantation???? (blastocyst)
????,?????1000???,?3?????????,???????????,?????,??
?????????????,???????? DDT, nicotine,
methylmethane?body and/or brain weight deficits
and embryo lethality but not malformation ??,
Methylnitrosourea, cyproterone?malformation
34
(No Transcript)
35
3. Implantation ?? ?6-13days 4.
Gastrulation-?????, ?3?
?????????????????????
36

• 5. Organogenesis ????,?3-8?
• ??????????,????
• Cell proliferation
• Cell migration
• Cell-cell interactions
• Morphogenetic tissue remodeling

37
6. Fetal period??? ?8wk-birth ?????,??????????,??
??????????????????????????????????,?????,?????????
???????????? ????????????????????deformation,????
?malformation
38
Critical periods of sensitivity for induction of
various defect by retinoic acid in the hamster
39
Dose-response Patterns and the threshold concept
40
Mechanisms and pathologenesis of developmental
toxicology

• Mutations
• Chromosomal breaks
• Altered mitosis
• Altered nucleic acid integrity or function
• Diminished supplies or precursors of substrates
• Decreased energy supplies
• Altered membrane characteristics
• Osmolar imbalance
• Enzyme inhibition

41
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
42
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
43
(No Transcript)
44
Cell death in the neural tube by CP
Sensitivity to CP-induced cell death Neuroepitheli
um gtheart Cell cycle length 9.5 hr vs 13.4 hr
(longer Go/G1)
45
(No Transcript)
46
(No Transcript)
47
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
48
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-acetylaminofluo
rene (proteratogen) 7-hydroxyl
metabolites(proximate teratogen) 4.Maternal
metabolism of xenobiotics 2-methoxyethanol
2-methoxyacetic acid
49

• 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

50

• Placental toxicity
• Metals, Cd, As, Hg, ethanol, cocaine, cigaratte,
  sodium salicylate
• Maternal injection vs fetal injection of Cd
• Production of metallothionein
• Interaction with Zn

51

• 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

52
(No Transcript)
53
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.
54
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
55
(No Transcript)
56
Fetal Basis and Transgenerational Transmission of
Reduced Fertility
Endocrine Disruptor

F0
F1
F2
F3
F4
Vinclozolin (antiandrogenic fungicide)
Methoxychlor (estrogenic pesticide)
F0gestating mother F11st generation
57
Environmental Epigenetics
Anway. Science 2005 3081466
58
Decreased spermatogenic capacity and decreased
fertility ..as well as increased prevalence of
other diseases transferred via MALE germ line
59
Summary

• A transient embryonic exposure to endocrine
  disruptors at the time of gonadal sex
  determination can cause epigenetic
  transgenerational disease state of subfertility
  and spermatogenic defects in F1 through F4
  generations
• Transgenerational disease phenotype was primarily
  transmitted through the male germ line
• Exposure appears to have caused an epigenetic
  reprogramming of the germ cell line that is
  permanent and transferred transgenerationally
  to subsequent generations

60
Modern safety assessment

• Regulatory guidelines for in vivo testing
• Multigeneration tests
• Childrens health and the food quality protection
  act
• Tenfold safety factor for children
• Alternative testing strategies
• Epidemiology
• Concordance of data (among species)
• Elements of risk assessment
• use-in pregnancy rating A, B, C, D, X

61
In Vivo Regulatory Protocol Guideline
62
The 17 intercellular signaling pathways by most
metazoans

• Early development and later
• 1. Wnt pathway
• 2. Receptor serine/threonine kinase (TGFb)
  pathway
• 3. Hedgehog pathway
• 4. Receptor tyrosine kinase (small G proteins)
  pathway
• 5. Notch/Delta pathway
• Mid-development and later
• 6. Cytokine receptor (cytoplasmic tyrosine
  kinases)
• pathway
• 7. IL1/Toll NFkB pathway
• 8. Nuclear hormone receptor pathway
• 9. Apoptosis pathway
• 10. Receptor phosphotyrosine phosphatase pathway
• Larval/adult physiology
• 11. Receptor guanylate cyclase pathway
• 12. Nitric oxide receptor pathway
• 13. G-protein coupled receptor (large G proteins)
  pathway
• 14. Integrin pathway
• 15. Cadherin pathway

63
Sonic Hedge-hog signal pathway
Cholesterol synthesis inhibitor
cyclopamine jervine
Holoprosencephaly
64
(No Transcript)
65
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

66
(No Transcript)
67
(No Transcript)
68
Homework

• 1. Describe the possible mechanisms for
  teratogenic effects of the following chemicals.
• a. aminoglycosides
• b. ethylene oxide
• c. captopril
• d. danazol
• e. aminopterin
• f. Accutane