Transcript Developmental Toxicology

Developmental Toxicology
4 manifestations of developmental
toxicity
•Structural malformations
•Growth retardation
•Functional impairment
•Death of the organism
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”
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•Environmental conditions (1200)
•Maternal nutritional deficiencies (1930)
•Rubella virus infection (1941)
•Thalidomide (1961)
Adverse Outcomes in Pregnancy
1. 57% of pregnancies detected by hCG at 8-9 days
after fertilization do not develop as clinically
detectable pregnancies
2. 15-20% of recognisable pregnancies end in
spontaneous abortion – 90% in the first trimester
3. 2% of pregnancies end in miscarriage >20 weeks.
4. 2-3% of newborn have a major malformation
severe enough to require hospitalisation.
Cause of human birth defects
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15-25% genetic cause
4% maternal conditions
3% maternal infections
1-2 deformation (mechanical problem)
<1% chemical and environmental
influences
• 65% unknown etiology
Chemical teratogenicity
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>4100 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)
Pregnancy Risk Categories
From: A Textbook of
Psychopharmacology. Ed. by
Schatzberg and Nemeroff. American
Psychiatric Publishing Company, 2004.
Therapeutic Drugs Teratogenic to Humans
• Anticonvulsants
– Phenytoin, primidone, trimethadione, valproic acid,
carbamazepine
• Anticancer agents
– Alkylating agents –busulfan, cyclophosphamide,
chlorambucil, mechlorethamine
– Antimetabolites-aminopterin, methotrexate, cytarabine
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Androgenic hormones-danazol
Coumarin anticoagulants-warfarin
Retinoids-accutane, isotretinoin, etretinate, acitretin
Antihyperlipidemic agents-lovastatin, atorvastatin
• Other drugs-diethystilbestrol, thalidomide, penicillamine,
lithium, fluconazole, misoprostol
Thalidomide
Thalidomide
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Susceptible period- 20-36 days after fertilization
Proposed mechanisms (>24)
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.
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.
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
Tobacco smoke
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Spontaneous abortions
Perinatal deaths
Lower birth weight
Increased risk of
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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
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
Retinoic Acid
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, 7001000 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.
Retinoids
• Malformations of the face, limbs, heart,
CNS, and skeleton
• RXR α receptor
• Schizophrenia
Retinoid Therapies
Use
Drugs
Psoriasis
Tazartene (Zorac),
Etritinate (Tegison)
Adapalene (Differin),
Tretinoin (Renova),
Isotretinoin (Accutane)
Tretinoin/ATRA (Vesanoid)
Acne
Leukemia
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
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).
Valproic Acid
• spina bifida with menigomyelocele or
menigocele
• The proposed mechanism of action is that
valproic acid influences folate metabolism
Angiotensin Converting enzyme
inhibitors and angiotensin antagonists
• 2-3 trimester
• related reduced amniotic fluid volume and
impaired fetal renal function
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Oligohydromnios
Fetal growth retardation
Pulmonary hypoplasia
Renal failure
Hypotension
Death
• First trimester
– Congenital malformation
Wilson’s General Principles of Teratology (Table 10-2)
1. Susceptibility to teratogenesis depends on the genotype of
the conceptus and the manner in which this interacts with
environmental factors.
2. Susceptibility to teratogenic agents varies with the
developmental stage at the time of exposure.
3. Teratogenic agents act in specific ways (mechanisms) on
developing cells and tissues to initiate abnormal
embryogenesis (pathogenesis).
4. The final manifestations of abnormal development are
death, malformation, growth retardation, and functional
disorder.
5. The access of adverse environmental influences to
developing tissue depends on the nature of the influences
(agent).
6. Manifestations of deviant development increase in degree
as dosage increases from the no-effect to the totally lethal
level.
Critical periods of susceptibility and
endpoints of toxicity
1. Gametogenesis and Fertilization
Mechanism unclear, may be related to
imprinting
Cytosine methylation and change in chromatin
conformation
受精後6hr暴露ethylene oxide, ethylmethane
sulfonate, ethylnitrosourea→malformed fetus
Early development: ovulation to implantation
– 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:
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•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
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 > 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
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
Blastocyst
The developing embryo becomes a
hollow ball of cells and is called a
blastocyst.
Group of cells within the hollow space forms
the inner cell mass (ICM).
develops into the embryo.
The cells around the ICM become the
extraembryonic membranes
role in implantation
supports embryo’s growth
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
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
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
Critical periods of sensitivity for induction of various
defect by retinoic acid in the hamster
Dose-response Patterns and the threshold concept
Mechanisms and pathologenesis of
developmental toxicology
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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
Example of cyclophosphamide (CP)
A teratogenic
chemotherape
utic agent
Damage to
DNA inhibit
cell cycle
progression
cell cycle
arrest too long
apoptosis
Bind to protein
Single strand DNA break
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
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)
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
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
2-methoxyacetic acid
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
Placental toxicity
• Metals, Cd, As, Hg, ethanol, cocaine, cigaratte,
sodium salicylate
• Maternal injection vs fetal injection of Cd
• Production of metallothionein
• Interaction with Zn
Maternal toxicity•
acetazolamide inhibits carbonic anhydrase
forelimb ectrodactyly
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diflunsial results in anemia
skeleton defects in rabbits
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phenytoin affects folate metabolism and
heart rates
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metallothionein synthesis inducer-urathane,
mercaptopurine, valproic acid
Zn deficiency
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.”
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
Fetal Basis and Transgenerational
Transmission of Reduced Fertility
Endocrine Disruptor
F0
F1
F2
F3
F4
Vinclozolin (antiandrogenic fungicide)
Methoxychlor (estrogenic pesticide)
F0=gestating mother
F1=1st generation
Environmental Epigenetics
Anway. Science 2005; 308:1466
Decreased spermatogenic capacity and decreased
fertility ..as well as increased prevalence of other diseases
transferred via MALE germ line
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
Modern safety assessment
• Regulatory guidelines for in vivo testing
• Multigeneration tests
• Children’s health and the food quality protection
act
– Tenfold safety factor for children
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Alternative testing strategies
Epidemiology
Concordance of data (among species)
Elements of risk assessment
use-in pregnancy rating: A, B, C, D, X
In Vivo Regulatory Protocol Guideline
The 17 intercellular signaling
pathways by most metazoans
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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
16. Gap junction pathway
17. Ligand-gated cation channel pathway
Sonic Hedge-hog signal pathway
Cholesterol
synthesis
inhibitor
cyclopamine
jervine
Holoprosencephaly
The signalling molecule
Sonic hedgehog Shh.
Homologue of Drosophila hedgehog gene
(involved in compartmentalisation of wing).
Three vertebrate homologues, Sonic hedgehog,
Indian hedgehog and Desert hedgehog.
Secreted molecules, recognised by
transmembrane receptors (e.g. Patched1,2).
Sonic hedgehog expressed first in notochord, then floor plate.
Shh misexpression in dorsal neural tube leads to ectopic floor plate and motor
neurons.
Inhibition of Shh, or gene knockout, leads to absence of floor plate and motor
neurons.
Cells ‘read’ the Shh concentration to which they are exposed. Hi [Shh]
induces floor plate. 5x lower [Shh] induces motor neurons.
Consequences of Folate Deficiency
• Result of low dietary intake, genetic error of folate
metabolism, lifestyle exposures
1. DNA Hypomethylation
− Gene overexpression, uncontrolled cell growth,
genomic instability
2. Hyperhomocysteinemia
− Excessive accumulation of Hcy
3. Base Misincorporation
− Decrease in thymine synthesis; replaced by uracil
− DNA strands prone to nicks, breaks and vulnerable
to mutagen insertion
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