Transcript Developmental toxicology Structural malformations Growth retardation Functional impairment

Developmental toxicology
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”

•Environmental conditions (1200)
•Maternal nutritional deficiencies (1930)
•Rubella virus infection (1941)
•Thalidomide (1961)
(65%)
35-40 /4100 chemicals alter prenatal development, Table 10-1
Thalidomide
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was later (1961) withdrawn from the market once it was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.
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
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.
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
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.
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.
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.
Retinoic Acid
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, 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.
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.
Retinoid Actions in vivo
Myeloid differentiation
 Epithelial growth – keratinocytes
 Embryo development
 Anti-oxidants

Retinoid Therapies
Use
Drugs
Psoriasis
Tazartene (Zorac),
Etritinate (Tegison)
Adapalene (Differin),
Tretinoin (Renova),
Isotretinoin (Accutane)
Tretinoin/ATRA (Vesanoid)
Acne
Leukemia
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
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

Molecular biology
Target cell
RBP–ROH
Receptor
ROH–CRBP
RA–CRABP
RA
RA
Nucleus
RA–RXR
Regulated
gene
transcription
RA
= Retinoic acid
RBP
= Retinol binding
protein
CRBP
= Cellular retinol
binding protein
CRABP
= Cellular retinoic acid
binding protein
RXR, RAR = Nuclear retinoic acid
receptors
Intracellular
Extracellular
S17
= Retinol
RAR–RA
DNA
ROH
ROH
Hormonal Targeting of Nuclear Complexes to Chromatin
SIGMA-ALDRICH.com/rbi
RAR and RXR types
Gene
RAR
Major Isoforms Endogenous
Ligand
ATRA, 9-cisRA
1, 2
RAR
1, 2, 3, 4
ATRA, 9-cisRA
RAR
1, 2
ATRA, 9-cisRA
RXR
1, 2
9-cis RA
RXR
1, 2
9-cis RA
RXR
1, 2
9-cis RA
Summarized from: Chambon, FASEB J., 1996
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).
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.
Congenital Minamata Disease
Methylmercury was used in the past as a fungicide on wheat
and grains. Cases have been documented in Iraq (19711972), 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.
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.
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.
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.
The 6 Principles of Teratology
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.
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
Critical periods of susceptibility and
endpoints of toxicity
1. 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
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:
•Inactivation of genes of one X chromosome in
female mammals occurs prior to a wave of
methylation
•Implantation – a new wave of methylation
occurs


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

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
Dose-response Patterns and the threshold concept
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
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
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, 6aminonicotinamide), 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
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, cigratte,
sodium salicylate
• Maternal injection vs fetal injection of Cd
• Production of metallothionein
• Interaction with Zn
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
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
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
Impact on screening and testing programs
1. 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
Tier I screening battery for EDS
In vitro:Estrogen receptor binding or transcriptional activation
assay
Androgen receptor binding or transcriptional activation
assay
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
Summary of in vivo regulatory protocol guidelines
for evaluation of developmental toxicity
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 (-)
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.
Birth Defect Prevention Measures

Folate supplementation

Healthy lifestyle

Genetic counseling; diagnostic testing
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
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