Developmental toxicology Structural malformations Growth

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

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35-40 /4100 chemicals alter prenatal development,
Table 10-1
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Thalidomide
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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 It was later (1961) withdrawn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.Symptomsmalformed 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.
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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
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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.
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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

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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 smokers 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.
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• 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.

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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.
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Retinoic Acid
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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.
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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.
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Retinoid Actions in vivo

• Myeloid differentiation
• Epithelial growth keratinocytes
• Embryo development
• Anti-oxidants

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Retinoid Therapies
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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
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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

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Hormonal Targeting of Nuclear Complexes to
Chromatin
SIGMA-ALDRICH.com/rbi
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RAR and RXR types
Summarized from Chambon, FASEB J., 1996
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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).
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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.
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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.
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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.
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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.
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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.
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• 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.

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Principle of developmental toxicology
1.  genetic influences-??????,??????????????? 2. 
critical periods-??????????????? 3.  initiating
mechanism-??????????????????????????? 4.  access
to embryo and fetus-????????????????? 5. 
abnormal development-??????????death,
malformation, growth retardation, and functional
disorder 6.  dose-response relationship-however,??
?????threshold level Attribution of threshold 1.
high restorative growth potential of mammalian
embryo 2. cellular homeostatic mechanisms 3.
maternal metabolic defenses
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• 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
• ???6hr??ethylene oxide, ethylmethane sulfonate,
  ethylnitrosourea?malformed fetus

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• 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

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• 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 gt 100 imprinted genes in
  clusters
• Imprinted due to selective methylation of one of
  the alleles

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2.Preimplantation???? (blastocyst)
????,?????1000???,?3?????????,???????????,?????,??
?????????????,???????? DDT, nicotine,
methylmethane?body and/or brain weight deficits
and embryo lethality but not malformation ??,
Methylnitrosourea, cyproterone?malformation
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3. Implantation ?? ?6-13days 4.
Gastrulation-?????, ?3?
?????????????????????
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• 5. Organogenesis ????,?3-8?
• ??????????,????
• Cell proliferation
• Cell migration
• Cell-cell interactions
• Morphogenetic tissue remodeling

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6. Fetal period??? ?8wk-birth ?????,??????????,??
??????????????????????????????????,?????,?????????
???????????? ????????????????????deformation,????
?malformation
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Dose-response Patterns and the threshold concept
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6 Principles of Teratology
1.  genetic influences-??????,??????????????? 2. 
critical periods-??????????????? 3.  initiating
mechanism-??????????????????????????? 4.  access
to embryo and fetus-????????????????? 5. 
abnormal development-??????????death,
malformation, growth retardation, and functional
disorder 6.  dose-response relationship-however,??
?????threshold level
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Mechanisms and pathologenesis of developmental
toxicology 1.   mutation ?? somatic mutation in
the early embryo, ex.mutagen 2.   chromosomal
abnormalities ????? ex. advanced maternal age,
viral infection, irradiation, and chemical
agents 3.   mitotic interference?????? slow or
arrest DNA synthesis (hydroxyurea or
irradiation), interfere with spindle formation
(colchicine, vincristine) 4.   interference with
nucleic acid function??????? including
replication , transcription, translation ex.
antibiotics and antineoplastic drugs 5.  
nutritional deficiencies???? ex. vitamins ,
minerals  
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6. deficient or alter energy supply
?????????? 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?????? ex. hypoxia, trypan blue,
hypertonic solutions, adrenal hormone?edema,
hematoma, and blisters 8.   changes in cell
membranes?????? ex. solvent, vitamin A 9.  
enzyme inhibition????? ??????,DNA repairing,
polymerase
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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
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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
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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)
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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
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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
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Maternal factors affecting development Genetics h
igh incidence of cleft lip/palate in white
mother Disease-chronic hypertension diabetes inf
ection-cytomegalovirus, Taoxoplasma
gondii Hyperthermia-CNS malformation Nutrition-fo
late neural tube defect Stress-noise,
restraint Placenta toxicity -46 toxicants, Cd
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• placental toxicity
• Metals, Cd, As, Hg, ethanol, cocaine, cigratte,
  sodium salicylate
• Maternal injection vs fetal injection of Cd
• Production of metallothionein
• Interaction with Zn

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• 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

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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.
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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
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Modern safety assessment Regulatory guidelines
for in vivo testing Multigeneration
tests Childrens health and the food quality
protection act Alternative testing
strategies Epidemiology Concordance of
data Elements of risk assessment use-in
pregnancy ratingA, B, C, D, X
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• 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

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Tier I screening battery for EDS In
vitroEstrogen receptor binding or
transcriptional activation assay Androgen
receptor binding or transcriptional activation
assay Steroidogenesis assay using minced
testis In vivoRodent 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
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Summary of in vivo regulatory protocol guidelines
for evaluation of developmental toxicity
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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 (-)

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Sonic Hedge-hog signal pathway
Cholesterol synthesis inhibitor
cyclopamine jervine
Holoprosencephaly
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Birth Defect Prevention Measures

• Folate supplementation
• Healthy lifestyle
• Genetic counseling diagnostic testing

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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

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