TERATOGENESIS AND
MUTAGENESIS
What is teratology?
Referred to as developmental toxicology
Study of _____________________ in embryo
Congenital defects and malformations
______________ = agent which overtly causes production of congenital defects and malformations
Effects of teratogens
Death/Resorption
__________—complete absence of organ
__________—reduced size of all or part of an organ
Dysraphic anomalies—failure of apposed structures to fuse (i.e. spinal bifida)
Division failures—i.e. syndactyly, fusion of fingers
Effects of teratogens
__________—incomplete formation of a lumen (i.e. esophageal atresia)
__________—presence of organ outside its normal site (i.e. ectopic heart, with heart outside thoracic cavity)
Developmental syndromes—involves multiple, but related, anomalies (i.e. fetal alcohol syndrome)
__________—absence of limb(s)
Phocomelia—absence or the shortening of a limb root, as the distal part is relatively well formed.)
Effects of teratogens
_____________—disturbance of formation, flow, or absorption of cerebrospinal fluid that leads to its increase in volume in CNS
_____________—condition in which skull is defective with the brain exposed or extruding (e.g Anencephaly)
Cleft palate, cleft lip
Growth retardation
Mode of Action of Teratogens
Interference with ________________
Replication, transcription or RNA translation targeted
Inhibition of _______________
5-fluorouracil—inhibit thymidylate synthetase
6-aminonicotinamide—inhibit glucose-6-phosphate dehydrogenase
____________ stress
Can undergo bioactivation
Lipid peroxidation, protein oxidation and protein degradation
Example: phenytoin
____________
Mutations, chromosomal breaks, altered mitosis
PRINCIPLES OF TERATOLOGY
Principle of Teratology #1
Species and strains of organisms differ in their sensitivity to teratogens
Teratogens affect different people in different ways
As a result, not every embryo exposed to a harmful substance will be harmed by it
A developing organism’s reaction to a teratogen is thought to be influenced by her or his genetic makeup
Principle of Teratology #2
Susceptibility to teratogen varies with stage of
______________ when exposure occurs
Developmental Stages
Early _____________ (pre-implantation) - lethal or no effects
Humans 1-2 wks, rodents 1-5 days
______________ - major malformations
Human first trimester, rodent 5-14 days
Characterized by rapid growth of cells and the differentiation of most organ systems
Most of organogenesis takes place
Fetal development – minor effects
Malformations, physiological/functional defects, growth retardation
Human 12-38 wks, rodent 15-21 days
Principle of Teratology #3
Exposure to teratogens can result in a _________ of related defects (syndrome)
Congenital rubella syndrome—eyes, ears, heart and brain
Fetal alcohol syndrome—craniofacial, skeletal, heart and genitals
Refer to fetal alcohol syndrome in notes
Principle of Teratology #4
As dose of teratogen increases, developmental defects ___________ as well (leads to dose-response)
A specific amount of a teratogen is usually required before embryo or fetus is at risk of harm
Once that threshold has been crossed, the risk of impairment tends to increase with repeated exposures
Example: ___________
TERATOGENIC AGENTS
Recognized Teratogens and Timeline
1930’s—Rubella, radiation (X-rays)
1950’s—Aminopterin, androgens
1960’s—Thalidomide, methylmercury, warfarin, PCBs
1970’s—Smoking, alcohol, DES
1980’s—Methadone, valproic acid, accutane (derived from vitamin A)
Probable human teratogens—quinine, amphetamines, aspirin, dioxins, cadmium, cocaine
Thalidomide
Identified as teratogen = _______
1956—introduced in Europe as sedative drug to alleviate symptoms of __________ ___________
i.e. nausea and vomiting during first trimester of pregnancy
Sudden increase in limb deformities in Germany and England
Phocomelia, amelia, small or missing ears, heart anomalies were among effects observed in over 7,000 children born to women who took thalidomide
Thalidomide
Sensitive period between __ and __ weeks of gestation—humans
Interesting—thalidomide does not exhibit teratogenic properties in toxicity tests with mice and rats
However teratogenesis discovered in primates
Precise mechanism of toxicity is still unknown
Now being investigated for use in treating AIDS, tuberculosis and other illnesses.
From: Patil, C.R., Bhise, S.B., 2003. Re-emergence of thalidomide. Indian J. Pharm. 35, 204-212.
Diethylstilbestrol (DES)
4,4'-(1,2-diethyl-1,2-ethenediyl)bisphenol
First synthetic __________ pill
Between 1940 and 1971, over 2 million women given DES
Prescribed as pharmaceutical treatment for high-risk pregnancies
Prescribed for pregnant women after Harvard researchers erroneously concluded, without proper testing, that it helped prevent miscarriages !
Diethylstilbestrol (DES)
―Delayed‖ ___________—Why?
Unlike thalidomide, whose teratogenic effects are manifested at birth,
the delayed teratogenic actions of DES are observed in daughters, most
commonly between ages of 17 to 22, whose mothers took DES during
pregnancy
Delayed teratogenesis includes an uncommon ___________
(i.e. new growth, cancer), called clear-cell adenocarcinoma
Mechanism is unknown
Alcohol
Consumption of alcohol during pregnancy results in morphological and developments anomalies
Fetal Alcohol syndrome (FAS)
Observable effects:
_________________
____________ (i.e. small head)
____________ (i.e. small jaw)
underdeveloped philtrum (i.e. depression on upper lip)
intrauterine growth retardation
CNS dysfunction
Alcohol
Prevalence in Europe and US is 1 to 3 per 1000 live births
HOWEVER some populations with high rates of alcoholism, incidence is 20 to 250 per 1000 live births
Approximately 30 to 50% of women consuming more than 450 mL (1 pint) of whiskey per day will give birth to child with FAS
Mechanism is unknown
Vitamin A
Different forms __________________________
Pre-formed vitamin A
Found in animal products like liver, fortified cereals and vitamin tablets
__________________________
Vitamin A precursor
Converted to vitamin A in body
Found in fruits and vegetables
Picture of retinol
Vitamin A
Teratogenicity of excess vitamin A was first observed
by Cohlan: ―Excessive intake of vitamin A as a cause of
congenital anomalies in rat‖ (1953)
Involved administrating 35,000 IU of vitamin per day to
pregnant rats on days 2-16 of gestation
Fetal anomalies: exencephaly, cleft lip and/or
palate, brachygnathia, and various eye defects
Vitamin A
Around late 70’s—emergence of increased vitamin
usage
―If its good for me than more of it will be better for
me‖
Studies started to help combat this mentality
Chronic intake of vitamin A, greatly exceeding the
recommended daily allowance, can lead to hypervitaminosis
A
Vitamin A
Intakes of 20,000 IU of retinyl esters or retinol during the first trimester of pregnancy may result in (< 20 %) spontaneous abortions and birth defects (J. Am. Med. Assoc. 257:1292-1297,1987)
US RDA (recommended daily allowance) established by the Food and Drug Administration is ________IU/day
Supplementation of 8,000 IU vitamin A (as retinol/retinyl esters) per day should be considered the recommended maximum prior to or during pregnancy
Vitamin A—other studies
One report stated urinary tract malformations seen in infant of women taking 25,000 IU during first three months of pregnancy and 50,000 IU during fourth through ninth months of gestation (Bernhardt et al., 1974)
Earlier report described infant born with gross urinary tract defects in which mother had ingested 40,000 IU of vitamin A per day from the sixth through tenth weeks of pregnancy (Pilotti et al., 1965)
Vitamin A—other studies
CNS defects were present after ingestion of
150,000 IU per day during pregnancy (Stange et
al., 1978)
Cleft lip and/or palate were consistently seen in all
infants exposed to 40,000 IU/day or more (Rosa,
1986)
MUTAGENESIS
Mutations
Mutation can be defined as any heritable change
Can be distinguished by the types of cells in which they occur:
___________ mutations occur in germ cells and can be passed on to future generations
___________ mutations occur in somatic cells and cannot be transmitted to offspring
Somatic mutations early in development may affect the entire development process of the organism
Somatic mutations may result in unusual growth, e.g. cancer
Two categories of mutations
Spontaneous
Induced
Spontaneous mutations
Mutations that are not under the control of man
Are relatively infrequent, and occur without a known
cause
Due to biochemical mistakes during DNA
replication, or intrinsic errors in DNA repair
Induced mutations
Mutations brought about by exposure to chemicals
or various forms of radiation
Mutagen can react with DNA or RNA
What is a mutagen?
Substance or agent that causes an increase in the rate
of change in ____________
These mutations (changes) can be passed along as cell
reproduces, sometimes leading to defective cells or
cancer
Do not confuse a mutagen with a carcinogen (a
substance that causes cancer)
Mutagens may cause cancer, but not always
Mutagens
Typical mutagens are:
____________:
gamma and X-rays
ultraviolet radiation
subatomic particles (high energy electrons)
____________:
cigarette smoke
asbestos
free radicals and oxidizing agents
formaldehyde
benzene
No matter what the mutagen the end result is a chemical change in
DNA—a ______________________!
MUTATION
Microlesions Macrolesions
Base-pair substitution
Frameshift mutations
Structural changes
in chromosomes
Numerical changes
in chromosomes
Microlesions
Rearrangement of the genetic sequence
Could be one gene or just a few nucleotides
2 types:
___________________
___________________
Base-Pair Substitutions
Replacement of one DNA base by another in DNA sequence. Replacement of nucleotide bases can have several possible consequences:
__________ mutation: An amino acid residue in original protein may be replaced by different one in mutated protein
__________ mutation: The codon for an amino acid residue within original protein is changed to stop codon, which leads to a premature termination of protein non-functional
__________ mutation: The codon for an amino acid is changed, but same amino acid is still coded for (possible because some amino acids are coded for by multiple codons, for example, UGC and UGU both code for cysteine)
Frameshift Mutation
Genetic code is translated three nucleotide bases (one codon) at a time
Addition or deletion of a single base pair in middle of a coding sequence will result in out-of-frame translation of all of the downstream codons
Result in a completely different amino acid sequence, which is often prematurely truncated by stop codons (UAG,UAA,UGA) generated by reading the coding sequence out-of-frame
Normal THE BIG RED DOG RAN OUT
Missense THE BIG RAD DOG RAN OUT
Nonsense THE BIG RED
Silent THE BIG RED DOG RAN OUT
Frameshift—deletion THE BRE DDO GRA…(IG
missing)
Frameshift—insertion THE BIG RED ZDO GRA…(Z
added in)
Microlesions: Examples
Sickle Cell Anemia
A to T—in beta-globulin gene
UV radiation
T-T dimers (covalent bond)
5-Bromouracil
Base analog—mimic appearance of bases
Substitutes for T but mRNA reads it as G
Macrolesions
Large-scale mutations (____________)—changes (# or structure) at chromosome level
Chromosomal rearrangements that affect long segments of DNA during cell divisions, and misdistributions of chromosomal numbers during gamete production in meiosis.
Examples include:
duplications
deletion
inversions
translocations
Duplication
Involves insertion of an extra copy of a region of chromsome into neighboring position
Zygotes produced from gametes involving duplications are often viable and may or may not have any serious problems
Example: color vision versus thalassemia
Deletion
Result when a gene is mistakenly removed from a chromosome
Often zygotes produced by gametes involving deletions are not viable since they do not have full compliment of genes
Inversion
Inversions happen when whole region of genes on a chromosome gets flipped around
Two types:
Paracentric—centromere is not included in the inversion
Pericentric—centromere is involved in the inversion
Translocation
Movement of part of chromosome to another part of the genome
May happen with same chromosome—_______________ translocation
Other translocations involve transfer of a region of chromosome to a non homologous chromosome—________________ translocation
Example: certain types of Down syndrome involve translocations between chromosome 14 and chromosome 21
Mutagenicity tests
___________ Mutagenesis Test Ames test
___________ Mutagenesis Tests In vitro mouse lymphoma assay
___________ TestsMicronucleus test
Screening for Mutagens:
Ames Test __________-mutation test
Bacterium used is a strain of Salmonella typhimurium that caries defective (mutant) gene making it unable to synthesize __________ from ingredients in its culture medium
However, in presence of xenobiotic, mutation can be reversed (back mutation) with the gene regaining its function
These revertants are able to grow on medium lacking histidine
Filter paper containing potential
mutagen
Screening for Mutagens:
Ames Test Since many mutagens are inactive
before bioactivation, this test can be carried out with a bioactivating system (____)
_____________ system = rat liver microsomal fraction (containing MFOs) Microsome = spherical particle, not
present in the living cell, derived from pieces of ER present in homogenates of tissues or cells
MFO activity enhanced by pre-treating animal with inducing agent—i.e. 3-methylcholanthrene, phenobarbital, or PCBs
Ames Test
Use different strains (four to five) of S. typhimurium
bacteria as well as one strain of E. coli
E.coli growth involves use of ______________, not histidine
Range of concentrations of chemical to be tested
Check for growth of revertants
Growth=mutation=mutagenic
Use of positive/negative controls
Ames Test: Pros and Cons
Pros:
Rapid
Cheap
~90% effective
Cons:
Salmonella typhimurium is a prokaryote and thus not a
perfect model of the human body
False positives
False negatives (miss mutagens)