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)