Historical Aspects
Malformed children have been treated as the result of witchcraft with mother and child being killed or sex with animals or cohabitating with demons. It was not that many years ago that abnormalities were thought to be caused by sights, sounds, or experiences of the mother when pregnant. One should listen to good music, not look at nasty things.
The first observations of any note occurred in Germany in the 1930's when a physician noted that there was a relationship between rubella (German measles) and cataract in the newborn. Up until that time, it was thought the the placental barrier was perfect and the child was protected by the separated blood supplies. It was also found that the extent of malformations depended upon the time during gestation that the disease occurred with 50% of children being affected during the first month, 30% the second month, and 10% and 5% the third and fourth months. Rubella can still affect the child after birth
Some diseases are transmitted at birth as syphilis (a bacterium). This is the reason that newborn eyes are treated after birth. Herpes simplex can cause encephalitis when a child is exposed going through the birth canal if the mother has an active infection and often Caesarian births are recommended.
Radiation was recognized as a teratogen before 1950. Chemicals were not recognized as a possible cause until after 1950 even though aminopterin which was used as an abortifacient and when unsuccessful was known to cause malformations in humans and a number of chemicals were recognized as teratogens in animals.
Thalidomide changed all this. Thalidomide was developed by a German pharmaceutical company for the relief of nausea, tension, sleeplessness and was advertised as having no side effects and was advertised especially for pregnant women. Between 1959 and 1962 8000 babies were born with malformations which included no arms, no legs, paralyzed faces, and death in early infancy. It was also found to cause nerve damage in some adults.
Its mechanism of action is just now being discovered. It interferes with the chemicals released by rapidly growing cells that induces blood vessels to grow into new tissue. It is now being tested as a tumor reducing drug and cancer treatment.
It was never approved for distribution in the U.S. because Frances Kelsey at the FDA refused to approve a license until tests were done on pregnant animals especially as it was being touted for pregnant women. There was a great deal of political pressure to approve the drug. Few babies in the US were affected and those that were got the drug in trials for the licensing application or from Canada or Mexico. The drug company, Chemie Gruenenthal, pushed the use in pregnancy because they came to know that long term use in adults would lead to nerve damage and one gets over pregnancy. The company also tried to silence physicians who were reporting damage to adults.
Since Thalidomide, numerous chemicals have been shown to cause birth defects. It has been found frustrating to use animal studies to predict human teratogenicity. There is great variation in animals and the dose and timing of exposure is very important.
The following chemicals are known to cause birth defects:
Rubella 1930's
Radiation 1930's
Toxoplasmosis 1950's
Aminopterin 1952
Androgens 1961
Methylmercury 1960's
Thalidomide 1960's
Warfarin 1968
PCB's 1970's
Smoking 1970's
Alcohol 1974
DES 1974
Diphenhydantoin (Dilantin) 1970's
Methadone 1980's
Valproic acid 1981
Acutane 1983
Vitamin A derivatives
LeadThe following are strongly suspected, but not yet proven:
Quinine
Amphetamines
Hypoglycemics
Insulin
Tranquilizers
Cocaine
Aspirin
Marijuana
Cadmium
Dioxin
Barbiturates
Narcotics
Basic Principles of Teratology
A few chemicals interfere with such basic development that they cause defects in all animals. There are genes that are highly conserved and have been found in fruit files and humans. One group of these are called homeobox genes that deal with limb development and bilateral symmetry. Then also there are animals that are limbless or from simple appendages to the human hand. There are obviously differences in the genes and processes.
A fetal liver is like able to metabolize chemicals than the mother's. This means that concentrations that do not affect the mother can affect the fetus. On the other hand some chemicals require metabolism by the liver to be transformed into teratogens.
A fetus has essentially no fat stores so whereas the mother may rid herself of a chemical by storing it in fat, a fetus can not do that.
A teratogen usually must be able to cross the placenta and it is somewhat of a barrier. Although some effects of smoking my be a depletion of fetal oxygen by depleting maternal oxygen.
Sensitivities vary between species, between strains within species, and even litter mates. Mothers of one spinal bifida infant have 10X the background risk to produce another. However 95% of all cases are born to women with no history of spinal bifida in their families showing a strong environmental component also.
Incidence per 100,000 births Population Spinal bifida Anencephaly American Indians British Columbia 24 8 US 74 30 Negroes Pretoria 110 50 US 53 40 Caucasians Belfast 426 440 New York 117 80 Hawaii 100 60 Japanese Japan 21 60 Hawaii 64 50 Hawaiians Caucasians 100 60 Japanese 64 50 Relative sensitivities of Species to Induction of Structural Malformations by Thalidomide.
Species Smallest Dose Inducing
Malformations (mg/kg/day)Largest Dose Not Producing
Malformations (mg/kg/day)Human > 1.0 ? Monkey 5 -- Rabbit 30 50 Mouse 31 4000 Rat 50 4000 Armadillo 100 -- Dog 100 200 Hamster 350 8000 Cat --- 500 There are other influences of environment. Down's Syndrome is completely genetic. It is due to an extra chromosome 21. Even so, cases increase with the age of the mother or age of the egg that is fertilized.
Cases of malformations involve about 2% of human births, but can vary with time as changing environmental factors. From 1945-1959 Scotland went from
Cases per 100,000 from 1945-1959
19451959 Anencephalus Scotland
200 300 New York 150 50 Spina bifida Scotland 175 150 New York 175
75 Hydrocephalus Scotland 275 150 New York 150 50 Manifestations of Developmental Toxicology
1. Death - reduced fertility or infertility
2. Malformation
3. Growth retardation - small birth weight
4. Functional deficit - mental retardation, dyslexia, motor dysfunction (clumsiness)
Increasing dose usually increases malformations until death obscures this result. But it depends upon the seriousness of the malformation and survival of offspring.
The fungicide Caftan causes no malformations in hamster fetuses at levels that kill 50% of fetuses.
Retinoic acid causes no prenatal mortality at doses that kill 50% post natally.
Thresholds
Structural Teratogenesis is a threshold phenomenon. There is argument about thresholds with lead and radiation, but none for organic chemicals. During development, there are cells that can take the place of damaged cells as this happens normally. In some tissues (nervous), we produce many more cells than we need and then those that don't hook-up die. Some cells are programed to die as the diminishing tail of a tadpole. This natural cell death is called apoptosis.
The very nasty teratogens are much more toxic to the fetus than to the mother so that the mother has no symptoms. Thalidomide and Dilantin are in this category.
Most malformations in the human fetus occur in the first 7 weeks of gestation when the organs are being formed. At these times a mother may not know that she is pregnant and know to take precautions.
Susceptibility varies with age
For the first few cell divisions, each cell is totipotent which means that if it is moved around it can take over the specializations of its new position. As time goes on, the cells become more specialized and limited as to what they can become. Chemical gradients determine this specialization and the association between cells and the induction of the cornea by an outpouching of the brain, etc. There are so many communications by chemical concentration that one wonders how any embryo develops normally.
In rats, 100-rad radiation on day 8 of gestation kills 100% of embryos, one day 10, 75% are deformed, on day 11 no deformations..
Three major periods are
1. Preimplantation when cells are totipotent. Any damage here is usually lethal
2. Organogenesis and early differentiation. Very rapid growth and differentiation. of most organ systems. The rapid growth is susceptible to nutrient loss and since more cells are present, not as susceptible to death. This is when most structural teratogens act.
3. Fetal period. Characterized by growth and maturation. more susceptible to functional deficits. Growth retardation can occur here. Methyl mercury, alcohol, cigarette smoking, sulfa drugs act during the last trimester.
Mechanisms
In rats, there are 10 mitoses between days 8 and 10. This produces N x 210 cells. There is a cell division every 5 hours. This is also the time of most inductions. So tissues need to grow enough to come into mutual contact for induction. (If the ureter does not reach the kidney, the kidney does not develop. If it does reach the kidney, its size depends upon the amount of branching of the ureter.
Lack of nutrients can lead to too slow growth as can lack of energy. In late stages, it may just make a small fetus, in early stages it can lead to malformations. These functions are time dependent as:
limbs on day 10-12
palate on day 12
Identifying Human Developmental Toxins
We use a two species test currently. Usually rat and rabbit. The requirements are a rodent and non-rodent. There is no animal that has human development except humans and animal tests only work for teratogens that hit developmental sites we have in common. Even the difference between rat and mouse does not one to predict consequences in the other.
Even new chemicals that are negative in pre-market screening should be followed for several years after exposure to humans. A central registry independent of manufacturer is essential. This type of surveillance has caught two teratogens.
In practice all teratogens, but 2, have been identified by alert physicians. Only smoking and the antisiezure valprioic acid were discovered by surveillance
Alcohol was declared a teratogen 10 years after Thalidomide for fetal alcohol syndrome. What would have happened if thalidomide caused a decrease in IQ of 10 points? We would never know.
Acutane was discovered before is was licensed.
The herbicide nitrofen was marketed for 10 years before it was discovered as a teratogen. Premarket tests proved negative. It causes no maternal morbidity or prenatal mortality at doses that cause 50% postnatal fatality.
Teratology Testing
We need to test for "any chemical, given at the right time in the right dose to the right species that causes malformations. Thalidomide is a potent teratogen in humans, but not in rodents or chickens. Cortisone is a potent teratogen in mice and rats, but not in humans.
Testing is designed with Thalidomide in mind and interpreted with cortisone in mind. Or regulations are based on the fear that one might miss an important human teratogen, but with the realization that there are 700 known animal teratogens and only 30 are known for humans.
Usually mice, rats and rabbits are the test animals. Regulations require testing in two species, one of which is a non-rodent. Primates may be used in situations where exposure of pregnant women is expected. But the expense means that few animals will be tested.
There is no non-mammalian developmental assay that is reliable. Tissue and embryo culture may be useful for determining mechanisms, but their costs and expertise of labor make them expensive and they are not reliable for screening. Mammalian in vivo remain the most reliable for human safety studies.
In Vivo Studies
Sample size - usually 20 pregnant females per dose and control. One must have enough females to test whether the chemical is reducing fertility.
Dose selection - One dose should cause female morbidity. One should cause no apparent effect on fetus or dam. The third dose is placed logarithmically between the other 2 doses.
Controls must be concurrent
Route of administration - should be the same as exposure route to humans.
Multigeneration test - for long term exposure or bioaccumulation studies. Should be used for any drug used therapeutically during pregnancy as shown by the results of DES use.
Each group of animals is exposed at its dosage in the food.
Fo generation is exposed usually in food, bred after 60 days of diet 1 st Mating 2nd Mating F1a Generation F1a Generation Necropsy at weaning Select F1 Parental Animals F2a Necropsy at weaning F2b Select F2 parental animals F3a - Necropsy at weaning F3b Necropsy at weaning Histopathology One could collect the following information in such a study:
Fertility index = pregnancies / mating X 100
Gestation index = liveborn / litter
Sex ratio at ages 1,4,7,14,21 days
Weaning index = number alive day 21 / number alive day 4
Growth index = mean weights of two generations of male and female offspring at days 1,4,7,14,21This test could have caught DES a second-generation reproductive poison, bioaccumulative chemicals, post-natally lethal agents.
This assay Takes more than 3 generations, is expensive (>$500,000 per species), requires tedious, expensive labor.
There are other modifications of this scheme to shorten the time period. One major teratogen has been introduced into commerce since developmental testing, Acutane and its effects were known at the time of introduction
Even these tests may not pick up all human teratogens.
DES not only causes cancer much later in the offspring, but also birth defects in the F2 generation, an abnormality that is diagnosed long after birth.
Reproductive Toxicity in Natural Populations
Chemical types of interest are: pesticides, metals, long-lived lipophilic chemicals as PCB's and hexachlorobenzenes. Even after identification, chemicals can remain for long periods in the environment.
One must use uncontrolled experiments and work backwards to the causes.
Varied insensitivity to laboratory animals is a problem
Fish are often used for testing as eggs are plentiful at hatcheries
Chick embryos are often used as they are plentiful at all times of year.
It is impossible to study all the species and there is no reason to believe that they are not at least as variable in their responses as mammals.