In this article we will discuss about:- 1. Causes of Teratogenicity 2. Stage Sensitivity for Teratogenicity 3. Mode of Action of Teratogens 4. Administration of Teratogenic Agent 5. Evaluation of Teratogenic Effects.
Causes of Teratogenicity:
The toxicants which cause teratogenesis are known as teratogenic agents.
A gestating-embryo exhibits great dynamicity of the living cells. The embryonic cells multiply and differentiate at a tremendous rate making the embryo more susceptible to the drugs.
Stage Sensitivity for Teratogenicity:
i. Pre-Differentiation Stage:
During this stage the embryo is not susceptible to teratogenic agents. These agents either cause death to the embryo by killing all or most of the cells, or have no apparent effect on the embryo. Even when some widely harmful effects have been produced, the surviving cells can compensate and form a normal embryo. This resistant stage varies from 5-9 days depending on the species.
ii. Embryonic Stage:
In fact this is the period when the cells undergo intensive differentiation, mobilization and organization. It is during this period that most of the organogenesis takes place. As a result, the embryo becomes most susceptible to the effects of various teratogens.
This period generally ends sometimes from the 10th-14th day in rodents and in the 14th week of the gestation period in humans. All organs are, however, not susceptible in the same period of the pregnancy. Rat embryo is most susceptible between days 8 and 12 for most organs, but the palate and urinogenital organs are more susceptible at a later stage for teratogens.
J. G. Wilson (1965) observed teratogenic treatment on the 10th day of gestation which resulted in the following incidences of malformations in rat:
Brain defects – 35%
Eye defects – 33%
Heart defects – 24%
Skeletal defects – 18%
Urinogential defects – 6%
iii. Fetal Stage:
This stage is characterized by growth and functional maturation. Teratogens are thus unlikely to cause morphological defects during this stage, but they may induce functional abnormalities. Whereas, morphologic defects are, in general, readily detected at birth or shortly thereafter functional abnormalities, viz., CNS impairment, may not be diagnosed for some time even after birth.
Mode of Action of Teratogens:
Various mechanisms are involved in teratogenic effects:
i. Interference with Nucleic Acids:
Various teratogenic agents interfere with nucleic acid replication, transcription, or RNA translation. These include alkylating agents, antimetabolites, intercalating agents and amino acid antagonists.
ii. Inhibition of Enzymes:
Inhibitors of enzymes, e.g. 5-flourouracil, may induce malformation through interference with differentiation or growth by inhibiting thymidylate synthatase. Other examples include 6-aminonicotinamide, which inhibits glucose-6-phosphate dehydrogenase, and folate antagonists which inhibit dihydrofolate reductase.
iii. Deficiency of Energy Supply and Osmolarity:
Certain teratogens can affect the energy supply for the metabolism by restricting the availability of substrates either directly (e.g., dietary deficiencies) or through the presence of analogs for antagonists of vitamins, essential amino acids, and others.
In addition, hypoxia and agents i.e., CO and CO2, can be teratogenic by depriving the metabolic process of the required O2 and probably also by the production of osmolar imbalances. These can induce edema, which, in turn, cause mechanical distortion and tissue ischemia. Physical agents that can cause malformations include radiation, hypothermia, hyperthermia and mechanical trauma.
It shall not be out of place to mention that the mode of action of many teratogens is yet uncertain. Furthermore, a potential teratogen may or may not exert teratogenic effects depending on such factors as bio-activating mechanism, stability and detoxifying capability of the embryonic tissues. Appropriate experimental testing for the teratogenicity of toxicants is, therefore, essential.
Animals:
For teratogenic tests, the animals should be young, mature and healthy. Usually, Prima gravida females are preferred. Rats, rabbits and hamsters are the commonly used animals, because of their ready availability, easy handling, little size and short gestational period. Pigs, are sometimes also used because they are phylogenetically more similar to humans. WHO (1967) suggested the use of nonhuman primates because of their phylogenetic proximity to humans. Other animals such as dogs and cats have also been used by some investigators.
With rats and rabbits, at least 20 and 12 females, respectively, are placed in each dose group of teratogenic agent. Smaller numbers of large animals such as dogs and nonhuman primates are also used.
Administration of Teratogenic Agent:
Dosage:
At least three dosages are usually used. The lowest dosage should be approximately interspersed between the two extremes.
In addition, two control groups are included. One of these is given the vehicle or physiologic saline and the other receives a substance of known teratogenic activity. These groups provide information on the incidence of spontaneous malformation and the sensitivity of the specific lot of animals under the existing experimental conditions. In addition to these contemporary controls, data from historical controls are also useful.
Route and Timing:
The test compounds should be administered through route that stimulates the human exposure situations. For food additives and contaminants, the chemical is preferably incorporated in the animal feeds. Oral drugs are generally administered by gastric gavage.
The timing of administering the substance is of great importance. For routine teratologic studies, it is customary to administer the substance during the entire period of organogenesis when the embryo is most susceptible. This period varies from one species to another.
The Pregnant Animals:
The animals should be examined daily for gross signs of toxicity and many females that show signs of impending abortion or premature delivery (e.g., vaginal bleeding) should be examined.
The Fetuses:
Fetuses are usually surgically removed from the mother about one day prior to the expected delivery. This procedure is intended to avoid cannibalism and permit counting of resorption sites and dead fetuses.
Following observations are to be made and recorded:
i. Number of corpora lutea
ii. Number and position of implantations
iii. Number and position of resorptions
iv. Number and position of dead fetuses
v. Number and position of live fetuses
vi. Sex of each live fetus
vii. Weight of each live fetus
viii. Length of each live fetus, and
ix. Abnormalities of each fetus.
To determine the different types of abnormalities, each fetus is examined for external defects. In addition, about 2/3rd of random sampled fetuses are closely examined for skeletal abnormalities after staining with Alizarin Red. The remaining one-third of the fetuses are examined for visceral defects after fixations in Bowin’s fluid and sectioned by microtome. With larger animals, e.g., dogs, pigs, and non-human primates, the skeletal structure is generally examined with X-ray instead of staining.
With toxicants that are suspected of having effects on the central nervous system or genitourinary system, a sufficient number of pregnant females are allowed to deliver their pups. These pups are nursed either by their biological mothers — thus possibly being exposed to the toxicants via the milk — or by foster mothers. In the latter case, the potential effects of postnatal exposure are eliminated.
Neuromotor and behavioural tests may be applied to detect CNS effects. These include posture, mother activity, coordination, endurance, vision, hearing, learning ability, response to foreign environment, mating behaviour and maternal behaviour.
Evaluation of Teratogenic Effects:
Categories and Relative Significance:
Aberrations:
In addition to functional abnormalities, morphologic defects may involve external/or internal structure. Not all types of aberrations have the same significance. For example, supernumerary ribs decrease, or abnormal sternal ossification might have little or no visible effect on external morphology, functional activity, or survival of the fetus. These have been considered as deviations.
Malformations of doubtful significance include curly tail, straight legs, malrotated limbs and paws, wrist drop, protruding tongue, enlarged atria and/or ventricles, abnormal renal pelvic development, and translucent skin. In general, these have been characterized as minor anomalies. There are, at the other extreme, major malformations that are incompatible with survival, growth, development, fertility, and longevity, e.g., Spina bifda, hydrocephalus.
In practice, the distinction between these categories is not always clear cut. It is then necessary to take other factors into consideration:
(i) Resorption:
This is a manifestation of death of the conceptus. Although the site of resorption can be readily identified with a close examination of the uterus; the number of resorptions is more reliably determined by subtracting the total near term offsprings from the total implantations, as indicated by the number of corpora lutea.
If there is an appreciable increase in the number of resorptions in the treated group, it may be necessary to alter the testing procedure to differentiated embryotoxicity from teratogenicity, e.g. by lowering the dose used to reduce the toxicity or shorten the exposure period.
(ii) Fetal Toxicity:
This may manifest as reduced body weight on non-viable fetus. This type of data is often useful in assessing the teratogenicity of the toxicant in question. With rabbits, the viability of fetus, if in question, may be determined by incubating it for 24 hours.
i. The animals used may exhibit an excessive number of spontaneous malformation or may be resistant to teratogenic effects. These errors can usually be assessed by the response of the animals to the negative and positive control agents.
ii. Poor animal husbandry and mishandling of the animals may also result in an increased incidence of malformations.
iii. The food consumption can.be affected by the toxicant used. This fact may then alter the body weight of the mothers and indirectly affect the fetuses.
iv. Excessively large doses can result in many resorptions but few or no malformations. On the other hand, if the doses are too small, there may not be any evidence of teratogenicity.
In comparing the treated and control groups, the proper experimental unit is the litter rather than the individual fetuses. The number of litters with malformed fetuses, resorptions, or dead fetuses are the parameters to be used in statistical analysis. However, an increase in the average number of fetuses with defects per litter may provide evidence of teratogenicity.
If the results indicate a relationship between the doses and the response (incidence of malformation), it is generally justifiable to conclude that the agent is teratogenic under the specific experimental conditions.
When the incidence of malformation does not provide a definite conclusion, an analysis of the data from the historical controls may be valuable. Furthermore, a close examination of the data on other parameters of the fetus and on the mother is sometimes useful.
The results obtained in teratogenesis studies in animals cannot be readily extrapolated to humans. The lack of a suitable animal model is evidenced by the fact that the most potent human teratogen, thalidomide, which is effective at a dose of 0.5 -1.0 mg/kg, has no teratogenic effect in rats and mice at 4,000 mg/kg. Only moderate embyropathy is noted in rabbits. On the other hand, acetylsalicylic acid has a long history of safe use in human pregnancy but is a potent teratogen in rat, mice and hamsters.
The mechanism of teratogenesis and the differences in response among various species of animals are poorly understood. The cause of spontaneous congenital malformations in humans are unknown. More basic animal studies and prospective epidemiologic studies are essentially required.
Nevertheless, since all chemicals that are teratogenic in humans were found to be active in certain animals as well, it is, therefore, prudent to carry out appropriate animal tests on all chemicals to which females of child-bearing age are usually exposed.
If positive results are achieved with a substance — especially this is so in more than one species of animal — exposure of females of childbearing age to this substance should be avoided, if possible. In assessing the teratogenic effects of a chemical, not only the incidence but also the severity of the aberrations should be taken into account.
Actually, these tests are not in routine use as yet, though they may show the mode of actions of teratogens. Some of these tests are — cell culture, organ culture, etc.