Nothing can cause greater fear in an expectant mother than the prospectus of her unborn child being defective. In recent years, the public has become alarmed that inadvertent or careless exposure to chemicals in the environment might pre-dispose birth defects or reproductive failure. This fear and awareness is reflected by the titles of the articles such as The Poisoning of America, Love Canal, Agent Orange, dioxin, PCB's. Chemicals from a range of environmental sources have been implicated in birth defects and reproductive failure. Birth defects are known to occur in 2-3% of all births. Of these, 25% have underlying genetic causes, while 5-10% results from the influence of the teratogens. The remaining 60-65% arise from an interplay of multiple environmental agents with genetic factors. Of those birth defects caused by environmental agents, an estimated 4-6% are due to chemical. To date, only about 25% chemicals are known to be teratogenic in humans compared to over 800 in laboratory animals. It is known if this discrepancy rests with a greater  resistance of human to those agents or a failure in our ability to determine sources of teratogencity in humans. The potential danger is great and growing, however, because of the estimated 2000 new chemicals synthesized each year and introduced into the environment.

In terms of the development of a new-born; there are three periods of development during which the foetus is at risk for injury (Bozzoli etal., 1978):  fertilization and implantation, the embryonic period and the foetal period. The period of fertilization and implantation starts from conception to 17 days, through the blastocyst and early gastrula stages. An injury during this stage results in cell death, from which the aggregate of still totiopotential cells can recover and multiply, or if a lethal does is given absorption or resorption of the developing organism results. If recovery follows, no structural deformity in the embryo is seen. The embryonic period (18-55 days) is when organogenesis takes place; therefore, this period is one of extreme sensitivity to toxic agents (Monie, 1963). Within this time each organ system or morphological trait has its own critical period of development. In later stages, the foetus is less sensitive but not immune to environmental influences. During the foetal period (56 days to term) toxicity is mainly manifested as a reduction in cell size and number. Growth retardation or functional deficits (e.g. central nervous system injury or retarded development) usually result from injury during this stage of development, since growth in size is the primary process active at this time.

A theoretical curve depicting the susceptibility of the human embryo to teratogenesis. Organogenesis (about 18-60 days) marks the period of maximal sensitivity to morphological defects from teratogens. After this the incidence of anatomical defect diminishes but it is likely that minor structural effects (often very important in nervous system development, etc..) can be generated in certain instances until after birth. Defects generated during the late foetal and neonatal period are more likely to involve growth and function because these factors predominate at that time. Developing brain is still prone to injury, since its development. (e.g. myelination) is incomplete even at birth.
 
Over the years, a number of factors have been determined to play a role in the susceptibility of a foetus to the teratogenic effects of an agent. These include:

1. The time of administration of the agent during gestation, with the most sensitive period being that of organ differentiation.

1. The route of administration and dose, which involve the chemistry of the compound, its toxicity, metabolism and toxicity of its degradation products.

The unborn foetus is exposed to chemical agents by hematogenous transplacental transfer. The routes of administration to the mother, on the other hand, are by the usual routes, including inhalation, absorption by contact with the skin and mucous membranes, and most importantly ingestion.
 
1. The number of doses, with a single does being most effective in producing defects. It is assumed with the single high dose that the mother is not given the chance to adjust to the agent and to mobilize her detoxification mechanisms. These mechanisms may be protective in chronic low level exposure.

1. Species variation, most strikingly demonstrated by the thalidomide incident, in which exposure produced limb defects in man, monkeys and rabbits but not in mice and rats, on which the drug was tested. Therefore, the susceptibility to a chemical depends on the genotype of the conceptus, with different effects being seen in different species and different strains of the same species. The difficulty thus poses in the animal testing of compounds and in the interpretation and extrapolation of the results to human is obvious. In humans, racial and familial differences may even play a role in susceptibility

1. Other factors, including the extrinsic effects of maternal metabolic diseases, maternal age (the young are more likely to have defects) and stresses such as nutritional deficiencies (protein or vitamin A, E and B₂ deficiencies). Lastly, an embryo may be more susceptible than the mother to the effects of interaction between multiple agents to which they have been concurrently exposed.

Manifestation of damage to the developing organism caused by a chemical agent follows the injury or death of certain cells; this interferes with the orderly sequence of development, giving rise to morphologic aberrations. Depending on the does and stage of embryonic development, the effect may be teratogenic to foetotoxic. Foetotoxicity is usually seen at doses higher than those expected for teratogenicity; at high doses, foetotoxicity may culminate in foetal death. Although teratogenic agents are usually also foetotoxic, but not all foetotoxic agents are teratogenic.

The mechanisms by which chemicals induce teratogenic actions are in general unknown. Unlike mutagenic chemicals, which usually belong to the alkylating class, teratogens may belong to many classes of chemicals; their mechanisms of action are, therefore, different. Malformations and mutations thus cannot be equated, nor can carcinogens and teratogens. Chemical may act directly on the embryo, foetus, or neonate or indirectly via interference with material, placental, or foetal membrane function.

It has been proposed that a foetus may be more susceptible to damage by chemical exposure than an adult, depending in part on the development of the liver enzyme conjugating systems and in its greater blood brain permeability. Indeed, for a given exposure there may be no maternal signs of toxicity and the foetus may be the only victim of the toxicant. This has been verified in animal studies as well as in human accidents, for example, chronic low level exposure to methyl mercury. Since all substances cross the placenta to some extent, susceptibility to foetal damage may be related to the effective dose reaching the foetus. Some chemicals, such as methyl mercury, are preferentially concentrated in the foetus, resulting in injury to the central nervous system. In most other instances, however, the foetus is separated from maternal circulation by the placenta, which permits only a fraction of the concentration of chemical to reach the developing offspring. The placenta, therefore, serves a protective role in reducing the dose to the foetus. Transfer of a compound across the placenta decreases with increasing molecular weight, increasing electrical charge, and decreasing lipid solubility. Transfer is by simple diffusion for unbound chemicals or for those with high dissociation constants; in other cases, the mechanisms of transfer include active transport and pinocytosis. In some cases, the placenta may also be involved in biotransformation of the compound.

The foetus may also be protected by maternal homeostatic mechanisms that lower the dose reaching the foetus via detoxification by the liver (although some metabolites may be more toxic), excretion by the kidneys or in the bile, or removal of the chemical via binding to plasma proteins which are less likely to cross the placenta.  Peak concentrations in maternal circulation may also be decreased by deposition into maternal storage depots. Organochlorine pesticides and other lipid soluble compounds, for example, are deposited in their adipose tissue, which some heavy metals are deposited in bone. It has been suggested that variation in the dose of chemicals that reaches the foetus, controlled by the factors outlined above, may underline the variability in observed species susceptibility.

Much of what we know about teratology in humans comes from clinical observations and epidemiological studies. The bulk of information, however, derives from animal studies, which may or may not be true in case of humans. Unreliability of animal experimentation and the extrapolation of such results to humans can be best explained by quoting Thalidomide story. 

1. Thalidomide did not produce teratological effects in mice but was found to produce serious teratological effects on human systems. 
2. The dose response relationship may not be valid because the levels present in human systems or in the environment are to be considered in reality.
3. Effects of various toxicants is also dependent besides other, on the nutritional status of the individual and therefore, may vary from individual to individual. Indians are reported to be malnourished or underccnouccccrished, the screening of general masses for such studies, therefore, assumes significance. 
4. The prescribed acceptable daily intakes (ADIs) have been chalked out for adults may not be valid for the prenates, neonates and the developing conceptus. The study may help in elucidating foetal acceptable daily intake (FADIs) under the Indian tropical conditions.
5. Frequency of abortions and neonatal mortality rate is highest among the Indians for the reasons unknown so far. The study shall be of great importance in exploring the involvement of environmental contaminants.
6. Most of the animal experiments are designed to extrapolate the findings on human beings. But this study is proposed in such a way that we would be examining human beings, without administering them any toxic substances and following all moral ethical norms.

Death may also follow in the post-partum period due to either severe life threatening malformations or to cumulative toxicity to vital organs. Reproductive impairment in females may take the form of pre implantation  loss (damage to the blastocyst, or even damage to the zygote) or menstrual irregularities. In males, impaired fertility may take the form of decreased sperm counts, decreased motility, or abnormal morphology. In addition, reproductive failure may be manifested as an increased frequency of spontaneous abortions in unexposed females due to mutations in male gamete.

More concern is expressed about the chemicals, which persist in the environment for longer duration and individuals are easily susceptible to these chemicals through direct or indirect exposure. Organochlorine pesticides figure among those environmental chemicals which are harmful to man in some way or the other and enter our food supply as an inadvertent additives. Not only this they are resistant for their degradation.

Pesticides become pollutants when they enter our food supplies as inadvertent additives, they are present in trace amounts on grains, in our surface and ground drinking water and even in the tissues of lower animal farms. Pesticides by definition include insecticides, herbicides and fungicides. These in turn can be variously sub classified by chemical groups, such as the methyl carbonates, organophosphates, organchlorides, ureas and phenoxyacetic acids. The teratogenic capabilities of pesticides is not limited to any particular grouping, but rather transcends all groups; the same is true of pesticides and their mutagenic action. 

That pesticides were associated with reproductive failure in lower animals became apparent in the 1960s with decreased hatch ability of avian species (The Silent Spring). Since then the teratogenic action of pesticides has been confirmed and extensive listings of pesticides and their embryo toxic and teratogenic effects on various lower species have been published. Good studies and well documented cases of teratogenic effects of pesticides in humans, on the other hand, are lacking. Only the organomercurials are conclusively known to be teratogenic in humans. 

Because of their lipid solubility and resistance to degradation, the presence of organochlorine pesticides have also been well documented in human adult tissues. In 1969, Curley et al. reported that DDT, DDE, lindane, dieldrin, and heptachlorpepoxide were present in the cord blood (foetal) and tissues of 10 still born infants, and further the has also confirmed that various organochlorine pesticides are capable of crossing the so called placental barrier and reach the foetus at a considerably high concentration. A significant correlation between clinical history of pregnant mother and amount of these toxicants transported across the placenta has been documented. Pregnant women who underwent spontaneous abortion or premature labour were found to contain high amount of these environmental toxicants, as compared to controls. In a detailed study conducted in our laboratory, it was found that when statistically sound number of cases of premature labour and spontaneous abortion were compared with sizeable controls, the concentration of pesticides found in placenta and maternal blood was fund inversely proportional to the weeks of gestation. A hypothesis based on the findings has spelled out as to how the pesticides can bring about the termination of pregnancy before term. Specimens of maternal blood, placenta and umbilical cord of still births were found to contain significant amounts of  these pesticides. The foregoing facts suggest that the developing human embryo/foetus is inadvertently exposed to these chemicals. Whether these compounds receiving the foetus exert toxic or teratogenic effects is speculative. One Japanese report notes increased foetal deaths and malformations following acute high level spraying of organophosphorus insecticides in field workers.

An interesting observation by O'Leary et al. nothing several pesticide levels in premature infants, may have some implications with respect to human foetal toxicity.

Measuring DDE levels in foetal whole blood, they found that premature infants had levels elevated there to fourfold relative to control, term infants. Although these elevations could be explained by the absence of body fat to store the compound, which is common in preterm infants, a causative role of chlorinated pesticides in inducing premature deliveries cannot be ruled out. Other supporting data comes from animals studies. DeLong reported that California sea lions which delivered their pups prematurely lead an eight-fold increase in fat levels of chlorinated pesticides (DDT, DDD, DDE and PCB) relative to those which delivered pups at term. The premature pups themselves had a twofold elevation in fat levels of these compounds. Similarly, in New Zealand, in white rabbits fed DDT (50 mg/kg), a premature delivery rate of 57% was noted, with a 25% early foetal loss.

Another pesticide that gives evidence of human neonatal toxicity is hexachlorobenzene, a fungicide. Although human fetotoxicity or teratogenicity has not been established, the compounds presence in breast milk in known, and incidents of new born poisonings following breast feeding have been documented in Turkey. Skin pigmentation and a syndrome of toxicity resembling porphyria cutania tarda was described.

This present study of environmental impact of pesticides on foetal growth retardation is an effect to detect those cases in which there is growth retardation because of environmental pollution by pesticides and try to restrict their further use, in a manner in which they pose a threat to the environment.

The placenta is a temporary organ that establishes a functional union between mother and foetus. This unique organ transfers all the nutrients needed for development, eliminates foetal metabolic waste, synthesizes hormones essential to pregnancy, and carries out many other anabolic and catabolic functions.

The placenta behaves almost like a complete human body, like an animal itself. It is virtually like another body and every placenta is, of course, unique. It is far more like a human than an inbred laboratory animal, for example, a full-term placenta behaves like many different organs, therefore, we can treat it as kidneys, liver or lungs, and it corresponds accordingly. It secretes many hormones, and protects the baby from adverse reactions to any foreign chemicals if mother might be taking. So it can also yield information about immunological processes. Increasingly, people are becoming concerned about the possible effects of foreign chemicals and a placenta can tell us so much more about likely reactions that a rat or mouse even could. Relevance of animal data and their extrapolation to humans has now become a debatable subject, underlying the need to look for systems which could mimic a situation in humans. Human placenta, a discarded living tissue, may provide valuable information relating to different human systems. Various phenomena occurring in placenta, are predominant in the foetus and vice versa. This similarity may be utilized to modulate human term placenta as a suitable model to assess the foetotoxicity of pesticides. Successful demonstrations have been achieved in our laboratory to provide useful information concerning transport of amino acids and carbohydrate metabolism in human term placenta under the influence of organochlorine pesticides, having potential to cross the human placental barrier.

Nothing can cause greater fear in an expectant mother than the prospects of her unborn child being defective. In recent years, the public has become alarmed that inadvertet or careless exposure to chemicals might predispose birth defects or foetal abnormality. The awareness to these facts are well documented in publications such as "Love Canal", "Agent Orange", Dioxin, PCB's and the most famous "Silent Spring".

Incorporation of pesticides as the latest innovation in the management of pests has resulted in huge consumption of pesticides all over the globe, more so in developing countries. India, in its ambitious plan to produce more food and agricultural products, has intensified the usage of pesticides totalling 51,630 tonnes during the year 1981-82, still higher figures yet to achieve during the course of time. 

As a consequence of higher consumption of pesticides, the problem of pesticide residues was magnified during the last one decade or so. Degradability of organochlorine pesticides, biochemically and chemically is still a big question resulting into their persistence in nature and biota. Lipid susceptibility is another major factor for the residue built-up in food chain resulting in their deposition in adipose tissue of humans and animals. Pesticides as a whole and organochlorine pesticides in particular such as isomer and metabolites of DDT (Dichloro diphenyl trichloro ethane), isomers of HCH (Hexachlorocyclohexane), aldrin, dieldrin, etc. have been implicated in various toxic manifestations including carcinogenecity, mutagenecity and teratogenecity both in humans and animals. Because of these findings, the use of DDT and some developed countries. Sustained use of DDT in developing countries has posed various problems and the following points need scientific attention:

1. Indians have been reported to carry the highest body burden of pesticides that reach the developing foetus via placenta at a considerably higher concentration.
2. Rate of mis- carriage is quite high among Indians. A fairly good percentage of the aborted foetuses is malformed in some way or the other, 4-6 percent of the malformations are attributed to the environmental chemicals.
3. The developing foetus in more vulnerable to toxic insults by environmental chemicals such as pesticides.
4. There are about 25 chemicals reported to be teratogenic in man as compared to 800 chemicals reported against animals. It is unknown if this discrepancy is due to the greater resistance of humans to these agents or failure on the part of scientists to determine the sources of teratogenencity.
5. Much of what we know about teratology in humans comes from clinical observations and epidemiological studies. The information derived from animal experimentations may not be true in the case of humans. This is well exemplified by "Thalidomide Tragedy" where the compound did not produce any effect in mice but did so in humans.
6. The dose response relationship may not be valid because the levels present in human systems or the susceptible environmental levels are to be considered in reality.
7. The prescribed acceptable daily intakes (ADI values) have been chalked out for adults and may not be valid for the prenates, neonates and the developing conceptus.
8. Most of the animal experiments are designed to extrapolate the findings on humans beings. But studies involving human being are difficult to carry out specially in determining the toxicities and following all moral and ethical norms.

Age, dietetic habits and area of residence of expectant mothers were found to influence the transfer of pesticides across the placenta. Further studies revealed that pregnant mothers undergoing spontaneous abortions and premature labour had higher concentrations of OCP's in their venous blood, placenta/conceptus as compared to those delivering full-term babies. Reports have also been adduce that DDT has an oestrogenic effect. Other incriminating results have led to explore the role of OCP's in the homeostasis of pregnancy.

There are reports on the presence of organochlorine  pesticides in the tissues of skill born infants. Studies carried out in our laboratory also showed significant difference between the content of OCP's in planceta, umbilical cort and venous blood of still born and live born cases.

This was considered to be a significant finding because in beagle dogs, experimentally exposed to capsulated aldrin and DDT, the reproduction was severely affected in all the treated groups, as evidence by reduce fertility, still births, increased mortality in pups and impaired lactation. Similar results were also obtained in a separate study carried out with aldrin/dieldrin.

Since Indians have been reported to carry the highest body burden of pesticides, these toxins reach the developing foetus via placenta in a significant amount. Interestingly, the rate of miscarriage is quite high among Indians. Therefore, this study was planned to investigate the role of organochlorine pesticides on the intrauterine growth retardation (IUGR) in humans by monitoring levels of these pesticides in the venous blood, placenta and cord blood of women delivering intrauterine retarded babies and in women having normal deliveries. A correlation was also found between these pesticide levels in control as well as in IUGR cases.

The Placenta -Suitable Model For the Assessment of Foetotoxicity in Humans

Relevance of animal data and their extrapolation to humans has now become a debatable subject, underlying the need to look for systems which could mimic a situation in humans. Human placenta, a discarded living tissue, may provide valuable information relating to different human systems. The placenta behaves almost like a complete human body, like an animal itself. it is virtually like another body and every placenta is, of course, unique. It is far more like a human than an inbred laboratory animal, for example, a full-term placenta behaves like many different organs, it can be treated as kidneys, liver or lungs and it will correspond accordingly. It secretes many hormones, and protects the foetus from adverse reactions to any drug or foreign chemical. The potential of the placenta for research is enormous. Because of these properties, human placenta may be utilized as a suitable model to assess the developmental toxicity of pesticides and other environmental chemicals.

Successful demonstrations have been achieved to provide useful information concerning carbohydrate metabolism and transport of amino acids in human term placenta under the influence of organochlorine pesticides, known to cross the human placental barrier.

The placenta, like other tissues, derives most of its energy from carbohydrate and lipid substrates. The regulations of metabolism of carbohydrate and lipids in the human placenta seems of special importance, since placental functions, responsible for normal foetal growth, are dependent on adequate energy supply through glucose or lipid dissimulation. The absence of hexokinase activity in foetal liver is suggestive to the fact that an alternate mechanism to provide chemical energy to the foetus is existent in the placenta. Evidences are accumulating on the presence of fructose in foetal plasma in higher concentrations than in maternal plasma and is derived from the fructose synthesized in placenta by an alternate mechanism, the Her's-Ginsberg pathway (polyol pathway), by the reduction of glucose to sorbitol in the presence of an enzyme aldose reductase followed by conversation of sorbitol into fructose. Fructose thus, formed is further metabolized through Glycolysis and Tricarboxylic acid cycle. In the present study, some enzymes viz. aldose reductase, adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and diaphorase associated with the carbohydrate metabolism in human term placenta have been essayed and the effect of some dorganochlorine pesticides, known to cross the human placenta has been studied.

p,p'-DDE, a metabolite of DDT which is more persistent than the parent compound and equally toxic has been shown to increase the aldose reductase activity. Aldrin and HCH also increased the aldose reductase activity. The Michaelis-Menten constant (Km) was also found to be affected by these pesticides using DL-glyceraldehyde as a substrate of this enzyme. ATPase activity was also found to be affected by these chlorinated pesticides. p,p'-DDE decreased the enzyme activity. HCH, p,p'-TDE, aldrin and lindane also inhibited the ATPase activity while dieldrin, an epoxide of aldrin was found to increase the enzyme activity.

SDH activity was significantly inhibited at lower concentrations of p,p'-DDE whereas at higher concentrations the enzyme activity was increased. HCH also increased the enzyme activity at higher concentrations. p,p'-TDE showed significant increase in SDH activity at lower concentrations while at higher concentrations the enzyme activity was found to be inhibited. Dieldrin increased the enzyme activity whereas aldrin inhibited the SDH activity. Lindane, inhibited the enzyme activity at lower concentrations however, the enzyme activity was increased at higher concentrations of this pesticide. 

MDH activity was also found to be affected by these pesticides. p,p'-DDE increased the MDH activity while p,p'-TDE inhibited the enzyme activity. Dieldrin, increased the enzyme activity whereas aldrin was found to inhibit the MDH activity. Lindane inhibited the enzyme activity at higher concentrations while HCH did not show any significant change. 

The behaviour of diaphorase in the presence of organochlorine pesticides has also been observed. p,p'-DDE, aldrin and lindane did not show  any significant change in diaphorase activity while p,p'-TDE and dieldrin significantly inhibited the enzyme activity. In contrast, HCH was found to increase the diaphorase activity at highest concentration used in the study.

Placental transport of amino acids has also been studied in human term placenta using radio labelled amino acids (¹⁴C-glycine and ¹⁴C-methionine).

Since amino acids are the building blocks of protein, therefore, they are required by the foetus for the formation of new tissue protein. The human placenta concentrates all amino acids intracellularly for transfer to the foetus. Transport mechanism comprises of mainly two steps, concentrative uptake by the release into foetal plasma. Dancis et al. (1968) have reported that the injection of ³⁵S-methionine into a pregnant guinea pig is followed by rapid and efficient incorporation of the amino acid into foetal proteins.

Tissue slices provide a well controlled experimental situation for the study of amino acid transfer. The placental uptake of radio labelled amino acids has been studied. Since organochlorine pesticides are also reported to be present in human placenta, therefore, the effect of some organochlorine pesticides on the placental uptake of amino acids and their incorporation into placental proteins has been studied. p,p'-DDE was found to decrease significantly the incorporation of ¹⁴C-glycine into placental proteins at lower concentrations while at higher concentrations was found to be significantly increased. HCH was found to show significant increase in the ¹⁴C-glycine incorporation at lower concentrations whereas at higher concentrations, gradual decrease was observed. Aldrin showed significant decrease, only at lower concentrations.

In another series of experiments we have investigated the effect of some chlorinated pesticides on the total uptake of ^{14C-methionine} by the placental tissue and its incorporation into placental protein. p,p'-DDE significantly inhibited the total uptake and incorporation into placental protein of ^{14C-methionine}. In contrast, aldrin significantly increased the uptake and incorporation. HCH and lindane, showed different pattern of change. In case of HCH, the total uptake was significantly decreased at lower concentrations while at a particular concentration the uptake was increased. Similar was the case in incorporation of ^{14C-methionine} into placental protein. Lindane also showed significant increase in the total uptake at a particular concentration while at higher concentrations the uptake was significantly decreased. Similarly, incorporation into protein of ^{14C-methionine} was significantly increased at particular concentration of lindane whereas at highest concentration, it was found to be significantly decreased. This observation is in conformity with the principle of teratology.  It is widely known that the synthesis of all proteins begin with the amino acid methionine. Since organochlorine pesticides are also reported to be transferred across the human placenta, the presence of such pesticides in placenta may affect the ribosomal incorporation of methionine at this stage, thereby affecting the protein synthesis. An altered methionine uptake due to chlorinated pesticides may also affect the synthesis of another amino acid cysteine for which methionine is the precursor. Besides, methionine is a glycogenic amino acid, thus providing energy to the foetus. This may essentially suggests that any alteration in the methionine uptake in the placenta, or its transfer to the foetal side may impair the energy fulfilment of the foetus. 

Apart from all the intrauterine factors, our study suggests that the environmental exposure of pesticides can also be one of the important factors for altered amino acid uptake for its utilization in various metabolic pathways. 

A slight variation in the availability of amino acid to the foetus may trigger off multidimensional chain reactions resulting in causing some irreparable damages to the developing foetus. Genetic defects of amino acid transfer have been reported for kidney and intestine. It may be assumed that if such defects affect the foetus, the placenta will also be involved. 

The present study may, therefore, suggests that the environmental exposure to chlorinated pesticides can also be considered as one of the important factors, influencing the energy metabolism of the placenta/foetus and placental amino acid transport -an interesting subject matter leading to the emergence of foetotoxicity of environmental chemicals.