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Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000.
Developmental Biology. 6th edition.
Show detailsFrom what has been said so far in this chapter, it is clear that the instructions for development do not reside wholly in the genes or even in the zygote. The developing organism is often sensitive to cues from the environment. However, this sensitivity makes the organism vulnerable to environmental changes that can disrupt development.
If you think it is amazing that any one of us survives to be born, you are correct. It is estimated that one-half to two-thirds of all human conceptions do not develop successfully to term (Figure 21.20). Many of these embryos express their abnormality so early that they fail to implant in the uterus. Others implant but fail to establish a successful pregnancy. Thus, most abnormal embryos are spontaneously aborted, often before the woman even knows she is pregnant (Boué et al. 1985). Edmonds and co-workers (1982), using a sensitive immunological test that can detect the presence of human chorionic gonadotropin (hCG) as early as 8 or 9 days after fertilization, monitored 112 pregnancies in normal women. Of these hCG-determined pregnancies, 67 failed to be maintained.
Figure 21.20
The fates of 20 hypothetical human eggs in the United States and western Europe. Under normal conditions, only 6.2 eggs of the original 20 would be expected to develop successfully to term. (After Volpe 1987.)
It appears, then, that many human embryos are impaired early in development and do not survive long in utero. Defects in the lungs, limbs, face, or mouth, however, are not deleterious to the fetus (which does not depend on those organs while inside the mother), but can threaten life once the baby is born. About 5 percent of all human babies born have a recognizable malformation, some of them mild, some very severe (McKeown 1976).
Congenital (“present at birth”) abnormalities and the loss of embryos and fetuses prior to birth are caused both intrinsically and extrinsically. Those abnormalities caused by genetic events (mutations, aneuploidies, translocations) are called malformations. For instance, aniridia (absence of the iris), caused by a mutation of the PAX6 gene, is a malformation (see Chapter 4). Down's syndrome, caused by trisomy of chromosome 21, is likewise a malformation. Most early embryonic and fetal demise is probably due to chromosomal abnormalities that interfere with normal developmental processes.
Abnormalities caused by exogenous agents (certain chemicals or viruses, radiation, or hyperthermia) are called disruptions. The agents responsible for these disruptions are called teratogens.* Most teratogens produce their effects only during certain critical periods of development. The most critical time for any organ is when it is growing and forming its structures. Different organs have different critical periods, but the time from period from day 15 through day 60 of gestation is critical for many human organs. The heart forms primarily during weeks 3 and 4, while the external genitalia are most sensitive during weeks 8 and 9. The brain and skeleton are always sensitive, from the beginning of week 3 to the end of pregnancy and beyond.
Teratogenic Agents
Different agents are teratogenic in different organisms. A partial list of agents that are teratogenic in humans is given in Table 21.2. The largest class of teratogens includes drugs and chemicals.
Table 21.2
Some agents thought to cause disruptions in human fetal developmenta.
Some chemicals that are naturally found in the environment can cause birth defects. Even in the pristine alpine meadows of the Rocky Mountains, teratogens are found. Here grows the skunk cabbage Veratrum californicum, upon which sheep sometimes feed. If pregnant ewes eat this plant, their fetuses tend to develop severe neurological damage, including cyclopia, the fusion of two eyes in the center of the face (see Figure 6.25). Two products made by this plant, jervine and cyclopamine, inhibit cholesterol synthesis in the fetus and prevent Sonic hedgehog from functioning. Indeed, this resembles human genetic conditions (discussed in Chapters 6 and 12) in which the genes for either Sonic hedgehog or cholesterol synthesising enzymes are mutated (Beachy et al. 1997; Opitz 1998). The affected organism dies shortly after birth (as a result of severe brain defects, including the lack of a pituitary gland).
Quinine and alcohol, two substances derived from plants, can also cause congenital malformations. Quinine can cause deafness, and alcohol (when more than 2–3 ounces per day are imbibed by the mother) can cause physical and mental retardation in the infant. Nicotine and caffeine have not been proved to cause congenital anomalies, but women who are heavy smokers (20 cigarettes a day or more) are likely to have infants that are smaller than those born to women who do not smoke. There is controvery over whether cigarette smoking enhances the risk of facial anomalies. Smoking also significantly lowers the number and motility of sperm in the semen of males who smoke at least four cigarettes a day (Kulikauskas et al. 1985).
In addition, our industrial society produces hundreds of new artificial compounds that come into general use each year. Pesticides and organic mercury compounds have caused neurological and behavioral abnormalities in infants whose mothers have ingested them during pregnancy. Moreover, drugs that are used to control diseases in adults may have deleterious effects on fetuses. For example, valproic acid is an anticonvusant drug used to control epilepsy. It is known to be teratogenic in humans as it can cause major and minor bone defects. Barnes and colleagues (1996) have shown that valproic acid decreases the level of Pax1 transcription in chick somites. This causes the malformation of the somite and the corresponding malformations of vertebrae and ribs.
Retinoic acid as a teratogen
In some instances, even a compound that is involved in normal development can have deleterious effects if present in large amounts at particular times. Retinoic acid is important in forming the anterior-posterior axis of the mammalian embryo and also in forming the limbs (see Chapters 11 and 16; Morriss-Kay and Ward 1999). In these instances, retinoic acid is secreted from discrete cells and works in a small area. However, if retinoic acid is present in large amounts, cells that normally would not receive such high concentrations of this molecule will respond to it.
Inside the developing embryo, vitamin A and 13-cis-retinoic acid become isomerized to the developmentally active forms of retinoic acid, all-trans-retinoic acid and 9-cis-retinoic acid (Creech Kraft 1992). Retinoic acid cannot bind directly to genes. To effect gene regulation, RA must bind to a group of transcription factors called the retinoic acid receptors (RARs). These proteins have the same general structure as the steroid and thyroid hormone receptors, and they are active only when they have bound retinoic acid (Linney 1992). The RARs bind to specific enhancer elements in the DNA called retinoic acid response elements. Retinoic acid response elements contain at least two copies of the sequence GGTCA (Ruberte et al. 1990, 1991). Some of the Hox genes have retinoic acid response elements in their promoters (Yu et al. 1991; Pöpperl and Featherstone 1993; Studer et al. 1994). There are three major types of retinoic acid receptors: RAR-α, RAR-β, and RAR-γ. They each bind both forms of retinoic acid, and they each bind to the same retinoic acid response element.
Retinoic acid has been useful in treating severe cystic acne and has been available (under the name Accutane) since 1982. Because the deleterious effects of administering large amounts of vitamin A or its analogues to pregnant animals have been known since the 1950s (Cohlan 1953; Giroud and Martinet 1959; Kochhar et al. 1984), the drug carries a label warning that it should not be used by pregnant women. However, about 160,000 women of childbearing age (15 to 45 years) have taken this drug since it was introduced, and some of them have used it during pregnancy. Lammer and his co-workers (1985) studied a group of women who inadvertently exposed themselves to retinoic acid and who elected to remain pregnant. Of their 59 fetuses, 26 were born without any noticeable anomalies, 12 aborted spontaneously, and 21 were born with obvious anomalies. The malformed infants had a characteristic pattern of anomalies, including absent or defective ears, absent or small jaws, cleft palate, aortic arch abnormalities, thymic deficiencies, and abnormalities of the central nervous system.†
This pattern of multiple congenital anomalies is similar to that seen in rat and mouse embryos whose pregnant mothers have been given these drugs. Goulding and Pratt (1986) have placed 8-day mouse embryos in a solution containing 13-cis-retinoic acid at a very low concentration (2 × 10-6 M). Even at this concentration, approximately one-third of the embryos developed a very specific pattern of anomalies, including dramatic reduction in the size of the first and second pharyngeal arches (see Figure 11.40). In normal mice, the first arch eventually forms the maxilla and mandible of the jaw and two ossicles of the middle ear, while the second arch forms the third ossicle of the middle ear as well as other facial bones.
Retinoic acid probably works by several mechanisms. One basis for the teratogenicity of retinoic acid appears to reside in the drug's ability to alter the expression of the Hox genes and thereby respecify portions of the anterior-posterior axis and inhibit neural crest cell migration from the cranial region of the neural tube (Moroni et al. 1994; Studer et al. 1994). Radioactively labeled retinoic acid binds to the cranial neural crest cells and arrests both their proliferation and their migration (Johnston et al. 1985; Goulding and Pratt 1986). The binding seems to be specific to the cranial neural crest-derived cells, and the teratogenic effect of the drug is confined to a specific developmental period (days 8–10 in mice; days 20–35 in humans). Animal models of retinoic acid teratogenesis have been extremely successful in elucidating the mechanisms of teratogenesis at the cellular level.
WEBSITE
21.9 Mechanisms of retinoic acid teratogenesis. Within the cell, numerous retinoid binding proteins interact to influence the ability of retinoic acid to transcribe particular genes. http://www.devbio.com/chap21/link2109.shtml
WEBSITE
21.10 Thalidomide as a teratogen. The drug Thalidomide caused thousands of babies to be born with malformed arms and legs, and it provided the first major evidence that drugs could induce congenital anomalies. The mechanism of its action is still hotly debated. http://www.devbio.com/chap21/link2110.shtml
Alcohol as a teratogen
In terms of frequency and cost to society, the most devastating teratogen is undoubtedly ethanol. In 1968, Lemoine and colleagues noticed a syndrome of birth defects in the children of alcoholic mothers. This fetal alcohol syndrome (FAS) was also noted by Jones and Smith (1973). Babies with FAS were characterized as having small head size, an indistinct philtrum (the pair of ridges that runs between the nose and mouth above the center of the upper lip), a narrow upper lip, and a low nose bridge. The brain of such a child may be dramatically smaller than normal and often shows defects in neuronal and glial migration (Figure 21.21; Clarren 1986). There is also prominent extra cell death in the frontonasal process and in cranial nerve ganglia (Sulik et al. 1988). Fetal alcohol syndrome is the third most prevalent type of mental retardation (behind fragile X syndrome and Down syndrome) and affects 1 out of every 500–750 children born in the United States (Abel and Sokol 1987).
Figure 21.21
Comparison of a brain from an infant with fetal alcohol syndrome (left) with a brain from a normal infant of the same age (right). The brain from the infant with FAS is significantly smaller, and the pattern of convolutions is obscured by glial cells (more...)
Children with fetal alcohol syndrome are developmentally and mentally retarded, with a mean IQ of about 68 (Streissguth and LaDue 1987). FAS patients with a mean chronological age of 16.5 years were found to have the functional vocabulary of 6.5-year-olds and to have the mathematical abilities of fourth graders. Most adults and adolescents with FAS cannot handle money or their own lives, and they have difficulty learning from past experiences. Moreover, in many instances of FAS, the behavioral abnormalities exist without any gross physical changes in brain or head size (J. Opitz, personal communication). There is great variation in the ability of mothers and fetuses to metabolize ethanol, and it is thought that 30 to 40 percent of the children born to alcoholic mothers who drink during pregnancy will have FAS. It is also thought that lower amounts of ethanol ingestion by the mother can lead to fetal alcohol effect, a less severe form of FAS, but a condition that lowers the functional and intellectual abilities of the sufferer.‡
A mouse model system has been used to explain the effects of alcohol on the face and nervous system. When mice are exposed to ethanol at the time of gastrulation, it induces the same range of developmental defects as in humans. As early as 12 hours after the mother ingests the alcohol, abnormalities of development are observed. The midline structures fail to form, allowing the abnormally close proximity of the medial processes of the face. Forebrain anomalies are also seen, and the more severely affected fetuses lack a forebrain entirely (Sulik et al. 1988). Studies on these mice suggest that ethanol may induce its teratogenic effects by more than one mechanism. First, anatomical evidence suggests that neural crest migration is severely impaired. Instead of migrating and dividing, ethanol-treated neural crest cells prematurely initiate their differentiation into facial cartilage (Hoffman and Kulyk 1999). Second, ethanol can cause the apoptosis of neurons. One way it can cause apoptosis is to generate superoxide radicals that can oxidize cell membranes and lead to cytolysis (Figure 21.22A-CDavis et al. 1990; Kotch et al. 1995). Another pathway to apoptosis involves the activation of GABA receptors and simultaneous inhibition of glutamate receptors (Ikonomidou et al. 2000). Ethanol-induced apoptosis can delete millions of neurons from the developing forebrain, frontonasal (facial) process, and cranial nerve ganglia. Third, alcohol may directly interfere with the ability of cell adhesion molecule L1 to function in holding cells together. Ramanathan and colleagues (1996) have shown that ethanol can block the adhesive functions of L1 proteins in vitro at levels as low as 7 mM, a concentration of ethanol produced in the blood or brain with a single drink (Figure 21.22D). Moreover, mutations in human L1 genes cause a syndrome of mental retardation and malformations similar to that seen in severe cases of fetal alcohol syndrome.
Figure 21.22
Possible mechanisms producing fetal alcohol syndrome. (A-C) Cell death caused by ethanol-induced superoxide radicals. Staining with Nile blue sulfate reveals areas of cell death. (A) Control 9-day mouse embryo head region. (B) Head region of ethanol-treated (more...)
WEBSITE
21.11 Our knowledge of alcohol's teratogenicity. A hundred years ago, alcohol was considered dangerous to the fetus. Fifty years ago, it was considered harmless, and today it is considered very dangerous. Sociological studies look at how these assessments were made. http://www.devbio.com/chap21/link2111.shtml
Other teratogenic agents
Chemicals
Over 50,000 artificial chemicals are currently used in our society and about 200 to 500 new materials being made each year (Johnson 1980). In the former Soviet Union, the unregulated “industrial production at-all-costs” approach leaves a legacy of soaring birth defects. In some regions of Kazakhstan, teratogens such as lead, mercury, and zinc are found in high concentrations in drinking water, vegetables, and the air. In these places, nearly half the people tested have extensive chromosome breakage. In some areas, the incidence of birth defects has doubled since 1980 (Edwards 1994).
Although teratogenic compounds have always been with us, the risks increase as more and more untested compounds enter our environment each year. Most industrial chemicals have not been screened for their teratogenic effects. Standard screening protocols are expensive, long, and subject to interspecies differences in metabolism. There is still no consensus on how to test a substance's teratogenicity for human embryos.
Pathogens as teratogenic agents
Another class of teratogens includes viruses and other pathogens. Gregg (1941) first documented the fact that women who had rubella (German measles) during the first third of their pregnancy had a 1 in 6 chance of giving birth to an infant with eye cataracts, heart malformations, or deafness. This was the first evidence that the mother could not fully protect the fetus from the outside environment. The earlier the rubella infection occurred during the pregnancy, the greater the risk that the embryo would be malformed. The first 5 weeks appear to be the most critical, because this is when the heart, eyes, and ears are being formed. The rubella epidemic of 1963–1965 probably resulted in about 20,000 fetal deaths and 30,000 infants with birth defects in the United States. Two other viruses, Cytomegalovirus and Herpes simplex, are also teratogenic. Cytomegalovirus infection of early embryos is nearly always fatal, but infection of later embryos can lead to blindness, deafness, cerebral palsy, and mental retardation.
Bacteria and protists are rarely teratogenic, but two of them can damage human embryos. Toxoplasma gondii, a protozoan carried by rabbits and cats (and their feces), can cross the placenta and cause brain and eye defects in the fetus. Treponema pallidum, the cause of syphilis, can kill early fetuses and produce congenital deafness in older ones.
Ionizing radiation
Ionizing radiation can break chromosomes and alter DNA structure. For this reason, pregnant women are told to avoid unnecessary X-rays, even though there is no evidence for congenital anomalies resulting from diagnostic radiation (Holmes 1979). Heat from high fevers is also a possible teratogen.
While we know the causes of certain malformations, most congenital abnormalities are not yet able to be explained. For instance, congenital cardiac anomalies occur in about 1 in every 200 live births. Genetic causes are responsible for about 8 percent of these heart abnormalities, and about 2 percent can be explained by known teratogens. That leaves 90 percent of the them unexplained (O’Rahilly and Müller 1992). We still have a great deal of research to do.
WEBSITE
21.12 Other teratogenic agents. Certain behavior-modifying drugs are teratogenic, while others appear not to be. This is an area of great concern, as conflicting studies debate whether caffeine, cannabis, and cocaine are teratogenic. http://www.devbio.com/chap21/link2112.shtml
WEBSITE
21.13 Maternal effects on later disease. Recent epidemiological data suggests that nutritional stress to a pregnant mother may predispose her offspring to certain diseases when they are adults. http://www.devbio.com/chap21/link2113.shtml
Genetic-Environmental Interactions
The observation that a substance may be teratogenic in one strain of mice but not in another strongly suggests that there is a genetic component to whether a substance can produce changes in normal development. Recent evidence suggests that different alleles in the human population can influence whether a substance is benign or dangerous to the fetus. For example, among the general population, there is only a slight risk that heavy smoking by the mother will cause facial malformations in her fetus. However, if the fetus has a particular allele (A2) of the gene for growth factor TGF-β, tobacco smoke absorbed through the placenta can raise the risk of cleft lip and palates tenfold (Shaw et al. 1996). Similarly, different alleles encoding the enzyme alcohol dehydrogenase-2 result in differing abilities to degrade ethanol. Whether heavy maternal alcohol consumption leads to fetal alcohol syndrome or fetal alcohol effect may be due to the types of alcohol dehydrogenase isozymes in the mother and fetus (McCarver-May, 1996). Thus, whether or not a compound is “teratogenic” depends on many things, including the genes of the individuals exposed to it.
Coda
Development usually occurs in a rich environmental milieu, and most animals are sensitive to environmental cues. The environment may determine sexual phenotype, may induce remarkable structural and chemical adaptations according to the season, may induce specific morphological changes that allow an individual to escape predation, and can induce caste determination in insects. The environment can also alter the structure of our neurons and the specificity of our immunocompetent cells. Unfortunately, the environment can also be the source of chemicals that disrupt normal developmental processes.
The developmental plasticity of the nervous system assures that each person is an individual. Our brain adds experience to endowment. Fears that cloning could produce “thousands of Hitlers” are unfounded. Not only have there been no genes identified for bigotry, demagoguery, or political canniness, but one would have to reconstruct Hitler's personal, social, and political milieus to even come close to replicating the dictator's personality. Wolpe (1997) has pointed out that thinking that a genetically identical clone of Hitler would become a bigoted dictator is buying into the same genetic essentialism that made Hitler so evil. Similarly, Gould (1997) points out that even Eng and Chang Bunker, the well publicized conjoined twins who most likely had both the same heredity and the same environment, became very different people. One was cheerful and abstained from all liquor. The other was morose and alchoholic (which was a problem, since they shared the same liver). The plasticity of our nervous system enables us to be individuals and “allows us to escape the tyranny of our genes” (Childs 1999).
While development usually occurs in a complex natural environment, it can most easily be studied in the laboratory. Indeed, our “model systems” are animals that are readily domesticated and whose development is least affected by environmental factors (Bolker 1995). However, as we become aware of the complexity of development, we are realizing that development is critically keyed to the environment. It can take a community to develop an embryo. Ecologists have known about “life history strategies” of organisms for over a century. However, the proximate causes of these histories (such as how a fish becomes male in one environment and female in another) are just beginning to be understood. The exploration of environmental regulation of development is just beginning.
Snapshot Summary: The Environmental Regulation of Development
- 1.
The environment can affect development in several ways. Development is sometimes cued to normal circumstances that the organism can expect to find in its environment. The larvae of many species will not begin metamorphosis until they find a suitable substrate. In other instances, symbiotic relationships between two or more species are necessary for the complete development of one or more of the species.
- 2.
Developmental plasticity makes it possible for environmental circumstances to elicit different phenotypes from the same genotype. Many species have a broad reaction norm, wherein the genotype can respond in a graded way to environmental conditions.
- 3.
Some species exhibit polyphenisms, in which distinctly different phenotypes are evoked by different environmental cues.
- 4.
Seasonal cues such as photoperiod, temperature, or type of food can alter development in ways that make the organism more fit. Changes in temperature also are responsible for determining sex in several organisms, including many types of reptiles and insects.
- 5.
Predator-induced polyphenisms have evolved such that the prey species can respond morphologically to the presence of a specific predator. In some instances, this induced adaptation can be transmitted to the progeny of the prey.
- 6.
The differentiation of immunocompetent cells and the formation of synapses in the visual system are examples where experience influences the phenotype.
- 7.
Compounds found in the environment (teratogens) can disrupt normal development. Teratogens can be naturally occurring substances or synthetic ones.
- 8.
Alcohol and retinoic acid are two of the most intensively studied human teratogens. They may produce their teratogenic effects through more than one pathway.
- 9.
It is possible that numerous compounds may be acting as hormone mimics or antagonists disrupt normal development by interfering with the endocrine system.
- 10.
Genetic differences can predispose individuals to being affected by teratogens.
Endocrine Disruptors
Endocrine disruptors are exogenous chemicals that interfere with the normal function of hormones. They can disrupt hormonal function in many ways.
- 1.
Endocrine disruptors can mimic the effects of natural hormones by binding to their receptors. DES (diethylstilbesterol; Chapter 17), is one such example.
- 2.
Endocrine disruptors may block the binding of a hormone to its receptor, or they can block the synthesis of the hormone. Finasteride, a chemical used to prevent male pattern baldness and enlargement of the prostate glands, is an anti-androgen, since it blocks the synthesis of dihydrotestosterone. Women are warned not to handle this drug if they are pregnant, since it could arrest the genital development of male fetuses.
- 3.
Endocrine disruptors can interfere with the transport of a hormone or its elimination from the body. For instance, rats exposed to polychlorinatedbiphenyl pollutants (PCBs; see below) have low levels of thyroid hormone. The PCBs compete for the binding sites of the thyroid hormone transport protein. Without being bound to this protein, the thyroid hormones are excreted from the body (McKinney et al. 1985; Morse et al. 1996).
Developmental toxicology and endocrine disruption are relatively new fields of research. While traditional toxicology has pursued the environmental causes of death, cancer, and genetic damage, developmental toxicology/endocrine disruptor research has focused on the roles that environmental chemicals may have in altering development by disrupting normal endocrine function of surviving animals (Bigsby et al. 1999).
WEBSITE
21.14 Environmental endocrine disruptors. The Wingspread Consensus Statement of 1991 began a move by scientists to influence government policy concerning potential endocrine disruptors. This site looks at that statement and at some of the policies presently being implemented. http://www.devbio.com/chap21/link2114.shtml
Environmental estrogens
There is probably no bigger controversy in the field of toxicology than whether chemical pollutants are responsible for congenital malformations in wild animals, the decline of sperm counts in men, and breast cancer in women. One of the sources of these pollutants is pesticide use. Americans use some 2 billion pounds of pesticides each year, and some pesticide residues stay in the food chain for decades. Although banned in the United States in 1972, DDT has an environmental half-life of about 100 years (Nature Genetics 1995). Recent evidence has shown that DDT (dichloro-diphenyl-trichloroethane) and its chief metabolic by-product, DDE (which lacks one of the chlorine atoms), can act as estrogenic compounds, either by mimicking estrogen or by inhibiting androgen effectiveness (Davis et al. 1993; Kelce et al. 1995). DDE is a more potent estrogen than DDT, and it is able to inhibit androgen-responsive transcription at doses comparable to those found in contaminated soil in the United States and other countries. DDT and DDE have been linked to such environmental problems as the decrease in the alligator populations in Florida, the feminization of fish in Lake Superior, the rise in breast cancers, and the worldwide decline of human sperm counts (Carlsen et al. 1992; Keiding and Skakkebaek 1993; Stone 1994; Swan et al. 1997). Guillette and co-workers (1994; Matter et al. 1998) have linked a pollutant spill in Florida's Lake Apopka (a discharge including DDT, DDE, and numerous other polychlorinated biphenyls) to a 90% decline in the birthrate of alligators and to the reduced penis size in the young males.
Dioxin, a by-product of the chemical processes used to make pesticides and paper products, has been linked to reproductive anomalies in male rats. The male offspring of rats exposed to this planar, lipophilic molecule when pregnant have reduced sperm counts, smaller testes, and fewer male-specific sexual behaviors. Fish embryos seem particularly susceptible to dioxin and related compounds, and it has been speculated that the amount of these compounds in the Great Lakes during the 1940s was so high that none of the lake trout hatched there during that time survived (Figure 21.23; Hornung et al. 1996; Zabel and Peterson 1996; Johnson et al. 1998).
Some estrogenic compounds may be in the food we eat and in the wrapping that surrounds them, for some of the chemicals used to set plastics have been found to be estrogenic. The discovery of the estrogenic effect of plastic stabilizers was made in a frightening way. Investigators at Tufts University Medical School had been studying estrogen-responsive tumor cells. These cells require estrogen in order to proliferate. Their studies were going well until 1987, when the experiments suddenly went awry. Then the control cells began to show the high growth rates suggesting stimulation comparable to that of the estrogen-treated cells. Thus, it as if someone had contaminated the medium by adding estrogen to it. What was the source of contamination? After spending four months testing all the components of their experimental system, the researchers discovered that the source of estrogen was the plastic tubes that held their water and serum. The company that made the tubes refused to tell the investigators about its new process for stabilizing the polystyrene plastic, so the scientists had to discover it themselves. The culprit turned out to be p-nonylphenol, a chemical that is also used to harden the plastic of the plumbing tubes that bring us water and to stabilize the polystyrene plastics that hold water, milk, orange juice, and other common liquid food products (Soto et al. 1991; Colburn et al. 1996). This compound is also the degradation product of detergents, household cleaners, and contraceptive creams. A related compound, 4-tert-pentylphenol, has a potent estrogenic effect on human cultured cells and can cause male carp (Cyprinus carpis) to develop oviducts, ovarian tissue, and oocytes (Gimeno et al. 1996).
Some other environmental estrogens are polychlorinated biphenyls (mentioned earlier). These PCBs can react with a number of different steroid receptors. PCBs were widely used as refrigerants before they were banned in the 1970s when they were shown to cause cancer in rats. They remain in the food chain, however (in both water and sediments), and they have been blamed for the widespread decline in the reproductive capacities of otters, seals, mink, and fish. Some PCBs resemble diethylstilbesterol in shape, and they may affect the estrogen receptor as DES does, perhaps by binding to another site on the estrogen receptor. Another organochlorine compound (and an ingredient in many pest- icides) is methoxychlor. Pickford and colleagues (1999) found that methoxychlor blocked progesterone-induced oocyte maturation in Xenopus at concentrations that are environmentally relevant. This would severely inhibit the fertility of the frogs, and it may be a component of the worldwide decline in amphibian populations.
Some scientists, however, say that these claims are exaggerated. Tests on mice had shown that litter size, sperm concentration, and development were not affected by environmental concentrations of environmental estrogens. However, recent work by Spearow and colleagues (1999) has shown a remarkable genetic difference in the sensitivity to estrogen among different strains of mice. The strain that had been used for testing environmental estrogens, the CD-1 strain, is at least 16 times more resistant to endocrine disruption than the most sensitive strains such as B6. When estrogen-containing pellets were implanted beneath the skin of young male CD-1 mice, very little happened. However, when the same pellets were placed beneath the skin of B6 mice, their testes shrunk, and the number of sperm seen in the seminiferous tubules dropped dramatically (Figure 21.24). This widespread range of sensitivities has important consequences for determining safety limits for humans.
Environmental thyroid hormone disruptors
The structure of some PCBs resembles that of thyroid hormones (Figure 21.25), and exposure to them alters serum thyroid hormone levels in humans. Hydroxylated PCB were found to have high affinities for the thyroid hormone serum transport protein transthyretin, and can block thyroxine from binding to this protein. This leads to the elevated excretion of the thyroid hormones. Thyroid hormones are critical for the growth of the cochlea of the inner ear, and rats whose mothers were exposed to PCBs had poorly developed cochleas and hearing defects (Goldey and Crofton in Stone 1995; Cheek et al. 1999).
Deformed frogs: pesticides mimicking retinoic acid?
Throughout the United States and southern Canada there is a dramatic increase in the number of deformed frogs and salamanders in what seem to be pristine woodland ponds (Figure 21.26A; Ouellet et al. 1997). These deformities include extra or missing limbs, missing or misplaced eyes, deformed jaws, and malformed hearts and guts. Some of these malformations (especially the limb anomalies, see Figure 3.23) may be due to trematode infestation, but other malformations do not seem to be explainable by that route. Some lakes containing high proportions of malformed frogs do not appear to be infested with trematodes, and water from these lakes is able to disrupt development in frog eggs that are placed into it. It is not known what is causing these disruptions, but there is speculation (see Hilleman 1996, Ouellet et al. 1997) that pesticides (sprayed for mosquito and tick control) might be activating or interfering with the retinoic acid pathway. The spectrum of abnormalities seen in these frogs resembles those malformations caused by exposing tadpoles to retinoic acid (Crawford and Vincenti 1998; Gardiner and Hoppe 1999).
New research has focused on compounds such as methoprene, a juvenile hormone mimic that inhibits mosquito pupae from metamorphosing into adults. Since vertebrates do not have juvenile hormone, it was assumed that this pesticide would not harm fish, amphibians, or humans. This has been found to be the case: methoprene, itself, does not have teratogenic properties. However, upon exposure to sunlight, methoprene breaks down into products that have significant teratogenic activity in frogs (Figures 21.26B,C). These compounds have a structure similar to that of retinoic acid and will bind to the retinoid receptor (Harmon et al. 1995; La Claire et al 1998). When Xenopus eggs are incubated in water containing these compounds, the tadpoles are often malformed, and show a spectrum of deformities similar to those seen in the wild (La Claire et al. 1998).
Chains of causation
Whether in law or science, establishing chains of causation is a demanding and necessary task. In developmental toxicology, numerous endpoints must be checked, and many different levels of causation have to be established (Crain and Guillette 1998; McNabb et al. 1999). For instance, one could ask if the pollutant spill in Lake Apopka was responsible for the feminization of male alligators. To establish this, one has to ask how might the chemicals in the spill contributed to reproductive anomalies in males alligators and what would be the consequences of that happening. Table 21.3 shows the postulated chain of causation. After observing that the population level of the alligators has declined, at the organism level one discovers the unusually high levels of estrogens in the female alligators, the unusually low levels of testosterone in the males, and the decrease in the number of births among the alligators. On the tissue and organ level, the decline in birth rate can be explained by the elevated production of estrogens from the juvenile testes, the malformation of the testes and penis, and the changes in enzyme activity in the female gonads. On the cellular level, one sees ovarian abnormalities that correlate with unusually elevated estrogen levels. These cellular changes, in turn, can be explained at the molecular level by the finding that many of the components of the pollutant spill bind to the alligator estrogen and progesterone receptors and that they are able to circumvent the cell's usual defenses against overproduction of steroid hormones (Crain et al. 1998).
While there is little dispute about the damage to wildlife being wrought by endocrine disrupting chemicals, it is difficult to document the effects of environmental compounds on humans. There is enormous genetic variation in the human species, and one cannot perform controlled experiments to determine the effect of any particular compound on a human population. Rather, we are exposed to “cocktails” consisting of different compounds ingested at different times. There is a great deal more research that needs to be done on the biochemistry of these compounds, their effects on development, and the epidemiology of developmental abnormalities. At the moment, evidence coming from animal studies suggests that humans and natural animal populations are at risk from these hormonal modulators, but not all the needed data are in. ▪
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21.15 Deformed frogs and salamanders. Considerable efforts are being made to find the causes for both the recent decline of amphibian populations and for the developmental anomalies being discovered in these animals. Parasites, fungus, ultraviolet radiation, and pesticides may all be playing a role. http://www.devbio.com/chap21/link2115.shtml
Figure 21.23
Lake trout 4 weeks after hatching. (A) Normal larva with its golden yellow yolk sac. (B) Dioxin-exposed larva exhibiting a blue yolk sac. The yolk sac has swelled with water and has numerous sites of hemorrhage. Such fish often have reduced growth, as (more...)
Figure 21.24
Effects of estrogen implants on different strains of mice. The graph shows the percentage of seminiferous tubules containing elongated spermatozoa. (The mean standard ± error is for an average of six individuals). The photographs show cross sections (more...)
Figure 21.25
Structures of hormones and endocrine disruptors.
Figure 21.26
Teratogenesis in frogs. (A) Wild green frog (Rana clamitans) with an eye deformity, collected in New Hampshire in 1999 by K. Babbitt. (B) Xenopus tadpole with eye deformities caused by incubating newly fertilized eggs in water containing methoprenic acid, (more...)
Table 21.3
Chain of causation linking contaminant spill in Lake Apopka to endocrine disruption in juvenile alligators.
Footnotes
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In some cases, the same condition can be caused by a disruption (from an exogenous agent) or a malformation (from the nucleus). For instance, certain axial malformations in mice can be produced either by the administration of retinoic acid or by mutations in certain Hox genes. In some instances, the mutation and the teratogen are known to affect the same enzyme. Chondroplasia punctata is a congenital defect of bone and cartilage, characterized by abnormal bone mineralization, underdevelopment of nasal cartilage, and shortened fingers. It is caused by a defective gene on the X chromosome. An identical phenotype is produced by the ingestion of the rat-killing compound, warfarin. It appears that the defective gene is normally responsible for producing an arylsulfatase protein necessary for cartilage growth. The warfarin compound inhibits this same enzyme (Franco et al. 1995).
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This is a critical public health concern, because there is significant overlap between the population using acne medicine and the population of women of childbearing age, and because it is estimated that half of the pregnancies in America are unplanned (Nulman et al. 1997). Vitamin A is itself teratogenic when injected in megadose amounts. Rothman and colleagues (1995) found that pregnant women who took more than 10,000 international units of preformed vitamin A per day (in the form of vitamin supplements) had about a 2 percent chance of having a baby born with disruptions similar to those produced by retinoic acid.
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For a sensitive account of raising a child with fetal alcohol syndrome, as well as an analysis of FAS in Native American culture in the United States, read Michael Dorris's The Broken Cord 1989. The personal and sociological effects of FAS are well integrated with the scientific and economic data.
- Environmental Disruption of Normal Development - Developmental BiologyEnvironmental Disruption of Normal Development - Developmental Biology
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