Lead+and+Thalidomide+as+Teratogens

=**Teratogenic Effects of Lead and Thalidomide**=

Introduction
Teratogens are compounds which can cause abnormal development to occur upon exposure to a developing embryo. In non-mammalian development, these compounds can affect the embryo through direct contact. In mammalian development, teratogens must be taken in by the mother and cross the placenta in order to gain access to the embryo. Lead and thalidomide are teratogens which affect brain and limb development respectively. They are quite dangerous to developing embryos and exposure to them must be minimized during development (Gilbert, 2010).

Normal Development
Brain after 12 weeks (lead)

Between weeks 12 and 23 of gestation the brain of the human fetus is developing into various sections. From weeks 24-26 the fetus' brain begins to form sulci making the formerly smooth brain grooved and ridged. This increases surface area within the brain resulting in increased ability of the fetus to perceive stimuli. Additionally, myelination has been seen as early at 20 weeks, which further increases the ability of the nerves to respond to outside stimuli (Lan, Yamashita, Tang, Sugahara, Takahashi, Ohba, and Okamura, 2000).

Limb bud development (thalidomide)

Limb development in vertebrates is controlled by the homeobox or Hox genes. These genes determine which portions of the embryo will form which structures along the anterior/posterior axis. The concentration of retinoic acid (vitamin A) determines the expression of Hox genes, meaning that the concentrations of retinoic acid in vertebrate embryos is responsible for determining proper limb development. Additionally, the apical ectodermal ridge is responsible for the production of signaling molecules which control limb development among other developmental processes (Tickle, 1991).

The above examples show a few of the normal developmental processes which might be affected through exposure to the teratogens lead and thalidomide.

Lead
Lead is a heavy metal that is used for making pottery, battery manufacturing, printing and auto repair. Lead can be found in the ground, water, and in paints produced before 1978. Because lead is widespread in the environment, many people have a minimal amount of lead in their blood. Levels of lead that exceed 10 µg/dl in the blood indicate high exposure to lead and are a cause of concern. Lead can enter the body mainly through the lungs and gut and to a lesser extent through the skin. Long term exposure to lead causes the metal to accumulate in the bones and to remain there for an extended period of time (Organization of Teratology Information Specialists, 2010). For pregnant women, high levels of lead in the blood can be problematic because at the start of the twelfth week of pregnancy, lead can cross the placenta and can be absorbed by the developing fetus (Carpenter, 1974). In the developing fetus, lead can be found in the fetal blood, bone and soft tissue. The developing fetus is especially vulnerable to lead because the fetal blood-brain barrier is more permeable. During pregnancy, high levels of lead have been associated with increased risk of miscarriage, still birth, low birth weight and neurobehavioral abnormalities of exposed babies. High levels of paternal lead exposure have also been linked to reduced fertility, preterm delivery and low birth weight (OTIS, 2010).

The fetal blood-brain barrier is undeveloped and studies carried out in rats have shown that the uptake of lead during the prenatal period is greater than after birth. Immature endothelial cells form the capillaries of the brain are more sensitive to the effects of lead as compared to the capillaries of mature brains. These immature endothelial cells facilitate and enhance the movement of fluid and small metal ions like lead to the developing brain structures, particularly neurons and astrocytes. There is a direct relationship between fetal brain weight and the quantity of lead present. A direct relationship between fetal brain weight and calcium levels exists. Goyer (1996) has suggested the movement of calcium in the fetal brain induces the movement of lead into the brain.
 * Exposure to the fetal brain**

Multiple studies have shown that increased prenatal lead exposure is negatively correlated to neurobehavioral abnormalities during infancy and childhood. A study conducted by Bellinger et al. (1987) showed that prenatal lead exposure inversely affects early cognitive development. Children were placed into groups based on the quantity of lead they were exposed to during prenatal development. The Mental Developmental Index of the Beyley Scales of Infant Development was use to assess infant development twice a year. Infants that were exposed to the highest quantity of lead ( > 10µg/dl) during development scored lower on developmental tests as compared to the other groups of infants who had been exposed to lower levels of lead.
 * Neurobehavioral Effects**

Another study conducted by Wasserman et al. (2000), also showed that prenatal lead exposure is associated with a decreased performance on standardized tests. The study was conducted in Kosovo and involved 422 children whose mothers were recruited during pregnancy from a smelter town and a non-lead exposed town. Prenatal blood lead levels were assessed at mid-pregnancy. Blood lead levels were also assessed at delivery and periodically after birth. The intelligence of the children were measured at ages 3, 4, 5, or 7. The tests were administered to the children by bilingual trained psychologists. For the 3 and 4 year olds, McCarthy Scales of Children’s Abilities test was administered. This test assesses general intellectual functioning, memory and gross motor skills. The 5 and 7 year olds were administered The Wechsler Preschool and Primary Scale of Intelligence which provides a full scale IQ score. The overall results from this study indicate that prenatal elevations in blood lead levels were associated with decrements in intelligence. Postnatal blood levels varied proportionally with prenatal blood lead levels. Postnatal elevations in blood lead levels that occurred prior to age two were associated with the most significant decrements in child intelligence. This phenomenon was likely to have occurred because prior to age two, there is rapid brain growth and significant changes take place in dendritic pruning and synaptogenesis (Wasserman et al., 2000).

The effects of lead on the developing brain have been categorized as morphological and pharmacological.
 * Mechanisms of Neurological Effects**

Neuropharmacological Effects:
 * Lead substitutes for calcium
 * Neurotransmitter release
 * Protein kinase C
 * Na-Ca ATPase
 * Energy metabolism

Neuromorphological Effects:
 * Interference with adhesion molecules
 * Impairing programming of cell-cell connections
 * Miswiring of the central nervous system

During early postnatal development, a significant amount of brain growth takes place. Brain growth and development is facilitated by an overgrowth of neuronal processes, followed by deletion of synapses in accord with the infants’ specific environmental needs. During this critical period of brain development, synaptic connections form on a continuous basis. Lead inhibits the production of new synapses and as a result a modification in the neuronal circuit can be seen. Impairment of cell-cell interactions causes neuronal cells to produce less sialic acid, which leads to the formation of abnormal synaptic structures. Lead has also been shown to induce the differentiation and migration of premature glial cells during structuring of the brain, inducing further abnormalities in brain development (Goyer, 1996).

Lead can substitute for calcium and perhaps zinc in ion-dependent events that occur at the synapse leading to the impairment of multiple neurotransmitter systems, such as cholinergic and dopaminergic systems. During the neonatal period, the //N-//methyl-D-aspartate (NMDA) receptor-ion channel seems to be most affected by the inhibitory effects of lead. The NMDA receptor is a non- specific cation channel that is associated with memory. Protein kinase C, an important secondary messenger that regulates cellular metabolism can be activated by very small concentrations of lead. Lead also disrupts the release of calcium from the mitochondria adversely affecting energy metabolism (Goyer, 1996).

Lowering of blood levels involves removal from the source of lead exposure or chelation therapy. Chelation therapy involves the use of molecules such as ethylenediaminetetraacetic acid ( EDTA), and Dimercaptosuccinic acid (DMSA). There are fundamental characteristics that a good lead chelator should exhibit. These include (i) the ability to reduce cell burden in target cells for lead toxicity, (ii) the ability to restore or prevent lead induced loss of functions, (iii) absence of adverse effects which affect homeostasis and utilization of trace elements and (iv) no intrinsic toxicity. Though DMSA and EDTA are effective in reducing blood levels, there are side effects associated with the use of these molecules. For example, the administration of EDTA can lead to nephrotoxicity, increased excretion of essential metal ions and it has to be administered by injection or intravenously. On the other hand, DMSA can be administered orally but its use has been associated with increased excretion of copper from the body (Goyer, 1995). The use of chelating therapy has been largely restricted to children and as such there is no definite outcome regarding the effects of lead removal with regard to fetal development.
 * Possible treatments for outcomes**

The use of chelating therapy has been largely restricted to children. The use of chelation as a technique to lower blood lead levels during pregnancy has been limited. Evidence from case studies, for which chelation was used to lower blood lead levels in pregnant women and thus the fetus have shown that overall blood lead levels were reduced after a course of chelation therapy and that neonatal blood lead levels were lower at birth than peak maternal levels during pregnancy. The information with regard to the long term benefits and effects associated with chelation therapy during pregnancy is very limited. Chelation therapy may help protect both mother and the unborn fetus from high blood levels. However, because there have been no long term studies with regard to the treatment, pregnant women should exercise caution when making a decision with regard to chelation therapy (The Centers for Disease Control and Prevention, 2010).

Figure 1. Published experience of chelating agents during pregnancy in humans. (The Centers for Disease Control and Prevention, 2010).

There is no universally recognized or accepted medical treatment which was been suggested for pregnant women with high levels of lead in their blood. (Chelation therapy has been used for limited number of cases, but it is yet to be accepted as a universal treatment option). A simple blood test can be used to determine the amount of lead in the blood. If the levels of lead in the blood are elevated, the source of exposure in the environment needs to be identified, and the pregnant woman should try to avoid or eliminate that source of lead. Healthy eating habits and refraining from all contact with lead are measures which pregnant women can take to help protect their babies from the effects of lead (LEAD SAFE Illinois, 2012).
 * Ways to avoid impacts of lead exposure**

The Centers for Disease Control and Prevention (2010) has provided specific guidelines for pregnant women to help reduce the effects of lead exposure and the adverse effects that can result from it. These measures include: I. Being aware of lead in the home. Many homes that were built before 1978 may contain lead paints. If this paint peels or cracks, it can be breathed in. Pregnant women should avoid cleaning or remodeling a house with lead paint. II. Avoid certain hobbies or jobs. Activities such as construction or home repair in old homes, and battery recycling are associated with lead exposure. III. Consume foods with iron, vitamin C and calcium. Foods rich in these nutrients can help protect an unborn baby from the effects of high lead exposure. IV. Proper storage of food. Pregnant women should avoid storing food in containers such as ceramic pottery and brass containers that may contain traces of lead. They should also avoid cooking with utensils that have lead and using dished that are cracked or chipped.

Thalidomide Thalidomide was created in 1957 and was marketed as a sedative and antiemtic in more than 40 countries. Typically, a woman took thalidomide during weeks 3-8 of her pregnancy to treat morning sickness. The effects of thalidomide were not recognized until 1961. Over 10,000 children were estimated to have developmental defects including ear malformations, glaucoma, micropthalmia, kidney malformations and most commonly, limb malformations. Thalidomide has since been used to treat erythema nodosum leprosum, chronic graft versus host disease, rheumatoid arthritis, tumor angiogenesis and multiple myeloma. Thalidomide exists as two enantiomers, both of which were teratogenic in the murine model (Ito, Ando, and Handa, 2011).

The understanding of the mechanism of thalidomide teratogenicity has been hindered because non-primate animals are typically used in these experiments. Non-primate animals have different limb anatomy and seem to exhibit a limited range of malformations when exposed to thalidomide. In humans, phomocelia and amelia are the most common congenital malformations associated with thalidomide. Phomocelia and amelia are defined as malformation of the limbs and absence of limbs, respectively. Limb truncation has been observed in chickens and rabbits although embryo death is more likely. In chickens, angiogenesis was inhibited. Each species of lab animal is able to "withstand" different amounts of oxidative stress. The effects of thalidomide seem to be most significant during organogenesis (Reichard-Brown, Spinner, and McBride, 2009).
 * Description/Mechanism of Phenomenon**





After the seventh radial holoblastic cleavage, sea urchin cleavage becomes irregular allowing formation of macromeres in the animal hemisphere and micromeres in the vegetal hemispheres. The blastocoel begins forming at the 128-cell blastula stage. The blastula hatches from the fertilization envelope and primary mesenchyme cells (PMCs) form the synctial ring which then forms the skeleton matrix. Formation of the archenteron marks gastrulation. Then, arms grow (Reichard-Brown, Spinner, and McBride, 2009).
 * Sea urchin model**

Reichard-Brown et al. (2009) showed that sea urchins (//Lytechinus pictus//) exposed to thalidomide at 0, 6 and 12 hours post-fertilization exhibited abnormal archenteron formation, exo-gastrulation, elongated and misshapen anterior regions (when compared to the control), and arms positioned at wider angles than in the control. The arms also were asymmetrical when comparing left and right arms in a single organism. The arms also had varying lengths when comparing each of the embryos. Depending on the timing of administration of thalidomide, the number of embryo malformations varied.

The number of abnormal embryos was reduced by one-third when thalidomide was administered at 12 hours post-fertilization when compared to earlier administration of thalidomide. Administration of thalidomide at the blastula stage increased the number of abnormal embryos by 10% when compared to the control. When embryos were only exposed to thalidomide for 6 hours after post-fertilization, 55% (at 24 hours) to 72% (at 72 hours) of the embryos were abnormal. The embryos exposed the thalidomide from hours 2-6 after fertilization exhibited the highest percentage of abnormalities, 66% (at 24 hours) to 81% (at 72 hours). Morphogenesis of endoderm and mesoderm seem to be most disrupted in gastrulation and skeletogenesis. The sea urchin embryos were most sensitive to thalidomide between the 4 and 16 cell stage. The number of abnormal embryos increased as development progressed even when cleavage seemed to be normal (Reichard-Brown, et. al., 2009).

The exact processes which causes the developmental abnormality are unknown, but one possibility is that beta catenin is inhibited by thalidomide in some way. Beta catenin is responsible for signaling formation of the vegetal, rather than animal, hemisphere in the sea urchin. The formation of separate hemispheres is essential to later development. The vegetal hemisphere is responsible for production of both the endoderm and the mesoderm. Disruption of their formation can cause issues with musculo-skeletal formation as well as gut and respiratory system abnormalities. Another possibility set forth by Reichard-Brown includes an upregulation of BMP and DKK1, which are beta catenin and wnt antagonists. Up-regulation of these compounds could also cause the abnormalities seen in the sea urchin embryos as a result of thalidomide exposure ( Reichard-Brown, et. al., 2009; Beurel and Jope, 2006).

A third proposed mechanism is the oxidative stress hypothesis which suggests thalidomide causes reactive oxygen species (ROS) to develop. The ROS increase NF-kB activity. NF-kB then enters the nucleus of the cell and induces expression of //Fgf8// and //Fgf10//. Reduction in the amount of //Fgf8// and //Fgf10// has been shown to cause abnormal limb development in rabbits, though not in mice. It is believed that thalidomide inhibits the activity of the apical ectodermal ridge (produces signaling molecules essential for limb development) in rabbits, chicks and zebrafish through inhibition of //Fgf8//. The mechanism of inhibition has not been fully elucidated and needs further study to confirm current suspicions about the involvement of the apical ectodermal ridge (Hansen and Harris, 2004). Less than 3,000 peoploe born with birth defects caused by thalidomide are still alive. There is no way to reverse the effects of thalidomide so many are living with shortened and malformed limbs. Some of the severe limb malformations have been prevented by treating animals with alpha-phenyl-N-tertbutylnitrone (PBN). PBN was shown to inhibit the thaliomide limb malformations in rabbits by preventing inhibtion of //Fgf8// and //Fgf10//, which are both required for cell survival (Parman, Wiley, and Wells, 1999). PBN has also been shown to reverse the upregulation of bone morphogenic proteins (BMP) and Dickkopf-1 (Dkk1) induced by thalidomide. BMPs are expressed in and beneath the AER and pattern the limb. BMPs stabilize phosphotase and tensin homologue (PTEN) which inhibits the Akt survival pathway (Knobloch, Shaughness, and Ruther, 2007). BMPs aso inhibit //Fgfs// (in mice). NF-kB inhibits BMPs.

The half-life of thalidomide is between 2.4 and 5 hours (Neubert and Neubert, 1997). Doctors recommend women wait at least one month after thalidomide before trying to become pregnant. Embryo susceptibility to thalidomide is estimated to be 10-50% (Reichard-Brown, et. al., 2009). Since thalidomide was only used during the first trimester of pregnancy when experiencing morning sickness, no studies have been conducted to observe the effects of thalidomide on second and third trimester development. In addition, thalidomide can be ejaculated along with semen, thus being transmitted to the pregnant woman and ultimately the fetus. Additionally, thalidomide may also be transmitted through breast milk from mother to infant. Although there are no studies on the effects thalidomide may have on embryos, fetuses or infants in these situations, precautionary measures should be taken to ensure the safety of the children or potential children.
 * Ways to avoid impact**

The only way to reduce the impact of thalidomide on first trimester embryos and fetuses is by avoiding thalidomide altogether. Dietary changes, emotional support, acupuncture, ginger and pyridoxine (Vitamin B6) are often encouraged by health care providers as treatment for morning sickness before prescribing antiemtics. Antiemetics have been synthesized and shown effective in treatment of morning sickness with limited side effects, including prochloperazine (Compazine), chlorpromazine (Thorazine), promethazine (Phenergan) and ondansetron (Zofran). Other antihistamines and anticholinergics like diphenhydramine (Benadryl) and dimenhydrinate (Dramamine) have alternative uses which include the treatment of morning sickness. Metoclopramide (Reglan) and corticosteroids like methylprednisolone (Medrol) have also been used to reduce the nausea and vomiting associated with first trimester pregnancy (Quinlan and Hill, 2003). ** Conclusion ** Lead and thalidomide behave teratogenically by inhibiting normal development. Lead may affect brain development after 12 weeks of gestation while thalidomide inhibits limb formation. Contact with either substance during pregnancy should be avoided, particularly when the teratogenic effects are most potent (2-3 weeks and later for thalidomide and 12 weeks or later for lead). Because of the birth defects caused by thalidomide, drugs now must be tested on pregnant animals before they are given to pregnant humans. Post-drug administration surveillance systems have also been established which will hopefully notice problems like the ones seen with thalidomide much sooner than previous methods. These practices will hopefully lead to a reduced number of teratogens being marketed as morning sickness pills to unsuspecting pregnant women. 20% of women who had taken thalidomide between days 37-54 of their pregnancy delivered an infant with birth defects, which shows that teratogens should probably be avoided by anyone planning to become pregnant within the next few months to fully avoid the effects of certain teratogens like thalidomide.

Resources Primary resources in bold Bellinger D, Leviton A, Waternaux C, Needleman H & Rabinowitz M (1987). Longitudinal analyses of prenatal and postnatal lead exposure and early cognitive development. //New England Journal of Medicine,// 316:1037-1043 Beurel, E., and Jope, R.S. (2006). The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. //Progress in Neurobiology// 79:173–189.

The Centers for Disease Control and Prevention(2010). Guidelines for the identification and management of lead exposure in pregnant and lactating women. Retrieved from []

Carpenter, S. J. (1974). Placental permeability of lead. //Environmental Health Perspectives//, 7: 129-131.


 * Gilbert, S. F. (2010). //Developmental biology// (9th ed.). Sunderland, MA: Sinauer Associates, Inc.**

Goyer, R. A, Cherian, M.G, Jones, M. M & Reigart JR (1995). Meeting report on the Role of chelating agents for prevention, intervention, and treatment of exposures to toxic metals. //Environmental Health Perspectives//., 103 :1048-1053 Goyer, R. A (1996). Results of lead research: Prenatal exposure and neurological consequences. //Environmental Heath Perspectives,// 104: 1050-1054. Ito, T., Ando, H., and Handa, H. (2011). Teratogenic effects of thalidomide: molecular mechanisms. //Cellular and Molecular Life Sciences//, 68: 1569-1579. Knobloch, .J, Shaughnessy, J. D. Jr., and Ruther, U. (2007). Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway. //FASEB Journal//, 21:1410–1421.
 * Hansen, J.M., and Harris, C. (2004). A novel hypothesis for thalidomide induced limb teratogenesis: redox misregulation of the NF-kB pathway. //Antioxidants & Redox Signaling//, 6:1–14.**

Lan, L. M., Yamashita, Y., Tang, Y., Sugahara, T., Takahashi, M., Ohba, T., & Okamura, H. (2000). Normal fetal brain development: MR imaging with a half-fourier rapid acquisition with relaxation enhancement Sequence1. //Radiology,// //215//(1), 205-210.**

=LEAD SAFE Illinois. (2012)Effects of lead exposure during pregnancy. Retrieved from [] = Neubert, R., and Neubert, D. (1997). Peculiarities and possible mode of actions of thalidomide. //Handbook of Experimental Pharmacology,// 124(2): 506.

Organization of Teratology Information Specialists, OTIS (2010). Lead and Pregnancy.

Parman, T., Wiley, M. J., and Wells, P. G. (1999). Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. //Nature Medicine// 5:582–585.

Quinlan, J. D., and Hill, D. A. (2003). Nausea and vomiting in pregnancy. //American Family Physician,// 68(1): 121-128.
 * Reichard-Brown, J. L., Spinner, H., McBride, K. (2009). Sea urchin embryos exposed to thalidomide during early cleavage exhibit abnormal morphogenesis later in development. //Wiley InterScience//, 86: 496-505**

Tickle, C. (January 1991). Retinoic acid and chick limb bud development. //Development,// //113//(Supplement 1), 113-121.

** Wasserman, G. A, Liu X., Popovac ,D., Factor-Litvak, P., Kline, J., Waternaux, C., LoIacono, N. & Graziano, J. H2000. The Yugoslavia Prospective Lead Study: contributions of prenatal and postnatal lead exposure toearly intelligence. //Neurotoxicology and Teratolology,// 22:811– 818. **