Thalidomide as a teratogen
Thalidomide was developed as a sedative for pregnancy and a means of preventing nausea. When tested on rodents, there seemed to be no teratogenic consequences to the offspring. In the early 1960s, McBride and Lenz independently made the association of thalidomide and limb defects, and it is now known that over 10,000 children were been affected by this drug. The most obvious anomalies are in the limbs and ears, but abnormalities of eye, heart, kidney, and neuronal development are also seen.
The mechanisms for thalidomide’s mode of action in causing these congenital anomalies remain controversial (see Vargesson 2009; Ito et al. 2011). This is because:
- There may be multiple actions. (As we saw in our discussion of fetal alcohol syndrome, ethanol can cause malformations in several ways.)
- Thalidomide is interconverted rapidly into two isomeric forms. The R (+) isomer appears to have sedative effects but is not teratogenic; the S (–) form appears to be teratogenenic.
- Thalidomide is metabolized into over a dozen different products. Some of these products may be teratogenic.
- Thalidomide can produce congenital anomalies in chicks, zebrafish, and rabbits, as well as in primates. (However, it did not cause birth defects in mice and rats, which were the predominant test animal.) The manner in which thalidomide produces these results may be different in the different species.
The mechanism of thalidomide action was postulated to include:
- Inhibition of angiogenesis
- Induction of cell death by reactive oxygen species (oxidative stress)
- Antagonism of cell adhesion
- Misregulation of paracrine factor signaling
- Specific effects on chondrogenesis
The two prevalent hypotheses are presently the oxidative stress hypothesis and the angiogenic disruption hypothesis.
The oxidative stress hypothesis
This hypothesis was first put forth by Parman et al. (1999), is based on the ability of thalidomide to induce reactive oxygen species (ROS) chemicals in the limb. These chemicals oxidize DNA and proteins, leading to cell death. When a scavenger of ROS (phenyl-tert-butylnitrone, PBT) was added in vitro to the cells receiving thalidomide, both the ROS agents and the limb defects were alleviated. Moreover, thalidomide does not cause ROS increases or limb cell death in mice.
Further support for the oxidative stress hypothesis came from studies of paracrine factors involved in limb development. Hansen et al. (2002; Hansen and Harris 2004) showed that thalidomide blocked the production of Fgf8 and Fgf10 in the limb. This loss could be produced by cell death or by the activation of the transcription factor NF-kb by oxidative stress. This transcription factor blocks Fgf gene expression. The effects of thalidomide on this expression of Fgf can be alleviated by PBN. Subsequent research showed that thalidomide also blocks Wnt signaling and upregulates BMP signaling (Knobloch et al. 2007; 2008), and that its ability to perturb these signals involves oxidative stress. Thus, thalidomide may create reactive oxygen species that misregulate the paracrine factors in the limb bud. This can lead to cell death and/or the stalling of limb growth.
The angiogenesis inhibition hypothesis
In 2009, Therapontos and colleagues (2009) demonstrated that the main target of thalidomide were the immature blood vessels. They showed that thalidomide blocked the stabilization of new blood vessels and that the time-sensitivity of thalidomide defects correlated with the time that these immature blood vessels were forming. They were able to use the chick as a model system for limb development, and were also able to use a single form of thalidomide, the tetrafluorinated analogue, CPS49. This analogue, which resembles certain metabolic breakdown products of thalidomide, is known to produce severe limb defects. When the blood vessels were beginning to form (and before they had a smooth muscle shell), CPS49 could induce the disaggregation of the blood vessels. This occurred about a day before limb growth was obviously stunted. Vessels were seen to have regressed 6 hours after treatment with CPS49, and by 24 hours, the limb bud was severely stunted. The defects in vascular development were seen prior to cell death and paracrine signaling abnormalities. It appears the CPS49 prevents the endothelial cells from migrating and forming the tubes that are critical in angiogenesis. Once the smooth muscles cover the tubes, CPS49 has no effect. This period of angiogenesis correlates with the temporal window of time where CPS49 can do damage. It appears that blood vessel loss may be the trigger leading to increased cell death and misregulation of the paracrine factor pathways necessary for limb bud formation.
Identification of a thalidomide-binding protein
The mechanism(s) by which thalidomide causes congenital defects would be easier to study if the cell moiety that bound thalidomide could be discovered. One such molecule has been is isolated. When thalidomide-coated beads are incubated with human cell extracts, they collect a protein called cereblon (CRBN). Cereblon is a part of the E3 ubiquitin ligase complex that adds ubiquitin protein to other proteins, thus targeting them for destruction. Thalidomide may block this protein-destruction pathway. There is some evidence that CRBN may play a specific role in the antioxidant pathway, and there is also evidence that it might contribute to angiogenesis (see Ito et al. 2011.) The presence and quantity of CRBN has been associated with the ability of thalidomide to improve the clinical outcomes of cancer patients treated with thalidomide, as well.
The identification of the thalidomide-binding protein, and the ability to synthetically produce analogues of thalidomide that are anti-inflammatory (i.e., that can help cancer patients and patients with Crohn’s disease and leprosy) but not teratogenic may allow more people to benefit from this drug (Mahony et al. 2013). It may also allow us to return to explain the older observations that don’t fit into either of the current models (see Lash and Saxén 1971, 1972). Moreover, it would be fascinating if science were to convert one of the major villains of embryos into a hero of adults.
Hansen, J. M., Gong, S. G., Philbert, M., and Harris, C. 2002. Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide. Dev Dyn. 225: 186–194.
Hansen, J. M., and Harris, C. 2004. A novel hypothesis for thalidomide-induced limb teratogenesis: redox misregulation of the NF-kappaB pathway. Antioxid Redox Signal. 6: 1–14.
Heintel, D., Rocci, A., Ludwig, H., Bolomsky, A., Caltagirone, S., Schreder, M., Pfeifer, S., Gisslinger, H., Zojer, N., Jäger, U., and Palumbo, A. 2013. High expression of cereblon (CRBN) is associated with improved clinical response in patients with multiple myeloma treated with lenalidomide and dexamethasone. Br J Haematol. 161: 695–700.
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Knobloch, J., Shaughnessy, J. D. Jr, and Rüther, U. 2007. Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway. FASEB J. 21: 1410–1421.
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Parman, T, Wiley, M. J., and Wells, P. G. 1999. Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nature Med. May; 5(5): 582–5.
Therapontos, C., Erskine, L., Gardner, E. R., Figg, W. D., and Vargesson, N. 2009.
Thalidomide induces limb defects by preventing angiogenic outgrowth during early limb formation. Proc Natl Acad Sci U S A. 106: 8573–8578.
Vargesson, N. 2009. Thalidomide-induced limb defects: resolving a 50-year-old puzzle. Bioessays. 31: 1327–1336.