Mechanisms of Teratogenesis

1. Mechanisms of Teratogenesis Lynda B. Fawcett, Ph.D. Assistant Professor of Pediatrics Thomas Jefferson University & Alfred I duPont Hospital For Children Wilmington, DE

2. Structural Anomalies/Dysmorphology • Malformation:the normal developmental process is altered such that a given structure cannot form or forms improperly, the error is intrinsic to the morphogenetic process itself. • Deformation: alterations to already existing structures. Alterations in body shape, form, or position. Often due to uterine constraint (twinning, oligohydramnios etc). ex: dislocation of the hip, clubfoot, and some facial anomalies • Disruption:alterations to already existing structures, often involving extensive destruction of tissue, and/or prevention of later tissue or organ formation. Can arise from mechanical or physiological factors. ex: amniotic band syndrome, fetal vascular occlusion, placental emboli, localized or general hypoxia, vascular insults.

3. Disruption: Syndactyly/adactyly Hypoxia, hemorrhage, necrosis ROS Usually asymmetrical EX: Cocaine CVS

4. Mechanism of action Mode of Action Outcomes cell death altered cell-cell interactions cell signaling reduced biosynthesis impaired cell migration reduced/impaired proliferation altered differentiation ROS/Lipid peroxidation DNA damage Altered gene expression Metabolic disturbance Enzyme inhibition Growth factor imbalance Receptor agonist/antagonist Malformation Deformation Death Functional deficit Growth retardation Pathogenesis

5. Cytotoxic Teratogens • Irradiation • 2’Deoxyadenosine • 4-CP (metabolites) • Hyperthermia • Ethanol Whether a toxicant induces cell death depends on dose and timing of exposure as well as the nature of the toxicant

6. Apoptosis Necrosis The blebs fuse and become larger, no organelles in blebs The nucleus begins to break apart, DNA breaks into small pieces organelles in to blebs Blebs The cell membrane ruptures and releases the cells contents, the organelles are not functional The cell breaks into several apoptotoc bodies the organelles are still functional

7. Apoptosis SIGMA-ALDRICH Receptor mediated - Extrinsic (TNF Signaling) Mitochondrial – Intrinsic CytC, caspase 9

8. Characteristics of Cell Death Mediated Teratogenesis: Replication associated cytotoxicity • Tissue susceptibility changes over time, however usually rapidly dividing undifferentiated tissues are most affected with agents that induce direct cytotoxicity • Tissue sensitivity greatest during organogenesis in the neuroepithelium, limb buds and least in heart and yolk sac • Characteristic malformations include NTDs, anopthalmia, limb defects but, generally, not heart defects (time dependent). • Occurs because • DNA more accessible in S phase • Defects manifest sooner, critical mass, windows of oportunity • Molecular mechanisms

9. Tissue sensitivity: Irradiation/mouse studies/organogenesis • Molecular basis for tissue sensitivity may be due to differential expression of P53 in tissues: • Temporal/spacial expression of P53 mirrors that of tissues most effected by cytotoxic teratogens • Irradiation caused activation of P53 in rapidly proliferating, undifferentiated tissues (used a lac Z reporter model). Neuroepithelium, limb buds, arches (Gottli et al, 1997)

10. P53 Cell death Repair

11. P53 KO studies Consistent findings: +/+ apoptosis, cell cycle check G1/S +/- apoptosis and necrosis -/- necrosis, no cell cycle check (M,S)

12. Replication associated cytotoxicity Open NT, reduced tissues Anopthalmia Growth retardation Control 11.5 d AA deficiency (Met)

13. Demethylating agents. Whole Mount Immunostaining: 165 kD NF Control -AA (Met) CL (-SAM) NTD only ?165 Kd NF Mechanism probably more specific, not general cytotoxicity NTD GR, Reduced: Size Protein Somite Pairs limb buds Anopthalmia No heart defects

14. Effects of Homocysteine on Rat Embryos Cultured in Vitro from 9.5-11.5 p.c; Cardiac Defects Control Hcy Hcy Control

15. Effects of Hcy on Rat Embryos Cultured in Vitro from 9.5-11.5 p.c: Somite/Vertebral/Arch Abnormalities (450 g/ml) control

16. Although defects such as NTDs may result from death, other mechanisms can also result in NTD • Specific mechanisms of action • Receptor mediated - directly or indirectly • Display some type of ligand specificity (enzyme) • downstream alterations in cell signaling (growth factors; differentiation) • altered expression of key genes involved in morphogenesis. • Closure defects have discrete window of susceptibility, and less chance for recuperation. for NTD reduced proliferation in neuroepithelium, failure of neural fold elevation, failure of neural fold fusion due to specific mechanisms may resemble cytotoxicity at term

17. Tissue specificity and TeratogenesisSpecific Mechanism: Usually receptor mediated Only affect tissues bearing appropriate receptors: ex: sex steroids - genitourinary

18. Receptor-Mediated Teratogenesis

19. Retinoic Acid • Accutane 13-cis retinoic acid, used for dematological and oncology therapies RAE: Spectrum of defects and potential for all organ systems to be affected Most common defects are craniofacial, external ear (BA 1-2)

20. Specificity & Teratogenesis: RA • Vitamin A derivatives involved in signaling pathways during normal development - Morphogen; directly alter/control developmental programs. • 1) Induces differentiation or apoptosis of various cell types; breast carcinoma, prostate cancer, AL • 2) Produces homeotic mutations in experimental animals (limb duplications) • Proposed Mode of action for defects: • Promotes excessive cell death in regions undergoing programmed cell death (Alles & Sulik, 89, 90 etc) • Alters specification of tissues (NC); HOX genes • Alters expression of other RARE genes

21. RA receptors • RAR  (all trans and 9-cis); RXR(9 cis) • Multiple isoforms exist for all RAR • RAR/RXR form heterodimers; RXR homodimers, heterodimers with thyroid hormone R, peroxisome proliferators and others • RAR, RXR differentially expressed in embryo; temporal and spatial pattern • RA interacts with other receptors (CRABP)

22. CRABP1 RA RA RA RA RA RAR/RXR CRABP-II () RA Other involvment CRABP1, II Biosynthesis RA ADH - ETOH hypervitamitosis vitamin deficiency Transcription RARE AP-1 transcription factor block RAR/RXR Nucleus

23. Limb • RAR -/- Vitamin A deficiency, Resistant to RA induced limb defects. Agonists RAR>RAR>RAR for inducing limb defects in wild type • RAR -/- Normal, Resistant to BA 1-2 fusion by RA +/+ RAR agonist caused BA fusions RAR upregulated in limbs following RA • RAR -/- Vitamin A deficiency defects, normal craniofacial and posterior axial skeleton; resistant to RA induced posterior axial skeleton truncation but not cranial, partial resistance to other except limb • RXR -/- Lethal, cardiac. Resistant to RA induced limb defects Craniofacial Posterior Cardiomorphogenesis (Matt et al., 2003; Collins and Mao, 1999)

24. Chromatin condensation suppresses RAREs (RARß2) VPA Potential for increased RARß2; destabilization of chromatin; Inappropriate expression of other genes (p21; p53 – cell death) RARE

25. Summary • Teratogenic mechanisms can be broadly separated into specific (receptor mediated) and non-specific (cell death) • Teratogens can have both specific and non-specific mechanisms (alcohol) • Even general cytotoxic/genotoxic teratogens may have a molecular component that alters tissue susceptibility • Initial molecular mechanisms have the potential to result in myriad responses; cell death, differentiation, altered signaling. Pathogenesis must be considered when evaluating the action of teratogens • Understanding the molecular mechanisms of teratogens furthers our understanding of development, and allows for the development of therapeutics with reduced chance of teratogenicity