Prenatal Toxicity and Molecular Mechanisms of Teratogens

Introduction to Prenatal Toxicity and Teratogenicity

• Prenatal toxicity is the focus of this second lecture on toxicology, specifically examining how exposure to human teratogens can cause irreversible structural defects in newborns.

• A human teratogen refers to any agent capable of causing changes to the structure or growth of an embryo or fetus, leading to birth defects.

• The lecture emphasizes the molecular mechanisms underlying two specific human teratogens: Thalidomide and Alcohol.

• Contextualizing the field:

  • Embryology: The study of the development of embryos and fetuses.

  • Teratology: A branch of embryology that specifically studies the abnormal development of embryos and fetuses.

  • Developmental Toxicity: Studies the toxicities caused to developing embryos and fetuses, which includes embryo toxicity, embryo lethality, and teratogenicity.

  • Teratogenicity: The ability of an agent to cause irreversible structural alterations linked to birth defects.

Types and Examples of Human Teratogens

• There are various categories of human teratogens, similar to how there are different types of toxicants.

• Examples of human teratogens include:

  • Drugs: Specifically thalidomide and certain antibiotics.

  • Foodstuff contaminants: Lead is a prominent example. It has been used to adulterate turmeric powders to improve appearance and increase the sale price. Children consuming this turmeric are exposed to lead toxicity.

  • Viruses.

  • Physical constraints.

  • Diabetes: This maternal condition can act as a human teratogen.

• Antibiotics Caveat: One particular class of antibacterial drugs acts as a human teratogen. These should not be used during pregnancy or prescribed to children under the age of $8$ because they bind with high affinity to calcium-rich tissues such as teeth and bones.

Gestational Periods and Sensitivity Windows

• The sensitivity of a developing embryo or fetus to teratogens depends heavily on the specific period of gestation.

• The first two weeks: During this stage, cells are dividing. The outcome of exposure is typically "all or nothing," meaning the embryo either survives with no harm or it dies.

• Week $3$ to Week $8$: This is the period of organogenesis (the formation of organs). This period is the most sensitive to human teratogens.

  • Different organs have specific windows of sensitivity.

  • For thalidomide, the critical window is between Day $21$ and Day $36$ post-conception.

• Week $9$ to Birth: This period is generally less sensitive. However, exposure during this time most likely results in functional disturbances, where organs may not be fully functional.

• Central Nervous System ($CNS$) Development: The development of the nervous system spans the majority of gestation. Consequently, there is no safe window for alcohol consumption during pregnancy; the best practice is total avoidance.

Fetal Exposure and the Placental Barrier

• The placenta is only a partial barrier and does not completely prevent the exposure of the fetus to xenobiotics (foreign chemicals).

• Chemicals can cross the placenta through passive diffusion or the use of transporters.

• Transporters on the fetal capillary can pick up xenobiotics, while efflux transporters can return drugs back to the maternal side for protection.

• Fetal Metabolism: The activity of drug-metabolizing enzymes (such as $CYP2E1$, Alcohol Dehydrogenase, and Aldehyde Dehydrogenase) is very low or non-existent in the developing fetus.

• Because of this limited elimination capacity, the fetus has a limited ability to remove harmful chemicals, though some enzymes may be induced by maternal alcohol consumption.

• Therapeutic Categorization: The Therapeutic Goods Administration ($TGA$) classifies medicines based on safety during pregnancy. Category $X$ is the classification for drugs definitely known to cause birth defects and must not be used during pregnancy (e.g., thalidomide).

Thalidomide: History and Landmark Studies

• History: Introduced in the late $1950s$ as a safe alternative to barbiturates (which were addictive at the time). It was used to treat morning sickness in pregnant women.

• Emergence of Toxicity: Early reports of peripheral neuropathy (nerve damage) occurred, but the major crisis began in the early $1960s$ with reports of severe birth defects in Germany, Australia, and the UK.

• Global Impact: Over $10,000$ babies were born with defects. The drug was withdrawn between $1962$ and $1967$. The delay was partly due to the drug being marketed under various brand names in different countries, which highlights the clinical importance of using generic names.

• United States Exception: The $FDA$, specifically Doctor Francis Kelly, was dissatisfied with the quality of animal safety data and never approved the drug in the $US$, preventing widespread defects there.

• Typical Defects: The primary defect is limb malformation, including shortened limbs or no limb formation (amelia). It can also affect the eyes, ears, and internal organs like the heart and kidneys.

Molecular Mechanisms of Thalidomide Teratogenicity

• Early Hypotheses:

  • Anti-angiogenesis: Inhibition of new blood vessel formation, cutting off blood supply to developing limbs.

  • Reactive Oxygen Species ($ROS$): Development of oxygen radicals causing cell damage.

• The $2010$ Science Study (Zebrafish Model):

  • Researchers used zebrafish to study fin malformation and identified the thalidomide-binding protein as Cereblon ($CRBN$).

  • Cereblon is part of an $E3$ ubiquitin ligase complex comprised of four proteins: Cereblon ($CRBN$), Cullin-$4$ ($CUL4$), DNA Damage-Binding Protein $1$ ($DDB1$), and Regulator of Cullins $1$ ($ROC1$).

  • Mechanism of Action: Thalidomide acts as a "molecular glue." When it binds to Cereblon, it causes a conformational change in the $E3$ ubiquitin ligase complex. This change causes the complex to recognize a completely different set of substrates, known as "neo-substrates."

  • Ubiquitination and Proteolysis: The primary function of this complex is to ubiquitinate proteins so they can be degraded via proteolysis.

  • Transcription Factor $SALL4$: This factor is critical for fetal limb development. In humans, thalidomide binding leads to the ubiquitination and subsequent degradation of $SALL4$.

  • Inter-species Differences: Rodent $SALL4$ and human $SALL4$ differ by a single amino acid. This single difference makes rodent $SALL4$ resistant to degradation by the complex, explaining why thalidomide appeared safe in rodent studies.

• Repurposing Thalidomide: The drug and its derivatives are now used as immunomodulatory drugs to treat multiple myeloma. They cause the degradation of transcription factors $IKZF1$ and $IKZF3$, which reduces plasma cell differentiation.

Alcohol: Fetal Alcohol Spectrum Disorder ($FASD$)

• Alcohol exposure is the leading cause of preventable birth defects.

• Fetal Alcohol Syndrome ($FAS$): First recognized in $1973$ and diagnosed extensively by $1990$, it is the most severe form of $FASD$.

• Three Clinical Domains for $FAS$ Diagnosis:

  1. Facial defects (e.g., a thin upper lip).

  2. Growth retardation.

  3. Impaired neurodevelopment (often evident at school age).

Metabolism and Biomarkers of Alcohol Consumption

• Diagnosis of alcohol exposure often relies on biomarkers because maternal self-reporting is frequently inaccurate due to under-reporting.

• Four key biomarkers formed through specific metabolic pathways:

  1. Ethyl glucuronide: Formed via $UGT$ (UDP-glucuronosyltransferase); detected in maternal urine.

  2. Ethyl sulfate: Formed via sulfotransferase; detected in maternal urine.

  3. Fatty acid ethyl esters ($FAEEs$): Formed by $FAEE$ synthase (non-oxidative mechanism); detected in hair and meconium (fecal matter). These have a longer duration in the body.

  4. Phosphatidylethanol: Formed via Phospholipase $D$ ($PLD$). Ethanol is incorporated into phospholipids on red blood cell membranes. It has a half-life of $5$ to $12$ days and can be detected in maternal blood $2$ to $4$ weeks after consumption.

Oxidative and Non-Oxidative Mechanisms of Ethanol Toxicity

• Oxidative Mechanisms:

  • Involves $CYP2E1$ and Alcohol Dehydrogenase ($ADH$).

  • Leads to the formation of Reactive Oxygen Species ($ROS$) and hydroxyl radicals.

  • Results in lipid peroxidation (cell membrane damage), protein adducts (malfunction), and $DNA$ adducts (mutagenesis).

• Non-Oxidative Mechanisms:

  • Involves the formation of Fatty Acid Ethyl Esters ($FAEEs$) and Phosphatidylethanol.

  • $FAEEs$ cause direct cell damage.

  • Phosphatidylethanol causes disruption in cell signaling due to its incorporation into phospholipids.

• Both pathways ultimately lead to cell death and the structural or functional birth defects characterized as teratogenicity.

Questions & Discussion

• Brand Names vs. Generic Names: The speaker noted that brand names are not used by pharmacologists because they vary by country. The first lecture test included a question assessing the understanding of the three types of drug names.

• Future Assessments: The speaker mentioned that for the second lecture test and final exam, complex short-answer questions would represent the majority of the marks, covering mechanisms like those discussed today.