4 manifestations of developmental toxicity • Structural malformations • Growth retardation • Functional impairment • Death of the organism
Teratology 1. the study of malformations or serious deviations from the normal type in organisms 2. the branch of science concerned with the production, development, anatomy, and classification of malformed fetuses. • Teratogen • Any agent that causes a birth defect • After Greek “monster creating”
Environmental conditions (1200) • Maternal nutritional deficiencies (1930) • Rubella virus infection (1941) • Thalidomide (1961)
Adverse Outcomes in Pregnancy • 57% of pregnancies detected by hCG at 8-9 days after fertilization do not develop as clinically detectable pregnancies • 15-20% of recognisable pregnancies end in spontaneous abortion – 90% in the first trimester • 2% of pregnancies end in miscarriage >20 weeks. • 2-3% of newborn have a major malformation severe enough to require hospitalisation.
Cause of human birth defects • 15-25% genetic cause • 4% maternal conditions • 3% maternal infections • 1-2 deformation (mechanical problem) • <1% chemical and environmental influences • 65% unknown etiology
Chemical teratogenicity • >4100 chemicals have been tested • 66% non-teragenic • 7% teratogenic more than one species • 18% teratogenicin most species tested • 9% equivocal experimental results • 50-60 chemicals or conditions alter prenatal development in humans (Table 10-1)
Pregnancy Risk Categories From: A Textbook of Psychopharmacology. Ed. by Schatzberg and Nemeroff. American Psychiatric Publishing Company, 2004.
Therapeutic Drugs Teratogenic to Humans • Anticonvulsants • Phenytoin, primidone, trimethadione, valproic acid, carbamazepine • Anticancer agents • Alkylating agents –busulfan, cyclophosphamide, chlorambucil, mechlorethamine • Antimetabolites-aminopterin, methotrexate, cytarabine • Androgenic hormones-danazol • Coumarin anticoagulants-warfarin • Retinoids-accutane, isotretinoin, etretinate, acitretin • Antihyperlipidemic agents-lovastatin, atorvastatin • Other drugs-diethystilbestrol, thalidomide, penicillamine, lithium, fluconazole, misoprostol
Thalidomide • Susceptible period- 20-36 days after fertilization • Proposed mechanisms (>24) • Embryonal DNA oxidation (PBN can prevent) • Misregulation of the expression of genes critical for outgrowth of limb • The inability of NF-kappaB, a redox-sensitive transcription factor, to bind to its DNA promoter results in the failure of limb cells to express fibroblast growth factor (FGF)-10 and twist in the limb progress zone mesenchyme, which in turn attenuates expression of FGF-8 in the apical ectodermal ridge.
Diethylstilbesterol (DES) • DES was prescribed between 1940 and 1970 to prevent miscarriages in high risk pregnancies. • DES increases estrogen and progesterone synthesis by the placenta. • In the mid 1970 cases of vaginal adenocarcinoma in women ages 16-20 were linked to fetal exposure through maternal DES ingestion early in the pregnancy. • Female children - vaginal and cervical carcinomas, uterine anomalies. • Male offspring - epididymal cyst, hypotrophic testes, decreased semen volume and poor semen quality.
Alcohol (Ethanol) Fetal Alchohol Syndrome (FAS) Fetal Alchohol Effects (FAE) • Cranial facial dysmorphism • Intrauterine and postnatal growth retadation • Retarded psychomotor and intellectual development • IQ 68
Tobacco smoke • Spontaneous abortions • Perinatal deaths • Lower birth weight • Increased risk of • Sudden infant death syndrome • Behavioral attention disorders • Orofacial cleft (particular xenobiotic gene polymorphisms) • Gastroschisis (with variant alleles N053, ICAM1, NPPA) • Branching morphogenesis and maturation of the lung • Nicotine-related adverse nerodevelopmental outcomes
Cocaine • At risk for premature labor, spontaneous abortion, increased perinatal mortality and fetal death. • intrauterine growth retardation, microcephaly, altered presencephalic development, decreased birth weight, a neonatal neurologic syndrome of abnormal sleep, tremor, poor feeding, irritability, and occasional seizures. • Genitaouinary tract malformation • Impaired uditory process
c acid Retinoic acid is the active ingredient in “Accutane”, a drug used to treat severe acne. Since its introduction in September of 1982, an estimated 160,000 women of child bearing age have ingested the drug. Between 1982 and 1987, approximately 900-1300 malformed children, 700-1000 spontaneous abortions and 5000-7000 elective abortions are due to Accutane exposure. Exposed children may have hydrocephaly, ear malformations, cardiovascular defects and decreased IQ. Accutane carries a pregnancy category X warning, meaning it is a known human teratogen.
Retinoids • Malformations of the face, limbs, heart, CNS, and skeleton • RXR α receptor • Schizophrenia
RAR and RXR (Simple Version) • Nuclear Receptors (like ER, PPAR, VDR and others) • RXR/RAR Heterodimer is functional unit • Bind selectively to REs in genome • Act as transcription factors • Up-regulate or Repress the expression of particular genes
Valproic acid was released in 1967 in Europe and in 1978 in the United States to treat epilepsy. Approximately 11,500 epileptic women become pregnant each year, many of which use valproic acid. By 1980, publications began linking malformed children to in utero exposure to valproic acid (greater than 500 mg/day).
Valproic Acid • spina bifida withmenigomyelocele or menigocele • The proposed mechanism of action is that valproic acid influences folate metabolism
Angiotensin Converting enzyme inhibitors and angiotensin antagonists • 2-3 trimester • related reduced amniotic fluid volume and impaired fetal renal function • Oligohydromnios • Fetal growth retardation • Pulmonary hypoplasia • Renal failure • Hypotension • Death • First trimester • Congenital malformation
Wilson’s General Principles of Teratology (Table 10-2) • Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with environmental factors. • Susceptibility to teratogenic agents varies with the developmental stage at the time of exposure. • Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis). • The final manifestations of abnormal development are death, malformation, growth retardation, and functional disorder. • The access of adverse environmental influences to developing tissue depends on the nature of the influences (agent). • Manifestations of deviant development increase in degree as dosage increases from the no-effect to the totally lethal level.
Critical periods of susceptibility and endpoints of toxicity • Gametogenesis and Fertilization • Mechanism unclear, may be related to • imprinting • Cytosine methylation and change in chromatin conformation • 受精後6hr暴露ethylene oxide, ethylmethane sulfonate, ethylnitrosourea→malformed fetus
DNA Methylation • Methyl groups may be attached to cytosine (C5 position) • Methyltransferases • Methyl groups provide a tag concentrated in CG-rich domains, often in promoter regions • Maintains a gene in inactive state rather than initiating gene repression – Example: • Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation • Implantation – a new wave of methylation occurs • Early Zygote – most methylation tags removed
DNA Methylation vs Genomic Imprinting • Certain genes are active or inactive during early development • Depending on whether they are paternal or maternal genes • Eg – IGF-2 is only active in the gene from the male parent • The gene is imprinted according to parental origin • Mammalian genome has > 100 imprinted genes in clusters • The majority of imprinted genes in mammals have been found to have roles in the control of embryonic growth and development, including development of the placenta • Imprinted due to selective methylation of one of the alleles
2.Preimplantation著床前期 (blastocyst) 囊胚形成，細胞分裂到1000個細胞，僅3個細胞將發育成胎兒，餘發育成胎盤等支持組織，在此期暴露，理論上不影響或稍微影響胎兒生長，不然就導致死胎。 DDT, nicotine, methylmethane→body and/or brain weight deficits and embryo lethality but not malformation 然而, Methylnitrosourea, cyproterone→malformation
3. Implantation 著床 第6-13days 4. Gastrulation-三胚層形成, 第3週 在此期暴露有害物質將造成眼、腦及臉部的畸形
5. Organogenesis 器官形成，第3-8週 • 為最容易受影響的時期，因為本期 • Cell proliferation • Cell migration • Cell-cell interactions • Morphogenetic tissue remodeling
6. Fetal period胎兒期 第8wk-birth 在此期暴露，影響生長和功能的成熟，需要在出生後仔細觀察才能察覺。如中樞神經的異常包括行為、智力、運動的缺失，生殖力降低，以及免疫系統、心臟、肺臟、腎臟功能受損等。 *若有構造的改變乃是破壞原本正常的構造稱為deformation，不同於前述malformation
Critical periods of sensitivity for induction of various defect by retinoic acid in the hamster
Mechanisms and pathologenesis of developmental toxicology • Mutations • Chromosomal breaks • Altered mitosis • Altered nucleic acid integrity or function • Diminished supplies or precursors of substrates • Decreased energy supplies • Altered membrane characteristics • Osmolar imbalance • Enzyme inhibition
Example of cyclophosphamide (CP) A teratogenic chemotherapeutic agent Damage to DNA inhibit cell cycle progression cell cycle arrest too long apoptosis Bind to protein Single strand DNA break
CP induces DNA damage (predominant occur in S phase) leading to Cell cycle perturbation Cell death Sensitivity is determined by cell cycle length and cell predisposition to apoptosis
Cell death in the neural tube by CP Sensitivity to CP-induced cell death Neuroepithelium >heart Cell cycle length 9.5 hr vs 13.4 hr (longer Go/G1)
Advances in the Molecular basis of dysmorphogenesis 1.Using either singly or double gene knockout Retinoic acid receptor family (syndactyly) 2. Antisense oligonucleotide Wnt-1, Wnt-3a (mid and hindbrain malformation) 3. Reporter transgenes RA activate hoxb-1-lacZ
Pharmacokinetics and metabolism in pregnancy 1.Changes in maternal physiology hepatic metabolism, GI tract, cardiovascular system, excretory system, respiratory system 2.Overall decrease in hepatic xenobiotic transformation 3.Roles of placenta in influence embryonic exposure help to regulate blood flow -offer a transport barrier-pH gradient, weak acid rapidly transfer -metabolize chemicals 2-acetylaminofluorene (proteratogen) 7-hydroxyl metabolites(proximate teratogen) 4.Maternal metabolism of xenobiotics 2-methoxyethanol 2-methoxyacetic acid
Maternal factors affecting development • Genetics • high incidence of cleft lip/palate in white mother • Disease-chronic hypertension • diabetes • infection-cytomegalovirus, Taoxoplasma gondii • Hyperthermia-CNS malformation • Nutrition-folate neural tube defect • Stress-noise, restraint • Placenta toxicity -46 toxicants, Cd
Placental toxicity • Metals, Cd, As, Hg, ethanol, cocaine, cigaratte, sodium salicylate • Maternal injection vs fetal injection of Cd • Production of metallothionein • Interaction with Zn