Copyright 2008, The Johns Hopkins University and James Yager. All rights reserved. Use of these materials permitted only in accordance with license rights granted. Materials provided “AS IS”; no representations or warranties provided. User assumes all responsibility for use, and all liability related thereto, and must independently review all materials for accuracy and efficacy. May contain materials owned by others. User is responsible for obtaining permissions for use from third parties as needed. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike License . Your use of this material constitutes acceptance of that license and the conditions of use of materials on this site.
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Lecture 7: Reproductive and Developmental Toxicology (Yager)
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Copyright 2008, The Johns Hopkins University and James Yager. All rights reserved. Use of these materials permitted only in accordance with license rights granted. Materials provided “AS IS”; no representations or warranties provided. User assumes all responsibility for use, and all liability related thereto, and must independently review all materials for accuracy and efficacy. May contain materials owned by others. User is responsible for obtaining permissions for use from third parties as needed.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike License. Your use of this material constitutes acceptance of that license and the conditions of use of materials on this site.
Primary FollicleThis section is from an ovary of a woman of childbearing age. It demonstrates typical swirling ovarian stroma and primary follicles, each of which consists of a primary oocyte surrounded by a single layer of cuboidal epithelial cells (granulosa cells). Two of the primary follicles are well- preserved and 2 have been distorted in tissue processing.
Image used with permission from Brown Medical School Digital Pathology. All Rights Reserved.
16
Reproductive Tissue - Ovary
17Exposure
Control
60
50
40
30
20
10
300 mg/kg 500 mg/kg
Number of small folliclesNumber of medium folliclesNumber of large follicles
Ovarian Follicle counts in MiceFed Nitrofurantoin for 43 weeks
– Illness during pregnancy/parturition : toxemia: Hemorrhage
– Early fetal loss (to 28 weeks)
– Late fetal loss (after 28 weeks)/stillbirth
– Intrapartum death– Death in first week– Decreased birth
weight
– Gestational age at delivery; prematurity; postmaturity
– Altered sex ratio; chromosome abnormalities
– Multiple births; birth defects
– Infant death– Childhood morbidity;
childhood malignancies
Section B
General Principles of Developmental Toxicity
21
A birth defect is "any anomaly, functional or structural, that presents in infancy or later in life and is caused by events preceding birth, whether inherited, or acquired."
From: Whorton et al. Infertility in Male Pesticide Workers, The Lancet, Dec. 17, 1977, 1259-1261.
38
Comparison of Non-Vasectomized DBCP Workers with Very Low (A)
and Normal (B) Sperm Counts
Group A Group B
Subjects (n) 11 11
Age (yr) 37.7 26.7
Exposure (yr) 8.0 0.08
Sperm Count (x106/ml) 0.2 93
FSH (mIU/ml) 11.3 2.6
LH (mIU/ml) 28.4 14.0
Testosterone (ng/dL) 459 463
39
Neuroendocrine Feedback
Hypothalamus
40
Proposed mechanism of prokaryotic and eukaryotic
toxicity
1,2-Dibromo-3-Chrolopropane (DBCP) Metabolism and Mechanisms of Toxicity
Prokaryotic mutations2-BromoacroleinP450
CH2 Br CHBr CH2 R R: Cl or
GSH S-Transferase
Episulfonium ion Eukaryotic DNA damage
Cell necrosis
C C Ch2Br
OH
O P (O CH2 Br CH2 Br)1 or 2
O
H C C C ClH H H
S+ HG
(S-9)
Section D
Estrogens
42
Molecular Structures
OHHO
HO
OH
Estradiol (E2)
Diethylstilbestrol (DES)
43
Diethylstilbesterol (DES)
Clinical use and adverse human health effects– Prevention of spontaneous abortion– Appearance of clear cell
adenocarcinoma of the vagina and cervix in exposed offspring of mothers treated during pregnancy
– Case-control study
44
No.Maternal Age (yr) Bleeding In This
PregnancyAny Prior
Pregnancy LossEstrogen Given In
This Pregnancy
Case Mean of 4 Controls Case Control Case Control Case Control
1 25 32 No 0/4 Yes 1/4 Yes 0/4
2 30 30 No 0/4 Yes 1/4 Yes 0/4
3 22 31 Yes 0/4 1/4 Yes 0/4
4 33 30 Yes 0/4 Yes 0/4 Yes 0/4
5 22 27 No ¼ 1/4 0/4
6 21 29 Yes 0/4 Yes 0/4 Yes 0/4
7 30 27 No 0/4 Yes 1/4 Yes 0/4
Total Mean 26.1 29.3 3/8 1/32 6/8 5/32 7/8 0/32
P <0.05 <0.01 <0.00001
DES: Case-Control Study – Summary of Data Comparing
Patients With Matched Controls
45
Diethylstilbesterol (DES)Incidence by Age of Clear Cell Adenocarcinoma at Diagnosis
among Native-born White Resident Female Subjects
46
Diethylstilbesterol (DES)
Effects of in utero exposure on human female progeny– Clear cell adenocarcinoma (malignant)– Adenosis (benign)—ectopic location of
glandular epithelium of the vagina and cervix
47
Diethylstilbesterol (DES)
Structural abnormalities of the cervicovaginal area including cervical collars, hoods, hypoplasia of the cervix– The significance of these abnormalities
is not known and some spontaneously regress with time
Structural abnormalities of the uterus
48
Diethylstilbesterol (DES)
Menstrual irregularitiesPossible adverse pregnancy outcomes including spontaneous abortions, ectopic pregnancies, and premature deliveryPossible increased risk for breast cancer (recent)
49
Diethylstilbesterol (DES)
Effects of in utero exposure on human male progeny– Anatomic abnormalities of the
reproductive tract– Altered semen including decreased
sperm density, count, and motility– Neoplasms of the reproductive tract not
widely reported
50
Human
Est
roge
n C
once
ntra
tion
Pregnancy (trimester)1 2 3
CP
Diethylstilbesterol (DES)
Mechanisms of DES toxicity: Critical period for estrogen exposure—toxicity window
51
Metabolism of Diethylstilbesterol (DES)
Mechanisms: Animal models– MouseSpecies differences in metabolism– Qualitatively similar, quantitatively
different
52
Female Genital Tract Tissue
OHHO
Phase I Metabolism
Reactive Metabolites
Robust Phase II Metabolism
Excretion
Conjugated Inactive Metabolites
OHHO
Phase I Metabolism
Reactive Metabolites
Little Phase II
Metabolism
Little conjugation and Excretion -
Retention of reactive metabolites
Liver Tissue
Metabolism of Diethylstilbesterol (DES)
Metabolism in male and female genital tract tissues– Data from mouse tissue in culture
53
Estrogenicity of DESDES is a potent estrogen
O2-•
Estrogen
2/4-OH-E2
ER
mRNAsP450sPeroxidases
ER
ER MAPK
ER = Estrogen receptor
54
Endocrine disruptors defined as:– An exogenous chemical substance or
mixture that alters the structure or function(s) of the endocrine system and causes adverse effects at the level of• Organism and its progeny• Populations• subpopulations
Environmental Endocrine Disruptors
55
1. Endocrine Active chemicals—natural and synthetic product
2. Adverse human health effects—very controversial
Cl
Cl
C
CCL3
H
o,p-DDT
Environmental Endocrine Disruptors
OHOH
HOO
O
Genistein
HO
O
EquileninBisphenol A
56
Effects on wildlife– Alligators—Abnormal gonad
development and sex hormoneEffects on humans– Cancer?– Reproductive/developmental?Effects of combinations of environmental chemicals
61 studies over 52 years suggest declining sperm counts. Circle size reflects relative number of subjects.
Source: adapted by CTLT from British Medical Journal, 1992.
Sper
m c
ou
nt
(x10
6 /m
l)
150
100
50
01930 1950 1970 1990
Presenter
Presentation Notes
Source: adapted by CTLT from British Medical Journal, 1992.
58
Environmental Endocrine Disruptors
1996 Congress passed the Food Quality Protection Act and amendments to the Safe Drinking Water ActBoth laws contained provisions requiring screening and testing of chemicals and pesticides for possible endocrine-disrupting effects– Estrogens (agonist/antagonist)– Androgens– Thyroid hormones
Environmental Endocrine Disruptors Prioritization for Testing
>
60
The Tier 1 Screening assays: To detect chemical substances capable of interacting with the estrogen, androgen, and thyroid hormonal systems. All known endocrine disruptor mechanisms for the estrogen, androgen and thyroid systems were included in the Tier 1 Screening battery. In Vitro assays include:
an estrogen receptor binding or reporter gene assay; an androgen receptor binding or reporter gene assay; and a steroidogenesis assay with minced testis.
In Vivo assays include: a rodent 3-day uterotrophic assay; a rodent 20-day pubertal female assay with enhanced thyroid endpoints; a rodent 5 to 7-day Hershberger assay; a frog metamorphosis assay; and a fish reproductive screening assay.
Results ->move the chemical into Tier 2 Testing or
conclude that no further screening or testing is needed at that time (Hold and periodically review).
Tier 2 Testing: is designed to determine whether a chemical may have an effect similar to that of naturally occurring hormones and to identify, characterize, and quantify those effects for estrogen, androgen, and thyroid hormones. Conducting all five tests in the Tier 2 battery will provide the type of information necessary for endocrine disruptor hazard assessments for human health, fish, and other wildlife species.
Tier 2 tests will usually encompass two generations and will include effects on fertility and mating, embryonic development, sensitive neonatal growth and development, and transformation from the juvenile life stage to sexual maturity.
Tier 2 tests include: •a two-generation mammalian reproductive toxicity study or a less comprehensive alternative mammalian reproductive toxicity test; •an avian reproduction toxicity test; •a fish life cycle toxicity test; •an opossum shrimp (Mysidacea) or other invertebrate life cycle toxicity test; and •an amphibian development and reproduction test.
Environmental Endocrine Disruptors Tier 2 Testing
Section E
Thalidomide
63
Structure and Uses
Structure
64
Structure and Uses
Chemical properties: Lipophilic, insoluble, and unstableClinical effects in rats– Sedation– Toxicity—LD 50
65
Comparison to Other Sedatives
Thalidomide was very potent, long-lasting and safe by these results
Cutaneous Hemangioma of the upper lip, glabella, and forehead
Ocular Microphthalmia; anophthalmia; coloboms
Auditory Deformed or absent pinna; atresia of the external canal; aplasia of the drum; low-set ears
Respiratory Choanal atresia; saddle nose; cleft palate; bilobular right lung
70
Congenital Malformations Attributed to Thalidomide
In Humans
System Affected Type of Malformation
Cardiovascular Aortic hypoplasia; transposition of the great vessels; atrial and ventricular septal defects; stenosis of the pulmonary artery; abnormal pulmonary veins
Gastrointestinal Atresia of the esophagus; pyloric stenosis; imperforate anus; absence of the gall bladder; atresia of the common bile duct
Urogenital Hypoplasia and aplasia of the kidney; horshoe kidney; hypoplasia and aplasia of the ureter; atresia of the vagina; bicornate uterus; aplasia of the uterus; recto-vaginal fistula
71
Timetable of Human Malformations with Thalidomide2
Drug Administration Defect (days after menstruation)Duplication of thumbs 34-38Abnormal ears (anotia)Heart and vessel anomalies 36-45Renal defects 38Amelia, arms 38-43Phocomelia, arms 38-47Duodenal and gall bladder atresia 40-45Urogenital and respiratory defects 41-43Phocomelia, legs 42-47Rectal stenosisTriphalangism, thumbs 49-50
The Thalidomide Syndrome Period of Sensitivity
72
The Thalidomide Syndrome
Laboratory studies: Sensitive periods of embryogenesisSpecies differences but positive in primates
73
Thailomide Syndrome
Thalidomide Teratogenesis in Primates Specie Teratogenic