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Reproductive and Developmental Toxicology

James D. Yager, PhD Johns Hopkins University

3

Outline

Introduction and general principles– Reproductive toxicology– Developmental toxicologyCase studies– 1,2-Dibromo-3-chloropropane– Estrogens– Thalidomides

Section A

General Principles of Reproductive Toxicology

5

Frequencies of Selected Reproductive Failures

Frequency Event

Per 100 Unit

Failure to conceive after one year 10–15 Couples

Spontaneous abortion 8–28 weeks 10–20 Pregnancies or women

Chromosome anomalies in spontaneous abortions, 8–28 weeks

30–40

Chromosome anomalies from amniocentesis, > 35 years 2 Amniocentesis specimen

Stillbirths 2–4 Stillbirths and live births

Birth weights < 2,500 g 7 Live births

Birth defects 2–3 Live births

Chromosome anomalies, live births 0.2 Live births

Severe mental retardation 0.4 Children to age 15 years

6

General Principles Reproductive Toxicology

What is it?– Study of the recurrence, causes,

manifestations, and sequalae of adverse effects of exogenous agents on reproduction

Reproductive ToxinsTargets for chemical toxicityReproductive toxicity endpoints

7

Reproductive Toxins: Drugs, Environmental Chemicals

Drugs that Are Gonadotoxic in Humans

Males Females

Bisulfan Bisulfan

Chlorambucil Chlorambucil

Cyclophosphamide Cyclophosphamide

Nitrogen mustard Nitrogen mustard

Adriamycin

Corticosteriods

Cystosine arabinoside

Methotrexate

Procarbazine

Vincristine

Vinblastine

8

Environmental Chemical ExposureEnvironmental Chemical Exposure Associated with Reproductive Function

Males Females Carbon disulfide Anesthetic gas (OR personnel) Chlordecone (Kepone) Aniline Chloroprene Benzene Dibromochloropropane (DBCP) Carbon disulphide Ethylene dibromide Chloroprene Ethylene oxide Ethanol consumption Ethanol consumption Ethylene oxide Glycol ethers Glycol ethers Hexane Formaldehyde Inorganic lead (smelter emissions) Inorganic lead (smelter emissions) Organic lead Organic lead Pesticides (occupational exposure) Methyl mercury Vinyl chloride Pesticides (occupational exposure) Phthalic acid esters (PAEs) Polychlorinated biphenyls (PCBs)

9

Neuroendocrine Targets for Reproductive Toxins

CNS

Hypothalamus

AnteriorPituitary

Testis/Ovary

+ GnRH

Trophic HormonesFSHLH +

Testis/Ovary-derived HormonesTestosterone

EstrogenInhibin

-

GnRH Inhibitor

-

10

Reproductive Tissue

Sertoli cells (blood-testes barrier)Leydig cells (production of testosterone)

11

Reproductive Tissue: Testis/Seminiferous Tubules

Public Domain

12

Reproductive Tissue—Testis

Histological section through testicular parenchyma of a boar.

1 = Lumen of seminiferous tubules2 = spermatids3 = spermatocytes4 = spermatogonia5 = Sertoli cell6 = Myofibroblasts7 = Leydig cells8 = capillaries

Creative Commons BY-SA. Mickael Haggestrom.

13

Reproductive Tissue

Leydig cells locatedIn this layer

Form the Blood-TestisBarrier

14

Reproductive Tissue - Testis

15

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

Num

ber o

f Fol

licle

s

0

Reproductive Tissue - Ovary

18

Reproductive Toxicity Endpoints

Reproductive endpoints indicating reproductive dysfunction– Decreased libido:

Impotence– Sperm abnormalities:

Decreased number/motility: Morphology

– Subfecundity• Abnormal

gonads/ducts of external genitalia

• Abnormal pubertal development

• Infertility of male/female

• Amenorrhea• Anovulatory cycles• Delay in

conception

19

Reproductive Toxicity Endpoints

– 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."

--March of Dimes

Causes of Birth Defects

22

Pregnancy& Infant Health

Social Factors

Biological Factors Environmental Factors Genetics, gender, age Diet, tobacco, chemicals, radiation

Behavior, community, medical care

Birth Defects & Developmental Disorders

March of Dimes

Causes of Birth Defects

23

Hereditary disease

20%

Cytogeneticdisease 4%

Unknowncauses 70%

Birth trauma and uterine factors 1%

Maternal metabolic factors 1%

Maternal infection 2%

Drugs, chemicals and radiation 2%

Causes of Birth Defects

24

Toxic WindowsReproductive Exposure

Gametes Blastocyst Embryo FetusNEWBORN

(normal)

Sterility Death

Death Functional Abnormalities

NEWBORN(anomalies)

REPRODUCTIVEOUTCOME

Death

Structural Abnormalities

25

Toxic Windows: Stages of Embryogenesis

26

Expected Incidence of Malformation of Different Organs and Systems

27

Radiation Infections– Rubella virus– Cytomegalovirus– Herpes virus

hominis– Toxoplasmoisis– Syphilis

Maternal metabolic imbalances– Alcoholism– Cretinism– Diabetes– Folic acid

deficiency– Hyperthermia– Phenylketonuria– Rheumatic disease

Human Developmental Toxicants

28

Human Developmental Toxicants

Drugs and chemicals– Androgenic

chemicals– Antibiotics

Anticancer drugsAnticonvulsants– Diphenylhydantoin

, trimethadione

Anti-thyroid drugsChelators– Penicillamine

Chlorobiphenyls

29

Human Developmental Toxicants

Cigarette smokeCocaineCoumarinanticogulantsDiethylstilbesterolEthanolEthylene oxide

IodidesLithiumMetals– Mercury (organic)– Lead

RetinoidsThalidomide

30

PlacentaParent compound(Lipid soluble)

Parent compound(Lipid soluble)

Parent compound(Lipid soluble)

Parent compound(Lipid soluble)

Polar metabolites

Polar metabolites

Non-polar metabolites Non-polar metabolites

Maternal compartment Fetal compartment

Distribution of Xenobiotics Between the Fetal and Maternal Compartments

31

Placenta

Parent compound(Lipid soluble)

Parent compound(Lipid soluble)

Parent compound(Lipid soluble)

No compound

Polar metabolites

Maternal compartment Fetal compartment

Non-polar metabolites

Polar metabolites

Non-polar metabolites

Elimination

Elimination

Distribution of Xenobiotics Between the Fetal and Maternal Compartments

32

Developmental Toxicity Endpoints

Type I changes – Outcomes permanent, life-threatening and

frequently associated with gross malformations• Reduction of number of live births

(litter size)• Increased number of stillbirths• Increased number of resorptions• Increased number of fetuses with

malformations

33

Developmental Toxicity Endpoints

Type II changes – Outcomes, non-permanent, non-life

threatening, and not associated with malformations• Reduced birth weights• Reduced postnatal survival• Decreased postnatal growth, reproductive

capacity• Increased number of fetuses with retarded

development

34

Developmental Toxicology – Dose-Response Patterns

Dose-Response Patterns for Different Types of Developmental Toxicants

Section C

1,2-Dibromo-3-Chloropropane

36

Structure of DBCP and Tris-BP

1,2-Dibromo-3-Chrolopropane (DBCP) Structure and Uses

Br C C C Cl

H Br H

H H HO P O

O

H C HH C BrH C H

Br

C C C Br

H Br H

H H H

Br C C C

H Br H

H H H

O

DBCP

Tris-BP

37

1,2-Dibromo-3-Chrolopropane (DBCP): Reproductive Effects

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

Environmental Endocrine Disruptors

57

Environmental EstrogensAdverse reproductive effects

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

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

59

Endocrine Disruptor Screening Program

Revised Sept. 10, 1999; URL:http://www.epa.gov/scipoly/oscpendo/chart.htm

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).

Environmental Endocrine Disruptors Tier 1 Screening Assays

61

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

Comparison of Thalidomide to Other Sedatives

Smallest Hypnotic

Dose (mg/kg)

Start

of Effect (mins)

Duration

(mins)

Smallest Narcotic

Dose (mg/kg)

Initial

Excitation Stage

LD30

Thalidomide 100 5 240-300 >5,000 None >5,000 Luminal 40 30 300 150 ++ 300 Valamin 50 10 60 375 + 725 Doriden 75 30 200 400-500 ++ 600 Methylpentynol 150 15 210 500 + 750 Sodium bromide

500 80 360 7,000 None 800

66

Discovery of Side Effects

IntolerancePeripheral neuropathyBirth defects

67

Discovery of Side Effects

68

The Thalidomide Syndrome

10,000 childrenLimb defectsOther defects

69

Congenital Malformations Attributed to Thalidomide

In Humans System Affected Type of Malformation

Locomotor Amelia; phocomelia; micromelia, oligodactyly, syndactyly; club-hand; club-foot

Nervous Hydrocephalus; meningomyelocele; spina bifida

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

dose mg/mg (oral)

Gestation days treated

Offspring with defects (type 1)

Cynomolgous monkey (crab eating) Macoca fascicularis

10 22-32 6/7 Limbs, teratomas

Rhesus monkey M. mulatta

12 24-30 3/6 Limbs

Stump-tailed monkey M. arciodes

5 24-30 5/5 Limbs and tail

Bonnet monkey M. radiata

5 24-44 13/29

Limbs and visceral

Japanese monkey M. fuscata

20 24-26 6/6 Limbs, tail and CNS

74

Thailomide Syndrome

Thalidomide Teratogenesis in Primates Specie Teratogenic

dose mg/mg (oral)

Gestation days treated

Offspring with defects (type 1)

Baboon Papio cynocephalus

5 18-44 4/10 Limbs and tail

Marmoset Callithrix jacchus

45 25-35 11/11

Limbs, ear and jaw

Green Monkey Cercopithecus aethiops

40 25-48 15/18

Limbs, skeleton and visceral

Bushbaby Galago crassicaudatus

_a 16-42 Limbs, ears

Human

2 21-36

75

Mechanisms of Action

Metabolism—Numerous spontaneous hydrolysis productsMechanisms—Trapped in fetal compartment?Current clinical uses

76

Current Clinical Uses

Thalidomide was once banned because it caused several birth defects, but researchers are discovering new uses; it:– Clears up lesions caused by a

complication of leprosy

77

– Seems to cure graft-versus-host disease in bone-marrow transplant recipients

– Appears to stop drastic weight loss in TB and AIDS patients

– Mechanism?• Immunomodulatory by unknown

mechanism

Current Clinical Uses

78

– Inhibits replication of the HIV-1 virus in vitro

– Could prevent the two leading causes of blindness• Glaucoma; Diabetic retinopathy

– May inhibit tumors– Mechanism of effect?

• Inhibition of blood vessel growth

Current Clinical Uses