Developmental Toxicology

Human Reproduction in the U.S.• Loss of conceptuses

– 50%-80% die before term• Most before implantation

– Chromosomal abnormalities very common

• Live births– Major malformations: 2% to 5%: 20 to 50 per 1000 live births

• Genetic syndromes: 20% or ≤ 10 per 1000 live births• Environmental origins:

– Maternal infections: 2%-3%: ≤ 1.5 per 1000 live births– Maternal metabolic imbalance 1%-2%: ≤ 1 per 1000 live births

» Diabetes– Maternal infections 2%-3%: ≤ 1.5 per 1000 live births

» Herpes, toxoplasmosis, rubella– Drugs and other chemicals: 4-6%: ≤ 3 per 1000 live births

» Alcohol» Smoking

• The causes of 60%-70% of birth defects are unknown

SCORE CARD• Proven positive– Rubella 1930s– Radiation 1930s– Aminopterin 1952– Toxoplasmosis 1950s– Androgens 1959– Thalidomide 1961– Methylmercury 1960s– Warfarin 1960s– Coumarin 1966– PCBs 1968– Lithium 1970– Smoking 1970s– DES 1974– Alcohol 1973– Diphenylhydantoin 1981– ACE inhibitors 1981– Isotretinoin (Accutane) 1983– Toluene (abuse) 1980s– Cocaine 1987– Carbamazepine 1988– Hyperthermia 1990s– Lead ongoing– Vit A derivatives ongoing

• Probably negative– LSD– Sulfonamides– Adrenocortical steroids (cortisone)– Antihistamines (meclizin)– Bendectin– Hypoglycemics (insulin)

• ?????– Aspirin– Dioxin– Tranquilizers– Quinine– Marijuana– Barbiturates– Narcotics– Caffeine– Methadone 1980s– Misoprostol 1991– Fluconazole (antifungal) 1992

Identifying Human Developmental Toxicants• Animal studies

• 65% concordance between species

• Karnofsky’s law• Hundreds of animal teratogens

known• ~ 30 human agents

• Alert clinicians• Rubella • Aminopterin• Thalidomide• Diphenylhydantoin• Warfarin• Alcohol• Valproic acid• DES

• Epidemiology– X-rays– Smoking– Valproic acid

• Dramatic discordance– Human/Rodent

• Thalidomide• Cortisone

– Rat/Mouse• Ethylene thiourea

Developmental Stages

• Preimplantation Period– Totipotent cells– Damage is all-or-nothing

• Possible exception: mutagens

• Organogenesis– Pattern formation– Organ formation– Physical defects

• Fetal Period– Maturation and growth– Functional deficits

• Birth to maturity– Cognitive development

• Brain continues to add neurons until ~ age 12

• Connections continue to be made until early 20s

– Growth• Adequate nutrition• Gene expression

– Sexual development• Adequate nutrition• Hormonal balance

• Senescence– Long-term effects of early

nutrition?– Environmental effects of childhood

exposures?• PCBs?

Human Organogenesis

end of thalidomide sensitivity

W. His: Day 15 human embryo

W. His Day 18-21 human embryo

(start of thalidomide-sensitive period)

W. His Day 27-30 human embryo

W. His Day 31-34 human embryo:(almost at the end of the thalidomide-sensitive period)

6 week old W. His: human embryos 8.5 week old

~ 60 days~ 40 days old

Principles of Developmental Toxicology1. The developmental toxicant must have access to the target

– Physical agents must penetrate maternal tissues– Chemicals must cross the placenta

2a. Dose-response relationships differ from other forms of toxicityMore malformationsMore severe malformations

2b. There is a threshold dose below which there is no fetal damage– Most developmental toxicants are not mutagens

(But most mutagens are developmental toxicants.)– Remember Paracelsus!

3. Genetics interacts with the fetal environment – Most developmental toxicants affect only some exposed fetuses

• ~ 30% of offspring of alcoholic mothers have fetal alcohol syndrome• Thalidomide and possibly retinoic acid are exceptions to this rule• Anencephaly and spina bifida (ASB) illustrate this rule

– Genetics acts• Across species

– Examples: thalidomide, cortisone• Between strains or individuals within species

– Example: cortisone

Interaction of Genetics and Environment: Anencephaly and Spina Bifida (ASB)

Principles of Developmental Toxicology

4. Time of exposure is very important in determining– Type of malformation– Incidence of malformation– Most birth defects must occur before a tissue or organ is

fully formed• Exception: cocaine• The most sensitive period for malformations is during

organogenesis– Well before the end of the 3rd month of pregnancy

Timetable of Thalidomide-Induced Malformations in Humans

(Timing is from the start of the last menstrual period)

Malformations Induced by a Hypothetical Teratogen

Principles of Developmental Toxicology

5. Manifestations of developmental toxicity include– Death– Malformation– Growth retardation– Functional deficit

• A single chemical may cause several effects by different mechanisms

Functional Deficits• Often occur in an already formed organ• Are not readily apparent at birth• Chemicals causing neurological deficits

– Alcohol– Mercury– PCBs– Isotretinoin (AcutaneTM)– Diphenylhydantoin (DilantinTM)– All psychoactive drugs?

Growth Retardation • Temporary– Catch-up growth

• Permanent– Fetal Alcohol Syndrome

• Small until puberty

Current Test Requirements• 2 species

– 1 must be non-rodent• 2 generation assay:

– Continuous dosing from before mating of parents (F0) to weaning of F2.

– Look at • Survival• Weight• Pathology

• Highest dose must affect dam adversely• Additional studies to examine effects on

– Fertility and mating– Organogenesis– Parturition and lactation

In Germany: ConterganIn Britain: Distaval

Reproductive Toxicology

• Fertilization• Early cleavage• Gastrulation• [Implantation & organogenesis

– role of placenta in fetal development]

• Growth and maturation• Birth• Childhood: growth and maturation• Puberty & sexual maturity [or

metamorphosis]• Mating

Reproductive Cycle: Egg Layers

All necessary nutrients must be in the egg when it is laid.All wastes must be processed or excreted by the embryo.

The egg and its embryo are not buffered by the maternal organism.Metamorphosis adds another layer of complexity - even more than puberty.

Requirements for Successful Reproduction

• Male– Hormones

• Testosterone• Estrogen

– Testes• Sperm maturation

– Accessory organs• Epididymis• Prostate

– Sperm• Number • Motility

– Behaviour• Fenarimol in rats

• Female– Hormones

• Estrogen• Progesterone• Prolactin

– Ovary• Ovulation

– Fallopian tubes• Transport of ova

– Uterus• Receptivity• Maintenance of

pregnancy– Ova

• Offspring– Growth– Maturation

• Puberty• Sexual development

Reproductive Toxicity Observed

• In ecosystems: – Species-wide epidemiology

• There are no juveniles … because …?– Male or female does not produce gametes OR– Gametes are not functional OR– Male or female does not (cannot) mate OR– Female cannot sustain embryos to independent life OR– Offspring do not mature sexually

• In humans:– Epidemiology or clinical observation– Followed by medical documentation– Males provide most examples because

• System is simpler– Count sperm, check motility

• More occupational exposures

Assorted Reproductive Toxicants• Wildlife

– DDT in some species of birds• Greater sensitivity + greater exposure• Eggshell thinning + normal embryos• Pelicans, peregrine falcons

– TCDD equivalents in Great Lakes• PCBs, dioxins• Fish: lake trout

– Death of early life stages• Birds: gulls, eagles

– Malformed chicks, poor parenting

– Pulp mill effluents• Fish

– Temperature?• Alligators, turtles in Lake Apopka

– Sewage outflow (phthalates, hormones)• Feminizing fish

• Human Male– DBCP– Chlordecone (KeponeTM)– Phthalates and other

“endocrine disruptors”?• Human Female

– DES– Major focus on

developmental tox

diethylphthalate

Protocol for 2-Generation AssayNB: No known reproductive or developmental toxicants are missed by this assay.

F0: parental animals

1st mating

F1A

Necropsy at weaning

2nd mating

F1B

Select F1 parental animals

F2BF2A

Necropsy at weaning

Necropsy F2B at weaning; including complete histopathology

Continue feeding chemical to each group at the appropriate dosing level throughout the study (progeny,

too!)

Necropsy parents [F0 and F1] aftertheir 2nd litter is weaned.

Current Test Requirements• 2 species

– 1 must be non-rodent• 2 generation assay:

– Continuous dosing from before mating of parents (F0) to weaning of F2.

– Look at • Survival• Weight• Pathology

• Highest dose must affect dam adversely• Additional studies to examine effects on

– Fertility and mating– Organogenesis– Parturition and lactation– Behaviour

DES

Cholesterol

Simplified diagram outlining points of entry of estrogenic chemicals into the water supply, adapted from Velicu et al., 2009 (12).

Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45, 51-60.DOI: 10.1021/es1014482Copyright © 2010 American Chemical Society

Environmental Endocrine Disruptors: Hormones

Estimated contribution of total natural estrogens (E1+E2+E3) and EE2 excretion to total estrogen excretion in the Dutch population. OCs are estimated to be 1% of total. Figure adapted from ref 51. This figure does not account for the higher potency of EE2 compared to natural estrogens.

Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45, 51-60.DOI: 10.1021/es1014482Copyright © 2010 American Chemical Society

Estimates of fate and excretion of EE2 in the body, adapted from Johnson and Williams, 2004(57). All numbers are approximate and final numbers do not add to 100% of the original ingested EE2, as a complete mass-balance analysis has yet to be performed.

Published in: Amber Wise; Kacie O’Brien; Tracey Woodruff; Environ. Sci. Technol.  2011, 45, 51-60.DOI: 10.1021/es1014482Copyright © 2010 American Chemical Society

“Environmental Endocrine Disruptors”:Phthalates

DEHP:diethylhexylphthalate

General Structure

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY OF PHTHALATES

Jan L. Lyche et al, Journal of Toxicology and Environmental Health,

Part B, 12:225–249, 2009

• “The present human toxicity data are not sufficient for evaluating the occurrence of reproductive effects following phthalate exposure in humans, based on existing relevant animal data. This is especially the case for data on female reproductive toxicity, which are scarce. Therefore, future research needs to focus on developmental and reproductive endpoints in humans. It should be noted that phthalates occur in mixtures but most toxicological information is based on single compounds. Thus, it is concluded that it is important to improve the knowledge of toxic interactions among the different chemicals and to develop measures for

com- bined exposure to various groups of phthalates.”

Current Concerns:Environmental Endocrine Disruptors

• Phthalates– and other plasticizers

• Agricultural and pharmaceutical hormone use– Steroids (estrogens)– Recombinant Bovine Growth Hormone

• Pesticides– Atrazine– Vinclozolin