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Genetic and Environmental Factors AsDeterminants of Susceptibility to Disease

Initiated during Development

James D. Yager, PhDJohns Hopkins University

3

Lecture Outline

General principles of toxicology and of developmentaltoxicologyEffects of exposure to diethylstilbestrol duringpregnancyCytochrome P450 biotransformation enzymes

General functionCYP 2E1

Function in alcohol metabolismGenetic polymorphismsChanges in expression during development

Genetic, environmental life-style factors and risk

Section A

General Principles of Toxicology and ofDevelopmental Toxicology

5

Susceptibility Factors

Exposure

AlteredStructure/Function

Disease

Exposure Assessment Risk Assessment

InternalDose

Biologically EffectiveDose

Early BiologicalEffect

ToxicodynamicsToxicokinetics

The Toxicological Paradigm

6

Toxicological Process in Vivo

Adapted from: Frazier. (1990) In Vitro Toxicology, 3, 349–357.

7

Developmental Toxicology

A birth defect is "any anomaly, functional orstructural, that presents in infancy or later in life andis caused by events preceding birth, whetherinherited, or acquired.”

March of Dimes

8

Pregnancy & Infant Health

Social Factors

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

Behavior, community, medical care

Birth Defects & Developmental

Disorders

Factors That Affect Pregnancy and Infant Health

Source: March of Dimes

9

Selected Categories of Preventable Birth Defects

Birth defects Estimated incidence Prevention measure

I. Structural/metabol i c

Neural tube defect s 1 in 1,600 births Folic acid

II. Congenital infecti o n s

Congenital syphilis 1 in 2,000 births Safe sex prior to conception/ treatment

Congenital HIV 1 in 2,700 births Safe sex prior to conception/ avoid risks

Congenital rubella syndr o m e 1 in 100,000 births Immunization

III. Other

Rh disease 1 in 1,400 births Immunoglobi n

Fetal alcohol syndrome 1 in 1,000 births Avoid alcohol

Note: All numbers are based on the best available estimates, which underestimate the incidence of many birth defects.Source: March of Dimes, Metropolitan Atlanta Congenital Defects Program and California Birth Defects Monitoring Program

10

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

11

Effects of Alcohol on Pregnancy

Fetal Alcohol Spectrum Disorder (FASD)MiscarriagePreterm birthLow birth weightFASARBD

alcohol-related birth defectsARND

alcohol-related neurodevelopmental disordersBirth complications

12

Principles of Developmental Toxicity: Toxic Windows

13

Critical Periods of HumanDevelopment for Teratogen Sensitivity

Critical Periods of Human Development

Source: Jones RE. Human Reproductive Biology. Academic Press, Inc. New York (1991)

14

Expected incidence of malformation of different organs and systems,the susceptibility of which varies according to the days of gestation

Principles of Developmental Toxicity: Toxic Window

15

RadiationInfections Rubella virus Cytomegalovirus Herpes virus hominis Toxoplasmoisis SyphilisMaternal metabolic imbalances Alcoholism Cretinism Diabetes Folic acid deficiency Hyperthermia Phenylketonuria Rheumatic diseaseDrugs and chemicals Androgenic chemicals AntibioticsAnticancer drugs

Anticonvulsants Diphenylhydantoin,trimethadioneAntithyroid drugsChelators PenicillamineChlorobiphenylsCigarette smokeCocaineCoumarin anticogulantsDiethylstilbesterolEthanolEthylene oxideIodidesLithiumMetals (Mercury(organic) LeadRetinoidsThalidomide

Human Developmental Intoxicants

Section B

Effects of Exposure to Diethylstilbestrol during Pregnancy

17

HO

OH

Estradiol

OH

HO

Diethylstilbestrol

Estrogens

18

Diethylstilbestrol (DES)

Given to about 4.8 million pregnant women toprevent miscarriage

Effects of in utero exposure on human female progeny

Vaginal cancer in young women(1.4/1,000–10,000) Reproductive problems (18%)

Effects of in utero exposure on human male progeny (3 times normal incidence)

Anatomic abnormalities of the reproductivetract Altered semen—including decreased sperm

density, count, and motility

19

1 2 3Critical period

Human

Estr

ogen

con

cent

ratio

n

Pregnancy (trimester)

Critical Period for Exposure to DES

20

How Does DES Work?

DES is a potent estrogen

21

Estrogens

Oxidative metabolites

Estrogen receptors

mRNAs

Specific P450s

Specific proteins

Nucleus

DNA strand

Mitochondria

Mechanisms of Estrogen Receptor–Mediated Effects

22

OHHO

Diethylstilbestrol

OO

DES Quinone very reactive

P450-mediated

Species differencesTissue differences

DES Is Also Metabolized

23

Estradiol Is Also Metabolized

O

HO

A B

C D115

14

13

12

11

98

7

65

43

2

18

17

16

OHCH3

10

OA B

C D115

14

13

12

11

98

7

653

2

18

17

16

OHCH3

10

HOA B

C D115

14

13

12

11

98

7

653

2

18

17

16

OHCH3

10

OH Estradiol Catechol

Estradiol Quinone

P450sEstradiol

Source: Adapted from Zhu and Conney. (1998) Carcinogenesis.

24

OHOH

HOO

O

GenisteinHO

OH

Estradiol

Cl

Cl

C

CCL3

H

o,p-DDTHO

O

Equilenin

Environmental Chemicals with Hormone Activity

Continued

25

Environmental Chemicals with Hormone Activity

OHOH

HOO

O

GenisteinHO

OH

Estradiol

Cl

Cl

C

CCL3

H

o,p-DDTHO

O

Equilenin

Section C

Cytochrome P450 Biotransformation Enzymes: General Function and CYP 2E1 in AlcoholMetabolism

27

Cytochrome P450 (CYP) Enzymes

Phase I biotransformation of xenobiotics(environmental chemicals) and endogenousmoleculesFunctions of CYPs

Add or expose functional groups

28

TissueAccumulation

Elimination

No Biotransformation

Expose/Add Functional

Groups

Conjugation

Phase IProduct

Phase IIProduct

Elimination

P 450s

Xenobiotic

Biotransformation of Xenobiotics

29

NO2

OCH3 OH

NO2

+ HCHO

p- Nitroanisole p- Nitrophenol

OCH2OH

NO2

Biotransformation Reactions

Biotransformation reactions: exposing a functionalgroup

Continued

30

Benzene

Functional group introduced

OH

Phenol

OSO3H

O

Glucuronide conjugation

Glucuronic acid

Phenyl glucuronide

Phenyl sulfate

Sulfate conjugation

Biotransformation Reactions

Biotransformation reactions: adding a functionalgroup

31

P450 GENE CHARACTERISTIC CHARACTERISTICSUBFAMILY INDUCER REACTION

IA Polycyclic aromatic Benzo(a)pyrene hydroxylation hydrocarbons

IIA Steroid hydroxylation IIB Phenobarbital Benzphetamine demythelation IIC Steroid hydroxylation IID Debrisoquine hydroxylation IIE Ethanol Ethanol hydroxylation IIIA Steroids Steroid hydroxylation

Multiple Forms of P450

Major Mammalian Cytochrome P450 Gene Families

32

Functions of CYP 2E1

Metabolism of ethanolMetabolism of certain drugs and environmentalchemicals

DrugsAcetaminophenChlorzoxazone

Environmental chemicalsChloroformBenzeneToluene

Inducible

33

Ethanol Acetaldehyde AcetateAlcohol dehydrogenase

CYP 2E1

Acetaldehyde dehydrogenase

Damage to proteinsDamage to mitochondria

Reactive oxygen species

Reactive oxygen species

Metabolism of Ethanol

Section D

Cytochrome P450 Genetic Polymorphisms:Changes in Expression during Development

35

CYP 2E1 Gene Structure

Lee et al. (1996). J Biol Chem, 12063–12067.

36

Cytochrome P450 2E1

Genetic polymorphism that affects expressionChanges in expression during embryonic/fetaldevelopment

37

Human CYP 2E1 Polymorphism

Use of restriction endonucleases and Southernblots to detect polymorphismDetection of 100 bp insertion in regulatory region ofP450 2E1 geneDetermination of functional effect of thepolymorphism

Metabolism of chlorzoxazone metabolism to 6-hydroxychlorzoxazone in human subjects

38

5'-T^C T A G A-3'3'-A G A T C^T-5

XbaI1500u#ER0681

5'-T T T^ A A A-3'3'-A A A^T T T-5'

DraI (AhaIII)1500u#ER0221

Restriction Endonucleases

39

Polymorphism in the Upstream Region of CYP 2E1

Source: McCarver D. G., Byun R., Hines R. N., et al. (1998) A genetic polymorphism in the regulatory sequences of human CYP2E1: associationwith increased chlorzoxazone hydroxylation in the presence of obesity and ethanol intake, Toxicol Appl Pharmacol, 152, 276–281.

40

Polymorphism in the Upstream Region of CYP 2E1


41

Chlorzoxazone Metabolism

Administered 500 mgTook blood sample three hours later

42

Polymorphism vs. CYP 2E1 Metabolic Ability

Chlo

rzoxazone H

ydro

xyla

tion Index


43

CYP 2E1 Expression vs. Age

Source: Johnsurd E. K., Koukouritaki S. B., Divakaran K., et al. (2003). Human hepatic CYP2E1expression during development. J Pharmacol Exp Ther, 307, 402–407.

44

CYP 2E1 Expression: First Six Postnatal Months


45

Summary of CYP 2E1 Changes in Expression During Development

Changes in Expression during Development


46

Electrophoretic Determination

CYP 2E1 *1C and *1D genotypes—electrophoreticdetermination


47

CYP 2E1 Genotype vs. Expression during Development


48

RISK

Genetic & Environmental/Life-Style Susceptibility Factors

Genetic & Environmental/Life-StyleProtective Factors

Genetic, Environmental, and Life-Style Factors and Risk

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