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Genetic and Environmental Factors AsDeterminants of Susceptibility to Disease
Initiated during Development
James D. Yager, PhDJohns Hopkins University
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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
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Section A
General Principles of Toxicology and ofDevelopmental Toxicology
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5
Susceptibility Factors
Exposure
AlteredStructure/Function
Disease
Exposure Assessment Risk Assessment
InternalDose
Biologically EffectiveDose
Early BiologicalEffect
ToxicodynamicsToxicokinetics
The Toxicological Paradigm
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6
Toxicological Process in Vivo
Adapted from: Frazier. (1990) In Vitro Toxicology, 3, 349–357.
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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
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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
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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
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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
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Effects of Alcohol on Pregnancy
Fetal Alcohol Spectrum Disorder (FASD)MiscarriagePreterm birthLow birth weightFASARBD
alcohol-related birth defectsARND
alcohol-related neurodevelopmental disordersBirth complications
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Principles of Developmental Toxicity: Toxic Windows
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Critical Periods of HumanDevelopment for Teratogen Sensitivity
Critical Periods of Human Development
Source: Jones RE. Human Reproductive Biology. Academic Press, Inc. New York (1991)
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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
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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
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Section B
Effects of Exposure to Diethylstilbestrol during Pregnancy
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HO
OH
Estradiol
OH
HO
Diethylstilbestrol
Estrogens
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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
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1 2 3Critical period
Human
Estr
ogen
con
cent
ratio
n
Pregnancy (trimester)
Critical Period for Exposure to DES
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How Does DES Work?
DES is a potent estrogen
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Estrogens
Oxidative metabolites
Estrogen receptors
mRNAs
Specific P450s
Specific proteins
Nucleus
DNA strand
Mitochondria
Mechanisms of Estrogen Receptor–Mediated Effects
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OHHO
Diethylstilbestrol
OO
DES Quinone very reactive
P450-mediated
Species differencesTissue differences
DES Is Also Metabolized
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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.
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OHOH
HOO
O
GenisteinHO
OH
Estradiol
Cl
Cl
C
CCL3
H
o,p-DDTHO
O
Equilenin
Environmental Chemicals with Hormone Activity
Continued
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25
Environmental Chemicals with Hormone Activity
OHOH
HOO
O
GenisteinHO
OH
Estradiol
Cl
Cl
C
CCL3
H
o,p-DDTHO
O
Equilenin
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Section C
Cytochrome P450 Biotransformation Enzymes: General Function and CYP 2E1 in AlcoholMetabolism
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Cytochrome P450 (CYP) Enzymes
Phase I biotransformation of xenobiotics(environmental chemicals) and endogenousmoleculesFunctions of CYPs
Add or expose functional groups
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TissueAccumulation
Elimination
No Biotransformation
Expose/Add Functional
Groups
Conjugation
Phase IProduct
Phase IIProduct
Elimination
P 450s
Xenobiotic
Biotransformation of Xenobiotics
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NO2
OCH3 OH
NO2
+ HCHO
p- Nitroanisole p- Nitrophenol
OCH2OH
NO2
Biotransformation Reactions
Biotransformation reactions: exposing a functionalgroup
Continued
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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
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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
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Functions of CYP 2E1
Metabolism of ethanolMetabolism of certain drugs and environmentalchemicals
DrugsAcetaminophenChlorzoxazone
Environmental chemicalsChloroformBenzeneToluene
Inducible
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Ethanol Acetaldehyde AcetateAlcohol dehydrogenase
CYP 2E1
Acetaldehyde dehydrogenase
Damage to proteinsDamage to mitochondria
Reactive oxygen species
Reactive oxygen species
Metabolism of Ethanol
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Section D
Cytochrome P450 Genetic Polymorphisms:Changes in Expression during Development
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CYP 2E1 Gene Structure
Lee et al. (1996). J Biol Chem, 12063–12067.
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Cytochrome P450 2E1
Genetic polymorphism that affects expressionChanges in expression during embryonic/fetaldevelopment
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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
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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
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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.
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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.
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Chlorzoxazone Metabolism
Administered 500 mgTook blood sample three hours later
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Polymorphism vs. CYP 2E1 Metabolic Ability
Chlo
rzoxazone H
ydro
xyla
tion Index
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.
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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.
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CYP 2E1 Expression: First Six Postnatal Months
Source: Johnsurd E. K., Koukouritaki S. B., Divakaran K., et al. (2003). Human hepatic CYP2E1expression during development. J Pharmacol Exp Ther, 307, 402–407.
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Summary of CYP 2E1 Changes in Expression During Development
Changes in Expression during Development
Source: Johnsurd E. K., Koukouritaki S. B., Divakaran K., et al. (2003). Human hepatic CYP2E1expression during development. J Pharmacol Exp Ther, 307, 402–407.
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Electrophoretic Determination
CYP 2E1 *1C and *1D genotypes—electrophoreticdetermination
Source: Johnsurd E. K., Koukouritaki S. B., Divakaran K., et al. (2003). Human hepatic CYP2E1expression during development. J Pharmacol Exp Ther, 307, 402–407.
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CYP 2E1 Genotype vs. Expression during Development
Source: Johnsurd E. K., Koukouritaki S. B., Divakaran K., et al. (2003). Human hepatic CYP2E1expression during development. J Pharmacol Exp Ther, 307, 402–407.
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RISK
Genetic & Environmental/Life-Style Susceptibility Factors
Genetic & Environmental/Life-StyleProtective Factors
Genetic, Environmental, and Life-Style Factors and Risk