Transcript
Effect of Genetic Variation of the Formation of Hepatocarcinogenic Metabolites of Trichloroethylene
Using Chloral Hydrate Studies (An Interdisciplinary Approach)
Apryl DeLancey Department of Biostatistics, Bioinformatics and Epidemiology
May 12, 2005
Outline
Introduction Specific Aims Hypotheses Background
Data Conclusions
Introduction
Specific Aims
Specific Aim 1: (Pharmacokinetics) to determine variability in the kinetics of formation of the chloral hydrate metabolites, TCA and TCEOH using primary human hepatocyte cultures. Within this, experimental model validation is needed:
– To validate assay with rat model for intra-individual variability.
– To validate assay with mouse model for inter-individual (with same genetic profile) variability.
Specific AimsSpecific Aim 2: (Genetics) to determine the
relationship between the kinetics of metabolite formation and hepatic alcohol and aldehyde dehdrogenase genotypes.
Specific Aim 3: (Quantitative) to generate valid tools to incorporate/make inferences to the degree of human variability in metabolite formation into a regression model using demographic, genetic, and metabolic covariates
Study Hypotheses1. Chloral hydrate metabolism will show variability
among humans with respect to the toxic and non-toxic metabolite pathway; exhibiting sensitivity in certain individual genotypes.
2. Each genotype for the alcohol and aldehyde dehydrogenase genes in humans will have specific kinetics that can be used to predict sensitivity to chloral hydrate which can be extrapolated to trichloroethylene.
Background
Common metal degreasing solvent
In over ½ of EPA hazardous waste sites to receive $$
Long-term effects disputed
Background
Background
Epidemiology vs. Pharmacology
Occupational Studies vs. Animal Studies
PBPK Model of Risk
Specific Aim 1: (Pharmacokinetics)
Collect metabolism data from cell or
homogenate 10’ incubations with
chloral hydrate by gas chromatogram
Standard curve to determine amount
Specific Aim 1: (Pharmacokinetics)
Specific Aim 1: (Pharmacokinetics)
Experiment Date
TCEOH Vmax/Km
TCA Vmax/Km
03/21/05 0.09 0.0704/21/05 0.07 0.0804/22/05 0.07 0.07Std Dev ±0.009 ±0.007
Summary for genetically similar individual male B6 mouse validation experiments
Specific Aim 2: (Genetics)
Isolate DNA from samples used in metabolic data and then:
PCR Restriction Enzyme Gel Electrophoresis Band Identification
← 100 bp
(-) ZAG HL10 KTG AOK Marker
ALDH2 91bp ProductSpecific Aim 2: (Genetics)
Specific Aim 3: (Quantitative)
Excel to store, export, some graphing
Stata for robust regression
Use Vmax for risk assessment
SAS for prediction model construction
Specific Aim 3: (Quantitative)
ND = not determined
Vm
ax (n
mol
es/m
in/1
06 c
ells
)
0.010
0.100
1.000
10.000
100.000
1000.000
Km (mM)
0.001 0.010 0.100 1.000 10.000 100.000
typicalatypicalAOK
EJR
HL12
HL6
ZAG DAD
CEC
CHD
IOEHL10
KTG
Log-Log plot
ALDH2 Genotype
Underlined Non-drinker
Bolded Drinker
Specific Aim 3: (Quantitative)
TCEOH
Specific Aim 3: (Quantitative)
Underlined Non-drinker
Bolded Drinker
ALDH2 Genotype
EJR
AOK
IOECEC
HL10
ZAGCHDHL12DAD
HL8HL7
KTG
Prediction Models
ALDH2 most important for TCA model p = 0.0002
EJR in: Both TCA and TCEOH: ALDH2 (TCEOH p = 0.03, TCA p = 0.0061)
Followed by other genes
Vmax to risk extrapolation
Specific Aim 3: (Quantitative)
Conclusions - Study Hypotheses1. Chloral hydrate metabolism will show variability
among humans with respect to the toxic and non-toxic metabolite pathway; exhibiting sensitivity in certain individual genotypes. Supported by atypical ALDH2!
2. Each genotype for the alcohol and aldehyde dehydrogenase genes in humans will have specific kinetics that can be used to predict sensitivity to chloral hydrate which can be extrapolated to trichloroethylene. ALHD2 most important here!
Conclusions
Best predictors for both: ALDH2 genotype
Limitation - Sample size considerations
BUT - Framework for future directions
Thank you!DB2E
Dr. David Hoel Dr. Jonas Almeida
Dr. Lawrence Mohr
Pharmacology Dr. David Jollow
Dr. David McMillan Dr. JoEllyn McMillan
and a really, really big special thank you to the Ogretmen Lab
and, of course, thank you EBP for $$
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