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 $$