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DTEx 02042009
1. DTExtm Gene Expression Analysis
Microarray based method - 768 element array
145 genes - 4 elements per gene
12 grids - 64 elements [16 x 4] per grid
All DTExtm genes are involved in drug metabolism, conjugation or transport
* interrogate for both reported and unreported drug effects on gene expression
* investigate coordinate regulation of gene expression, protein expression and functional activity
* all genes are RefSeq entries verified by NCBI with published literature history
All DTExtm genes are members of coordinate regulatory pathways
* internal control for induction or suppression of gene expression
* control for drug selectivity or specificity in the modulation of gene expression
DTExtm microarray is an ADME gene expression survey and screening method
* analysis of ADME gene expression signature(s) for cell or cell line characterisation and comparison
* analysis of ADME gene expression signature(s) and associated effects in drug treated cells or cell lines
- isogenic cancer cell lines
- hepatocytes
- tissue surrogates [HepaRG, Fa2N-4, etc.]
- tissues [liver, kidney, brain, eye, etc.]
DTExtm microarray analysis is reliable and reproducible
* either biological replicates or experimental replicates
2. ADME and DTExtm Microarray Analysis
The regulation of gene expression of various phase I enzymes, phase
II enzymes and phase III transporters has significant potential impact
on the metabolism, elimination, pharmacokinetics / dynamics,
toxicokinetics / dynamics and drug-drug interactions of many
therapeutic agents, as well as their ability to protect the human body
against exposure to environmental xenobiotics.
(Kong 2002, Guengerich 2003, LeCluyse 2003, Kong 2005)
4. DTExtm Microarray Analysis
What is DTExtm?
A method that allows the simultaneous analysis of changes in the levels
of gene expression of 145 ADME associated genes.
How is DTExtm done?
Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA
converted to labelled cDNA > hybridise to DTExtm microarray > scan >
quantitation > first pass data analysis > second pass data analysis >
differential gene expression analysis [induction, suppression].
Why use DTExtm?
Survey basal level gene expression in various primary cells, cell lines or
tissues as a prelude to induction, inhibition or toxicity testing.
Survey coordinated changes in the levels of gene expression in various
primary cells, cell lines or tissues as a consequence of drug treatment.
5. DTExtm Microarray Gene Set
Gene Group Phase Group Members Number
Cytochrome P450s I 1A, 1B, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 18
[CYPs] 4B, 7A, 8B, 19A, 27A, 27B
Nuclear Xenobiotic I AHR, AR, CAR, FXR, HNF, LXR, 19
Receptors [NXRs] NFE, NRF, PPAR, PXR, RXR, VDR
Sulfotransferases II 1A, 1B, 1C, 1E, 2A, 2B 6
[SULTs]
UDP glucuronosyl II 1A, 2A, 2B, 8 6
transferases [UGTs]
SLC (uptake) III CNT, ENT, LST, NTCP, OAT, OATP, 36
transporters [SLCs] OCT, OCTN, OST, PEPT, PGT,
URAT
ABC (efflux) III A, B, C, D, E, F, G 49
transporters [ABCs]
Controls 18S, 28S, ACT, B2M, GDH, S28, 11
SH1, SH2, RPLP0, TUB, VIL1
6. DTExtm Microarray Analysis
What is DTExtm?
A method that allows the simultaneous analysis of changes in the levels
of gene expression of 145 ADME associated genes.
How is DTExtm done?
Total RNA converted to cDNA > cDNA converted to aRNA > aRNA
converted to labelled cDNA > hybridise to DTExtm microarray > scan >
quantitation > first pass data analysis > second pass data analysis >
differential gene expression analysis [induction, suppression].
Why use DTExtm?
Survey basal level gene expression in various primary cells, cell lines or
tissues as a prelude to induction, inhibition or toxicity testing.
Survey coordinated changes in the levels of gene expression in various
primary cells, cell lines or tissues as a consequence of drug treatment.
7. DTExtm Microarray Analysis
What is DTExtm?
A method that allows the simultaneous analysis of changes in the levels of gene
expression of 145 ADME associated genes.
How is DTExtm done?
Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA
converted to labelled cDNA > hybridise to DTExtm microarray > scan >
quantitation > first pass data analysis > second pass data analysis >
differential gene expression analysis [induction, suppression].
Why use DTExtm?
Survey basal level gene expression of all 145 ADME associated genes in various
primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity
testing.
Survey coordinated changes in the levels of gene expression in various primary
cells, cell lines or tissues as a consequence of drug treatment.
8. Basal level DTExtm gene expression in human hepatocytes
Matrix plot of relative level of gene expression data
Cluster plot of relative level of gene expression data
9. DTExtm Microarray Analysis
What is DTExtm?
A method that allows the simultaneous analysis of changes in the levels of gene
expression of 145 ADME associated genes.
How is DTExtm done?
Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA
converted to labelled cDNA > hybridise to DTExtm microarray > scan >
quantitation > first pass data analysis > second pass data analysis >
differential gene expression analysis [induction, suppression].
Why use DTExtm?
Survey basal level gene expression of all 145 ADME associated genes in various
primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity
testing.
Survey coordinated changes in the levels of gene expression for all 145 ADME
associated genes in various primary cells, cell lines or tissues as a consequence
of drug treatment.
10. DTExtm gene expression in six rifampicin treated human hepatocyte lots
Matrix plot of relative level of gene expression data [range = 0-60x actin]
Cluster plot of relative level of gene expression data [range = 0-60x actin]
11. Induction of DTExtm gene expression in six rifampicin treated human hepatocyte lots
Matrix plot of relative level of gene expression induction [range = 0-4x vehicle treated]
Cluster plot of relative level of gene expression induction [range = 0-4x vehicle treated]
17. Induction of CYP3A4 gene expression and activity in RIF treated human hepatocytes
9
8
7
6
5
4
7.8
3
2
2.4 2.3
1
0
DTEx QPCR P450-Glo
18. Induction of CYP3A4 gene expression and activity in RIF treated HepaRG cells
5
4.5
4
3.5
3
2.5
4.5
4.1
2
1.5
1
1.8
0.5
0
DTEx QPCR P450-Glo
19. DTExtm Gene Expression Analysis
All DTExtm genes are involved in drug metabolism, conjugation or transport
* interrogate for both known and unknown drug effects on gene expression
** CYP3A4 induction by RIF (PXR mediated effects on ABCB1 & UGT1A1)
** different levels of CYP3A4 gene expression and induction in different donors
** CYP1A2 induction by BNF (AHR mediated effects on ABCC2 & UGT1A1)
** RIF (PXR) effects on SULT1A1, 1E1, 2A1, UGT2B, ABCA9, A13 and C13 gene expression and activity?
All DTExtm genes are members of coordinate regulatory pathways
* internal control for induction or suppression of gene expression
** coordinate regulation of CYP3A4, UGT1A1 and ABCB1 via PXR activation by RIF
** coordinate regulation of CYP1A2, UGT1A1 and ABCC2 via AHR activation by BNF
* control for drug selectivity or specificity in the modulation of gene expression
* concordance between DTExtm microarray, Q-PCR and functional activity assays for CYP induction studies
DTExtm microarray analysis is reliable and reproducible
* either biological replicates or experimental replicates