Date post: | 06-Jul-2015 |
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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
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)
Regulation of Cytochrome P450 gene expression
CYPs Ligands NXRs Modulators
CYP1 Polycyclic AHs,
Halogenated AHs
AHR [Arnt] 3MC, BNF, OMP
CYP2B Steroids CAR [RXR] PBB, CITCO
CYP2C Prostaglandins,
Steroids
CAR [RXR]
PXR [RXR]
PBB, RIF
CYP3A Steroids PXR [RXR] DEX, RIF [hu],
PCN [rt]
CYP4A Prostaglandins,
FAs
PPAR [RXR] DEX, CLF,
Wy14,643
CYP7A Cholesterol,
BAs
LXR [RXR]
FXR [RXR]
CDCA, APD,
22(R)HC,
T0901317
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.
DTExtm Microarray Gene Set
Gene Group Phase Group Members Number
Cytochrome P450s
[CYPs]
I 1A, 1B, 2A, 2B, 2C, 2D, 2E, 3A, 4A,
4B, 7A, 8B, 19A, 27A, 27B
18
Nuclear Xenobiotic
Receptors [NXRs]
I AHR, AR, CAR, FXR, HNF, LXR,
NFE, NRF, PPAR, PXR, RXR, VDR
19
Sulfotransferases
[SULTs]
II 1A, 1B, 1C, 1E, 2A, 2B 6
UDP glucuronosyl
transferases [UGTs]
II 1A, 2A, 2B, 8 6
SLC (uptake)
transporters [SLCs]
III CNT, ENT, LST, NTCP, OAT, OATP,
OCT, OCTN, OST, PEPT, PGT,
URAT
36
ABC (efflux)
transporters [ABCs]
III A, B, C, D, E, F, G 49
Controls 18S, 28S, ACT, B2M, GDH, S28,
SH1, SH2, RPLP0, TUB, VIL1
11
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.
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.
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
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.
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]
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]
Induction of DTEx gene expression in six rifampicin treated human hepatocyte lots
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00abcA7
abcC8
cyp2C9
NTCP1
PGT
UGT2B17
SLC22A3
abcA1
abcB7
abcF2
RXRG
OATPD
UGT2A1
OATPRP4
abcA8
abcC9
cyp2C19
OAT1
UGT2B15
ORCTL4
abcB2
abcD2
cyp27B1
OATPE
AHR
SULT2B1
abcC4
cyp1B1
ENT2
cyp4A11
abcA3
abcB9
abcG1
PPARA
OATPF
cyp7A1
abcA10
abcC11
cyp2E1
OAT3
cyp8B1
abcA4
abcB10
abcG2
PPARB
cyp4A22
abcA12
abcC12
cyp3A4
OAT4
cyp4B1
abcA5
abcB11
abcG4
PPARG
OCT_1
NRF1
abcC7
cyp2C8
LST2
CAR
OSTB
URAT1
abcB6
abcF1
RXRB
OCT_2
Induction of CYP450 gene expression in six rifampicin treated human hepatocyte lots
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
cyp2C9
cyp27A1
cyp1A2
cyp2C19
cyp27B1
cyp1B1
cyp2D6
cyp4A11
cyp7A1
cyp2A6
cyp2E1
cyp8B1
cyp4A22
cyp2B6
cyp3A4
cyp4B1
cyp2C8
cyp19A1
Induction of DTExtm gene expression in six rifampicin treated human hepatocyte lots
Cluster plot and histogram of CYP gene expression induction ratios
[range = 0-4x vehicle treated]
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
cyp3A4
cyp2E1
cyp2C19
cyp2C9
cyp2C8
cyp1B1
cyp1A2
Coordinate regulation of DTExtm gene expression in RIF treated human hepatocytes
1.261.37
1.28
1.621.44
2.11
0.87
2.86
0.95
1.54
1.07
1.45
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
PXR CYP3A4 ABCB1 UGT1A1
Coordinate regulation of DTExtm gene expression in BNF treated human hepatocytes
0.90 0.92
1.44
2.51
2.132.25
2.56
2.96
0.98 1.00
0.78
1.64
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
AHR CYP1A2 ABCC2 UGT1A1
Induction of CYP3A4 gene expression and activity in RIF treated human hepatocytes
2.4
7.8
2.3
0
1
2
3
4
5
6
7
8
9
DTEx QPCR P450-Glo
Induction of CYP3A4 gene expression and activity in RIF treated HepaRG cells
1.8
4.5
4.1
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
DTEx QPCR P450-Glo
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