#327 THE EFFECTS OF TRIS (2-ETHYLHEXYL) TRIMELLITATE (TOTM) ON GENE EXPRESSION
ASSOCIATED WITH TESTICULAR MAL-DEVELOPMENT (TMD) IN RAT FOETAL TESTES
Elcombe, Clifford R.1; Dhritiman, Dan1; Farrar, David G .2 and Plummer, Simon M.1.
CXR Biosciences Ltd, Dundee, Scotland1 and INEOS Chlor Limited, Runcorn, UK 2
Introduction
To assess the potential of tris(2-ethylhexyl)trimellitate (TOTM) to induce
testicular mal-development (TMD) in the rat, we studied its effects on the
expression of genes in steroidogenesis and testes development
pathways that are involved in the induction of TMD by some phthalates.
The in utero effects of TOTM were compared with those of di(2ethylhexyl) phthalate (DEHP), mono(2-ethylhexyl)phthalate (MEHP), an
“active” metabolite of DEHP, and with 2-ethylhexanol (EHO), considered
to be an ‘inactive’ DEHP metabolite. A low-dose DEHP treatment group
was also included as DEHP can be a minor contaminant (maximum
0.05%) of the technical grade of TOTM.
Results
DEHP
Bioinformatic analysis using Ingenuity Pathways AnalysisTM (IPA)
software was used to identify genes in the signature lists that are
associated with the TMD target pathways of testosterone synthesis and
cryptorchidism. Lists of genes in these pathways relevant to TMD were
constructed using from data derived from previous transcript profiling
studies (Plummer et al. 2007)
NM_013134
NM_017268
NM_031541
NM_031558
NM_012941
NM_019238
NM_031840
NM_178866
NM_012590
NM_053680
MEHP
NM_016989
NM_012738
NM_138828
NM_017244
NM_057137
NM_138508
NM_022519
XM_213329
Control vs MEHP
Control vs DEHP
Control v 0.05%DEHP
Control vs EHO
Control vs TOTM
Symbol Entrez Gene Name
Fold Change p-value Fold Change p-value Fold Change p-value Fold Change p-value Fold Change p-value
Steroidogenesis
CYP11A1 cytochrome P450, family 11, subfamily A, polypeptide 1
0.00
0.00
0.96
0.18
0.21
-5.3
-1.7
1.0
-1.2
1.2
CYP17A1 cytochrome P450, family 17, subfamily A, polypeptide 1
0.00
0.01
0.00
0.00
-4.4
-1.5
1.5
1.4
SC4MOL sterol-C4-methyl oxidase-like
0.07
0.00
0.01
0.11
0.69
-1.3
-1.3
-1.5
-1.8
1.0
0.00
2
0.00
0.00
0.00
0.34
HSD3B2 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase
-4.3
-1.5
2.5
2.3
-1.1
NR5A1
nuclear receptor subfamily 5, group A, member 1
0.01
0.16
0.04
0.11
0.40
-1.3
-1.1
-1.3
-1.5
-1.1
Cholesterol Transport / Biosynthesis
HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase
0.01
0.00
0.32
0.39
0.97
-2.0
-1.4
-1.4
1.1
-1.0
HMGCS1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) -2.6
0.00
0.00
0.01
0.02
0.00
-1.9
-1.2
-1.4
-1.5
SCARB1 scavenger receptor class B, member 1
0.00
0.00
0.00
0.00
0.00
-6.2
-2.6
-1.5
-1.9
-1.3
STAR
steroidogenic acute regulatory protein
0.00
0.00
0.92
0.00
0.76
-10.1
-2.8
-1.0
-1.6
1.0
CYP51A1 cytochrome P450, family 51, subfamily A, polypeptide 1
0.00
0.00
0.45
0.06
0.00
-2.4
-2.1
-1.0
-1.2
-1.4
FDFT1
farnesyl-diphosphate farnesyltransferase 1
0.00
0.04
0.14
0.11
0.00
-1.4
-1.2
-1.1
-1.1
1.1
farnesyl diphosphate synthase (farnesyl pyrophosphate synthetase,
0.00
0.00
geranyltranstransferase)
0.06
0.36
0.07
FDPS
-2.0 dimethylallyltranstransferase,
-1.5
-1.3
-1.5
1.1
Testes Development / Cryptorchidism
IGF1
insulin-like growth factor 1 (somatomedin C)
0.09
0.04
0.00
0.00
0.00
1.5
-1.2
2.0
2.5
-1.3
INHA
inhibin, alpha
0.00
0.00
0.97
0.02
0.46
-2.3
-1.5
-1.0
-1.3
1.0
INSL3
insulin-like 3 (Leydig cell)
0.00
0.04
0.88
0.33
0.71
-4.4
-1.4
1.0
-1.1
-1.0
Miscellaneous
ADCYAP1 adenylate cyclase activating polypeptide 1 (pituitary)
0.00
0.19
0.11
0.01
0.49
1.6
-1.2
1.4
1.6
-1.1
APOA1
apolipoprotein A-I
0.25
0.09
0.06
0.20
0.41
-1.4
1.3
2.3
1.4
-1.2
APOE
apolipoprotein E
0.17
0.05
0.01
0.00
0.76
1.2
1.2
1.3
1.4
-1.0
CRABP2 cellular retinoic acid binding protein 2
0.00
0.00
-1.5
-1.2
EBP
emopamil binding protein (sterol isomerase)
0.00
0.00
0.21
0.00
0.11
-2.4
-2.0
-1.1
-1.4
-1.1
SCP2
sterol carrier protein 2
0.38
0.17
0.52
0.29
0.13
-1.1
-1.1
1.0
1.1
-1.1
0.27 1
0.91
0.16
0.52
0.26
SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),
member
-1.2
-1.0
2.0
1.2
-1.1
SREBF1 sterol regulatory element binding transcription factor 1
0.01
0.24
0.08
0.06
0.21
1.5
1.2
1.3
1.3
-1.4
Gene expression changes (P<0.01) at GD19 in TMD pathways involved in
steroidogenesis and testes development affected by in-utero exposure to
phthalates. MEHP and DEHP, Green = repression; EHO, light green = mild
repression; TOTM and DEHP low dose, white = no repression of
steroidogenesis pathway.
Conclusions
•
EHO
MEHP & DEHP (500 mg/Kg) caused a repression of genes in TMD
pathways involved in cholesterol synthesis and transport (HMGCS, HMGCR,
STAR, SCARB1, FDFT1, FDPS), Steroidogenesis (Cyp11a, HSD3B1,
SC4MOL) and testes development (INSL3, INHA).
• Neither TOTM nor low-dose DEHP caused significant repression of
genes in TMD-relevant pathways.
• EHO caused significant but weak repression of some previously identified
steroidogenesis genes involved in TMD.
TOTM
Questions
Does TOTM-mediate changes in the expression of genes
involved in testosterone synthesis and cryptorchidism
pathways that have been previously identified as targets for
TMD induced by certain phthalates?
Does low-dose DEHP (0.25mg/Kg) treatment
affect
steroidogenesis pathways at the level of transcription?
GenBank
NM_017286
NM_012753
AA924473
NM_017265
AA850212
The microarray analysis involved labelling RNA from foetal testes of four
control-, DEHP-, TOTM-, DEHP (0.25mg/Kg) -, EHO- and MEHP- in utero
treated litters. Total RNA (100ng-1mg) was labelled prior to microarray
hybridisation using the Agilent Quick Amp Labelling Kit One Colour.
Agilent 4x44K Whole Rat Genome Oligo Microarray slides were
hybridised, washed and then scanned on an Agilent Microarray Scanner.
Images from the scanner were processed using Agilent Feature
Extraction Software v9.1. Rosetta ResolverTM 6 software was used to
define a list of significantly altered genes (the “Signature List”). Signature
lists of significantly (p<0.01) altered genes were filtered to remove low
intensity genes.
Comparison of the effects of
DEHP, MEHP, TOTM & EHO on genes
involved in TMD
Effect of test compounds on pathways
involved in TMD
Methods
Pregnant dams were treated daily with vehicle (corn oil), TOTM, DEHP,
EHO or MEHP at doses of 500 mg/Kg by oral gavage on gestation days
12 to 19 inclusive. A further group received 0.25mg/Kg DEHP. On GD19,
testes from a minimum of 5 litters per treatment group were microdissected and snap frozen in liquid nitrogen. Pools of foetal testes from a
minimum of three pups per litter (6 foetal testes) were disrupted using a
qiashredder column and purified using RNeasy mini columns.
Abstract # 1048
• Our recent work on the mechanisms of phthalate-induced testicular maldevelopment indicate that the ability of certain phthalates to repress
steroidogenic genes including STAR is mediated by direct binding of
peroxisome proliferator activated receptor alpha (PPARα) to the promoter
regions of steroidogenic genes (Plummer et al. 2010; 2011).
Plummer et al. (2007). Time-dependent and compartment-specific effects of in utero exposure to
di(n-butyl)phthalate on gene/protein expression in the fetal rat testis as revealed by transcription
profiling and laser capture microdissection. Toxicol. Sci. 97, 520–532
Fold change data derived from the microarray analyses were superimposed on the
genes shown in the above pathways using IPA software. Fold changes (black
lettering under gene icons) greater than 1.5 fold cause gene icons to be coloured
either red = up-regulated or green = down-regulated. Icons in white signify
genes unchanged by the treatment.
Plummer et al. (2010). The effects of dibutylphthalate (DBP) on transcription factor-DNA binding to
fetal rat testes genes relevant to phthalate-induced testicular maldevelopment. The Toxicologist.
Abstract No.1488
Plummer et al. (2011). Immunohistochemical and ChIP microarray analysis of PPARa in fetal rat
testes exposed in utero to dibutylphthalate The Toxicologist. Abstract No.1041
This research was supported by INEOS Enterprises Limited, Polynt SpA,
Oxea Deutschland GmBh and Novasol N.V/S.A.