The Toxicologist - Society of Toxicology

345 PEROXISOME PROLIFERATOR ACTIVATED
RECEPTOR γ IS ACTIVATED BY 15-DEOXY-PGJ 2 -G, A
PUTATIVE METABOLITE OF THE
ENDOCANNABINOID, 2-ARACHIDONYL GLYCEROL.
P. Raman 1, 2 , B. Kaplan 1, 2 , J. Thompson 3 , J. Vanden Heuvel 3 and N. Kaminski 1,
2 . 1 Pharmacology & Toxicology, Michigan State University, East Lansing, MI,
2 Center for Integrative Toxicology, Michigan State University, East Lansing, MI and
3 Veterinary Science & Biomedical Sciences, Center for Molecular Toxicology and
Carcinogenesis, Pennsylvania State University, University Park, PA.
2-Arachidonyl glycerol (2-AG), an endocannabinoid, suppressed IL-2 secretion in
activated T cells by the activation of peroxisome proliferator activated receptor γ
(PPARγ) independently of the cannabinoid receptors, CB1 and CB2. One of the
putative metabolites of 2-AG is a glycerol ester of a prostaglandin, 15-deoxy
prostaglandin J 2 -glycerol (15d-PGJ 2 -G). Since one of the known ligands for PPARγ
is 15-deoxy-prostaglandin J2 (15d-PGJ 2 ), 15d-PGJ 2 -G was investigated as a putative
PPARγ ligand. Previously, it was demonstrated that 15dPGJ 2 -G transcriptionally
activated PPARγ in HEK293T cells and it has been demonstrated that 2-AG
and other PPARγ agonists suppress interleukin-2 (IL-2) in activated T cells.
Therefore, we investigated if 15d-PGJ 2 -G activates PPARγ in a T cell line. In Jurkat
T-Ag cells transfected with PPARγ reporter, 15d-PGJ 2 -G treatment induced PPARγ
reporter. Time of addition studies using activated Jurkat T cells demonstrated that
addition of 15d-PGJ 2 -G either 30 min before or after Jurkat cell activation produced
marked IL-2 suppression whereas addition of 15d-PGJ 2 -G at later times (1,
2, 4, 8 h post stimulation) resulted in an attenuation of 15d-PGJ 2 -G-mediated IL-
2 suppression. In addition, 15d-PGJ 2 -G decreased the transcriptional activity of
nuclear factor of activated T cells (NFAT). Importantly, 15d-PGJ 2 -G-mediated IL-
2 suppression and the decrease in NFAT reporter activity were attenuated in the
presence of a PPARγ antagonist, T0070907. We hypothesize that 15d-PGJ 2 -G is a
metabolite of 2-AG and is responsible for IL-2 suppression through the activation
of PPARγ and the decrease in NFAT transcriptional activity. (Support in part by
NIH RO1 DA12740 and Pfizer Global Research).
346 PHARMACOLOGICAL STUDY OF SEROTONIN AND
DOPAMINE POST-SYNAPTIC RECEPTORS OF THE
LATERAL CILIATED CELLS OF THE GILL AND
VISCERAL GANGLIA OF CRASSOSTREA VIRGINICA.
C. Brown, Z. Bandaogo, E. J. Catapane, M. A. Carroll, A. Hernandez and D.
Adebesin. Biology, Medgar Evers College, Brooklyn, NY.
Lateral cilia of gill of Crassostrea virginica are controlled by serotonergic–dopaminergic
nerves from their visceral and cerebral ganglia. Serotonin (HT) and dopamine
(DA) are the neurotransmitters that increase and decrease ciliary beating rates, respectively.
We undertook a pharmacological study of the HT receptor types. HT receptor
families are classified as HT1 - 7, with subtypes. All but HT3 are G protein
metabotrophic; HT3 are iontotrophic. DA receptors are classified as D1-like and
D2-like, each with respective subtypes. D1 receptors are coupled to G protein Gαs
and activates adenyl cyclase. D2 receptors are coupled to the G protein Gαi, and
directly inhibits the formation of cAMP by inhibiting the enzyme adenyl cyclase.
Beating rates of cilia were measured by stroboscopic microscopy of isolated gill
preparations and VG-gill preparations in which the innervation of the gill by the
visceral ganglia was kept intact. Agonists and antagonists of different receptor families
(5-carboxamidotryptamine, alpha methyl serotonin, BIMU8, 1-(3chlorophenyl)biguanide
methysergide, NAN-190, GR127935 and clozapine Noxide,
A68930, propylpiperdine, piribedil, BHT920, 2-bromo-ergocryptine,
SKF89626, supiride, droperidol, metoclopramide, ergonovine and chlorprothixene)
were tested at the gill and visceral ganglia to determine their efficacy in altering
cilia beating rates. Analysis of the receptor families and subtypes for the agonists
and antagonists indicates that HT4 or HT7 receptors are present in the gill and visceral
ganglia, and that the DA receptors present in the gill and visceral ganglia are
the D2 type. The study also shows that this preparation is a good model for pharmacological
studies of serotonin and dopamine function as well as the pharmacology
of drugs affecting biogenic amines in nervous systems. This work was supported
by 2R25GM06003 of the Bridge Program of NIGMS, 0516041071 of
NYSDOE and 0622197 of the DUE Program of NSF.
74 SOT 2011 ANNUAL MEETING
347 SPECIFIC AMINO ACID RESIDUES WITHIN THE
LIGAND BINDING DOMAIN CONFER LOW
RESPONSIVENESS TO 2, 3, 7, 8-
TETRACHLORODIBENZO-P-DIOXIN (TCDD) TO AN
ARYL HYDROCARBON RECEPTOR (AHR) FROM THE
FROG XENOPUS LAEVIS.
C. Odio 1 , S. A. Holzman 1 , M. S. Denison 2 , L. Bonati 3 and W. H. Powell 1 .
1 Biology, Kenyon College, Gambier, OH, 2 Environmental Toxicology, University of
California-Davis, Davis, CA and 3 Scienze dell’Ambiente e del Territorio, Universita`
degli Studi di Milano-Bicocca, Milano, Italy.
Frogs are remarkably insensitive to TCDD toxicity. Previous studies in Xenopus laevis
revealed that frog AHRs bind TCDD with low affinity. In other vertebrates, differences
in TCDD toxicity and AHR binding affinity result from differences in specific
amino acids within the ligand binding domain (LBD). We sought to identify
structural determinants within X. laevis AHR1β associated with low TCDD responsiveness
using transactivation assays. Substitution of the entire LBD with the
corresponding sequence from mouse AHR b-1 restored TCDD responsiveness.
Surprisingly, the frog LBD contains all residues previously associated with high
affinity TCDD binding in mouse AHR alleles, suggesting a role for other amino
acids in the low-affinity phenotype. To seek additional candidate residues, we constructed
a comparative model of the AHR1β LBD using the homologous domains
of HIF2α and ARNT. The model revealed an internal cavity similar in dimensions
to the putative binding pocket of mouse AHR b-1 , suggesting the importance of
side-chain interactions over cavity size. Of residues with side chains clearly pointing
into the pocket, only two differed from the mouse sequence. When A354, located
within a conserved β-strand, was changed to serine, the homologous mouse
residue, the EC 50 for TCDD decreased more than 15-fold. When N325, in a long
α-helical connector, was changed to serine, EC 50 declined 3-fold. The combined
mutations resulted in a 22-fold reduction in EC 50 , from 18.75 nM to 0.83 nM, a
large but incomplete recovery of TCDD responsiveness. The location of these
residues suggests that low responsiveness of AHR1β in transactivation assays is substantially
attributable to low affinity binding of TCDD. [NIH ES011130 (WHP),
ES07685 (MSD)]
348 THE NOVEL DAPHNIA NUCLEAR RECEPTOR, HR97G,
IS ACTIVATED BY JUVENILE HORMONE ANALOGS.
G. Ginjupalli 2 , Y. Li 2 and W. S. Baldwin 2, 1 . 1 Biological Sciences, Clemson
University, Clemson, SC and 2 Environmental Toxicology Program, Clemson
University, Clemson, SC.
Daphnia magna are a commonly used sentinel specie in toxicity testing. The
Daphnia pulex genome sequenced recently revealed three novel nuclear receptors related
to the CAR/PXR/VDR group and were called as HR97a/b/g, because of their
similarity to the HR96 nuclear receptors in Drosophila and Daphnia. We cloned
HR97g from Daphnia magna, and then made a Gal4-expression vector containing
the LBD of HR97g for transactivation assays. These assays found only three activators,
cortisol, pyriproxyfen, and methyl farnesoate; two of which are key juvenile
hormone analogs that induce the production of males in the otherwise female
parthenogenic Daphnia. Dose-response studies found that pyriproxyfen and
methyl farnesoate have EC50’s of 3.4 and 2.2 uM, respectively.
Immunohistochemistry (IHC) revealed that HR97g is primarily expressed in the
gut, ovaries, and mandibular organ, a key organ in juvenile hormone production.
RT-PCR and IHC demonstrated that HR97g is predominantly expressed in young,
pre-reproductive daphnids less than 8 days old similar to juvenile hormone expression.
In addition, treatment of adult female Daphnia magna with 200 or 400ng/L
pyriproxyfen induces HR97g expression providing further evidence that HR97g is
associated with juvenile hormone function. In summary, the juvenile hormone receptor
is an ongoing search and HR97g is a candidate juvenile hormone receptor in
the crustacean, Daphnia magna, because it is activated by juvenoids, its expression
is associated with juvenoids, and it is induced by juvenile hormone analogs such as
the pesticide pyriproxyfen.
349 LIGAND-ACTIVATED AHR ALTERS THE EXPRESSION
OF GENES IN THE HUMAN EPIDERMAL
DIFFERENTIATION COMPLEX.
C. H. Sutter, S. Bodreddigari, C. Campion, R. Wible and T. Sutter. Biological
Sciences, University of Memphis, Memphis, TN.
In humans exposed to dioxin (TCDD), the most often observed and best studied
toxic response is chloracne. Chloracne is manifested in the skin as hyperkeratinization
of the interfollicular squamous epithelium, hyperproliferation and hyperkera-