| dc.description.abstract |
<p>Accumulating evidence is suggesting that exposure to some environmental contaminants
may alter adipogenesis, resulting in accumulation of adipocytes, and often significant
weight gain. Thus these types of contaminants are often referred to as obesogens.
Many of these contaminants act via the activation (i.e. agonism) of the peroxisome
proliferator activated receptor γ (PPARγ) nuclear receptor. To date, very
few chemicals have been identified as possible PPAR ligands. In the thesis,
our goal was to determine the PPARγ ligand binding potency and activation of
several groups of major semi-volatile organic compounds (SVOCs) that are ubiquitously
detected in indoor environments, including flame retardants such as polybrominated
diphenyl ethers (PBDEs) and Firemaster 550 (FM550), and other SVOCs such as phthalates,
organotins, halogenated phenols and bisphenols. Additional attention was also given
to the potential activity of the major metabolites of several of these compounds.
Since the primary sink for many of these SVOCs is dust, and dust ingestion has been
confirmed as an important pathway for SVOCs accumulation in humans, the potential
PPAR binding and activation in extracts from environmentally relevant dust
samples was also investigated. </p><p> Previous studies have also shown that
SVOCs sorbed to organic matrices (e.g., soil and sediment), were only partially bioaccessible
(bioavailable), but it was unclear how bioaccessible these compounds are from indoor
dust matrices. In addition, bioactivation of SVOCs (via metabolism) could exacerbate
their PPAR potency. Therefore, to adequately assess the potential risk of
PPARγ activation from exposure to SVOC mixtures in house dust, it is essential
that one also investigates the bioaccessibility and bioactivation of these chemicals
following ingestion. </p><p> In the first research aim of this thesis, the bioaccessibility
and bioactivation of several important SVOCs in house dust was investigated. To accomplish
this, Tenax beads (TA) encapsulated within a stainless steel insert were used as an
infinite adsorption sink to estimate the dynamic absorption of a suite of flame retardants
(FRs) commonly detected in indoor dust samples, and from a few polyurethane foam samples
for comparison. Experimental results demonstrate that the bioaccessibility and stability
of FRs following ingestion varies both by chemical and by matrix. Organophosphate
flame retardants (OPFRs) had the highest estimated bioaccessibility (~80%) compared
to brominated compounds (e.g. PBDEs), and values generally decreased with increasing
Log Kow, with <30% bioaccessibility measured for the most hydrophobic compound tested,
BDE209. In addition, the stability of the more labile SVOCs that contained ester groups
(e.g. OPFRs and 2-ethylhexyl-tetrabromo-benzoate (TBB)) were examined in a simulated
digestive fluid matrix. No significant changes in the OPFR concentrations were observed
in this fluid; however, TBB was found to readily hydrolyze to tetrabromobenzoic acid
(TBBA) in the intestinal fluid in the presence of lipases. </p><p> In research
aims 2 and 3, two commercially available high-throughput bioassays, a fluorescence
polarization PPAR ligand binding assay (PolarScreenTM PPARγ-competitor
assay kit, Invitrogen, Aim 2) and a PPAR reporter gene assay (GeneBLAzer PPARγ
non-DA Assay, Invitrogen, Aim 3) were used to investigate the binding potency and
activation of several groups of SVOCs and dust extracts with human PPARγ LBD;
respectively. In the PPAR binding assay (Aim 2), most of the tested compounds
exhibited dose-dependent binding to PPARγ. Mono(2-ethylhexyl) tetrabromophthalate
(TB-MEHP), halogenated bisphenol/phenols, triphenyl phosphate and hydroxylated PBDEs
were found to be potent or moderate PPARγ ligands, based on the measured ligand
binding dissociation constant (Kd). The most potent compound was 3-OH-BDE47, with
an IC50 of 0.24 μM. The extent of halogenation and the position of the hydroxyl
group strongly affected binding. Of the dust samples tested, 21 of 24 samples showed
significant PPAR binding potency at a concentration of 3 mg dust equivalents
(DEQ)/mL. In the PPAR reporter assay (Aim 3), many SVOCs or their metabolites
were either confirmed (based on previous reports) or for the first time were found
to be potential PPARγ agonists with various potency and efficacy. We also observed
that 15 of 25 dust extracts examined showed an activation percentage more than 8%
(calculated activation threshold) of the maximal activation induced by rosiglitazone
(positive control). In some cases, activation was as high as 50% of the rosiglitazone
activation for the dust extracts with the highest efficacy. Furthermore, the correlation
between the reporter assay and the ligand binding assay among the house dust extracts
was significant and positive (r = 0.7, p < 0.003), suggesting the binding potency
was predicting activation. In research aim 2, the effect of bioactivation on the PPARγ
binding potency was also investigated. In vitro bioactivation of house dust extracts
incubated with rat and human hepatic S9 fractions was used to investigate the role
of in vivo biotransformation on PPAR gamma activity. The result showed that metabolism
may lead to an increased binding affinity, as a 3-16% increase in PPARγ binding
activity was observed following bioactivation of the dust extracts.</p><p> In research
aim 4, an effect-directed analysis (EDA) was used to identify compounds likely contributing
to the observed PPAR activity among the dust extract. Three dust extracts
which showed significant PPAR activity with approximately 25, 30, and 50%
of the maximal response induced by rosiglitazone at the highest efficacy were fractionated
using normal phase high-performance liquid chromatography (NP-HPLC) and each fraction
was individually tested for PPAR activity. Active fractions were then analyzed
using gas-chromatography mass spectrometry (GC-MS) and possible compounds identified.
Three dust extracts showed a similar PPAR activity distribution among the
NP-HPLC fractions. In the most active fractions, fatty acids (FAs) were identified
as the most active chemicals. The concentrations of four FAs were measured in the
house dust extracts, and the concentrations were found to be highly correlated with
the observed PPAR activity. These four FAs were also tested for PPAR
activity and found to be partial PPAR agonists, particularly oleic and myristic
acid. To tentatively identify sources of FAs, FAs in human/animal hair, dead skin
cells, and two brands of cooking oil were analyzed. We found the same FAs in those
samples and there concentrations were relatively abundant, ranging from 186 to 14,868
µg/g. Therefore, these results suggest that FAs are likely responsible for the observed
PPAR activity in indoor dust. Also, this is the first study reporting on the
level of FAs in dust samples. The source of these FAs in dust may be either from the
cooking or accumulation of human/animal cells in indoor dust.</p><p> In conclusion,
this research demonstrates that many SVOCs ubiqutiously detected in house dust, and/or
their metabolites, can be weak or moderate PPAR ligands. In addition, chemical
mixtures in house dust can effectively bind to and activate PPAR. However,
our results suggest FAs are probably responsible for these observations, and likely
outcompeting the synthetic environmental contaminants present in the dust extract.
Furthermore, bioactivation of contaminants present in house dust can potentially increase
their affinity for PPAR. And lastly, the bioaccessibility and stability of
SVOCs in house dust after ingestion are likely to modulate the PPAR activity
in the environmental mixtures and should be considered in future risk assessments.</p>
|
|
