Impacts of Chemical Mixtures Isolated from Household Dust on - - PowerPoint PPT Presentation

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Impacts of Chemical Mixtures Isolated from Household Dust on Metabolic Health Chris Kassotis, PhD Postdoctoral Fellow Duke University @cdkassotis Young EDC Scientists Showcase Seminar July 1, 2020 Prevalence and Consequences of

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Young EDC Scientists Showcase Seminar July 1, 2020

Impacts of Chemical Mixtures Isolated from Household Dust on Metabolic Health

Chris Kassotis, PhD Postdoctoral Fellow Duke University @cdkassotis

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SLIDE 2

Prevalence and Consequences of Obesity Epidemic in US, Globally

Ø Currently ~40% of US adult

population is obese.

Ø ~9% infants/toddlers Ø ~19% of 2-19 year-olds

Ø >$265B in US health care costs on

  • besity related illnesses (2015)

Ø ~8% of total US health care costs

(>12% in NC, OH, WI; 2018)

Ø Increased comorbidities

Ø T2D, CVD, hypertension

Ø Interventions have produced only

modest effects

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SLIDE 3

Potential Role of Chemicals in Increasing Obesity Rates in Humans

Ø First posited in 2002, despite

decades of experimental evidence.

Ø Challenges caloric intake,

activity, genetics as sufficient factors to explain magnitude/speed of observed trend.

Ø Summarizes wealth of animal

evidence on antibiotics, PCBs, plastics, pharmaceuticals, pesticides, organophosphates, heavy metals, etc.

Baillie-Hamilton et al. 2002, J Alt Comp Med

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SLIDE 4

Potential Mechanisms of Metabolic Dysfunction

Ø Numerous potential mechanisms

  • f metabolic disruption:

Ø Adipocyte commitment from

MSCs

Ø Adipocyte differentiation from

precursor cells

Ø

Increased pre-adipocyte proliferation

Ø

Increased lipid uptake Ø Shifting energy balance to

favor calorie storage

Ø Altering basal metabolic rate Ø Altering hormonal control of

appetite and satiety

Ø Altering brain circuitry that

controls food intake, energy expenditure

Heindel et al. 2017, Repro Tox

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SLIDE 5

Adipocyte Differentiation Process

Nagy et al. 2011, Mol Med

Other pathways: Myoblasts Osteoblasts Chondroblasts Resemble brown/developing white adipose cell Resemble mature white adipose cell

Adipocyte commitment Adipocyte differentiation

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SLIDE 6

3T3-L1 Pre-adipocyte Adipogenesis Assay

Ø Swiss albino mouse embryonic fibroblast cell line – committed

pre-adipocytes

Ø Extensively used over decades to evaluate adipogenesis

Ø

Mechanisms of adipocyte differentiation well understood

Ø

This assay, particularly coupled with PPARγ reporter gene assays, has proven a reliable in vitro model for metabolic disruption in vivo.

Differentiation cocktail: 5% NCS -> FBS, 1 µg/mL insulin, 800 mM IBMX

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SLIDE 7

Adipogenesis Assay Measures

§

Triglyceride accumulation

§

AdipoRed - hydrophilic fluorescent dye (Nile Red)

§

Partitions into lipid droplets in the cells, fluoresces

§

Cell proliferation/cytotoxicity

§

NucBlue DNA dye (Hoechst 33342)

§

Partitions into nuclei and fluoresces upon binding DNA

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SLIDE 8

Cancer in the Environment (CIE) Cohort

Ø N=137 adult participants recruited from

central NC.

Ø Demographic, lifestyle, and environment

information collected via questionnaire.

Ø Clinical data abstracted from medical records. Ø Visited participants’ homes and collected dust

samples as a measure of long-term exposure.

Ø ~200 mg dust sieved to <500 μm, solvent extracted

in 50:50 DCM:hexane, concentrated under N2 gas.

Ø Half of extract evaporated and reconstituted in

DMSO for bioassays, half purified further for mass spec analysis.

photo credit: Jared Lazarus Duke Photography

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SLIDE 9

Chemical Exposure Markers: Indoor House Dust

Ø Household dust is a well-described reservoir for

chemicals leaching from consumer products and materials in home.

Ø Hundreds of contaminants have been measured

in dust globally – a complex environmental mixture

Ø Previous research has measured endocrine

bioactivities for various receptors by household dust extracts

Ø Residents chronically exposed to chemicals

present in dust via oral, dermal, and inhalation exposure routes.

Ø Research has demonstrated strong positive

correlations between chemicals in dust and internal chemical/metabolite concentrations in serum/urine.

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SLIDE 10

Majority of Dust Extracts Promote Adipocyte Development at Low Concentrations (<1 mg)

Ø Majority of dust extracts promoted significant adipogenic activity

(~90%).

Ø >60% exhibited significant triglyceride accumulation Ø >70% exhibited significant pre-adipocyte proliferation

Kassotis et al. 2019, STOTEN

Rosiglitazone (10 uM) DMSO control Dust extract A 200 ug/well Dust extract B 110 ug/well

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SLIDE 11

Adipogenesis Endpoints Shared and Distinct Across Dust Extracts

1000 1 10 100 1000 30 60 90 120 Dust Extract Quantity (µg/well) DNA Content Relative to Vehicle (%)

Dust-Induced Proliferation / Cytotoxicity

1000 10 100 1000

  • 60
  • 30

30 60 Dust Extract Quantity (µg/well) % DNA Content Relative to Vehicle

Dust-Induced Proliferation / Cytotoxicity

1000

ell

0.1 1 10 100 1000 10 20 30 40 50 60 Dust Extract Quantity (µg/well) DNA Content Relative to Vehicle (%)

Dust-Induced Proliferation / Cytotoxicity

High triglyceride accumulation High pre-adipocyte proliferation High triglyceride accumulation Minimal pre-adipocyte proliferation Minimal triglyceride accumulation High pre-adipocyte proliferation Kassotis et al. 2019, STOTEN

1 10 100 1000 50 100 150 200 250 Dust Extract Quantity (µg/well) Triglyceride Accumulation Per Cell (%)

Dust-Induced Triglyceride Accumulation per Cell

1000 1 10 100 1000 30 60 90 120 150 Dust Extract Quantity (µg/well) Triglyceride Accumulation Per Cell (%)

Dust-Induced Triglyceride Accumulation per Cell

1000 0.1 1 10 100 1000 10 20 30 40 50 Dust Extract Quantity (µg/well) Triglyceride Accumulation Per Cell (%)

Dust-Induced Triglyceride Accumulation per Cell

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SLIDE 12

BFR and PFR Flame Retardants Associated with Increased Triglyceride Accumulation

Correlation Coefficients BFRs/PFRs Triglyceride Accumulation Pre-adipocyte Proliferation

BDE-47 0.244**

  • 0.096

BDE-99 0.294**

  • 0.124

BDE-100 0.339**

  • 0.043

BDE-153 0.385**

  • 0.049

BDE-154 0.394**

  • 0.073

BDE-209 0.462** 0.060 TBB 0.324** 0.006 TBPH 0.341** 0.025 TCEP 0.343**

  • 0.013

TDCIPP 0.397**

  • 0.099

TCIPP 0.290**

  • 0.041

TPHP 0.199*

  • 0.011

Kassotis et al. 2019, STOTEN Spearman’s correlations: * p<0.05; ** p<0.01

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Regression Analyses of Health Outcomes and House Dust Extract Bioactivities

Ø Thyroid stimulating hormone in adult

residents positively correlated with adipogenic activity of their house dust (normalized by concentration); free triiodothyronine (T3) and thyroxine (T4) negatively correlated.

Ø TRβ antagonism promoting

adipogenesis a likely factor in the TH suppression

Ø Performed regressions controlling

for sex, age, race, and education as potential confounders.

Ø Triglyceride accumulation efficacy

was significantly associated with resident BMI.

Kassotis et al. 2019, STOTEN

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SLIDE 14

Putative Role of Thyroid Receptor β Antagonism in Adipogenic Activity

Ø GR (dexamethasone) and PPARγ

(rosiglitazone) are potent and efficacious regulators of adipogenesis.

Ø 1-850 (non-specific TRβ isoform antagonist)

also significantly promotes adipocyte differentiation.

Kassotis et al. 2017, Sci Rep

20 40 60 80 50 100 150 200 350 400

TRβ Antagonism (% Inhibition of EC80 T3) Triglyceride Accumulation Per Cell (%) TRβ Antagonism and 3T3-L1 Dust-Induced Triglycerides

rs = 0.447 p < 0.0001

Ø Triglyceride accumulation (3T3-L1

cells) significantly correlated with TRβ antagonism in dust extracts.

Ø

Not correlated with pre-adipocyte proliferation

Kassotis et al. 2019, STOTEN

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SLIDE 15

Contributory Role of TRβ Antagonism in Adipogenic Activity

Ø Two experiments bolster causative

link between TRβ and triglyceride accumulation in 3T3-L1 cells:

Ø Ligand recovery experiment. Dust +

T3 (TR agonist):

Ø

Addition of T3 inhibited dust- induced triglyceride accumulation for 7 of 9 samples. Ø siRNA knock-down of TRa/β:

Ø

TR knock-down inhibited dust- induced triglyceride accumulation for 7 of 9 samples (two trending).

CE095 CE143 CE134 CE124 CE151 CE152 CE114 CE117 CE133 50 100 150 200 250

Dust Sample % Triglycerides to Dust Control

Dust-Induced Triglycerides - TR siRNA Knock-Down

* **

#

*

#

*

# Each grouping: Dust alone, Dust+Negative Control siRNA, Dust+TRα/β siRNA CE095 CE114 CE117 CE143 CE133 CE134 CE124 CE151 CE152 25 50 75 100 125 150 Dust Sample Triglyceride Accumulation Per Cell (%)

Low-Dose Dust-Induced Triglycerides - T3 Recovery

** ** * ** ** **

#

*

Kassotis et al. 2019, STOTEN

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SLIDE 16

Ethoxylated Surfactants are Common Environmental Contaminants

Ø

High-production volume chemicals

Ø

>13 million metric tons, 2008

Ø

>$33 billion global revenues, 2014

Ø

Used widely in laundry detergents, hard- surface cleaners, paints, cosmetics, agriculture.

Ø

Common environmental contaminants

Ø

Widely reported at μg/L conc. in water column (wastewater)

Ø

Detected with high frequency in indoor house dust samples

Ø

Tested the ability of various ethoxylated surfactants to promote adipogenesis

Ø

6 APEO/AEO surfactants with varying alkyl chain lengths (carbon backbones C11-16)

Ø

Select NPEOs with varying average ethoxylate chain lengths (2, 4, 6, 10, 20)

Alkyl chain length Ethoxylate chain length

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SLIDE 17

Various Alkyl Chain Length Surfactants Induce Adipogenesis to Varying Degrees

  • 4
  • 10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4

50 100 150 200

Concentration (M) Triglyceride Accumulation Per Cell (%)

Triglyceride Accumulation per Cell

* * ** * *

C E

ylated Surfactants

Kassotis et al. 2019, Tox Sci

  • 4

Cetyl alcohol ethoxylate Nonylphenol ethoxylate (4) Octylphenol ethoxlate (3) Lauryl alcohol ethoxylate Tomadol 1-9 Tridecyl alcohol ethoxylate

Ø Six ethoxylated surfactants (alkyl lengths 11-16) all induced triglyceride

accumulation in 3T3-L1 cells.

Ø

Cetyl alcohol and NPEO induced greater maximal accumulation than the rosiglitazone control.

Ø 4/6 surfactants induced pre-adipocyte proliferation.

  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 50

50 100 150 200

Concentration (M) DNA Content Relative to Vehicle (%)

Cell Proliferation/Cytotoxicity

* * * *

B C E

yl Length Ethoxylated

(C11) (C12) (C13) (C14) (C15) (C16) (C15) (C16) (C15) (C16)

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SLIDE 18

Nonylphenol Ethoxylates Induce Chain- Length Dependent Adipogenic Effects

  • 4
  • 10
  • 9
  • 8
  • 7
  • 6
  • 5
  • 4

25 50 75 100 125 150

Concentration (M) Triglyceride Accumulation Per Cell (%)

Triglyceride Accumulation per Cell

* * * ** *

C E

Kassotis et al. 2019, Tox Sci

Ø NPEOs induced varied adipogenic responses. Ø Maximal response for medium-length (4/6) ethoxylate chains;

decreasing activity with decreasing or increasing chain number.

Ø

Activity for NPEO(20) indistinguishable from base (0).

  • 4

Nonylphenol ethoxylate (0) Nonylphenol ethoxylate (1-2) Nonylphenol ethoxylate (4) Nonylphenol ethoxylate (6) Nonylphenol ethoxylate (9-10) Nonylphenol ethoxylate (20) ll

  • 9
  • 8
  • 7
  • 6
  • 5
  • 4
  • 50

50 100 150 200

Concentration (M) DNA Content Relative to Vehicle (%)

Cell Proliferation/Cytotoxicity

* * * * *

B C E

5 10 15 20 25 50 100 150

Ethoxylate Chain Length & Adipogenicity

Ethoxylate Chain Length (EO #) Triglyceride Accumulation Per Cell (%)

(4) (6) (1.5) (4) (6) (1.5)

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SLIDE 19

Next Steps: K99/R00 Research Aims

Ø Utilization of the zebrafish model to assess whether select

polyethoxylated surfactants (alcohol and alkylphenol) induce metabolic health effects following developmental exposure.

Ø Weight gain (gross), adipose depot development (adipocyte staining

and depot-specific quantification) Ø Identification of molecular mechanisms driving the adipogenic

effects of polyethoxylated surfactants across species.

Ø Human and zebrafish in vitro models, cell-based and cell-free

Ø Utilize affinity-directed analysis and HRMS to identify causative

adipogenic ligands in environmental samples.

Ø Confirmation in pre-adipocyte models; role of APEOs/AEOs

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SLIDE 20

The Zebrafish Model (Danio rerio) for Metabolic Health Research

Ø

High genetic fidelity to humans – endocrine system is highly conserved, as is metabolic system

Ø

84% of genes known to be associated with human disease have zebrafish counterpart Ø

Molecular mechanisms underlying adipocyte and lipid depot development are highly conserved

Ø

Energy storage functions and morphology of adipose tissue

Ø

Genes associated with adipocyte differentiation, lipolysis, and endocrine function

Ø

Control of adipose distribution into anatomically/ physiologically/molecularly distinct depots Ø

Fish adipose tissue also contains a heterogeneous cell population, including adipocyte progenitor cells – similar to mammals

Ø

Imaging of whole-animal adipose imaging in mammals is limited, technically challenging, and generally low resolution

Minchin and Rawls 2017, Disease Mod Mech

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SLIDE 21

Summary: Environmental Contaminants as Metabolic Disruptors

Ø Numerous common environmental contaminants and complex

environmental mixtures can disrupt metabolic health in vitro at environmentally-relevant concentrations.

Ø

Evidence that some environmental mixtures might promote adipogenesis through mechanisms other than PPARγ Ø In many mixtures, the causative chemicals promoting the activity have

yet to be determined.

Ø

Need for new analytical tools to isolate and identify

Ø

Need for better application of molecular databases to ease translation of in vitro data to potential in vivo health effects Ø Seems to be an association between the adipogenic activity exhibited by

house dust and the metabolic health of residents living in those homes.

Ø

This is not necessarily causative; could be a measure of altered behavior in individuals who are already overweight, contributing to different chemical burdens in the indoor environment

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SLIDE 22

Acknowledgements

Ø Stapleton Lab

Ø

Heather Stapleton, PhD

Ø

Kate Hoffman, PhD

Ø

Nick Herkert, PhD

Ø

Erin Kollitz, PhD

Ø

Ellen Cooper, PhD

Ø

Allison Phillips, PhD

Ø

Stephanie Hammel, PhD

Ø

Matt Ruis

Ø

Kirsten Overdahl

Ø

Sam Hall

Ø

Jessica Levasseur

Ø

Emina Hodzic

Ø

Sharon Zhang

Ø

Funding

Ø

NIEHS R01 ES016099

Ø

NIEHS F32 ES027320

Ø

NIEHS K99 ES030405

Ø Collaborators

Duke:

Ø

Lee Ferguson, PhD NC State:

Ø

Seth Kullman, PhD

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SLIDE 23

I’m Recruiting!

Ø Incoming Assistant

Professor in Institute of Environmental Health Sciences and Department

  • f Pharmacology at

Wayne State University in Detroit, starting September 1!

Ø Recruiting grad

students, technician(s), postdoc, etc. Email me for more information!