investigating chemical- microbiota interactions in zebrafish...apr 26, 2018 · u.s....
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Investigating chemical-microbiota interactions in zebrafish
Tamara Tal, PhDU.S. EPA/ORD/NHEERL/ISTD
EPA’s Computational Communities of Practice
April 26, 2018
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Image credit: Chuck Gaul, US EPA
This presentation does not necessarily reflect EPA policyNo conflicts of interest to disclose
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Outline
• Background• Triclosan case study• Estradiol case study• BPA and BP replacements case study• Summary • Challenges
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Microbiota
Image source: http://www.umassmed.edu/microbi
ome/microblog1/publications/
• Consists of all the bacteria, viruses, and fungi external to the body
• Colonization begins at birth and continues throughout life
• Required for development of host organs and systems
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Microbiota-gut-brain axis
4Source: Rea et al. 2016
• Bidirectional communication
• Colonization status modifies neurodevelopmental events
• Imbalances in gut microbiota composition are associated behavioral disorders
• Microbiota has not be assessed as a modifying factor for the developmental neurotoxicity (DNT) of environmental chemicals
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Microbiota-chemical interactions
Adapted from: http://www.oecd.org/chemicalsafety 5
Adverse outcomes• Cancer• Developmental
neurotoxicity (DNT)• Immunotoxicity• Pulmonary toxicity• Reproductive toxicity
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Microbiota-chemical interactions
Adapted from: http://www.oecd.org/chemicalsafety 6
Parent chemical
• Bioactivation• Detoxification
Microbiota
1. Toxicokinetic hypothesis
Adverse outcomes• Cancer• Developmental
neurotoxicity (DNT)• Immunotoxicity• Pulmonary toxicity• Reproductive toxicity
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Microbiota-chemical interactions
Adapted from: http://www.oecd.org/chemicalsafety 7
Parent chemical
• Bioactivation• Detoxification
Microbiota
1. Toxicokinetic hypothesis
2. Toxicodynamic hypothesis
Adverse outcomes• Cancer• Developmental
neurotoxicity (DNT)• Immunotoxicity• Pulmonary toxicity• Reproductive toxicity
Microbiota
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Hypothesis
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Host-associated microbiota:1. Modify the toxicity of environmental chemicals via biotransformations; and/or2. Is a target of chemical exposures during sensitive windows of early
development.
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Zebrafish as a model system for microbiota research
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• External and rapid development• Majority of genes conserved with humans• Complex resident microbiota• Control colonization status• Methods for rearing axenic (microbe-free)
zebrafish through early development• Simple conventionalization (add microbes
to axenic embryos)
A. Veronii:dTomato, gift from K. Guillemin, University of Oregon
Phelps et al. 2017, Scientific Reports
days post fertilization (dpf)
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Does microbiota modify the toxicokinetics or toxicodynamicsof xenobiotic exposures?
10Phelps et al. 2017, Scientific Reports
• CC = conventionally colonized
• AX = Axenic or microbe-free
• AC1 = Axenic larvae colonized on day 1
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Microbiota & DNT: Zebrafish neurobehavioral toxicity assay
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Developmental antibiotic exposure mimics AX hyperactivity phenotype at 10 dpf
Phelps et al. 2017, Scientific Reports
• AB = amphotericin B (0.25 ug/mL), kanamycin (5 ug/mL), and ampicillin (100 ug/mL)
B.A.
C C A C 1 A X C C + A B0
5
1 0
1 5
2 0
2 5
3 0
a a
bb
Me
an
dis
tan
ce
mo
ve
d (
cm
)/1
0 m
in d
ark
pe
rio
d
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
0
5
1 0
1 5
2 0
2 5
3 0
3 5 T im e , p < 0 .0 0 0 1T im e x g ro u p , p = 0 .0 0 0 1
T im e (m in )
Dis
tan
ce
mo
ve
d (
cm
)/2
min
pe
rio
d
C C (n = 2 4 )
A C 1 (n = 2 4 )
A X (n = 2 4 )
C C + A B (n = 2 4 )
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Examine microbiota-chemical interactions: Triclosan case study
13Phelps et al., In preparation
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Chemical dependent effects on host-associated microbiota begin to emerge at 6 dpf
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Chemical dependent effects on host-associated microbiota begin to emerge at 6 dpf
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Widespread changes in microbiota coalesce at 10 dpf
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Triclosan exposure changes relative family-level taxonomy
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Triclosan exposure changes relative family-level taxonomy
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No status-dependent differences in parent tissue dose observed at 6 dpf
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Colonized zebrafish contain higher concentrations of triclosan at 10 dpf
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Do triclosan resistant microbes biotransformtriclosan?
A.
B.
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Microbial colonization changes 78 features ≥ 2 fold
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A. B.
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Colonized larvae contain higher concentrations of parent triclosan by NTA
A. B.
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Link microbiota to phenotype: 17-β estradiol (E2) case study
24Catron et al., In preparation
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Exogenous E2 exposure does not affect microbial community structure
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A. B.
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E2 exposures triggers behavioral hypoactivity in colonized zebrafish
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0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2
0
5
1 0
1 5
2 0
2 5
3 00 .1 % D M S O (n = 2 3 )
0 .0 5 µM E 2 (n = 2 4 )
0 .1 4 µM E 2 (n = 2 4 )
0 .4 µM E 2 (n = 2 3 )
1 .2 µM E 2 (n = 2 2 )
3 .5 µM E 2 (n = 2 4 )
T im e (m in )
Dis
tan
ce m
ove
d (
cm)
/2
min
ep
och
C o n c e n t ra t io n * p h a s e , p = 0 .0 8 3 1
C o n c e n t ra t io n * p h a s e * t im e , p = 0 .0 0 0 3
0
5
1 0
1 5
2 0
2 5
3 0
E 2 (µM)
Me
an
dis
tan
ce m
ove
d (
cm)
/1
0 m
inu
te e
po
ch
0 .1%
DM
S O 0 .0 5
0 .1 4 0 .
41 .
23 .
5
a a abb c b c
c
a abab ab
b b
L ig h t , p < 0 .0 0 0 1 D a rk , p = 0 .0 1 1 0
E 2 (µM)0 .
1% D
MS O 0 .
0 50 .
1 4 0 .4
1 .2
3 .5
A. B.
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Link microbiota to phenotype: 17-β estradiol case study
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Link microbiota to phenotype: 17-β estradiol case study
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Microbe-free zebrafish contain higher concentrations of parent compound
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00 .
41 .
2 00 .
41 .
2 00 .
41 .
2
0
1
2
3
4
N o m in a l w a te rb o rn e E 2 c o n c e n tra tio n (µM )
Tis
sue
co
nce
ntr
atio
n(p
mo
le E
2/la
rva
)
C C (n = 4 )
A C 1 (n = 4 )
A X (n = 4 ,2 ,5 )
a a
c
b cb
b
S ta tu s , p < 0 .0 0 0 1D o se , p < 0 .0 0 0 1S ta tu s *d o se , p < 0 .0 0 0 1
b
d
a
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Examine chemical-dependent changes in microbial communities: Bisphenol A (BPA) and BPA replacement compounds case study
Bisphenol A (BPA)
Bisphenol B (BPB)
Bisphenol S (BPS)
Bisphenol F (BPF)
Bisphenol AF (BPAF)
Catron et al., Under revision
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BPS, BPA, or BPF exposure disrupted global microbial community structure
NMDS Axis 1
NM
DS
Axis
2
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Family level taxonomy
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Predicted microbial functions by linear discriminant analysis (PICRUSt)
Recreated from: http://www.genome.jp/kegg-bin/show_pathway?map00363; Kanehisa et al. 2000
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Differential chemical effects: Host developmental toxicity vs. microbiota disruption
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A. B.
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Summary
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1. We developed an experimental system to test whether microbiota affects the kinetics and/or dynamics of xenobiotic exposures
2. Axenic zebrafish are hyperactive3. Antibiotic exposure phenocopies hyperactivity in colonized zebrafish4. Triclosan resistant taxa increase host parent tissue dose and
perform a sulfation reaction5. Exogenous E2 exposure triggers hypoactivity in the light period in
colonized zebrafish, possibly via a bioactivation event6. Inverse relationship between host toxicity and microbiota disruption
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Microbiota-triclosan interaction take home
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Parent chemical
1. Toxicokinetic hypothesis
2. Toxicodynamic hypothesis
TOXICOKINETIC• Biotransformation; triclosan: Phelps et al. In preparation.• Biotransformation; estradiol (E2): Catron et al. In
preparation.
TOXICODYNAMIC• Antibiotics: Phelps et al. Scientific Reports. 2017.• Bisphenol compounds: Catron et al. Submitted.
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Outstanding questions
• Do chemical-induced compositional changes affect other aspects of development or predispose the organism to future insults?
• Do microbiota-mediated biotransformations broadly affect chemical toxicity?
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Acknowledgements
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U.S. EPA• Emily Anneken (NERL)• Doris Betancourt (NERL)• Nichole Brinkman (NERL)• Scott Keely (NERL)• James McCord (NERL)• Judy Schmid (NHEERL)• Jon Sobus (NERL)• Mark Strynar (NERL)• Adam Swank (NHEERL)• Leah Wehmas (NHEERL)• Charles Wood (NHEERL)• U.S. EPA Zebrafish Facility (Kim Howell, Joan
Hedge, Ned Collins)
Tal lab • Tara Catron (ORISE)• Shaza Gaballah (ORISE)• Allison Kvasnicka (Meredith College)• Drake Phelps (ORISE)
Funding • U.S. EPA Office of Research and
Development• Pathfinder Innovation Project Award
Investigating chemical-microbiota interactions in zebrafishOutlineMicrobiotaMicrobiota-gut-brain axisMicrobiota-chemical interactionsMicrobiota-chemical interactionsMicrobiota-chemical interactionsHypothesisZebrafish as a model system for microbiota researchDoes microbiota modify the toxicokinetics or toxicodynamics of xenobiotic exposures?Microbiota & DNT: Zebrafish neurobehavioral toxicity assayDevelopmental antibiotic exposure mimics AX hyperactivity phenotype at 10 dpfExamine microbiota-chemical interactions: Triclosan case studyChemical dependent effects on host-associated microbiota begin to emerge at 6 dpfChemical dependent effects on host-associated microbiota begin to emerge at 6 dpfWidespread changes in microbiota coalesce at 10 dpfTriclosan exposure changes relative family-level taxonomyTriclosan exposure changes relative family-level taxonomyNo status-dependent differences in parent tissue dose observed at 6 dpfColonized zebrafish contain higher concentrations of triclosan at 10 dpfDo triclosan resistant microbes biotransform triclosan?Microbial colonization changes 78 features ≥ 2 foldColonized larvae contain higher concentrations of parent triclosan by NTALink microbiota to phenotype: 17-β estradiol (E2) case studyExogenous E2 exposure does not affect microbial community structureE2 exposures triggers behavioral hypoactivity in colonized zebrafishLink microbiota to phenotype: 17-β estradiol case studyLink microbiota to phenotype: 17-β estradiol case studyMicrobe-free zebrafish contain higher concentrations of parent compoundExamine chemical-dependent changes in microbial communities: Bisphenol A (BPA) and BPA replacement compounds case studySlide Number 31BPS, BPA, or BPF exposure disrupted global microbial community structureSlide Number 33Family level taxonomyPredicted microbial functions by linear discriminant analysis (PICRUSt)Differential chemical effects: Host developmental toxicity vs. microbiota disruptionSummaryMicrobiota-triclosan interaction take homeOutstanding questionsAcknowledgements