Investigating chemical- microbiota interactions in zebrafish...Apr 26, 2018 · U.S. EPA/ORD/NHEERL/ISTD. EPA’s Computational . Communities of Practice. April 26, 2018. 1 Image

Investigating chemical-microbiota interactions in zebrafish

Tamara Tal, PhDU.S. EPA/ORD/NHEERL/ISTD

EPA’s Computational Communities of Practice

April 26, 2018

1

Image credit: Chuck Gaul, US EPA

This presentation does not necessarily reflect EPA policyNo conflicts of interest to disclose

Outline

• Background• Triclosan case study• Estradiol case study• BPA and BP replacements case study• Summary • Challenges

2

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

3

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

Microbiota-chemical interactions

Adapted from: http://www.oecd.org/chemicalsafety 5

Adverse outcomes• Cancer• Developmental

neurotoxicity (DNT)• Immunotoxicity• Pulmonary toxicity• Reproductive toxicity



Parent chemical

• Bioactivation• Detoxification

Microbiota

1. Toxicokinetic hypothesis





Parent chemical

• Bioactivation• Detoxification

Microbiota


2. Toxicodynamic hypothesis



Microbiota

Hypothesis

8

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.

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)

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

12

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 )

Examine microbiota-chemical interactions: Triclosan case study

13Phelps et al., In preparation

Chemical dependent effects on host-associated microbiota begin to emerge at 6 dpf

14

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

16

Triclosan exposure changes relative family-level taxonomy

17

Triclosan exposure changes relative family-level taxonomy

18

19

No status-dependent differences in parent tissue dose observed at 6 dpf

20

Colonized zebrafish contain higher concentrations of triclosan at 10 dpf

21

Do triclosan resistant microbes biotransformtriclosan?

A.

B.

Microbial colonization changes 78 features ≥ 2 fold

22

A. B.

23

Colonized larvae contain higher concentrations of parent triclosan by NTA

A. B.

Link microbiota to phenotype: 17-β estradiol (E2) case study

24Catron et al., In preparation

Exogenous E2 exposure does not affect microbial community structure

25

A. B.

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

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ove

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ch

0 .1%

DM

S O 0 .0 5

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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.

Link microbiota to phenotype: 17-β estradiol case study

27

Link microbiota to phenotype: 17-β estradiol case study

28

Microbe-free zebrafish contain higher concentrations of parent compound

29

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

30

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

BPS, BPA, or BPF exposure disrupted global microbial community structure

NMDS Axis 1

NM

DS

Axis

2

32

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

35

Differential chemical effects: Host developmental toxicity vs. microbiota disruption

36

A. B.

Summary

37

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

Microbiota-triclosan interaction take home

38

Parent chemical


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.

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

Investigating chemical- microbiota interactions in zebrafish...Apr 26, 2018 · U.S. EPA/ORD/NHEERL/ISTD. EPA’s Computational . Communities of Practice. April 26, 2018. 1 Image

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