Developing the Zebrafish Developing the Zebrafish ((Danio rerioDanio rerio) As a Model to ) As a Model to

Evaluate the Toxicity of Evaluate the Toxicity of Chlorinated and Non-Chlorinated and Non-Chlorinated SolventsChlorinated Solvents

Matthew Wenning Matthew Wenning Department of Bioresource ResearchDepartment of Bioresource Research

Robert L. Tanguay Ph.D. and David E. Williams Robert L. Tanguay Ph.D. and David E. Williams Ph.D.Ph.D.

Department of Environmental and Molecular Department of Environmental and Molecular ToxicologyToxicology

OverviewOverview• What are these chemicals?What are these chemicals?

• Why do we care?Why do we care?

• What are the advantages of using the What are the advantages of using the zebrafish?zebrafish?

• What happens to these chemicals?What happens to these chemicals?

• MethodsMethods– Embryo assays and adult assay (1,2 DAB)Embryo assays and adult assay (1,2 DAB)

• ResultsResults– Embryo assays and adult assay (1,2 DAB)Embryo assays and adult assay (1,2 DAB)

• ConclusionConclusion

What Are These Chemicals?What Are These Chemicals?Chlorinated SolventsChlorinated Solvents

• Trichloroethylene (TCE) – (degreasing Trichloroethylene (TCE) – (degreasing agent, polyvinylchloride)agent, polyvinylchloride)

• Dichloroacetic Acid (DCA)*Dichloroacetic Acid (DCA)*

* Metabolite of parent compound shown above it* Metabolite of parent compound shown above it

What Are These Chemicals?What Are These Chemicals?Non-Chlorinated SolventsNon-Chlorinated Solvents

• Hexane - (extractions, adhesives)Hexane - (extractions, adhesives)• 2,5-Hexanedione (2,5-HD)*2,5-Hexanedione (2,5-HD)*

• 1,2-Diethylbenzene (1,2-DEB) - (fuel additive)1,2-Diethylbenzene (1,2-DEB) - (fuel additive)• 1,2-Diacetylbenzene (1,2-DAB)*1,2-Diacetylbenzene (1,2-DAB)*

• 1,3-Diethylbenzene (1,3-DEB)1,3-Diethylbenzene (1,3-DEB)• 1,3-Diacetylbenzene (1,3-DAB)*1,3-Diacetylbenzene (1,3-DAB)*

* Metabolite of parent compound shown above it* Metabolite of parent compound shown above it

Why Do We Care?Why Do We Care?• Neurotoxic agentsNeurotoxic agents

• Occupational Occupational exposureexposure

• Environmental Environmental contamination and contamination and exposureexposure

• Developmental Developmental toxicity toxicity

• Toxicity in adultsToxicity in adults

Mechanism???Mechanism???

What Are the Advantages of What Are the Advantages of Using the ZebrafishUsing the Zebrafish

• Embryos are transparentEmbryos are transparent

• Embryonic development is similar between Embryonic development is similar between all species, especially in early stagesall species, especially in early stages

• They can be spawned and raised at a lower They can be spawned and raised at a lower costcost

• Mechanism of toxicity likely conserved Mechanism of toxicity likely conserved between zebrafish and humansbetween zebrafish and humans

What Happens With These What Happens With These Chemicals?Chemicals?

Cytochrome P450 (CYP)Cytochrome P450 (CYP)

O

O

CYP 2E1

Hexane 2,5-Hexanedione

Cl

Cl ClOH

O

Cl

Cl

Trichloroethylene Dichloroacetic Acid

CYP 2E1

NeurologicalNeurologicaleffectseffects

NeurologicalNeurologicaleffectseffects

What Happens With These What Happens With These Chemicals?Chemicals?

1,3-DAB

O

O

1,3-DEB

CYP 2E1

1,2-DAB

O

O1,2-DEB

CYP 2E1

No ResponseNo Response

Product forms Product forms adducts with adducts with macromoleculesmacromolecules

Molecular targets: Molecular targets: Undefined at this Undefined at this pointpoint

• Adult fish setup for spawning 15 -17 hours Adult fish setup for spawning 15 -17 hours prior to exposureprior to exposure

MethodsMethods

Spawning setupSpawning setup Recirculating systemRecirculating system

Embryos:Embryos:• Embryos selected for exposureEmbryos selected for exposure• Exposures started 6-8 hours post fertilizationExposures started 6-8 hours post fertilization

MethodsMethods

Non-viable Non-viable embryoembryo

Viable Viable embryoembryo

MethodsMethodsEmbryos:Embryos:• Placed in 96-well (200 Placed in 96-well (200 µL)µL) plates plates • One embryo per wellOne embryo per well• Exposed in 100 Exposed in 100 µL of solution µL of solution at various concentrations at various concentrations • Broad range of concentrations evaluated Broad range of concentrations evaluated

– Dimethylsulfoxide (DMSO) used as a co-solvent for non-polar Dimethylsulfoxide (DMSO) used as a co-solvent for non-polar solventssolvents

• N = 24 for each concentrationN = 24 for each concentration

ResultsResultsTrichloroethylene Not Developmentally ToxicTrichloroethylene Not Developmentally Toxic

Trichloroethylene Data (TCE)X-axis: TCE concentration in millimolar

Y-axis: Percent mortality @ 48 hours post fertilization

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

0 0.001 0.01 0.1 1 10

Conc. in mM

% M

ort

alit

y @

48

hp

f

TCEData

ResultsResultsDichloroacetic Acid Not Highly ToxicDichloroacetic Acid Not Highly Toxic

Dichloroacetic Acid Data (DCA)X-axis: DCA concentration in millimolar

Y-axis: Percent mortality @ 48 hours post fertilization

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

0 1 10 100

Conc. in mM

% M

ort

alit

y @

48

hp

f

DCA

ResultsResultsDichloroacetic Acid Representative ImagesDichloroacetic Acid Representative Images

Representative images of (A) control and (B) 100 mM DCA exposed embryos. The DCA exposed larva exhibits mild pericardial edema (*) and unlike the control animals the swim bladder (sb) is not inflated.

*

sb

*

sb

ResultsResults2,5-Hexanedione Not Highly Toxic2,5-Hexanedione Not Highly Toxic

2,5-Hexanedione Data (2,5-HD)X-axis: 2,5-HD concetraion in millimolar

Y-axis: Percent mortality @ 48 hours post fertilization

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

0 1 10 100

Conc. in mM

% M

ort

alit

y @

48

hp

f

2,5-HD

ResultsResults2,5-Hexanedione Representative Images 2,5-Hexanedione Representative Images

Representative images of (A) control and (B) 10 mM 2,5-HD exposed embryos. The 2,5-HD exposed larva

A

B

Control

2,5-HD (10mM)

1,2-Diacetylbenze Toxic But Not 1,3-Diacetylbenze1,2-Diacetylbenze Toxic But Not 1,3-Diacetylbenze

ResultsResults

O

O

O

O

1,2 DAB, but not 1,3 DAB is developmentally toxic to zebrafish. Cumulative mortality was collected for 2 days (48 hpf). A total of 24 animals were assessed for each concentration.

0%

20%

40%

60%

80%

100%

0 ppb 40 ppb 400 ppb 4000 ppb Concentration

% M

orta

lity

@ 4

8 hp

f

1,2-DAB

1,3-DAB

O

O

O

O

ResultsResults

1,2 DAB developmental toxicity. Cumulative mortality was collected for 5 days (120 hpf). A total of 24 animals were assessed for each concentration, and the experiment was repeated 3 times.

0.00

20.00

40.00

60.00

80.00

100.00

0 ppb 62.5 ppb 125 ppb 250 ppb 500 ppb

1,2 DAB Concentration

% M

ort

alit

y @

72

hp

f

1,2-Diacetylbenzene Concentration-Dependent1,2-Diacetylbenzene Concentration-Dependent ResponseResponse

ResultsResults1,2-Diacetylbenzene Representative Images1,2-Diacetylbenzene Representative Images

1,2 DAB developmental toxicity. Representative images of a control and 1,2 DAB exposed embryos. Note the development of cardiovascular edema , curved body axis and the characteristic “blue” appearance.

62.5 ppbcontrol

125 ppb125 ppb

62.5 ppb62.5 ppbcontrolcontrol

125 ppb125 ppb125 ppb125 ppb

Chromogenic effectChromogenic effect

MethodsMethodsAdults:Adults:

• Two trials testing 1,2-diacetylbenzene Two trials testing 1,2-diacetylbenzene (1,2-DAB)(1,2-DAB)

• Chronic low-dose (100 ppb for 52 days) Chronic low-dose (100 ppb for 52 days)

• Acute high-dose (1000 ppb days for 6 Acute high-dose (1000 ppb days for 6 days)days)

• 1,2-DAB solvated in dimethylsulfoxide 1,2-DAB solvated in dimethylsulfoxide (DMSO)(DMSO)

• DMSO concentration in exposure and DMSO concentration in exposure and control groups was 0.1%control groups was 0.1%

• Control and dosing solutions Control and dosing solutions prepared and changed dailyprepared and changed daily

MethodsMethods

MethodsMethods

• Placed in 600 ml of solutionPlaced in 600 ml of solution

• Each zebrafish placed in individual tankEach zebrafish placed in individual tank

MethodsMethods• Each trial contained two groupsEach trial contained two groups

• Control and experimental groups Control and experimental groups N=4N=4

• Transfer to clean 10 gallon tank for Transfer to clean 10 gallon tank for behavioral observationbehavioral observation

ResultsResultsRepresentative Images Of Adults Representative Images Of Adults

Control (top) and 1,2 DAB (bottom) adult fish image three days post exposure. It was a dramatic blue appearance of the 1,2 DAB exposed fish. The appearance of the blue correlated with behavioral and motor deficits.

Control

1,2 DAB

Control

1,2 DAB

ConclusionConclusion• Trichloroethylene is not developmentally toxic Trichloroethylene is not developmentally toxic

under these conditionsunder these conditions

• Dichloroacetic acid is not developmentally Dichloroacetic acid is not developmentally toxic under these conditionstoxic under these conditions

• 2,5-Hexanedione is not developmentally toxic 2,5-Hexanedione is not developmentally toxic under these conditions under these conditions

• 1,3-Diacetylbenzene showed no toxic response 1,3-Diacetylbenzene showed no toxic response

• 1,2-Diacetylbenzene is developmentally toxic1,2-Diacetylbenzene is developmentally toxic

ConclusionConclusion

• Similar responses in zebrafish as in Similar responses in zebrafish as in mammals with 1,2-DABmammals with 1,2-DAB

• Zebrafish can be used as a model to Zebrafish can be used as a model to evaluate toxicity of these compoundsevaluate toxicity of these compounds

• Future studies can be done to Future studies can be done to determine “how” the chemicals determine “how” the chemicals produce toxicity produce toxicity

AcknowledgementsAcknowledgements• Robert L. Tanguay Ph.D.Robert L. Tanguay Ph.D.

• Stacey Harper Ph.D.Stacey Harper Ph.D.

• Ms. Jane La DuMs. Jane La Du

• Mr. Lijoy K. MathewMr. Lijoy K. Mathew

• Ms. Sumitra SenguptaMs. Sumitra Sengupta

• Ms. Crystal UsenkoMs. Crystal Usenko

• Ms. Lisa DuongMs. Lisa Duong

• Ms. Kate SailiMs. Kate Saili

• David E. Williams Ph.D.David E. Williams Ph.D.

• Katherine G. Field Ph.D.Katherine G. Field Ph.D.

• Ms. Wanda K. Crannell M.S. Ms. Wanda K. Crannell M.S.

Tanguay LabTanguay Lab

Questions?Questions?

Proposed Mechanism for 1,2-Proposed Mechanism for 1,2-DABDAB