INTRODUCTION

HYPOTHESIS

CONCLUSIONS

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METHODS

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ACKNOWLEDGEMENTS

Measuring Tissue-specific Glutathione (GSH) Utilization in the Developing Embryo

Sarah Brown, Karen Melendez, Alicia Timme-Laragy

• Humans are continuously exposed to oxidative stress-inducing environmental contaminants each day.

• Embryonic development is extremely sensitive to oxidative stress due to the extreme changes in cell growth, development and differentiation that occur.

• Therefore antioxidant functions during development are essential.

• The most abundant antioxidant is glutathione (GSH). • Oxidation of GSH causes the formation of glutathione disulfide

(GSSG), which can then be recycled back to GSH through the enzyme glutathione disulfide reductase.

We aim to discover tissue-specific utilization of GSH to identify sensitive targets of oxidative stress during development

No spatial analysis of GSH utilization has yet been performed in the zebrafish.

Glutathione Synthesis

GSH Redox Potential in the developing Zebrafish Embryo

Timme-Laragy, Alicia R., et al. "Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo." Free Radical Biology and Medicine 65 (2013): 89-101.

Tissue-specific utilization of GSH will change during embryonic development.

FUTURE DIRECTIONS

• Tissue-specific utilization of GSH changes in the developing embryo. • The yolk sac retained consistently high levels of GSH throughout all

developmental stages. • Other structures such as the eye, tail bud, somite 5, esophagus, heart

and kidney region all displayed transient peaks depending on the developmental stage.

• Do chemical exposures affect GSH distribution and utilization? • Can we predict target tissues that will be sensitive to oxidative stress

during different stages of development?

Members of the Timme-Laragy Lab: Dr. Karilyn Sant, Michelle Rousseau, Derek Luthi, Christopher Sparages, Katrina Borofski, Kaylee-Anna Williams, Shana Fleischman, Jiali Xu

Department of Environmental Health Sciences, University of Massachusetts Amherst, Amherst MA 01003

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Yolk Eye Tailbud

Yolk Eye Tailbud Cerebellum Somite 1/2

Yolk Eye Tailbud

Yolk Eye Pectoral Fin Bud

Somite 5

Yolk Eye Somite 5 Aortic Arches

Yolk Eye Somite 5 Heart Otolith

Yolk Eye Swim Bladder

Pharynx/ Esophagus

Yolk Eye Intestine

Stomach Eye (pupil)

Gallbladder Kidney Region

Liver

ABSTRACT Humans are exposed to oxidative stress-inducing environmental contaminants every day. Embryonic development is extremely sensitive to oxidative stress due to the extreme changes in cell growth, development and differentiation that occur during this life stage. These changes depend on reduction and oxidation potentials that influence cell signaling pathways; therefore antioxidant functions during development are essential. The most abundant non-protein antioxidant is glutathione (GSH), however the development of GSH concentration during early life stages is still not well understood. The GSH S-transferase catalyzed reaction that occurs with the dye Monochlorobimane (MCB) and GSH creates a fluorescent adduct that can be seen and measured. Here, we propose that incubating zebrafish embryos (Danio rerio) of the Mitfa strain that lack pigment, at different developmental stages in .02 mM MCB will allow for a reliable method showing tissue-specific sensitivity to oxidative stress by observing critical windows of organ development and understand the mechanism as to how embryonic stages are effected by oxidative stress. We examined GSH concentrations at 6 developmental stages, using NAC and Menadione as models to manipulate GSH content in the embryos. Images were quantified using Image J. Here, stage specific staining of GSH showed that the certain organs using GSH change during development. The yolk sac retained consistently high levels of GSH throughout all developmental stages whereas the cerebellum, eye, and heart displayed transient peaks depending on the developmental stage. We conclude that MCB is a sufficient method in measuring tissue-specific GSH concentrations and oxidative stress during embryonic development.

• T-tests and F-tests were performed using Excel to determine significance.

Timme-Laragy, Alicia R., et al. "Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo." Free Radical Biology and Medicine 65 (2013): 89-101.

Bright Field Overlay

Fluorescence Only

Heat Map

GST + GSH

MCB Fluorescence