Nicotine Developmental Toxicity in Zebrafish

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Nicotine Developmental Toxicity in Zebrafish

• By Andrew Kiemnec
• Mentor Dr. Robert Tanguay
• HHMI Summer Internship 2004

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Background

• Approximately 18-20 of mothers still smoke
  during pregnancy.
• Tobacco smoke contains thousands of chemicals.
• Nicotine passes easily through the placenta and
  becomes concentrated in amniotic fluid and fetal
  blood to levels as much as 15 higher than in the
  mother.
• Problems related to smoking mothers
• Spontaneous abortions, low birth weight, and
  sudden infant death. Nicotine related to
  intellectual, behavioral and cognitive
  impairments in offspring.1

1. Svododa, K., Vijayaraghavan, S., Tanguay, R.
(2002) The Journal of Neuroscience.
22(24)10731-10741
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Research Question

• What are the effects of nicotine on the
  development of zebrafish, specifically behavioral
  abnormalities?

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Hypothesis

• Nicotine will have effects on developing
  zebrafish embryos due to stimulation of nicotinic
  receptors.
• Goals
• Gain further understanding of nicotine behavioral
  effects in zebrafish.
• Extend previous studies.
• Provide a base for further research.
• Gain valuable lab skills and experience.

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Why Use Zebrafish?

• Zebrafish are not humans and they do not use
  tobacco.
• There are many benefits to using zebrafish
  including
• Ease of use Easily bred, short life cycle, fast
  development, and they are cheap.
• Advantages over mice and frogs.
• Cheap and easy to care for compared to other
  models.
• Their embryos are clear and facilitate easy
  observation as they develop externally.
• Mechanistically, the process in which nicotine
  effects zebrafish is similar to humans.

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Zebrafish Development
6d
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How does nicotine cause the effects seen in
humans?

• Acts like acetylcholine
• Binds to neuronal nicotinic acetylcholine
  receptors (nAChRs).
• Activation allows more Ca2 and Na into the cell
  than Ka out causing a depolarization and
  excitement of the muscle cell or neuron.

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Nicotine Dose Response Experiments
4hpf

• Dose response with varying concentrations of
  nicotine.
• 0.3µM-3000µM control
• Two Exposure times
• 4-48hpf
• 20-48hpf
• Other variables studied
• Role of chorion
• Minerals

20hpf
48hpf
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Methods

• Set up fish to spawn.
• 20 embryos per concentration.
• Spontaneous behavior observed at 24hpf.
• Embryos washed at 48hpf and chorions removed.
• Touch response tested at 48,72,96,120hpf.

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Spontaneous Behavior
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Types of Touch Response
Normal
Unresponsive
Abnormal
Twitch
Spasms
Odd
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4-48hpf Nicotine Exposure

• Spontaneous Behavior
• Beginning study used a yes/no response.

24hpf zebrafish
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Touch Response and Travel Distance
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Focus of studies

• Occasional consequences
• Reduced length
• Unable to maintain upright position
• Failure to straighten
• Edema
• Tailless
• Death
• However, we want to focus on sub-lethal exposure
  and neurological responses.

Picture Source Water Permeability and
TCDD-Induced Edema in Zebrafish Early-Life
Stages. Adrian J. Hill, et al.
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New window 20-48hpf Nicotine Exposure
Use of new equipment
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Touch Response
4-48hpf Nicotine Exposure
20-48hpf Nicotine Exposure
4-48hpf exposure appears to have more affect.
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While the chorion is permeable to nicotine, does
it offer any protection?
Zebrafish are able to develop normally without
their chorion, it is not essential.
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The chorion does not appear to offer any
protection against nicotine
20-48hpf nicotine exposure
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Embryo medium

• Other studies have seen paralysis at only 30µM
  nicotine solutions. However, they used a
  different medium.

20-48hpf exposure in normal R.O. fish water.
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Embryo medium
In embryo medium
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Calcium Effect?
Increased calcium levels do not appear to
influence nicotine response.
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Antibodies for the alpha-2 and alpha-7 subunit
proteins

• Goal find antibodies so nAChRs can be detected
  in developing zebrafish.
• Results
• alpha 7

• Procedure
• The a2 and a7 subunit genes were inserted into a
  plasmid.
• The corresponding proteins were made using TNT
  Reticulolysate System.
• The proteins were then ran on a western and
  screened for a useable antibody.

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Alpha 2 subunit
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Negative
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Nicotine Metabolism in Humans
Diagram source Nicotine Psychopharmacology
Molecular, Cellular, and Behavioural Aspects,
1990.
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What about nicotine metabolites?

• Question Does cotinine affect zebrafish
  development.
• Hypothesis There may be some affects from
  cotinine, but they will not be as pronounced as
  nicotine.

Cotinine Nicotine
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Cotinine Experiment
Cotinine vs. Nicotine

• 0-30000uM, 4-48hpf cotinine exposure.
• Results
• Spontaneous behavior All but highest showed
  movement.
• Touch Response High precent responsive expect
  highest conc.
• Eventual death at very high concentration.

30000uM Cotinine exposure, 48hpf
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Conclusions

• Nicotine does affect behavioral responses in
  developing zebrafish.
• This occurs at both exposure windows, however, it
  seems more pronounced from 4-48hpf.
• Chorions do not affect zebrafish reaction to
  nicotine.

• Calcium alone does not appear responsible for
  increased zebrafish reaction to nicotine but it
  is unclear why the embryo medium caused an
  increases abnormal response.
• There is more work to be done with the subunit
  proteins.
• Cotinine does not affect behavioral response at
  the same concentrations as nicotine.

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Future Directions

• Continue analysis of alpha sub-unit receptor
  proteins.
• Investigate neuronal consequences of nicotine.
• Grow up fish to view long term consequences of
  nicotine.

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Acknowledgements
Dr. Robert Tanguay The Tanguay Lab Mark
Reimers Fred Tilton Lijoy Mathew Dr. Eric
Andreasen Jane La Du Amber Young Meng Vue
Rachelle Hasson
Howard Hughes Medical Institute Dr. Kevin Ahern
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Questions?