Nicotine Developmental
Toxicity in Zebrafish
By Andrew Kiemnec
Mentor: Dr. Robert Tanguay
HHMI Summer Internship 2004
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
Research Question

What are the effects of nicotine on the
development of zebrafish, specifically
behavioral abnormalities?
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.
Why Use Zebrafish?


Zebrafish are not humans
and they do not use
tobacco.
There are many benefits to using
zebrafish including:



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
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.
Zebrafish
Development
4.3hr
8.4hr
1.25hr
19hr
3 min
24hr
6d
48hr
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.
Nicotine Dose Response
Experiments

Dose response with varying
concentrations of nicotine.


20hpf
0.3μM-3000μM + control
Two Exposure times:



4hpf
4-48hpf
20-48hpf
Other variables studied:


Role of chorion
Minerals
48hpf
Methods


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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.
Spontaneous Behavior
Types of Touch Response
Normal
Abnormal
Unresponsive
Twitch
Spasms
Odd
4-48hpf Nicotine Exposure


Spontaneous Behavior
Beginning study used a yes/no response.
Show Spontaneous Behavior @ 24 hpf
100
90
24hpf zebrafish
80
70
Percent
60
50
40
30
20
10
0
0
0.3
3
30
300
Concentration in µM
3000
Touch Response and Travel Distance
Average Travel Distance @ 72 hpf
Touch Response @ 72 hpf
1.2
100
90
1
80
70
0.8
60
Distance
[inches] 0.6
Percent 50
40
0.4
30
20
0.2
10
0
0
0
0.3
3
30
300
0
3000
0.3
3
30
300
3000
Concentration in µM
Concentration in µM
Touch Response @ 120 hpf
Average Travel Distance @ 120 hpf
100
1.2
90
1
80
70
0.8
60
Percent
50
Distance
[inches]
40
0.6
0.4
30
20
0.2
10
0
0
0
0.3
3
30
Concentration in µM
300
3000
0
0.3
3
30
Concentration in µM
300
3000
Focus of studies

Occasional consequences:
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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.
New window: 20-48hpf Nicotine
Exposure
Use of new equipment
Flicks/fish/30sec
Spontaneous Behavior @ 24hpf
3
2
1
0
0
3
30
Concentration in µM
300
Touch Response
4-48hpf Nicotine Exposure
20-48hpf Nicotine Exposure
Touch Response @ 48 hpf
Touch Response @ 48hpf
100
90
80
70
Percent
Percent
Percent
60
50
40
30
100
90
80
80
70
70
60
60
50
50
40
40
30
Control
3 µM
30 µM
300 µM
30
20
10
20
0
10
20
10
0
0
0.3
3
30
Concentration in µM
300
3000
0
0
Normal
3
30
Concentration
inUnresponsive
µM
Abnormal
4-48hpf exposure appears to have more affect.
300
While the chorion is permeable to
nicotine, does it offer any
protection?
Chorion
Developing
Embryo
Yolk
Zebrafish are able
to develop normally
without their
chorion, it is not
essential.
The chorion does not appear to offer
any protection against nicotine
Response with Chorion @ 120hpf
100
90
80
Precent
70
Control
3
30
300
60
50
40
30
20
10
0
Normal
Abnormal
Unresponsive
Response without Chorion @ 120hpf
100
90
80
Precent
70
60
Control
30
300
50
40
30
20
10
0
normal
abnormal
20-48hpf
nicotine
exposure
Embryo medium

Other studies have seen paralysis at only 30μM
nicotine solutions. However, they used a
different medium.
Touch Response @ 48hpf
100
90
80
70
Percent
Control
3 µM
30 µM
300 µM
60
50
40
30
20
10
0
Normal
Abnormal
Unresponsive
20-48hpf
exposure
in normal
R.O. fish
water.
Embryo medium
In embryo medium
Touch Response @ 48hpf
100
90
80
70
Percent
Control
0.3 µM
3 µM
30 µM
300 µM
60
50
40
30
20
10
0
Normal
Abnormal
Unresponsive
Calcium Effect?
Touch Response @ 48hpf
100
90
80
Percent
70
Control
0.3 µM
3 µM
30 µM
300 µM
60
50
40
30
20
10
0
Normal
Abnormal
Unresponsive
Increased calcium levels do not appear to
influence nicotine response.
Antibodies for the alpha-2 and
alpha-7 subunit proteins

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Goal: find antibodies so
nAChRs can be detected
in developing zebrafish.

Procedure:

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Results:
alpha 7

The α2 and α7 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.
Alpha 2 subunit
Negative
Nicotine Metabolism in Humans
4%
Nicotine-Noxide
Nicotine
9%
Urine
70%
17%
Other
metabolites
Diagram source: Nicotine Psychopharmacology: Molecular, Cellular, and Behavioural Aspects, 1990.
Cotinine
What about nicotine
metabolites?

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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
Cotinine Experiment
Cotinine vs. Nicotine

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Spontaneous behavior: All but
highest showed movement.
Touch Response: High precent
responsive expect highest conc.
Eventual death at very high
concentration.
Positive Touch Response @ 48 hpf
100
90
80
70
Percent
0-30000uM, 4-48hpf cotinine
exposure.
Results:
60
50
Nicotine
40
Cotinine
30
20
10
0
0
0
3
30
300
3000
30000
Concentration in µM
Positive Touch Response @ 120 hpf
100
90
80
70
Percent
30000uM
Cotinine
exposure,
48hpf
60
Nicotine
50
Cotinine
40
30
20
10
0
0
0.3
3
30
300
Concentration in µM
3000
30000
Conclusions



Nicotine does affect behavioral
responses in developing
zebrafish.
This occurs at both exposure
windows, however, it seems
more pronounced from 448hpf.
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.
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.
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
Questions?