BIOLOGY 220 RESEARCH PROPOSAL

TEAM CELLULICIOUS
Central Washington University

Fall 2010

INTRODUCTION

Alcohol and marijuana are two of the most widely used psychoactive drugs in the world
( Heisman et al 1997). Alcohol is the drug most frequently paired with marijuana
(Earleywine & Newcomb 1997). The recent escalation in the movement toward
legalizing marijuana for medical and recreational use in the United States has revived
discussions about the possible cytotoxic effects of each substance (Gertis et al (2002)
as well as the extent to which the drugs have additive effects (Liguori et al 2002).

Although surveys and studies have begun to scratch the surface of these issues, there
are still gaps in our knowledge of the specific cellular effects of these substances.
Several studies have focused on comparing the effects on behavior or perceived
intoxication such as the driving study in Accident Analysis & Prevention (Ronen et al
2010), (Wright & Terry 2002), but further research is needed to reveal the mechanistic
details of how THC and alcohol affect the body at the cellular level.

The extent to which they

RESEARCH QUESTION:

How does alcohol effect the morphology, viability and density of cells compared to THC
and how do they compare individually?

RESEARCH HYPOTHESES

NULL : There will be no individual or combined effect on the morphology, viability and
density of the cells due to administration of experimental of treatments of THC and
alcohol.

ALTERNATIVE: There will be a statistically significant difference in the effects on cell

morphology, viability and density measureable between each of the three treatment
cultures as compared to the control cells.

RESEARCH PREDICTIONS
Administering a treatment of 0.16% alcohol to the C2C12 mouse myoblasts will
negatively distort the morphology of the cells, decrease their viability, and decrease
their density due effects of alcohol.

Administering the experimental preparation of THC to the second culture will have no
effect on the morphology of the cells, their viability, or their density as compared to the
control culture.
 
In the culture receiving both THC and alcohol the detrimental effects will be most
severe.

EXPERIMENTAL DESIGN

A C2C12 mouse myoblast culture will be grown in a Falcon T-25 tissue culture flask and
will be passages in a biological safety cabinet by washing cells with 1X phosphate
buffered saline (PBS), removing cells with 1X Trypsin/EDTA (sigma), and centrifuging
resulting in cell suspension at 1000x g for seven minutes at room temperature. The cell
pellet will be resuspended in complete Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 4mM L-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10%
fetal bovine serum, and grown in a humidified incubator at 5% CO 2 and 37 ̊C. Prior to
cell passage, a Falcon 24-well polystyrene tissue cultures will be prepared with
alcohol/phosphate-buffered saline(PBS) solutions in a concentration of 0.16%, THC/
PBS solution in a concentration of 100 nanograms per milliliter, alcohol at a
concentration of 0.16% and THC at 100 nanograms per milliliter/PBS solution for 2
hours at room temperature. PBS with no additives will be placed in additional wells and
used a control group. Treatments will be applied to glass coverslips so that fluorescent
microscope examination of myoblasts morphology of the different treatments can be
achieved. Immediately following passage, cell viability and density will be examined by
using 0.4% Trypan Blue dye exclusion and a Brightline hemacytometer. The
alcohol/PBS solution, THC/PBS solution and the combination/sterile water solution will
be aspirated from the Falcon 24-well plate and glass coverslips and seeded with
passaged and resuspended cells. Complete and Fresh DMEM media will be added to
each seeded well and coverslip and grown in culture conditions. After 3 days, cell
morphology, viability and density will be examined using the protocols determined
above and will then be compared between initial and final cultures to determine the
effects of alcohol at 0.16%, THC at two times the legal limit and the combined effects of
THC and alcohol on the cells.

REFERENCES
Cohen, Peter J. “Medical marijuana: the conflict between scientific evidence and
political ideology.” Utah Law Review, 2009, Vol. 2009 Issue 1, p35-104.

Gaber, Mark. “State Constitutions.” Stanford Law Review, Jun2010, Vol. 62 Issue 6,
p1513-1514.

Pérez, Anna; Ariza, Carles; Sánchez-Martínez, Francesca; Nebot, Manel.”Cannabis

consumption initiation among adolescents: A longitudinal study.” Addictive
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Kleiman, Richard; Nickle, Richard; Schwartz, Michael, “Medical Toxicology and Public
Health--Update on Research and Activities at the Centers for Disease Control
and Prevention, and the Agency for Toxic Substances and Disease Registry.” J
ournal of Medical Toxicology, Sep2009, Vol. 5 Issue 3, p158-164.

Ronen, Adi; Chassidim, Hadas Schwartz; Gershon, Pnina; Parmet, Yisrael; Rabinovich,
Alex; Bar-Hamburger, Rachel; Cassuto, Yair; Shinar, David. “The effect of
alcohol, THC and their combination on perceived effects, willingness to drive and
performance of driving and non-driving tasks.” Accident Analysis & Prevention,
Nov2010, Vol. 42 Issue 6, p1855-1865.

Mikos, Robert A. “On the Limits of Supremacy: Medical Marijuana and the States'
Overlooked Power to Legalize Federal Crime.” Vanderbilt Law Review, March 9,
2009.

Wright, K.A.; Terry, P “Modulation of the effects of alcohol on driving-related

psychomotor skills by chronic exposure to cannabis.” Psychopharmacology,
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