Post on 15-Apr-2017
Background
Mechanism of Action of Sulfur and Nitrogen Mustard Blistering Agents on Skin in vitroEric Zhu1, Jennifer Schaefer2, Michael P. Shakarjian3,4
1Rutgers Aresty Research Center for Undergraduates and 2School of Environmental and Biological Sciences, Rutgers University, Piscataway, NJ3Department of Medicine, Rutgers Robert Wood Johnson Med. School, Piscataway, NJ
4Department of Environmental Health Science, School of Public Health, New York Medical College, Valhalla, NY
Abstract Adhesion and Migration Assays
Despite its continued use since World War I, the exact mechanism of
mustard gas induced blistering remains unknown. Since Laminin 5 is an
important attachment protein for skin cells, specifically keratinocytes, a
sulfur or nitrogen mustard (SM or NM) agent could trigger blistering by
directly binding the protein, or indirectly, by inducing production of the
enzyme gelatinase, causing the destruction of the protein. Through the
use of adhesion assays, where cells are coaxed to stick to a treated plate,
we are testing whether keratinocytes and HT-1080 cells, a type of
cancerous fibroblast, could replicate cell adhesion with and without
Laminin 5 or a mustard agent in a lab setting. Preliminary results of these
experiments support that Laminin 5 encourages cell adhesion, but we
anticipate that a mustard agent will reverse this effect. In the future, the
project will use cell migration assays, a test of the cells’ ability to close
gaps in the presence of these substances, to further elucidate the effects
that mustard agents have on keratinocytes and the HT-1080 cells.
However, initial results support the role of Laminin 5 in cell adhesion.
Data Analysis
Figure 3: Adhesion of HT1080 and mouse keratinocytes in laminin 5 Fig 1: Rat Laminin induced Adhesion Assay
with Mouse Keratinocytes
Laminin 5 plays an important role in the adhesion of
keratinocytes. Adhesion assays tested MEK, and
cancerous cell fibroblast, HT1080’s ability to adhere
in 96 well plate in a variety of concentrations of Rat
and Human Laminin 5. After each successive
adhesion assay, the cells were stopped with 0.1%
glutaraldehyde and stained with the dye crystal violet.
Using a light microscope (40X), these pictures were
taken after the an assay comparing adhesion of MEK
and Rat Laminin 5.
Low concentration (C3) produced the most
adherence. C4 are negative controls with no Laminin,
We predict that a mustard agent will reduce the
adhering effects of Laminin, and reverse the results
seen in C1-3. Note: C3 contained some human
laminin but the effect should be negligible.
Figure 2: Migration Assay with Mouse Keratinocytes
C1 2.5ug/ml
C2 1.25ug/ml
C3 0.625ug/ml
C4 Control
Mustard agents may negatively effect HT1080 and keratinocytes’ ability to
migrate or heal skin wounds. Using Platypus Technologies’ Oris Migration
Assay, we will be measuring the cells’ ability to fill gaps in the presence of
nitrogen mustard. In each well of the plate, an insert was used to block the
center of each well, after which cells were added. After they grew, the
inserts were removed.
This initial experiment lasted 4 days (stained on day 4) and measured the
MEKs ability to migrate when treated with a negative control, EGF
inhibitor AG1478, in different concentrations. After each experiment, the
cells were stopped with 0.1% glutaraldehyde and dyed with crystal violet.
Observations were made with a light microscope (40X).
F2 0uM AG1478/No
Stain/Day 0
F2 0uM AG1478/
Stained/Day 4
G11 0uM AG1478/Capped
F2 0uM AG1478/Stained
F11 10uM AG1478/StainedF9 3uM AG1478/Stained
F6 1uM AG1478/Stained
After each plate assay was completed, a comparison between the concentration of Laminin
used and the absorbance detected by the plate reader set at the absorbance of 570nm allowed
the detection of the effect Laminin 5 has on cell adhesion.
Due to a manufacturing shortage of Laminin 5, the experiment was cut short before
completion of validation and any application of mustard agents.
Figure 5: Migration of MEKsFigure 4: Viability Assay
After each migration assay, the cells were
fixed with 0.1% glutaraldehyde, stained
with crystal violet, washed, and read in
the fluorescent plate reader.
Before each migration assay was
stopped, a viability assay was run to
check how well the cells survived assay
conditions.
Future Direction
• Future adhesion assays will find the specific range which rat laminin 5 best promotes
adhesion in mouse keratinocytes and fibroblasts like HT1080s. Additionally, analyzing and
using human laminin can lead these experiments out of the animal model and into the
human model.
• Also, additional migration assays can help determine a differential between theory 1 and 2,
revealing whether SM causes blistering directly or indirectly and can lead to a greater
understanding the role MMP-9 may play in blistering.
• Finally, with the results of these adhesion and migration assays, we will have an
understanding of mustard blistering, and can look forward to developing an antidote to
counter them.
AcknowledgementsI would like to thank Dr. Shakarjian, Jennifer Schaefer, and Ariana Acosta for starting me on
this project and their many contributions.
Mustard agents alkylate
proteins like Laminin 5.
• Theory 1: SM damages
Laminin, causes blistering.
• Theory 2: SM causes skin
fibroblasts to release the
gelatinase, MMP-9, damaging
Laminin and causing
blistering.
Anoikis, “loss of home”,
describes how
keratinocytes die from a
lack of adhesion.
• Appears like
Epidermolysis bullosa
acquista (a type of
blistering disease) 2
References1. Okamoto, O. ( 1 ), et al. "Normal Human Keratinocytes Bind To The Α3lg4/5 Domain Of Unprocessed Laminin-
5 Through The Receptor Syndecan-1." Journal Of Biological Chemistry 278.45 (2003): 44168-44177. Scopus®.
Web. 18 Sept. 2015.
2. Ries, Christian, et al. "Matrix Metalloproteinase-9 Expression And Release From Skin Fibroblasts Interacting
With Keratinocytes: Upregulation In Response To Sulphur Mustard." Toxicology 263.Clinical Picture of Sulfur
Mustard Poisoning (2009): 26-31. ScienceDirect. Web. 18 Sept. 2015.
Laminin 5r LG4/5
NHK
HT1080
Keratinocytes in epidermis
adheres to the basement
membrane through Laminin 5
at the a3, b3, y2 chains1.Nitrogen Mustard
Sulfur Mustard
0.000
0.100
0.200
0.300
0.400
0.500
0.600
Capped
+ 0uM
0uM 1uM 3uM 10uM
Abso
rban
ce D
iffe
rence
AG1478 Concentration
0
0.5
1
1.5
2
2.5
3
3.5
Capped
+ 0uM
0uM 1uM 3uM 10uM
Flo
ure
sece
ne
AG1478 Concentration
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.625 1.25 2.5
Abso
rben
ce
Laminin Coating (ug/ml)
MEK on Rat Laminin
HT1080 on Rat Laminin
MEK on Human Lamin
HT1080 on Human Laminin