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

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AG1478 Concentration

0

0.5

1

1.5

2

2.5

3

3.5

Capped

+ 0uM

0uM 1uM 3uM 10uM

Flo

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AG1478 Concentration

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

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Abso

rben

ce

Laminin Coating (ug/ml)

MEK on Rat Laminin

HT1080 on Rat Laminin

MEK on Human Lamin

HT1080 on Human Laminin