Toxicogenetic Analysis of Livers Collected from Rats with Subcutaneous

Chitosan Patch Formulations

Andrew Colburn1, Leslie Dyal, Ph.D.2 and John Mata Ph.D.2

Oregon State University, Bioresource Research1, Dept. Biomedical Sciences2,

Corvallis, OR 97331

AcknowledgementsAcknowledgements

Dr. John MataDr. John Mata Dr. Tim HazzardDr. Tim Hazzard Dr. Kate FieldDr. Kate Field Dr. Scott GustafsonDr. Scott Gustafson Wanda CrannellWanda Crannell

Dr. Lisa DyalDr. Lisa Dyal Mata LabMata Lab

Lisa Poland Lisa Poland Deena RelucioDeena Relucio

Hemcon Inc. Hemcon Inc. Jeri ColburnJeri Colburn

IntroductionIntroduction

Hemostasis Current uses for the patch

Military Recently for Rx

Chitosan based Chitosan based hemorrhage control hemorrhage control

patchespatches Currently being used by US Armed Currently being used by US Armed

Forces Forces Recently available to the publicRecently available to the public Few side effectsFew side effects Contains endotoxins due to bacterial Contains endotoxins due to bacterial

contamination during processingcontamination during processing Endotoxins may cause Endotoxins may cause in vivoin vivo

responseresponse

HemostasisHemostasis

Stoppage of bleedingStoppage of bleeding Steps of coagulationSteps of coagulation DisordersDisorders

Coagulation Coagulation PlateletPlatelet

18% Potentially Preventable

82%Non-preventable

Causes of death on the conventional battlefield:

2% 11 % 4%

2%

81%

CNS

Airway

MSOF*

Other

Hemorrhage

*MSOF (major systems organ failure)

How does it work?

Becomes sticky Positively charged Coagulation occurs Anti-Bacterial Console

Vocabulary

Chitosan Endotoxin Hemostatic Microarray EU Cytocrome P450

ChitosanChitosan

N-acetylglucosamineN-acetylglucosamine

N-acetylglucosamineN-acetylglucosamine Glucose

ChitinChitin

A more familiar view

Chitin vs. ChitosanChitin vs. Chitosan

EndotoxinsEndotoxins

Found in bacteria cell walls Heat Stable Lipopolysaccharide complexes Increase capillary permeability

Cytochrome P450Cytochrome P450

Found in all Found in all lineageslineages

Catalyze Catalyze monooxygenase monooxygenase reactionreaction

Drug metabolismDrug metabolism Hormone Hormone

metabolismmetabolism

Immune ResponseImmune Response

Differences vs. Differences vs. toxic responsetoxic response

Cellular threatsCellular threats ProteinsProteins

Hypothesis Hypothesis

Endotoxin contamination causes Endotoxin contamination causes changes in gene expressionchanges in gene expression

Changes would indicate systemic Changes would indicate systemic endotoxin exposure endotoxin exposure

Materials and MethodsMaterials and Methods

Implanted patches Implanted patches in ratsin rats

Patches had Patches had varying levels of varying levels of endotoxin endotoxin contaminationcontamination

8 day & 28 day test 8 day & 28 day test groupsgroups

Extract RNA from Extract RNA from liver for analysisliver for analysis

Rats!

Cause and Effect

Swelling Skin lesions Alopecia (hair loss)

7/12/06

Rat 26 (100 Eu)

Rat 31 (100 EU)

7/12/06

7/13/06

Rat 33 (1070 EU)

7/12/06

Crusty lesions over dorsum

Cracking/ulcerated lesions over patch area

Alopecic (dehaired) area over the upper jaw around the nose

7/12/06

Rat 34 (500 EU)

Skin crusting over dorsum Crusting around tail base

Hair loss over lower jaw Hair loss over upper jaw

7/13/06

7/17/06

Alopecic (dehaired) area over the

upper and lower jaw with mild exudate

from the lesions

Large scabs over the back have lifted off

revealing healthy healing tissue below

Rat 34 (500 EU

Ears From Best to Worst

HistologyHistology

AnalysisAnalysis

RNA quantified via NanoDrop ND-RNA quantified via NanoDrop ND-1000 UV-Vis Spectrophotometer 1000 UV-Vis Spectrophotometer

Biotin-labeled cRNA generated for Biotin-labeled cRNA generated for microarray microarray

cRNA assessed with Agilent cRNA assessed with Agilent Bioanalyzer for purityBioanalyzer for purity

Affymetrix AnalysisAffymetrix Analysis

Affy GeneChip RU43 usedAffy GeneChip RU43 used Significance Analysis of Microarrays Significance Analysis of Microarrays

(SAM) performed using “R”(SAM) performed using “R”

MicroarrayMicroarray

StatisticsStatistics

Significance Analysis of Microarrays Significance Analysis of Microarrays (SAM) gene expression analysis(SAM) gene expression analysis

Two-class unpaired case assuming Two-class unpaired case assuming unequal variancesunequal variances

Output included values for deltas of Output included values for deltas of 0.1 to 1.30.1 to 1.3 The cost of lowering DeltaThe cost of lowering Delta

ResultsResults

Control vs. 1700 EUControl vs. 1700 EU Delta 1.3Delta 1.3 Delta 0.8Delta 0.8

Delta 1.3Delta 1.3

Delta 0.8Delta 0.8

-4 -3 -2 -1 0 1 2 3 4

-4-3

-2-1

01

23

4

SAM Plot for Delta = 0.8

Expected d(i) values

Ob

serv

ed

d(i

) va

lue

s

cutlow: -3.32

cutup: Inf

p0: 0.963

Significant: 9

False: 2.143

FDR: 0.229

100 eu

0.01

0.1

1

10

100

1000

False genes

Called genes

Delta

Nu

mb

er

of

Gen

es

500 eu

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.01

0.1

1

10

100

Called genes

False genes

Delta

Nu

mb

er o

f G

enes

1070 eu

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.01

0.1

1

10

100

1000

Called genes

False genes

Delta

Nu

mb

er o

f G

enes

1700 eu

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.01

0.1

1

10

100

1000

Called genes

False genes

Delta

Nu

mb

er o

f G

enes

Graphical summery of Graphical summery of False and Called GenesFalse and Called Genes

ConclusionsConclusions

Genes do not appear to be related to Genes do not appear to be related to toxic responsetoxic response

Under expression due to false Under expression due to false discovery ratediscovery rate

Future studies investigating proteins Future studies investigating proteins as a source of immunotoxicityas a source of immunotoxicity

Not Endotoxin!?Not Endotoxin!?

Investigate Investigate proteins as sourceproteins as source

Test for proteinsTest for proteins

SDS-PAGESDS-PAGE

Western BlotWestern Blot

Two antibodiesTwo antibodies 11stst for your protein for your protein 22ndnd for the 1 for the 1stst

Blot animation

New ConclusionsNew Conclusions

Proteins cause immune responseProteins cause immune response Future studies to identify protein Future studies to identify protein

contaminatescontaminates

Questions?Questions?

Thank you for coming!Thank you for coming! Enjoy the Shrimp!Enjoy the Shrimp!

References Liu H. et al., Chitosan kills bacteria through cell

membrane damage, International Journal of Food

Microbiology 95 (2004) 147-155. HemCon Medical Technologies Inc. White blood cell picture

www.abc.net.au/science/photos/whatis/answer4.htm

PLoS Pathogens Vol. 1(3) November 2005. Scale

bar is 5 micrometers.