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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.