Blood Parameters as Biomarkers in Brown Bullhead Catfish, Ameiurus nebulosus

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Blood Parameters as Biomarkers in Brown Bullhead
Catfish, Ameiurus nebulosus

• Michael W. Rowan
• Environmental Science Graduate Program
• Proposal for Ph.D. Research
• Committee
• Dr. Paul Baumann, Adviser
• Dr. Susan Fisher
• Dr. David Johnson
• Dr. David Stetson

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Problems with Classical Blood Variables

• Teleost erythrocyte differs from mammalian
  erythrocyte therefore use of hematocrit,
  hemoglobin, etc. does not convey the same
  information as it does for humans
• Teleost erythrocytes released from
  erythro-poietic sites at an early stage of
  development
• Mature over time, changing physiologically and
  morphologically

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Blood Parameters as Biomarkers

• Biomarker a xenobiotically-induced variation in
  cellular or biochemical processes or functions
  that is measurable in a biological system or
  sample
• Blood parameters are sensitive to environmental
  stressors
• Blood biomarkers are less invasive
• Fish can be sampled and released without altering
  population or community structure

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Brown Bullhead (Ameiurus nebulosus)

• Ubiquitous benthic catfish distributed throughout
  the great lakes
• Vulnerable to many hydrophobic contaminants
  (PAHs)
• Sediment PAHs linked to high tumor rates and
  changes in blood variables

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BROWN BULLHEAD (Ameiurus nebulosus)
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Lake Erie Areas of Concern
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Goals of the Study

• To describe in detail the circulating blood cells
  of adult brown bullhead
• To compare blood parameters of brown bullhead
  populations from contaminated and uncontaminated
  sites around Lake Erie
• To investigate the use of various blood
  parameters as indicators of toxic stress

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Study Sites

• Ottawa River
• Black River
• Cuyahoga River (upstream)
• Cuyahoga River (harbor)
• Presque Isle Bay
• Huron River
• Old Woman Creek
• reference sites

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Methods

• Bullhead collected using fyke nets fished
  overnight
• Anesthetized with tricaine methylsulfonate
  (MS-222)
• Mixed arterio-venous blood drawn from caudal
  vessels into vacutainer tubes
• Blood smears stained with Wright-Giemsa
• Whole blood on ice
• Blood plasma frozen in liquid nitrogen

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Potential Blood Biomarkers

• Erythron profiles
• Leukocyte (white blood cell) ratios
• Plasma cortisol levels
• Genetic damage indicators
• (micronuclei analysis, comet assay)

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Erythron Profile

• An estimation of the relative abundances of red
  blood cells in various developmental stages
• Mature, intermediate, immature, dividing,
  enucleate, karyorrhetic (degenerating)

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Methods Erythron Profile Image Analysis

• Mocha Image Analysis software was used to capture
  random images at 100X
• Measurements cell nuclear area cell nuclear
  major axis length cell and nuclear minor axis
  length cell and nuclear shape factor
• Shape factor(4?)(area)/(perimeter2)
  (perfect circle has a shape factor 1)

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Brown bullhead erythrocytes
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M
1
3
1) Extremely immature erythrocyte 2) 3)
Slightly more developed immature erythrocytes M)
Mature erythrocyte
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Brown bullhead erythrocytes
M
Int
Int
Int
M
Int) Intermediate erythrocytes M) Mature
erythrocytes
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Maturity Ratio Estimates
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Methods (continued)

• To determine the relative number of karyorrhetic,
  enucleate, dividing cells, seven fields on the
  slide are randomly selected
• The total number of each cell type is counted

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Karyorrhetic erythrocytes
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4
1
3
2
1) 2) Cytoplasmic swelling 3) 4) Membrane
disintegration nuclear deformation 5) Nuclear
shadow or smudge cell
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Enucleate Erythrocyte
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Division of immature erythrocytes
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Leukocyte Ratios

• Leukocyte numbers are affected by a variety of
  physiological and environmental factors
• Chronic exposure to contaminants
• - increases number of neutrophils
• - decreases number of lymphocytes
• Methods count 100 total leukocytes per slide,
  and record percent neutrophils, lymphocytes,
  monocytes

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Leukocytes
Neutrophil
Lymphocyte
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Plasma Cortisol Levels

• Cortisol is a hormone that prepares organisms for
  the fight-or-flight response
• Studies have shown that chronic environmental
  stress may reduce the cortisol response to acute
  stressors
• Experiment designed to determine how capture
  methods affect blood parameters

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Methods Cortisol Experiment

• Capture fish via fyke net acclimate to an
  aerated holding tank for 24 hours
• Draw blood from each fish four times, at time
  0min, 60min, 120min, or 180min
• Fish stressed before either first or second
  bleeding
• Analyze blood smears for erythron profiles /
  leukocyte ratios
• Freeze blood plasma in liquid nitrogen,
  determine cortisol levels by an ELISA

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Micronuclei Analysis

• Fish erythrocytes contain nuclei
• Easy to obtain DNA samples from small amount of
  fish blood look for genetic damage
• Micronuclei small abnormalities of the
  erythrocyte nucleus, usually caused by
  chromosomal damage
• Methods
• Fix blood smears in methanol, then stain in
  acridine orange
• Use fluorescent microscope to record the number
  of micronuclei-containing erythrocytes per 2000
  erythrocytes

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Comet Assay

• Lysed blood cells are placed in an
  electrophoresis buffer, DNA is allowed to
  unwind
• Cells with genetic damage have chromosome
  fragments that migrate during electrophoresis,
  leaving a tail of damaged DNA
• Image analysis used to quantify the length of DNA
  migration the percentage of migrated DNA

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Significance

• Determining the efficacy of various fish blood
  biomarkers
• Developing non-invasive methods of acquiring
  data, thus preventing further community
  disruption in already stressed ecosystems

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Blood Parameters as Biomarkers in Brown Bullhead
Catfish, Ameiurus nebulosus

• Michael W. Rowan
• Environmental Science Graduate Program
• Proposal for Ph.D. Research