USE OF BLOOD PARAMETERS AS BIOMARKERS
IN BROWN BULLHEADS (AMEIURUS NEBULOSUS) FROM
LAKE ERIE TRIBUTARIES AND CAPE COD PONDS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Michael William Rowan, M.S.
*****
The Ohio State University
2007
Dissertation Committee:
Approved by
Dr. Susan W. Fisher, Adviser
______________________________
Dr. Paul C. Baumann
Dr. David Johnson
Adviser
Environmental Science Graduate Program
Dr. David L. Stetson
1
Copyright by Michael William Rowan
2007
2
ABSTRACT
In an attempt to describe the blood parameters of brown bullheads
(Ameiurus nebulosus) and to evaluate the use of blood parameters as non-lethal
biomarkers of contaminant exposure, brown bullheads were collected from five
contaminated sites around Lake Erie, inc luding the Ottawa River (OTT), the
Black River (BLA), the Cuyahoga River harbor (CRH), the Cuyahoga River
upstream (CRU), and Presque Isle Bay (PIB). Fish were also collected from two
relatively uncontaminated reference sites around Lake Erie, the Huron River
(HUR) and Old Woman Creek (OWC). Brown bullheads were also sampled from
Ashumet Pond (ASH), a contaminated site on Cape Cod, and Great Herring
Pond (GHP), a less contaminated site near Cape Cod. Image analysis was used
on fish blood smears to measure the major axis length, minor axis le ngth, area,
and shape factor of erythrocytes and their nuclei. These data were used to
determine the average numbers of mature, intermediate, and immature
erythrocytes at each site. Blood smears were also used to qua ntify other types
of erythrocytes (karyorrhetic, dividing, and enucleate) and leukocytes
(neutrophils, monocytes, and lymphocytes). To determine what effects, if any,
that capture and handling stress had on fish blood variables, an experiment was
conducted to mimic typical handling stress, and fish were bled serially. An ELISA
ii
was used to determine plasma cortisol levels, which were compared to other
blood variables. Lastly, two genetic assays were used to assess genotoxic
exposure. The comet assay and micronucleus assay were performed to
compare fish from contaminated and uncontaminated sites. Additionally, the
micronucleus assay provided another method to quantify the number of
polychromatic (immature) erythrocytes in circulation.
Fish from P IB had the lowest cellular area. Fish from OTT and OWC had
higher cellular shape factors than fish from HUR, BLA, and PIB. Fish from CRH
had lower nuclear area than fish from HUR, OWC, BLA, and PIB. Fish from
OTT, OWC, CRH, and CRU had higher nuclear shape factors than fish from
HUR, BLA, and PIB. The percentage of immature cells in fish from BLA was
significantly lower than those from OTT, HUR, CRH, and PIB; and the
percentages of immature cells in fish from HUR and OWC were significantly
lower than those from PIB. Although rare, dividing and enucleate erythrocytes
were present in circulation. Concentrations of sediment PCBs were negatively
associated with nuclear area. Concentrations of sediment heavy metals were
positively associated with the proportion of immature erythrocytes and negatively
associated with nuclear area. Nuclear shape factor was positively associated
with concentrations of sediment DDTs and PAHs .
Brown bullheads from BLA had significantly lower percent monocytes than
iii
fish from P IB and HUR. HUR fish had the highest percent monocytes, which was
significantly different from all of the other sites. Percent lymphocytes in OWC
fish was significantly higher than fish from PIB and OTT. Percent neutrophils
was highest in PIB and BLA, which were significantly different from OWC and
HUR. Sediment contaminants were not associated with percent monocytes,
lymphocytes, or neutrophils.
The stress experiment was plagued by a small sample size, and the
cortisol ELISA produced several readings below the detection limit. The results
were inconsistent and difficult to interpret.
Fish from CRH had a significantly higher frequency of polychromatic
(immature) erythrocytes fish from OWC. This study suggests that the acridine
orange/PCE method of determining red cell maturity may be more suitable than
the cell and nuclear morphology method. OWC fish erythrocytes had 0‰
micronuclei, while CRH had 0.43 ‰. The comet assay showed significantly more
genetic damage at CRH and ASH than their respective reference sites, OWC
and GHP.
This research suggests that erythrocyte nuclear morphology, percentage
of immature erythrocytes in circulation, and the comet assay are suitable nonlethal biomarkers of contaminant exposure in brown bullheads.
iv
Dedicated to my wife Maria,
and my daughters Sophia and Maya
v
ACKNOWLEDGMENTS
I wish to thank my adviser, Dr. Paul Baumann, for his financial support,
scientific knowledge and experience, intellectual advice, and most of all, his
patience. This dissertation and research would not be possible without his
guidance and encouragement over the past several years.
I also thank my committee members Dr. David Johnson, Dr. David
Stetson, and especially Dr. Susan Fisher, who graciously agreed to take over as
advisor quite late in my travails. I thank each of them for their constructive
criticism and their willingness to remain on the committee.
I thank my colleagues Xuan Yang and Dan Peterson for their assistance in
the field and for their ability to laugh when things weren’t going our way. I thank
Xuan Yang specifically for her assistance with statistical analysis. I am also
indebted to Xiaosong Li and Rongfang Gu, graduate students in the department
of statistics at Case Western Reserve University. I thank them for donating
hours of their time to assist me with my statistical analyses, and cheerfully
accomplishing our work in spite of looming deadlines.
A variety of other scientists have been important in the completion of my
field research, including Steve Smith, John Hickey, Dora Passino-Reader, Roger
Thoma, Dave Klarer, and Dennis LeBlanc. In the lab, I thank Christine
vi
Densmore of the USGS for running the cortisol ELISA. I especially thank John
Meier of the USEPA for running the comet assay, training me on the
micronucleus assay and PCE/NCE counts, coauthoring a publication, and for his
overall collegiality and expertise.
I wish to thank my students, from both Ohio State and Cuyahoga
Community College. Their enthusiasm, questions, and positive feedback have
motivated me to constantly strive to be a better teacher. I have learned more
from them than they have from me. I also thank my employer, Cuyahoga
Community College, for supporting my academic growth and paying me for doing
something I love.
I am forever thankful to my parents and family for their love and support. I
wish to especially thank my father, whose financial support, blunt
encouragement, and unwavering faith in me have kept me on track and
sustained my progress.
To my bright daughter Sophia, thank you for making me smile and
warming my heart. You have been the most worthwhile distraction! To my
beautiful baby Maya, your arrival has made me a better father. Thank you both
for inspiring me to succeed.
Lastly, I thank my wife Maria. She has made countless sacrifices to
provide me the time to accomplish this work. She has nourished my mind and
vii
body with her unending love and support. I am truly blessed to have such a
wonderful woman as a life partner and as mother of my children. To her I am
eternally grateful.
This research was funded in part by grants from the United States
Geological Survey and the United States Environmental Protection Agency.
viii
VITA
26 November 1974……………………….Born – Clevela nd Ohio
1997………………………………………..B.S. Biology, John Carroll University
1997 - 2002…………..…………………...Graduate Teaching and Research
Associate, The Ohio State University
2000………………………………………..M.S. E nvironmental Science, The Ohio
State University
2002 – present……………………………Assistant Professor of Biology,
Cuyahoga Community College
PUBLICATIONS
1. Yang, X., Meier, J., Chang, L., Rowan, M., and Baumann, P. (2006). DNA
damage and external lesions in brown bullheads (Ameiurus nebulosus) from
contaminated habitats. Environmental Toxicology and Chemistry 25: 3035-3038.
Smith, S.B., Passino-Reader, D.R., Baumann, P.C., Nelson, S.R., Adams,
J.A., Smith, K.A., Powers, M.M., Hudson, P.L., Rasolofoson, A.J., Rowan, M.,
Peterson, D., Blazer, V.S., Hickey, J.T., and Karwowski, K. (2003). Lake Erie
Ecological Investigations: Summary of Findings. Part 1: Sediment, Invertebrate
Communities, Fish Communities 1998-2000. Administrative Report: 2003-001.
U.S. Geological Survey, Great Lakes Science Center, Ann Arbor, MI, USA.
2.
FIELDS OF STUDY
Major Field: Environmental Science
ix
TABLE OF CONTENTS
Page
Abstract………………………………………………………………………………......ii
Dedication………………………………………………………………………………..v
Acknowledgments…………………………………………………………………..…..vi
Vita………………………………………………………………………………….……ix
List of Tables……………………………………………………………………….......xii
List of Figures……………………………………………………………….……...….xiv
Chapters:
1.
Introduction………………………………………………………………………1
2.
Circulating Erythrocytes in Brown Bullheads from
Lake Erie Tributaries………………………………………………………..…20
2.1. Introduction……………………………………………………………….20
2.2. Methods…………………………………………………………………..22
2.3. Results……………………………………………………………………26
2.4. Discussion………………………………………………………………..30
3.
Circulating Leukocytes in Brown Bullheads from
Lake Erie Tributaries…………………………………………………………..49
3.1. Introduction……………………………………………………………….49
3.2. Methods…………………………………………………………………..51
3.3. Results……………………………………………………………………54
3.4.Discussion………………………………………………………………….55
x
4.
Plasma Cortisol in Brown Bullheads from Old
Woman Creek, a Tributary of Lake Erie …………………………………...70
4.1. Introduction………………………………………………………………70
4.2. Methods…………………………………………………………………..72
4.3. Results……………………………………………………………………76
4.4. Discussion………………………………………………………………..77
5.
Genetic Damage in Erythrocytes of Brown Bullheads
from Lake Erie Tributaries and Cape Cod Ponds………………………….89
5.1. Introduction……………………………………………………………….89
5.2. Methods…………………………………………………………………..91
5.3. Results……………………………………………………………………95
5.4. Discussion………………………………………………………………..96
Complete Literature Cited………………………………………...…………………115
xi
LIST OF TABLES
Table
Page
2.1
Major and minor axis length, area, and shape factor of
red blood cells and nuclei in fish from Ottawa River (OT), Huron
River (HU), Old Woman Creek (OW), Black River (BL), Cuyahoga
River harbor (CH), Cuyahoga River upstream (CU), and Presque
Isle Bay (PI)…………………………………………………………………………….46
2.2
Mean number of karyorrhetic, dividing, and enucleate
erythrocytes per seven randomly selected fields for brown bullheads
from Cuyahoga River Harbor, Cuyahoga River Upstream, Ottawa
River, Black River, Presque Isle Bay, Old Woman Creek, and
Huron River…………………………………………………………………………….47
2.3
Concentrations of dissolved oxygen in water (mg/L)
and selected chemicals in sediments (µg/g dry weight) of Ottawa
River (OT), Huron River (HU), Old Woman Creek (OW), Black
River (BL), Cuyahoga River harbor (CH) and Cuyahoga River
upstream (CU), and Presque Isle Bay (PIB) (Smith et al. 2003;
Passino-Reader et al., in press). …………………………………………………….48
3.1
Mean percent monocytes, lymphocytes, and neutrophils
for brown bullheads from Cuyahoga River Harbor, Ottawa River,
Black River, Presque Isle Bay, Old Woman Creek, and Huron River…………...65
3.2
Mean percent monocytes, lymphocytes, and neutrophils
for brown bullheads from Black River and Old Woman Creek. Data
from Danielle Hermann (1999)……………………………………………………….66
3.3
Mean percent monocytes and neutrophils for male and
female brown bullheads from Black River and Old Woman Creek.
Data from Vanessa Murchake (1987)……………………………………………….67
3.4
Mean counts of activated neutrophils for brown bullhead
collected from the Black River and Old Woman Creek in May and
September, 1993. Black River fish were found to have significantly
higher activation (p < 0.05, Student's t-test). Data from Stasiak
and Baumann (1996)………………………………………………………………….68
xii
3.5
Concentrations of dissolved oxygen in water (mg/L)
and selected chemicals in sediments (µg/g dry weight) of
Ottawa River (OT), Huron River (HU), Old Woman Creek
(OW), Black River (BL), Cuyahoga River harbor (CH) and
Cuyahoga River upstream (CU), and Presque Isle Bay (PIB)
(Smith et al. 2003). ……………………………………………………………………69
4.1
Plasma cortisol, erythrocyte, and leukocyte data from 4
group A fish and 1 control fish from Old Woman Creek.…………………………..86
4.2.
Plasma cortisol, erythrocyte, and leukocyte data from 1
control fish and 4 group B fish from Old Woman Creek…………………………...87
4.3
Mean cortisol levels (ng/ml) for brown bullheads from
the Toussaint River, Old Woman Creek, and the Black River
collected in May 1995. Data from Stasiak (1995)…………………………………88
5.1
Number of polychromatic erythrocytes (PCEs), normochromatic
erythrocytes (NCEs), total (both types) erythrocytes (total Es),
and frequency of polychromatic erythrocytes in brown bullheads
from the Cuyahoga River and Old Woman Creek (Ohio). ……………………....111
5.2
Frequencies of micronuclei in polychromatic erythrocytes
(MNPCEs), normochromatic erythrocytes (MNNCEs), and total
(both types) erythrocytes (MN) of brown bullheads from the
Cuyahoga River and Old Woman Creek (Ohio)…………………………………..112
5.3
Measurements of DNA damage in erythrocytes of brown
bullheads from the Cuyahoga River and Old Woman
Creek (Ohio)……………………………………………………...........................…113
5.4
Measurements of DNA damage in erythrocytes of brown
bullheads from Ashumet Pond and Great Herring Pond
(Massachusetts)……………………………………………………………………...114
xiii
LIST OF FIGURES
Figure
Page
1.1
Seven study sites around Lake Erie in Ohio and
Pennsylvania…………………………………………………………………….….….18
1.2
Two study sites in Massachusetts…………………………………………………...19
2.1
Map locations of the sampling sites, Ottawa River (OT), Huron
River (HU), Old Woman Creek (OW), Black River (BL),
Cuyahoga River harbor (CH), Cuyahoga River upstream (CU),
and Presque Is le Bay (PI)…………………………………………………………….39
2.2
Immature (1: extremely immature, 2 and 3: slightly more
developed immature), intermediate (Int), and mature (M) brown
bullhead erythrocytes at 1000X magnification, stained with
Accustain  Camco  Stain Pak…………………………………………………….40
2.3
Karyorrhetic brown bullhead erythrocytes at 1000X
magnification, stained with Accustain  Camco  Stain Pak.
1 and 2 show cytoplasmic swelling; 3 and 4 show membrane
disintegration and nuclear deformation; and 5 shows the
“nuclear shadow” or smudge cell remaining after cell death……………………...41
2.4
Dividing brown bullhead erythrocytes at 1000X magnification,
stained with Accustain  Camco  Stain Pak……………………………………...42
2.5
Enucleate brown bullhead erythrocytes at 1000X magnification,
stained with Accustain  Camco  Stain Pak……………………………………...43
2.6
Variation in area, shape factor, and minor axis length with major
axis length of brown bullhead red blood cells and nuclei………………………….44
2.7
Relative abundance of immature (slanted), intermediate (dotted),
and mature (open) erythrocytes in brown bullheads from Ottawa
River (OT), Huron River (HU), Old Woman Creek (OW), Black
River (BL), Cuyahoga River harbor (CH) and Cuyahoga River
upstream (CU), and Presque Isle Bay (PI)………………………………………….45
xiv
3.1
Map locations of the sampling sites, Ottawa River (OT), Huron
River (HU), Old Woman Creek (OW), Black River (BL),
Cuyahoga River harbor (CH), Cuyahoga River upstream (CU),
and Presque Isle Bay (PI)…………………………………………………………….63
3.2
Brown bullhead lymphocyte (L), neutrophil (N), and monocyte
(M) at 1000X magnification, stained with Accustain  Camco 
Stain Pak………………………………………………………………………………..64
4.1
Map location of Old Woman Creek near Huron, Ohio……………………………..83
4.2
Change in plasma cortisol in group A fish stressed at time =60
minutes. Due to a damaged sample, fish 3 has only two data
points. Fish 5 (control) was not stressed…………………………………………...84
4.3
Change in plasma cortisol in group B fish stressed at time =0
minutes. Fish 5 (control) was not stressed…………………………………………85
5.1
Fluoresced fish erythrocytes stained with acridine orange. Cells
with orange-red cytoplasm are polychromatic erythrocytes (PCEs),
and cells with barely visible cytoplasm are normochromatic
erythrocytes (NCEs). The PCE in the center contains a
green-yellow micronucleus………………………………………………….………107
5.2
Lysed brown bullhead erythrocytes showing
electrophoresis-induced migration of damaged DNA for use in
the comet assay………………………………………………………………………108
5.3
Map locations of the Ohio sampling sites, Old Woman Creek
and Cuyahoga River harbor…………………………………………………………109
5.4
Map locations of the Massachusetts sampling sites, Great
Herring Pond and Ashumet Pond………………………………………….……….110
xv
CHAPTER 1
INTRODUCTION
Hematological parameters are widely used indicators of environmental
stress in fish. Indices such as hemoglobin, hematocrit, red and white cell counts,
erythrocyte sedimentation rates, and differential blood smears have all been
used as indicators of disease and stress (Blaxhall & Daisley 1973; Wedemeyer &
Yasutake 1977). Many studies have demonstrated changes in blood variables
as a result of environmental conditions such as temperature (Houston & Murad
1992; Houston & Schrapp 1994), radiation (Schultz et al. 1993), hypoxia (Scott &
Rogers 1981), and presence of contaminants (Zbanyszek & Smith 1984;
Houston et al. 1993).
Since blood parameters are influenced by a variety of environmental
stressors, they have the potential to be used as biomarkers. A biomarker can be
defined as a xenobiotically induced variation in cellular or biochemical processes,
structures, or functions that is quantifiable in a biological system or sample
(Everaarts et al. 1993). Biomarkers are used as indicators of an organism’s
response to environmental stressors. There are several biomarkers commonly
1
used to indicate environmental stress in fish, including MFO assays (EROD),
hepatic tumor frequencies, and bile metabolites. Most of these biomarkers,
however, are intrusive and require (in most studies) for large numbers of fish to
be sacrificed. This causes further disruption to populations in already disturbed
ecosystems. Even for fish species not considered endangered, there is concern
about the effects of sacrificing large numbers of fish for the purpose of
environmental research and monitoring (Fossi & Leonzio 1994). With blood
biomarkers, however, fish can be sampled and released without affecting
population or community structure.
A commonly used fish species in environmental studies is the brown
bullhead catfish, Ameiurus nebulosus (Lesueur). This is a ubiquitous benthic
catfish distributed throughout the great lakes (Trautman 1981). Its almost
constant association with the sediment renders it vulnerable to many
hydrophobic contaminants such as polycyclic aromatic hydrocarbons (PAHs).
Numerous studies have shown a direct link between PAH-contaminated
sediment and tumors in Great Lakes bullhead (Black 1983; Baumann et al. 1990;
Baumann & Harshbarger 1995; Baumann & Harshbarger 1998). PAHs can also
affect fish blood. Murchake (1987) observed a higher number of smaller,
immature erythrocytes in brown bullhead taken from a PAH-contaminated site
than from an uncontaminated site. Zbanyszek and Smith (1984) found that
erythrocyte counts in rainbow trout decreased significantly after acute exposure
to PAH, and Stasiak and Baumann (1996) reported an increase in activated
2
neutrophils in brown bullhead from the PAH-contaminated Black River in Lorain,
Ohio.
There are three primary goals of this research: 1. to describe in detail the
circulating blood cells of brown bullhead; 2. to compare the blood parameters of
brown bullhead populations from contaminated and uncontaminated sites around
Lake Erie; and 3. to investigate the use of various blood parameters as indicators
of toxic stress in brown bullhead. The four potential blood parameters that will be
investigated as biomarkers for brown bullhead are erythron profiles, differential
leukocyte counts (white blood cell ratios), plasma cortisol levels, and genetic
damage indicators.
Erythron Profiles
In a review, Houston (1997) questioned the validity of the “classical”
hematological variables listed above as indictors of fish health. Because the
teleost erythrocyte differs so much from the mammalian erythrocyte, the use of
red blood cell counts, hematocrit, and hemoglobin does not convey the same
useful information as it does for humans. This is due to the nature of the teleost
erythrocyte—maturing over time, changing physiologically and morphologically.
Houston instead proposes the use of an erythron profile: an estimation of the
relative abundances of red blood cells in various developmental stages. These
stages include immature cells, intermediately developed cells, and mature cells,
as well as dividing cells, enucleate cells, and karyorrhetic (degenerating) cells.
3
Formation of teleost red blood cells, or erythropoiesis, occurs in several tissues,
including the kidney, thymus, and spleen. Erythrocytes are released from these
sites into adjacent capillaries at an early stage in their development, and
complete their maturation within the circulation. Various forms of stress stimulate
the release of erythrocytes into circulation. Thus, the proportion of immature red
cells i n circulation can be used as an indicator of environmental stress.
Murad et al. (1990) exposed goldfish to three distinct forms of respiratory
stress (reduced blood oxygen-carrying capacity, transient hypoxia, and
temperature-forced increases in oxygen demand), and noted an increase in the
number of immature erythrocytes in circulation. These respiratory stresses also
increased the number of karyorrhetic (degenerating) and dividing red cells.
Houston et al. (1993) produced similar results by exposing goldfish to sub lethal
levels of cadmium, a pollutant known to cause anemia by damaging
erythropoietic tissues. While many laboratory studies such as these have
demonstrated the dynamic nature of the fish erythrocyte, few (if any) field studies
have considered the effect of environmental contaminants on the erythron profile
of wild fish populations.
To construct erythron profiles, image analysis is used to record the
following erythrocyte measurements: cell and nuclear major axis length, cell and
nuclear minor axis length, cell and nuclear area, and cell and nuclear shape
factor. These measurements of morphology are used to estimate the proportion
of immature, intermediate, and mature erythrocytes for each fish. Erythron
4
profiles also quantify the abundance of karyorrhetic (dying or degenerating) cells,
enucleate cells, and dividing cells.
Differential Leukocyte Counts (White Blood Cell Ratios)
The next potential blood biomarker for brown bullheads is the leukocyte
ratio. White blood cell numbers are a ffected by a variety of physiological and
environmental factors. Murchake (1987) found that bullhead from a
contaminated site (Black River) had higher numbers of neutrophils and lower
numbers of monocytes than fish from a reference site (Old Woman Creek).
Similarly, Stasiak (1995) reported higher numbers of activated neutrophils in fish
from contaminated sites, and lower numbers in fish from reference sites. Other
studies have found that exposure to chronic stress, such as contaminants and
infectious agents, caused a decrease in circulating lymphocytes while increasing
the number of neutrophils (Siwicki & Studnicka 1987; Murad & Houston 1988).
To determine leukocyte ratios, 100 total leukocytes are counted per slide, and
data are recorded as percent neutrophils, lymphocytes, and monocytes.
Plasma Cortisol Levels
One question concerning fish research is whether the sampling methods
themselves affect blood parameters. Bullheads are usually captured in fyke nets
that are set in the evening and checked the next morning. Wedemeyer and
Yasutake (1977) suggested that this capture method causes stress. However,
5
other capture methods, such as electroshocking, have also been shown to cause
even physiological changes. In order to further examine the use of blood
parameters as biomarkers, it is necessary to determine how such sampling
techniques affect these parameters. Although stress may manifest itself in a
variety of blood parameters, one of the most quickly affected measurements is
plasma cortisol level.
The hypothalamo-pituitary-intrarenal axis is composed of several
hormonal pathways that can be affected by artificial and environmental stressors
(Hontela et al. 1992). The hypothalamus secretes corticotropin releasing factor,
which stimulates the anterior pituitary to secrete adreno -corticotropin (ACTH),
which stimulates the head kidney to secrete glucocorticoids (such as cortisol)
that lead to glucose production. This is an energy-mobilizing event that enables
an organism to mount the necessary fight-or-flight response. Several studies
have shown that chronic environmental stress may reduce the cortisol response
to acute stressors (Hontela et al. 1992, Stasiak 1995, Hontela et al. 1995,
Brodeur et al. 1997). This may have a detrimental effect o n overall fish health,
since cortisol also has a regulatory role in metabolism, osmoregulation,
reproduction, and immune function.
Since cortisol is such a direct measure of stress, and since it affects many
aspects of fish health, it is a well-suited biomarker (especially when considered
along with other blood parameters). Experiments that measure blood cortisol
levels while simulating “handling stress” can be used to investigate the use of this
6
biomarker. Blood from these experiments can also be analyzed for complete
erythron profiles (immature, intermediate, mature, karyorrhetic, enucleate, and
dividing red blood cells) and leukocyte ratios to determine if (and to what degree)
these indices change over time as the fish responds to handling stress.
Genetic Damage Indicators
Unlike mammalian red blood cells, fish erythrocytes contain nuclei (and
therefore DNA). It is therefore easy to obtain DNA samples form very small
amounts of fish blood. Since many of the contaminants found in the study sites
are known mutagens, an index that measures genetic damage could be a useful
biomaker. Furthermore, genetic damage indicators are direct measurements of
the effects of contaminants, and would not be affected by sampling methods or
handing stress. Two genetic damage indicators used in this study are the
micronuclei analysis and the comet assay.
Micronuclei are small abnormalities of the erythrocyte nucleus, usually
caused by chromosomal damage. They are visible as either a protuberance from
the nucleus itself or as a separate piece of genetic material within the cytoplasm.
When stained with acridine orange, micronuclei are identified as bright-yellow
fluorescing, circular bodies, occupying 1/20 to 1/3 the cell size. Acridine orange
also differentiates polychromatic erythrocytes (PCEs) from normochromatic
erythrocytes (NCEs) due to differences in the amount of RNA in the cytoplasm.
With acridine orange stain, PCEs (immature red blood cells) fluoresce a bright
7
orange, compared to the dull, khaki-green fluorescence of NCEs (mature red
blood cells).
Hose et al. (1987) found an elevated frequency of erythrocyte micronuclei
in two marine fish species from contaminated sites. Schultz et al. (1993)
described an increased frequency of erythrocyte micronuc lei due to X -ray
radiation. Similarly, Metcalfe (1988) induced the formation of erythrocyte
micronuclei in brown bullhead by injecting fish with benz[a]pyrene.
The benefits of the micronuclei analysis are twofold. First, the micronuclei
frequency is quick and direct measure of genetic damage caused by
environmental contaminants—and is therefore a well-suited biomarker. Second,
this analysis also provides an alternative method for determining erythrocyte
maturity ratios. This new method (acridine orange staining) can be compared to
the previously described method (red cell morphology measurements) to check
for similarities and differences.
Perhaps a more sensitive genetic damage indicator is the comet assay.
In this analysis, lysed blood cells are placed in an electrophoresis buffer and the
DNA is allowed to unwind. The DNA then migrates under electrophoresis, and
the cells are scored under a microscope. The cells with genetic damage are
visible with long tails of DNA migrating from the center of the cells (hence the
name “comet assay”). Comet assays are widely used in laboratory studies
(Mitchelmore & Chipman 1998; Nacci et al. 1996; Anitha et al. 2000; Sastre et al.
2001). Only a few studies have investigated the use of the comet assay in field
8
studies (Devaux et al. 1998; Bombail et al. 2001). This research will further
develop methods to make the comet assay suitable for field studies
The comet assay is a potentially suitable biomarker for several reasons.
First, it is a direct measure of the biological effects of mutagenic environmental
contaminants. Secondly, it is not affected by sampling methods or handling
stress. Thirdly, it is a relatively easy and inexpensive procedure. For these
reasons, the comet assay has the potential to be an extremely practical and
beneficial biomarker for brown bullhead.
Study Sites
Seven sites investigated in this study are all tributaries or embayments of
Lake Erie (Figure 1). They are similar freshwater ecosystems, but differ both in
size and in the way they have been altered by various anthropogenic factors.
The first site is Old Woman Creek, a relatively small waterway with an
agricultural watershed that served as one of two reference sites for this study.
The lower section of Old Woman Creek includes a large wetland contained in the
Old Woman Creek National Estuarine Preserve (and is therefore protected from
development). It is from within this estuary that the bullheads were collected.
Some low-level PAH contamination has been recorded in sediments near a
railway and highway bridge (Johnston & Baumann 1989), but there are no
industrialized sources of pollution.
9
The Huron River reaches Lake Erie in Huron, Ohio, about 6 km west of
Old Woman Creek. This system is slightly more developed than Old Woman
Creek, with residential neighborhoods and agricultural areas comprising most of
the watershed. A small marina for recreational boaters is likely the primary
source of contaminants; Smith et al. (1994) documented a 300,000 gallon fuel oil
spill o n the Huron in the spring of 1986. The lack of industrial development,
however, makes the Huron a second suitable reference site.
The remaining five sites have been designated by the International Joint
Commission as a Great Lakes Area of Concern (AOC) because of in-place
pollutants and various municipal and industrial discharges (International Joint
Commission 1985). Recently, however, the Black River has undergone
significant remediation and has been reclassified as an Area of Recovery.
The first AOC in this study is Presque Isle Bay, a natural embayment of Lake
Erie near Erie, Pennsylvania. Although a state park surrounds the bay at Lake
Erie, its watershed collects runoff from agricultural and municipal areas, as well
as industrial discharges. Bullhead from Presque Isle have historically shown
elevated tumor incidence, presumably due to high levels of PAH’s in the
sediment (Pennsylvania Department of Environmental Protection 1997).
The Cuyahoga River in Cleveland, Ohio, is a heavily industrialized system
in an urban setting. To maintain shipping lanes, the river channel is frequently
dredged and has permanently modified banks. Steel manufacturing is a major
source of PAH’s, and storm sewer discharges and urban runoff carry a variety of
10
contaminants into the river. Like in Presque Isle, studies here have shown
elevated incidences of liver tumors in brown bullhead (Baumann et al. 1991).
Because of its physical characteristics, the Cuyahoga was divided into two sites:
Cuyahoga upstream and Cuyahoga harbor. The upstream site, around 7 km
from the river mouth, is proximal to several steel processing plants. Water flow
and dissolved oxygen are normal for an urban river. The harbor site, however, is
located near the river mouth as it enters Clevela nd Harbor, and includes a
backwater area that was formerly part of the original river channel. This low-flow
area is relatively shallow and stagnant, and has low levels of dissolved oxygen.
The Ottawa River runs through northwest Ohio and flows into Maumee
Bay near Toledo, Ohio. Its watershed is both industrial and agricultural, and it is
polluted with an assortment of contaminants including PCB’s, PAH’s, and metals
(chromium, lead, zinc, copper, arsenic, and cadmium). PCB concentrations in
the tributa ry have been reported as high as 74,000 ppm, and fish have contained
PCBs at concentrations over 500 ppm (US EPA 1998). Discharges from
upstream industrial sites in Lima, Ohio include wastewater from a sewage
treatment plant, a chemical plant, and an oil refinery (Ohio EPA 1992).
The Black River, situated about 45 km west of Cleveland, meets the lake
in Lorain, Ohio. It too has a history of PAH contamination from the steel industry.
Studies began in 1980 by Dr. Paul Baumann of the United States Geological
Survey at The Ohio State University have shown high levels of PAH
contamination in Black River sediments corresponding with increased incidences
11
of internal and external tumors in brown bullhead (Baumann et al. 1987). In 1983
the steel plant closed its coking facility, which caused sediment PAH levels and
bullhead tumor frequencies to decline (Baumann & Harshbarger 1995). In 1990,
however, the most contaminated sediments were dredged, which resuspended
the previously buried PAH’s. This led to an increase in tumor frequencies to the
previously high levels of the early 1980s. Yet only those fish present during the
dredging were affected; the first age group born after the dredging showed a
neoplasm incidence of zero. As mentioned above, the Black River has recently
been reclassified as an Area of Recovery.
Two other sites used for the genetic damage portion of this research are
located in Massachusetts. Ashumet Pond is located on Cape Cod next to the
Massachusetts Military Reservation (MMR) (formerly Otis Air Force Base) near
Mashpee, MA. The MMR is a Superfund site. The United States Geological
Survey has detected groundwater plumes of untreated sewage, trichloroethylene,
and various other solvents migrating south and east from the MMR. Some of
these have entered Ashumet Pond through a spring (Savoie et al. 2000). Brown
bullheads surveyed at this site had high incidences of skin and liver neoplasms.
Great Herring Pond is located near Cedarville MA, on the mainland of
southeastern Massachusetts. It was selected as a reference location by the
Massachusetts Division of Fisheries and Wildlife, because it had boat traffic and
cranberry beds similar to those of Ashumet Pond but no known industrial
contaminants. Brown bullheads form this site had lo w incidences of neoplasms.
12
Conclusion
Biomarkers are an invaluable tool in assessing the biological effects of
environmental stressors. This project explores new areas of biomarker research,
and will hopefully establish the use of non-invasive (and non-lethal) techniques
for use in wild fish populations. This will assist scientists as well as state and
federal agencies in their task of monitoring the effects of environmental
disturbances.
13
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Baumann, P.C. and Harshbarger, J.C. 1998. Long term trends in liver neoplasm
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Baumann, P.C., Mac, M.J., Smith, S.B., and Harshbarger, J.C. 1991. Tumor
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Baumann, P.C., Smith, W.D., and Parkland, W.K. 1987. Tumor frequencies
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and a recreational lake. Trans. Am. Fish. Soc. 116: 79-86.
Black, J.J. 1983. Epidermal hyperplasia and neoplasia in brown bullheads
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Blaxhall, P.C. and Daisley, K.W. 1973. Routine haematological methods for
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Devaux, A., Flammarion, P., Bernardon, V., Garric, J., and Monod, G. 1998.
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Everaarts, J.M., Shugart, L.R., Gustin, M.K., Hawkins, W.E., and Walker, W.W.
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Fossi, M.C. and Leonzio, C. 1994. Nondestructive Biomarkers in Vertebrates.
Lewis Publishers, Florida, USA.
Hontela, A., Rasmussen, J.B., and Audet, C. 1992. Impaired cortisol response
in fish from environments polluted by PAHs, PCBs, and mercury. Arch. Environ.
Contam. Toxicol. 22:278-283.
Hontela, A., Dumont, P, Duclos, D., and Fortin, R. 1995. Endocrine and
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Hose, J.E., Cross, J.N., Smith, S.G., and Diehl, D. 1987. Elevated circulating
erythrocyte micronuclei in fishes from contaminated sites off Southern California.
Marine Environmental Research. 22: 167-176.
Houston, A.H. 1997. Review: Are the classical hematological variables
acceptable indicators of fish health? Transactions of the American Fisheries
Society. 126: 879-894.
Houston, A.H., Blahut, S., Murad, A., and Amirtharaj, P. 1993. Changes in
erythron organization during prolonged cadmium exposure: an indicator of heavy
metal stress? Can. J. Fish. Aquat. Sci. 50: 217-222.
Houston, A.H., and Murad, A. 1992. Erythrodynamics in goldfish, Carassius
auratus L.: temperature effects. Physiological Zoology. 65: 55-76.
Houston, A.H., and Schrapp, M.P. 1994. Thermoacclimatory hematological
response: Have we been using appropriate conditions and assessment
methods? Canadian Journal of Zoology. 72: 1238-1242.
International Joint Commission. 1985. Report on Great Lakes water quality.
Great Lakes Water Quality Board, Windsor, Ontario, Canada.
Johnston, E.P. and Baumann, P.C. 1989. Analysis of fish bile with HPLC—
fluorescence to determine environmental exposure to benzo(a)pyrene.
Hydrobiologica. 188: 561-566.
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Lowe-Jinde L, Niimi AJ (1983) Influence of sampling on the interpretation of
haematological measurements of rainbow trout,Salmo gairdneri. Can J Zool
61:396–402
Metcalfe, C.D. 1988. Induction of micronuclei and nuclear abnormalities in the
erythrocytes of mudminnows (Umbra limi) and Brown Bullhead (Ictalurus
nebulosus). Bull. Environ. Contam. Toxicol. 40: 489-495.
Mitchelmore, C.L. and Chipman, J.K. 1998. Detection of DNA strand breaks in
brown trout (Salmo trutta) hepatocytes and blood cells using the single cell gel
electrophoresis (comet) assay. Aquatic Toxicology. 41: 161-182.
Murad, A., Houston, A.H., and Samson, L. 1990. Haematological response to
reduced oxygen-carrying capacity, increased temperature and hypoxia in
goldfish, Carassius auratus L. Journal of Fish Biology. 36: 289-305.
Murchake, V.K. 1987. Hematological parameters of brown bullhead (Ictalarus
nebulosus) taken from polluted and non-polluted tributaries of Lake Erie.
Master’s Thesis. The Ohio State University, Columbus, Ohio.
Nacci, D.E., Cayula, S., Jackim, E. 1996. Detection of DNA damage in
individual cels from marine organisms using the single cell gel assay. Aquatic
Toxicology. 35: 197-210.
Ohio EPA. 1992. Biological and Water Quality Study of the Ottawa River, Hog
Creek, Little Hog Creek, and Pike Run (Hardin, Allen, and Putnam Counties,
Ohio). OEPA Technical Report. EAS/1992-9-7.
Pennsylvania Department of Environmental Protection. 1997. The 1997
Presque Isle Bullhead Tumor Study. The Pennsylvania State University,
University Park, Pennsylvania.
Sastre, M.P., Vernet, M., and Steinert, S. 2001. Single -cell gel/comet assay
applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp.
(Cryptophyta). Photochemistry and Photobiology. 74: 55-60.
Schultz, N., Norrgren, L., Grawe, J., Johannisson, A., and Medhage, O. 1993.
Micronuclei frequency in circulating erythrocytes from rainbow trout
(Oncorhynchus mykiss) subjected to radiation, an image analysis and flow
cytometric study. Comp. Biochem. Physiol. 105C: 207-211.
Scott, A.L., and Rogers, W.A. 1981. Haematological effects of prolonged
sublethal hypoxia on channel catfish Ictalarus punctatus (Rafinesque). Journal of
Fish Biology. 18: 591-601.
16
Siwicki, A.K. and Studnicka, M. 1987. The phagocytic ability of neutrophils and
serum lysozyme activity in experimentally infected carp Cyprinus carpio L.
Journal of Fish Biology 31A: 57– 60.
Smith, S.B., Blouin, M.A., and Mac, M.J. 1994. Ecological comparisons of Lake
Erie tributaries with elevated incidence of fish tumors. Journal of Great Lakes
Research. 20: 701-716.
Stasiak, S.A. 1995. Implications of chronic exposure to polynuclear aromatic
hydrocarbons: effects on immune and endocrine functions in brown Bullhead,
Ameiurus nebulosus, from a contaminated river. Masters thesis, The Ohio State
University.
Stasiak, S.A. and Baumann, P.C. 1996. Neutrophil activity as a potential
bioindicator for contaminant analysis. Fish and Shellfish Immunology. 6: 537539.
Trautman, M.B. 1981. The Fishes of Ohio. The Ohio State University Press,
Columbus, Ohio. pp. 484-487.
US EPA 1998. Ottawa River, Ohio: Contaminated Sediment Remediation Project
Completed. Contaminated Sediments News, Issue 22. EPA-823-N-98-007.
Wedemeyer, G.A. and Yasutake, W.T. !977. Clinical methods for the
assessment of the effects of environmental stress on fish health. United States
Fish and Wildlife Technical Papers. 89:1-18.
Zbanyszek, R. and Smith, L.S. 1984. The effect of water-soluble aromatic
hydrocarbons on some haematological parameters of rainbow trout, Salmo
gairdneri Richardson, during acute exposure. Journal of Fish Biology. 24: 545552.
17
Figure 1.1. Seven study sites around Lake Erie in Ohio and Pennsylvania.
18
Figure 1.2. Two study sites in Massachusetts.
19
CHAPTER 2
CIRCULATING ERYTHROCYTES IN BROWN BULLHEADS
FROM LAKE ERIE TRIBUTARIES
INTRODUCTION
Hematological indices such as erythrocyte count, hematocrit, and
hemoglobin concentration have been used as markers for evaluating fish health
(Houston, 1997). The hypothesis has been that these indices could provide
information comparable to that given by human blood variables. Fish
erythrocytes, however, are more responsive to environmental stresses, and often
vary in morphology and effectiveness of oxygen transport. Lowe -Jinde and Niimi
(1983) suggested that differential erythrocyte ratios, since they are slow to
change in response to handling stress, may be a suitable index for fish laboratory
and field studies.
Because of the heterogeneity of erythrocytes in circulation, Houston
(1997) proposed the use of an erythron profile as an alternative measure of
contaminant exposure. Fish red blood cells are released from erythropoietic
sites (spleen and head kidney) at an early developmental stage, and mature
within the circulation (Fange, 1986). During maturation, cells increase in area and
20
become less spherical, nuclei become more elliptical, and cytoplasmic organelles
decrease with a corresponding accumulation of hemoglobin in the cytoplasm
(Lane et al., 1982). An erythron profile describes the relative abundances of
various developmental stages of red blood cells , including mature, intermediate,
immature, karyorrhetic (degenerating), and dividing erythrocytes. Houston
(1997) argued that such a profile provides a more sensitive means for assessing
contaminant exposure than the classic blood indices. Although normal fish
erythrocytes are nucleated, this author has observed enucleate erythrocytes
(lacking a nucleus) in the circulating blood of brown bullheads. Although
enucleate cells were not included in the erythron profile described by Houston
(1997), they were investigated in this study.
A number of studies have been conducted on the relative abundances of
immature and mature red blood cells in fish and their relations to environmental
conditions and contamination (Hardig, 1978; Lane & Tharp, 1980; Keen et al.,
1989; Murad et al., 1990; Houston & Murad 1991; Murad & Houston 1992;
Houston et al., 1993). Respiratory stresses such as reduction in blood oxygencarrying capacity, transient hypoxia, and temperature-forced increases in oxygen
demand can elicit increased rates of erythropoiesis, immature cell division,
karyorrhexis, and release of stored cells (Murad et al., 1990). Each of these
increases the proportion of immature cells in circulation. Other stressors have
been shown to stimulate teleost erythropoiesis, including starvation, bleeding,
exposure to heavy metals, and environmental changes in temperature and
21
dissolved oxygen (Lane & Tharp, 1980; Murad & Houston, 1992; Houston et al.,
1993).
Few studies are available regarding the circulating blood cells of the
brown bullhead, Ameiurus nebulosus (Lesueur), a widely used indicator species
ubiquitous in Lake Erie. The bullhead’s almost constant association with the
sediment renders it vulnerable to many hydrophobic contaminants. It is limited in
home range (Pennsylvania Department of Environmental Protection, 1997) and it
is widely used in tumor surveys (Baumann 1992; Baumann and Harshbarger
1995). The current study was intended to develop and compare erythron profiles
of brown bullhead populations from Lake Erie tributaries and embayments, and
to investigate their use as an indicator of contaminant exposure in brown
bullheads.
METHODS
Field Procedures
Brown bullheads were collected using standard fyke nets from the Ottawa
River (OT), Huron River (HU), Old Woman Creek (OW), Black River (BL),
Cuyahoga River harbor (CH), Cuyahoga River upstream (CU), and Presque Isle
Bay (PI) in the spring and early summer of 1998 and 1999 (Figure 2.1). Two of
these sites, HU and OW, received agricultural runoff but had little to no industrial
sources of pollution. The remaining five sites were industrially contaminated, and
each was designated as a Great Lakes Area of Concern by the International
22
Joint Commission. Recently, however, the Black River has undergone significant
remediation with reduced PAH contamination, and has been reclassified as an
Area of Recovery for the 'Fish Tumors and Other Deformities' use impairment.
The EPA acceptance letter for the re-designation can be found at:
http://www.noaca.org/FishBUI.PDF.
Bullheads longer than 250mm (about 3 years old or older) were removed
from the nets and immediately placed in an aerated tub of river water. Fish were
removed from the tank one at a time and anesthetized in a bucket containing
100mg/L tricaine methylsulfonate (MS-222). Mixed arteriovenous blood was
drawn from the caudal vasculature of each fish using the lateral approach
described by Schmitt et al. (1999). A non-heparinized vacutainer tube was used
to collect a small amount of blood (< 2mL). Two blood smears were immediately
made with drops of this blood and allowed to air dry. Slides were stained using
an Accustain  Camco  Stain Pak (Sigma Diagnostics ), a rapid differential
stain for blood smears comparable to Wright-Giemsa stains. All slides were
prepared and stained by the same individual.
Dissolved oxygen was measured at each sampling site, both at the water
surface and near the sediment. Because the water depth at each site was usually
shallow and the surface-to-bottom differences in oxygen were very small (less
than 0.1 ppm), the oxygen measurements were averaged yielding one dissolved
oxygen reading per site.
At least five surface sediment samples were randomly collected using a
23
stainless steel Ekman dredge from each site and analyzed as described by Smith
et al. (2003) and Passino-Reader et al. (2004?). Polynuclear aromatic
hydrocarbons (PAHs) in sediments were measured by capillary gas
chromatography (CGC) with a mass spectrometer detector in the SIM mode.
Polychlorinated biphenyls (PCBs) and pesticides were quantified by CGC with an
electron capture detector. Metals in sediments were determined by atomic
absorption spectrometry and atomic emission spectrometry.
Slide Analysis
Slides were examined using an Olympus oil-immersion light microscope
with 1000X magnification. Images were captured from random areas of the slide
using a COHU high performance color camera attached to a Targa image
capture board on an IBM PC and analyzed using Mocha  Image Analysis
software (version 1.2.10; Jandel Scientific). The major and minor axis length,
area, and shape factor (= 4π × area / perimeter2) of erythrocyte cells and nuclei
were measured. The last measurement, shape factor, is a measure of circularity,
with values ranging from 0 to 1 (a perfect circle having a shape factor equal to1).
To determine the numbers of karyorrhetic, dividing, and enucleate
erythrocytes, seven fields were randomly selected on each slide. The numbers
of each cell type were counted, and data were recorded as number of cells per
seven fields.
24
Statistical Analysis
In total, 9010 erythrocytes from 208 brown bullheads were measured from
the seven study sites. To examine the distribution and variation of erythrocyte
morphology data, area, shape factor, and minor axis length of cells and nuclei
were regressed on their major axis length. Tukey’s multiple comparisons were
used to see if the mean measurements of cells and nuclei differed among sites.
Because numbers of cells measured in fish varied from one to another, the cell
and nucleus data were averaged over each individual fish first to assure an equal
weight. Spearman’s rank correlation coefficients were calculated between the
mean cell and nucleus measurements and concentrations of dissolved oxygen
and sediment contaminants in the study sites to explore the effects of oxygen
and contamination.
Tukey’s multiple comparisons were also used to check for significant
differences between the mean numbers of karyorrhetic erythrocytes at each site.
Since the dividing and enucleate erythrocyte data were not normally distributed
and included many zeros, the Wilcox rank -sum test was used to determine
significant differences. Spearman’s rank correlation coefficients were calculated
to see if dissolved oxygen and sediment contaminants affected the number of
karyorrhetic, dividing, and enucleate erythrocytes.
To determine the developmental stages of erythrocytes, a random
subsample of 56 erythrocytes was visually classified by shape as either
immature, intermediate, or mature (Fig. 2.2), and measured for cellular and
25
nuclear area and shape factor. A proportional odds model (log [P(Y ≤ i) / (1-P(Y ≤
i))] = α i + X'β) was developed to predict classifications using the subsample
measurements. In this model, Y was the developmental stage of red blood cells,
with 1 indicating immature, 2 indicating intermediate, and 3 indicating mature
level. Cellular and nuclear area and cellular and nuclear shape factor were used
as independent variables, along with all cross products and squares of these
terms. Area and shape factor were chosen for building the model because they
measure changes in both size and shape. A stepwise procedure was used to
choose significant independent variables at a significant entry level of 0.2.
According to the proportional odds model established, the developmental
stage of each cell observed (9010 cells from 208 fish) was determined. The
proportions of immature, intermediate, and mature erythrocytes for each fish and
the mean proportions for each site were calculated. The nonparametric KruskalWallis test, followed by Dunn’s test, was used to compare the mean proportions
of immature red blood cells between different sites.
RESULTS
The following erythrocytes were found in the circulating blood of brown
bullhead: mature, intermediate, and immature erythrocytes (Figure 2.2);
karyorrhetic or degenerating erythrocytes (Figure 2.3); dividing erythrocytes
(Figure 2.4); and enucleate erythrocytes (Figure 2.5).
26
Measurements of cellular and nuclear major axis length, cellular and
nuclear minor axis length, cellular and nuclear area, and cellular and nuclear
shape factor demonstrated that erythrocytes varied in size and shape (Table
2.1). The major and minor axis ranged from 7.15µm to 19.6 µm and from 4.50µm
to 12.9µm for cells, and from 2.21µm to 6.59µm and from 1.54µm to 5.20µm for
nuclei. The minimum and maximum of cellular area were 33.6µm2 and 141µm2
and the minimum and maximum of nuclear area were 3.57µm2 and 25.4µm2,
respectively. Among the 9010 cells measured, 51 cells had perfectly circular
nuclei (nuclear shape factor = 1.0) but none had perfectly circular cells (the
largest cellular shape factor = 0.938).
Cells and nuclei increased in area (Figure 2.6 a,b) and became less
circular (smaller shape factor) (Figure 2.6 c,d)as their major axis length
increased. Minor axis length of cells and nuclei also increased as cells and
nuclei elongated but the minor axis of cells tended to decrease after the
elongation obtained a certain degree (Figure 2.6 e,f).
Significant differences were found between sites in cellular and nuclear
area and cellular and nuclear shape factor (Table 2.1). On average, fish from
Presque Isle Bay had the lowest cellular area. Fish from Ottawa River and Old
Woman Creek had higher cellular shape factors than fish from Huron, Black, and
Presque Isle. Fish from C uyahoga harbor had lower nuclear area than fish from
Huron, Old Woman Creek, Black, and Presque Isle. Fish from Ottawa, Old
27
Woman Creek, Cuyahoga harbor, and Cuyahoga upstream had higher nuclear
shape factors than fish from Huron, Black, and Presque Isle.
On the basis of the subsample measurements, the proportional odds
model, log [P(Y ≤ i) / (1-P(Y ≤ i))] = α i - 0.5795 × Nuclear Area - 0.3023 ×
Cellular Area + 29.9319 × Cellular Shape Factor, with α 1 = 1.7115, α 2 = 7.2525,
was developed for characterization of developmental (immature, intermediate,
and mature) stages of erythrocytes. Estimated using this model, the relative
abundances of immature red blood cells varied from 4% at Black River to 37% at
Presque Isle (Figure 2.7). Proportions of immature red blood cells varied among
different sites (p < 0.001, Kruskal-Wallis Test). The percentage of immature cells
in fish from the Black River was significantly lower than those of Ottawa, Huron,
Cuyahoga harbor, and Presque Isle; and the percentages of immature cells in
fish of Huron and Old Woman Creek were significantly lower than that of Presque
Isle (p < 0.05, Dunn’s test).
Few significant differences were found between sites for mean numbers of
karyorrhetic and dividing erythrocytes (Table 2.2). Fish from Cuyahoga harbor
had a significantly lower number of karyorrhetic erythrocytes than fish from Old
Woman Creek (p < 0.05, Tukey’s test).
Dividing erythrocytes were rare. No
dividing cells were found in the circulating blood of bullheads from the Cuyahoga
upstream site. This was significantly lower than the number of dividing cells from
Presque Isle Bay (p < 0.05, Wilcox rank-sum test). Out of 20 fish from Presque
Isle, 15 fish had 0 dividing cells, 4 fish had 1 dividing cell, and 1 fish had 6
28
dividing cells. Enucleate erythrocytes were also rare, but all sites had at least 3
fish with enucleate erythrocytes. There were no significant differences in the
mean number of enucleate erythrocytes between sites (p < 0.05, Wilcox ranksum test).
Dissolved oxygen measured at each site (Table 2.3) was not a significant
factor affecting the number of karyorrhetic, dividing, and enucleate erythrocytes
(p > 0.5, Spearman’s rank correlation procedure). Likewise, sediment PAHs,
PCBs, DDTs, and heavy metals (Table 2.3) were not associated with the number
of karyorrhetic, dividing, and enucleate erythrocytes (p > 0.5, Spearman’s rank
correlation procedure).
Dissolved oxygen was also not a significant factor affecting the shape and
area of fish erythrocytes and nuclei (p > 0.5, Spearman’s rank correlation
procedure). Concentrations of contaminants in sediments (Table 2.3) were
related to the mean nuclear measurements and the proportions of immature red
cells across the sampling sites. Briefly, concentrations of sediment PCBs were
negatively associated with nuclear area (rSpearman’s = -0.79, p < 0.05).
Concentrations of sediment heavy metals were positively associated with the
proportion of immature erythrocytes and negatively associated with nuclear area
(rSpearman’s = 0.75, p = 0.05 and rSpearman’s = -0.68, p < 0.1). Nuclear shape factor
was positively associated with concentrations of sediment DDTs and PAHs
(rSpearman’s = 0.82, p < 0.05 and rSpearman’s = 0.68, p < 0.1). There were no
statistically significant relationships between sediment contamina nts and any
cellular measurements.
29
DISCUSSION
Morphology of brown bullhead erythrocytes has been described by Haws
et al. (1962) and Weinberg et al. (1972). The ranges for length (long axis) and
width (short axis) of red blood cells reported by Haws et al. (1962) (11.4µm 15.9µm and 7.6µm – 11.4µm) and Weinberg et al. (1972) (9.0µm – 13.0µm and
7.0µm – 10.0µm) are narrower than those (7.1µm - 19.6µm and 4.5µm - 12.9 µm)
observed in the present study. The increased variation in cell size could be
caused by the bigger sample size used in this study (208 fish, 9010 cells)
compared with the previous studies (10 fish, 300 cells in Haws et al., 32 fish in
Weinberg et al.). The differing environmental conditions at the locations sampled
in this study may also help explain the increased variation.
Similar to a study by Houston & Murad (1991) on goldfish (Carassius
auratus L.) erythrocytes, second order polynomials generally fit erythrocyte
measurement data well (Fig. 2.6). As expected, increase in area and decrease in
shape factor were associated with increase in major axis of cells and nuclei
during maturation. However, minor axis did not decrease with cell elongation as
observed by Houston & Murad (1991). Instead, minor axis tended to increase
(Fig. 2.6), suggesting that growth in width of cells and nuclei is also involved in
the maturation of brown bullhead erythrocytes.
Difficulties in discriminating between immature and mature red cells have
caused differences in estimates of their relative abundances (Houston & Murad,
1991). Various methods have been used to calculate proportions of immature
30
erythrocytes in teleosts. Hardig (1978) categorized 16.8% of the erythrocytes of
Baltic salmon (Salmo salar) as immature. Keen et al. (1989) suggested that at
least 10% of rainbow trout (Oncorhynchus mykiss) erythrocytes were immature,
based on variations in hemoglobin of cells fractioned by velocity sedimentation.
Lane & Tharp (1980) reported 28.3% immature cells in rainbow trout by using
polysome abundance as an index. On the basis of cytoplasmic staining
properties, only 5% of goldfish (Carassius auratus) erythrocytes were classified
as immature (Murad et al., 1990). Houston & Murad (1991) exposed goldfish to
different temperatures, and reported 23% - 57% immature cells based on nuclear
shape factors.
Cell and nucleus data of brown bullhead erythrocytes were distributed
continuously (Fig. 2.6 e,f). No discontinuities were potentially useful for
distinguishing immature from mature erythrocytes, as reported by Houston &
Murad (1991). In order to classify developmental stages of red cells, a
proportional odds model was developed. Using this model, 4 - 37 % of
erythrocytes were identified as immature in brown bullheads from the seven
sites. This range is comparable to those reported for other fish species.
Comparisons of cellular and nuclear measurements showed low cellular
area for fish from the Presque Isle Bay; high cellular and nuclear shape factor for
fish from the Ottawa River; and low nuclear area and high shape factor for fish
from the Cuyahoga River harbor. These suggested relatively high numbers of
immature cells were present at these three sites. This was confirmed by the
31
immature ratios of red cells estimated for these sites (Fig. 2.7 ).
In a laboratory experiment Houston et al. (1993) reported an increased
number of karyorrhetic erythrocytes in goldfish (Carassius auratus) due to
chronic exposure to cadmium. Similarly, Murad et al (1990) described an
increase in degenerating red cells in goldfish exposed to phenylhydrazine
hydrochloride (which reduces blood oxygen-carrying capacity), transient hypoxia,
and transient temperature-induced elevation of oxygen demand. This study,
however, showed no significant correlations between karyorrhetic cells and
sediment contaminants or dissolved oxygen. Furthermore, the number of
karyorrhetic erythrocytes was not correlated with the percent of immature and
mature erythrocytes (R2 < 0.01). More field and laboratory studies are needed
to determine what factors increase karyo rrhetic red cell counts, and what
contaminant exposures, if any, contribute to this process.
Murad et al. (1993) reported the presence of dividing erythrocytes in
rainbow trout (Oncorhynchus mykiss); carp (Cyprinus carpio); goldfish (Carassius
auratus); carp × goldfish hybrids; emerald shiner (Notropis atherinoides); and
freshwater drum (Aplodinotus grunniens). Similar findings were reported by Ellis
(1982) and Benfrey and Sutterlin (1984) on the blood of plaice (Pleuronectes
platessa), and Atlantic salmon (Salmo salar), respectively. To this author’s
knowledge, this is the first study to report the presence of dividing erythrocytes in
the circulating blood of the brown bullhead (Ameiurus nebulosus). As shown in
Figure 2.4, the dividing cells do not show the classic characteristics of eukaryotic
32
mitosis. Neither chromosomes nor a mitotic spindle are visible, and the nuclear
membrane is still intact. More research is needed to determine the exact
mechanism of erythrocyte division. Since the numbers o f dividing cells were not
correlated with sediment contaminants, and given that their numbers (if present
at all) are extremely low, it seems unlikely that dividing cells could be useful as
an indicator of contaminant exposure.
The cause of enucleated erythrocytes is difficult to ascertain. Perhaps
they result from abnormal cell division. Division, however, has only been seen in
relatively immature erythrocytes, whose cytoplasm is often basophilic. The
enucleate red cells found in this study stained similarly to mature erythrocytes, as
seen in Figure 2.5. Furthermore, in mammals, a nucleus is present in
erythrocytes at their site of production, but is expunged during maturation before
release into the circulation. For these reasons I believe the enucleate cells found
in this study are mature cells, possibly in some abnormal stage of karyorrhexis.
More research is needed to identify the stimuli that create these cells. As above,
since enucleate cells were not correlated with sediment contaminants, a nd since
their numbers are relatively low, it seems unlikely that they could be useful as an
indicator of contaminant exposure.
Concentrations of sediment contaminants were negatively associated with
the erythrocyte nuclear area and positively associated with the nuclear shape
factor and ratio of immature erythrocytes. These relationships indicate that toxic
stress might have stimulated erythropoiesis and increased the abundance of
33
immature red blood cells in the circulation of brown bullheads from contaminated
locations. Witeska (1995) reported similar results. He found an increase in the
percent of circulating erythrocytes in common carp, Cyprinus carpio, exposed to
various heavy metals. The positive correlation between the ratio of immature
cells and concentrations of heavy metals in sediments is also consistent with the
finding by Houston et al. (1993) that exposure to sublethal levels of cadmium led
to slower maturation of goldfish (Carassius auratus) red cells.
It was not surprising that dissolved oxygen had little effect on brown
bullhead erythrocytes. Bullheads are fairly tolerant to low-oxygen conditions.
Since the study sites were open systems, fish could simply migrate to better
environments when conditions became stagnant. Bullheads can also gulp air to
acquire oxygen. For most teleosts, short-term fluctuations in dissolved oxygen
can also be compensated for without major changes in erythrocyte maturity
proportions. Through simple adjustment of cardiac, ventilatory, and
branchiovascular activities, fish can quickly counterbalance the effects of mild to
moderate hypoxia (Cameron & Davis, 1970; Wood et al., 1979).
Houston & Murad (1991) proposed that nuclear changes, not changes in
cellular morphology, are most correlated with proliferation of hemoglobinsynthesizing organelles and accumulation of hemoglobin in the cytoplasm. They
postulated that nuclear morphology is a better indicator of the onset of maturation
than cellular morphology. Moreover, Jagoe & Welter (1995) proposed that
changes in DNA content caused by genotoxic environmental contaminants may
34
be observable as alterations in nuclear size, shape, or structure. Schultz et al.
(1993) increased the micronuclei frequency in erythrocytes of rainbow trout
(Oncorhynchus mykiss) by exposure to radiation. Similar results were obtained
by Hose et al. (1987), who correlated increased micronuclei frequency in white
croaker (Genyonemus lineatus) and kelp bass (Paralabrax clathratus) to
environmental PCBs and PAHs. These studies, togethe r with the present study,
support the hypothesis that nuclear morphology could be an indicator of both
erythrocyte maturity level and environmental exposure to contaminants.
To conclude, this study suggests that relative abundance of immature
erythrocytes as well as nuclear morphology may be suitable indicators of
contaminant exposure in wild brown bullhead populations. The number of
karyorrhetic, dividing, and enucleate erythrocytes may not be as efficacious for
use as biomarkers in this species.
Further research is needed to more
precisely identify the stimuli of brown bullhead erythropoiesis, including
laboratory studies that establish models of normal and contaminant-related
hematological changes in this species.
35
REFERENCES
Baumann, P. C. & Harshbarger, J. C. (1995). Decline in liver neoplasms in wild
brown bullhead catfish after coking plant closes and environmental PAHs
plummet. Environmental Health Perspectives 103, 168-170.
Baumann, P. C. (2000). Health of bullhead in an urban fishery after remedial
dredging. Final Report, U.S. EPA Grant No. GL985635-01-0. Chicago, IL,
U.S.A.: U.S. Environmental Protection Agency, Great Lakes National Program
Office.
Benfey, T.J. & Sutterlin, A.M. (1984). Binucleated red blood cells in the
peripheral blood of an Atlantic salmon, Salmo salar L. Journal of Fish Dis. 7,
415-420.
Cameron, J. N. & Davis, J. C. (1970). Gas exchange in rainbow trout (Salmo
gairdneri) with varying blood oxygen capacity. Journal of the Fisheries Research
Board of Canada 27, 1069-1085.
Ellis, A.E. (1984). Bizarre forms of erythrocytes in a specimen of plaice,
Plueronectes platessa. Journal of Fish Dis. 7, 411-414.
Fange, R. (1986). Physiology of haemopoiesis. In Fish Physiology: Recent
Advances (Nilsson & Holmgren, eds.), pp. 1-23. London, U.K.: Croom Helm.
Hardig, J. (1978). Maturation of circulating red blood cells in young Baltic salmon
(Salmo salar L.). Acta physiologica Scandinavica 102, 290-300.
Haws, G. T. & Clarence, G. J. (1962). Some aspects of the hematology of two
species of catfish in relation to their habitats. Physiological Zoology 35-36, 8-17.
Hose, J. E., Cross, J. N., Smith, S. G. & Diehl, D. (1987). Elevated circulating
erythrocyte micronuclei in fishes from contaminated sites off Southern California.
Marine Environmental Research 22, 167-176.
Houston, A. H. (1997). Review: Are the classical hematological variables
acceptable indicators of fish health? Transactions of the American Fisheries
Society 126, 879-894.
Houston, A. H. & Murad, A. (1991). Hematological characterization of goldfish,
Carassius auratus L., by image analysis: effects of thermal acclimation and heat
shock. Canadian Journal of Zoology 69, 2041-2047.
Houston, A. H. & Murad, A. (1995). Erythrodynamics in fish: recovery of the
goldfish Carassius auratus from acute anemia. Canadian Journal of Zoology 73,
411-418.
36
Houston, A. H., Blahut, S., Murad, A. & Amirtharaj, P. (1993). Changes in
erythron organization during prolonged cadmium exposure: an indicator of heavy
metal stress? Canadian Journal of Fisheries and Aquatic Sciences 50, 217-222.
Jagoe, C. H. & Welter, D. A. (1995). Quantitative comparisons and ultrastructure
of erythrocyte nuclei from seven freshwater fish species. Canadian Journal of
Zoology 73, 1951-1959.
Keen, J. E., Calarco Steele, A. M. & Houston, A. H. (1989). The circulating
erythrocytes of rainbow trout (Salmo gairdneri). Comparative Biochemistry and
Physiology A 94, 699-711.
Lane, H. C. & Tharp, T. P. (1980). Changes in the population of polyribosomal
containing red cells of peripheral blood of rainbow trout, Salmo gairdneri
Richardson, following starvation and bleeding. Journal of Fish Biology 17, 75-81.
Lane, H. C., Weaver, J. W., Benson, J. A. & Nichols, H. A. (1982). Some age
related changes of adult rainbow trout, Salmo gairdneri Rich., peripheral
erythrocytes separated by velocity sedimentation at unit gravity. Journal of Fish
Biology 21, 1-13.
Lowe-Jinde, L. & Niimi, A.J. (1983). Influence of sampling on the interpretation of
haematological measurements of rainbow trout, Salmo gairdneri. Can. Journal of
Zoology 61, 396-402.
Murad, A. & Houston, A. H. (1992). Maturation of the goldfish (Carassius
auratus) erythrocyte. Comparative Biochemistry and Physiology A 102, 107-110.
Murad, A., Houston, A. H. & Samson, L. (1990). Haematological response to
reduced oxygen-carrying capacity, increased temperature and hypoxia in
goldfish, Carassius auratus L. Journal of Fish Biology 36, 289-305.
Passino-Reader, D. R., Rasolofoson, A. J., Nelson, S. R. & Smith, S. B. (in
press). Lake Erie ecological investigations: chemical contaminants in sediments.
Open File Report. Reston, VA, U.S.A.: U.S. Geological Survey.
Pennsylvania Department of Environmental Protection. 1997. The 1997
Presque Isle Bullhead Tumor Study. The Pennsylvania State University,
University Park, Pennsylvania.
37
Schmitt, C. J., Blazer, V. S., Dethloff, G. M., Tillitt, D. E., Gross, T. S., Bryant, W.
L., DeWeese, L. R., Smith, S. B., Goede, R. W., Bartish, T. M. & Kubiak, T. J.
(1999). Biomonitoring of environmental status and trends (BEST) program: field
procedures for assessing the exposure of fish to environmental contaminants.
Information and Technology Report USGS/BRD-1999-0007.iv + 35pp. +
appendices. Columbia, MO, U.S.A.: U.S. Geological Survey, Biological
Resources Division.
Schultz, N., Norrgren, L., Grawe, J., Johannisson, A. & Medhage, O. (1993).
Micronuclei frequency in circulating erythrocytes from rainbow trout
(Oncorhynchus mykiss) subjected to radiation, an image analysis and flow
cytometric study. Comparative Biochemistry and Physiology C 105, 207-211.
Smith, S. B., Passino -Reader, D. R., Baumann, P. C., Nelson, S. R., Adams, J.
A., Smith, K. A., Powers, M. M., Hudson, P. L., Rasolofoson, A. J., Rowan, M.,
Peterson, D., Bla zer, V. S., Hickey, J. T. & Karwowski, K. (2003). Lake Erie
ecological investigations: summary of findings. Part 1: sediment, invertebrate
communities, fish communities 1998-2000. Administrative Report: 2003-001. Ann
Arbor, MI, U.S.A.: U.S. Geological Survey, Great Lakes Science Center.
Weinberg, S. R., Siegel, C. D., Nigrelli, R. F. & Gordon, A. S. (1972). The
hematological parameters and blood cell morphology of the brown bullhead
catfish, Ictalurus nebulosus (Le Sueur). Zoologica 57, 71-78.
Witeska, M. (2005). Stress in fish – hematological and immunological effects of
heavy metals. Electronic Journal of Ichthyology 1: 35-41.
Wood, C. M., McMahon, B. R. & McDonald, D. G. (1979). Respiratory, ventilatory
and cardiovascular responses to experimental anemia in the starry flounder,
Platichthys stellatus. The Journal of Experimental Biology 82, 139-162.
Yang, X., Peterson, D. S., Baumann, P. C. & Lin, E. L. C. (2003). Fish biliary
PAH metabolites estimated by fixed-wavelength fluorescence as an indicator of
environmental exposure and effects. Journal of Great Lakes Research 29, 116123.
38
Figure 2.1. Map locations of the sampling sites, Ottawa River (OT), Huron River
(HU), Old Woman Creek (OW), Black River (BL), Cuyahoga River harbor (CH),
Cuyahoga River upstream (CU), and Presque Isle Bay (PI).
39
Figure 2.2. Immature (1: extremely immature, 2 and 3: slightly more developed
immature), intermediate (Int), and mature (M) brown bullhead erythrocytes at
1000X magnification, stained with Accustain  Camco  Stain Pak.
40
Figure 2.3. Karyorrhetic brown bullhead erythrocytes at 1000X magnification,
stained with Accustain  Camco  Stain Pak. 1 and 2 show cytoplasmic
swelling; 3 and 4 show membrane disintegration and nuclear deformation; and 5
shows the “nuclear shadow” or smudge cell remaining after cell death.
41
Figure 2.4. Dividing brown bullhead erythrocytes at 1000X magnification, stained
with Accustain  Camco  Stain Pak.
42
Figure 2.5. Enucleate brown bullhead erythrocytes at 1000X magnification,
stained with Accustain  Camco  Stain Pak.
43
a.
160
25
120
100
80
60
40
y = -98.01 + 23.03x - 0.65x2
R2 = 0.567
20
Nucleus Area (µm2)
Cell Area (µm2)
b.
30
140
20
15
10
y = -1.04 + 2.32x + 0.12x 2
R2 = 0.698
5
0
0
6
8
10
12
14
16
Cell Major Axis (µm)
18
20
2
c.
1
3
4
5
6
Nucleus Major Axis (µm)
7
1.2
0.8
0.7
0.6
0.5
0.4
0.3
0.2
y = 0.75 + 0.032x - 0.0023x 2
R2 = 0.145
0.1
Nucleus Shape Factor
Cell Shape Factor
0.9
1
0.8
0.6
0.4
0
0
6
8
10
12
14
16
Cell Major Axis (µm)
18
20
2
14
6
12
5
10
8
6
4
y = -2.75 + 1.87x - 0.074x
2
R = 0.061
2
2
0
Nucleus Minor Axis (µm)
Cell Minor Axis (µm)
y = 1.44 - 0.22x + 0.014x 2
R2 = 0.156
0.2
3
4
5
6
Nucleus Major Axis (µm)
7
4
3
2
y = 0.27 + 1.07x - 0.087x 2
R2 = 0.205
1
0
6
8
10
12
14
16
Cell Major Axis (µm)
18
20
2
3
4
5
Nucleus Major Axis (µm)
6
7
Figure 2.6. Variation in area, shape factor, and minor axis length with major axis
length of brown bullhead red blood cells and nuclei.
44
100%
Abundance
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
OT
HU
OW
BL
CH
CU
PI
Site
Figure 2.7. Relative abundance of immature (slanted), intermediate (dotted), and
mature (open) erythrocytes in brown bullheads from Ottawa River (OT), Huron
River (HU), Old Woman Creek (OW), Black River (BL), Cuyahoga River harbor
(CH) and Cuyahoga River upstream (CU), and Presque Isle Bay (PI).
45
Site
Sample
Cell
size
Major axis (µm)
Minor axis (µm)
Area (µm 2 )
Shape factor
OT
37
11.30±0.10
9.00±0.08
79.91±1.14 abc
0.834±0.004 a
HU
29
11.37±0.12
8.89±0.09
78.04±1.47 c
0.795±0.010 b
OW
22
11.71±0.12
9.14±0.10
84.37±1.58 ab
0.835±0.004 a
BL
33
12.14±0.11
9.15±0.09
85.63±1.39 a
0.757±0.012 c
CH
37
11.42±0.08
8.85±0.09
79.43±1.23 bc
0.824±0.004 ab
CU
15
11.60±0.12
9.07±0.09
82.54±1.42 abc
0.822±0.006 ab
PI
35
10.92±0.11
8.31±0.08
70.27±1.15 d
0.797±0.005 b
Site
Sample
Nucleus
size
Major axis (µm)
Minor axis (µm)
OT
37
3.81±0.05
3.01±0.04
HU
29
4.12±0.03
3.21±0.03
Area (µm 2 )
Shape factor
9.78±0.22 ab
10.27±0.16 a
10.47±0.37
a
0.859±0.006 a
0.666±0.010 b
OW
22
3.99±0.08
3.09±0.06
10.36±0.16 a
0.861±0.007 a
BL
33
4.15±0.03
3.23±0.03
8.99±0.25 b
0.674±0.013 b
CH
37
3.60±0.05
2.94±0.04
9.88±0.27 ab
10.08±0.18
a
0.849±0.007 a
CU
15
3.77±0.05
3.12±0.05
0.837±0.011 a
PI
35
4.07±0.04
3.20±0.03
0.667±0.008 b
Values reported as mean ± SE. Means with the same letter are not significantly different (p > 0.05, Tukey’s
multiple comparisons).
Table 2.1. Major and minor axis length, area, and shape factor of red blood cells and
nuclei in fish from Ottawa River (OT), Huron River (HU), Old Woman Creek (OW), Black
River (BL), Cuyahoga River harbor (CH), Cuyahoga River upstream (CU), and Presque
Isle Bay (PI).
46
SITE
N
mean #
karyorrhetic
erythroctes per
7 fields
± S.E.
mean # dividing
erythroctes per
7 fields
± S.E.
mean # enucleate
erythroctes per
7 fields
± S.E.
0.05 ± 0.05 ab
0.35 ± 0.13 a
0±0a
0.27 ± 0.15 a
CUYAHOGA RIVER
HARBOR
20
15.30 ± 1.25 a
CUYAHOGA RIVER
UPSTREAM
15
17.40 ± 3.05
OTTAWA RIVER
20
19.40 ± 2.02 ab
0.40 ± 0.20 ab
0.40 ± 0.17 a
BLACK RIVER
20
15.15 ± 1.52 ab
0.20 ± 0.09 ab
0.35 ± 0.25 a
PRESQUE ISLE
BAY
20
19.25 ± 1.81 ab
0.50 ± 0.30 b
0.35 ± 0.17 a
OLD WOMAN
CREEK
20
23.90 ± 1.75 b
0.25 ± 0.12 ab
1.15 ± 0.72 a
HURON RIVER
20
21.60 ± 1.92 ab
0.15 ± 0.08 ab
0.80 ± 0.37 a
ab
Means with the same letter are not significantly different (p < 0.05,
tukey's multiple comparisons for mean # karyorrhetic erythrocytes; p < 0.05, wilcox
rank test for
mean # dividing and enucleate erythrocytes)
Table 2.2. Mean number of karyorrhetic, dividing, and enucleate erythrocytes
per seven randomly selected fields for brown bullheads from Cuyahoga River
Harbor, Cuyahoga River Upstream, Ottawa River, Black River, Presque Isle Bay,
Old Woman Creek, and Huron River.
47
a
Site
OT
HU
OW
BL
CH
CU
PI
Oxygen
12.5
7.8
6.7
11.0
3.7
8.7
9.9
PAHsa
9.41
1.01
5.25
5.42
19.07
3.33
2.28
PCBs
2.93
0.04
0.08
0.15
0.46
0.19
0.13
DDTsb
0.081
0.012
0.033
0.030
0.023
0.025
0.013
Metalsc
0.88×103
0.66×103
0.33×103
0.69×103
1.17×103
0.46×103
1.12×103
,
Sum
of
concentrations
benzo[b]fluoranthene,
of
chrysene,
anthracene,
benz[a]anthracene,
dibenz(a,h)anthracene,
benzo[a]pyrene,
fluoranthene,
fluorene,
naphthalene, C1-, C2-, C3-, and C4- naphthalene, perylene, phenanthrene, and pyrene.
b
, Sum of concentrations of o,p'-DDD, o,p'-DDE, o,p'-DDT, p,p'-DDD, p,p'-DDE, and p,p'-
DDT. Half of detection limit was used for concentrations below detection limit.
c
, Sum of concentrations of heavy metals, Ba, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Sr, and V.
Half of detection limit was used for concentrations below detection limit.
Table 2.3. Concentrations of dissolved oxygen in water (mg/L) and selected
chemicals in sediments (µg/g dry weight) of Ottawa River (OT), Huron River
(HU), Old Woman Creek (OW), Black River (BL), Cuyahoga River harbor (CH)
and Cuyahoga River upstream (CU), and Presque Isle Bay (PIB) (Smith et al.
2003; Passino-Reader et al., in press).
48
CHAPTER 3
CIRCULATING LEUKOCYTES IN BROWN BULLHEADS
FROM LAKE ERIE TRIBUTARIES
INTRODUCTION
Previous studies have described the normal blood parameters of several fish
species. Wedemyer and Yasutake (1977), Zybanyszek and Smith (1984), and
McKim et al. (1987a and 1987b) described the blood of rainbow trout,
Oncorhynchus mykiss. Munkittrick and Leatherland (1983) studied the goldfish,
Carassius auratus, while Haws and Goodnight (1962), Wedemyer and Yasutake
(1977), and Ellsaesser and Clem (1986) studied the channel catfish, Ictalarus
punctatus. The blood of brown bullhead, Ameiurus nebulosus (Lesueur), has
been studied by Haws and Goodnight (1962), Weinberg et al. (1972), Murchake
(1987), and Stasiak (1995). These studies indicate that the relative numbers of
leukocyte types vary from one species to the next.
Lowe-Jinde and Niimi (1983) suggested that differential leukocyte ratios,
since they are slow to change in response to handling stress, may be a suitable
index for fish laboratory and field studies. White blood cell numbers are affected
by a variety of physiological and environmental factors. Ellsaesser & Clem
(1986) found that fish exposed to chronic stress, such as contaminants and
49
infectious agents, had a decreased number of lymphocytes. Similar studies have
also reported a not only this decrease in fish lymphocytes but a corresponding
increase in the number of neutrophils (Siwicki & Studnicka 1987; Murad &
Houston 1988).
Witeska (2005) subjected common carp, Cyprinus carpio, to different
concentrations of various heavy metals, and reported a decrease in total
leukocyte count due to a large drop in lymphocyte count. He also reported that
exposure to cadmium and lead corresponded with an increase in the percentage
of neutrophils. Donaldson and Dye (1975) reported that heavy metal-induced
increases in fish cortisol are responsible for a decrease in leukocyte numbers,
especially in the number of lymphocytes and their activity.
The brown bullhead, Ameiurus nebulosus, is a widely used indicator
species ubiquitous in Lake Erie. The bullhead’s almost constant association with
the sediment renders it vulnerable to many hydrophobic contaminants. It is
limited in home range, and it is commonly used in tumor surveys (Baumann et al.
1996; Baumann and Harshbarger 1998). Murchake (1987) found that bullhead
from a contaminated site (Black River) had higher numbers of neutrophils and
lower numbers of monocytes than fish from a reference site (Old Woman Creek).
Similarly, Stasiak (1995) reported higher numbers of activated neutrophils in fish
from contaminated sites, and lower numbers in fish from reference sites.
Anderson (1990) suggested several immunological indicator assays that
can be used to determine fish immunosuppression, inc luding passive
50
hemagglutination, ELISA, chemiluminescence through oxidative burst, and
fluorescent antibodies. While all of these are sensitive indices, they require
specific training and laboratory equipment. Conversely, differential leukocyte
counts that determine the percentage of each cell type are easy to prepare in the
field and require minimal laboratory skills and equipment. This practicality makes
them worthy for investigation for use as a potential biomarker
The purpose of this study is to: describe the relative proportions of
leukocytes in brown bullheads from several sites around Lake Erie; compare the
means for percent leukocytes from one site to the next; and investigate their
efficacy as an indicator of contaminant exposure in brown bullhead.
METHODS
Field Procedures
Brown bullheads were collected using standard fyke nets from the Ottawa
River (OT), Huron River (HU), Old Woman Creek (OW), Black River (BL),
Cuyahoga River harbor (CH), and Presque Isle Bay (PI) in the spring and early
summer of 1998 and 1999 (Figure 3.1). Two of these sites, HU and OW,
received agricultural runoff but had little to no industrial sources of pollution. The
remaining five sites were industrially contaminated, and each was designated as
a Great Lakes Area of Concern by the International Joint Commission. Recently,
however, the Black River has undergone significant remediation with reduced
PAH contamination, and has been reclassified as an Area of Recovery for the
51
'Fish Tumors and Other Deformities' use impairment. The EPA acceptance letter
for the re-designation can be found at: http://www.noaca.org/FishBUI.PDF.
Bullheads longer than 250mm (about 3 years old or older) were removed
from the nets and immediately placed in an aerated tub of river water. Fish were
removed from the tank one at a time and anesthetized in a bucket containing
100mg/L tricaine methylsulfonate (MS-222). Mixed arteriovenous blood was
drawn from the caudal artery and vein of each fish using the lateral approach
described by Schmitt et al. (1999). A non-heparinized vacutainer tube was used
to collect a small amount of blood (< 0.5mL). Two blood smears were
immediately made with drops of this blood and allowed to air dry. Slides were
stained using an Accustain Camco Stain Pak (Sigma Diagnostics), a rapid
differential stain for blood smears comparable to Wright-Giemsa stains. All slides
were prepared and stained by the same individual.
Dissolved oxygen was measured at each sampling site, both at the water
surface and near the sediment. Because the water depth at each site was usually
shallow and the surface-to-bottom differences in oxygen were very small (less
than 0.1 ppm), the oxygen measurements were averaged yielding one dissolved
oxygen reading per site.
At least five surface sediment samples were randomly collected using a
stainless steel Ekman dredge from each site and analyzed as described by Smith
et al. (2003). Polynuclear aromatic hydrocarbons (PAHs) in sediments were
measured by capillary gas chromatography (CGC) with a mass spectrometer
52
detector in the SIM mode. Polychlorinated biphenyls (PCBs) and pesticides were
quantified by CGC with an electron capture detector. Metals in sediments were
determined by atomic absorption spectrometry and atomic emission
spectrometry.
Slide Analysis
Slides were examined using an Olympus oil-immersion light microscope
with 1000X magnification. A random area of the slide was chosen to begin
counting neutrophils, lymphocytes, and monocytes. The slide was moved in one
direction until 100 total leukocytes were counted. Data were recorded as percent
neutrophils, lymphocytes, and monocytes.
Statistical Analysis
Tukey’s multiple comparisons were used to check for significant
differences between the percent neutrophils, percent lymphocytes, and percent
monocytes at each site. Spearman’s rank correlation coefficients were
calculated to determine if percent neutrophils, percent lymphocytes, and percent
monocytes were correlated with dissolved oxygen and sediment contaminants
(PAHs, PCBs, DDTs, and metals).
53
RESULTS
Figure 3.2 shows microscopic images of the three leukocyte types
counted in this study. Cells were distinguished by their morphology and staining
characteristics. In general, lymphocytes were the smallest in area but had the
largest nucleus-to-cytoplasm ratio. Neutrophils were the most abundant
lymphocyte and were characterized by their multi-lobed nucleus. In monocytes,
both the nucleus and the cytoplasm stained darker purple than the other
leukocytes. They are usually larger in area than other circulating blood cells.
Monocytes were the least abundant of the three leukocytes. Thrombocytes were
also found in the blood of brown bullhead. These leukocytes function in blood
clotting, and were not quantified as part of this study. No eosinophils or
basophils were found in fish from this study, which corresponds with the findings
of Murchake (1987) and Weinberg et al. (1972).
Blood smears from twenty brown bullheads from each of the six sites were
analyzed to determine leukocyte ratios. Table 3.1 shows the mean percent
monocytes, lymphocytes, and neutrophils for each site. For the six sites in this
study, mean percent monocytes ranged from 3.65% - 15.90%; mean percent
lymphocytes ranged from 25.25% - 41.65%; and mean percent neutrophils
ranged from 48.70% - 64.65%.
Brown bullheads from the Black River had significantly lower percent
monocytes than brown bullheads from Presque Isle Bay and the Huron River
(p<0.05, Tukey’s test). Fish from the Huron River had the highest percent
54
monocytes, which was significantly different from all of the other sites (p<0.05,
Tukey’s test). For percent lymphocytes, brown bullheads from Old Woman
Creek had the highest percent lymphocytes, which was significantly different
from brown bullheads from Presque Isle Bay and the Ottawa River. (p<0.05,
Tukey’s test). Percent neutrophils was highest in fish from Presque Isle Bay and
the Black River, which were significantly different from fish from Old Woman
Creek and the Huron River.
Dissolved oxygen measured at each site (Table 3.5) was not a significant
factor affecting the mean percent monocytes, lymphocytes, or neutrophils (p >
0.5, Spearman’s rank correlation procedure). Similarly, sediment PAHs, PCBs,
DDTs, and heavy metals (Table 3.5) were not associated with mean percent
monocytes, lymphocytes, or neutrophils (p > 0.5, Spearman’s rank correlation
procedure).
DISCUSSION
Some of the slides analyzed in this study were also analyzed by Ohio
State student Danielle Hermann for an undergraduate research project. Using
the same techniques (and slides) described above, she calculated the mean
percent monocytes, lymphocytes, and neutrophils for 15 brown bullheads from
the Black River and 15 brown bullheads from Old Woman Creek. Her results are
shown in table 3.2. These data confirm the results from this study (Table 3.1): a
lower percent lymphocytes and a higher percent neutrophils in fish from the
55
Black River, as compared to fish from Old Woman Creek, the historical reference
site due to lack of anthropogenic contaminants.
Murchake (1987) also reported leukocyte ratios in brown bullheads from
Lake Erie tributaries. She used the same technique as this study, but only
included monocytes and neutrophils in her percent leukocyte calculations. Her
results are shown in Table 3.3. She found no significant differences between
male and female brown bullheads, but reported significantly lower percent
monocytes and significantly higher percent neutrophils in fish from the Black
River, as compared to fish from Old Woman Creek. These data compare
favorably to some of the results from this study (Table 3.1). My results show that
two contaminated sites, the Black River and Cuyahoga River, had the lowest
percent monocytes, while the Huron River, another historical reference site, had
the highest percent monocytes. My results also show that fish from Old Woman
Creek and the Huron River had the lowest percent neutrophils of all six sites in
this study.
Stasiak and Baumann (1996) used the nitroblue tetrazolium (NBT) assay
to determine the number of activated neutrophils in brown bullheads from a
contaminated site (Black River) and a reference site (Old Woman Creek). Their
results are shown in Table 3.4. They reported a significantly higher number of
activated neutrophils in fish from the Black River as compared to fish from Old
Woman Creek. They concluded that this could indicate that the bullheads’ nonspecific immune response may have been stimulated by the presence of PAHs.
56
Their results compare favorably to this study and the studies described above,
which showed higher number of neutrophils in fish from contaminated sites.
Dissolved oxygen was not significantly correlated with percent monocytes,
lymphocytes, or neutrophils (p > 0.5, Spearman’s rank correlation procedure).
This is similar to results obtained by Scott and Rogers (1981), who reported that
differential leukocyte counts in channel catfish, Ictalurus punctatus, were not
sensitive indicators of prolonged sublethal hypoxia. Brown bullheads are fairly
tolerant to low-oxygen conditions. Since the study sites were open systems, fish
could simply migrate to better environments when conditions became stagnant.
Through simple adjustment of cardiac, ventilatory, and branchiovascular
activities, fish are able to quickly counterbalance the effects of mild to moderate
hypoxia (Cameron & Davis, 1970; Wood et al., 1979).
Karrow et al. (1999) exposed rainbow trout, Oncorhynchus mykiss, to
PAH-containing creosote and found a reduction in the oxidative burst of head
kidney leukocytes as well as a reduction in peripheral leukocytes positive for
surface immunoglobulins. This study, however, found no significant correlations
between leukocyte percentages and sediment PAHs (p > 0.5, Spearman’s rank
correlation procedure).
In fact, percent monocytes, lymphocytes, and neutrophils were not
significantly correlated with any specific sediment contaminant (p > 0.5,
Spearman’s rank correlation procedure). Yet as stated above, the results of this
study are similar to previous studies of brown bullheads at historical
57
“contaminated” and “reference” sites. There are several hypotheses to account
for this. First, many field and laboratory studies (Anderson et al. 1988; Murad &
Houston 1988; Sanchez-Dardon et al. 1999; Karrow et al. 1999) showed a
relationship between fish leukocyte parameters and exposure to single types of
contaminants. The sites in this study are contaminated with a variety of
anthropogenic pollutants (Table 3.5). For example, Old Woman Creek sediment
had very low levels of PCBs and metals, but moderate levels of PAHs and DDTs.
Presque Isle Bay sediment contained low levels of PAHs and DDTs, moderate
levels of PCBs, and high levels of metals. Huron River sediment had low levels
of all contaminants except metals, while Ottawa River sediment contained high
levels of all contaminants except metals. It is likely that synergistic effects of
multiple contaminants are affecting leukocyte ratios in brown bullheads from
these study sites.
In addition, the fish immune system responds to a variety of stimuli,
including temperature, pH, and starvation (Mahajan & Dheer 1983; Anderson
1994). Many of the fish collected in this study, especially from the more
contaminated sites, had barbel deformities, skin and liver parasites, and open
sores and lesions on their skin, fins, and oral cavities. These abrasions
compromise the physical barrier that acts as the immune system’s first line-ofdefense. Thus the fish are more vulnerable to viral and bacterial infections,
further complicating the analysis of the differential leukocyte counts.
58
In conclusion, the results of this study suggest that differential leukocyte
counts determined by light microscopy may not be suitable as stand-alone
biomarkers of contaminant exposure in brown bullhead. While other indices are
more sensitive at detecting immunological changes in fish, differential leukocyte
counts, which require minimal skills and equipment, can still provide useful
information if combined with other non-invasive biomarkers.
59
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Baumann, P.C. and Harshbarger, J. (1998). Long term trends in liver neoplasm
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Mahajan, C.L. and Dheer, T.R. (1983). Haematological and hematopoietic
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Environmental Toxicology and Chemistry 6: 295-312.
McKim, J.M., Schmieder, P.K., Niemi, G.J., Carlson, R.W., and Henry, T.R.
(1987b). Use of respiratory-cardiovascular responses of rainbow trout (Salmo
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Munkittrick, K.R. and Leatherland, J.F. (1983). Hematocrit values in feral
goldfish, Carassius auratus, as indicators of the health of the population. Journal
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Murchake, V.K. (1987). Hematological parameters of brown bullhead (Ictalurus
nebulosus) taken from polluted and non-polluted tributaries of Lake Erie.
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Boermans, H., Blakely, B., and Fournier, M. (1999). Immunomodulation by
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(1999). Biomonitoring of environmental status and trends (BEST) program: field
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Scott, A.L. and Rogers, W.A. (1981). Haematological effects of prolonged
sublethal hypoxia on channel catfish, Ictalarus punctatus (Rafinesque). Journal
of Fish Biology 18: 591-601.
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Siwicki, A.K. and Studnicka, M. (1987). The phagocytic ability of neutrophils and
serum lysozyme activity in experimentally infected carp Cyprinus carpio L.
Journal of Fish Biology 31A: 57– 60.
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Smith, K.A., Powers, M.M., Hudson, P.L., Rasolofoson, A.J., Rowan, M.,
Peterson, D., Blazer, V.S., Hickey, J.T., and Karwowski, K. (2003). Lake Erie
Ecological Investigations: Summary of Findings. Part 1: Sediment, Invertebrate
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Stasiak, S.A. (1995). Implications of chronic exposure to polynuclear aromatic
hydrocarbons: effects on immune and endocrine functions in brown Bullhead,
Ameiurus nebulosus, from a contaminated river. Masters thesis, The Ohio State
University.
Stasiak, S.A. and Baumann, P.C. (1996). Neutrophil activity as a potential
bioindicator for contaminant analysis. Fish & Shellfish Immunology 6: 537-539.
Wedemeyer, G.A. and Yasutake, W.T. (1977). Clinical methods for the
assessment of the effects of environmental stress in fish health. United States
Fish and Wildlife Technical Papers. 89: 1-18.
Weinberg, S. R., Siegel, C. D., Nigrelli, R. F. & Gordon, A. S. (1972). The
hematological parameters and blood cell morphology of the brown bullhead
catfish, Ictalurus nebulosus (Le Sueur). Zoologica 57, 71-78.
Witeska, M. (2005). Stress in fish – hematological and immunological effects of
heavy metals. Electronic Journal of Ichthyology 1: 35-41.
Wood, C. M., McMahon, B. R. & McDonald, D. G. (1979). Respiratory, ventilatory
and cardiovascular responses to experimental anemia in the starry flounder,
Platichthys stellatus. The Journal of Experimental Biology 82, 139-162.
Zbanyszek, R. and Smith, L.S. (1984). The effects of water-soluble aromatic
hydrocarbons on some haematological parameters of rainbow trout, Salmo
gairdneri, during acute exposure. Journal of fish Biology 24: 545-552.
62
Figure 3.1. Map locations of the sampling sites, Ottawa River (OT), Huron River
(HU), Old Woman Creek (OW), Black River (BL), Cuyahoga River harbor (CH),
Cuyahoga River upstream (CU), and Presque Isle Bay (PI).
63
N
M
L
Figure 3.2. Brown bullhead lymphocyte (L), neutrophil (N), and monocyte (M) at
1000X magnification, stained with Accustain  Camco  Stain Pak.
64
Site
Cuyahoga River
Harbor
Ottawa River
Black River
Presque Isle Bay
Old Woman Creek
Huron River
N
20
20
20
20
20
20
mean % monocytes
± S.E.
7.45 ± 0.90
mean % lymphocytes
± S.E.
ab
35.30 ± 1.60
ab
27.35 ± 1.79
ab
32.6 ± 2.22
a
25.25 ± 1.47
b
41.65 ± 2.67
ab
35.35 ± 1.01
9.8 ± 1.61
a
3.65 ± 0.52
bc
10.05 ± 0.93
ab
9.25 ± 1.71
c
15.9 ± 1.50
mean % neutrophils
± S.E.
ab
56.75 ± 1.70
ab
a
62.85 ± 2.08
b
63.75 ± 2.13
b
64.65 ± 1.91
a
49.1 ± 3.38
a
48.7 ± 1.97
ab
Means with the same letter are not significantly different (Tukey's test, p < 0.05).
Table 3.1. Mean percent monocytes, lymphocytes, and neutrophils for brown
bullheads from Cuyahoga River Harbor, Ottawa River, Black River, Presque Isle
Bay, Old Woman Creek, and Huron River.
65
N
mean % monocytes
± S.D.
mean %
lymphocytes
± S.D.
mean % neutrophils
± S.D.
Black River
15
0.73 ± 1.67
39.4 ± 12.78
59.9 ± 13.04
Old Woman Creek
15
8.30 ± 5.81
44.30 ± 14.94
47.3 ± 18.74
Site
Table 3.2. Mean percent monocytes, lymphocytes, and neutrophils for brown
bullheads from Black River and Old Woman Creek. Data from Danielle Hermann
(1999).
66
Sex
N
Black River
F
19
mean %
monocytes
± S.E.
9.00 ± 3.52a
Black River
Old Woman Creek
Old Woman Creek
M
F
M
18
21
19
12.50 ± 3.62a
31.43 ± 3.35b
28.16 ± 3.52b
Site
mean %
neutrophils
± S.E.
91.00 ± 3.61a
87.61 ± 3.70a
67.81 ± 3.43b
71.84 ± 3.61b
Means within each column that are not significantly different from each other (p<0.05)
are indicated by identical letters.
Table 3.3. Mean percent monocytes and neutrophils for male and female brown
bullheads from Black River and Old Woman Creek. Data from Vanessa
Murchake (1987).
67
Site
Black River
Old Woman Creek
N
21
12
May 1993
mean
activated
neutrophils
160.2
30.2
S.E.
43.8
9.82
N
22
19
Sept 1993
mean
activated
neutrophils
101.7
17.8
S.E.
13.4
1.44
Table 3.4. Mean counts of activated neutrophils for brown bullhead collected from the
Black River and Old Woman Creek in May and September, 1993. Black River fish
were found to have significantly higher activation (p < 0.05, Student's t-test). Data
from Stasiak and Baumann (1996).
68
OT
HU
OW
BL
CH
CU
PI
Oxygen
12.5
7.8
6.7
11.0
3.7
8.7
9.9
PAHs a
9.41
1.01
5.25
5.42
19.07
3.33
2.28
PCBs
2.93
0.04
0.08
0.15
0.46
0.19
0.13
DDTs b
0.081
0.012
0.033
0.030
0.023
0.025
0.013
Metals c
0.88×103
0.66×103
0.33×103
0.69×103
1.17×103
0.46×103
1.12×103
a
,
Sum
of
concentrations
of
anthracene,
benz[a]anthracene,
benzo[a]pyrene,
benzo[b]fluoranthene, chrysene, dibenz(a,h)anthracene, fluoranthene, fluorene, naphthalene, C1, C2-, C3-, and C4- naphthalene, perylene, phenanthrene, and pyrene.
b
, Sum of concentrations of o,p'-DDD, o,p'-DDE, o,p'-DDT, p,p'-DDD, p,p'-DDE, and p,p'-DDT.
Half of detection limit was used for concentrations below detection limit.
c
, Sum of concentrations of heavy metals, Ba, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Sr, and V. Half of
detection limit was used for concentrations below detection limit.
Table 3.5. Concentrations o f dissolved oxygen in water (mg/L) and selected
chemicals in sediments (µg/g dry weight) of Ottawa River (OT), Huron River
(HU), Old Woman Creek (OW), Black River (BL), Cuyahoga River harbor (CH)
and Cuyahoga River upstream (CU), and Presque Isle Bay (PIB) (Smith et al.
2003).
69
CHAPTER 4
PLASMA CORTISOL IN BROWN BULLHEADS
FROM OLD WOMAN CREEK, A TRIBUTARY OF LAKE ERIE
INTRODUCTION
One question concerning fish research is whether the sampling methods
themselves affect blood parameters. Bullheads are usually captured in fyke nets
that are set in the evening and checked the next morning. Wedemeyer and
Yasutake (1977) suggested that this capture method causes stress. However,
other capture methods, such as electroshocking, have also been shown to cause
physiological changes. In order to further examine the use of blood parameters
as biomarkers, it is necessary to determine how such sampling techniques affect
these parameters. Although stress may manifest itself in an assortment of blood
variables, one of the most quickly affected parameters is plasma cortisol level.
The hypothalamo-pituitary-intrarenal axis is composed of several
hormonal pathways that can be affected by artificial and environmental stressors
(Hontela et al. 1992). The hypothalamus secretes corticotropin releasing factor,
which stimulates the anterior pituitary to secrete adreno -corticotropin (ACTH),
70
which in turn stimulates the head kidney to secrete glucocorticoids (such as
cortisol) that lead to glucose production. This is an energy-mobilizing event that
enables an organism to mount the necessary fight-or-flight response (Barton &
Iwama 1991). Several studies have shown that chronic environmental stress
may reduce the cortisol response to acute stressors (Hontela et al. 1992, Stasiak
1995, Hontela et al. 1995, Brodeur et al. 1997). This may have a detrimental
effect on overall fish health, since cortisol also has a regulatory role in
metabolism, osmoregulation, reproduction, and immune function. In an
aquaculture study, Small (2004) administered dietary cortisol to channel catfish
(Ictalurus punctatus) and reported not only an increase in plasma cortisol and
plasma glucose, but also an increase in reproductive output (measured in
number of spawns per pond) without effects on fecundity and hatching success.
Gendron et al. (1997) reported significantly lower corticosterone levels in
an aquatic salamander, the mudpuppy (Necturus maculosus), from contaminated
sites compared to mudpuppies from reference sites. Because corticosterones
function in hepatic glycogen storage, this led to significantly lower levels of liver
glycogen in these amphibians from contaminated sites (Gendron et al. 1997).
Witeska (2005) reported that short-term exposures to high levels of heavy metals
induced stress reactions in fish. Other fish studies showed similar results.
Cortisol levels were relatively low in fish exposed to hydrogen peroxide (De La
Fuente 1998); cadmium (Brodeur et al. 1998; Hontela et al. 1996); copper and
cadmium together (Laflamme et al. 2000); mercury (Bleau et al. 1996); mercury,
71
cadmium, arsenic, and zinc together (Hontela et al. 1995); DDT compounds
(Benguira and Hontela 2000); and other organic contaminants (Girard et al.
1997; Hontela et al. 1995). These changes in the cortisol stress response in fish
from contaminated sites are caused, at least in part, by a dysfunction of the
interrenal tissue (Brodeur et al. 1997a; Brodeur at al. 1997b).
Several studies have suggested that the impaired ability to raise blood
cortisol levels in response to acute stress may be used as an indicator of toxic
stress in wild fish populations (Hontela et al. 1995; Girard et al. 1998; Stasiak
1995). This biomarker would be strengthened when used along with other blood
parameters. Experiments that measure blood cortisol levels while simulating
“handling stress” can be used to determine if (and to what degree) this stress
affects other blood parameters, including erythron profiles (immature,
intermediate, mature, karyorrhetic, enucleate, and dividing red blood cells) and
differential leukocyte ratios.
METHODS
Fish Collection
Brown bullheads were collected using standard fyke nets from Old
Woman Creek in October of 2001 (Figure 4.1). Fish longer than 250mm (about 3
years old or older) were tagged on the dorsal fin and placed in an aerated tub of
creek water. Five days of field effort produced only 9 brown bullheads. They
were transported to the laboratory at Old Woman Creek National Estuarine
72
Research Reserve and were left undisturbed in the aerated holding tank for 24
hours for acclimation.
Fish were separated into two test groups, A and B. The four fish in group
A were bled three times, at times 0, 60, and 180 minutes. These fish were
“stressed” immediately prior to the second bleeding (t=45 minutes). The “stress”
was designed to mimic the effects of capture and handling; fish were lifted from
the holding tank with a dip net, and repeatedly raised and lowered for 1 minute.
The four fish in group B were also bled three times, at times 0, 120, and 210
minutes. These fish were “stressed” immediately prior to the first bleeding (t=0
minutes). One fish served as a control. It was bled three times (at times 0, 150,
and 210 minutes) but was not “stressed”.
When removed from the tank, fish were a nesthetized in a bucket
containing 100mg/L tricaine methylsulfonate (MS-222). Mixed arteriovenous
blood was drawn from the caudal vasculature using the lateral approach
described by Schmitt et al. (1999). A non-heparinized vacutainer tube was used
to collect a small amount of blood (< 2mL). Two blood smears were immediately
made with drops of this blood and allowed to air dry. Slides were stained using
an Accustain  Camco  Stain Pak (Sigma Diagnostics ), a rapid differential
stain for blood smears comparable to Wright-Giemsa stains. All slides were
prepared and stained by the same individual. The remaining blood was
centrifuged at approximately 1200 rpm for 10 minutes. Blood plasma was then
transferred to cryovials which were frozen in liquid nitrogen. Samples were
73
stored in a -80°C freezer, and shipped on dry ice to the United States Geological
Survey Fish Health Laboratory of the Leetown Science Center in Kearneysville ,
West Virginia for the enzyme-linked immunoassay (ELISA).
Enzyme-Linked Immunoassay (ELISA)
The ELISA procedure was based on the methods described by Barry et al.
(1993) with the following modifications. Each plate was coated with 50 µL of
1:8500 rabbit anti-cortisol (30 µL diluted Ab in 5.1 mL coating buffer [0.05M, pH
9.6]), sealed, and incubated for 2 hours at 37°C. Plates were washed three
times with wash solution (1.5M NaCl, 0.5% Tween-20) using a Molecular Devices
plate washer. Plates were blocked by adding 300 µL of EIA buffer per well and
incubated at room temperature for 30 minutes. Fifteen µL of sample were added
to 135 µL of 1:30,000 cortisol-HRPO conjugate, and 50 µL were added per well
(in duplicate) per sample. Plates were incubated overnight at room temperature
and washed five times with wash buffer using a Molecular Devices plate washer.
One hundred µL of ABTS (2,2’-azino -di[3-ethylbenzthiazoline sulfonate]) solution
were added and the plates were read at 10 and 20 minutes.
Slide Analysis
Slides were examined using an Olympus oil-immersion light microscope
with 1000X magnification. Images were captured from random areas of the slide
using a COHU high performance color camera attached to a Targa image
74
capture board on an IBM PC and analyzed using Mocha  Image Analysis
software (version 1.2.10; Jandel Scientific). The major and minor axis length,
area, and shape factor (= 4π × area / perimeter2) of erythrocytes and their nuclei
were measured. The last measurement, shape factor, is a measure of circularity,
with values ranging from 0 to 1 (a perfect circle having a shape factor equal to1).
To determine the numbers of karyorrhetic, dividing, and enucleate
erythrocytes, seven fields were randomly selected on each slide. The numbers
of each cell type were counted, and data were recorded as number of cells per
seven fields. For the differential leukocyte counts, a random area of the slide
was chosen to begin counting neutrophils, lymphocytes, and monocytes. The
slide was moved in one direction until 100 total leukocytes were counted. Data
were recorded as percent neutrophils, lymphocytes, and monocytes.
Data Analysis
Plasma cortisol data from each test group were graphed on a scatterplot
to look for trends over time. Spearman’s rank correlation coefficients were
calculated to see if plasma cortisol affected erythrocyte nuclear area, erythrocyte
nuclear shape factor, the number of karyorrhetic, dividing, and enucleate
erythrocytes, and the percent monocytes, lymphocytes, and neutrophils.
75
RESULTS
One plasma sample (from fish 3 of group A) was damaged at the USGS
Fish Health Laboratory and was unable to be analyzed for plasma cortisol.
Additionally, the ELISA results for 8 plasma samples were below the detection
limit (40 ng/ml). These 8 samples were assigned a value of one-half the
detection limit (20 ng/ml) for data analysis.
Results from group A brown bullheads, which were stressed immediately
prior to the second bleeding (at t = 60 minutes), were not consistent (Figure 4.2).
Two fish showed an overall increase in plasma cortisol over the time period, but
the other two fish showed a decrease in plasma cortisol. The control fish (which
was not stressed) showed an increase in plasma cortisol over time, and exhibited
the highest overall plasma cortisol reading of 157.2 ng/ml at time = 210 minutes
(Figures 4.2 and 4.3). Results from group B fish, which were stressed
immediately prior to the first bleeding (at time = 0 minutes), were also
inconsistent (Figure 4.3). While two fish from this group showed overall
increases in plasma cortisol, plasma cortisol for the other two fish fluctuated up
and down.
Plasma cortisol results were compared to erythrocyte and leukocyte data
(Table 4.1). Previous studies (Houston & Murad 1991; Chapter 2 of this
dissertation) indicated that of all the measurements of cell and nuclear
morphology, nuclear area and nuclear shape factor were best suited for
determining red cell maturity. Thus, the other erythrocyte parameters determined
76
by image analysis (cell major axis length, cell minor axis length, cell area, cell
shape factor, nuclear major axis length, and nuclear minor axis length) were not
included in this data analysis. Plasma cortisol was not correlated with
erythrocyte nuclear area, erythrocyte nuclear shape factor, or the number of
karyorrhetic, dividing, and enucleate erythrocytes (Spearman's rank correlation
procedure, p > 0.05). Similarly, plasma cortisol was not correlated with percent
monocytes, percent neutrophils, or percent lymphocytes (Spearman's rank
correlation procedure, p > 0.05).
DISCUSSION
The overall increase in plasma cortisol in the unstressed control fish
indicates that the serial bleeding technique stimulates the release of this
hormone in brown bullheads. Efforts to account for this procedural effect by
subtracting the control data from the experimental data were not effective. The
control fish exhibited the highest overall plasma cortisol reading (157.2 ng/ml at
time = 210 minutes) which, upon subtraction, masked any experimentallyinduced hormone changes. Furthermore, the inconsistent results within each
test group were not able to be clarified by this transformation via subtraction.
Stasiak (1995) also used an ELISA to determine plasma cortisol levels in
brown bullheads (Table 4.2). He found no significant differences between male
and female fish. Because of his results, as well as the small sample size of this
study, gender differences were not investigated here. Stasiak reported a mean
77
plasma cortisol level of 69.7 ng/ml in 12 fish from Old Woman Creek. This was
consistent with the results of this study, where the mean plasma cortisol level
was 65.58 ng/ml for 26 samples from 9 Old Woman Creek fish. Stasiak also
found that the mean plasma cortisol level was significantly higher in fish from the
Toussaint River compared with fish from Old Woman Creek and the Black River.
He reported that the Toussaint River was the lowest in anthropogenic
contaminants, followed by Old Woman Creek and the Black River.
Even though Old Woman Creek has been used as a reference site for fish
studies around Lake Erie, it is likely that the brown bullheads in this study were
adapted to some environmental contaminants. Chapter two of this dissertation
reported a sediment PAH dry weight concentration of 5.25 µg/g. The source of
these PAHs is most likely a number of creosote-treated wooden ties near the
railroad bridge that crosses the southern end of the estuary. Johnston and
Baumann (1989) reported moderately elevated PAH metabolites in the bile of Old
Woman Creek brown bullheads. Karrow et al. (1999) exposed rainbow trout,
Oncorhynchus mykiss, to PAH-containing creosote and found that plasma
cortisol levels were not significantly different across creosote concentrations.
It is difficult to ascertain if the base-line cortisol levels in this study were affected
by PAHs or other environmental pollutants.
Chapter 2 of this dissertation suggested that erythrocyte nuclear
morphology measurements are suitable indicators of contaminant exposure.
Here, these morphology measurements were not correlated with plasma cortisol
78
and showed no drastic changes over time. This could imply that short-term
capture and handling stress have no effect on the proportion of immature
erythrocytes in circulation. However, the lack of trends in and the sparcity of the
data make it difficult to support any conclusions.
Plasma cortisol also showed no associations with the number of
karyorrhetic, dividing, or enucleate erythrocytes. Similarly, cortisol was not
correlated with percent monocytes, percent lymphocytes, or percent neutrophils.
These results are consistent with Lowe-Jinde and Niimi (1983), who suggested
that differential leukocyte ratios, since they are slow to change in response to
handling stress, may be a suitable index for fish laboratory and field studies.
Again, a lack of association with cortisol would have suggested that these
variables were not affected by capture and handling stress, if the data had not
been so inconsistent.
The small sample size and BDL (below detection limit) ELISA readings in
this study limit the conclusions that can be drawn. The ELISA was performed by
Christine Densmore, a scientist at the USGS Fish Health Lab. From my
communications with her, BDL readings are not uncommon, especially when
analyzing a fish species’ samples for the first time. With more fish, more time,
and more money, this cortisol study could be expanded and standardized for the
brown bullhead (Ameiurus nebulosus) species, providing more reliable data.
79
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microtitre plate ELISA for measuring cortisol in fish and comparison of stress
responses of rainbow trout (Oncorynchus mykiss) and lake trout (Salvelinus
namaycush). Aquaculture 117: 351-363.
Barton, B.A. and Iwama, G.K. 1991. Physiological changes in fish from stress in
aquaculture with emphasis on the response and effects of corticosteroids.
Annual Review of Fish Disease 1: 3-26.
Benguira, S. and Hontela, A. (2000). Adrenocorticotropin- and cyclic adenosine
3’,5’-monophosphate- stimulated secretion in interrenal tissue of rainbow trout
exposed in vitro to DDT compounds. Environmental Toxicology and Chemistry
19: 842-847.
Bleau, H., Daniel, C., Chevalier, G., van Tra, H., and Hontela, A. (1996). Effects
of acute exposure to mercury chloride and methylmercury on plasma cortisol, T3,
T4, glucose and liver glycogen in rainbow trout (Oncorynchus mykiss). Aquatic
Toxicology 34: 221-235.
Brodeur, J.C., Girard, C., and Hontela, A. (1997a). Use of perifusion to assess
in vitro the functional integrity of interregnal tissue in teleost fish from polluted
sites. Environmental Toxicology and Chemistry 16: 2171-2178.
Brodeur, J.C., Sherwood, G., Rasmussen, J.B., and Hontela, A. (1997b).
Impaired cortisol secretion in yellow perch (Perca flavescens) from lakes
contaminated by heavy metals: in vivo and in vitro assessment. Canadian
Journal of Fish. Aquat. Sci. 54: 2752-2758.
Brodeur, J.C., Daniel, C., Ricard, A.C., and Hontela, A. (1998). In vitro response
to ACTH of the interrenal tissue of rainbow trout (Oncorhynchus mykiss) exposed
to cadmium. Aquatic Toxicology 42: 103-113.
Donaldson, E.M. and Dye, H.M. (1975). Corticosteroid concentrations in
sockeye salmon (Oncorhynchus nerka) exposed to low concentrations of copper.
J. Fish. Res. Bd. Can. 32: 533-539.
Gendron, A.D., Bishop, C.A., Fortin, R., and Hontela, A. (1997). In vivo testing
of the functional integrity of the corticosterone-producing axis in mudpuppy
(amphibian) exposed to chlorinated hydrocarbons in the wild. Environmental
Toxicology and Chemistry 16: 1694-1706.
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Girard, C., Brodeur, J.C., and Hontela, A. (1998). Responsiveness of the
interrenal tissue of yellow perch (Perca flavescens) from contaminated sites to an
ACTH challenge test in vivo. Canadian Journal of Fish. Aquat. Sci. 55: 438-450.
Hontela, A., Dumont, P., Duclos, D., and Fortin, R. (1995). Endocrine and
metabolic dysfunction in yellow perch, Perca flavescens, exposed to organic
contaminants and heavy metals in the St. Lawrence River. Environmental
Toxicology and Chemistry 14: 725-731.
Hontela, A., Daniel, C., and Ricard, A.C. (1996). Effects of acute and subacute
exposures to cadmium on the interrenal and thyroid function in rainbow trout,
Oncorhynchus mykiss. Aquatic Toxicology 35: 171-182.
Houston, A. H. & Murad, A. (1991). Hematological characterization of goldfish,
Carassius auratus L., by image analysis: effects of thermal acclimation and heat
shock. Canadian Journal of Zoology 69: 2041-2047.
Johnston, E.P. and Baumann, P.C. (1989). Analysis of bile with HPLC
fluorescence to determine environmental exposure of fish to B(a)P.
Hydrobiologica 188/189: 561-566.
Karrow, N.A., Boermans, H.J., Dixon, D.G., Hontella, A., Solomin, K.R., Whyte,
J.J., and Bols, N.C. (1999). Characterizing the immunotoxicity of creosote to
rainbow trout (Oncorynchus mykiss): a mesocosm study. Aquatic Toxicology 45:
223-239.
Laflamme, J.S., Couillard, Y., Campbell, P.G.C., and Hontela, A. (2000).
Interrenal metallothionein and cortisol secretion in relation to Cd, Cu, and Zn
exposure in yellow perch, Perca flavescens, from Abitibi lakes. Canadian
Journal of Fish. Aquat. Sci. 57:1692-1700.
Lowe-Jinde L, Niimi A.J. (1983). Influence of sampling on the interpretation of
haematological measurements of rainbow trout, Salmo gairdneri. Canadian
Journal of Zoology 61:396–402.
Ruane, N.M., Wendelaar-Bonga, S.E., and Balm, P. ( 1999). Differences
between rainbow trout and brown trout in the regulation of the pituitary-interrenal
axis and physiological performance during confinement. General and
Comparative Endocrinology 115: 210-219.
Small, B.C. (2004). Effect of dietary cortisol administration on growth and
reproductive success of channel catfish. Journal of Fish Biology 64: 589-596.
81
Stasiak, S.A. (1995). Implications of chronic exposure to polynuclear aromatic
hydrocarbons: effects on immune and endocrine functions in brown Bullhead,
Ameiurus nebulosus, from a contaminated river. Masters thesis, The Ohio State
University.
Witeska, M. (2005). Stress in fish – hematological and immunological effects of
heavy metals. Electronic Journal of Ichthyology 1: 35-41.
82
Figure 4.1. Map location of Old Woman Creek near Huron, Ohio
83
180
fish 1
fish 2
fish 3
fish 4
fish 5 (control)
160
140
plasma cortisol (ng/ml)
120
100
80
60
40
20
0
0
50
100
150
200
250
time (minutes)
handling stress
Figure 4.2. Change in plasma cortisol in group A fish stressed at time =60 minutes. Due to a damaged sample, fish 3 has
only two data points. Fish 5 (control) was not stressed.
1
180
fish 6
fish 7
fish 8
fish 9
fish 5 (control)
160
140
plasma cortisol (ng/ml)
120
100
80
60
40
20
0
0
50
100
150
200
250
time (minutes)
handling stress
Figure 4.3. Change in plasma cortisol in group B fish stressed at time =0 minutes. Fish 5 (control) was not stressed.
2
sample
(group-fish-time)
mean plasma
cortisol (ng/ml)
A-01-0
A-01-60
A-01-180
A-02-0
A-02-60
A-02-180
A-03-0
A-03-60
A-04-0
A-04-60
A-04-180
control-05-0
control-05-150
control-05-210
20.0*
20.0*
106.1
43.0
60.7
76.3
116.6
41.3
128.5
68.2
20.0*
70.1
98.3
157.2
mean
erythrocyte
nuclear area
(µm 2)
9.94
9.97
9.89
9.57
9.62
9.61
10.06
10.16
9.86
9.93
9.81
9.94
9.92
10.23
mean
erythrocyte
nuclear
shape factor
0.847
0.853
0.865
0.865
0.858
0.867
0.856
0.849
0.851
0.852
0.861
0.859
0.846
0.851
number of
karyorrhetic
erythrocytes
per 7 fields
21
19
26
26
29
17
9
22
26
23
20
18
16
29
number of
dividing
erythrocytes
per 7 fields
0
0
0
0
0
1
0
0
0
0
0
0
1
0
number of
enucleate
erythrocytes
per 7 fields
1
0
0
3
0
1
0
0
0
5
0
1
0
0
number of
monocytes
per 100
leukocytes
7
12
16
4
8
16
22
11
16
16
8
3
7
11
number of
lymphocytes
per 100
leukocytes
39
38
32
28
26
29
32
30
45
40
37
32
40
28
number of
neutrophils
per 100
leukocytes
54
50
52
68
66
55
46
59
39
44
55
65
53
61
*denotes plasma cortisol readings below the 40 ng/ml detection limit and assigned a value of 20 ng/ml.
Table 4.1. Plasma cortisol, erythrocyte, and leukocyte data from 4 group A fish and 1 control fish from Old Woman
Creek.
3
sample
(group-fish-time)
mean plasma
cortisol (ng/ml)
mean
erythrocyte
nuclear area
(µm 2)
mean
erythrocyte
nuclear
shape factor
number of
karyorrhetic
erythrocytes
per 7 fields
number of
dividing
erythrocytes
per 7 fields
number of
enucleate
erythrocytes
per 7 fields
number of
monocytes
per 100
leukocytes
number of
lymphocytes
per 100
leukocytes
number of
neutrophils
per 100
leukocytes
control-05-0
control-05-150
70.1
98.3
9.94
9.92
0.859
0.846
18
16
0
1
1
0
3
7
32
40
65
53
control-05-210
157.2
10.23
0.851
29
0
0
11
28
61
B-06-0
B-06-120
53.8
69.1
10.00
10.15
0.847
0.861
25
21
0
0
0
1
8
7
40
38
52
55
B-06-210
125.6
10.08
0.866
22
0
0
8
36
56
B-07-0
B-07-120
20.0*
82.2
10.16
10.03
0.837
0.857
18
23
0
0
0
0
16
8
37
31
47
61
B-07-210
20.0*
9.69
0.849
18
0
0
7
29
64
B-08-0
B-08-120
106.3
20.0*
9.94
10.07
0.860
0.853
17
16
0
0
1
0
5
13
36
38
59
49
B-08-210
57.8
10.12
0.872
23
0
1
6
38
56
B-09-0
B-09-120
20.0*
20.0*
10.16
10.31
0.847
0.854
18
16
0
0
0
1
6
8
48
42
46
50
B-09-210
84.1
10.16
0.833
8
44
65.58 ± 8.00
9.98 ± 0.04
0.854 ± 0.002
20.2 ± 1.02
1
0.615 ±
0.222
12
Mean ± S.E.
0
0.077 ±
0.053
10 ± 0.92
35.9 ± 1.13
44
54.1 ±
1.46
*denotes plasma cortisol readings below the 40 ng/ml detection limit and assigned a value of 20 ng/ml.
Table 4.2. Plasma cortisol, erythrocyte, and leukocyte data from 1 control fish and 4 group B fish from Old Woman
Creek.
4
Toussaint River
Old Woman Creek
Black River
N
20
12
13
mean ± S.E.
125.4 ± 8.7
69.7 ± 10.3*
42.8 ± 9.3*
*Values are significantly lower than those from the Toussaint River (p <0.05). There was no
significant difference between the Black River and Old Woman Creek.
Table 4.3. Mean cortisol levels (ng/ml) for brown bullheads from the Toussaint
River, Old Woman Creek, and the Black River collected in May 1995. Data from
Stasiak (1995).
88
CHAPTER 5
GENETIC DAMAGE IN ERYTHROCYTES OF BROWN BULLHEADS
FROM LAKE ERIE TRIBUTARIES AND CAPE COD PONDS
INTRODUCTION
Unlike mammalian red blood cells, fish erythrocytes contain nuclei (and
therefore DNA). It is relatively easy to obtain large DNA samples from very small
amounts of fish blood. Since many environmental contaminants are known
mutagens, an index that measures genetic damage could be a useful biomarker.
Furthermore, genetic damage indicators are direct measurements of the effects
of contaminants, and would not be affected by sampling methods or handling
stress.
Micronuclei are small abnormalities of the erythrocyte nucleus, usually
caused by chromosomal damage. They are visible as either a protuberance from
the nucleus itself or as a separate piece of genetic material within the cytoplasm.
In fish, they are likely formed in the erythropoietic organs, especially the head
kidney. When stained with acridine orange, micronuclei are identified as brightyellow fluorescing, circular bodies, occupying 1/20 to 1/3 the cell size. Acridine
89
orange also differentiates polychromatic erythrocytes (PCEs) from
normochromatic erythrocytes (NCEs) due to differences in the amount of RNA in
the cytoplasm. With acridine orange stain, the cytoplasm of PCEs (immature red
blood cells) fluoresce a bright orange-red, compared to the darker, sometimes
khaki-green fluorescence of NCEs (mature red blood cells) (Figure 5.1). Hose et
al. (1987) found an elevated frequency of erythrocyte micronuclei in two marine
fish species from contaminated sites. Schultz et al. (1993) described an
increased frequency of erythrocyte micronuclei due to X-ray radiation. Similarly,
Metcalfe (1988) induced erythrocyte micronuclei formation in brown bullhead by
injecting fish with benz[a]pyrene.
Another sensitive indicator of genetic damage is the comet assay. In this
analysis, lysed blood cells are placed in an electrophoresis buffer and the DNA is
allowed to unwind. The DNA then migrates under electrophoresis, and the cells
are scored under a microscope. In the cells with genetic damage, DNA
fragments are visible as long tails migrating from the center of the nuclear mass,
hence the name “comet assay” (Figure 5.2). Comet assays are widely used in
fish laboratory studies (Mitchelmore & Chipman 1998; Nacci et al. 1996; Anitha
et al. 2000; Sastre et al. 2001). However, there is a paucity of studies that have
investigated the use of the comet assay in the field (Devaux et al. 1998;
Pandrangi et al. 1995; Yang 2004).
The purpose of this study is to compare the genetic damage in the
circulating erythrocytes of brown bullheads (Amieurus nebulosus) from paired
90
contaminated and reference sites, and to investigate the use of genetic damage
assays as indicators of contaminant exposure. The paired Ohio sites are the
Cuyahoga River in Cleveland (contaminated) and Old Woman Creek in Huron
(reference) (Figure 5.3). The paired Massachusetts sites are Ashumet Pond
near Mashpee on Cape Cod (contaminated) and Great Herring Pond on the
mainland near Cedarville (reference) (Figure 5.4).
METHODS
Field Procedures
Brown bullheads were captured using standard fyke nets from Old Woman
Creek (Ohio) and Cuyahoga River harbor (Ohio) in May and June 2002.
Although 10 fish were eventually captured from each site, the first three days of
field effort at Old Woman Creek yielded only four brown bullheads. Thus blood
samples of only four fish from Old Woman Creek were able to be analyzed with
the time-sensitive comet assay (detailed procedure described below). Blood
smears from all twenty fish from these Ohio sites were analyzed with the
micronucleus assay.
Fish were captured by electro-shocking from Great Herring Pond
(Massachusetts) and Ashumet Pond (Massachusetts) in June and July of 2002.
Madden and Houston (1976) reported that electroanesthesia is a useful
alternative to chemical anesthesia, with minimal effects on hematological
parameters. Capture effort produced eleven fish from Great Herring Pond and
91
ten fish from Ashumet Pond, and all twenty-one blood samples were analyzed
with the comet assay.
Brown b ullheads longer than 250mm (about 3 years old or older) were
placed in an aerated tub of river/pond water and transported to shore. Fish were
removed from the tank one at a time and anesthetized in a bucket containing
100mg/L tricaine methylsulfonate (MS-222). Mixed arteriovenous blood was
drawn from the caudal vasculature using the lateral approach described by
Schmitt et al. (1999). A non-heparinized vacutainer tube was used to collect a
small amount of blood (< 2mL). A few drops of blood were used to make two
blood smears. The smears were fixed in absolute methanol for 10 minutes, then
stored for use in the micronucleus assay. Approximately 1 mL of whole blood
was transferred into a small plastic vial. Vials were shipped in ice-containing
coolers (0-4°C) by overnight mail to the United States Environmental Protection
Agency National Exposure Research Laboratory in Cincinnati, Ohio. The comet
assay was performed within 24 hours of delivery.
Comet Assay
The comet assay was performed at the USEPA National Exposure
Research Laboratory, Cincinnati, Ohio, based on the method developed by Singh
et al. (1988) and modified by Tice (1995). Briefly, 3 µl of fish blood were diluted
with 1 ml of cold “mincing” solution (10% dimethyl sulfoxide in Hank’s balanced
salt solution [Invitrogen, Carlsbad, CA, USA] with 5mM disodium ethylene-
92
diaminetetra-acetate [EDTA]). Ten microliters (in duplicate) were mixed with 75
µL of 0.5% low melting point agarose at 37°C and pipetted onto a microscope
slide precoated with a layer of 1.2% normal-melting agarose. After solidification
at 4°C, a third layer of 75µL of 0.5% low melting point agarose (75 uL LMPA) was
added to the slide. Slides were then placed in ice-cold, freshly made lysing
solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, 10%
dimethylsulfoxide, pH 10) and refrigerated at 4°C in the dark for a minimum of 1
hour. After lysing, the slides were drained and placed on a horizontal
electrophoresis tray with freshly made alkaline buffer (300 mM NaOH, 1 mM
EDTA, pH >13) for 15 minutes at room temperature (21°C) to allow the DNA to
unwind. Electrophoresis was carried out in the same buffer for 10 minutes at 25
V. After electrophoresis, the slides were neutralized by three 5-minute washes in
a neutralizing solution (0.4 M Tris, pH 7.5), then fixed by immersion in cold
methanol for 5 minutes, air-dried, and stored at room temperature.
After staining with ethidium bromide (50 µl of a 2 µg/ml solution), slides
were coded and scored using a fluorescence microscope (BH-2; Olympus
Optical) under 400X magnification. Fifty cells per slide and a total of 100 cells
per fish were analyzed using image analysis software (Komet 4.0, Kinetic
Imaging), which recorded the length of DNA migration (tail length), percentage of
DNA in tail (% migrated DNA), tail extent moment (tail length × % DNA in tail /
100), and Olive tail moment ([tail mean – head mean] × % DNA in tail / 100).
These last two parameters are measures of the extent of DNA migration (Olive et
al. 1990).
93
Micronucleus Assay
The micronucleus assay was also performed at the USEPA National
Exposure Research Laboratory, Cincinnati, Ohio, based on the method
described by Meier et al. (1999). One of the two blood smears prepared for each
fish was stained in acridine orange according to the method described by Tinwell
and Ashby (1989). The unstained smear was saved as a backup. Slides were
analyzed at 400 X magnification using an Olympus BH-2 microscope with
fluorescence attachment and blue excitation tube (excitation range of 435490nm). Approximately 2000 total erythrocytes were scored as normochromatic
(NCE), polychromatic (PCE), micronucleus -containing normochromatic
(MNNCE), and micronucleus-containing polychromatic (MNPCE).
Statistical Analyses
S-PLUS statistical analysis software was used for data analysis. For the
micronucleus assay, the two-sample t-test was performed to determine if the
frequency of polychromatic erythrocytes was significantly different between
Cuyahoga River bullheads and Old Woman Creek bullheads. The Wilcoxon
rank-sum test was performed to test for significant differences between sites for
the frequencies of MNNCEs, MNPCEs, and total MNs (which were not normally
distributed and included several zeroes). For the comet assay, the small sample
size at Old Woman Creek (N=4) required the use of the Wilcoxon rank-sum test
to compare the tail length, % tail DNA, tail extent moment, and Olive tail moment
94
of erythrocytes of fish from the Ohio sites, Cuyahoga River and Old Woman
Creek. The two-sample t-test was used to compare the tail length, % tail DNA,
tail extent moment, and Olive tail moment of erythrocytes of fish from the
Massachusetts sites, Ashumet Pond and Great Herring Pond.
RESULTS
For the micronucleus assay, most of the slides were scored by the author.
However, due to travel limitations, a few slides were scored by a laboratory
assistant at the USEPA Exposure Research Laboratory. The data for four of the
slides scored by this individual were unusual and of questionable validity, and
were therefore discarded. Thus the data analysis contained eight fish from the
Cuyahoga River and eight fish from Old Woman Creek. Brown Bullheads from
the contaminated Cuyahoga River had a significantly higher frequency of
polychromatic (immature) erythrocytes than brown bullheads from the Ohio
reference site, Old Woman Creek (two -sample t-test, p<0.03)(Table 5.2). No
erythrocytes from fish from Old Woman Creek contained micronuclei, while
0.43 ‰ of the erythrocytes from eight Cuyahoga River fish contained micronuclei
(Table 5.2). One half of the Cuyahoga River fish had at least one micronucleus containing erythrocyte, and three of those had two micronucleus-containing
erythrocytes.
For the comet assay, erythrocytes from brown bullhead from the
Cuyahoga River showed significantly more genetic damage (using all four
95
parameters) than erythrocytes from Old Woman Creek fish (Wilcoxon rank-sum
test, p<0.03) (Table 5.3). Similarly, erythrocytes in brown bullheads from the
contaminated Ashumet Pond showed significantly more genetic damage (using
all four parameters) than erythrocytes from fish from the Massachusetts
reference site, Great Herring Pond (two -sample t-test, p<0.03) (Table 5.4).
DISCUSSION
Polychromatic Erythrocyte Frequency
One benefit of the micronucleus assay is that is provides not only a
measure of genetic damage, but also a measure of the frequency of immature
erythrocytes in circulation. In this study 21.19% of the circulating erythrocytes of
brown bullhead from the Cuyahoga River were polychromatic, or immature. This
compares favorably to the results in chapter two of this dissertation, where
erythrocyte morphology was used to classify 25.0% of the circulating
erythrocytes of Cuyahoga River fish as immature (Figure 2.7). Similarly, this
study showed that Old Woman Creek fish had 12.90% immature erythrocytes in
circulation, while the erythrocyte morphology method (chapter two) showed an
immature erythrocyte frequency of 14.2% (Figure 2.7). These studies sampled
brown bullhead populations in different years (morphology method in 1999, PCE
method in 2002). However, no known changes occurred in point sources or
remedial actions during this time period for either system. Therefore it is safe to
assume that the levels of sediment contaminants did not change significantly
96
over these three years, and that these populations were responding to essentially
the same cocktail of anthropogenic pollutants during both studies. The similar
results for frequency of immature erythrocytes lend credibility to both procedures.
Most fish studies have utilized cell and nuclear morphology measurements
to determine red cell maturity. This research demonstrates several benefits of
using the PCE method with acridine orange over the commonly used morphology
method. First, the morphology technique provides data for at least eight
variables (major axis, minor axis, area, and shape factor for both cell and
nucleus). This requires a more in-depth data analysis, such as the proportional
odds model described in Chapter 2. The PCE method provides data for only two
variables, PCEs and NCEs, which can be analyzed with a simple t-test. Second,
the PCE method is a better monitor of intracellular conditions, since it selectively
stains RNA in the cytoplasm. In vertebrates, translation only occurs in immature
erythrocytes. A cell that stains positive for cytoplasmic RNA should certainly be
classified as immature, regardless of size and shape (which, although rare, can
be influenced by blood sample storage conditions and slide preparation).
Additionally, the PCE method does not require a significantly greater amount of
training or equipment than the morphology method. For these reasons the PCE
method with acridine orange appears to be a more effective measurement of
erythrocyte maturity, which, as stated in chapter two, could be a suitable indicator
of contaminant exposure in feral brown bullhead populations .
97
Micronucleus Assay
Many studies on various animal species have investigated the use of the
micronuclei assay to detect genetic damage. Meier et al. (1999) observed an
increase in micronucleus frequency in mice (Peromyscus leucopus) and shrews
(Cryptotis parva) exposed to methyl methanesulfonate and 4-nitroquinoline 1oxide. A similar laboratory study on a different mouse species (Mus domesticus)
exposed to various mutagens, including benzo[a]pyrene, reported comparable
results (Vrzoc & Petras, 1997). A range of micronuclei studies have also focused
on fish. Schultz et al. (1993) subjected rainbow trout (Oncorhynchus mykiss) to
radiation, and observed an increased micronuclei frequency. Nepomuceno et al.
(1997) exposed carp (Cyprinus carpio) to mercury, which increased the
micronuclei frequency. In a field study, Bombail et al. (2001) reported a higher
frequency of micronuclei in butterfish (Pholis gunnellus) from contaminated areas
in Scotland.
While erythrocytes of Old Woman Creek brown bullheads in this study had
zero micronuclei, the frequency of micronuclei in Cuyahoga River fish (0.43 ‰)
was still very low. Xuan Yang of our research group reported similar findings in
brown bullheads from the Ashtabula River, another tributary of Lake Erie in
northeast Ohio (Yang 2004). She sampled 24 fish and found that only 0.17 ‰ of
erythrocytes contained micronuclei. Because of this low frequency (with high
standard error), and because her data were not correlated with external lesions
98
or barbel deformities, she suggested that the micronucleus assay may not be a
suitable biomarker of contaminant exposure in brown bullhead populations.
Carrasco et al. (1990) also found no correlations between micronuclei
frequency in wild white croaker (Genyonemus lineatus) and levels of chemical
contamination. Yet Hose et al. (1987) found that micronuclei frequencies from
contaminated sites were four times higher in the same species of white croaker
(Genyonemus lineatus), and eleven times higher in kelp bass (Paralabrax
clathratus).
While Smith (1990) reported no significant differences in the frequency of
micronuclei in Great Lakes brown bullheads (Amieurus nebulosus) between a
contaminated site and reference site, other studies on this species describe
discordant results. Metcalfe (1988) reported an elevated frequency of
erythrocyte micronuclei in brown bullheads injected with benz[a]pyrene. Two
studies (Rao et al. 1997; Arcand-Hoy & Metcalfe 2000) observed higher
frequencies of hepatic micronuclei in brown bullheads from contaminated sites.
The latter of these studies compared fish from the contaminated Black River with
fish from the reference site used in this study, Old Woman Creek.
More research is needed to determine the efficacy of the micronucleus
assay as a biomarker of contaminant exposure in brown bullheads, including field
studies with large sample sizes. It is possible that micronucleated cells are being
removed from fish circulation by the spleen (Smith 1990). This has been
observed in other species (rats and humans) and would weaken the
99
effectiveness of the micronucleus assay (Meier et al. 1999). Smith (1990)
suggests that because many carcinogens and mutagens affect the liver but not
the erythropoietic organ (head kidney), they are not likely to induce micronuclei in
erythrocytes. Laboratory studies that expose fish to specific contaminants and
analyze changes in blood, spleen, and erythropoietic tissues would provide
valuable information for environmental scientists and government agencies.
Comet Assay
Collins (2004) suggests that percent tail DNA is the best comet assay
parameter, and that tail length has some limitations. This study showed
significant differences between sites for both percent tail DNA and tail length.
Tail extent moment and Olive tail moment are essentially redundant, since both
are measures of the extent of DNA migration (both parameters are calculated
using tail length and percent tail DNA). They are both reported here for
comparison to previous studies.
Many laboratory studies have utilized the comet assay to detect genetic
damage in aquatic organisms exposed to genotoxic contaminants. Sastre et al.
(2001) observed genetic damage in marine unicellular flagellates. Nacci et al.
(1996) reported DNA damage in flounder (Pleuronectes americanus) and oysters
(Crassostrea virginica) exposed to genotoxic chemicals. The comet assay was
also able to detect significant increases in DNA damage in erythrocytes and liver
cells of carp (Cyprinus carpio) exposed to genotoxic textile dye effluent (Sumathi
100
et al. 2001). Mitchelmore and Chipman (1998) exposed brown trout (Salmo
trutta) to sub-cytotoxic concentrations of several types of genotoxic
contaminants. The direct genotoxic agents produced a significant concentrationdependent increase in % tail DNA in both hepatocytes and blood cells. The
genotoxic agents that required metabolic activation, such as benzo[a]pyrene,
produced a significant concentration-dependent increase of hepatocytes but not
blood cells.
The results of this field study show that brown bullheads from the
Cuyahoga River and Ashumet Pond had greater DNA damage than brown
bullheads from their respective reference sites, Old Woman Creek and Great
Herring Pond. Similar results were described by Xuan Yang of our research
group, who used the comet assay and reported that brown bullheads from the
industrially contaminated Ashtabula River had greater DNA damage that brown
bullheads from the relatively clean Conneaut River. The data from all six of
these sites were analyzed for a publication in Environmental Toxicology and
Chemistry (Yang et al. 2006). Percent tail DNA was highest in fish from the
Ashtabula River (25%), lower in fish from Ashumet Pond (19.22%), and lowest in
fish from the Cuyahoga River (17.83%). Conneaut River fish had the lowest
percent tail DNA (9.0%).
Yang (2004) also investigated the relationships between fish sex and size
with the four comet assay parameters, and reported no significant correlations.
She did, however, find that relatively high DNA damage in fish from the
101
Ashtabula River was associated with relatively higher incidence of raised lesions
(grossly visible tumors) and barbel deformities.
The few field studies that have used the comet assay in feral fish
populations support the findings of this research. Pandrangi et al. (1995) found
that bullheads from contaminated sites had significantly greater DNA damage
than bullheads from reference sites. DNA damage in Chub, Leuciscus cephalus,
was higher at contaminated sites relative to cleaner reference sites (Devaux et
al. 1998). These previous studies, along with the results of this study, support
the suitability of the comet assay as an indicator of genotoxic contaminant
exposure for field studies of brown bullhead populations. The methods and
results of this field study have served as the basis for other research projects on
wild fish populations. Scientists from the United States Geological Survey
applied these methods in a study on white suckers, Catostomus commersoni,
from the contaminated Charles River near Boston, Massachusetts (results
pending). U.S. Environmental Protection Agency researchers are using these
methods for the ongoing sampling of brown bullheads from the Ashtabula River.
Since the Ashtabula is currently undergoing remedial dredging, this study will
provide genetic damage data from fish before and after exposure to resuspended
sediment contaminants. Lastly, the Ohio Environmental Protection Agency,
along with the USEPA, will be using the methods described in this stud y on white
suckers from the Little Scioto River in a project scheduled for this summer (July
102
2007). These researchers will be comparing DNA damage to bile metabolites
and other IBI (index of biological integrity) endpoints.
103
References
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evaluation of heat shock in gold fish (Carassius auratus). Mutation Research /
Genetic Toxicology and Environmental Mutagenesis 469: 1-8.
Arcand-Hoy, L.D. and Metcalfe, C.D. (2000). Hepatic micronuclei in brown
bullheads (Ameiurus nebulosus) as a biomarker for exposure to genotoxic
chemicals. Journal of Great Lakes Research 26: 408-415.
Bombail, V., Aw, D., Gordon, E., and Batty, J. (2001). Application of the comet
and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the
Firth of Forth, Scotland. Chemosphere 44: 383-392.
Carrasco, K.R., Tilbury, K.L., and Myers, M.S. (1990). Assessment of the piscine
micronucleus test as an in situ biological indicator pf chemical contaminant
effects. Can. Journal Fish. Aquat. Sci. 47: 2123-2136.
Devaux, A., Flammarion, P., Bernardon, V., Garric, J., and Monod, G. (1998).
Monitoring of the chemical pollution of the River Rhone through measurement of
DNA damage and cytochrome P4501A induction in chub (Leuciscus cephalus).
Marine Environmental Research 46: 257-262.
Hose, J.E., Cross, J.N., Smith, S.G., and Diehl, D. (1987). Elevated circulating
erythrocyte micronuclei in fishes from contaminated sites off Southern California.
Marine Environmental Research. 22: 167-176.
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104
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gel (comet) assay and genotoxicity monitoring using bullheads and carp.
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105
Yang, X. (2004). Use of fish biomarkers to assess the contaminant exposure
and effects in Lake Erie tributaries. Ph.D. dissertation, The Ohio State
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106
Figure 5.1 . Fluoresced fish erythrocytes stained with acridine orange. Cells with
orange-red cytoplasm are polychromatic erythrocytes (PCEs), and cells with
barely visible cytoplasm are normochromatic erythrocytes (NCEs). The PCE in
the center contains a green-yellow micronucleus.
107
Figure 5.2. Lysed brown bullhead erythrocytes showing electrophoresis-induced
migration of damaged DNA for use in the comet assay.
108
Figure 5.3. Map locations of the Ohio sampling sites, Old Woman Creek and
Cuyahoga River harbor.
109
Figure 5.4. Map locations of the Massachusetts sampling sites, Great Herring
Pond and Ashumet Pond.
110
Site
N
PCEs
(mean ± SE)
NCEs
(mean ± SE)
total Es
(mean ± SE)
Cuyahoga River
8
429.8 ± 53.73 1600.5 ± 58.12 2031 ± 9.82
21.19 ± 2.67
a
Old Woman Creek
8
262.5 ± 38.81 1777 ± 42.85
12.90 ± 1.92
b
2039 ± 7.23
%PCEs
(mean ± SE)
Means with the same letter are not significantly different (two-sample t-test, p < 0.03).
Table 5.1. Number of polychromatic erythrocytes (PCEs), normochromatic
erythrocytes (NCEs), total (both types) erythrocytes (total Es), and frequency of
polychromatic erythrocytes in brown bullheads from the Cuyahoga River and Old
Woman Creek (Ohio).
111
Site
N
MNPCEs (‰)
(mean ± SE)
Cuyahoga River
8
0.62 ± 0.44
Old Woman Creek
8
0±0
a
MNNCEs (‰)
(mean ± SE)
0.40 ± 0.17
b
0±0
b
a
MN (‰)
(mean ± SE)
0.43 ± 0.17
0±0
a
b
Means with the same letter are not significantly different (Wilcox rank-sum
test, p < 0.05).
Table 5.2. Frequencies of micronuclei in polychromatic erythrocytes (MNPCEs),
normochromatic erythrocytes (MNNCEs), and total (both types) erythrocytes
(MN) of brown bullheads from the Cuyahoga River and Old Woman Creek
(Ohio).
112
Site
Cuyahoga River
Old Women Creek
N
Tail Length
(µm)
(mean ± SE)
10
42.90 ± 3.02
4
36.94 ± 3.25
Tail DNA (%)
(mean ± SE)
a
17.83 ± 1.70
b
11.51 ± 1.47
Tail Extent
Moment (µm)
(mean ± SE)
a
8.29 ± 1.06
b
5.00 ± 0.85
Olive Tail
Moment (µm)
(mean ± SE)
a
2.58 ± 0.21
a
b
1.80 ± 0.19
Means with the same letter are not significantly different (Wilcoxon rank -sum test, p < 0.03).
Table 5.3. Measurements of DNA damage in erythrocytes of brown bullheads
from the Cuyahoga River and Old Woman Creek (Ohio).
113
b
N
Tail Length
(µm)
(mean ± SE)
Ashumet Pond
10
46.95 ± 3.11
Great Herring Pond
11
40.89 ± 2.90
Site
Tail DNA (%)
(mean ± SE)
a
19.22 ± 1.90
b
15.28 ± 1.64
Tail Extent
Moment (µm)
(mean ± SE)
a
10.27 ± 1.34
b
7.06 ± 1.03
Olive Tail
Moment (µm)
(mean ± SE)
a
3.04 ± 0.23
a
b
2.14 ± 0.18
Means with the same letter are not significantly different (two-sample t-test, p < 0.03).
Table 5.4. Measurements of DNA damage in erythrocytes of brown bullheads
from Ashumet Pond and Great Herring Pond (Massachusetts).
114
b
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