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Fluorescence
Based Detection of Chemically Induced DNA Damage
Kumaran Ramanathan, US EPA, Las Vegas, NV
Kim R. Rogers, US-EPA, Las Vegas, NV
Interpretation
of Vitellogenin Gene Expression and Protein Production as
Predictors of Exposures to Xenoestrogens and Potential
Adverse Reproductive Effects in Fish
Patricia Cline, Golder Associates, Gainsville, FL
Biosensors
and Bioanalytical Microsystems for the Rapid and Specific
Detection of Pathogenic Organisms
Antje
J. Baeumner, Cornell
University, Ithaca, NY
An
Overview of Ecological Indicators: Putting Biomarkers and
Bioindicators in Context
James R. Bernard, Environmental Management Consulting,
Harpswell, ME
Residue-effect
Relationships as Indicators of Ecological Stress
Ed Zillioux, FPL
Environmental Services, Juno Beach, FL
John H. Gentile, Brewster, MA
Sediment
Characterization Data as Indicators for Dredged Material
Disposal Suitability
Ann B. Shortelle, Harding ESE, Gainsville, FL
Glenn Schuster, USACE – Jacksonville District,
Jacksonville FL
Tom
Park, PPB Laboratories, Gainesville FL.
Biomonitoring
Environmental Contaminants Near a Municipal Solid-Waster
Combuster: A Decade Later
Darren
G. Rumbold, South
Florida Water Management District, West Palm Beach, FL
Mary
Beth Mihalik, Solid Waste Authority Palm Beach County,
West Palm Beach Fl
Museum
Specimens as Historic Biomarkers and their Utility in the
Interpretation of Current Site Contamination
Jim Newman, Pandion Systems,
Gainesville, FL
Ed Zillioux, Florida Power & Light Co., Juno Beach, FL
Fluorescence
Based Detection of Chemically Induced DNA Damage
Kumaran
Ramanathan, NRC Associate, US-EPA, 944 E. Harmon Ave,
Las Vegas, NV 89119, Tel:
702-798-2663, Fax: 702-798-2107
Kim R. Rogers, Scientist, Branch Chief, US-EPA/HERB,
944 E. Harmon Ave, Las Vegas, NV 89119, Tel:
702-798-2299, Fax: 702-798-2107
Chemically
induced DNA damage for double stranded (ds) DNA is a topic
of continuing interest. In this report we demonstrate a
rapid fluorescence based multi-analytical approach for
detection and measurement of DNA damage. Water soluble and
insoluble chemical molecules induced the damage. These
included styrene oxide, styrene sulfonate, chlorophyllin,
glutaraldehyde, pyrene, benzo[a]pyrene, 4-nitroquinoline
N-oxide, 1,3-butadiene diepoxide and griseofulvin.
Identical concentration of calf thymus or pUC19 plasmid
dsDNA (100 ng/mL) was challenged with equimolar
concentrations (1 mM) of each of these compounds. The
consequent damage on the dsDNA was monitored
simultaneously (at least for 32 samples) by a dye (PicoGreen)
binding assay. The emission from the DNA-PG complex was
monitored at 530 nm. The induced DNA damage was also
enhanced by repeated melting/annealing cycles (5 to 10
cycles) within a pre-defined temperature region (30oC
to 95 oC) using a lightcycler instrument. The
results demonstrate the ease of performing such screening
assays, not limited to the above compounds, in microliter
(10 to 20 mL)
volume capillaries with automatic logging of the data
points. In addition to screening potential carcinogens,
mutagens or chemicals for DNA damage, the assay could be
extended for probing the mechanism of damage induction.
The assay is sensitive to 1 ng/mL DNA concentration with a
precision of 2-5% (CV%).
Interpretation
of Vitellogenin Gene Expression and Protein Production as
Predictors of Exposures to Xenoestrogens and Potential
Adverse Reproductive Effects in Fish
Pat
Cline,
Golder Associates, 6241 NW 23rd Street, Suite 500,
Gainesville, FL 32653-1500, Tel: 352-336-5600, Email:
pcline@golder.com
Plasma
vitellogenin (Vtg) in male fish is a frequently measured
biomarker to evaluate potential exposure to xenoestrogens
in aquatic systems. Vtg is the egg yolk precursor protein,
produced in the liver in response to estrogen with
concentrations in females fluctuating with spawning and
seasonal cycles as Vtg is produced and deposited into
eggs. The protein is rather long lived; lasting from 2-6
weeks in the plasma of males exposed to estrogenic
compounds and can accumulate with repeated exposures.
Elevated Vtg in males or reduced levels in females may be
considered biomarkers of exposure to estrogenic or
anti-estrogenic compounds respectively.
In
the liver, the Vtg protein is produced after the estrogen
binds to the estrogen receptor, activating Vtg gene
expression. This promotes the synthesis of mRNA, which is
then translated into the protein. The mRNA, which could be
detectable before the protein has accumulated, has a much
shorter half-life (on the order of days). The Vtg mRNA and
protein represent different potential exposure regimes.
The measurement of these biomarkers and their correlation
to adverse impacts on reproduction was evaluated based on
fathead minnow exposures to estrogen and anti-estrogen.
Biosensors
and Bioanalytical Microsystems for the Rapid and Specific
Detection of Pathogenic Organisms
Antje
J. Baeumner, Assistant Professor of Biotechnology,
Dept. of Biological & Environmental Engineering, 318
Riley-Robb Hall, Cornell
University, Ithaca, NY 14853, Tel: 607-255-5433, Fax:
607-255-4080
Research
in the Bioanalytical Microsystems and Biosensors Lab at
Cornell University focuses on the development of
biosensors for the detection of pathogens in the
environment, food and for clinical diagnostics. Several
biosensor strategies are being developed, all of them
involve the use of liposomes as instantaneous signal
amplification system.
The
simple single-use, inexpensive and rapid optical
biosensors are designed for field-tests. While they
demonstrate fantastically low detection limits, they also
are extremely easy to use. Signals are generated in less
than 20 minutes with limits of detection in the range of a
femtomol. When combined with Nucleic Acid Sequence Based
Amplification (NASBA) single cell detection is possible.
Biosensors under investigations focus on the detection of Cryptosporidium
parvum, Dengue virus, B. anthracis and E.
coli. They will find their application in routine
drinking water testing, environmental water testing, food
analysis and in clinical diagnostics.
More
sophisticated microfluidic devices are developed, that
will transfer the technology of the simple strip
biosensors into more automated and sophisticated
microchannel designs. The same biological principles are
used, however, novel sample preparation systems, novel
molecular biology amplification chambers, and intricate
hybridization channel patterns are being developed: for
example, a laser-induced cell lysis system that allows
that instantaneous and effective lysis of bacterial, yeast
and mammalian cells.
Electrochemical detection is accomplished using
ultramicroelectrode arrays that allow an even more
sensitive detection. This research aims toward the
creation of an easy-to-use but sophisticated micro-Total
Analysis System.
We
present today the principles and techniques of our
different biosensor systems, the simple optical biosensors
and the more complex microfluidic-based sensors. We
demonstrate the application of the optical biosensors for
the detection of pathogens in real-world samples and
provide an out-look regarding the impact of microfluidic-based
biosensing systems on the detection of pathogens in food,
the environment and for medical diagnostics.
An
Overview of Ecological Indicators:
Putting Biomarkers and Bioindicators in Context
James
R. Bernard, Environmental Management
Consulting, 36 Cundy’s Point Road, Harpswell, Maine
04079-4607, Tel: 207-729-3169, Fax: 207-729-3169,
Email: jbernard@clinic.net
Often
management objectives are stated in broad terms such as
“sustaining environmental quality” or “maintaining
historical biological integrity.”
These broad statements need to be more explicitly
defined so that they can provide a clear focus for
ecological risk assessment activities. During the problem
formulation stage of risk assessment, risk assessors and
managers determine what is to be protected, establish what
information is needed, and plan the assessment, including
the selection of indicators.
Specifically, each of the ecological resources of
concern needs to be identified (e.g., salmon) and the
specific attribute that is of interest (e.g., numbers in a
spawning run in a particular stream) should be stated.
The combination of resource and attribute is called
an assessment endpoint.
Assessment endpoints, or bioindicators,
are identified based on their relevance to management
objectives, their susceptibility to stressors, and their
ecological relevance.
It
is important to differentiate between bioindicators
and biomarkers in
the context of ecological risk assessment.
Bioindicators are multiple measures of organism
health related to environmental stressors that include
several levels of biological organization and time scales
of response. Multiple measures of health are needed in
order to identify and separate the effects of
human-induced stressors (e.g., contaminants) from the
effects of natural stressors (e.g., food and habitat
availability). Bioindicators
can range from biomolecular/biochemical responses to
population and community-level responses.
Biomarkers
of exposure are biochemical or physiological changes which
indicate that an organism has received an internal dose of
a chemical (Suter, 1993).
Biomarkers can be considered as bioindicators if
they are causally linked to ecologically-relevant
endpoints (McCarty and Munkittrick, 1996).
The fundamental difference is that biomarkers
concentrate on measurement attributes, while bioindicators
require validation in addition to measurement (McCarty, et
al, 2002). Biomarkers
are generally used to indicate exposure of organisms to
contaminants at lower levels of biological organization
while bioindicators are typically used to reflect effects
of stressors on biological systems at higher levels of
organization (ORNL, 2000).
Residue-effect
Relationships as Indicators of Ecological Stress
Edward
J. Zillioux, Florida Power & Light Co.,
Environmental Services Dept., 700
Universe Boulevard, Juno Beach, Florida
33408, Tel: 561-691-7063,
Fax: 561-691-7070
John H. Gentile, 582 Airline Road, Brewster, MA
02631, Tel: 508-385-4572,
Fax: 508-385-6888
Environmental
concentrations of contaminants are of increasing
ecological concern whether these concentrations are at
"background" levels or significantly elevated.
However, toxicological and regulatory endpoints are more
often set in accordance with human health concerns and
their corresponding endpoints. Monitoring approaches and
regulatory compliance typically focus on ambient media
although biomonitoring becomes the method of choice when
the contaminant is strongly bioaccumulative but present in
the surrounding media only near levels of detection. This
is the case for mercury where fish monitoring has formed
the basis of human health consumption advisories in most
of the United States and elsewhere.
Zillioux et al. (1993) evaluated existing data on
mercury residues and related residue levels with specific
effects of ecological significance. Since then,
considerable additional work has accumulated on mercury
residue-effect relationships using a variety of species.
This paper presents a review of this expanded
database of environmental exposures to mercury.
In addition, it will provide an overview of a wider
range of residue-effect relationships that have proven
useful in ecological assessment. These include setting of
national and international benchmarks for persistent
organochlorine contaminants (e.g., PCBs, PAHs, log P
>3., etc.) in fresh and marine waters as well as the
use of residue-effects relationships in the management of
wildlife risks at hazardous waste and superfund sites and
others. Residue-effects
relationships are particularly important benchmarks for
managing top predators, often threatened and/or endangered
species whose primary route of exposure is through
ingestion of contaminated prey and whose ambient
concentrations are often below routine analytical
detection limits. Most importantly, the residue-effect relationship integrates
exposure and effects over both time and space which, for
wide ranging, valued ecological populations, would
otherwise be impossible to measure.
Sediment
Characterization Data as Indicators for Dredged Material
Disposal Suitability
Ann
B. Shortelle, Harding ESE, Gainesville, FL
Glenn Schuster, USACE – Jacksonville District,
Jacksonville FL
Tom Park, PPB Laboratories, Gainesville, FL
Evaluations
under Section 103 of the Marine Protection, Research, and
Sanctuaries Act of 1972 consider potential dredged
material proposed for disposal in Ocean Dredged Material
Disposal Sites for maintenance dredging projects.
The determination of suitability for ocean disposal
is based upon physical, chemical, and biological
evaluations. Currently,
a tiered testing procedure is used for evaluating the
potential dredged material.
After completion of laboratory analyses and
toxicity tests, the concentration of allowable dredged
material in the water column can be calculated that will
not cause unreasonable toxicity or bioaccumulation. Our study compares available data for sediments from several
Florida and Caribbean sites to sediment screening criteria
or concentrations that may predict adverse environmental
effects. These data are also compared to actual observed toxicity in
bulk sediment and elutriate testing for the same samples. Results support the tiered testing procedure that allows for
site-specific testing and interpretation.
These analyses result in significantly fewer toxic
responses than suggested by simple but conservative
screening values. These
site-specific data provide direct estimates of the
potential for environmental impact rather than relying
upon potential adverse impacts inferred from other
studies. Thus,
site-specific data enable ocean disposal of dredged
materials with significantly more confidence that adverse
impacts will not occur.
In addition, due to the conservative nature of
screening criteria, more sites can be permitted for ocean
disposal with site-specific data in hand.
Biomonitoring
Environmental Contaminants Near a Municipal Solid-Waste
Combustor:
A Decade Later
Darren
G. Rumbold, South Florida Water Management District,
Mail code 4642, 3301 Gun Club Road, West Palm Beach, FL
33406, Tel: 561 682-2132, Fax: 561 687-6442
Mary Beth Mihalik, Solid Waste Authority Palm Beach
County, 7501 North Jog Road, West Palm Beach Fl 33412,
Tel: 561-640-4000, Fax: 561-683-4067
During
the 1999 nesting season, eggs and nestlings of anhingas
(Anhinga anhinga)
and white ibises (Eudocimus
albus) were collected from a colony located on
the site of a municipal
solid-waste (MSW) combustor and analyzed for residues of chemicals potentially released from this facility.
Concentrations of most residues, including tetrachlorodibenzo-p-dioxin (TCDD), tetrachlorodibenzofuran (TCDF),
arsenic, beryllium, cadmium and lead, were at levels
comparable to those found during a similar survey done in
1989, prior to facility start-up. Further, results from a
recombinant cell line bioassay system (CALUXTM)
found residues of all dioxin-like chemicals to be at
background concentrations in birds collected in 1999.
Nickel residues were detected only sporadically in
anhingas and in eggs from ibises, and decreased
significantly in concentration in ibis nestlings in 1999
compared to 1989. While
concentrations of mercury in anhinga nestlings and in eggs
of both species were comparable to 1989 levels, its
concentration was significantly greater in ibis nestlings
in 1999. However,
levels of mercury in the ibises remained relatively low
when compared to birds from other areas of Florida and did
not appear to represent a health threat.
While lead did not increase in 1999, its
concentration in ibis nestlings remained a concern. The most notable temporal trend observed in birds at this
site was a general monotonic decrease in levels of
selenium residues during the ten-year monitoring period.
Museum
Specimens as Historic Biomarkers and their Utility in the
Interpretation of Current Site Contamination
Jim
Newman,
Pandion Systems, 5200 NW 43rd Street, Suite 102-314,
Gainesville, FL 32606-4482
Ed Zillioux, Florida Power & Light Co., 700
Universe Blvd., Juno Beach, FL 33408, Tel: 561-691-7063
Understanding
the historical source of contamination is often necessary
in determining the source and cause of biological
contamination as well as for evaluating the trends in
contamination over time.
Archived museum tissues have frequently been used
to as a biomarkers to determine source and trends of
contamination. For
example, hair and feathers have been used as biomarkers
for mercury, lead, arsenic and other heavy metals.
Although museum tissues can be useful as historical
biomarkers care must be taken in interpreting the results.
There are a number of factors such as the
preservation techniques of tissues, the representativeness
of species populations in a museum as well as biological
characteristics of the species than can affect the
results. This
paper examines the use of museum tissues as historical
biomarkers of contamination and provides recommendations
on their use as biomarkers.
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