Case Study: DNA Damage in Plaice (Pleuronectes platessa)
Red Blood Cells from Two Contrasting Scottish Marine Sites
Introduction
Detection of unintended or unacceptable biological
responses to hazardous substances in the aquatic
environment is of increasing importance, as reflected in
the 2003 Oslo and Paris Commission Joint Assessment
and Monitoring Programme and in investigative monitoring
in the context of Water Framework Directive. Scientists at
Fisheries Research Services (FRS) are interested in
detecting damage at a genetic level, to provide information
to assist with ecotoxicological monitoring and
environmental assessment.
DNA: The recipe of life
Deoxyribonucleic acid (DNA), is essentially the recipe of
life, a molecule carrying a set of genetic instructions which
determine how cells grow. DNA consists of two adjoining
strands of DNA, linked in a twisting formation structure
called a double helix (Fig. 1). These strands can be
damaged if cells are exposed to harmful chemicals of
types of radiation including ultraviolet light. If the cell is
unable to repair accurately the damage, the information
contained in the DNA can be disrupted leading to errors
in the genetic recipe.
Figure 2.
Comet assay image. Fragments of broken strands of DNA are
transported by an electric current away from the main mass of
undamaged DNA during gel electrophoresis. When viewed by
microscope, the stained intact DNA and the DNA fragments
resemble the head and diffuse tail of a comet
Pioneering work at FRS
The Comet assay is traditionally a laboratory-based
technique carried out on a level surface. In 2005 FRS took
the method to sea during a research cruise on FRV Scotia
to investigate DNA damage in flat fish (Fig. 3). This posed
particular difficulties due to the movement of the vessel.
A containerised laboratory was fully equipped for the
work. It included dark room lights, microscope, fume
hood and a custom built gimbal mounting to reduce the
effect of ship movement on the electrophoresis
equipment.
Plaice as biological indicators
Plaice (Pleuronectes platessa) is a commercial species of
marine flat fish. Flatfish live on the seabed which brings
them into contact with any chemical pollutants in the
sediment, making them ideal ‘biological indicators’ of
environmental quality.
DNA damage was compared in plaice from two contrasting
marine sites: a control site by the Hebridean island of
Figure 1.
Figure 1. DNA molecule forming the double helix structure of two
entwined strands of DNA linked by base molecules. Image: Peter
Artymiuk / Wellcome Photo Library.
Quantifying DNA strand brake damage
Sometimes, disruption will cause stands of DNA to break,
and it is this ‘strand break’ damage that FRS has been
investigating. Using a technique called single cell gel
electrophoresis, commonly known as the Comet assay,
DNA strand break damage can be visualised (Fig. 2) and
quantified (Fig. 5).
Figure 3 .
Plaice were caught by trawl using FRV Scotia. Plaice live on the
seabed and can come into direct contact with contaminated
sediment.
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen
tel +44 (0)1224 876544 fax +44 (0)1224 295511
[email protected] http://www.frs-scotland.gov.uk
AB11 9DB
UK
Table 1.
Concentrations of contaminants in sediments and fish
from Colonsay and Garroch Head, 1999 to 2002.*
Chemical
Contaminant
Sampling Site
Colonsay
Garroch Head
PAHS*
(µg/kg dry weight
sediment)
20.1 to 137.6
(n=10)
2544 to 16263
(n=10)
ICES7**
chlorobiphenyl
congeners
(µg/kg lipid plaice
livers)
23.3 to 864.3
(n=16)
762 to 2187
(n=17)
*PAHS: 2- to 6-ring parent and branched polycyclic aromatic
hydrocarbons found in crude oil and its derivatives
**ICES7 chlorobiphenyl congeners: a group of seven compounds
representative of this large group of persistent and
bioaccumulative environmental pollutants
Colonsay
Garroch Head
Figure 4.
The two plaice sampling sites. Colonsay has low levels of
pollutants whereas Garroch Head in the Firth of Clyde is impacted
by both historic disposal of waste and current controlled and
accidental releases of contaminants from a variety of industrial,
military and urban sources.
Colonsay and the historic sewage disposal site off Garroch
Head in the Firth of Clyde (Fig. 4). On-going FRS monitoring
has revealed contrasting contaminant concentrations at
these two sites (Table 1).
Comet tail intensity
Plaice were caught by beam trawl and blood collected
and processed at sea. The red blood cells were embedded
Figure 5.
Images analysis of cellular DNA. Image of a normal plaice cell
collected from Colonsay and a damaged plaice cell. Green vertical
line identifies centre of comet head. Red vertical line marks end
of comet tail. Red area second image indicates the damaged
DNA. DNA damage is reported as Percentage Tail Intensity.
on slide gels and after electrophoresis were returned to
the FRS Marine Laboratory, for image analysis (Fig 5). DNA
damage was expressed as percentage Comet Tail Intensity.
A difference was observed in the level of DNA damage at
the two sites. The median tail intensity of pooled data
from Colonsay was 1.1%, while a significantly (p<0.014)
elevated level of damage (3.3%) was found in fish from
the Garroch Head site. Variation between fish within sites
was significant (p<0.001).
Conclusions
Quantification of DNA damaged revealed significantly
elevated levels in plaice red blood cells from the Garroch
Head dump site compared to fish from the less
contaminated waters by the island of Colonsay. These
results correlated with FRS’ on going monitoring of the
sites. Considerable variation in DNA damage was observed
between fish at each sampling site. It is currently unknown
if the observed degree of DNA damage confers any
detrimental effect in the plaice.
FRS has demonstrated that it was possible to prepare
fish cells for measurement of DNA damage by the Comet
assay at sea during fair weather.
AE23|06|06
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen
AB11 9DB
UK
tel +44 (0)1224 876544 fax +44 (0)1224 295511
[email protected] http://www.frs-scotland.gov.uk
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