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Project Title
Altered Gene Expression in Marine Organisms.
Executive Summary
1. National and international commitments to monitoring programmes on the
state of the marine environment require ever more sophisticated tools to
detect cause and effect.
2. Biological measurements are an increasingly important part of monitoring
and biomarkers provide evidence for exposure and indicate risk for adverse
effects at individual and population level.
3. Historically, biomarker assays have been limited to species and class of
hazardous chemical and this presents difficulties of interpretation in
environmental situation where there are likely to be multiple classes of
chemical input.
4. There is a need for assays that are independent of species and type of
chemical and techniques based on molecular biology offer to provide this.
The project aims were thus to develop a species-independent biologicaleffects monitoring tool which is diagnostic of exposure to a range of
contaminants and to test this tool against a range of organisms and
contaminants with controls.
5. A variety of approaches are available including DDRT-PCR and various
array-based media. At the outset of the study Differential-Display, ReverseTranscriptase, Polymerase Chain Reaction (DDRT-PCR) was the chosen
method because of its potential for use in a general laboratory environment.
6. DDRT-PCR uses RNA from exposed and non-exposed animals,
experimental or environmental (test site cf reference site) and converts these
to cDNA libraries. These are then amplified with random sequence,
oligonucleotide primers and the products then resolved by denaturing
polyacrylamide, gel electrophoresis. Comparison of exposed/non-exposed
for the resulting banding patterns identify products representing genes with
altered expression because of up- or down-regulation.
7. A standard operating protocol was developed after investigation of a variety
of parameters (RNA isolation, cDNA synthesis conditions and enzyme,
template concentration, PCR parameters and type of enzyme used,
electrophoresis conditions, staining conditions). The standard procedure
used a one-base pair anchored oligodT primer (3 variants) along with a
10mer random sequence primer (26 variants). It was found that the random
primers alone generated PCR products and we are not aware that this
possibility has been considered previously.
8. DDRT-PCR was used to investigate gene responses to hazardous chemicals
under controlled laboratory conditions and for animals from contaminated
environmental sites. A variety of fish (dab, turbot, plaice, sand goby) and
invertebrate (mussel, hermit crab, shore crab) species were used.
9. DDRT-PCR products typically generated banding patterns with ~30 bands
of differing mobility and intensity. Differential bands were found for most
test comparisons with many combinations of primers. Banding patterns and
differentially expressed bands were specific to animal species, to the
exposure chemical and to the random primers used.
10. Differential displays were produced for experimentally exposed animals as
follows: turbot/ -naphthaflavone (BNF), dab/benzo[a]pyrene (BaP),
plaice/BNF, dab/phenanthrene, sand goby/ethynyl oestradiol (EE2), sand
goby /octylphenol (OP), mussel/BaP, mussel/cadmium, hermit crab/BaP.
For each species/chemical, informative, differential displays were produced
with a wide range of random primers.
11. Problems of inter-individual variation in the displays were identified at an
early stage. This manifested itself by some animals not showing a diagnostic
band seen in a majority of tested individuals and/or the appearance of
additional bands not seen in a majority of others. Tests to identify the origins
of such variability indicated it was largely biological. For work with
experimentally exposed animals, individual variation in response to a
chemical could be detected by assay for a single biomarker such as CYP1A
mRNA and was found to be significant. However, even when animals were
selected for equal response by such single biomarker assay, some interindividual variation was still observed in subsequent DDRT-PCR.
12. DDRT-PCR does not lend itself readily to the generation of dose-response
relationships because of the non-linear nature of the PCR process. However,
the technique is very sensitive and work with a dose-response study of dab
treated with BaP showed differential bands at the lowest treatment dose and
an increase in band intensity in one of the differentially displayed bands.
13. Work with sand goby treated with either EE2 or OP demonstrated that the
method was capable of generating diagnostic changes in banding specific to
either of the two xeno-oestrogens.
14. Differential displays were also generated for invertebrate species from
environmental sites. Shore crab and mussel from Loch Leven, which is
heavily contaminated with PAHs from an aluminium smelter, were
compared to crabs from Loch Ewe or mussel from Loch Etive. Mussel from
the Holy Loch, subject to many contaminants including PAHs and heavy
metals from the disused US navy base, were compared to mussel from Loch
Etive.
15. For each of the species tested altered banding patterns were found in animals
from the contaminated sites compared to the reference sites. However,
interpretation of the data was complicated by inter-individual variation and
diagnostic signals were not found in all test animals.
16. Despite the wealth of information provided by DDRT-PCR it cannot identify
the genes with altered expression revealed by the gel-banding pattern. In
principle it is possible to excise and clone differential bands but this is not
easy and can lead to artefacts. As an alternative an array strategy was tested
and two approaches for cloning of informative genes was used. The arrays
were then tested as monitoring tools.
17. The simplest variant of the array approach was to identify stress-response
genes for which sequence data exists in a sentinel species or species related
to it. For this purpose mussel was chosen since biomarkers for this organism
are limited despite the use of the species as a world-wide sentinel. Using
RT-PCR clones were produced and characterized for genes likely to be
responsive to PAHs, heavy metals, endocrine disruptors and general stress.
The clones were used to construct a macro-array, along with reference and
control nucleic acids and used to assess gene expression in mussel from
Loch Leven and the Holy Loch. Samples from Loch Etive, our reference
site, proved to be degraded making interpretation of the macroarray data
difficult. Despite this clear signals were obtained on the arrays and it
appeared that the metallothionein signal was greater in test samples from
metal-contaminated Holy Loch than the PAH-contaminated Loch Leven. In
contrast and as expected GST expression was higher in test samples from
Loch Leven.
18. The second method of generating arrays was Suppression Subtractive
Hybridisation. This produces cDNA libraries that are greatly enriched for
genes with altered gene expression consequent on exposure to a hazardous
chemical. Individual genes can be isolated from such libraries and identified
by sequence determination and BLAST searches of the databases. The
individual cDNAs can then be arrayed and used for gene expression
profiling with RNA from test and reference animals.
19. SSH was used to investigate the endocrine disruptor, ethynylestradiol in
plaice and forward and reverse libraries were generated. More than 200
differentially expressed clones were isolated and 140 of these were
sequenced. A large number of these encoded vitellogenin (2 genes) and zona
radiata proteins (3 genes) spanning different regions of those genes. Almost
40 of the sequenced clones encoded other proteins including some which are
cryptic and could not be identified by BLAST search.
20. A macro-array with 88 individual plaice cDNAs along with 4 reference
genes and control DNA was constructed. These were used to analyse altered
gene expression in male plaice treated for various time periods with EE2 and
during subsequent depuration after removal of the oestrogen. The expression
profiles of each gene could be clustered into three distinct time-course
patterns based upon a peak expression at 16 days and subsequent decline
(pattern A), a peak at 16 days and decline after withdrawal of oestrogen
(pattern B) or a peak at 21 days and decline after withdrawal of oestrogen
(pattern C). The various VTG clones appeared to co-cluster in that one
group (VTG1) all followed pattern A while another (VTG2) followed
pattern B. Similarly for ZRP all ZRP1 and 3 followed pattern A while ZRP2
followed pattern C. It is speculated that genes following pattern A may be
associated with adverse reproductive effects and thus provide better
predictive biomarkers.
21. This project has developed a SoP for DDRT-PCR that can be used in a
standard laboratory environment, with marine organisms and illustrated the
wealth of information that can be generated with the technique. The
technique is sensitive and as required of the Aims not limited by animal
species or class of hazardous chemical. Problems of inter-individual
variation are biological in origin and thus not different from assessment of
field samples with single biomarker end points.
22. Additionally it has been shown that other current molecular techniques are
capable of generating species- and chemical-independent macro-array
monitoring tools. These have been produced by two methodologies and have
the advantage over DDRT of providing information on identified gene
targets and thus directly point to likely environmental contaminants causing
altered gene expression.
23. DDRT-PCR could be readily adapted to routine monitoring purposes. The
array techniques could be further developed and ideally would be transposed
from macro to micro-array format. All three techniques could be used in
responsive mode for analysis of suitable organisms following environment
incidents.