Biomarkers
Dream or Reality?
Ecotoxicity Testing
• Integral Part of Environmental Risk Assessment
• Plant and Animal Communities Consist of a Huge
Diversity of Species, Which Vary in Their
Sensitivity to Toxicants
• Thus, a Battery of Bioassays That Reflect
Different Trophic Levels and Varied Habitats,
Rather Than Single Species Assays, Should Be
the Backbone of Toxicity Evaluation to
Represent the Range of Sensitivities of Field
Organisms
A schematic bio-reporter
Microtox™ System
• Bioassay Based on the Reduction of Bioluminescent
Light Emitted by the Marine Bacterium Vibrio fischeri
to a Toxic Substance
• Bacterial Test System was Unique in that Test
Organisms Were Hydrated, Freeze Dried
Preparations
• The More Toxic the Sample, the Greater the %
Light Loss From the Test Suspension of
Luminescent Bacteria
Pseudokirchneriella subcapitata (freshwater alga)
Growth Inhibition Test
• Aquatic Plant Toxicity Tests Frequently Conducted to Determine The
Potential Impacts of Toxicants on Primary Producers.
• Phytoplankton often represent the 1st Trophic Level in a Food Chain
•
Classic Assay Using Flasks (Miller et al, 1978)
• Unicellular, Non-motile
• Measure Algal Growth -
Dry Weight, ATP,
Chlorophyll
• Microplate Assays (Arensberg et al, 1995, Blaise et al, 1986)
“Blue Book” – manual of tox.
testing proceedures
• http://www.google.ca/url?sa=t&rct=j&q=&e
src=s&source=web&cd=1&ved=0CDIQFjA
A&url=http%3A%2F%2Fwww.environment
agency.gov.uk%2Fstatic%2Fdocuments%
2FResearch%2Fbluebook219_2060295.p
df&ei=lrbnUqDYFLPisATNmYHABQ&usg=
AFQjCNFsJO995ZQfxm8VhnBVxBK9C1T
PrA&sig2=wU38tvwDJULWlBMWZi_JWA
&bvm=bv.59930103,d.cWc
Allium cepa Growth Inhibition Test
• Allium Test System First Used in 1938
• Plants Easy to Store, Handle, Plentiful and Inexpensive
• Macroscopic And Microscopic Effects May Be Observed
• Root Length Reduction Appears to be the Most Sensitive
Parameter
• Microscopic Evaluation Allows the Genotoxic Assessment
• Recovery Study Has Been Introduced
Artemia Salina Lethality Assay
• 24-h LC50 Bioassay Performed in 24 Multiwell Plate Using
Larvae of the Brine Shrimp Artemia Salina
• Often Used as an Indicator Organism to Determine the
Toxic Effects of a Chemical in Marine Aquatic Systems
• Portability, Miniaturisation, Minimal Training and Equipment
Requirement
Artemia salina, cyst and recently hatched shrimp beside it
L. Lewan, M. Anderrson & P. Morales-Gomez (1992).
"The use of Artemia salina in toxicity testing".
Alternatives to Laboratory Animals 20: 297–301.
Toxkits
• New Generation of Low Cost User-Friendly Microbiotests
• Exist for Freshwater, Marine and Estuarine Environments
• Most Toxkits Contain the Test Animals in the Form of Resting Eggs Eliminates the Need for Continuous Recruitment and Stock Culturing of Test
Organisms
• A Number of Toxkits Incorporating Microalgae, Protozoa and Invertebrates
Native to Different Environments are Available
• Daphtoxkit F™, Algatoxkit F™ , Thamnotoxkit F™ , Artoxkit M™ ,
Rototoxkit (both F and M)
Ceriodaphtoxkit F™ , and Fluotoxkit F™ are Under Development
Thamnotoxkit F
Thamnocephalus
platyurus cysts
Tube with
Thamnocephalus platyurus
cysts
4
HATCHING OF THE CYSTS
Thamnocephalus platyurus
larvae
Example of tox assessment
data
Daphnia magna Immobilisation Assay
• Represent an Important Link in Many Aquatic Food Chains
• Primary Consumers - Widely Used as a Freshwater Toxicity Test Organism
• Sensitive to a Broad Range of Aquatic Contaminants
• Daphnia for Testing are Neonates That are Released from a Mothers
Brooding Chamber During The Preceding 24-h
•
Exposed for 24 and 48-h to Toxicant
• Percentage Immobilisation after 24 and 48-h recorded
Daphnia magna neonate
Assessment of Cytotoxicity as
Determined by Morphological
Alterations and Cytotoxicity Assay
Endpoints
• Use of Fish Cells Began in 1960’s in the Hopes of Finding an Alternative to
Whole Animal Testing
• Good Correlations Between Toxicity on Cell Culture and the in vivo Fish
Acute Toxicity Test Have Been Reported
• Advantages Include Low Cost, Rapidity, Versatility, Small Amounts of the
Toxic Chemical Required
• Four Endpoints for Detecting
Cytotoxicity Were Investigated on
Three Fish Cell Lines (RTG-2,
CHSE, and EPC)
• Cell Morphology - Estimation of the HTD
• Cell Viability (Neutral Red Uptake and the MTT Assay)
• Cell Proliferation (Increase in Total Protein)
Morphological Changes
• Morphological Changes Have Been Repeatedly Regarded as
one of the Earliest Responses of Isolated Cells to Chemical
Injury
• Determination of the Highest Tolerated Dose (HTD)
• Following Treatment of Cells for 24-h cells are examined
under phase contrast microscope
• HTD i.e. The Test Substance Concentration That Causes
Minimum Morphological Defects to the Cells is Then
Estimated and Recorded
• Non Invasive Manner of This Test Allows other Cytotoxicity
Endpoints to be Investigated
Neutral Red Cytotoxicity Assay
• Established by Borenfreund and Puerner (1985)
• An Early Effect of Many Toxic Substances is Manifested as Damage at the
Level of the Cell Membrane
• Dye is Preferentially Taken Up and Accumulated by Lysosomes in Cells.
Damaged Cells Have Therefore Altered Rates of NR Uptake Whereas Dying
and Dead Cells Do Not Retain The Dye
• Absorbance of Solubilised Dye is Then Determined Using a Scanning Well
Spectrophotometer Equipped With a 560 nm Filter
• Determination of IC50 Concentration (i.e. the Concentration Producing 50%
Inhibition of Growth)
MTT Cytotoxicity Assay
• MTT Assay Determines the Ability of Viable Cells to Convert a Soluble
Tetrazolium Salt into an Insoluble Formazan Precipitate
• NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined
conditions, reflect the number of viable cells present. These enzymes are
capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to its insoluble formazan
• The Reaction Converts the Yellow Salts to Purple Coloured Formazan
Crystals That Can Be Dissolved in an Organic Solvent
• MTT Has Shown Good Correlations With NR and LDH Release
• Measurements of Cell Growth by MTT Reduction Correlated Well With
Indices of Cellular Protein and Viable Cell Number
Microtitre plate showing increasing viability as
toxin is diluted
Total Protein Cytotoxicity Assay
• Endpoint Based on Cell Growth as Determined by Total Cell
Protein Accumulation
• Measurement of Total Cell Protein is Based on a Direct
relationship Between Protein Content, Cell Number and Binding
of a Dye
• Advantages:
» Much Faster to Perform and Less Variation than
Lowry
» Repeat Test More Than Once on Same Cells
» Performed on Same Plate of Cells Following NR
Uptake Analysis (Liebsch and Spielmann, 1995)
Decomposer
Microtox®*
Primary Producer
T. suecica
S. costatum
Producer (Macrophyte)
C. tenuicorne
Primary Consumer
T. battagliai
A.tonsa
Secondary Consumer
C. volutator
Fig. 1 Multi-trophic battery of marine test species employed in many regulatory
agencies
Rainbow trout gill proteomics: Example of useful technique where doses
are too low for the previous assays to show effects
What is “proteomics”?
“Proteomics collectively analyses the proteins that are regulated,
expressed or modified in the cell under different conditions”
Liebler DC (2002). Introduction to proteomics: tools for the new biology.
Humana Press Inc, Totowa, NJ
• PROTEOME
Rather as “the genome” is the collective noun for the total genetic material,
the “proteome” describes the proteins expressed in any given cell, tissue,
organ or whole animal.
Note: whereas the genome is fixed, irrespective of cell / tissue / organ,
the proteome is a very fluid entity, responding to a variety of
endogenous and exogenous stimuli.
PROTEOMIC ANALYSIS
Image Analysis
Spot removal
and digestion
2-D Gel
...ADFEGTTHCCEFL
LHPQLRHGLSAMQ
LPDGQRATYMCHQ ...
Peptide analysis by MS
Peptide mapping
Spot
Identification
GEL HYDRATION AND ISOELECTRIC FOCUSSING
Protein application
Rehydration
Isoelectric focussing
2nd DIMENSION (SIZE) ELECTROPHORESIS
Large molecules
Small molecules
TWO-DIMENSIONAL GEL ELECTROPHORESIS
1st Dimension – iso-electric charge
pH 4.0
2nd Dimension – molecular size (kDa)
100
15
pH 7.0 – 11.0
2D GEL IMAGE ANALYSIS WITH PHORETIX PROTEOMICS
SOFTWARE
Reference gel
Comparative gel
TWO-DIMENSIONAL GEL COMPARISON USING PHORETIX
SOFTWARE
PROTEIN SPOTS PICKED FROM 2D GEL
Rainbow trout as sentinel species
• Universal distribution - allows comparison of data with
other research groups.
• Demanding of high water quality - sensitive to any
change in aquatic environment.
• Commercially important species - inclusion in human
food chain.
• Acts a a surrogate for other salmonids
X-RAY AND BYSTANDER EFFECT INDUCED CHANGES TO THE
TROUT GILL PROTEOME
76.0
Hemopexin-like
protein
66.2
Molecular size (kDa)
Pyruvate dehydrogenase (PDH)
43.0
Chromosome 1
SCAF protein
36.0
Annexin II
31.0
RhoGDP dissociation inhibitor (RhoGDI)
21.5
17.5
4.5
5.1 5.4 5.6
6.0
7.0
8.5
Isoelectric point (pH units)
SCAF = SR-like CTD-associated factor (SR = Serine – argenine Rich, CTD = C Terminal Domain)
ELISA – Enzyme-Linked ImmunoSorbent Assay
(or Enzyme Immuno Assay; EIA)
Technique used mainly to detect the presence of an antigen or an antibody
in a sample.
Also used as a diagnostic tool in medicine, plant pathology and as a quality
control check in various industries.
ELISA - definition
Enzyme - involves an enzyme reaction as one step in the assay
Linked
- the enzyme reaction is directly proportional to the concentration of
the unknown substance that is being detected by the assay
Immuno - the assay involves an antibody of known specificity
Sorbent - a reagent is adsorbed to a plastic surface (reaction thus can be
"trapped") on the surface
Assay - clinical term for a test to detect the presence of a substance in a test
sample
ELISA – PRINCIPLE
Exploits the precision of antibody
/ antigen binding
Non-radioactive derivative of the Radio ImmunoAssay (RIA), developed by
Rosalyn Yallow.
Instead of using a radio-labelled antibody, the antibody is linked to
peroxidase.
Peroxidase can then react with an appropriate substrate resulting in a
change in colour. Colour change is proportional to the amount of antigen.
96 well ELISA plate
ELISA plate is not a tissue culture plate.
ELISA plate is treated to promote adsorption of the antigen to the well
surface
ELISA components
Antigen
Adsorption. Gas or liquid solute accumulates on the
surface of a solid or a liquid (adsorbent), forming a
molecular or atomic film. Differs from absorption, in which a
substance diffuses into a liquid or solid to form a solution.
Primary antibody. Ig molecule raised specifically
against the antigen under detection. Can be
monoclonal or polyclonal.
Secondary antibody. Antibody which binds with the
primary antibody (or antibody fragments). May be
polyclonal or monoclonal. Anti-Ig antibody
Enzyme - bioconjugated to the secondary antibody.
Bioconjugation is the process of coupling two
biomolecules together in a covalent linkage. Acts on a
substrate resulting in a colourometric reaction.
Elements of the ELISA assay
Antigen
Primary Secondary antibody Peroxidase
antibody
(anti Ig)
conjugate
96 well plate is thoroughly washed before
adding each successive component.
Unbound molecules are therefore
removed
Antigen serial dilution
Primary & secondary antibody, & enzyme = constant and present in excess
Extent of colour development is therefore proportional to the amount of
antigen in the sample under test.
Controls – positive and negative
Positive control
Negative control
Positive control – confirms all elements of assay are functional
Negative control – confirms no non-specific binding of primary antibody.
Particularly important with polyclonal antibodies
Positive
Control
Negative
Control
Patient A
Patient B
Patient C
Assay Control
1.689
0.153
O.055
0.412
1.999
0.123
Methological variations. 1. Sandwich ELISA
1. Plate is coated with a capture antibody.
2. sample is added, and any antigen present
binds to capture antibody.
3. detecting antibody is added, and binds to
antigen.
4. enzyme-linked secondary antibody is added,
and binds to detecting antibody.
5. substrate is added, and is converted by
enzyme to detectable form.
Methological variations. 2. Competition ELISA
Antibody / antigen
incubated together
Antibody / antigen
complex added to
pre-coated antigen
wells (fixed amount)
More antigen in
original sample = less
binding to antigen
coated to well
INCREASED APOLIPOPROTEIN AI EXPRESSION IN ASYMMETRICAL
GILL CELL CULTURES
Apolipoprotein AI
Molecular size (kDa)
95
200
60
45
150
30
100
20
4.0
4.4
4.8
5.2
pI (pH units)
5.6
6.0
6.4
Normalised spot volu
15
50
0
Symmetrical
culture
Asymmetrical
culture