NATIONAL POLYTECHNIC UNIVERSITY OF ARMENIA
MASTER IN BIOTECHNOLOGY
Basics of Biotechnology in Environmental Protection
Biomarkers of Environmental Contamination
Rafael Diego Macho Reyes (rafael.d.macho@gmail.com)
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
INDEX
1. INTRODUCTION.
2. PRACTICAL CASE: AZNALCLLAR CASE.
3. CONCLUSSIONS.
4. REFERENCES.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
1. INTRODUCTION
One of the main points at the preservation of environment comes with the requirement of measuring
the presence of contaminants of different kind and the effects they cause. As usual, this starting
point of protection and work may be accomplished by physical, chemical, and as is in this referrat
biological measures.
Generally, a biomarker is considered as a measurable indicator of a biological state condition for
biological, pathological, ecological or pharmacological response terms, through a direct effect or
through the expression of secondary answers based on the methabolism of the molecules or physical
interactions related.
At Ecologycal Studies, the terms biomarkers and bioindicators are quite offenly used as
synonyms, although they show a point difference through the view of many experts and studies. In
general terms we can analyze them using the following structure:
1. Bioindicator: refers to the use of a entire organism, culture or group as a qualitative
indicator of the environmental quality of the place of study. It acts as a primary analysis
structure of the changes of the environment and the effect they provoque on the organisms.
They are used as a starting sample for creating Ecological Risk Analysis.
Table 1: Extracted from Reference N. 1
2. Biomarker: related to molecular biology of the organisms that is affected by the
interaction with xenobiotic compounds or physical events (UV light, radiations, etc),
inducing the expression of proteins (enzymes, receptors), the activation of methabolical
routes of synthesis (antioxidants) or apoptosis. They also allow to creat dose-effect
relationship, therefore they present quantitative measures that can track the effect of
xenobiotics during the whole transit through the living organism which includes ADME
(absorption, distribution, methabolis, excretion).
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
Biomarkers can be classified in three groups:
1. Biomarkers of exposure: expressed at point places in
the organism, after the interaction with xenobiotics, as an
example of this it can be 8-oxodiguanidine (8- oxodG) or
glutation dissulfide (GSSG) that shows that reducing
power in the cell.
Illustration 1: Structure of
Glutathion sulfide.
2. Biomarkers of effect: they describe changes in the biochemistry of the organism in a
dose related relationship within the xenobiotics, here there are defensive proteins (HSP,
heat shock proteins), antioxidants enzymes (super oxide dismutases, SOD), defensive
systems such as Lipids or DNA repair enzymes, or the specific inhibition of enzymes such
as AchE (that may create effects such as heartstrokes or tetanization of skeleton muscles.
Illustration 2: Structure of Human SOD and
reaction mechanism.
3. Biomarkers of susceptibilitty: indicator of the ability of the organism to respond to the
presence of xenobiotics (induceable molecules), inside these group there are all the inducible
enzymes such as P450 cytochromes (CYPs), or glutathione sulfide transferases (GSTs).
Illustration 3: CYP 450 1OG2
As xenobiotics it can be defined all the molecules that create relationships with the methabolism,
and may create the proliferation of reactive oxygen species that could undergo into the degradation
of cell structures and could create apoptosis or necrosis processes.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
The main compounds that can affect the methabolism of cells and subcellular structure are heavy
metals (Fe, Cu, Cd, Cr, As), P-Biphenils (poli-Cl, poli-Br), P-Quinones (PAHs, antitumorals), PBipiridiles(paraquat)
.
For instance, metalotioneines are small proteins (6-8 Kda), that appear after heat induction and
metals such as cadmium, mercury or arsenic, they present 12-20 Cys, that can be used for the
assimilation of high metals.
Illustration 4: Structure of Beta-Edomain of wheat Ec-1 metallothionein.
One of the interesting points at the analysis of biomarkers comes through the analysis itself. Since
all the molecules that can be traced are mainly proteins, nucleotides or lipidic molecules, this makes
no difference respect to the extraction and study of these molecules for basic or applied research,
and the way of studying them has changed within the years, the development of technicals and the
appearence of omics techniques. As an example, ProteoEcoToxicology study protein
biomarkers attending to two generations:
1. First Generation.
After the interaction of xenobiotics with the organism, some samples are collected (animals,
plants, water cultures for purifying bacteria),and their proteins are extracted and separated
through SDS-PAGE in 2 dimensions with high resolution for separating the large number of
proteins, and therefore accessing to more information. After this, the dots from that
experiment are treated with tripsine and
put
through
mass
spectrometry
(MALDI-TOF) to get the sequences of
the proteins and compare them with
model organisms and non-model
organisms to check the proteins that are
expressed under different conditions.
After
this,
using
different
bioinformatical tools, such as PEAKS
(http://www.bioinfor.com/peaks/features/
overview.html),
or
MASCOT
(http://www.matrixscience.com/)
for
further studies. This way of studying
provides with good data, nevertheless,
they can suffer from many experimental
errors and non accurated protocols that
lead to more qualitative results with low
reproducibility.
Illustration 5: Example of workflow.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
2. Second Generation.
These systems are based on robotics and computing systems directly and they offer more
accurate results (turning likely proteins into directly tags that identify the presence of
polluting xenobiotics in the cells). Amongst many of these techniques, iTRAQs systems
based on the labelling of N-terminus of peptides with different reagents that are sent to
nanoliquid chromatography and analyzed through tandem mass spectrometry (MS/MS) and
the fragmentation of the data to identify labeled peptides.
Illustration 6: iTRAQ workflow.
2. PRACTICAL CASE: AZNALCLLAR CASE.
The disaster of Aznalcllar is an ecological disaster
produced by the release of toxical residues (most of
them heavy metals: As, Ba, Be, Bi, Cd, Co, Cr, Cu,
Hg, In, Mn, Mo, Ni, Pb, Sb, Sc, Se, Sn, Th, Tl, U,
V, Y, Zn) in 1998, caused by the breaking of a press
in the residues pond of the Mines of Aznalcllar.
These release transmitted from Aznalcllar until the
National Park of Doana, the largest one in
European Union, dissolved into waters and as mud.
This ecological problem, supposed to the
Government of Andaluca the investment ofnear
240 millions , and it has negatively affected the
area of Huelva, Sevilla and Cdiz, giving to that
area a classification between moderate to
dangerously high in the environment of the affected
area (map below).
Illustration 7: Mark on a tree of the levels of mud in the
riverside are of Guadiamar river.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
For the analysis of the environmental effects of the catastrophe it were collected organisms, Mus
spretus at the starting point of the release, Procambarus clarkii for the estuary of Guadalquivir, and
different bivalves (Crassostrea angulata, Mytilus galloprovincialis and Scrobicularia plana).
Mus spretus: express mainly GSSG, and there was shown
a medium increase inthe presence of ROS treatment
enzymes. The respond was caused by the presence of Cd
and Hg, with related high expression of SeGSHPx
enzymes. The expression of these factors had a time-dose
correlation showing that after some years from the release,
the levels of them decreased.
Bivalves: mainly oysters and mussels that express
SOD, GST, cathalases, methallotioneins, in
response to the presence of metals, as it is shown
in the following table.
Accumulation of metals in bivalves.
Oysters
Sampling Site
Isla Cristina
Punta
Umbra
Mazagn
Whole body
Mussels
Gills
Hemocytes
Whole body
Cu
Zn
Cu
Zn
Cu
Zn
Cu
Zn
153
553
223
892
365
1077
25
1352
2240
1729
3103
1274
1831
26
73
1611
3016
1907
4275
5642
15494
nd
nd
Units in mg/mL.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
Procambarus clarkii: this croustaceous was used as
a monitoring bioindicator, and the biomolecules of
them were analyzed for tracking down either the
adaptation of the organisms to the environment or
the reduction of xenobiotics.
Illustration 8: SDS-PAGE 2E analysis and likely proteins expressed per dot.
3. CONCLUSSIONS.
1. Biomarkers and bioindicators are the use of biological molecules ans living organisms to
detect and/or quantify the pressence and effects of xenobiotics and how these molecules may
change the performance of them.
2. Appart from their use, the detection and quantification of these molecules and effects has
evolved like biosciences themselves, including the use of omics for the analysis.
3. At the Aznalcllar catastrophe it were used different species to quantify heavy metals, not
only for the detection but also for the monitoring of the process.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
4. REFERENCES.
1. Hyne, Maher, Invertebrate biomarkers: links to toxicosis that predict
population decline, Ecotoxicology and Environmental Safety 54 (2003) 366374.
2, Bartell, Biomarkers, Bioindicators, and Ecological Risk
AssessmentA Brief Review and Evaluation Environmental Bioindicators, 1:6073, 2006
3. Lpez Barea, Jurado Carpio, Michn Doa, Tema 8. Biomarcadores, University of
Crdoba, Department of Biochemistry and Molecular Biology.
4. Z. Garban, et al. Scientifical Researches, Biomarkers: theoretical aspects and
applicatives peculiaritiesAgroalimentary Processes and Technologies, Volume XI, No. 1
(2005), 139-146.
5. Biomarkers for Environmental Monitoring Suggestions for Norwegian monitoring
programs, M88-2013.
Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.
