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 measuringthe presence of contaminants of different kind and the effects they cause. As usual, this startingpoint of protection and work may be accomplished by physical, chemical, and as is in this referratbiological measures.

Generally, a biomarker is considered as a measurable indicator of a biological state condition forbiological, pathological, ecological or pharmacological response terms, through a direct effect orthrough the expression of secondary answers based on the methabolism of the molecules or physicalinteractions related.

At Ecologycal Studies, the terms biomarkers and bioindicators are quite offenly used assynonyms, although they show a point difference through the view of many experts and studies. Ingeneral 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.

2. Biomarker: related to molecular biology of the organisms that is affected by theinteraction with xenobiotic compounds or physical events (UV light, radiations, etc),inducing the expression of proteins (enzymes, receptors), the activation of methabolicalroutes of synthesis (antioxidants) or apoptosis. They also allow to creat dose-effectrelationship, therefore they present quantitative measures that can track the effect ofxenobiotics 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.

Table 1: Extracted from Reference N. 1

Biomarkers can be classified in three groups:

1. Biomarkers of exposure: expressed at point places inthe organism, after the interaction with xenobiotics, as anexample of this it can be 8-oxodiguanidine (8- oxodG) orglutation dissulfide (GSSG) that shows that reducingpower in the cell.

2. Biomarkers of effect: they describe changes in the biochemistry of the organism in adose related relationship within the xenobiotics, here there are defensive proteins (HSP,heat shock proteins), antioxidants enzymes (super oxide dismutases, SOD), defensivesystems such as Lipids or DNA repair enzymes, or the specific inhibition of enzymes suchas AchE (that may create effects such as heartstrokes or tetanization of skeleton muscles.

3. Biomarkers of susceptibilitty: indicator of the ability of the organism to respond to thepresence of xenobiotics (induceable molecules), inside these group there are all the inducibleenzymes such as P450 cytochromes (CYPs), or glutathione sulfide transferases (GSTs).

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 degradationof cell structures and could create apoptosis or necrosis processes.

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.

Illustration 1: Structure of Glutathion sulfide.

Illustration 2: Structure of Human SOD and reaction mechanism.

Illustration 3: CYP 450 1OG2

The main compounds that can affect the methabolism of cells and subcellular structure are heavymetals (Fe, Cu, Cd, Cr, As), P-Biphenils (poli-Cl, poli-Br), P-Quinones (PAHs, antitumorals), P-Bipiridiles(paraquat) .

For instance, metalotioneines are small proteins (6-8 Kda), that appear after heat induction andmetals such as cadmium, mercury or arsenic, they present 12-20 Cys, that can be used for theassimilation of high metals.

One of the interesting points at the analysis of biomarkers comes through the analysis itself. Sinceall the molecules that can be traced are mainly proteins, nucleotides or lipidic molecules, this makesno 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 theappearence of omics techniques. As an example, ProteoEcoToxicology study proteinbiomarkers 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 separatedthrough SDS-PAGE in 2 dimensions with high resolution for separating the large number ofproteins, and therefore accessing to more information. After this, the dots from that

experiment are treated with tripsine andput through mass spectrometry(MALDI-TOF) to get the sequences ofthe proteins and compare them withmodel organisms and non-modelorganisms to check the proteins that areexpressed under different conditions.After this, using differentbioinformatical tools, such as PEAKS(http://www.bioinfor.com/peaks/features/overview.html), or MASCOT(http://www.matrixscience.com/) forfurther studies. This way of studyingprovides with good data, nevertheless,they can suffer from many experimentalerrors and non accurated protocols thatlead to more qualitative results with lowreproducibility.

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.

Illustration 4: Structure of Beta-E-domain of wheat Ec-1 metallothionein.

Illustration 5: Example of workflow.

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.

2. PRACTICAL CASE: AZNALCLLAR CASE.

The disaster of Aznalcllar is an ecological disasterproduced by the release of toxical residues (most ofthem 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 pressin the residues pond of the Mines of Aznalcllar.

These release transmitted from Aznalcllar until theNational Park of Doana, the largest one inEuropean Union, dissolved into waters and as mud.

This ecological problem, supposed to theGovernment of Andaluca the investment ofnear240 millions , and it has negatively affected thearea of Huelva, Sevilla and Cdiz, giving to thatarea a classification between moderate todangerously high in the environment of the affectedarea (map below).

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.

Illustration 6: iTRAQ workflow.

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.

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 showna medium increase inthe presence of ROS treatmentenzymes. The respond was caused by the presence of Cdand Hg, with related high expression of SeGSHPxenzymes. The expression of these factors had a time-dosecorrelation showing that after some years from the release,the levels of them decreased.

Bivalves: mainly oysters and mussels that expressSOD, GST, cathalases, methallotioneins, inresponse to the presence of metals, as it is shownin the following table.

Accumulation of metals in bivalves.Oysters Mussels

Sampling Site Whole body Gills Hemocytes Whole body

Cu Zn Cu Zn Cu Zn Cu ZnIsla Cristina 153 553 223 892 365 1077 5 25Punta Umbra

1352 2240 1729 3103 1274 1831 26 73

Mazagn 1611 3016 1907 4275 5642 15494 nd ndUnits in mg/mL.

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.

Procambarus clarkii: this croustaceous was used asa monitoring bioindicator, and the biomolecules ofthem were analyzed for tracking down either theadaptation of the organisms to the environment orthe reduction of xenobiotics.

3. CONCLUSSIONS.1. Biomarkers and bioindicators are the use of biological molecules ans living organisms todetect and/or quantify the pressence and effects of xenobiotics and how these molecules maychange the performance of them.

2. Appart from their use, the detection and quantification of these molecules and effects hasevolved 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, notonly for the detection but also for the monitoring of the process.

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.

Illustration 8: SDS-PAGE 2E analysis and likely proteins expressed per dot.

4. REFERENCES.

1. Hyne, Maher, Invertebrate biomarkers: links to toxicosis that predictpopulation decline, Ecotoxicology and Environmental Safety 54 (2003) 366374.

2, Bartell, Biomarkers, Bioindicators, and Ecological RiskAssessmentA Brief Review and Evaluation Environmental Bioindicators, 1:6073, 2006

3. Lpez Barea, Jurado Carpio, Michn Doa, Tema 8. Biomarcadores, University ofCrdoba, Department of Biochemistry and Molecular Biology.

4. Z. Garban, et al. Scientifical Researches, Biomarkers: theoretical aspects andapplicatives peculiaritiesAgroalimentary Processes and Technologies, Volume XI, No. 1(2005), 139-146.

5. Biomarkers for Environmental Monitoring Suggestions for Norwegian monitoringprograms, M88-2013.

Funded in the framework of the Erasmus Mundus TEMPO Program Action 2.