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Thèse de doctorat/ PhD Thesis Citation APA:
De Angelis, F. (2010). Characterization of proteins involved in RND-driven heavy metal resistance systems of Cupriavidus metallidurans CH34 (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté des Sciences – Ecole Interfacultaire des Bioingénieurs, Bruxelles.
Disponible à / Available at permalink : https://dipot.ulb.ac.be/dspace/bitstream/2013/210154/4/62ab0525-bfb8-49ac-aaf1-7bbd53ccb5a0.txt
(English version below)
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Université Librede Bruxelles
Centrede Biologie Structurale
ET de Bioinformatique
Servicede Structureet Fonction des Membranes Biologiques
BIBLIOTHEQUE DES SCIENCES ET CP 174 Av. A. Der^^ 3^ B-1000 3RüX5lL£S
Téi B5ij.20.54 o5C.40.36
C haracterization of proteins involved
IN RND-DRIVEN HEAVY METAL RESISTANCE SYSTEMS OF CUPRIAVIDUS METALLIDURANS
Docteuren Sciences Agronomiqueset Ingénierie Biologique
Fabien De Angelis
Mars 2010 Promoteur : Erik Goormaghtigh
Co-promoteur : ]ean-Marie Ruysschaert
Co-promoteur : Guy Vandenbussche
Université Librede Bruxelles
Servicede Structureet Fonctiondes
C haracterization of proteins involved in
RND-DRIVEN HEAVY METAL RESISTANCE
SYSTEMS OF CUPRIAVIDUS METALLIDURANS CH34
Docteuren Sciences Agronomiqueset Ingénierie Biologique
Mars 2010 Promoteur : Erik Goormaghtigh
Co-promoteur : Jean-Marie Ruysschaert
Co-promoteur : Guy Vandenbussche
personnes - promoteurs, membres du sfmb, ucsf, sbb, irmw, sck-cen, ma famille, mes proches et amis - qui ont participé de près ou de loin à l'aboutissement de ce projet et qui m'ont épaulé au cours de sa réalisation.
TABLE OF CONTENTS
1 - INTRODUCTION...10
1.1 - Définitionof 'heavymétal'... 10
1.2 - Heavymétalinputsintotheenvironment...13
1.3 - RELATIONSHIPS BETWEEN ORGANISMS andheavy METALS...15
1.4 - BIOCHEMICAL MECHANISMS ofheavy METALS TOXICITY... 17
1.4.1 - Displacementofessentialcations... 17
1.4.2 - Interactionwithproteins... 17
1.4.3 - Oxidativestress... 18
1.5 - Heavymetalstoxicityforhumans...18
1.6 - IN-DEPTH DESCRIPTION OF SELECTED HEAVY METALS... 21
1.6.1- Zinc... 21
1.6.2 -COPPER... 21
1.7 - MICROORGANISMS heavy METALS RESISTANCE... 23
1.7.1 - ENZYMATIC TRANSFORMATION...24
1.7.2 - SEQUESTRATION... 24
1.7.3 - Activetransport...25
1.8 - EFFLUX-MEDIATED résistance SYSTEMS... 25
1.8.1 - P-TYPE ATPase... 26
1.8.2 - ATP-BINDING cassette...26
1.8.3 - Majorfacilitator... 26
1.8.4 - Cationdiffusionfacilitator...26
1.9 - RND RESISTANCE SYSTEMS... 27
1.9.1 - OVERVIEW OF THE RND TRANSPORTERS SUPERFAMILY...27
18.104.22.168 - HAE-RND Systems...28
22.214.171.124 - HME-RND Systems...28
1.9.2 - Theinner-membraneantiporter (RND)... 28
126.96.36.199 - Antiporters of the HAE class...28
188.8.131.52 - Antiporters of the HME class... 30
1.9.3 - Theouter-membraneprotein (OMF)...30
184.108.40.206 - OMF of the HAE class...30
220.127.116.11-OMFofthe HME class... 31
1.9.4 -Theperiplasmicprotein (MFP)... 32
18.104.22.168- MFPofthe HAE class... 32
22.214.171.124 - MFP of the HME class...33
1.9.5 - Mechanismofefflux...33
126.96.36.199 - Assembly of the tripartite System...33
188.8.131.52.1 - Docking...33
184.108.40.206.2 - Stoichiometry... 33
220.127.116.11 - Substrate transport mechanism of the antiporter...34
18.104.22.168.1 - Substrate specificity... 34
22.214.171.124.2 - Transport mechanism...34
126.96.36.199 - Opening of the OMF gâte... 35
188.8.131.52 - The rôles of the MFP... 36
184.108.40.206.1- MFP-mediated opening of the OMF gâte...36
220.127.116.11.2 - Substrate binding... 37
18.104.22.168.3 - MFP is mandatory for the antiporter to function...37
22.214.171.124 - The substrate capture issue...37
1.10 - CUPRIAVIDUS METALLIDURANS STRAIN CH34... 39
1.10.1 - CHARACTERISTICS OVERVIEW... 39
1.10.2 - CUPRIAVIDUS METALLIDURANS USE IN BIOTECHNOLOGY...41
1.10.3 - HEAVY METALS INHIBITION OF XENOBIOTICS DEGRADATION...43
1.10.4 - HEAVY METALS SELECTIVE PRESSURE ON DRUG RESISTANCE... 43
2 - PURPOSE OF THIS WORK...45
3 - EXPERIMENTAL BACKGROUND... 46
3.1 - WHY SELECTING THE SiL SYSTEM?...46
3.2 - WHY SELECTING THE ZNE SYSTEM?...47
4 - INSIGHTINTO MFP FUNCTION... 48
4.1 - Proteinspurification... 48
4.1.1 - Characterizationofpurified ZneB... 48
4.1.2 - Characterizationofpurified SilB... 50
4.2 - METAL BINDING PROPERTIES... 52
4.2.1 - ZneB METAL BINDING PROPERTIES...52
126.96.36.199 - ZneB Métal binding specificity...52
188.8.131.52 - Zinc-binding affinity... 54
184.108.40.206 - Conclusion...54
4.2.2 - SilB METAL BINDING PROPERTIES...55
220.127.116.11 - Silver and copper(l)... 55
18.104.22.168 - Copper(ll)...55
22.214.171.124 - Other cations... 55
126.96.36.199 - Conclusions... 57
188.8.131.52.1 - Selectivity... 57
184.108.40.206.2 - Binding sites... 57
4.3 - Crystallography... 57
4.3.1 - Crystal GROWTH... 57
4.3.2 - Crystalqualityimprovement... 58
220.127.116.11 - Zn-ZneB crystals...58
18.104.22.168 - Apo-ZneB crystals... 59
22.214.171.124 - Cu(l)-SilB crystals... 59
4.3.3 - Samplepurityimprovement...59
126.96.36.199 - Contaminant removal by affinity chromatography...60
188.8.131.52 - Contaminant removal by size exclusion chromatography...60
184.108.40.206 - Contaminant removal by ion exchange chromatography... 61
4.3.4 - Phasedétermination... 61
220.127.116.11 - Direct Methods...61
18.104.22.168 - Molecular replacement... 62
22.214.171.124 - AXRS based on heavy atom derivatization...62
126.96.36.199 - AXRS based on selenium-labeling of méthionine...62
4.3.5 - Structuredétermination... 62
4.3.6 - Structureanalysis... 63
188.8.131.52 - Structure overview...63
184.108.40.206 - Zinc coordination site... 64
220.127.116.11.1 - Architecture, selectivity and plasticity...64
18.104.22.168.2 - Validation by site-directed mutagenesis...66
22.214.171.124 - Molécule A vs Molécule B... 68
4.4 - ZneB behaviourinsolution... 69
4.4.1 - Rearrangementuponzincbinding (SEC)...69
4.4.2 - ZiNC-MEDIATED STRUCTURING OF MP DOMAIN (ATR-FTIR)... 70
4.4.3 - ZINC-MEDIATED STRUCTURING OF MP DOMAIN (TRYPTOPHAN QUENCHING)... 71
4.4.4 - ZINC-MEDIATED TWISTING OF HAIRPIN DOMAIN (CONTROLLED TRYPSINOLYSIS)...72
126.96.36.199 - Experimental procedure and observations... 72
4.5 - COMPARISON OF ZNEB, MEXA, AND CUSB CRYSTAL STRUCTURES... 75
4.5.1 - Generalarchitecture...75
188.8.131.52 - The a-helical hairpin domain...75
184.108.40.206 - The 3-barrel and MP domains... 75
220.127.116.11 - Rotation of the MP domain... 76
4.5.2 - Métalcoordinationsites... 78
4.6 - COMPARISON OF ZNEB AND HME-MFPS SEQUENCES... 79
4.6.1 - SEQUENCES ALIGNEMENT...79
18.104.22.168 - Conservation of the Zn-ZneB coordination site...79
22.214.171.124 -Copperand silver coordination sites... 80
4.6.2 - Phylogenetictree... 80
4.6.3 - Generalobservations...80
5 - RND-DRIVEN TRANSPORT ASSAYS... 87
5.1 - Membraneproteinsexpression...87
5.2 - Proteinspurification... 87
5.2.1 - SILA PURIFICATION...87
5.2.2 - ZneA purification... 88
5.3 - Membraneproteinreconstitutionintolipidbilayers... 89
5.3.1 - Protocoloverview... 89
5.3.2 - Determiningoptimalddmconcentration... 90
5.3.3 - VERIFICATION OF RECONSTITUTION EFFICIENCY... 91
5.4 - ESTABLISHING A PROTON GRADIENT...92
5.5 - RND-DRIVEN heavy METALS TRANSPORT ASSAYS...93
5.5.1 -SILA-DRIVEN METAL transport ASSAY... 93
5.5.2 -ZNEA-DRIVEN METAL transport ASSAY...95
5.6 - Generalremarks... 97
5.6.1- SilA... 97
5.6.2- ZneA... 97
6 - DISCUSSION... 98
6.1 - Therôleoftheperiplasmicprotein... 98
6.1.1 - Substratereleasetotheantiporter...98
126.96.36.199 - Key rôle of the MP domain... 98
188.8.131.52 - The coordination site architecture and plasticity...100
6.1.2 - OPENING OF THE OUTER MEMBRANE ASPARTATE GATE...100
184.108.40.206 - Theoretical reminder... 100
220.127.116.11 - Proposed mechanism... 100
6.1.3 - Thenecessityofmfpfortheeffluxmechanism...lOl 6.1.4 - MFP 'ADAPTOR' ROLE REVISED TO A DYNAMIC AND ACTIVE ROLE... 101
6.2 - Thesubstratecapturebytheantiporter...102
6.2.1 - Transmembraneeffluxhypothesis... 102
6.2.2 - OUTER MEMBRANE HYPOTHESIS... 104
6.2.3 - Transmembraneeffluxvsoutermembranehypothèses... 104
6.3 - The ZNE SYSTEM...105
6.3.1 - Rôleofthe Zne System... 105
7 - PERSPECTIVES...106
7.1 - CusB-likecrystallization...106
7.2 - Complexcrystallization...106
7.3 - InfluenceoftheMFP ontheantiportertransportactivity...106
8 - MATERIALS AND METHODS... 107
8.1 - Cloningof ZneA, ZneB, SilA and SilB... 107
8.1.1 - Cultureof Cupriavidusmetallidurans CH34... 107
8.1.2 - PCR AMPLIFICATION OF TARGET GENES... 107
8.1.1 - VERIFICATION OF GENE SEQUENCES... 108
8.1.2 - Insertionofgenesintoexpressionvectorsandtransformationintohosts... 109
8.2 - Proteinsoverexpression... 109
8.3 - Proteinspurification...110
8.3.1 - ZneA and SilA purification... 110
8.3.2 - ZneB purification...111
8.3.3 - SilB purification... 111
8.4 - ELEaROPHORESIS... 111
8.4.2 - Immunodetection...112
8.5 - Membraneproteinsreconstitution... 113
8.6 - Establishingaprotongradient... 113
8.7 - Fluorescencespectroscopy...113
8.7.1 - STABILITY of THE PROTON GRADIENT... 113
8.7.2 - HEAVY METAL TRANSPORT ASSAY...114
8.7.3 - ZneB tryptophanquenching...114
8.8 - Massspectrometry... 114
8.8.1 - ACCURATE MOLECULAR mass DETERMINATION...114
8.8.2 - Analysisofprotein-metalcomplexes... 114
8.8.3 - Proteinmicro-sequencing... 115
8.9 - Crystallography...115
8.9.1 - METHOD OVERVIEW...115
8.9.2 - Screeningofpositivecrystallizationconditions...117
8.9.3 - Zn-ZneB crystallization... 119
18.104.22.168 - Crystal size and resolution improvement... 119
22.214.171.124.1 - Crystallization conditions... 119
126.96.36.199.2 - Post-crystallization...119
188.8.131.52.3 - Seeding... 120
184.108.40.206.4 - Sample purity...120
220.127.116.11 - Growth of best diffracting Zn-ZneB crystals... 120
18.104.22.168 - Phase détermination...120
22.214.171.124.1 - Molecular replacement... 121
126.96.36.199.2 - Heavy atom derivatization... 121
188.8.131.52.3 - Sélénium labeling of méthionine residues... 121
184.108.40.206.4 - Anomalous X-ray scattering... 121
220.127.116.11 - Data collection...122
18.104.22.168 - ZneB structure détermination and refinement...122
8.9.4 - Apo-ZneB crystallizationtrials... 122
8.9.5 - SilB crystallizationtrials... 123
8.11 - SiZE EXCLUSION CHROMATOGRAPHY... 123
8.12 - INFRARED SPECTROSCOPY... 124
8.13 - ZneB Limitedtrypsinolysis...124
8.14 - HME-MFP SEQUENCES ALIGNMENT...124
9 - PUBLICATIONS...126
10 - ACKNOWLEGMENTS...137
11 - REFERENCE LIST... 138
Tripartite résistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug eind heavy métal résistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adapter protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active rôle in substrate efflux. To better understand the rôle of MFPs in RND-driven efflux Systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy métal RND-driven efflux complexes ffom Cupriavidus metallidurans CH34. We hâve identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we hâve solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Â resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the métal coordination site at the very flexible interface between the P-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active rôle of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy métal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co- exist; nevertheless, the substrate capture issue is a complex topic still needing conséquent efforts to understand it.
Ambic : Ammonium Bicarbonate ABC : ATP-Binding Cassette ACN : Acetonitrile
ADP : Adenosine Di-Phosphate AL : Annealing on the Loop ALS : Advanced Light Source APS : Ammonium Persulfate ATP : Adenosine Tri-Phosphate
ATR-FTIR : Attenuated Total Reflection - Fourier-Transformed Infrared
AXRS : Anomalous X-ray scattering BLAST ; Basic Local Alignment Search
CDF : Cation Diffusion Facilitator CMC : Critical Micelle Concentration DDM : n-Dodecyl-P-D-Maltoside DTT : Dithiothreitol
EDTA : Ethylenediaminetetraacetic Acid ESI : Electrospray Ionisation
EPA : Environmental Protection Agency ESRF : European Synchrotron Radiation
FA : Formic Acid FC: Fos-Choline
FCCP : Carbonyl Cyanide-p- trifluoromethoxyphenylhydrazone GSH : Glutathione
GS-SG : Bisglutathione or Glutathione Disulfide
HA : Hydroxyapatite
HAE : Hydrophobie / Amphiphile Efflux HEPES : 4-(2-hydroxyethyl)-l-
Piperazineethanesulfonic Acid HME : Heavy Métal Efflux HMR : Heavy Métal Résistance IM : Inner Membrane
IMP : Inner Membrane Protein IPTG : Isopropyl-P-D-1-
Thiogalactopyranoside LB : Luria-Bertani
MAD : Multiple wavelength Anomalous Dispersion
MCA : Macromolecular Crystal Annealing
MDR : Multidrug Résistance
MES : 2-(N-Morpholino)Ethanesulfonic Aeid
MFS : Major Facilitator Superfamily MIC : Minimal Inhibitory Concentration MIR : Multiple Isomorphous Replacement MLV : Multilamellar Vesicles
MFP : Membrane Fusion Protein MP : Membrane Proximal
MR : Molecular Replaeement MS : Mass Spectrometry
NAD^/NADH : Nicotinamide Adenine Dinucleotide
NADP”^/NADPH : Nicotinamide Adenine Dinucleotide Phosphate
NMR : Nuclear Magnetic Résonance NTA : Nitrilotriacetic Acid
OD : Optical Density OM : Outer Membrane
OMF : Outer Membrane Factor P AP: Periplasmic Adaptor Protein PEG : Polyethylene Glycol
PCR : Polymerase Chain Reaction
PMSF : Phenylmethanesulphonyl Fluoride RMSD : Root Mean Square Déviation RND : Résistance, Nodulation and cell-
ROS : Reactive Oxygen Species SAD : Single wavelength Anomalous
SDS-PAGE : Sodium Dodecyl Sulfate - Polyacrylamide Electrophoresis
SEC : Size Exclusion Chromatography SOD : Superoxide dismutase
SUV : Small Unilamellar Vesicles TBE : TRIS - Boric acid - EDTA
TBST : TRIS-Buffered Saline Tween-20 TC : Transport Classifieation
TEMED : N,N,N’,N'-
T etramethy lethylenediamine TFA : Trifluoroacetic Acid TMD : Transmembrane Domain
TRIS : Tris(Hydroxymethyl)Aminomethane
1 - INTRODUCTION
Heavy metals are natural components of our environment. They were created by nuclear fusion taking place into Stars'. Therefore, heavy métal atoms présent on Earth existed even before Earth itself, and will last after our planet will be no more.
Given heavy metals extraordinary lifetime, Life will aiways hâve to deal with them. It has leamed since its emergence to take advantage of those ions, and has incorporated them into its cellular metabolism. Humankind has aiso massively used heavy metals during its évolution, and they hâve now an essential part in its activities.
But ail heavy metals are potentially toxic. Since its most primitive forms, Life had to develop résistance mechanisms to protect itself against deleterious effects of those compounds. Bacteria were probably the first organisms to acquire heavy métal résistance déterminants. Among those microorganisms, Cupriavidus metallidurans CH34 is a bacterium especially well adapted in living on heavy métal contaminated biotopes. This résistance phenotype is mainly provided by the large number of genes coding for efflux Systems, which are able to transport heavy métal cations out of the cell.
> But what exactly are heavy metals? How can they be defined? Why are they ail toxic, while some are essential for life?
> Which kind of Systems living beings hâve developed to protect themselves against heavy métal deleterious effects? And more precisely how do efflux-based détoxifîcation Systems work?
> Which characteristics of Cupriavidus metallidurans CH34 provided this microorganism a référencé status in the bacterial heavy métal résistance issue?
1.1 - Définition of
Over the past two décades, the term ^ heavy métal’ has been increasingly used in varions scientific publications. Metallic éléments are often labeled as '‘heavy’ according to their density: most limits found in literature are arbitrarily set around 4-5 g/cm^ . Actually this archaic définition has often been criticized for its lack of scientific bases and its inability to fulfill basic characteristics one can expect from a définition (i.e. giving the précisé meaning of a word^). No relationship can be found between density and any of the varions physicochemical concepts that hâve been used to define heavy metals and the toxicity or ecotoxicity attributed to them.
' A variety of different nuclear fusion reactions take place inside the cores of stars, as part of stellar nucleosynthesis.
The net mass of the fused atomic nuclei is smaller than the sum of the constituents. This lost mass is released as electromagnetic energy, according to the mass-energy équivalence relationship E = mc^.
^ Defining the word définition is a very complex task; it is a major philosophical challenge and resolving its inhérent questionings motivated philosophers like Platon, Aristote, Locke and Hume. For example, given that a natural language contains a fmite number of words, any comprehensive list of définitions must either be circular or leave some terms undefined. If every term of every définition must itself be defined, "’where at last should we stop?"
Actually more than forty different définitions proposed in literature hâve been reported (and it is easily conceivable that each and every existing définition has not been identified) ; some are based on density, some on atomic number or atomic weight, and some on Chemical properties or even on toxicity . A lot of those définitions contradict each other, and none of them is satisfying since they are arbitrary, imprécise and several categories overlap. Définitions were sometimes even specifically created by the authors to fit their needs, and the term heavy métal was adapted to each spécifie purpose - no need to say this is a biased reasoning. Thus, any assumption of underlying functional similarity in biological or toxicological properties is bound to be wrong ,
Although metallic éléments hâve certain properties in common, each one is a distinct élément with its own physicochemical characteristics which déterminé its biological and toxicological properties and how it may move through the environment. Not only this, but each métal can exist as part of a wide range of compounds with properties at least as diverse as those of carbon compounds* .
This term "heavy metaV has been called ""meaningless and misleading’" , ""hopelessly imprécise and thoroughly objectionablé’’’  due to the contradictory définitions and its lack of a cohérent scientific basis. Some authors concluded that there is no Chemical basis for deciding which metals should be included in the heavy métal category . Besides, there is a tendency to assume that ail so-called heavy metals hâve highly toxic or ecotoxic properties. This immediately préjudices any discussion of the use of such metals, often without any real foundation.
With the aim to solve this problem, a new classification of metallic éléments designed for toxicity assessment has been proposed . This classification is based on Lewis^ acid^ behavior of Chemical éléments . Briefly, the interaction of metallic éléments with living Systems is dominated by the properties of métal ions as Lewis acids. Any elemental species with a net positive charge behaves as a Lewis acid because it can act as an électron acceptor, determining the possibilities of complex formation. Métal ions can be divided into Class A, Class B, or Borderline depending on their observed affinity for different ligands (Figure 1.1). The classification of metals by their Lewis acidity indicates the form of bonding in their complexes.
Class A métal ions (or hard Lewis acids) which are of smaller size and non-polarizable"^, preferentially form complexes with similar non-polarizable ligands, particularly oxygen donors, and the bonding in these complexes is mainly ionic. Class B métal ions (or soft Lewis acids) which are of larger size and highly polarizable preferentially bind to polarizable ligands to give rather more covalent bonding. Borderline are intermediate metals.
' For example, there is no similarity in properties between pure tin, which has low toxicity, and tributyltin, which is highly toxic . Nor is there any similarity in properties between chromium in stainless Steel which is essentially nontoxic, and in the chromate ion which has been associated with causing lung cancer .
^ Gilbert Newton Lewis (1875 - 1946) was an american physical chemist known for the discovery of the covalent bond, his purification of heavy water, his reformulation of Chemical thermodynamics in a mathematically rigorous manner accessible to ordinary chemists, his theory of Lewis acids and bases, and his photochemical experiments.
^ A Lewis acid (A) is a Chemical compound that can accept a pair of électrons (électron acceptor) ffom a Lewis base (B) that acts as an electron-pair donor, forming an adduct (AB) : A + :B —► A-B. Classification of both acids and bases into hard and soft Lewis-acids/bases is based on the strength of acid-base adducts.
Polarizability is the relative tendency of a charge distribution to be distorted from its normal shape by an extemal electric field, which may be caused by the presence of a nearby ion or dipole.
Most class B and borderline metals fit into the d-block of the periodic table (Figure 1.1). In each of the blocks, the members are related by valence' and hence by similarities in Chemical reactivity. Most heavy metals are transition éléments with incompletely filled d-orbitals. These d- orbitals (Error! Reference source not found.) provide heavy métal cations with the ability to form complex compounds which may or may not be redox-active. D-block éléments show an extremely wide range of both redox behavior and complex formation. These properties underlie their catalytic rôle in enzyme action. Thus, some heavy métal cations play an important rôle as trace éléments in sophisticated biochemical reactions [55,162].
The difficult task of defining what a 'heavy metaV is will not be challenged here; nevertheless, the füzziness of this term has to be mentioned. In this manuscript, the tenu “heavy métal” will refer to the Class B and Borderline metals as defined above according to their Lewis acid behavior. Those categories gather metals of most biological, toxicological and environmental relevance. Even if this définition has some drawbacks, it is still one of the most refined and has the advantage among any other one to classify metals according to their Chemical behavior in relation to their environment, thereby giving dues about the way they might interact with it.
s bkK'k d bicx’k P block
li Ik- Na Mg
K Ca Sc fi
Rb Si- 7.1
Cs Ba ♦
Fr Ra # Rf
10 11 V
Cr Mn Icilll»
Co Ni Cuilli Fcill) C'UlII Zn
Mo Te Ru Rh Pd Ag Cd
W Re Os Ir Pt Au Hg
Sg Bh Hs Mt 110
14 15 16 17 He
c : N O F Ne
Si P s Cl Ar
Ge As St* Bi Kr
Sn Sb Te I Xe
Pbi m Bi Po At Rn
Pr Nd Pm
1 Sm Eu Cd Tb Dy Ho Er
! Tm Yb ____ ^____ Lu Pa V Np Pu Am Cm Bk Cf Es Fm ---!---
Md i No ____ i____ Lr Figure 1.1: Periodic table showing classification of éléments based on the last électron subshell in the atom to be occupied (s-, d-, p- and f-blocks), superimposed to the classification of éléments according to their Lewis Acid behavior: Class A = hard metals, Class B = soft metals, and borderline = intermédiare metals. Revised ffom  using “The Chemical Thésaurus”
' Valence is a measure of the number of Chemical bonds formed by the atoms of a given element. As for the Lewis acids and bases theory, this concept has been introduced by Gilbert Newton Lewis.
1.2 - HEAVY METAL INPUTS INTO THE ENVIRONMENT
Metals and métal compounds are originally sequestered inside the Earth as ore deposits, and are naturally released into the biosphère mainly through geological processes like volcanic éruptions, érosion, spring water, etc., and can originate even from extra-terrestrial sources like météorites.
Microorganisms can aiso play a rôle in heavy metals biogeochemical cycles, as their metabolism can dissolve minerais . Those Chemical éléments are natural constituents of the abiotic and biotic components of all ecosystems, and under natural conditions they are cycled within and between the geochemical spheres at quite steady fluxes.
Humankind discovered heavy metals utility early in its évolution. Metallurgy has played such a significant rôle in human cultural évolution, that metals give their name to certain âges (i.e.
"Bronze Age’: IlT* *^ & 11"^* millennium BC - "Iron Age’: T* millennium BC) . Lead is one of the oldest known métal; metallic lead beads dating back to 6400 BC hâve been found in modem- day Turkey. It was widely used for more than 5000 years' because this métal is corrosion résistant, dense, ductile and malléable, and it was deployed for building materials, water pipes, ceramics pigments glazers, glass and crystals, paints (especially white)^, protective coatings, as a wine preservative^, etc. Early discovery and use of lead in human activities has made this métal one of the most widely studied occupational and environmental toxins.
Mercury has also been readily used in human history. It was used as a salve to alleviate teething pain in infants, and as a remedy for syphilis''. Mercurous chloride (calomel, Cl-Hg-Hg-Cl) is one of the oldest known pharmaceuticals and is continuously used for its antiseptie properties. It prevents seeds from fimgus contamination. Thimerosol^ is an antiseptie containing ethylmercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Until the 1970s, methylmercury was commonly used for control of fungi on seed grain.
Anthropogenic activities which emerged during the Industrial Révolution (i.e. fossil fuel combustion and industrial processes) brought about unprecedented demand for metals. At the same time heavy métal capability to affect microbial growth and survival was enhanced. Their conséquent massive use as bactéricides and fungicides (like mercury, silver, or copper in Bordeaux mixture^) during Agriculture Révolution combined with Industrial Révolution and production of energy from fossil fuel triggered an exponential increase in the intensity of métal émission, both in absolute masses and in the number and nature of toxic métal compounds released. Great amounts
‘ Lead was especially used during the time of the Roman Empire, where large quantities of several heavy metals (e.g.
copper, zinc, mercury, tin, zinc and lead) were required to sustain the high standard of living. Some Romans might hâve consumed as much as a gram of lead a day.
^ Lots of heavy métal compounds hâve readily been used as pigments (and are still used nowadays). For example cinnabar (red, mercury sulfide) was used in prehistoric caves for paintings, and Greeks used it as a cosmetic to lighten the skin. Claude Monet used cadmium pigments (yellow, orange or red) extensively.
^ Lead acetate stops fermentation.
'* Syphilis is a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Syphilis can nowadays be treated with antibiotics, including penicillin. If left untreated, syphilis can damage the heart, aorta, brain, eyes and bones, and in some cases can be fatal.
* Thiomersal is very toxic by inhalation, ingestion, and in contact with skin, with a danger of cumulative effects. It is believed to be responsible for autism. In the body, it is metabolized to ethylmercury (a neurotoxin). It is also very toxic to aquatic organisms and may cause long-term adverse effects in aquatic environments.
* Bordeaux mixture is a mixture of copper sulphate and hydrated lime used as a fungicide in vineyards. This fungicide has been used for over a century and is still used, although the copper can leach out and pollute streams.
of metals hâve been excavated and released on the surface of the Earth and dispersed into the pedosphere*. The development of large fumaces during the 16* century drastically extended the sphere of influence of smelters and industrial installations, provoking mass air pollution and heavy metals dispersion^. The cumulative industrial releases of heavy métal into our environment are indeed massive and pervasive (Figure 1.2) and hâve largely overwhelmed the natural biogeochemical cycles of the metals in many ecosystems, marking the beginning of an era of worldwide systematic pollution (Figure 1.3 & Table 1.1). As metal- recycling processes are limited and heavy metals cannot be degraded, excavated heavy metals will be in fine completely dispersed in the environment. Most parts of the Earth hâve been contaminated by anthropogenic-released heavy metals [17,24,87,92,100,101,103,171].
Table 1.2 summaries nowadays use of some heavy metals.
Figure 1.2 : Global mine production and anthropogenic émissions into the atmosphère of some heavy metals .
Natural Offgcuùng from volconot», hoispringt,
IndiMtrioi Raleoiei from minmg ocHv^cs, bummg cod ond oil, wosiv aitpetd
fluprvteant itgfth, ctrrmr/t bdori«$, crtmalorurm,
Océan & Land
Di$p«r»ion, dtpo»ition, rteyding and biemot* uptalc* irrvoKnng:
. ^..^1 in Id mtihylnwvcry C (mo»l dut to bodtnal ocHon)
BitPCtMWMfalitw in Itod chain ytfiCMliMW in fbh ond wildb^
(Up b 10^ X incrtott ovtr bocitground) Bücycol ■npwl/tfftet» in etil division ono itprodutfion, bthovior, and letalHy
Figure 1.3 : Biogeochemical cycling of mercury;
ffom subsurface to surface distribution and biomass uptake.
' The pedosphere is the outermost layer of the Earth that is composed of soil and subject to soil formation processes.
It exists at the interface of the lithosphère (the rigid rocky outermost shell), atmosphère, hydrosphère and biosphère.
^ Actually, air pollution is often regarded as the product of modem technological development. In fact, environmental pollution caused by heavy metals began with the domestication of fire; the déposition of small amounts of trace metals released during the bumlng of firewood altered the métal levels in the cave environment.
Table 1.1 : Annual world émissions (late 1980s) of some heavy métal into the atmosphère (10^ tons) .
Pb Zn Cu Cd
Natural source 19 4 19 1
Anthropogenic source 449 314 56 7
Table 1.2 : Examples of heavy métal uses in the industry.
Element Industrial uses
Cadmium Batteries, pigments, stabilizers, cernent, agro-chemical products, électrodes, electroplating, welding, etc.
Copper Piping, electrical applications (wires, magnets, etc.), agro-chemical products, biomédical applications, coinage, alloys, metallic coating, pigments, ceramic glazes, catalyst, etc.
Lead Ammunition, acid storage batteries, gasoline additives, cosmetics (face powders, lipstick, mascara, etc.), spermicidal, radiation shielding, etc.
Mercury Barometers, thermometers, electrical equipment, fimgicides, gold industry, dental amalgam, etc.
Silver Currency, jewelry, photography, electronics, antimicrobial agent, etc.
Zinc Anti-corrosion (galvanization), batteries, alloys, Tire retardant, etc.
1.3- RELATIONSHIPS BETWEEN ORGANISMSand heavy
<1 ■ir Because metals are natural components of our environment (the Earth's
crust), microorganisms hâve been exposed to them since basically the beginning of life, nearly four billion years ago. Life has evolved in this natural milieu and has leamed to use each and every possible element available (including potentially highly toxic components, namely heavy metals) to maximize its chance of survival. Now life requires some metals to be présent in appropriate levels and combinations (i.e. iron, cobalt, copper, manganèse, zinc, etc.). Too low métal concentrations can lead to physiological problems (and even death) as a resuit of nutrient
deficiencies, whereas too high métal concentrations can be toxic to any living being* [103,207].
Other heavy metals such as mercury, silver, and lead are toxic metals that hâve no known vital or bénéficiai effect to any organism. Nonetheless, certain widely nonessential éléments are, for certain organisms or under certain conditions, bénéficiai. Examples include vanadium^ [182,228], tungsten^ [9,98], and even cadmium"^ [115,116].
Je t nofi
' One might say that there is no toxic compound, but only toxic concentrations.
^ Vanadate can be used in place of molybdate in nitrogenase for nitrogen fixation if molybdate is not présent in the environment.
^ Tungsten seems to be used by the rare life forms that do not require molybdenum.
“ In marine diatoms, cobalt, cadmium and zinc can functionally substitute for one another in carbonic anhydrase (involved in acquiring inorganic carbon) in order to maintain optimal growth rates in depleted environments.
Proteins that must bind métal ions for their function (metalloproteins - Table 1.3) constitute a significant share of the proteome of any organism. A métal ion (or metal-containing cofactor) may be needed because it is involved in the catalytic mechanism and/or because it stabilizes or détermines the protein tertiary or quatemary structure. Redox-active métal ions may also be involved in electron-transfer processes .
Table 1.3 : Examples of biological flinctions of heavy metals.
Elément Examples of biological fonctions Cobalt Vitamin B12* cofactor
Copper Superoxide dismutase (SOD) cytochrome c oxidase^
cofactor, oxygen carrier (hemocyanin'*)
Iron Oxygen carrier (hemoglobin^ myoglobin^), cytochrome^
Manganèse DNA polymerase cofactor Niekel Urease*, hydrogenase^ cofactor
Zinc Zinc fmgers, DNA polymerase, dehydrogenase'®, carbonic anhydrase'*, SOD cofactor
' Vitamin B12, also called cobalamin, is a water soluble vitamin with a key rôle in the normal flinctioning of the brain and nervous System, and for the formation of blood. It is normally involved in the metabolism of every cell of the body, especially affecting DNA synthesis and régulation, but also fatty acid synthesis and energy production. It is structurally the most complicated vitamin and contains cobalt.
^ Superoxide dismutases catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly ail cells exposed to oxygen. They can be cofactored with copper and zinc, or manganèse, iron, or nickel.
^ Cytochrome c oxidase is the last enzyme in the respiratory électron transport chain of mitochondria and bacterla containing two copper centers. It receives an électron from each of four cytochrome c molécules, and transfers them to one oxygen molécule, converting molecular oxygen to two molécules of water. In the process, it binds four protons from the inner aqueous phase to make water, and in addition translocates four protons across the membrane, helping to establish a transmembrane différence of proton electrochemical potential that the ATP synthase then uses to synthesize ATP.
Hemocyanins are respiratory proteins containing two copper atoms that reversibly bind a single oxygen molécule.
Oxygénation causes a color change between the colorless Cu(I) deoxygenated form and the blue Cu(II) oxygenated form.
’ Hemoglobin is the oxygen-transport metalloprotein heme containing (iron-containing porphyrin) prosthetic group in the red blood cells of vertebrates, and the tissues of some invertebrates. In mammals, the protein makes up about 97% of the red blood cells dry content, and around 35% of the total wet content.
* Myoglobin is the primary oxygen-carrying heme containing protein of muscle tissues. High concentrations of myoglobin in muscle cells allow organisms to hold their breath longer.
^ Cytochromes are membrane-bound hemoproteins and carry out électron transport. They are found in the mitochondrial inner membrane and endoplasmic réticulum of eukaryotes, in the chloroplasts of plants, in photosynthetic microorganisms, and in bacteria.
* Urease is a metalloprotein containing nickel which catalyzes the hydrolysis of urea into carbon dioxide and ammonia.
’ Hydrogenases catalyze the réversible oxidation of molecular hydrogen, and play a vital rôle in anaérobie metabolism. Their active site might contain nickel and iron, iron only, or no métal at ail.
Dehydrogenases, which might contain zinc cation, oxidize a substrate by transferring one or more hydrides (H‘) to an acceptor, usually NAD^/NADP^.
" The carbonic anhydrases catalyze the conversion of carbon dioxide to bicarbonate and protons. The active site of most carbonic anhydrases contains a zinc ion.
1.4- BTOCHEMICAL MECHANISMS OF HEAVY METALS TOXICITY
Heavy métal cations are toxic at the biochemical level because of three main reasons: 1. displacement of essential cations, 2. interaction with proteins, and 3. oxidative stress. Spécifie différences in the toxicities of métal ions may be related to différences in solubility, absorbability, transport, Chemical reactivity, and the complexes that are formed within the body [215,235].
Bioavailability and toxicity are highly interconnected. Bioavailability is the key to assessment of the potential toxicity of metallic éléments and their
compounds. It dépends on biological parameters and on the physicochemical properties of metallic éléments, their ions, and their compounds. These in tum dépend upon the atomic structure of the metallic éléments .
1.4.1 - Displacement ofessentialcations
Métal cations are very similar with one another. Therefore intruder cations might displace physiological cations from spécifie binding sites (protein cofactors, cations transporters, zinc- finger DNA binding domain, etc.), thereby inhibiting the fünction of the respective physiological cations [162,202,225]. For example:
. Cd^"^ can replace Ca^^
. Ni^"^ and Co^"^ can replace Fe^^
. Zn^^, Al^"^ and Mn^"^ can replace Mg^"^
. Cu^"^ and Cd^"^ can replace Zn^”^
1.4.2 - Interaction withproteins
Soft métal ions (e.g. Hg^”^, Cu"^ or Ag^) form very strong bonds with ftinctional groups such as the thioyl, histidyl and carboxyl groups, causing the metals to target structural, catalytic and transport sites of the cell and triggering physiological disorders of any kind. Interestingly, the minimal inhibitory concentration (MIC)* of métal ions is in some way correlated to the complex dissociation constants of the respective sulfides (Figure 1.4) [17,162,188,202].
Zn2* Cd2» Cu*’ V Co2* Nfî» I iV’ I II
1 1 1 11
Figure 1.4: Heavy métal toxicity is related to the aflfînity of a cation to sulfur. The logarithm of the minimal inhibitory concentration of heavy métal cations for E. coli (open circles) and Cupriavidus metalliduram (closed circles) was plotted against the logarithm of the solubility for the respective métal sulfide as a measure of the affmity to sulfur [164,192],
20 30 40 50
' In microbiology, MIC is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after ovemight incubation. MIC is important to confirm résistance of microorganisms to an antimicrobial agent and to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism .
1.4.3- OXIDATIVE STRESS
Heavy métal cations may bind to glutathione in Gram-negative bacteria and the resulting bisglutathionato-complexes tend to react with molecular oxygen to form oxidized bisglutathione (GS-SG) and hydrogen peroxide (H2O2). Since the oxidized bisglutathione has to be reduced again in an NADPH-dependent reaction and the métal cations immediately bind another two glutathione molécules, heavy métal cations cause a considérable oxidative stress [106,162].
Heavy métal cations can also stimulate the génération of reactive oxygen species (ROS) that modify the antioxidant defense and provoke oxidative stress . The majority of ROS are hydroxyl radicals generated in vivo by the métal catalyzed breakdown of hydrogen peroxide', according to the Fenton reaction (where M is a métal cation):
+ H2O2 + *OH + OH“
The most realistic in vivo production of hydroxyl radical according to Fenton reaction occurs when M is iron, copper, chromium or cobalt because of their redox-activity (in contrast with physiologically non-redox métal cations such as zinc or cadmium) [202,235], It has been estimated that one human cell is exposed to approximately 1.5x10^ oxidative hits a day from hydroxyl radicals and other such reactive species.
The hydroxyl radical is known to react with ail components of the DNA molécule: damaging both the purine and pyrimidine bases and also the deoxyribose backbone. Permanent modification of genetic material resulting from these oxidative damages represents the first step involved in mutagenesis, carcinogenesis, diseases of the nervous System and aging [225,235]. Metal- catalyzed damage to proteins involves oxidative scission of peptide bond, loss of histidine residues, bityrosine crosslink, introduction of carbonyl groups, and création of oxygen radicals.
Metal-induced génération of oxygen radicals results in the attack of polyunsaturated fatty acid of phospholipids, which are extremely sensitive to oxidation. Metal-catalyzed oxidative stress is an autocatalytic process , and activâtes death-signaling pathways leading to apoptosis.
1.5 - Heavymetals toxicityforhumans
There is no common way in which metals interact with cells or body function. Metals and métal compounds can interfère with virtually any physiological function. They can damage the liver, kidneys, lungs, the skeleton, the haematopoietic System, the gastrointestinal System, the cardiovascular System, they can be carcinogen, and can even cause psychological damages such as tremor, changes in personality, restlessness, anxiety, sleep disturbance and dépréssion .
Furthermore, most metals are capable of forming covalent bonds with carbon, resulting in metal- organic compounds. Such a transformation (e.g. by méthylation or alkylation) makes them liposoluble and therefore influences their mobility, accumulation as well as their toxicity. As metal-organic compounds are able to easily penetrate the blood-brain barrier, they are neurotoxic and interfère with the central nervous System .
' Hydrogen peroxide necessary for Fenton reaction can corne from the above-mentioned metal-catalyzed gluthatione oxidation.
Although adverse Health effects of heavy metals hâve been known for a long time, exposure to heavy metals continues and is even increasing in some areas. For example, mercury is still used in gold mining'. Arsenic is still common in pesticides, wood preservatives, and in the production of glass, paper, and semiconductors. It is also is a common contaminant in pharmaceuticals and is used for therapeutic purposes, e.g. for the treatment of chronic myeloid leukemia^, leishmaniasis^
and trypanosomiasis^. Tetraethyl lead remains a common additive to petrol, although this use has decreased dramatically in the developed coimtries (Figure 1.5) .
Figure 1.5: Lead concentrations in petrol and children’s blood (USA) .
The Minamata disease was the conséquence of the biggest heavy métal pollution provoking massive human victims (~20.000 persons contaminated and ~2.000 deaths) around 1950’s. It was caused by the industrial release of methylmercury into wastewaters. This organic form of mercury is liposoluble and bioaccumulated by shellfish and fish living in the Minamata Bay, which when eaten by the local population resulted in mercury poisoning.
Methylmercury is almost completely absorbed into the bloodstream and is transported freely into the organism, including across the blood-brain barrier and across the placenta.
' Mercury was used extensively in hydraulic gold mining to form mercury-gold amalgam. Mercury is still used in small scale, often clandestine, gold prospection, especially in China. It is the second source of mercury pollution in the World, after the buming of fossil fuels. Mercury was also used in silver mining.
^ Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfère with the production of normal blood cells. Arsenic trioxide is used to treat relapsed AML.
^ Leishmaniasis is caused by protozoan parasites and is transmitted by the bite of certain species of sand fly. Arsenic- induced cell death in protozoan is due to oxidative stress .
'* Trypanosomiasis is a group of several diseases in vertebrates caused by parasitic protozoan trypanosomes of the genus Trypanosoma, including ‘sleeping sickness’ transmitted by the tsetse fly (Glossina Genus), which can be fatal if left untreated. The first effective treatment (arsenic-based drug) was introduced in 1910 but blindness was a serious side effect.
Methylmercury does not only corne from an anthropogenic source (although it was so in the case of the Minamata disease). Methylmercury is formed from inorganic mercury by the action of anaérobie organisms that live in aquatic Systems . This méthylation process (carried out in anaerobiose by methylcobalamin, a coenzyme form of vitamin B12) converts inorganic mercury into more toxic, bioaccumulated and liposoluble methylmercury, making this heavy métal extremely toxic for superior organisms . The most dangerous mercury compound, dimethylmercury, is so toxic that even a few microliters spilled on the skin or even latex, PVC or neoprene glove (which are dimethylmercury-permeable) can cause death'. It is one of the most potent neurotoxins known^.
The Hatter's disease. One important use of mercury compounds was in hat making; 200 years ago, the fürs used to make beaver felt hats was dipped into mercury nitrate solution as a preservative. But the workers in the felt hat trade absorbed mercury through their skins; the resulting mercury poisoning caused shaking and slurred speech, being known as "hatter's disease", which is believed to hâve inspired the character of the Mad Hatter in Lewis CarroU's Alice in Wonderland (Figure 1.6).
Figure 1.6; The Mad Hatter character in Lewis CarroU's Alice in Wonderland. Drawings by John Tenniel.
The Basra poison grain disaster is another example of mass methylmercury poisoning incident which originated in September 1971 in the Iraqi port of Basra. Grain treated with a methylmercury-based fongicide that was intended for planting only was used by the rural population to make bread. More than 10.000 persons were poisoned by eating this methylmercury-polluted bread, and several hundred people died as a conséquence of the poisoning .
Itai-itai (literally "ouch-ouch") disease was a case of mass cadmium poisoning in Japan.
Exposure was caused by the release of cadmium by mining company into rivers used for irrigation of rice fields. The cadmium poisoning caused softening of the bones and kidney failure.
The disease is named for the severe pains caused in the joints and spine .
' Karen Wetterhahn, a former professer of chemistry, died of dimethylmercury intoxication: tests showed that a drop on her gloved hand would hâve penetrated the glove and started entering her skin within 15 seconds. She died one year later after experiencing severe neurological troubles and coma.
^ Reference: U.S. Department of Labor, Occupatlonal Safety and Health Administration; Hazard Information Bulletin on Dimethylmercury - http://www.osha.gov/dts/hib/hib_data/hib 19980309.html
1.6 - IN-DEPTH DESCRIPTION OF SELECTED HEAVY METALS
Hereafter, some heavy metals, which will, later on, be revealed as relevant in this manuscript, hâve been described more thoroughly.
1.6.1 - Zinc
Zinc is essential for life and is the second most important transition métal ion in living organisms after iron. Zinc-binding proteins are an abundant fraction of organism proteomes (up to 10%).
The number of zinc-binding proteins correlates linearly with the total number of proteins encoded by an organism genome. Zinc plays ubiquitous biological rôles: it interacts with a wide range of organic ligands, and has rôles in the metabolism of RNA and DNA, signal transduction, and gene expression, protein structure, and can act as a catalyst [32,86]. It also régulâtes apoptosis. Zinc can modulate brain excitability and plays a key rôle in synaptic plasticity and also in leaming.
However it has been called "the brain's dark horse" since it also can be a neurotoxin, suggesting zinc homeostasis plays a critical rôle in normal fiinctioning of the central nervous System [30,86,156]. Signs of zinc deficiency in humans include loss of appetite, growth retardation, skin changes, and immunological abnormalities .
Zinc is a good Lewis acid and an electrophile, making it a useful catalytic agent in hydroxylation and other enzymatic reactions, like stabilizing anionic reaction intermediates in the case of certain peptidases and hydrogenases, and generating reactive nucleophiles such as hydroxyls in the case of carbonic anhydrase . The métal also has a flexible coordination geometry (tetra-, penta- or hexa-coordinated), which allows zinc metallo-proteins to rapidly shift conformations to perform biological reactions. In contrast to other transition métal ions, such as copper and iron, zinc(II) does not undergo redox reactions due to its filled d orbital .
Escherichia coli has a very tightly regulated zinc quota (zinc atoms per cell); the bacteria concentrate zinc by several orders of magnitude relative to the concentration in a typical growth medium to a quota of ~2xl0^ atoms per cell (~0.1 mM) .
In E. coli, two Zn-sensing metalloregulatory proteins (Zur and ZntR), hâve been shown to switch off expression of zinc uptake machinery or switch on production of zinc efflux pumps when [Zn^’^]free exceeds an extraordinarily low threshold of 0.5 fM [99,173]. Considering the small volume of a cell, this is six orders of magnitude less than one free cytosolic zinc atom per cell and is inconsistent with the presence of any pool of free zinc in the absence of stress. Taken together, this suggests that the intracellular milieu has an extraordinary chélation capacity for zinc [66.173] , which might therefore be présent and shuttled in the cell essentially in a bound form [41.66.173] .
1.6.2 - Copper
Copper is essential for ail organisms and at the same time very toxic. This métal cation can hâve two redox States (Cu"^ and Cu^"^) within the physiological range. This essential characteristic is used by many oxido-reductive active enzymes where this cation plays a critical rôle, the most important enzymes being cytochrome c oxidase and superoxide dismutase .
But the copper redox capacity makes in tum this cation very toxic by catalyzing the formation of ROS causing important oxidative stress .