Trace metals: physiological, environmental effects and mechanisms of action

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Trace metals physiological, environmental
effects and mechanisms of action

• Topic 10

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Subtopics

• Characteristics of metals and basis for their
  biological interactions
• Uptake and transport of metals
• Detoxification and elimination
• Biotransformation
• Factors affecting bioavailability
• Bioaccumulation and biomagnification

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What are metals and what is the basis of their
biological interactions?
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Learning objectives

• To distinguish a metal from non-metal
• To learn biochemical metal classification
• To understand the basis of metal toxicity

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What is a metal?

• A substance with high electrical conductivity,
  luster, and malleability, which readily loses
  electrons to form positive ions (cations)

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Periodic Table of Elements
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Characteristic of metals as environmental
pollutants

• Non-degradable
• Some are essential

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Anthropogenic enrichment factors (AEF) for metals
in the biosphere
(All values in 106 kg per year)
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Itai-itai disease Cadmium poisoning
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Target organs of itai-itai disease
Kidney
Bone Looser zones
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Progression of itai-itai disease
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Mortality of itai-itai patients
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Minamata disease mercury poisoning
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Sourse of methylmercury Acetaldehyde production
plants
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Target organ of minamata disease Brain
Symptoms ?)Gait disturbance, loss of balance
(Ataxia)Speech disturbance (Dysarthria) (?)Sight
disturbance of peripheral areas in the visual
fields (Constriction of visual fields) (?)Stereo
anesthesia (Disturbance of sensation) (?)Muscle
weakness, muscle cramp (Disturbance of
movement) (?)Hardness of hearing (Hearing
disturbance) (?)Disturbance of sense of pain,
touch or temperature (Disturbance of sensation)
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Histopathology of minamata
Cerebrum, minamata patient (775 g)
Cerebrum, normal brain (1,580 g)
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Hotspots for mercury pollution
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Childhood lead poisoning

• Symptoms of lead poisoning in children
• Neurological problems
• Learning disabilities
• Lowered intelligence
• Behavioral problems
• In severe cases - seizures, comas, and death

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Childhood lead poisoning
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Classification of metals
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Borderline and Class B metals and metalloids are
important pollutants

• Nitrogen- and sulphur-seeking
• High affinity to proteins and other biological
  ligands

Guanine
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Essential metals

• 1/3 of known enzymes require metals for their
  function
• Metalloenzymes
• Fe2, Fe3, Cu2, Zn2, Mn2, Co2
• Metal-activated enzymes
• Na, K, Mg2, Ca2

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Amino acids with high affinity for metals
Cysteine and histidine
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Cytochrome bc1 complex, mitochondrial
Rieske iron-sulphur cluster
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Carbonic anhydrase
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Too much of a good thing?Basis of toxicity of
metals

• Substitution of essential metals in active
  centers of enzymes
• Interference with intracellular signaling
  pathways and Ca2 metabolism
• Oxidative stress (excessive production of free
  radicals)
• Interference with DNA transcription, translation
  and repair

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Mechanisms of uptake and transport of metals
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Learning objective

• To understand how metals enter the cell and are
  transported within the cell

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Lipid route

• Plays limited role in metal transport
• Hg may diffuse through the membrane in the form
  of neutrally charged chlorocomplexes
• Hg22Cl- ? HgCl2

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Aqueous routes

• Simple diffusion
• Yes
• Facilitated diffusion
• Very much so
• Active transport
• Little or no role

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Simple and facilitated diffusion

• Ion channels
• Ca2 channels
• can transport Zn2, Cd2, Hg2, Pb2
• SH-rich Zn2 channels
• can transport Cd2,
• Carrier proteins
• Divalent cation transporter 1 (DCT1)
• Major carrier protein for uptake of Fe2, Zn2
  but can also transport Cd2, Hg2, Pb2
• Molecular mimicry
• MeHg-L-cysteine methionine transporters

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Endocytosis

• Receptor-mediated endocytosis
• Iron-binding proteins - transferrin, ferritin,
  lactoferrin
• Can bind other metals

Out In
Out In
Out In
Fe3
Apotransferrin
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Take home messages

• There are multiple pathways of metal uptake into
  the cell
• No specific pathways of uptake exist for toxic
  metals
• Toxic metals use uptake routes, which have
  evolved for uptake of essential metals such as
  iron, copper and zinc

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Mechanisms of detoxification and elimination of
metals
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Learning goals

• To understand the role of metal binding in
  detoxification

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Detoxification of metals

• Biotransformation
• Not possible for most metals
• Biotransformation (methylation) of Hg makes it
  more toxic
• Binding to intracellular ligands
• Reduces the amount of biologically active form
  (free ion)
• Deposition of insoluble metal granules

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Intracellular ligands for metal binding

• Metallothioneins
• Glutathione
• CRP

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Metallothioneins

• Low molecular weight (60-68 aa, 6-7 kDa)
• Cysteine-rich
• In mammals 20 Cys, bind eqiuvalent of 7
  bivalent metals
• Cys positions are highly conserved

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Rat MT I
Blue crab MT II
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Metallothionein is induced by exposure to heavy
metals
Ivanina, Cherkasov, Sokolova, 2008 JEB
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Metallothionein induction protects cells against
Cd stress
Ivanina, Cherkasov, Sokolova, 2008 JEB
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Metallothionein protects from Cd toxicity
Experimental exposure to toxic Cd
levels Survival Cd-pretreatedgtcontrol Liver
damage ControlgtCd-pretreated
Klaasen Liu (1998)
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MT-knockout mice studies support protective role
of MT against Cd toxicity
5 weeks 10 weeks
Liu et al., 1999
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Liu et al., 1999
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Cellular functions of metallothionein

• Storehouse for Zn
• Protection against Cd-toxicity
• Free-radical scavenger

Which function is the most important?
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Short peptide metal chelators

• Glutathione
• Phytochelatins

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Phytochelatines
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Cysteine-rich (intestinal) protein
Zn2
Zn2
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Deposition of insoluble granules (invertebrates
only)
Calcium phosphate granule in a snail Littorina
littorea
Marigomez et al. (2002)
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Deposition of insoluble granules (invertebrates
only)
Lysosome-derived granule in a snail Littorina
littorea
Marigomez et al. (2002)
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Metal granules in mollusks
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Metals are persistent contaminants

• Bioaccumulation (bioconcentration)
• Yes
• Biomagnificantion
• Typically does not occur except for mercury

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Take-home messages

• Specialized proteins (metallothioneins) and
  polypeptides can protect cells from heavy metal
  toxicity by binding metals
• Cysteine has high affinity for metals and
  therefore is a key amino acid in metal-binding
  proteins
• Some invertebrates (mollusks, crustaceans,
  annelids) can detoxify metals by deposition and
  excretion of insoluble metal-containing granules

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Physiological mechanisms of heavy metal toxicity
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Learning goal

• To understand general mechanisms of metal-induced
  cellular damage

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General mechanisms of metal toxicity

• Metals have multiple intracellular targets
• Proteins
• Substitution of essential metals in active
  centers of enzymes
• Binding to thiol (SH) groups
• Oxidative damage
• Membranes
• Membrane permeability
• Oxidative stress
• DNA
• Interference with transcription, translation and
  repair
• Oxidative damage
• Interference with intracellular signaling
  pathways and Ca2 metabolism

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Oxidative damage

• A hallmark of heavy metal toxicity

Free radical (ROS, RNS)
Increase in free radical production
Decrease in antioxidants
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Heavy metals increase ROS production

• Direct effects
• Haber-Weiss reactions
• MeoxO2-? MeredO2
• MeredH2O2? Meox OH OH-
• Net H2O2O2-? O2OH OH-
• Indirect (inhibition of the mitochondrial
  electron transfer chain)

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Oxidative damage to DNA

• Single Cell Comet Assay
• Detects DNA fragmentation

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Oxidative damage to DNA

• TUNEL (TdT-mediated X-dUTP nick-end labeling)
  assay
• Detects free OH groups created by strand breakage

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TUNEL-detected DNA damage in Cd-exposed zebra
fish embryos
Control
100 ?M Cd
Chan Cheng (2002)
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Cd-induced apoptosis in zebrafish
Control embryo
Cd-exposed embryos
Cd-exposed embryo
Chan Cheng (2002)
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Oxidative DNA damage may lead to mutations

• AT GC transitions
• Deamination of adenine or cytosine
• GC ? G - - U (deamination) ? GC A - -U
  (replication) ? GC AT (replication)
• GC-TA transversions
• 8-hydroxyguanine
• GC ? 8HOGC ? 8HOG - - A GC ? TA GC
  (replication)

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Oxidative damage to proteins and lipids

• Lipids
• Malondialdehyde (MDA)
• Lipofuscin
• Proteins
• Carbonylation
• Loss of iron from the active center

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Mutagenicity of cadmium
Jin et al., 2003 Nature Genetics
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Inhibition of DNA repair
Isolated human cells exposed to Cd in vitro
Jin et al., 2003 Nature Genetics
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DNA transcription
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Toxic metal can affect function of zinc-finger
proteins
Hartwig (2001)
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Take-home messages

• Heavy metals affect a wide variety of
  intracellular molecules and functions
• Two major mechanisms of heavy metal toxicity are
• Binding to SH and nitro-groups of biomolecules
• Cofactor substitution, conformational changes,
  etc.
• Oxidative damage due to direct catalysis of ROS
  production and/or to inhibition of ETC in
  mitochondria