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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Minamata disease mercury 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
Leung Furness (1998)
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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