Detoxification and biotransformation of xenobiotics in ... Detoxification and biotransformation

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  • Francesco Regoli

    Detoxification and biotransformation of

    xenobiotics in marine organisms

    Department of Life and Environmental Sciences

    Polytechnic University of Marche-Ancona f.regoli@univpm.it

  • Chemical pollutants in the marine environments

    Trace metals and organometallics

    Polycyclic Aromatic Hydrocarbons (PAHs)

    Halogenated hydrocarbons (Pesticides, PCBs, Dioxins)

    Organophosphates

    Estrogenic chemicals

    Pharmaceuticals and Personal Care Products PPCP

    Nanoparticles

    Microplastics.

  • As

    Cd Hg

    Cu

    Pb

    Mn

    Cr

    Bioaccumulation

    Integrated

    biological effects

    Time-integrated evaluation

    Bioavailability

    Use of bioindicator organisms and ecotoxicological

    approach

    http://ens.lycos.com/ens/pics5/pesticides.jpg

  • METALS AND

    XENOBIOTICS

    MIXTURES

    METALS AND

    XENOBIOTICS

    MIXTURES

    LYSOSOMES

    METALLOTHIONEINS

    ANTIOXIDANT

    DEFENCES CAT, SOD, GPX, GST, GR, GSH

    HO

    ROS

    O2-

    H2O2

    OXIDATIVE

    STRESS

    LYSOSOMAL MEMBRANE

    DESTABILIZATION

    HO

    DNA DAMAGE

    GENOTOXIC

    DAMAGE

    P-450 R

    Reductase

    NADPH NADP+

    O2

    ROH

    GSH metabolism

    BIOMARKERS

    http://www.unizh.ch/~mtpage/PIC/ratmt.jpghttp://images.google.it/imgres?imgurl=www.kidsregen.org/krrn/discoveries/nat_moves/1102/images/mitochondria.jpg&imgrefurl=http://www.kidsregen.org/krrn/discoveries/nat_moves/home.shtml&h=200&w=200&prev=/images%3Fq%3Dmitochondria%26svnum%3D10%26hl%3Dit%26lr%3D%26ie%3DUTF-8%26sa%3DG

  • Specific, sensitive exposure biomarkers

    Trace metals

    Cytochrome P-450 (biotransformation)

    Metallothioneins

    PAHs, TCDDs, PCBs

    Organophosphate

    carbammates

    Cholinesterase

    http://www.unizh.ch/~mtpage/PIC/ratmt.jpg

  • nucleus

    HRE DRE

    ARNT AhR

    Xe

    ARNT

    AhR

    Xe

    HIF-1

    ARNT

    HIF-1

    Vascular endothelial growth factors VEGF; Glucose transporters; Glycolitic enzymes

    HIF-1

    Hypoxia

    CYP 450 Enzymes Phase I (CYP1A, CYP1B)

    Phase II (GST, UDPGT, NQO1)

    nucleus

    cytoplasm

    PAHs, PCBs, TCDDs, OCPs

    HSP 90

    AhR

    HSP 90

    AhR

    Xe Cell cycle arrest Proliferation Apoptosis

    RB

    Bax

    Xe

    Xe

    DRE

    ARNT AhR

    Xe

    Other no-CYP proteins (some with unknown

    functions)

    Cytochrome P-450 (biotransformation)

  • Conjugated bile

    metabolites

    P450 (Fe3+)

    P450 (Fe3+)__RH

    RH (xenobiotic)

    P450 (Fe2+)__RH

    NADPH-P450 reductase

    O2

    P450 (Fe2+)__RH

    O2

    P450 (Fe2+)__RH

    O2

    2H+

    P450 (Fe3+) + H2O + R-OH

    e-

    NADPH-P450 reductase e-

    Toxic metabolites adduct to DNA

    (BaP 7,8-dihydrodiol-9,10-oxide)

    Phase II reactions, conjugation

    with glutathione, glucoronic acid, sulfate

    EXCRETION

    Hepatic biotansformation of xenobiotics

    (PAHs, dioxins, PCBs)

  • BaP

    BaP 7,8-oxide or

    BaP 9,10 oxide

    GSH conjugate of

    BaP 7,8-oxide or

    BaP 9,10 oxide

    BaP 7 sulphate or

    BaP 9 sulphate

    7or 9-OH BaP

    BaP 7 glucoronide or

    BaP 9 glucoronide

    BaP 7,8-dihydrodiol or

    BaP 9,10 dihydrodiol

    Glucoronide conjugate of

    BaP dihydrodiol

    Sulphate conjugate of

    BaP dihydrodiol

    BaP 7,8-dihydrodiol-9,10-oxide or

    BaP 7,9-oxide-9,10 dihydrodiol

    Adducts with macromolecules

    BaP 7,8,9,10-tetraols

    CYP1A

    Glutathione

    S-transferases

    Non enzymatic

    Sulpho-transferases

    Glucoronosyl-

    transferases

    Epoxide hydrolase

    and non enzymatic

    Glucoronosyl-

    transferases

    Sulpho-

    transferases

    CYP1A Epoxide hydrolase

    and non enzymatic

    The CYP450 biotransformation pathway

    Phase I

    Phase II

  • Excretion of conjugated aromatic metabolites

    conjugated

    metabolites

    Bile metabolites

    EROD activity

    0 0.1 1 10 50 mg/kg

    B[a]P

    Bile

  • Biotransformation-mediated oxyradical production

    P-450 R

    Reductase

    NADPH NADP

    +

    O2

    R-OH

    ROS

    ROS

  • Impairment of electron

    (i.e. mitochondrial)

    transfer chains

    Depletion of

    antioxidants

    (-SH)

    ROS

    Cellular damages

    decomposition of formed peroxides, release of metal ions

    from storage sites, haeme protein release, conversion of

    xanthine dehydrogenase to xanthine oxidase, impaired

    mitochondrial function and raised intracellular Ca2+ levels

    Metallothioneins

    Catalytic or enzymatic reactions (Fenton and Haber Weiss reactions,

    Oxido-Reductase reactions,

    Formation of thiol radicals,

    Changes of metal chemical speciation)

    metal n+ + H2O2 metal n+1 + OH + OH-

    Trace Metals

    http://www.unizh.ch/~mtpage/PIC/ratmt.jpg

  • Trace metal-mediated oxyradical production

    Fenton-type reactions

    metal n+ + H2O2 metal n+1 + OH + OH-

    Fe (II) Cu (I) Cr (III), (IV), (V) V(V) Co (II) very low* Ni(II) very low*

    * In the presence of chelating agents, such as Gly-Gly-His and thiol-containing agents,

    these metal ions react with H2O2 and lipid peroxides to generate OH and lipid radicals

    Haber-Weiss reactions

    metal n+1 + O2- metal n+ + O2

    Of particular importance during phagocytosis when a large amount of O2- is generated

    and a limited amount of metal is needed as catalyst

    metal n+ + H2O2 metal n+1 + OH + OH-

    O2- + H2O2 OH +O2 + OH-

    overall metal n+1/metal n+

  • metal n+1 + RSH metal n+ + RS

    Other mechanisms for trace metal-mediated ROS

    production

    - Thiol radicals are generated by reaction with some metals (i.e. Cr VI)

    leading to toxicity or other thiol radicals:

    RS + RSH RSSR + H+

    RSSR + O2 RSSR + O2-

    - Some metals (i.e. chromate and vanadate) are reduced by

    flavoenzymes, glutahione reductase, lipoyl dehydrogenase, ferroxin-

    NADP+ to generate active intermediates reacting with O2 to form O2- .

    - Arsenite activate NADH oxidase and produce O2-

  • The redox cycle: (PAHs metabolites, quinones, nitroaromatics, nitroamines, organometallics)

    toxicity

    prooxidant

    challenge..

    toxicity

    Parent compound

    ( R )

    Radical metabolite

    (R )

    O2 -

    NADPH + H+

    NADP+

    Flavoenzymes e-

    Cyclic generation

    of superoxide anion

    O2

  • Reduction of quinones

    O2

    O

    O

    O-

    O

    OH

    OH 1 e

    -

    2 e-

    O2-

    quinone

    semiquinone radical

    hydroquinone

    EXCRETION

    DNA damage

    Conjugation reactions

    DT-diaphorase

  • CYP3A

    CYP2B

    non-planar PCBs &

    Pesticides

    PXR CAR

    RXR

    PBRE

    Peroxisomal

    Proliferators (PP)

    PPRE

    PPAR,,

    RXR

    Peroxisomal

    enzymes

    DRE

    PAHs, PCBs, TCDDs,

    Pesticides

    AhR

    ARNT

    CYP450

    Ah gene battery

    (i.e. GST, UDPGT,

    NQO1..)

    ROS

    Trace metals

    direct

    reactions

    e- transfer

    chains

    Antiox.

    depletion

    cellular

    damages

    Xenobiotics metabolism

    Q

    Redox cycle

    PAHs

    metabolites

    DNA damage

    excretion

    Q

    HQ (NQO1)

    HIF-1

    Vascular endothelial

    growth factor VEGF;

    Glucose transporters;

    Glycolytic enzymes

    cell cycle arrest

    proliferation

    apoptosis

    Bax

    RB

    MAPK

    NF-kB

    iNOS

    HO-1

    inflammatory

    apoptosis

    Nrf2-Keap1 ARE

    conjugating enzymes

    antioxidant enzymes

    GSH metabolism

    reducing equivalents

    HO-1

  • PPRE ARE HRE DRE PBRE XRE p53 RE NF-KB RE

    PPAR

    RXR

    PXR CAR

    ARNT Nrf 2

    p53

    RXR

    PPAR

    RXR

    RXR RXR PXR CAR

    GRE MRE ARE

    MTF-1 AhR

    Xe

    ARNT

    AhR

    Xe

    HIF-1

    ARNT

    HIF-1

    DNA damage

    Nrf 2

    Nrf 2

    Keap 1

    Keap 1

    AP-1

    Binding site

    AP-1 NF-KB

    AP-1

    p53

    NFAT

    NF-KB

    NFAT

    NFAT

    PXR CAR

    PXR CAR

    HSP 90 HSP 90

    HSP 90 HSP 90

    Non planar PCBs and OCPs

    Peroxisomal proliferators

    (PP)

    Peroxisomal proliferation Release of trace metals from peroxisomes Fatty acid metabolism Lipid homeostasis Hepatocarcinogenesis

    Catalytic or enzymatic reactions; Haber-Weiss, Fenton, NADPH oxidase, formation of thiol radicals, changes of metal chemical speciation

    ROS

    Phase I (i.e. CYP3A, CYP2C, CYP2B, CYP2A) Phase II (i.e. GSTA2, UGT1A, SULT1A) Phase III and trasporters (i.e. MDR1, MRP1, MRP2, MRP4, OATP2)

    Vascular endothelial growth factors VEGF; Glucose transporters; Glycolitic enzymes

    HIF-1

    Hypoxia

    Trace metals

    Depletion of antioxidants (i.e. SH) Oxidative cell damages (i.e. decomposition of peroxides,

    leackage of metal io