Detoxification and biotransformation of xenobiotics in marine organisms

Transcription

1 Detoxification and biotransformation of xenobiotics in marine organisms Francesco Regoli Department of Life and Environmental Sciences Polytechnic University of Marche-Ancona

2 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.

3 Use of bioindicator organisms and ecotoxicological approach Cd Bioaccumulation Integrated biological effects Hg Time-integrated evaluation Bioavailability As Cr Cu Mn Pb

4 BIOMARKERS NADPH NADP + O 2 - DNA DAMAGE METALS AND XENOBIOTICS MIXTURES LYSOSOMES METALS AND XENOBIOTICS MIXTURES Reductase P-450 R O 2 ROH H 2 O 2 ROS HO GSH metabolism OXIDATIVE STRESS HO GENOTOXIC DAMAGE LYSOSOMAL MEMBRANE DESTABILIZATION METALLOTHIONEINS ANTIOXIDANT DEFENCES CAT, SOD, GPX, GST, GR, GSH

5 Specific, sensitive exposure biomarkers Trace metals PAHs, TCDDs, PCBs Organophosphate carbammates Cytochrome P-450 (biotransformation) Metallothioneins Cholinesterase

6 Cytochrome P-450 (biotransformation) PAHs, PCBs, TCDDs, OCPs Xe CYP 450 Enzymes Phase I (CYP1A, CYP1B) HSP 90 AhR RB Xe Phase II (GST, UDPGT, NQO1) HSP 90 AhR Xe Cell cycle arrest Proliferation Apoptosis cytoplasm Vascular endothelial growth factors VEGF; Glucose transporters; Glycolitic enzymes Bax Hypoxia Other no-cyp proteins (some with unknown functions) HIF-1α HIF-1α Xe AhR ARNT HIF-1α ARNT ARNT Xe AhR ARNT Xe AhR HRE DRE DRE nucleus nucleus

7 P450 (Fe 3+ ) Hepatic biotansformation of xenobiotics (PAHs, dioxins, PCBs) RH (xenobiotic) P450 (Fe 3+ ) RH e - NADPH-P450 reductase P450 (Fe 2+ ) RH O 2 P450 (Fe 2+ ) RH O 2 e - NADPH-P450 reductase P450 (Fe 2+ ) RH O 2 2H + Toxic metabolites adduct to DNA (BaP 7,8-dihydrodiol-9,10-oxide) P450 (Fe 3+ ) + H 2 O + R-OH Phase II reactions, conjugation with glutathione, glucoronic acid, sulfate EXCRETION Conjugated bile metabolites

8 The CYP450 biotransformation pathway Phase I BaP CYP1A Adducts with macromolecules Phase II Glutathione S-transferases GSH conjugate of BaP 7,8-oxide or BaP 9,10 oxide Sulpho-transferases BaP 7 sulphate or BaP 9 sulphate BaP 7,8-oxide or BaP 9,10 oxide 7or 9-OH BaP Non enzymatic Glucoronosyltransferases BaP 7 glucoronide or BaP 9 glucoronide Epoxide hydrolase and non enzymatic BaP 7,8-dihydrodiol or BaP 9,10 dihydrodiol Glucoronosyltransferases Glucoronide conjugate of BaP dihydrodiol BaP 7,8-dihydrodiol-9,10-oxide or BaP 7,9-oxide-9,10 dihydrodiol CYP1A Sulphotransferases Sulphate conjugate of BaP dihydrodiol Epoxide hydrolase and non enzymatic BaP 7,8,9,10-tetraols

9 Excretion of conjugated aromatic metabolites Bile conjugated metabolites Bile metabolites EROD activity B[a]P mg/kg

10 Biotransformation-mediated oxyradical production ROS NADPH NADP + Reductase P-450 R O 2 R-OH ROS

11 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 Trace Metals Metallothioneins Impairment of electron (i.e. mitochondrial) transfer chains Catalytic or enzymatic reactions (Fenton and Haber Weiss reactions, Oxido-Reductase reactions, Formation of thiol radicals, Changes of metal chemical speciation) metal n+ + H 2 O 2 metal n+1 + OH + OH - Depletion of antioxidants (-SH) Cellular damages ROS

12 Trace metal-mediated oxyradical production Fenton-type reactions metal n+ + H 2 O 2 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 H 2 O 2 and lipid peroxides to generate OH and lipid radicals Haber-Weiss reactions metal n+1 + O 2 - metal n+ + O 2 metal n+ + H 2 O 2 metal n+1 + OH + OH- overall O H 2 O 2 metal n+1 /metal n+ OH +O 2 + OH- Of particular importance during phagocytosis when a large amount of O 2 - is generated and a limited amount of metal is needed as catalyst

13 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: metal n+1 + RSH metal n+ + RS RS + RSH RSSR + H + RSSR + O 2 RSSR + O Some metals (i.e. chromate and vanadate) are reduced by flavoenzymes, glutahione reductase, lipoyl dehydrogenase, ferroxin- NADP + to generate active intermediates reacting with O 2 to form O Arsenite activate NADH oxidase and produce O 2 -

14 The redox cycle: (PAHs metabolites, quinones, nitroaromatics, nitroamines, organometallics ) prooxidant challenge.. toxicity O 2 - Cyclic generation of superoxide anion Parent compound ( R ) e - Radical metabolite ( R ) Flavoenzymes NADP + NADPH + H + O 2 toxicity

15 Reduction of quinones O O 2 - DT-diaphorase O O 2 1 e - O quinone 2 e - hydroquinone OH O - semiquinone radical OH Conjugation reactions DNA damage EXCRETION

16 non-planar PCBs & Pesticides Peroxisomal Proliferators (PP) PAHs, PCBs, TCDDs, Pesticides Bax PXR CAR PPARα,β,γ AhR cell cycle arrest proliferation apoptosis inflammatory apoptosis RXR PBRE RXR PPRE ARNT DRE RB HIF-1α Vascular endothelial growth factor VEGF; Glucose transporters; Glycolytic enzymes NF-kB CYP3A CYP2B Peroxisomal enzymes CYP450 Ah gene battery (i.e. GST, UDPGT, NQO1..) Q MAPK direct reactions inos e - transfer chains Antiox. depletion ROS Xenobiotics metabolism PAHs metabolites Redox cycle excretion HQ (NQO1) Q HO-1 Trace metals cellular damages Nrf2-Keap1 DNA damage ARE conjugating enzymes antioxidant enzymes GSH metabolism reducing equivalents HO-1

17 Me Depletion of antioxidants (i.e. SH) Me Zn-Cu MTs MTF-1 Zn Me/Zn Trace metals APO-MT NF-KB MAPKs i-nos, cell proliferation, differentation, apoptosis, inflammatory responses, survival, migration, carcinogenesis NFAT Oxidative cell damages (i.e. decomposition of peroxides, leackage of metal ions from storage, conversion of XDH to XO, increased Ca 2+, impaired mitochondrial function) Impairment of electron (i.e. mitochondrial) transfer chains Catalytic or enzymatic reactions; Haber-Weiss, Fenton, NADPH oxidase, formation of thiol radicals, changes of metal chemical speciation HO-1 Antioxidant enzymes Conjugating enzymes GSH metabolism Reducing equivalents AP-1 Cell cycle arrest DNA repair Apoptosis p53 Nrf 2 Keap 1 Nrf 2 Q HQ Keap 1 CYP 450 Enzymes Phase I (CYP1A, CYP1B) Phase II (GST, UDPGT, NQO1) Q REDOX CYCLE ROS Vascular endothelial growth factors VEGF; Glucose transporters; Glycolitic enzymes Hypoxia HIF-1α HSP 90 cytoplasm CAR HSP 90 Non planar PCBs and OCPs HSP 90 HSP 90 CAR HSP 90 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) PXR AhR Xe AhR Bax HSP 90 PXR PAHs, PCBs, TCDDs, OCPs RB Xe Xe Cell cycle arrest Proliferation Apoptosis Peroxisomal proliferation Release of trace metals from peroxisomes Fatty acid metabolism Lipid homeostasis Hepatocarcinogenesis Peroxisomal proliferators (PP) PPARα p53 HIF-1α ARNT Xe AhR CAR RXR PXR RXR PPARα GRE Me/Zn MTF-1 ARE MRE NF-KB NF-KB RE NFAT AP-1 NFAT AP-1 Binding site p53 p53 RE DNA damage Xe Nrf 2 HIF-1α ARNT ARNT AhR CAR RXR PXR RXR RXR PPARα ARE HRE DRE PBRE XRE PPRE nucleus

18 Gene expression mediated by Nrf2 pathway actin Nrf2 CuI3 Rbx1 Ub E2 (1) BASAL Nrf2 CuI3 Ub Rbx1 Ub E2 Ub Degraded Nrf2 (2) ACTIVATION Electrophiles ROS Nrf2 (4) POST-INDUCTION Nrf2 Nrf2 Nrf2 Maf ARE (3) INDUCTION Phase II genes Antioxidant Stress genes

19 mrna levels (A.U.) mrna levels (A.U.) mrna levels (A.U.) Gene expression and Nrf2 pathway in eels hepatocytes exposed to H 2 O 2 2,0 1,5 TARGET GENES mrna levels CAT 1,0 0,5 0,0 1,6 1,2 C 3h 6h 6h + 6h dep. GPx1 12h 12h + 12h dep. TARGET GENES Common transcriptional trends suggest common regulation 0,8 0,4 Induction after increased oxidative pressure: restoring redox balance 0,0 1,5 C 3h 6h 6h + 6h dep. GSTpi 12h 12h + 12h dep. Down-regulation after prolonged oxidative pressure: impairment 1,0 0,5 0,0 C 3h 6h 6h + 6h dep. 12h 12h + 12h dep.

20 mrna levels (A.U.) mrna levels (A.U.) Gene expression and Nrf2 pathway in eels hepatocytes exposed to H 2 O 2 NRF2 and KEAP1 mrna levels NRF2 1,6 1,2 NRF2 ARE-like sequence found in its promoter (Kwak et al, 2002) 0,8 0,4 0,0 C 3h 6h 6h + 6h dep. 12h 12h + 12h dep. A de novo synthesis of Nrf2 may be required to sustain the nuclear accumulation and Nrf2-mediated induction of target genes 1,8 1,2 KEAP1 KEAP1 0,6 0,0 C 3h 6h 6h + 6h dep. 12h 12h + 12h dep. ARE-sequence found in its promoter: Keap1 target of Nrf2 (Lee et al., 2007) possible feedback mechanism to balance level of Nrf2 and potentially switch off the response when oxidative pressure decreases (Keap1 as post-induction repressor)

21 Oxidative interactions between metabolism of different classes of chemicals EROD pmol/min/mg prot. NADPH Reductase NADP Ctrl BaP TCDD Cd Cd-TCDD R Cyt. P450 O Cadmium in liver µg/g dry weight ROH 0 Ctrl BaP TCDD Cd Cd-TCDD

22 Ca % distribution of cadmium S9 microsomi citosol Cd 2+ CTRL BaP TCDD Cd Cd-TCDD Cd 2+ Ah-R xenobiotics Cd 2+ Cd 2+ Ca 2+ Cd 2+ Ca 2+ Ca 2+ Cd 2+ Cd 2+ ROS CYP450 Cd 2+ Cd 2+ HO-1

23 Interactions between different classes of chemicals EROD activity 40 0 Protein (CYP1A1) CTRL BaP Cd+ BaP Hg+BaP Pb+BaP Ni+BaP Cu+BaP % BaP Gene expression (% mrna compared to BaP exposed organisms) % 55% 20% 43% 30% 0 mrna W.B EROD mrna W.B EROD

24 Variations during co-exposures Cd/BaP Hg/BaP Pb/BaP Ni/BaP Cu/BaP GPx GST GR GSH CAT TOSC HO TOSC ROO GPx GST GR TOSC ROO GPx GR TOSC ROO GPx GST GR TOSC ROO GST GR CAT BaP transcription mrna Cd 2+ ROS HO-1 -SH translation CYP450 activity Hg Pb Ni transcription mrna translation activity BaP Cu? transcription mrna translation activity Cu

25 how should antioxidants react to increased pro-oxidant pressure? Gene expression versus catalytic activities..do they really say the same? Frequently measured as alternative approaches Inconsistent trends often reported.. Molecular responses typically considered more sensitive and less variable.but the link between effects at different cellular levels is not easily elucidated which is the biological significance of transcriptional versus functional responses.??

26 mrna (copy n.) mrna (copy n.) Gene expression versus catalytic activities pmol/min/mg prot. µmol/min/mg prot. mrna (copy n.) mrna (copy n.) nmol/min/mg prot. nmol/min/mg prot CYP1A transcription EROD activity e 250 f d 50 e 14 d 12 cd 10 c 8 6 c c 4 b b a a 2 a a b 0 HP MP HP MP HP MP HP MP LIVER GILLS LIVER GILLS GSTpi transcription GST activity b a a a a d c a HP MP HP MP LIVER GILLS b b b b b a a a HP MP HP MP LIVER GILLS CAT transcription d c c c b ab ab a HP MP HP MP LIVER GILLS CAT activity d c b b a a a a HP MP HP MP LIVER GILLS GPx1 transcription d c b b a a a a HP MP HP MP LIVER GILLS GPx activity c c b b a a a a HP MP HP MP LIVER GILLS

27 Gene expression versus catalytic activities Delayed time-course responses due to different timing for post-transcriptional processes and protein synthesis, Generally shorter half life of mrna compared to corresponding protein. Tissue-dependent variations of transcriptional and catalytic responses. Compared to liver, gills have a less efficient cellular machinery to convert a greatly enhanced transcriptional signal into protein synthesis. Bi-phasic responses of enzymes which become target of toxicity at prolonged/acute exposures (not genes) Mixtures of xenobiotics/ros can differently affect transcriptional and catalytic responses

28 Gene expression versus catalytic activities Transcription factors (NF-kB, AP-1, Nrf2), but also posttranslational modifications of proteins modulate with nongenomic effects Post-translational modifications seem reversible at low dose or short term exposures, and irreversible at high dose or chronic exposures These effects are expected to have a greater influence after chronic exposures in field conditions when also adaptation mechanisms and confounding effects of environmental variables may likely occur

29 Conclusions Complex interactions occur between metabolism of chemicals, prooxidant and antioxidant mechanism, which are fundamental in adaptation to stressful conditions and in mediating biological effects and toxicity Oxidative pathways modulate effects with direct, indirect and cascade responses Importance of co-exposures to chemical mixtures Interactions can occur at different levels (from transcriptional to posttranslational). Gene expression responses do not necessarily parallel functional effects at cellular/catalytic level mrna levels of biotransformation and antioxidant enzymes could represent a snapshot of cell activity at a given time, not an effective endpoint of environmental pollutants