Detoxification and biotransformation of xenobiotics in...

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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 [email protected]

Transcript of Detoxification and biotransformation of xenobiotics in...

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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 [email protected]

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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….

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As

Cd Hg

Cu

Pb

Mn

Cr

Bioaccumulation

Integrated

biological effects

Time-integrated evaluation

Bioavailability

Use of bioindicator organisms and ecotoxicological

approach

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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

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Specific, sensitive exposure biomarkers

Trace metals

Cytochrome P-450 (biotransformation)

Metallothioneins

PAHs, TCDDs, PCBs

Organophosphate

carbammates

Cholinesterase

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nucleus

HRE DRE

ARNT AhR

Xe

ARNT

AhR

Xe

HIF-1α

ARNT

HIF-1α

Vascular endothelial growth factors VEGF; Glucose transporters; Glycolitic enzymes

HIF-1α

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)

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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)

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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

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Excretion of conjugated aromatic metabolites

conjugated

metabolites

Bile metabolites

EROD activity

0 0.1 1 10 50 mg/kg

B[a]P

Bile

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Biotransformation-mediated oxyradical production

P-450 R

Reductase

NADPH NADP

+

O2

R-OH

ROS

ROS

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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

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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+

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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-

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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

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Reduction of quinones

O2

O

O

O-

OH

OH 1 e

-

2 e-

O2-

quinone

semiquinone radical

hydroquinone

EXCRETION

DNA damage

Conjugation reactions

DT-diaphorase

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CYP3A

CYP2B

non-planar PCBs &

Pesticides

PXR CAR

RXR

PBRE

Peroxisomal

Proliferators (PP)

PPRE

PPARα,β,γ

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-1α

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

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

PPARα

RXR

PXR CAR

ARNT Nrf 2

p53

RXR

PPARα

RXR

RXR RXR PXR CAR

GRE MRE ARE

MTF-1 AhR

Xe

ARNT

AhR

Xe

HIF-1α

ARNT

HIF-1α

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-1α

Hypoxia

Trace metals

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

leackage of metal ions from storage, conversion of XDH to XO, increased Ca2+, impaired mitochondrial function)

Me Impairment of electron (i.e. mitochondrial) transfer chains

MAPKs

i-NOS, cell proliferation, differentation, apoptosis, inflammatory responses, survival, migration, carcinogenesis

Cell cycle arrest DNA repair Apoptosis

HO-1 Antioxidant enzymes Conjugating enzymes GSH metabolism Reducing equivalents MTF-1

APO-MT

Me/Zn

PAHs, PCBs, TCDDs, OCPs

HSP 90

AhR

HSP 90

AhR

Xe Cell cycle arrest Proliferation Apoptosis

RB

Bax

CYP 450 Enzymes Phase I (CYP1A, CYP1B) Phase II (GST, UDPGT, NQO1)

REDOX CYCLE

Q

Q•

HQ

Zn-Cu MTs

Zn

nucleus

cytoplasm

p53

Me/Zn

Me

Xe

Xe

PPARα

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Antioxidant Stress genes

Phase II genes

(1) BASAL

Degraded Nrf2

(2) ACTIVATION

(3) INDUCTION

(4) POST-INDUCTION

Electrophiles ROS

actin

Nrf2

E2

Ub

CuI3

Rbx1

Nrf2

E2

Ub

CuI3

Rbx1

Ub Ub

ARE

Maf Nrf2 Nrf2

Nrf2

Nrf2

Gene expression mediated by Nrf2 pathway…

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TARGET GENES mRNA levels

Gene expression and Nrf2 pathway in eels

hepatocytes exposed to H2O2…

CAT

0,0

0,5

1,0

1,5

2,0

C 3h 6h 6h +

6h dep.

12h 12h +

12h dep.

mR

NA

levels

(A

.U.)

*

* *

GPx1

0,0

0,4

0,8

1,2

1,6

C 3h 6h 6h +

6h dep.

12h 12h + 12h

dep.

mR

NA

le

ve

ls (

A.U

.)

*

*

GSTpi

0,0

0,5

1,0

1,5

C 3h 6h 6h +

6h dep.

12h 12h + 12h

dep.

mR

NA

le

ve

ls (

A.U

.)

*

*

* *

TARGET GENES

• Common transcriptional trends

suggest common regulation

• Induction after increased oxidative

pressure: restoring redox balance

• Down-regulation after prolonged

oxidative pressure: impairment

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NRF2 and KEAP1 mRNA levels

Gene expression and Nrf2 pathway in eels

hepatocytes exposed to H2O2…

KEAP1

0,0

0,6

1,2

1,8

C 3h 6h 6h +

6h dep.

12h 12h + 12h

dep.

mR

NA

le

ve

ls (

A.U

.) *

NRF2

0,0

0,4

0,8

1,2

1,6

C 3h 6h 6h +

6h dep.

12h 12h + 12h

dep.

mR

NA

le

ve

ls (

A.U

.)

* *

NRF2

• ARE-like sequence found in its promoter (Kwak et al, 2002)

• A de novo synthesis of Nrf2 may be

required to sustain the nuclear

accumulation and Nrf2-mediated induction

of target genes

KEAP1

• 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)

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0

10

20

30

40

50

60

Ctrl BaP TCDD Cd Cd-TCDD

*

*

Oxidative interactions between metabolism of

different classes of chemicals

EROD

pmol/min/mg prot. Reductase

NADPH

NADP+

ROH

R

O2

Cyt. P450

0

100

200

300

400

Ctrl BaP TCDD Cd Cd-TCDD

*

**

Cadmium in liver µg/g dry weight

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Ca2+

Ah-R

xenobiotics

Cd2+

Cd2+

Cd2+

Cd2+

Cd2+

Cd2+

Ca2+

Ca2+

Ca2+

CYP450

Cd2+

Cd2+

Cd2+

0

20

40

60

80

100

CTRL BaP TCDD Cd Cd-TCDD

S9

microsomi

citosol

% distribution of cadmium

ROS

HO-1

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Interactions between different classes of chemicals

0

40

80

120

160

*

Protein

(CYP1A1) CTRL BaP Cd+ BaP Hg+BaP Pb+BaP Ni+BaP Cu+BaP

EROD activity

0

20

40

60

80

100

120

mRNA W.B EROD mRNA W.B EROD

BaP

30% 20%

55%

43%

70%

94%

Gene expression

(% mRNA compared to

BaP exposed organisms)

*

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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

Cd/BaP Hg/BaP Pb/BaP Ni/BaP Cu/BaP

Variations during

co-exposures

Cd2+

ROS HO-1 -SH

translation

activity

CYP450 transcription

mRNA

transcription

mRNA

translation activity

Hg

Pb

Ni

BaP

Cu

?

BaP

Cu

transcription

mRNA translation activity

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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 ….??

how should antioxidants react to increased pro-oxidant pressure ?

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Gene expression versus catalytic activities…

HP MP HP MP

0

50000

100000

150000

200000

250000

300000

CYP1A transcription

mR

NA

(co

py n

.)

LIVER GILLS

a a b b

c c

d

e

HP MP HP MP

0

2

4

6

8

10

12

14

50

100

150

200

250

pm

ol/m

in/m

g p

rot.

EROD activity

LIVER GILLS

a a b

c

d

cd

e

f

HP MP HP MP

0

50000

100000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000GSTpi transcription

mR

NA

(co

py n

.)

LIVER GILLS

a a a

a a b

c

d

HP MP HP MP

0

200

400

600

800

1000

1200

1400

GST activity

nm

ol/m

in/m

g p

rot.

LIVER GILLS

a

a

a

b

b

b

b

b

HP MP HP MP

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

20000

30000

40000

50000

60000

70000

80000

CAT transcription

mR

NA

(copy n

.)

LIVER GILLS

a

b ab

ab

c

c c

d

HP MP HP MP

0

10

20

100

200

300

400

500

CAT activity

µm

ol/m

in/m

g p

rot.

LIVER GILLS

a a a a

b b

c

d

HP MP HP MP

0

100000

200000

300000

400000

500000

GPx1 transcription

mR

NA

(co

py n

.)

LIVER GILLS

a a a a

b b

c

d

HP MP HP MP

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500 GPx activity

nm

ol/m

in/m

g p

rot.

LIVER GILLS

a a a a b b

c

c

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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

Gene expression versus catalytic activities…

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Transcription factors (NF-kB, AP-1, Nrf2), but also post-

translational modifications of proteins modulate with non-

genomic 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

Gene expression versus catalytic activities…

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Conclusions

Complex interactions occur between metabolism of chemicals, pro-

oxidant 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 post-

translational)….

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