Francesco Regoli
Detoxification and biotransformation of
xenobiotics in marine organisms
Department of Life and Environmental Sciences
Polytechnic University of Marche-Ancona [email protected]
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
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
Specific, sensitive exposure biomarkers
Trace metals
Cytochrome P-450 (biotransformation)
Metallothioneins
PAHs, TCDDs, PCBs
Organophosphate
carbammates
Cholinesterase
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)
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
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
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
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
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…
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
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)
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
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
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)
*
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
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 ?
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
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…
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…
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