dieter schrenk lebensmittelchemie und toxikologie ......•2,3,7,8-tetrachlorodibenzo-p-dioxin...
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Schrenk 2010
Dieter Schrenk
Lebensmittelchemie und Toxikologie
Technische Universität Kaiserslautern
2010
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
1. Introduction
•2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been classified as a group I carcinogen by IARC
• TCDD exerts most (if not all) toxic effects through mechanisms involving the aryl hydrocarbon receptor (AHR)
• TCDD acts as a potent liver tumour promoter in two- step rodent carcinogenesis models (Campbell et al., 1992)
• when applied to rodents without pre-treatment with an initiator, TCDD also leads to increased cancer incidences (Kociba et al., 1976)
Schrenk 2010
1. Introduction
-0.5 0.0 0.5 1.0 1.5 2.0 2.5
0
10
20
30
40
50
60To
tal a
nim
als
with
neop
lasm
s(%
)
lg Dose (ng TEq/kg b.w. x day)
Aroclors
[Mayes
et al., 1998]TCDD [Kociba
et al., 1976]
Liver
neoplasms
in female
rats
treated
with
TCDD or
Aroclors
TEq
are
carcinogenic
in the
liver
of female
rats
Schrenk 2010
1. Introduction
OO
O
Ligand
CytoplasmA
hR
Chaperones AhR
binding
Nucleus
Translocati
on
AR
NT
Ah
R
AR
NT
Binding to DRE
DNA DRE
Transcripti
onCYP1A1 mRNA
O
O
O
O
Schrenk 2010
Signal transduction
I
AhR ARNT
XRE
CYP1A1 etc.
OO
O
OO
O
PAI-2
PAI-2fos/jun
PAI-2TGFα
PAI-2IL-1ß
transcriptional
1. Introduction
Schrenk 2010
cellular
effects
and signal
transduction
II
ROS
AhR ARNT
ERα/ßERα/ß XRE
CYP1A1 etc.
OO
O
OO
O
Metabolism
c-src
Phosphorylation
of target
proteins
A. Drug metabolismB. oxidative
stress
D. Protein phosphorylation
E. growth inhibition
F. suppression
of contact
inhibition
C. antiestrogenic
effects
D
A B
C
EGFR
?
p27*
E
E
*an inhibitor of cyclin-dependent kinases
cyclinA/cdk2
F
1. Introduction
?
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotors
Glutathione S-transferase
P –
positive preneoplastic
focus in rat liver (eight cell stage)
Nuclei
of normal hepatocytes
Tumor promotion
(rat liver) with
PCDDs*
*Schrenk et al. (1994) Carcinogenesis
15, 509
DEN
2 weeks 2 weeks
fr. interv. PCDDs13 weeks
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
initiation proliferati
on
apoptosis
progression
(tumor)DNA damage
hepatocyte/
hepatic
stem
cell
(?)
preneoplastic
cell
preneoplastic
focus
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
initiation proliferati
on
apoptosis
progression
(tumor)DNA damage
hepatocyte/
hepatic
stem
cell
(?)
preneoplastic
cell
preneoplastic
focus
2. ‚Dioxins‘
as tumor
promotors
+
Schrenk 2010
initiation proliferati
on
apoptosis
progression
(tumor)DNA damage
hepatocyte/
hepatic
stem
cell
(?)
preneoplastic
cell
preneoplastic
focus
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
1.
(Stinchcombe
et al., 1994):
TCDD inhibits
apoptosis
in preneoplastic
rat hepatocytes
in situ
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotorsExtrinsic and intrinsic pathway of
apoptosis
Schrenk 2010
2. (Wörner & Schrenk, 1994)
TCDD inhibits
apoptosis
in rat hepatocytes
in primary
culture
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
0 6 12 24
1
2
3
4
0
Time (h)
Apo
ptot
icnu
clei
(%)
control
(DMSO)
UV / TCDD 10-9
M
** *
*p<0.05
UV / DMSO
UV
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
3. (Wörner
& Schrenk, 1996):
TCDD suppresses the p53 response in rat
hepatocytes
in primary culture
2. ‚Dioxins‘
as tumor
promotors
UV dose (J/m2) 0 0 90 90 120 120 150 150
TCDD (10-9
M) -
+ -
+ -
+ -
+
Rat hepatocytes
in primary
culture
were
harvested
24 h after irradiation; TCDD was added
30 min after
irradiation.
*Wörner and Schrenk, 1996
53 kD
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
4. (Wörner
& Schrenk, 1998):
In rat hepatocytes, TCDD leads to hyper-phosphorylation
of p53. This effect shows the
same concentration-response characteristics as CYP1A1 induction.
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
[TCDD] (M) - 10-12 10-11 10-10 10-9 10-8
Rat hepatocytes
in primary
culture
were
harvested
6h after
TCDD treatment
*Woerner
and Schrenk, 1998
53 kD
2. ‚Dioxins‘
as tumor
promotors
0
200
400
600
800
-13 -12 -11 -10 -9 -8 -7
lg
[TCDD] (M)
ER
OD
act
ivity
Schrenk 2010
5. (Kunz et al., 2004):
TCDD does not suppress MNNG-induced activation of caspase3 but leads to an increase in caspase3 activity without MNNG treatment.
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
Rat hepatocytes: Effect
of TCDD on UV-induced
apoptosis
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
TCDD does
not suppress
caspase
activation
in rat hepatocytes
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
Pan-caspase
inhibition suppresses apoptosis
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
TCDD does
not inhibit
PARP cleavage
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
CAD pathway
2. ‚Dioxins‘
as tumor
promotors
Apoptotic
signal
Caspa se
3
DNA fragmentati
on factor (DFF)-
complex
ICADCAD
activeCAD
Nucl eus
Bild: DNA mit
Nukleosomen
Schrenk 2010
Nuclei from TCDD-treated hepatocytes
behave like untreated as CAD substrates
2. ‚Dioxins‘
as tumor
promotors
Caspase
3-
activated DFF action
is not affected by addition of TCDD in DNA
isolated from
untreated hepatocyte
s
Caspase
3-
activated DFF action
is not affected
by pretreatm
ent ofhepatocyte
s
with TCDD Chopra
et al., Tox. Sci., 2009
Schrenk 2010
Caspase-dependent/independent apoptosis?
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
UV light leads to a loss of AIF from mitochondria (M); TCDD does not suppress this release
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
The AhR-antagonist CH-223191 abrogates TCDD-mediated supression
of apoptosis
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotors10 nM
TCDD reduces Huh-7 cells in sub G1
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotorsTCDD does not suppress caspase
activation in Huh-7 cells
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotors
Schrenk 2010
catenin, H2A and NF-κB
p65 levels are not affected by TCDD
2. ‚Dioxins‘
as tumor
promotors
Chopra
et al., Tox. Sci., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotorsTCDD does
not inhibit
OTA-
or
CHX-initiated
apoptosis
Chopra
et al., Mol. Biol. Toxicol., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotorsOTA and CHX inhibit
protein
biosynthesis
Chopra
et al., Mol. Biol. Toxicol., 2009
Schrenk 2010
2. ‚Dioxins‘
as tumor
promotorsCHX reduces
UV light-induced
apoptosis
Chopra
et al., Mol. Biol. Toxicol., 2010
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
Mechanism
of dioxin
carcinogenicity–
tumor promotion
and/or
initiation?
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
Signal transduction and cellular effects
ROS
AhR ARNT
XRE
CYP1A1 etc.
OO
O
OO
O
ROS/genotoxic
metabolites?
c-srcc-src
Phosphorylation
of p53 (other
targets?)
A. Initiating
action?B. Suppression of apoptosis
A
BCaspase
3 ??
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
oxo8G per 106 dG
in hepatic
nuclear
DNA of intact
and ovariectomized female
Sprague-Dawley
rats
(Wyde
et al., Chem
Res Toxicol
14, 849, 2001)
Treatment intact
ovx ovx/estradiol
control
3.1 ±
1.5a
2.6 ±
1.4 2.8 ±
1.6
TCDDb
51 ±
22* 2.4 ±
1.5 32 ±
36*a
mean
± S.D.b
100 ng/kg per day, orally, for
30 weeks
* significantly
higher
than
control
(p<0.01)
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
HO
CH3OH
estradiol
Possible
consequences
of CYP1A1 induction: metabolic
activation
of estradiol*?
1
2
3
45
67
89
10
1112
14
13
15
16
17
18
HO
CH3OH
4-Hydroxy-estradiol
1
2
3
45
67
89
10
1112
14
13
15
16
17
18
HO[CYP1B1]
[CYP1A1?]
O
CH3OH
catechol
quinone estrogen
12
3
45
67
89
10
1112
14
13
15
16
17
18
O
.
O
CH3OH
semiquinone
radical
1
2
3
45
67
89
10
1112
14
13
15
16
17
18
OH
[Reductase]H+/e-
4-hydroxylation
oxidation
O2O2
-. reduction
*Wyde
et al., Chem. Res. Toxicol. 14, 849 (2001)
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
Possible
consequences
of CYP1A1 induction: increased
release
of superoxide?
N N
N N
FeIII
SCys
e - N N
N N
FeII
SCys
N N
N NFeII
SCys
O2
O
O
N N
N NFeIII
SCys
O
O-
e -2H+H2O
N N
N NFeV
SCys
O
N N
N NFeIII
SCys
O
HH
ROH
RH
O2- .
2H+
H2
O2
2H+
peroxo‚oxenoid‘
3. Are ‚Dioxins‘
genotoxic?
Fe2+/3+
1/2 O2
O2- . OH.
SOD/Fenton
reaction
DNA (e.g. guanine) N
NH
NH2
N
N
R
HO
O
8-oxo-deoxyguanosine
CYP1A1
Schrenk 2010
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
Determination of ROS formationin-vitro in the human cell line HepG2 and in primary rat hepatocytes
• after incubation with TCDD (1 nM)
2´,7´-dihydrodichloro-fluorescein
diacetate
(H2
DCFDA) assay
Quantification of 8-oxo-2´- deoxyguanosine (8-oxo-dG) with
HPLC-MS/MS.
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
10
20
30
0
oxo8 G
per
106
dG(m
ean
±S.
D.)
control
(DMSO)
TCDD (1 nM)
oxo8G per 106
dG
and H2
DCFDA assay
in rat hepatocytes
in primary
culture*significantly
higher
than
control
(p<0.01)
3. Are ‚Dioxins‘
genotoxic?
200
600
0
400
800
rela
tive
fluor
esce
nce
(%)
**
Schrenk 2010
10
20
30
0
oxo8 G
per
106
dG(m
ean
±S.
D.)
control
(DMSO)
TCDD (1 nM)
oxo8G per 106
dG
and H2
DCFDA assay
in Hep
G2 cells*significantly
higher
than
control
(p<0.01)
3. Are ‚Dioxins‘
genotoxic?
200
600
0
400
800
rela
tive
fluor
esce
nce
(%)
0
50000
1E+05
2E+05
2E+05
3E+05
3E+05
200
600
0
400
800
ERO
D a
ctiv
ity(p
mol
/min
x m
g pr
otei
n)
1000
3. Are ‚Dioxins‘
genotoxic?
Hep
G2 cells
rat hepatocytes
EROD actvity
and immuno-reactive
CYP1A1 in Hep
G2 cells
and rat hepatoytes
after
treatment
with
1 nM
TCDD
Schrenk 2010
Schrenk 2010
3. Are ‚Dioxins‘
genotoxic?
in-vivo: transgenic
mouse
model
(Andersson et al. 2001)
: constitutively
active
Ah-receptor
(CA-AHR):
deletion
of amino
acids
230–421 encompassing
the
minimal PAS B motif
generation of an activated
form of the
receptor
stimulates
transcription
in the absence
of ligand
schematic
representation
of the
structural
motifs
within
the
full-length
mouse
dioxin
receptor
(AHR) and the
dioxin
receptor
deletion
mutant
(CA-
AHR); (McGuire et al. 2001)
mAhR
CA-AhR
3. Are ‚Dioxins‘
genotoxic?
EROD activity
in microsomes
from
wildtype
C57Bl6 mice
and CA-AHR mice
Schrenk 2010
0
50
100
150
200
250
300
350
400
vehicle control TCDD wild type CA-AhR
ER
OD
(pm
ol/m
in*m
g)
3. Are ‚Dioxins‘
genotoxic?
8-oxo-dG in hepatic
DNA from
wildtype
C57Bl6 mice
and CA-AHR mice
Schrenk 2010
0
50
100
150
200
250
300
350
400
vehiclecontrol
TCDD wild type CA-AhR
8-ox
o-dG
(%)
n = 10n = 8n = 6n = 6
** unpaired t-test, one-tail p
value
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
0
50
100
150
200
250
Control TCDD 1nM E2 100nM TCDD 1nM + E2100nM
DC
F [%
of c
ontr
ol]
HepG2H4IIEHepa1
n ≥ 3*
**
*
0
50
100
150
200
250
Control TCDD 1nM E2 100nM TCDD 1nM + E2100nM
DC
F (%
of c
ontr
ol)
female-derived
male-derivedn ≥ 3
*
**
**
*
**
Effects of 1 nM
TCDD and 100 nM
17--estradiol on the formation of ROS, measured as increase in oxidized DCF in HepG2, H4IIE, and Hepa1 cells (left diagram) and in male-
and female-derived rat primary hepatocytes
(right diagram). Cells were incubated for a period of 48 h. Both TCDD and estradiol
were dissolved in DMSO. Controls represent cells treated with DMSO 0.5 % final concentration. Bars represent means ±
SD from n ≥
3 independent experiments; the asterisk indicates a significant
difference from the control. (paired t-test,; one tailed p value, *p≤
0,05, **p≤
0,001). E2, 17--estradiol; DMSO, Dimethylsulfoxide.
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
0
20
40
60
80
100
120
140
160
180
Medium DMSO E2 100nM TCDD 1nM TCDD 1nM +E2 100nM
ERO
D a
ctiv
ity (p
mol
/min
*mg)
female-derivedmale-derived
**
**
*
*n = 3
Effects of 1 nM
TCDD and 100 nM
E2 on EROD activity in male-
and female-derived rat primary hepatocytes. Cells were incubated for a period of 48 h. Both TCDD and Estradiol
were dissolved in DMSO. Bars represent means ±
SD from n = 3 independent experiments; the asterisk indicates a significant difference from the DMSO control.
3. Are ‚Dioxins‘
genotoxic?
Schrenk 2010
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
rat CYP1A1 rat CYP1A2 human CYP1A1 human CYP1A2 human CYP1B1 control supersomes
DCF
form
ation
[pm
ol/m
in*pm
ol P4
50]
0
10
20
30
40
50
60
P450
redu
ctas
e ac
tivity
[nm
ol/m
in*pm
ol P4
50]
.
no compound added DMSO TCDD
B[a]P 8-MOP 8-MOP + TCDD
E2 E2 + TCDD P450 reductase activity
n = 3
Effects of adding test compounds (1 nM
TCDD, 10 μM B[a]P, 100 μM 8-MOP, 100 nM
estradiol
(E2) or 0.5 % (v/v) DMSO) to SupersomesTM
on the formation of ROS, measured as increase in oxidized DCF (bars). The curve shows cytochrome
P450 reductase
activities of individual Supersomes.
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
What
about
NDL-PCBs?
ATHONAssessing
the
Toxicity
and Hazard
of Non-dioxin-like
PCBs
Present
in Food
Schrenk 2010
Cl
3,3‘,4,4‘,5-Pentachlorobiphenyl (PCB 126)Cl
Cl
ClClCl
Cl
3‘2‘4‘
5‘6‘654
3 21 1‘Cl Cl Cl
Cl
2,2‘,4,4‘,5,5‘-Hexachlorobiphenyl (PCB 153)
Cl
Cl
ClCl3‘2‘
4‘5‘6‘65
43 2
1 1‘Cl
Cl
a ‚non
-
dioxinlike‘
PCB congener:
ClCl
ClCl
Cl
a ‚dioxinlike‘
PCB congener:
4. What
about
NDL-PCBs?
Schrenk 2010
-0.5 0.0 0.5 1.0 1.5 2.0 2.5
0
10
20
30
40
50
60To
tal a
nim
als
with
neop
lasm
s(%
)
lg Dose (ng TEq/kg b.w. x day)
Aroclors
[Mayes
et al., 1998]TCDD [Kociba
et al., 1976]
Liver
neoplasms
in female
rats
treated
with
TCDD or
Aroclors
TEq
are
carcinogenic
in the
liver
of female
rats
4. What
about
NDL-PCBs?
Schrenk 2010
1.
Introduction
2.
‚Dioxins‘
as tumor
promoters
3.
are
‚Dioxins‘
genotoxic?
4.
what
about
non-dioxinlike
PCBs?
5.
Summary
Schrenk 2010
Summary
I
•The carcinogenicity of TCDD in rat liver is due (in part) to tumor promotion. Suppression of apoptosis seems to play a key role in this effect. A possible mechanism of suppression of apoptosis in irradiated rat hepatoyctes
in culture involves
suppression of the p53 response and a concomitant hyper- phosphorylation
of p53 (and other targets?)
•TCDD inhibits UV-induced apoptosis in a caspase-independent (!) manner
•TCDD-mediated suppression of apoptosis requires AhR activation and protein biosynthesis
Schrenk 2010
Summary
II •In rodent liver TCDD can lead to increased oxidative DNA damage measured as 8-
oxo-dG. In rats the latter effect is strongly estrogen-dependent.
•In rat hepatocytes
in primary culture TCDD enhances formation of ROS and 8-oxo- DG. In human HepG2 cells this effect is not
observed, which is in accordance with the much lower level of CYP1A1 induction.
•In transgenic mice bearing a constitutively active AHR, hepatic 8-oxo-dG levels were significantly higher than in wildtype
C57Bl6 mice, whereas EROD activity was not markedly increased (!).
•
In hepatoma
cell lines and rat hepatocytes
estradiol
did not significantly enhance TCDD-induced ROS formation. However, in rat hepatocytes
estradiol
only increased ROS formation significantly.
•In Supersomes, human CYP1A1 was the most effective source of ROS. Addition of estradiol
reduced ROS formation.
Schrenk 2010
•In rats, the carcinogenicity of techniocal
PCB mixtures is mainly, if not exclusively, due to DL-
compounds. •In mice, treatment with highly purified
PCBs 52,
101, 138, 153, and 180 induced CYP2B mRNAs indicating CAR activation ‘signature’, while PCB 28 did not. PCB 138 only (besides the DL-PCB 126) also seems to induce AhR-regulated CYP1A1.•In summary, PCBs 52 and 180 also have no DL properties in rats. PCB 138 is a ‘mixed’
DL/NDL-PCB
while PCB 28 is none of both in the mouse.
Summary
III
Schrenk 2010
Acknowledgment
EU (6. FP)Deutsche ForschungsgemeinschaftFederal Ministry of Education and ResearchStates of Rheinland-Pfalz
Food Chemistry
& Toxicology, University of Kaiserslautern:Martin ChopraAndreas KamyschnikowMaike GährsManuela GöttelMonika GroßStefanie KnerrRobert RoosHans-Joachim Schmitz
Institute of Toxicology
University of Würzburg:Barbara MallyWolfgang Dekant
Karolinska
Institute, Molecular Medicine, Stockholm
Lorenz PoellingerPatrick Andersson
Athon
Partners:Patrik de Boover, VITO, Mol, BelgiumMatti Viluksela, University of Kuopio, FinlandLeo van der Ven, RIVM, Bilthoven, The
Netherlands
Schrenk 2010
Thank
you
for
your
attention!
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