lri eco35 - interference of hepatotoxicity with...

LRI ECO35 - Interference of hepatotoxicity with endocrine activity in fish

Dr. Lisa Baumann

University of Heidelberg

Germany

The project team

16/11/2018LRI – ECO35

Thomas Braunbeck

Aquatic Ecology and ToxicologyCOS, University of Heidelberg

Henrik Holbech

Department of BiologyUniversity of Southern Denmark

Helmut Segner

Centre for Fish and Wildlife MedicineVetsuisse Faculty, University of Berne

Lennart Weltje

BASF SE Crop Protection – Ecotoxicology

Biomarker: Vitellogenin

16/11/2018LRI – ECO35

Hepatocyte

Oocyte

Estrogen receptor

agonist

VTG synthesis

Brain

16/11/2018


LRI – ECO35

Hepatocyte

Oocyte

Estrogen receptor

antagonist

VTG synthesis

Brain

16/11/2018


LRI – ECO35

Hepatocyte

Oocyte

Hepatotoxicant

VTG synthesis

Brain

☠️

?

16/11/2018

Model hepa totoxicants

LRI – E CO35

Paracetamol:

(Acetaminophen), ana lges ic and antipyretic drug, which is bio-activa ted by cytochrome P450 enzymes , leading to

the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) causes oxidative stress , thiol

oxidation, lipid peroxidation and results in mitochondrial injury and cellular necrosis of hepatocytes

Isoniazid:

Antibiotic drug aga inst tuberculos is , metabolized by hepatic N-acetyltrans ferase and cytochrome P450 2E 1 to form

reactive hepatotoxic metabolites that damage hepatocytes

Aspirin:

(Acetylsa licylic acid), non-steroidal anti-inflammatory and antipyretic drug, activa ted by cytochrome P450 isoforms

CYP2E 1 and CYP4A1 in the liver; might inhibit mitochondrial function and cause oxidative stress

16/11/2018

E xperimenta l set-up

LRI – E CO35

OECD TG 230 „21-day fish as say“:

10 adult fema les + 10 adult ma les per tank

3 replica tes for each exposure group

21 days flow-through exposure

Zebrafish (Danio rerio)

Paracetamol

(mg /L)

Isoniazid

(mg /L)

Aspirin

(mg /L)

Control Control Control

1 5 10

10 50 50

100 100 75

100

n = 240 n = 240 n = 300

Samples :

- Head/tail homogenates for vitellogenin

and hya luronic acid ana lys is (all fish)

- Liver samples for gene express ion

ana lys is (ha lf of the fish trunks)

- Liver samples for his topatholog ica l

ana lys is (ha lf of the fish trunks)

16/11/2018

Water ana lys is

LRI – E CO35

Paracetamol (mg/L) Isoniazid (mg/L) Aspirin (mg/L)

nomina l ∅ measured nomina l ∅ measured nomina l ∅ measured

0 0 0 0 0 0

1 1.5 5 4.2 10 8.0

10 13.9 50 47.1 50 32.9

100 129.7 100 85.4 75 50.4

100 65.9

16/11/2018

Water ana lys is

LRI – E CO35

Paracetamol (mg/L) Isoniazid (mg/L) Aspirin (mg/L)

nomina l ∅ measured nomina l ∅ measured nomina l ∅ measured

0 0 0 0 0 0

1 1.5 5 4.2 10 8.0

10 13.9 50 47.1 50 32.9

100 129.7 100 85.4 75 50.4

100 65.9

stopped continued at 75 mg /L

Mortality at day 10

16/11/2018

Liver histopathology: single cell necrosis

LRI – ECO35

16/11/2018

Sing le cell necros is of hepa tocytes

LRI – E CO35

Additiona l effects in aspirin-exposed fish:

- Bile duct hyperplas ia

- Hya linized hepatocytes

- Decreased vacuola tion

n = 30 per exposure group

16/11/2018

Hya luronic acid (biomarker for hepa totoxicity in humans )

LRI – E CO35

Contr

ol

1 m

g/L

10 m

g/L

0

1

2

3

4 Paracetamol

Hyalu

ronic

acid

/ p

rote

in (

ug/m

g)

**

Contr

ol

5 m

g/L

50 m

g/L

100

mg/L

0

1

2

3

4Isoniazid

Hyalu

ronic

acid

/ p

rote

in (

ug/m

g)

***

Contr

ol

10 m

g/L

50 m

g/L

75 m

g/L

100

mg/L

0

1

2

3

4 Aspirin

Hyalu

ronic

acid

/ p

rote

in (

ug/m

g)


16/11/2018

mRNA express ion of hepa totoxicity markers in the liver

LRI – E CO35

Paracetamol Isoniazid Aspirin

fema les ma les fema les ma les fema les ma les

fabp10 up - - down up -

apoa - - - down - up

cyp3a65 up - down down up -

cyp2k19 up down down down down -


16/11/2018

mRNA express ion of endocrine-rela ted genes in the liver

LRI – E CO35


fema les ma les fema les ma les fema les ma les

esr1 down - down up up -

vtg1 - - down - up -

vtg3 - - down - - -


16/11/2018LRI – ECO35

Hepatocyte

Oocyte

Hepatotoxicant

VTG synthesis

Brain

☠️

?

Interference of hepatotoxicity with VTG synthesis?

16/11/2018

VTG results : pa racetamol

LRI – E CO35

Con

trol

1 m

g/L

10 m

g/L

0

2×106

4×106

6×106

8×106

Acetaminophen concentration

VT

G (n

g/m

l hom

ogenate

)

***

Females

Con

trol

1 m

g/L

10 m

g/L

10

100

1000

10000

100000

Acetaminophen concentration

VT

G (n

g/m

l hom

ogenate

)

Males


16/11/2018

VTG results : isoniazid

LRI – E CO35

cont

rol

5 m

g/L

50 m

g/L

100

mg/

L104

105

106

107

108

Isoniazid concentration

VT

G (n

g/m

l hom

ogenate

)

Females

cont

rol

5 m

g/L

50 m

g/L

100

mg/

L10

100

1000

10000

100000

Isoniazid concentration

VT

G (n

g/m

l hom

ogenate

)

*Males


16/11/2018

VTG results : a spirin

LRI – E CO35

Con

trol

10 m

g/L

50 m

g/L

75 m

g/L

100

mg/

L105

106

107

108

Aspirin concentration

VT

G (n

g/m

l hom

ogenate

)

*

Females

Con

trol

10 m

g/L

50 m

g/L

75 m

g/L

100

mg/

L10

100

1000

10000

100000

1000000

Aspirin concentration

VT

G (n

g/m

l hom

ogenate

) *

*

**

Males


16/11/2018

Summary

LRI – E CO35


Liver

histopathology✓ ✓ ✓

Hya luronic

acid✓ ✓ -

Hepatotox

genes✓ ✓ ✓

Endocrine

genes✓ (f) ✓ (f&m) ✓ (f)

VTG ✓ (f: up) ✓ (m: up) ✓ (f&m: down)

16/11/2018

Conclusions and outlook

LRI – ECO35

• Simple question, but no simple answer!

• Endocrine system and liver physiology are closely related and able to compensate chronic toxic

impact to a certain extent

• Range between lethal effects and chronic hepatotoxicity is very small observed effects not

strong

• Effects depend on sex, age and exposure duration

• Results indicate that false VTG results in a typical EDC test scenario are unlikely to occur

• Important for regulatory decision making to distinguish between endocrine and non-endocrine

effects

16/11/2018

Thank you!

LRI – E CO35

Project team:

Thomas Braunbeck (University of Heidelberg)

Henrik Holbech (University of Southern Denmark, Odense)

Helmut Segner (University of Bern)

Lennart Weltje (BASF SE, Limburgerhof)

Co-workers:

TECOmedica l

Annette Duus (Odense)

Bente Holbech (Odense)

Angela Moiss l (Heidelberg)

Olga Litya g ina (Heidelberg)

Heike S chmidt-Posthaus (Bern)

Research liaison team:

Bruno Hubesch (Cefic)

Grace Panter (Syngenta)

Neil Wang (Arkema)

Arnd Weyers (Bayer)

James Wheeler (Dow AgroSciences)

Lucy Wilmot (ECE TOC)

THANK YOU!

lri eco35 - interference of hepatotoxicity with...

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