lnterference of hepatotoxicity with endocrine activity in fish - - PowerPoint PPT Presentation

lnterference of hepatotoxicity with endocrine activity in fish

lisa.baumann@uni-heidelberg.de SETAC Rome, May 2018

Research question:

He Hepatocyte Oo Oocyte

VTG synthesis

?

Hepatotoxicant

☠

Paracetamol: (Acetaminophen), analgesic and antipyretic drug, which is bio-activated by cytochrome P450 enzymes, leading to the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) à causes oxidative stress, thiol

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

Model hepatotoxicants

Isoniazid: Antibiotic drug against tuberculosis, metabolized by hepatic N-acetyltransferase and cytochrome P450 2E1 to form reactive hepatotoxic metabolites that damage hepatocytes Aspirin: (Acetylsalicylic acid), non-steroidal anti-inflammatory and antipyretic drug, activated by cytochrome P450 isoforms CYP2E1 and CYP4A1 in the liver; might inhibit mitochondrial function and cause oxidative stress

—> Impaired VTG synthesis in the liver?

Experimental set-up

Samples:

• Head/tail homogenates for vitellogenin and hyaluronic acid analysis (all fish)
• Liver samples for gene expression analysis (half of the fish)
• Liver samples for histopathological analysis (half of the fish)

OECD TG 230 „21-day fish assay“: 10 adult females + 10 adult males per tank 3 replicates 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

Water analysis

Paracetamol (mg/L) Isoniazid (mg/L) Aspirin (mg/L) nominal ∅ measured (n=8x3) nominal ∅ measured (n=8x3) nominal ∅ measured (n=8x3) 1 1. 1.5 5 4. 4.2 10 8. 8.0 10 13. 13.9 50 47. 47.1 50 32. 32.9 100 129.7 100 85.4 75 50. 50.4 100 65. 65.9

Mortality

stopped continued at 75 mg/L

Water analysis: NAPQI (toxic metabolite)

Paracetamol (mg/L) NAPQI (ng/L) nominal ∅ measured (n=8x3) ∅ measured (n=8x3) 1 1.5 0. 0.8 10 13.9 2. 2.4 100 129.7 6. 6.8

Liver histopathology: single cell necrosis

Single cell necrosis of hepatocytes

Ad Additional

• nal ef

effect ects in in as aspi pirin-ex expos posed ed fi fish:

• Bi

Bile duct duct hy hyper perpl plas asia

• Hy

Hyal alini nized ed hepat hepatocy

• cytes

es

• Decr

Decreas eased ed va vacuolation n n = = 30 30 per per ex expos posur ure gr group

• up

Hyaluronic acid (in head/tail homogenate)

= biomarker for hepatotoxicity (liver fibrosis) in humans

C

• n

t r

• l

5 m g / L 5 m g / L 7 5 m g / L 0.0 0.5 1.0 1.5 2.0 2.5

Isoniazid Hyaluronic acid / protein (ug/mg) *** Aspirin

Co 5 mg/L 50 mg/L 75 mg/L Control 10 mg/L 50 mg/L 75 mg/L 100 mg/L 1 2 3

Aspirin Hyaluronic acid / protein (ug/mg)

n n = = 60 60 per per ex expos posur ure gr group

• up

mRNA expression levels of hepatotoxicity markers in the liver

Par Paracet acetam amol

• l

Is Isoniazid As Aspirin fe females ma males fe females ma males fe females ma males fa fabp10 up

• down

up

• apoa

apoa

• down
• up

cy cyp3a65 p3a65 up

• down

down

• ngoing
• ngoing

cy cyp2k p2k19 19 up down down down

• ngoing
• ngoing

up up/d /down = = si significant change change vs vs cont control

• l, p

, p-va value < < 0. 0.05 05 n = = 15 15 per er ex expos posur ure gr group

• up and

and se sex

n = 15 per r ex expos

• sur

ure gr group and and se sex up up/d /down = = si significant chang change vs vs cont control

• l, p

, p-va value < < 0.05 n = 30 per r ex expos

• sur

ure gr group and and se sex

mRNA expression levels in the liver es esr1 vt vtg1 vt vtg2

Females Males Females Males Females Males Pa Paracetamo mol down

• Isonia

iazid id down up down

• down
• Aspir

irin in

• ngoing
• ngoing

up

• ngoing
• ngoing

Vitellogenin protein levels (head/tail homogenate)

Females Males Paracetamol slightly up

• Isoniazid
• Aspirin
• slightly down

Summary

Paracetamol Isoniazid Aspirin

Liver histopathology

✓ ✓ ✓

Hyaluronic acid

✓ ✓

• Hepatotox

genes

✓ ✓ ✓

Estrogenic genes

✓ ✓ ✓

VTG

✓(f: up)

• ✓(m: down)

Initial research question:

He Hepatocyte Oo Oocyte

VTG synthesis Hepatotoxicant

?

☠

Conclusions and outlook

• Simple question, but no simple answer!
• Endocrine system and liver physiology are closely related and able to

compensate toxic impact to a certain extent

• Range between lethal effects and chronic hepatotoxicity is very small à
• bserved effects not strong
• Effects depend on sex, age and species
• Results not conclusive yet à work ongoing (compare to historical data)
• Important for regulatory decision making to distinguish between

endocrine and non-endocrine effects

Thank you!!!

Project team: Thomas Braunbeck (University of Heidelberg) Henrik Holbech (University of Southern Denmark, Odense) Helmut Segner (University of Bern) Lennart Weltje (BASF SE, Limburgerhof) Research liaison team: Bruno Hubesch (Cefic) Grace Panter (Syngenta) Neil Wang (Arkema) Arnd Weyers (Bayer) James Wheeler (Dow AgroSciences) Lucy Wilmot (ECETOC) Co-workers: TECOmedical Annette Duus (Odense) Bente Holbech (Odense) Angela Moissl (Heidelberg) Olga Lityagina (Heidelberg) Heike Schmidt-Posthaus (Bern) Matthias Schuhmacher (Heidelberg)