SETAC Rome, May 2018 lnterference of hepatotoxicity with endocrine activity in fish lisa.baumann@uni-heidelberg.de
Research question: He Hepatocyte Hepatotoxicant ☠ ? VTG synthesis Oo Oocyte
Model hepatotoxicants 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 oxidation, lipid peroxidation and results in mitochondrial injury and cellular necrosis of hepatocytes 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 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 Isoniazid Aspirin (mg/L) (mg/L) (mg/L) Control Control Control 1 5 10 10 50 50 100 100 75 100 n = 240 n = 300 n = 240 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)
Water analysis Paracetamol (mg/L) Isoniazid (mg/L) Aspirin (mg/L) ∅ measured ∅ measured ∅ measured nominal nominal nominal (n=8x3) (n=8x3) (n=8x3) 0 0 0 0 0 0 1 1. 1.5 5 4. 4.2 10 8. 8.0 10 50 50 13. 13.9 47. 47.1 32. 32.9 100 129.7 100 85.4 75 50. 50.4 100 65. 65.9 stopped continued at 75 mg/L Mortality
Water analysis: NAPQI (toxic metabolite) Paracetamol (mg/L) NAPQI (ng/L) ∅ measured ∅ measured nominal (n=8x3) (n=8x3) 0 0 0 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 onal 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 ocytes es - Decr Decreas eased ed va vacuolation n n = = 30 30 per per ex expos posur ure gr group oup
Hyaluronic acid / protein (ug/mg) 0.0 0.5 1.0 1.5 2.0 2.5 C o n t r o l 5 m g = biomarker for hepatotoxicity (liver fibrosis) in humans / L Aspirin Isoniazid Hyaluronic acid (in head/tail homogenate) 5 0 m g / L *** 7 5 m g / L Hyaluronic acid / protein (ug/mg) 0 1 2 3 Control Co 10 mg/L n = n 5 mg/L = 60 50 mg/L 60 per Aspirin per ex 50 mg/L expos 75 mg/L posur ure gr 100 mg/L 75 mg/L group oup
mRNA expression levels of hepatotoxicity markers in the liver Par Paracet acetam amol ol Is Isoniazid As Aspirin fe females ma males fe females ma males females fe ma males fa fabp10 up - - down up - apoa apoa - - - down - up cy cyp3a65 p3a65 up - down down ongoing ongoing cy cyp2k p2k19 19 up down down down ongoing ongoing up up/d /down = = si significant change change vs vs cont control ol, p , p-va value < < 0. 0.05 05 n = = 15 15 per er ex expos posur ure gr group oup and and se sex
Vitellogenin protein levels (head/tail homogenate) Females Males Paracetamol slightly up - Isoniazid - - Aspirin - slightly down n = 30 per r ex expos osur ure gr group and and se sex mRNA expression levels in the liver esr1 es vt vtg1 vt vtg2 Females Males Females Males Females Males Pa Paracetamo mol down - - - - - Isonia iazid id down up down - down - Aspir irin in ongoing ongoing up - ongoing ongoing n = 15 per r ex expos osur ure gr group and and se sex up up/d /down = = si significant chang change vs vs cont control ol, p , p-va value < < 0.05
Summary Paracetamol Isoniazid Aspirin ✓ ✓ ✓ Liver histopathology ✓ ✓ Hyaluronic - acid ✓ ✓ ✓ Hepatotox genes ✓ ✓ ✓ Estrogenic genes ✓ (f: up) ✓ (m: down) - VTG
Initial research question: He Hepatocyte Hepatotoxicant ☠ ? VTG synthesis Oo Oocyte
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 observed 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) Co-workers: TECOmedical Annette Duus (Odense) Bente Holbech (Odense) Angela Moissl (Heidelberg) Olga Lityagina (Heidelberg) Research liaison team: Heike Schmidt-Posthaus (Bern) Bruno Hubesch (Cefic) Matthias Schuhmacher (Heidelberg) Grace Panter (Syngenta) Neil Wang (Arkema) Arnd Weyers (Bayer) James Wheeler (Dow AgroSciences) Lucy Wilmot (ECETOC)
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