1. NCT Acute Studies BromobenzeneHerbert 1, 2- DichlorobenzeneCesta 1, 4- DichlorobenzeneCesta N-NitrosomorpholineBoorman DiquatTurnier MonocrotalineNyska ThioacetamideHailey GalatosamineBoorman

2. Today’s Overview Chemical Mechanism - Rick Irwin Morphology - Gary Boorman

3. Bromobenzene

4. CYP P450 catalyzed formation of reactive epoxide metabolite – CYP expression predominates around the central vein quinone metabolites also formed metabolites react with cellular proteins detoxification by reaction with GSH cytosolic GSH depletion mitochondrial GSH depletion mitochondrial damage

5. Bromobenzene Acute centrilobular necrosis Rim of hepatocytes immediately adjacent to terminal hepatic venule Minimal inflammatory infiltrate Fairly uniform between animals/lobes Bromobenzene and 1, 2-dichlorobenzene appear similar; 1, 4-DCB - no lesions

6. Bromobenzene 24-hour Sac Example of centrilobular hepatocyte degeneration at 75 mg/kg; degenerative hepatocytes are swollen and have pale (rarefied) cytoplasm; note that there is also centrilobular glycogen depletion

7. Bromobenzene 24-hour Sac Example of centrilobular hepatocyte necrosis at 250 mg/kg; necrosis is massive coagulative and hemorrhagic effacing large areas of the hepatic parenchyma

8. N-Nitrosomorpholine CYP catalyzed hydroxylation at 2’ and 3’ position on morpholine ring protein and DNA adducts

9. Hepatotoxicity an initial wave of apoptosis which peaks at 12 hours but involves only 3-6% of hepatocytes followed by hepatocellular necrosis which is prominent by 24 hours no depeltion of GSH

10. N-Nitrosomorpholine, 300 mg/kg @ 24 hours, Rat # 34

11. N-Nitrosomorpholine 300 mg/kg at 24 hours, Rat # 34 Necrosis Hemorrhage THV PP

12. N-Nitrosomorpholine Acute centrilobular necrosis Mononuclear inflammatory infiltrate Fairly uniform between animals/lobes Prominent karyorrhexis of hepatocytes Prominent hemorrhage

13. Diquat (dibromide) widely used bipyridinium herbicide under goes one electron redox reaction catalyzed by NADPH cytochrome c reductase to produce high levels of ROS primary lesion reported to be centrilobular necrosis initial injury not associated with depletion of GSH characterized by formation of reactive protein carbonyls - aldehydes and ketones

14. Diquat 30 mg/kg at 24 hours, Rat # 7

15. Diquat Acute centrilobular necrosis At early times clearly demarcated zone of pale hepatocytes Rim of hemorrhage at margin of lesions Necrosis secondary to vascular damage and ischemia?

16. Monocrotaline requires bioactivation by CYP’s to reactive dehydro-MCT targets endothelial cells in the sinusoids and central vein hepatotoxicity characterized by hemorrhage and activation of coagulation system. may progress to hepatic veno-occlusive disease (HVOD); used as a model for similar human disease produces caspase dependent apoptosis

17. Monocrotaline 300 mg/kg at 24 hrs

19. Monocrotaline Acute Centrilobular Necrosis Prominent Hemorrhage Primary Endothelial Injury? Hepatocellular Injury Secondary to Ischemia? Diquat and Riddelliine may have similar mechanisms

20. Thioacetamide fungicide CYP2E1 bioactivation to reactive metabolites hepatocytes primary target resulting in necrosis

21. Thioacetamide 150 mg/kg @ 4x

22. Thioacetamide 150 mg/kg @10x

23. Thioacetamide Acute centrilobular necrosis Apoptosis? Mononuclear Cell Inflammatory Infiltrate Fairly uniform between animals/lobes

24. Galactosamine metabolized to UDP-hexoseamines and UDP-N-acetylhexosamines in hepatocytes and depletion of UTP causes a transient block in transcription and protein synthesis associated with UTP depletion accumulation of metabolites inhibit the synthesis of certain critical membrane proteins increases permeability of gut and potentially absorption of endotoxin produces oncotic necrosis, caspase dependent apoptosis, and induces oval cell proliferation

25. Galactosamine, 1500 mg/kg at 24 hours, Rat # 17

26. Galactosamine 1500 mg/kg at 24 hours, Rat # 17 THV PP

27. Galactosamine Patchy to Peripotal necrosis Granulocytes and Mononuclear Cell Infiltrates Hemorrhage is not prominent

28. NCT Acute Studies Very Rich Morphological Data Base Both Similarities and Differences with chemicals Bromobenzene and 1, 2-dichlorobenzene appear similar; 1, 4-DCB - Comparison Diquat, Monocrotaline and Riddelliine Comparison