One-month-old female Taconic line 8440 mouse (Mus musculus).Four mice were presented dead. Three were found dead and one was euthanized prior to submission. According to the history, they were treated with azoxymethane.

Gross Description:  

All mice have congested livers with light brown apical margins. Heart and kidneys are pale. The mice that were euthanized had blood tinged intestinal contents.

Histopathologic Description:

The liver has severe congestion of hepatic lobules with attenuation and loss of centrilobular hepatocytes. Portal zone hepatocytes are spared but have variably vacuolated cytoplasm. Other histology findings include lymphocytolysis of the thymus.  The mouse with intestinal hemorrhage has gastric ulcers.  One mouse has necrosis of the adrenal cortex.

Morphologic Diagnosis:  

Liver: Centrilobular to mid-zonal necrosis, and hemorrhage, severe, acute; micro-vesicular lipidosis mild to moderate.

Lab Results:  

Bacteriology: Small Intestine: Enterococcus faecalis, Staphylococcus sciuri, and E. coli; Negative for strict anaerobes.


Azoxymethane toxicity

Contributor Comment:  

Azoxymethane (AOM) is derived from the cycad palm nut of Guam.  Ingestion has been associated with acute liver failure in cattle.1 In mice and rats, it is used as a mutagen in the study of colon carcinogenesis and liver failure.  It is an alkylating agent that forms 06-methylguanine (06meG) adducts in DNA. 06meG formation has been found in human colon cancers. In the liver, AOM is oxidized to methylazoxymethanol by cytochrome P450 2E1 and transported to the colon where it methylates DNA which can cause G:C to A:T transition mutations.2,3 Methylazoxymethanol is also associated with acute and chronic liver toxicity.4

At reported doses of 100 ug/g in mice AOM causes acute liver injury.  It is characterized by hepatic necrosis and microvesicular lipidosis. The mechanism of action may be interference with beta-oxidation of fatty acids in the mitochondria1 Hemorrhage is an indication of damage to sinusoidal endothelial cells.4 Hepatic necrosis then progresses to liver failure and hepatic encephalopathy.

Acute liver failure is thought to lead to death via neurologic effects and hypotensive shock with multi-organ failure.  Neurologic effects were thought to be a result of ammonia and other metabolites directly causing hepatic encephalopathy.  More recent work has shown that inflammation-associated cytokine release contributes to brain edema and other clinical signs. Acute liver failure can result in cytokine storms with increased TNF-α, IL-1b, Il-6, and Il-12, There is also evidence of impaired neutrophil phagocytic activity and increased the risk of sepsis.  Portal hypertension may cause increased bacterial translocation5 cross the gut.5, 6, 7

JPC Diagnosis:  

Liver: Necrosis and hemorrhage, centrilobular to midzonal, acute, diffuse, severe with microvesicular lipidosis, Taconic line 8440 mouse, Mus musculus

Conference Comment:  

Despite some minor slide variability, the contributor provides a good example of the relatively stereotypical histologic changes associated with acute toxic hepatic injury. The liver is particularly susceptible to toxic injury due to constant exposure to ingested chemicals through the portal blood.4 Additionally, hepatocytes are responsible for the metabolism of most endogenous and exogenous substances, by a process called biotransformation. This process is broken down into three phases based on the hepatic enzymes involved. Phase I reactions involve oxidation, reduction, hydrolysis, cyclization, and decyclization of the compound via cytochrome P450 monooxygenases (CYP) utilizing NADPH and oxygen in the smooth endoplasmic reticulum of hepatocytes. Phase II involves conjugation of the metabolite produced in phase I via glucuronidation, sulphation, acetylation, or methylation, ultimately resulting in a water soluble metabolite that is then excreted through urine or bile. Phase III reactions involve transporting the conjugated substances through the hepatocyte and into the bile canaliculus.4,5,7

Conference participants discussed the various mechanisms of hepatotoxic liver injury, which are divided into six categories based on the mechanism of action and cellular targets of the toxin.4 The most common mechanism involves biotransformation of indirect-acting toxins by the CYP system, whichresults in bioactivated toxic metabolites that disrupt intracellular enzymatic pathways. CYP is abundant in the microsomes of the smooth endoplasmic reticulum in centrilobular zones which explains the prevalence of centrilobular to midzonal necrosis in some toxic hepatopathies, including this case of azoxymethane toxicosis.1,4,6 There is also inhibition of hepatic mitochondrial function, thus limiting beta-oxidation of fat and ATP generation via oxidative phosphorylation. This inhibition eventually leads to necrosis (due to production of damaging reactive oxygen species and lactic acid), as well as hepatocellular microvesicular lipid accumulation, as seen adjacent to areas of necrosis in this case.4 Readers are encouraged to review 2012 WSC Conference 18 Case 2 for further discussion of other mechanisms of hepatotoxic liver injury.

In addition to hepatic necrosis, acute and fatal hepatotoxicity causes destruction of the endothelium of the sinusoidal lining cells, resulting in zonal areas of hemorrhage. Widespread hemorrhage is often associated with acute hepatocellular toxicity due to consumption of platelets and decreased production of clotting factors by the liver.4 Centrilobular necrosis is the most common form of zonal hepatocellular necrosis observed in animals and may be caused by a variety of infectious, inflammatory, metabolic, and toxic insults. Although conference participants could not identify azoxymethane as the cause of the lesions in this mouse, most suspected a toxic etiology based upon the presence of centrilobular necrosis and hemorrhage combined with the lack of evidence of an infectious etiology.


1. Belanger M, et al. Neurobiological characterization of an azoxymethane mouse model of acute liver failure. Neurochem Int. 2006; (48):434-440.
2. Nyskolus LS, et al. Repair and removal of azoxymethane-induced 06-methyguanine in rat colon by06-methyguanine DNA methyltransferase and apoptosis.   Mutat Res. 2013; 758: 80-86.
3. Sohn OS, et al.  Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome p450IIE1.  Carcinogenesis. 1991; 12 (1):127-131.
4. Stalker MJ, Cullen JM.  Liver and biliary system, In: Maxie MG, ed. Jubb, Kennedy and Palmer´s. Pathology of Domestic Animals. Vol 2. 6th ed.  St Louis, MO: Elsevier Saunders; 2016:325-330.
5. Tranah TH, et al. Systemic inflammation and ammonia in hepatic encephalopathy.  Metab Brain Dis. 2013; 28:1-5.
6. Bemeur C, Desjardins P, Butterworth RF. Antioxidant and anti-inflammatory effects of mild hypothermia in the attenuation of liver injury due to azoxymethane toxicity in the mouse.  Metab Brain Dis. 2010; 25:23-29.
7. Chastre A, et al.  Inflammatory cascade driven by tumor necrosis factor-alpha play a major role in the progression of acute liver failure and its neurologic complications.  PLoS One. 2012; 7(11):e49670.

Click the slide to view.

4-1. Liver, mouse.

4-2. Liver, mouse.

4-3. Liver, mouse.

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