Conference 06 - 2012 Case: 01 20121031

Signalment:

4-month-old, female, C57BL/6 mouse (Mus muculus)This mouse received a single 400 mg/kg dose of acetaminophen (suspension in PBS) by oral gavage. The mouse was sacrificed 24 hours after.

Gross Description:

This mouse is presented alive in good body condition. There is mild, diffuse, red and tan mottling of the liver. All other organs and tissues are within normal gross limits.

Histopathologic Description:

Multiple sections of liver are examined, revealing moderate acute hepatocellular necrosis of hepatocytes around central veins (centrilobular necrosis; zone III necrosis). The necrosis is coagulative in nature, characterized by hypereosinophilia and swelling of affected hepatocytes with karyolysis and rarely pyknosis and/or karyorrhexis. Rare scattered individual hepatocytes throughout the liver are condensed, hypereosinophilic, round with absent nuclei (interpreted as apoptosis; Councilman bodies). Diffusely, remaining hepatocytes in other areas/zones are mildly swollen due to intracellular accumulation of small to moderate amounts of small- to medium-sized, clear, round vacuoles (interpreted as microvesicular lipid droplets).

Morphologic Diagnosis:

1. Liver, centrilobular necrosis, diffuse, moderate, acute.
2. Liver, microvesicular hepatic lipidosis, diffuse, mild.

Condition:

Acetominophen toxicity

Contributor Comment:

Acetaminophen (APAP; paracetamol) is a nonprescription drug used in humans. APAP is an aniline analgesic, and at therapeutic doses (500-1000 mg per human tid/qid), it has similar analgesic and antipyretic effects as ibuprofen and aspirin.(2,3) However, it is not classified as a NSAID anti-inflammatory agent because it is a weak COX inhibitor.(2,3)

In most mammalian species (including humans, mice and dogs), the biotransformation of APAP involves conjugation with glucuronic acid or sulfate in the liver, which are subsequently excreted by urinary system.(1,2,3,4,7) A small amount of APAP is metabolized by the P-450 (CYP) system to the reactive metabolite N-acetyl pbenzoquinone imine (NAPQI) in centrilobular zone hepatocytes, which is subsequently scavenged by glutathione.(2,3,4,5,6)

APAP toxicity manifests primarily as centrilobular hepatic necrosis that can lead to acute liver failure. In humans, a single or accumulative daily toxic dose is usually >15-25 g.2,3,4 The mechanism of APAP hepatotoxicity is as follows:(2,3,5,6)

At toxic doses, increased metabolism of APAP by the P-450 (CYP) system leads to higher concentrations of NAPQI formation in centrilobular zone hepatocytes.
Higher concentrations of NAPQI deplete glutathione concentrations in centrilobular zone hepatocytes, leading to increased formation of reactive oxygen and nitrogen species.
Increased oxidative stress in centrilobular zone hepatocytes results in alterations in calcium homeostasis and initiation of mitochondrial permeability transition.
Loss of mitochondrial membrane potential in mitochondria of centrilobular zone hepatocytes leads to loss of ATP synthesis subsequent necrosis.

Antidotes that can prevent or ameliorate APAP toxicity aim to prevent the loss/promote the synthesis of glutathione, such as with cysteine and methionine administration.(2,3,4)

Cats are more sensitive to APAP toxicity and have a different pattern of APAP toxicity due to a species deficiency in glucuronyl transferase, resulting in relatively higher conversion rates of APAP to the reactive NAPQI metabolite.(1,7) In addition to depleting glutathione in centrilobular hepatocytes, NAPQI can also deplete glutathione in erythrocytes, leading to methemoglobinemia, Heinz body hemolytic anemia, and methemoglobinuria.(1,7)

Currently, APAP toxicity is the leading cause of liver failure in humans in the US and UK. Since APAP biotransformation and toxicity is similar between humans and mice, this makes mice an attractive and clinically relevant animal model for research into acetaminophen hepatotoxicity and other toxicities leading to centrilobular necrosis of the liver via the P-450 (CYP) system.(2,4)

JPC Diagnosis:

Liver, centrilobular hepatocytes: Necrosis, coagulative, diffuse.

Conference Comment:

The contributor provided a very good summary of acetominophen (APAP; paracetamol) toxicity. During the discussion of this case, the moderator stressed the importance of phase 1 and phase 2 drug metabolism. Xenobiotics (chemicals not normally found or expected to be in the body) can undergo metabolism via various pathways. with the ultimate endpoint being excretion of water soluable metabolites through urine or bile. Many xenobiotic drugs are lipid-soluble and therefore pose a challenge to such excretion. Thus, a critical step in xenobiotic metabolism is the formation of water-soluble metabolites. Phase 1 drug metabolism includes oxidation, reduction and hydrolysis by various enzyme systems. If the resulting compound is sufficiently water soluble, then it is excreted via the kidneys or bile; however, if phase 1 does not render the drug excretable, it undergoes phase 2 metabolism. Phase 2 metabolism involves conjugation with ionized groups to significantly increase its water solubility and thus further enhance its excretion. Examples of phase 2 conjugation reactions include glucuronidation, sulphation, acetylation ad methylation.(8)

Drug metabolism occurs at several sites, including liver, intestines, lung, kidney, and plasma with the liver being the primary site. The liver contains a realtively large percentage of the bodys metabolizing enzymes, most importantly a group of proteins known as cytochrome P450 mixed function oxidases. These enzymes are found within microsomes in the smooth endoplasmic reticulum of centrilobular hepatocytes. The most common reaction catalyzed by the cytochrome P450 enzymes is a mono-oxygenase reaction in which a molecule of oxygen (O2) is split, with one oxygen atom oxidizing the xenobiotic and reduction of the other oxygen atom to produce a molecule of water. This oxidation-reduction reaction is responsible for the metabolism of a variety of drugs, to include paracetamol. Cytochrome P450 enzymes also catalyze reduction reactions to metabolize drugs such as prednisone, warfarin, and halothane. The third type of phase 1 metabolism reaction is hydrolysis, which is catalyzed by esterases and amidases (which also occur in hepatocytes and other extrahepatic sites, including plasma).(8)

Of the phase 2 reactions, glucuronidation and sulphation play a major role in the metabolism of paracetamol, with approximately 40% of the drug undergoing each reaction. As the contributor states, a small amount of paracetamol undergoes phase 1 Nhydroxylation which results in the toxic product Nacetyl-p-amino-benzoquinoneimine which is normally further metabolized (conjugated) by glutathione.(8)

Further discussion on this case centered on the use of severity modifiers (mild, moderate, severe) to quantify the extent of necrosis, which can be important in a case such as this in which there is a dose dependent response. Toxicologic pathologists often use such modifiers for quantification; however, this terminology is not traditionally used in diagnostic pathology at the AFIP/JPC, with the reasoning that necrosis in itself cannot be mild nor severe".

References:

1. Court MH, Greenblatt DJ. Molecular basis for deficient acetaminophen glucuronidation in cats. Biochem Pharmacol. 1997;53:1041-1047.
2. Hinson JA, Roberts DW, James LP. Mechanisms of acetaminophen-induced liver necrosis. Handb Exp Pharmacol. 2010;196:369-405.
3. James LP, Mayeux PR, Hinson JA. Acetaminophen h e p a t o t o x i c i t y. Dr u g Me t a b Di s p o s i t i o n . 2003;31:1499-1506.
4. Jaschke H, McGill MR, Williams CD, et al. Current issues with acetaminophen hepatotoxicity a clinically relevant model to test the efficacy of natural products. Life Sciences. 2011;88:737-745.
5. Kumar V, Abbas AK, Fausto N. Cellular adaption, cell injury, and cell death. In: Kumar V, Abbas AK, Fausto N, eds. Robbins and Coltran Pathologic Basis of Disease. 7th ed. Philadelphia, PA: Elsevier Saunders; 2005:25-26.
6. Maitra A, Kumar V. Environmental and nutritional pathology. In: Kumar V, Abbas AK, Fausto N, eds. Robbins and Coltran Pathologic Basis of Disease. 7th ed. Philadelphia, PA: Elsevier Saunders; 2005:424.
7. Savides MC, Oehme FW, Nash SL, et al. The toxicity and biotransformation of single doses of acetaminophen in dogs and cats. Toxicol Appl Pharmacol. 1984;74:26-34.
8. Schonborn JL, Gwinnutt C. The role of the liver in drug metabolism. Anesthesia tutorial of the week 179. ATOTW. 2010(179): 1-6. http://www.aagbi.org/sites/default/files/179-The-role-of-the-liver-in-drugmetabolism.pdf. Accessed 18 October 2012.