Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

Abstract

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2((-/-))). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2((+/+))) mice and GstP1/P2((-/-)) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2((+/+)) and GstP1/P2((-/-)) mice, GSH levels only recovered in the GstP1/P2((-/-)) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity.

Figure 1

Plasma ALT levels in control and GstP null mice after treatment with acetaminophen. (A) Male GstP1/P2^(−/−) and GstP1/P2^(+/+) mice (n = 5) were treated with a single dose of acetaminophen (250 mg/kg body weight) by gavage, and plasma ALT values were determined as detailed in Materials and Methods. ■, GstP1/P2^(+/+); ○, GstP1/P2^(−/−). **, P < 0.01; * P < 0.05. (B) Male GstP1/P2^(−/−) and GstP1/P2^(+/+) mice (n = 5–10) were treated with a single i.p. dose of acetaminophen ranging from 100 to 350 mg/kg body weight, and plasma ALT values were determined as detailed in Materials and Methods. ■, GstP1/P2^(+/+); ○, GstP1/P2^(−/−). *, statistical difference between controls and nulls (***, P < 0.001).

Figure 2

Liver histology in control mice compared with GstP null mice treated with a single oral dose of acetaminophen. Male GstP1/P2^(−/−) and GstP1/P2^(+/+) mice were treated with a single dose of acetaminophen 250 mg/kg body weight by gavage. At 24 and 48 h, the animals were killed and their livers removed, fixed, and sections (10 μm) cut and processed for staining with hematoxylin and eosin as detailed in Materials and Methods.

Figure 3

Correlation of plasma acetaminophen and alanine aminotransferase levels. Male GstP1/P2^(+/+) mice (n = 5) were treated with acetaminophen (300 mg/kg, i.p.), and plasma acetaminophen and ALT values were determined 5 h after dosing as detailed in Materials and Methods. Data were subject to a Pearson rank correlation test.

Figure 4

Metabolism of acetaminophen in control and GstP null mice. Male GstP1/P2^(−/−) and GstP1/P2^(+/+) mice (n = 3) were treated with ³H-acetaminophen (4–7 μCi; 150 mg/kg in saline, 10 ml/kg), administered i.v. as a bolus over 2 min. Bile and urine were collected from these animals, and the level of acetaminophen metabolites was determined as detailed in Materials and Methods. Data are presented as a percentage of the radioactive dose excreted. (A) Urinary metabolites. (B) Biliary metabolites. Filled columns, GstP1/P2^(+/+); open columns, GstP1/P2^(−/−).

Figure 5

Hepatic glutathione levels in control and GstP null mice following treatment with a single dose of acetaminophen. Male GstP1/P2^(−/−) and GstP1/P2^(+/+) mice (n = 6) were treated with acetaminophen i.p. at a dose of 300 mg/kg, and hepatic reduced and oxidized glutathione content was determined as detailed in Materials and Methods. (A) Reduced glutathione; ■, GstP1/P2^(+/+); ○, GstP1/P2^(−/−); *, statistical difference between controls and nulls (*, P < 0.05, **, P < 0.005). (B) Oxidized:reduced glutathione ratio (GSSG:GSH). Filled columns, GstP1/P2^(+/+); open columns, GstP1/P2^(−/−); *, statistical difference between pre- and post-acetaminophen treatment in control animals (*, P < 0.01; **, P < 0.005).