Hemizygosity of transsulfuration genes confers increased vulnerability against acetaminophen-induced hepatotoxicity in mice

• Published in: Toxicology and applied pharmacology Vol. 282; no. 2; pp. 195 - 206
• Main Authors: Hagiya, Yoshifumi, Kamata, Shotaro, Mitsuoka, Saya, Okada, Norihiko, Yoshida, Saori, Yamamoto, Junya, Ohkubo, Rika, Abiko, Yumi, Yamada, Hidenori, Akahoshi, Noriyuki, Kasahara, Tadashi, Kumagai, Yoshito, Ishii, Isao
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.01.2015
• Subjects: 2D DIGE; 60 APPLIED LIFE SCIENCES; Acetaminophen - toxicity; ADDUCTS; ALANINES; Analgesics, Non-Narcotic - toxicity; Animals; BIOSYNTHESIS; Chemical and Drug Induced Liver Injury - genetics; Chemical and Drug Induced Liver Injury - pathology; Chemical and Drug Induced Liver Injury - prevention & control; Cystathionine beta-Synthase - genetics; Cystathionine gamma-Lyase - genetics; Cystathionine β-synthase; Cystathionine γ-lyase; CYSTEINE; ELECTROPHORESIS; Female; FLUORESCENCE; GELS; GENES; Glutamate–cysteine ligase; GLUTATHIONE; Glutathione - metabolism; GUANINE; INJECTION; INJURIES; LIGASES; LIVER; MALDI-TOF/MS; MICE; Mice, Knockout; Mutation - genetics; Mutation - physiology; Oncosis; POISONING; Sulfates - metabolism; SUPEROXIDE DISMUTASE; TWO-DIMENSIONAL CALCULATIONS; VULNERABILITY; Zygote - drug effects; APAP; IPG; ALT; MDA; TUNEL; CBS; 2D DIGE; CYP; GSH; MALDI-TOF/MS; GSSG; Cystathionine γ-lyase; GST; PAGE; TBARS; HPRT; IEF; Cystathionine β-synthase; SOD1; GCL; GCLC; CTH; Oncosis; GCLM; NAPQI; Glutamate–cysteine ligase
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1016/j.taap.2014.11.015

The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs+/− or Cth+/−) and homozygous (Cth−/−) knockout mice. At 4h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth−/− mice at 150mg/kg dose, and also in Cbs+/− or Cth+/− mice at 250mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth−/− mice but not wild-type mice, although glutamate–cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth−/− mice with lower Km values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150mgacetaminophen/kg into Cth−/− mice; the profiles were similar to 1000mgacetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200–300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities. [Display omitted] •Cbs+/−, Cth+/−, and especially Cth−/− mice were susceptible to APAP hepatic injury.•Hepatic glutathione became rapidly depleted upon APAP injection in Cth−/− mice.•Hepatic glutamate–cysteine ligase was activated by APAP injection and CTH deletion.•2D DIGE identified 47 differentially expressed hepatic proteins by APAP injection.•Both transsulfuration enzymes are essential for protection against APAP injury.