Protective effects of hydrogen sulfide anions against acetaminophen-induced hepatotoxicity in mice.

The key mechanism for hepatotoxicity resulting from acetaminophen (APAP) overdose is cytochrome P450-dependent formation of N-acetyl-p-benzoquinone imine (NAPQI), a potent electrophilic metabolite that forms protein adducts. The fundamental roles of glutathione in the effective conjugation/clearance of NAPQI have been established, giving a molecular basis for the clinical use of N-acetylcysteine as a sole antidote. Recent evidence from in vitro experiments suggested that sulfide anions (S(2-)) to yield hydrogen sulfide anions (HS(-)) under physiological pH could effectively react with NAPQI. This study evaluated the protective roles of HS(-) against APAP-induced hepatotoxicity in mice. We utilized cystathionine γ-lyase-deficient (Cth(-/-)) mice that are highly sensitive to acetaminophen toxicity. Intraperitoneal injection of acetaminophen (150 mg/kg) into Cth(-/-) mice resulted in highly elevated levels of serum alanine/aspartate aminotransferases and lactate dehydrogenase associated with marked increases in oncotic hepatocytes; all of which were significantly inhibited by intraperitoneal preadministration of sodium hydrosulfide (NaHS). NaHS preadministration significantly suppressed APAP-induced serum malondialdehyde level increases without abrogating APAP-induced rapid depletion of hepatic glutathione. These results suggest that exogenous HS(-) protects hepatocytes by directly scavenging reactive NAPQI rather than by increasing cystine uptake and thereby elevating intracellular glutathione levels, which provides a novel therapeutic approach against acute APAP poisoning.

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

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 ß-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs(+/-) or Cth(+/-)) and homozygous (Cth(-/-)) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth(-/-) mice at 150 mg/kg dose, and also in Cbs(+/-) or Cth(+/-) mice at 250 mg/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 150 mg acetaminophen/kg into Cth(-/-) mice; the profiles were similar to 1000 mg acetaminophen/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.

Dietary deprivation of each essential amino acid induces differential systemic adaptive responses in mice.

SCOPE: Dietary deprivation of essential amino acids (EAAs) in mammals is known to cause reductions in food intake and body weight. The aim of this study was to determine whether and how mice respond to deprivation of individual EAA species. METHODS AND RESULTS: Dietary deprivation of any single EAA (not non-EAA) in mice led to progressive weight loss in the order of Ile(-) > Val(-) > Thr(-) > Leu(-) > Trp(-) > His(-) > Phe(-) > Met(-) > Lys(-) , which correlated with the reduction in food intake. Decreased levels of the deprived EAAs as well as increased levels of all or some of the other amino acids were detected in the serum, although these levels differed among the diets examined. Serum biochemistry identified significant increases in creatine phosphokinase, blood urea nitrogen, alanine aminotransferase, and aspartate aminotransferase, and decreases in glucose and triglycerides; computed tomography revealed a marked reduction in abdominal/femoral fat and muscle depots; histology identified diffuse myofiber atrophy in the rectus femoris muscle, all in that approximate order. In contrast, amino acid response, autophagy, and ubiquitination marker genes as well as amino acid transporter genes were induced in both deprived EAA-specific and tissue-specific manners. CONCLUSION: Dietary deprivation of individual EAAs induced systemic adaptive responses that differed in magnitude and molecular machinery.

Methionine excess in diet induces acute lethal hepatitis in mice lacking cystathionine γ-lyase, an animal model of cystathioninuria.

Physiological roles of the transsulfuration pathway have been recognized by its contribution to the synthesis of cytoprotective cysteine metabolites, such as glutathione, taurine/hypotaurine, and hydrogen sulfide (H(2)S), whereas its roles in protecting against methionine toxicity remained to be clarified. This study aimed at revealing these roles by analyzing high-methionine diet-fed transsulfuration-defective cystathionine γ-lyase-deficient (Cth(-/-)) mice. Wild-type and Cth(-/-) mice were fed a standard diet (1 × Met: 0.44%) or a high-methionine diet (3 × Met or 6 × Met), and hepatic conditions were monitored by serum biochemistry and histology. Metabolome analysis was performed for methionine derivatives using capillary electrophoresis- or liquid chromatography-mass spectrometry and sulfur-detecting gas chromatography. The 6 × Met-fed Cth(-/-) (not 1 × Met-fed Cth(-/-) or 6 × Met-fed wild type) mice displayed acute hepatitis, which was characterized by markedly elevated levels of serum alanine/aspartate aminotransferases and serum/hepatic lipid peroxidation, inflammatory cell infiltration, and hepatocyte ballooning; thereafter, they died of gastrointestinal bleeding due to coagulation factor deficiency. After 1 week on 6 × Met, blood levels of ammonia/homocysteine and hepatic levels of methanethiol/3-methylthiopropionate (a methionine transamination product/methanethiol precursor) became significantly higher in Cth(-/-) mice than in wild-type mice. Although hepatic levels of methionine sulfoxide became higher in 6 × Met-fed wild-type mice and Cth(-/-) mice, those of glutathione, taurine/hypotaurine, and H(2)S became lower and serum levels of homocysteine became much higher in 6 × Met-fed Cth(-/-) mice than in wild-type mice. Thus, transsulfuration plays a critical role in the detoxification of excessive methionine by circumventing aberrant accumulation of its toxic transamination metabolites, including ammonia, methanethiol, and 3-methylthiopropionate, in addition to synthesizing cysteine-derived antioxidants to counteract accumulated pro-oxidants such as methionine sulfoxide and homocysteine.