Novel protective mechanisms for S-adenosyl-L-methionine against acetaminophen hepatotoxicity: improvement of key antioxidant enzymatic function

Abstract

Acetaminophen (APAP) overdose leads to severe hepatotoxicity, increased oxidative stress and mitochondrial dysfunction. S-adenosyl-L-methionine (SAMe) protects against APAP toxicity at a mmol/kg equivalent dose to N-acetylcysteine (NAC). SAMe acts as a principle biological methyl donor and participates in polyamine synthesis which increase cell growth and has a role in mitochondrial protection. The purpose of the current study tested the hypothesis that SAMe protects against APAP toxicity by maintaining critical antioxidant enzymes and markers of oxidative stress. Male C57Bl/6 mice were treated with vehicle (Veh; water 15 ml/kg, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg, ip), and SAMe+APAP (SAMe given 1 h following APAP). Liver was collected 2 and 4 h following APAP administration; mitochondrial swelling as well as hepatic catalase, glutathione peroxidase (GPx), glutathione reductase, and both Mn- and Cu/Zn-superoxide dismutase (SOD) enzyme activity were evaluated. Mitochondrial protein carbonyl, 3-nitrotyrosine cytochrome c leakage were analyzed by Western blot. SAMe significantly increased SOD, GPx, and glutathione reductase activity at 4 h following APAP overdose. SAMe greatly reduced markers of oxidative stress and cytochrome C leakage following APAP overdose. Our studies also demonstrate that a 1.25 mmol/kg dose of SAMe does not inhibit CYP 2E1 enzyme activity. The current study identifies a plausible mechanism for the decreased oxidative stress observed when SAMe is given following APAP.

Fig. 1

ALT levels in blood plasma 2 and 4 h following APAP administration to C57Bl/6 mice. Mice were randomly allocated into Veh (water), SAMe (1.25 mmol/kg), APAP (250 mg/kg), and SAMe administered 1 h following APAP (S + A). Values represent mean ± S.E.M. with n=5 mice per group and superscripts denoting statistically significant differences (p<0.05).

Fig. 2

Catalase enzymatic activity in the liver 2 and 4 h following APAP overdose in C57Bl/6 mice was assessed with mice randomly divided into Veh (water), SAMe (1.25 mmol/kg), APAP (250 mg/kg), and SAMe administered 1 h post-APAP (S + A). Values represent mean ± S.E.M. with n=5 mice per group and superscripts denoting statistical differences (p<0.05).

Fig. 3

Liver GPx activity alterations when SAMe was administered 1 h after APAP overdose. C57Bl/6 mice were randomly divided into Veh (water), SAMe (1.25 mmol/kg), APAP (250 mg/kg), and SAMe administered 1 h following APAP (S + A). Livers were collected 2 and 4 h following APAP overdose. All values represent mean ± S.E.M. Each group represents 5 mice with superscripts denoting statistically significant differences (p<0.05).

Fig. 4

GSSG reductase activity in the liver following APAP overdose in C57Bl/6 mice was determined by enzymatic assay. Mice were randomly allocated into Veh (water), SAMe (1.25 mmol/kg), APAP (250 mg/kg), and SAMe administered 1 h following APAP (S + A) and livers collected 2 and 4 h following APAP overdose. Each group represents 5 experiments with different mice and superscripts denote statistically significant differences (p<0.05).

Fig. 5

SOD activity following APAP overdose in C57Bl/6 mice. Cu/Zn-SOD was inhibited with DDTC for determination of Mn-SOD. Panel A represents SOD levels 2 h following APAP overdose, while panel B represents SOD levels 4 h following APAP overdose. Superscripts denote statistically significant differences (p<0.05) with n=5 mice per group.

Fig. 6

Protein carbonyl formation in C57Bl/6 mouse mitochondria 2 and 4 h following APAP overdose. Protein carbonyls were assessed using an OxyBlot™ kit. Panel A contains densitometry from 2 and 4 h OxyBlots™ following APAP overdose. Densitometry was normalized to total protein staining (not shown) and is expressed as % Veh. All values represent mean ± S.E.M. with n=4 mice per group. Superscripts denote statistical difference (p<0.05). Panel B is a representative 4 h blot with lanes 1-2 corresponding to Veh, 3-4 SAMe, 5-6 APAP, and 7-8 SAMe administered 1 h following APAP (S + A).

Fig 7

Mitochondrial 3-NT protein adduct formation 4 h following APAP overdose. Panel A is a representative 4 h blot containing mitochondrial samples from the indicated groups. Panel B is the densitometry from panel A for whole lane 3-NT adduction normalized to total lane protein staining. Superscripts represent statistical significance, and values are mean ± S.E.M. with n=3-5 mice per group.

Fig 8

Mitochondrial swelling and cytochrome c leakage were used to assess mitochondrial function following APAP overdose. Mice were randomly allocated into Veh (water), SAMe (1.25 mmol/kg), APAP (250 mg/kg), and SAMe administered 1 h following APAP. Livers were collected 2 and 4 h following APAP administration and analyzed for mitochondrial swelling (Panel A and B). Panel C represents densitometry conducted on cytosolic cytochrome c levels following APAP overdose when analyzed at 2 and 4 h. Panel D is a representative 4 h cytosol cytochrome c blot depicting Veh (Lanes 1 and 2), SAMe (Lanes 3 and 4), APAP (Lanes 5 and 6), and S + A (Lanes 7 and 8). All values represent mean ± S.E.M. with at least 4 mice represented in each group. Super and subscripts denote statistical differences (p<0.05).

Fig. 9

CYP2E1 enzyme activity and expression in APAP treated mice. Panel A represent densitometry for CYP2E1 enzyme expression measured at 2 h (Panel B) and 4 h (Panel C) using a Western blot. APAP inhibited CYP2E1 expression while SAMe did not inhibit CYP2E1 expression. Panel D represent para-hydroxylation of aniline to p-aminophenol (PAP) measured 2 and 4 h after APAP treatment in groups as described in Fig. 1. Enzyme activity was expressed as nmol PAP/mg protein/20 min. All values represent mean ± S.E.M. with at least 4 mice represented in each group. Superscripts denote statistical differences between groups as differences between groups are denoted by different letters. (p<0.05).