Comparison of S-adenosyl-L-methionine (SAMe) and N-acetylcysteine (NAC) protective effects on hepatic damage when administered after acetaminophen overdose

Abstract

In the clinical setting, antidotes are generally administered after the occurrence of a drug overdose. Therefore, the most pertinent evaluation of any new agent should model human exposure. This study tested whether acetaminophen (APAP) hepatotoxicity was reversed when S-adenosyl-L-methionine (SAMe) was administered after APAP exposure, similar to what occurs in clinical situations. Comparisons were made for potency between SAMe and N-acetylcysteine (NAC), the current treatment for APAP toxicity. Male C57BL/6 mice were fasted overnight and divided into groups: control (VEH), SAMe treated (SAMe), APAP treated (APAP), N-acetylcysteine treated (NAC), SAMe or NAC administered 1h after APAP (SAMe+APAP) and (NAC+APAP), respectively. Mice were injected intraperitoneal (i.p.) with water (VEH) or 250 mg/kg APAP (15 ml/kg). One hour later, mice were injected (i.p.) with 1.25 mmol/kg SAMe (SAMe+APAP) or NAC (NAC+APAP). Hepatotoxicity was evaluated 4h after APAP or VEH treatment. APAP induced centrilobular necrosis, increased liver weight and alanine transaminase (ALT) levels, depressed total hepatic glutathione (GSH), increased protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins. Treatment with SAMe 1h after APAP overdose (SAMe+APAP) was hepatoprotective and was comparable to NAC+APAP. Treatment with SAMe or NAC 1h after APAP was sufficient to return total hepatic glutathione (GSH) to levels comparable to the VEH group. Western blot showed reversal of APAP mediated effects in the SAMe+APAP and NAC+APAP groups. In summary, SAMe was protective when given 1h after APAP and was comparable to NAC.

Figure 1. The effect of SAMe and NAC given 1 h after APAP treatment on hepatic tissue

Hepatic tissue collected 4 h after ip injection of 250 mg/kg acetaminophen (APAP) from male C57BL/6 mice. Groups were vehicle (VEH), SAMe treated (SAMe), NAC treated, acetaminophen treated (APAP) and SAMe or NAC administered 1 h after APAP injection (SAMe+APAP) and (NAC+APAP), resp. SAMe and NAC were injected (ip) at a dose of 1.25 mmol/kg (5 ml/kg). Normal morphology was observed in: vehicle (VEH, panel A), SAMe (panel B) and NAC (Panel E) treated animals. Centrilobular necrosis was evident in the APAP (panel C) group. Treatment with SAMe (SAMe+APAP) or NAC (NAC+APAP) 1 h after APAP treatment was associated with less extensive centrilobular necrosis (panels D and F, resp.). All slides are H&E stained at 10 μm slice and 200x magnification.

Figure 2. The Protective effect of SAMe and NAC on Total Hepatic Glutathione (GSH) Levels following APAP injection in C57BL/6 mice

Animals were randomly divided into vehicle (VEH), acetaminophen treated (APAP), SAMe (500 mg/kg, 1.25 mmol/kg) treated (SAMe), N-acetylcysteine treated (NAC), SAMe administered 1 h after APAP injection (SAMe+APAP) and NAC administered 1 h after APAP injection (NAC+APAP). APAP was injected (ip) at a dose of 250 mg/kg (15 ml/kg). VEH treated were injected with water (15 ml/kg). Values represent Mean ± SEM, n=5 mice/group. Total hepatic glutathione levels were measured 4 h after injection (ip) of 250 mg/kg APAP. Unlike superscript letters indicate groups with statistical difference (p<0.05).

Figure 3. Glutathione disulfide (GSSG) Levels, rescue effect of SAMe and NAC administered 1 h after APAP

Animals were randomly divided into vehicle (VEH), acetaminophen treated (APAP), SAMe (500 mg/kg, 1.25 mmol/kg) treated (SAMe), N-acetylcysteine treated (NAC), SAMe administered 1 h after APAP injection (SAMe+APAP) and NAC administered 1 h after APAP injection (NAC+APAP). APAP was injected (ip) at a dose of 250 mg/kg (15 ml/kg). VEH treated were injected with water (15 ml/kg). Glutathione disulfide (GSSG) levels were measured 4 h after APAP injection and 3 h after SAMe and NAC treatment. Unlike superscript letters indicate groups with statistical difference (p<0.05).

Figure 4. Lipid peroxidation was reduced by SAMe and NAC treatment 1 h after APAP injection

Animals were randomly divided into vehicle (VEH), acetaminophen treated (APAP), SAMe (500 mg/kg, 1.25 mmol/kg) treated (SAMe), N-acetylcysteine treated (NAC), SAMe administered 1 h after APAP injection (SAMe+APAP) and NAC administered 1 h after APAP injection (NAC+APAP). APAP was injected (ip) at a dose of 250 mg/kg (15 ml/kg). VEH treated were injected with water (15 ml/kg). Lipid peroxidation was reported as nmol malondialdehyde (MDA) per mg protein. Lipid peroxidation was measured in hepatic tissue collected 4 h after APAP injection. Values represent MEAN ± SE. Groups with dissimilar superscripts are different (p<0.05) from each other. SAMe and NAC administered 1 h after APAP diminished the extent of hepatic lipid peroxidation.

Figure 5. Protein carbonyls in hepatic tissue 4 h after APAP treatment and 3 h after SAMe or NAC treatment

Hepatic tissue was isolated 4 h after APAP or VEH injection. SAMe and NAC were administered 1 h after APAP injection (ip). The lanes within the gel represent VEH (lane 1), 1.25 mmol/kg SAMe (lane 2), 1.25 mmol/kg NAC (lane 3), 250 mg/kg APAP (lane 4), combination of NAC and APAP (NAC+APAP lane 5) and a combination of SAMe and APAP (lane 6) as described in the methods. Western blots were performed using an OxyBlot Kit to detect protein carbonyls. Each lane in the gel was loaded with the same volume and concentration of protein. Densitometry was performed and expressed in arbitrary units.

Figure 6. 4-HNE adducted hepatic proteins 4 h after APAP treatment and 3 h after SAMe or NAC treatment

Hepatic tissues were collected from mice 4 h after injection (ip) with VEH (lane 1), 1.25 mmol/kg SAME (lane 2), 1.25 mmol/kg NAC (lane 3), 250 mg/kg APAP (lane 4), 1.25 mmol/kg SAMe administered 1 h after 250 mg/kg APAP (SAMe+APAP, lane 5) and 1.25 mmol/kg NAC administered 1 h after 250 mg/kg APAP (NAC+APAP, lane 6) as described in the methods. Western blots were performed using an anti-4HNE primary antibody with a peroxidase conjugated secondary antibody. Gels were loaded with the same volume and concentration of protein based on mass. Bands were detected via chemiluminescence. Densitometry was performed and expressed in arbitrary units.