High methionine diet mediated oxidative stress and proteasome impairment causes toxicity in liver

Abstract

Methionine (Met) rich diet inducing oxidative stress is reported to alter many organs. Proteasome as a regulator of oxidative stress can be targeted. This study was performed to investigate if excessive methionine supplementation causes hepatotoxicity related to proteasome dysfunction under endogenous oxidative stress in rats. Male Wistar albino rats (n = 16) were divided into controls and treated groups. The treated rats (n = 08) received orally L-methionine (1 g/kg/day) for 21 days. Total homocysteine (tHcy), total oxidant status (TOS), total antioxidant status (TAS), hepatic enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), with total bilirubin (TBil), albumin (Alb), and C-reactive protein (CRP) were determined in plasma by biochemical assays. Liver supernatants were used for malondialdehyde (MDA), protein carbonyls (PC), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), 20S proteasome activities and their subunits expression, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) evaluation by appropriate methods and light microscopy for liver histological examination. Methionine treatment increased homocysteine, TOS, oxidative stress index (OSI), MDA and PC but decreased TAS, GSH, CAT, SOD, GPx with the 20S proteasome activities and their β subunits expression. Liver proteins: AST, ALT, LDH, ALP, TBil and CRP were increased but Alb was decreased. Liver histology was also altered. An increase in liver TNF-α and IL-6 levels were observed. These findings indicated that methionine supplementation associated oxidative stress and proteasome dysfunction, caused hepatotoxicity and inflammation in rat. Further investigations should be to better understand the relation between methionine, oxidative stress, proteasome, and liver injuries.

Figure 1

Effect of l-methionine supplementation on levels of Homocysteine (A), OSI (B), TAS (C), TOS (D), (MDA, PC,GSH) (E), CAT (F), SOD (G), GPx (H) in plasma of controls and treated rats (n = 08/group). Values were expressed as means ± SEM, (*p < 0.05, **p < 0.01, ***p < 0.001) vs controls. MDA malondialdehyde, PC protein carbonyl, GSH Reduced glutathione, CAT catalase, SOD Superoxide dismutase, GPx glutathione peroxidase.

Figure 2

Effect of L-methionine supplementation on 20S proteasome activities (A) and 20S β subunits expression levels normalized to β actin (B) in liver of controls and treated rats, A (n = 08/group) and B (n = 06/group). Proteins were quantified by densimetry using Image scanner III and Image Quant TL software (GE Healthcare). Blots images were cutting from the films scanned to eliminate other blots appeared in full length membranes, with membrane edges. Values were expressed as means ± SEM, (*p < 0.05, **p < 0.01) vs controls. CT-L chymotrypsin-like, T-L trypsin-like, C-L caspase-like.

Figure 3

Effect of l-methionine supplementation on levels of liver biomarkers (AST, ALT, LDH, ALP) (A), TBil (B), Alb (C) and CRP (D) in plasma of controls and treated rats (n = 08/group). Values were expressed as means ± SEM, (*p < 0.05, **p < 0.01) vs controls. (AST) aspartate amino transferase, (ALT) alanine amino transferase, (LDH) lactate dehydrogenase, (ALP) alkaline phosphatase, (TBil) total bilirubin, (Alb) albumin: (CRP) C-reactive protein.

Figure 4

Photomicrographs of H&E (X400 magnifications) stained rat liver longitudinal sections (4 slides/animal), from controls (n = 08) (A) (scale bar, 100 µm) and treated rats (n = 08) with 3 weeks L-methionine (B) (scale bar, 100 µm) and (C, D) (scale bar, 50 µm). H hepatocyte, S sinusoid, CV central vein, D dilatation, N necrosis, HB ballooned hepatocyte, V vacuoles, SH sinusoid hemorrhage, L lysis, ENDL endolysis, Inf Inflammatory cells infiltration.

Figure 5

Effect of l-methionine supplementation on levels of TNF-α (A) and IL-6 (B) in liver of controls and treated rats (n = 08/group). Values were expressed as means ± SEM, *p < 0.05 Vs controls. TNF-α tumor necrosis factor-α, IL-6 interleukin 6.