Journal of Nutrition LabDiet, Your World of Nutritional Answers

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplemental Material
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baumgardner, J. N.
Right arrow Articles by Ronis, M. J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baumgardner, J. N.
Right arrow Articles by Ronis, M. J. J.
© 2008 American Society for Nutrition J. Nutr. 138:1872-1879, October 2008

Biochemical, Molecular, and Genetic Mechanisms

N-Acetylcysteine Attenuates Progression of Liver Pathology in a Rat Model of Nonalcoholic Steatohepatitis1–3,

January N. Baumgardner4,8, Kartik Shankar4,8, Leah Hennings7, Emanuele Albano9, Thomas M. Badger4–68 and Martin J. J. Ronis4,6,8,*

Departments of 4 Pharmacology and Toxicology, 5 Physiology and Biophysics, 6 Pediatrics, and 7 Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205; 8 Arkansas Children's Nutrition Center, Little Rock, AR 72202; and 9 Department of Medical Sciences, University "Amedeo Avogadro" of East Piedmont, I-28100 Novara, Italy

* To whom correspondence should be addressed. E-mail: ronismartinj{at}uams.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
A "2-hit" model for nonalcoholic steatohepatitis (NASH) has been proposed in which steatosis constitutes the "first hit" and sensitizes the liver to potential "second hits" resulting in NASH. Oxidative stress is considered a candidate for the second hit. N-acetylcysteine (NAC), an antioxidant, has been suggested as a dietary therapy for NASH. We examined the effects of NAC in a rat total enteral nutrition (TEN) model where NASH develops as the result of overfeeding dietary polyunsaturated fat. Male Sprague-Dawley rats consumed pelleted AIN-93G diets ad libitum or were overfed a 9200 kJ·kg–0.75·d–1 liquid diet containing 70% corn oil with or without 2 g·kg–1·d–1 NAC i.g. for 65 d. Hepatic steatosis was not influenced by dietary supplementation with NAC; however, the liver pathology score was lower (P ≤ 0.05) and NAC provided partial protection against alanine aminotransferase release (P ≤ 0.05). NAC attenuated increased hepatic oxidative stress (TBARS; P ≤ 0.05) and prevented increases in cytochrome P450 2E1 apoprotein and mRNA and in tumor necrosis factor-{alpha} (TNF{alpha}) mRNA. Titers of auto-antibodies against proteins adducted to lipid peroxidation products were lower in serum of the NAC group than in the 70% corn oil group (P ≤ 0.05). NAC also decreased Picosirius red staining of collagen, a marker of fibrosis. However, markers of hepatic stellate cell activation were unaffected. Using NAC in a TEN model of NASH, we have demonstrated that NAC prevents many aspects of NASH progression by decreasing development of oxidative stress and subsequent increases in TNF{alpha} but does not block development of steatosis.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Nonalcoholic fatty liver disease is the most common of liver pathologies and is closely associated with obesity and the metabolic syndrome. There is a wide spectrum of pathologies covered by nonalcoholic fatty liver disease, ranging from simple reversible steatosis to nonalcoholic steatohepatitis (NASH),10 in which steatosis is accompanied by inflammation, necrosis, apoptosis, and fibrosis. The mechanisms leading to NASH are multifactorial. In patients with NASH, there is an initial development of steatosis, plus a second insult resulting in disease progression. The 2- hit model of NASH was proposed in 1998 (1) in which alterations in lipid homeostasis associated with obesity, insulin resistance, and adipokine disruption result in steatosis and constitute the first hit. This sensitizes the liver to potential second hits resulting in hepatocellular injury, inflammation, and fibrosis. Oxidative stress is frequently cited as a central mechanism of hepatocellular injury in NASH, correlating with the accumulation of lipid peroxidation products, appearance of mitochondrial dysfunction (2–4), and elevation of proinflammatory cytokines. It is considered one of the best candidates for the second hit, because it can explain all the recognized histological features of the disease (5–7). Glutathione (GSH) is a major endogenous antioxidant. N-acetylcysteine (NAC), a precursor of GSH, has been in clinical use for >30 y as an antidote for acetaminophen overdose (8). Potential protective effects of NAC are being studied in chronic diseases characterized by decreased GSH or oxidative stress such as alcoholic liver disease and NASH (9–12). Most of the beneficial effects of orally administered NAC are theorized to be a result of its ability to either reduce extracellular cystine to cysteine or to be a source of sulfhydryl metabolites. As a source of sulfhydryl groups, NAC can stimulate GSH synthesis, enhance GSH-S-transferase activity, promote detoxification, and act as a scavenger of free radicals as it interacts with reactive oxygen species (ROS) (13,14). We have developed a unique rat model in which liver pathology closely resembles clinical NASH using overfeeding of a high polyunsaturated fat diet via total enteral nutrition (TEN) (15). NASH is histologically indistinguishable from alcoholic steatohepatitis and it is proposed they may also have many similarities in pathogenesis, including oxidative stress and increased cytochrome P450 2E1 (CYP2E1), proinflammatory cytokines, and tumor necrosis factor-{alpha} (TNF{alpha}) (1,15,16). We have previously shown NAC to be successful in prevention of alcohol-induced liver disease in rats fed via TEN (12). The aim of the present study was to determine the effects of NAC treatment on the development of liver pathology in our TEN NASH model.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Reagents. All chemicals were purchased from Sigma-Aldrich unless otherwise specified. Potassium chloride, potassium phosphate, and potassium ferricyanide were purchased from Fisher Scientific. Enhanced chemiluminescence for chemiluminescent detection in western blotting was purchased from Amersham Biosciences. TRI Reagent used for RNA extraction was obtained from Molecular Research Center. Reagents for the assessment of RNA quality using the Agilent Bioanalyzer were acquired from Agilent Technologies.

    Experimental animals and diets. Male Sprague-Dawley rats (175 g) were purchased from Harlan Sprague-Dawley. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility. Animal maintenance and experimental treatments were conducted in accordance with ethical guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. Animals were randomly assigned to diet groups. Control rats (C) consumed ad libitum water and a pelleted AIN-93G diet in which the protein source (partially hydrolyzed whey) matched that of the liquid diets utilized for NASH development (Table 1) (17). Rats fed by TEN received 9200 kJ·kg–0.75·d–1 (which represents 17% greater energy intake than recommended by the NRC). TEN diets contained corn oil at a level equal to 70% of the total energy (15) with or without 2 g·kg–1·d –1 NAC [high fat (HF) or HF + NAC] (Table 1). TEN-fed rats had an i.g. cannula surgically inserted and were allowed 7 d to recover before diet infusion commenced as described previously (12,15,18–21). Vitamin and mineral content was the same in all diets (Table 1) (17,21). All diets met energy and nutritional levels recommended by the NRC (17,21). We recorded body weights twice a week and at the beginning and end of the study. Rats were killed after 65 d and serum and livers were collected and stored at –20°C and –70°C, respectively.


View this table:
[in this window]
[in a new window]

  		TABLE 1 Diet composition

 
    Pathological evaluation. Liver pathology was assessed by hematoxylin-eosin (H&E) and Oil Red O staining of liver sections and scored by a board-certified pathologist (L. Hennings). The pathology calculation was based on ballooning degeneration (0–2), presence or absence of serum markers of necrosis [alanine aminotransferase (ALT) score: cut-off was 55, based on data from Charles River Laboratories and our baseline values, <55 = 0, ≥55 = 1], the lipidosis score (based on evaluation of Oil Red O stained slides), and lobular inflammation/necrosis (0 = 0 foci, 1 = <2 foci, 2 = 2–4 foci, and 3 = >4 foci) as described previously (15). Portal fibrosis was detected by Picosirius red staining of collagen (22).

    Biochemical analysis. Serum ALT activity levels were measured when the rats were killed using the Infinity ALT liquid stable reagent (Thermo Electron) according to the manufacturer's protocols. Liver microsomes were prepared by differential centrifugation and stored at –70°C until analysis. Protein concentrations of the microsomes were determined by the Bradford method using the Bio-Rad Protein Assay. Liver lipid peroxidation was assessed as a measure of oxidative stress as described by Ohkawa et al. (23). Western immunoblot analysis of apoprotein expression for CYP2E1 was conducted as previously described (15,24). CYP2E1 was a gift from the laboratory of Dr. Magnus Ingelman-Sundberg (Karolinska Institute) (25). Western immunoblot analysis of protein expression for malondialdehyde (MDA) was conducted as described by Polavarupu et al. (26). Briefly, total proteins (30 µg) were loaded on SDS-PAGE and transferred to nitrocellulose membrane and incubated with 2% bovine serum albumin in Tris-buffered saline/Tween for blocking and subsequently incubated with the antibody (1 mg·L–1) (Cosmo Bio). This procedure was followed by the addition of horseradish peroxidase-linked goat antibody to mouse IgG and detected by enhanced chemiluminescence (27,28). The bands were visualized by autoradiography and entire lanes were quantified.

    Measurement of auto-antibodies to proteins adducted with MDA and arachidonate hydroperoxide. Circulating auto-antibodies to proteins adducted with the lipid peroxidation products MDA or arachidonate hydroperoxide (LOOH) were measured as described previously by Mottaran et al. (29).

    Real-time RT-PCR. Total RNA was extracted from livers using TRI Reagent and cleaned using RNeasy mini columns (Qiagen). RNA quality was ascertained spectrophotometrically (ratio of A260:A280) and also by checking ratio of 28S:18S ribosomal RNA using the RNA Nano Chip on a 2100 Bioanalyzer (Agilent Technologies). Total RNA (1 µg) was reverse transcribed using the iScript Reverse Transcription kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The reverse transcribed cDNA (10 ng) was utilized for real-time PCR using the 2x SYBR Green Master mix and monitored on a ABI Prism 7000 sequence detection system (Applied Biosystems). Gene-specific probes were designed using Primer Express Software (Applied Biosystems; Supplemental Table 1) and the relative amounts of gene expression were quantitated using a standard curve according to the manufacturer's instructions.

    Measurement of hepatic GSH concentrations. Hepatic GSH concentrations were quantified from C and NAC-treated rats using a commercially available kit (703002) from Cayman Chemical. The kit is based on enzymic recycling of GSH using GSH reductase followed by reaction of GSH with Ellman's reagent. The colored product is measured at 405 nm and quantified using a standard curve.

    Statistical analysis. Data are expressed as means ± SEM. Densitometric quantitation of Western blot autoradiograms was performed using Quantity One software (Bio-Rad). We used SigmaStat software package version 3.0 (SPSS) to perform all statistical tests. The data were tested using Levene's test for equality of variance. We performed Pearson product moment correlation using SigmaStat software. Group differences were evaluated via 1-way ANOVA followed by Student-Newman-Keuls post hoc comparisons unless the data were not normally distributed, in which case evaluation was using 1-way ANOVA of ranks. P-values ≤ 0.05 were considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Dietary supplementation with NAC partly protected against liver injury in NASH. The pellet-fed C group consumed 7800 kJ·kg–0.75.d–1 and had a growth rate of 3.83 ± 0.03 g·d–1. In comparison, the HF TEN group infused with 9200 kJ·kg–0.75.d–1 had a growth rate of 6.84 ± 0.1 g·d–1. The HF + NAC group infused with the same TEN diet + NAC had a growth rate of 5.89 ± 0.08 g·d–1. Growth rates for the HF groups were higher than that of the pellet-fed group (P ≤ 0.05). Pathological examination demonstrated no histological evidence of fat accumulation or inflammation in group C (Fig. 1D), but steatosis, hepatocellular ballooning, inflammation, necrosis, and portal/lobular fibrosis similar to that observed in Grade 3 clinical NASH was present in the HF group (Fig. 1E; Supplemental Fig. 1) consistent with previous observations for our laboratory (15). The total pathology score and pathology scores in excess of simple steatosis were lower in the HF + NAC group (Fig. 1F) than in the HF group (P ≤ 0.05) (Table 2). Development of NASH was accompanied by elevated serum ALT activity (P ≤ 0.05) (Table 3), indicating necrotic injury. The addition of NAC attenuated this increase; however, ALT values were still higher than in the C group (P ≤ 0.05) (Table 3). Picosirius red staining demonstrated an increase in portal/lobular fibrosis in the HF group (Fig. 1G,H) (P ≤ 0.05), which was attenuated by NAC administration (P ≤ 0.05) (Fig. 1I; Table 2). Oil red O staining and biochemical analysis of triacylglycerol showed an increase in hepatic steatosis in the HF group (Fig. 1A,B) (P ≤ 0.05) associated with an increase in relative expression of mRNA for the fatty acid transport-associated protein CD36 from 1.0 ± 0.08 to 12.1 ± 2.7 (P ≤ 0.05). NAC did not affect the overall level of fat accumulation in the liver or relative CD36 mRNA expression (14.1 ± 3.3) but did appear to attenuate progression of liver pathology beyond steatosis and result in formation of smaller lipid droplets (Fig. 1C; Table 3).


Figure 1
View larger version (144K):
[in this window]
[in a new window]

  		FIGURE 1  Representative Oil Red O (A–C), H&E (D–F), and Picosirius Red (G–I) stained liver sections from male rats overfed HF diets with or without NAC supplementation. Representative Oil Red O-stained liver sections: C (A); HF (B); and HF + NAC (C) (x20 magnification). Representative H&E-stained liver sections: C (D); HF(E); and HF + NAC (F) (x10 magnification). Representative Picosirius Red stained liver sections: C (G); HF(H); and HF + NAC (I) (x10 magnification).

 

View this table:
[in this window]
[in a new window]

  		TABLE 2 Pathology scores of rats fed C, HF, or HF + NAC diets for 65 d1

 

View this table:
[in this window]
[in a new window]

  		TABLE 3 Serum ALT and triacylglycerols and hepatic triacylglycerols and MDA protein in rats fed C, HF, or HF + NAC diets for 65 d1

 
    Dietary NAC alleviated oxidative stress and inflammation. Hepatic lipid peroxidation increased (TBARS; P ≤ 0.05) in the HF group (Fig. 2A), accompanied by decreased hepatic GSH concentrations (P ≤ 0.05) (Fig. 2B). Although it did not reach statistical significance, as a result of inter-animal variability, the mean expression of MDA-adducted proteins also increased in Western blots from liver homogenates in the HF group (P = 0.1) (Table 3). An increase in CYP2E1 apoprotein and mRNA (P ≤ 0.05) (Fig. 3A,B), a source of ROS, was also observed in the HF group as were increases in mRNA expression of the proinflammatory cytokine, TNF{alpha} (P ≤ 0.05) (Table 4). The level of mRNA for another proinflammatory cytokine, IL-1β, tended to be greater in the HF group than in the C group (P = 0.08). Expression of CD14 mRNA, an indicator of Kupffer cell activation (12), did not differ between the groups (Fig. 4A). The ratio of CD68:CD45, an indicator of leukocyte activation and infiltration (30), tended to be greater in the HF group than in the C group (P = 0.077) (Fig. 4B). These indictors of oxidative stress and inflammation, except IL-1β, were attenuated by NAC supplementation (P ≤ 0.05).


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 2  NAC prevents increases in hepatic TBARS (A) and partially restores hepatic GSH concentrations (B) reduced by overfeeding rats a HF diet. Values are means ± SEM; n = 4 (C), 7 (HF), or 8 (HF + NAC). Means without a common letter differ, P ≤ 0.05.

 

Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 3  NAC supplementation reverses induction of rat hepatic CYP2E1 apoprotein (A) and hepatic CYP2E1 mRNA (B) expression following the overfeeding of a HF diet. Values are means ± SEM; n = 4 (C), 7 (HF), or 8 (HF + NAC). Means without a common letter differ, P ≤ 0.05. In the representative western blot, each lane represents the liver microsomal protein from individual animals.

 

View this table:
[in this window]
[in a new window]

  		TABLE 4 Relative expression of hepatic proinflammatory cytokines in rats fed C, HF, or HF + NAC diets for 65 d1

 

Figure 4
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 4  Effects of overfeeding HF with or without NAC supplementation on rat hepatic CD14 mRNA (A) and the CD68:CD45 mRNA ratio (B) as an indicator of leukocyte activation and infiltration. Values are means ± SEM; n = 4 (C); 7 (HF), or 8 (HF + NAC). Means without a common letter differ, P ≤ 0.05.

 
    NAC supplementation reduced immune responses to protein adducts of lipid peroxidation products. Titers of antibodies directed against proteins adducted to the lipid peroxidation products MDA and LOOH were higher in the HF group than in the C group (P ≤ 0.05) and there were reduced by supplementation with NAC (Fig. 5).


Figure 5
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 5  NAC supplementation reduces immune responses to protein adducts of MDA (A) and lipid hydroperoxides (B) in rats overfed a HF diet. Values are means ± SEM; n = 4 (C), 7 (HF), or 8 (HF + NAC). Means without a common letter differ, P ≤ 0.05.

 
    Effects of NAC administration on early markers of hepatic stellate cell activation and the turnover of extracellular matrix. Transforming growth factor-β (TGFβ) is a cytokine linked to the activation of hepatic stellate cells (HSC) and development of fibrosis. Platelet-derived growth factor receptor-β (PDGFrβ) and {alpha}-smooth muscle actin ({alpha}-SMA) are also early markers associated with HSC activation (31). Hepatic mRNA for all 3 were higher in the HF group than in the C group (P ≤ 0.05). However, elevation in expression of these mRNA in the HF group was not affected by the addition of NAC (Fig. 6A–C). mRNA expression was not affected for connective tissue growth factor (Table 5) or the interstitial collagenase, matrix metallopeptidase 13 (MMP13) or its inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) (Table 5). In contrast, mRNA expression for CD44, the major cell-surface hyaluronan (HA) receptor (32), was higher in the HF group and was attenuated by NAC treatment (P ≤ 0.05) (Table 5).


Figure 6
View larger version (6K):
[in this window]
[in a new window]

  		FIGURE 6  Effects of overfeeding HF with or without NAC supplementation on relative mRNA expression of early markers of HSC activation: TGFβ (A), {alpha}-SMA (B), and PDGFrβ (C). Values are means ± SEM; n = 4 (C), 7 (HF), or 8 (HF + NAC). Means without a common letter differ, P ≤ 0.05.

 

View this table:
[in this window]
[in a new window]

  		TABLE 5 Relative expression of genes associated with fibrosis and modification of the extracellular matrix in rats fed C, HF, or HF + NAC diets for 65 d1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
One area of general agreement between investigators studying mechanisms of NASH is that steatosis is a necessary prerequisite for any further damage. In the rat TEN NASH model, where pathology is driven by overfeeding of polyunsaturated fats, development of hepatic steatosis results at least in part from increased fatty acid uptake (15). Consistent with this, expression of the fatty acid transport associated molecule CD36 significantly increased. However, neither CD36 expression nor steatosis was reversed by NAC supplementation. The lack of effect on steatosis is similar to our previous findings with NAC in a rat model of alcoholic steatohepatitis (12).

The progression from steatosis to steatohepatitis and fibrosis (the second hit) is complex and not well defined. Oxidative stress is thought to play an important role in the second hit. Peroxidation of lipids accumulated within steatotic hepatocytes has been demonstrated (5–7). Mitochondrial dysfunction and induction of hepatic CYP2E1 (33) leading to the formation of ROS (34) promote lipid peroxidation and oxidative stress. Increased staining for lipid peroxidation products in steatotic livers, with added increases in those with steatohepatitis (35), has been confirmed by immunohistochemistry. Disease progression may also involve immune responses to proteins adducted by lipid peroxidation products (36). Increases in proinflammatory cytokines, such as TNF{alpha} and IL-1β, could also mediate hepatic inflammation.

Interestingly, the hepatic accumulation of lipids in patients with NASH and in the current model is associated with increased mitochondrial β-oxidation of fatty acids (15,37,38), which has been suggested to be an adaptive response for limiting the accumulation of lipid. Respiratory chain complexes are reported to be poorly coupled in patients with NASH and increased mitochondrial FA oxidation may serve as a source of ROS (39). We observed hepatic lipid peroxidation, which correlates with development of steatosis, oxidative stress, increased TNF{alpha}, and increased necrosis (15). Bioactive aldehydes such as MDA are chemically reactive products of lipid peroxidation that result in chemotaxis and cytotoxicity (40,41) and the modulation of cell signaling cascades (21). Although the mechanisms whereby MDA and other aldehydes such as hydroxynonenal elicit these effects remain to be delineated, it is likely they occur as a consequence of protein modification. As has been described in NASH patients, proteins adducted with lipid peroxidation products such as MDA and hydroxynonenal and with LOOH also stimulate the host immune response, which could result in an autoimmune-like disease (39). The values for cellular injury and presence of auto-antibodies to LOOH-adducted proteins were weakly correlated (r2 = 0.34). That NAC reduces the presence of antibodies to these protein adducts coincidental with attenuated progression of hepatic pathology suggests that immune mechanisms might be involved. Consistent with this are observations in humans showing that elevated titers of circulating IgG toward MDA and LOOH adducted proteins in a large fraction of hepatitis and/or cirrhosis patients but only in a few subjects with simple steatosis alone (39).

Even though CD14 mRNA was not elevated in the HF group, indicating a lack of Kupffer cell activation, our data regarding the CD68:CD45 ratio and TNF{alpha} is consistent with the ability of NAC to reduce inflammation in this model. TNF-{alpha} is a cytokine proposed to play a central role in the chronic inflammatory response in NASH (42,43). Kupffer cells are the major source of increased TNF{alpha} secretion within the liver, with infiltrating monocytes and macrophages also contributing (42,43). TNF{alpha} mRNA is increased in both the liver and adipose tissues in NASH patients (44). In both steatosis and steatohepatitis, soluble TNF receptor levels are elevated (45,46), and enhanced TNF{alpha} secretion from circulating monocytes is seen in patients with NASH compared with healthy controls (47). TNF receptor knockout mice have less severe steatosis than wild-type littermates (48) and a significant reduction in liver injury in steatohepatitis after anti-TNF{alpha} therapy (49–51), supporting the pathogenic role of TNF{alpha}. In this study, the proinflammatory cytokine TNF-{alpha} mRNA was higher in the HF group but was attenuated by the addition of NAC to the diet. This suggests that TNF{alpha} elevation in NASH is the result of increased oxidative stress, perhaps secondary to an increase in CYP2E1 expression. It has recently been shown that CYP2E1 located in the mitochondrial fraction is associated with mitochondrial lipid peroxidation and damage and is associated with TNF{alpha} production in alcohol-induced steatohepatitis (52). NASH patients have also been shown to have upregulation of CYP2E1, which may be one of the most important sources of ROS.

Regardless of etiology, oxidative stress plays a major role in activation of HSC and in hepatic fibrogenesis (53). Lipid peroxidation has also been shown to stimulate collagen production in fibroblasts and HSC (54). GSH precursors or antioxidant agents have been shown to exert protective effects against activation of HSC (55); however, the role of oxidative stress and the beneficial effects of GSH precursors or antioxidant agents during the initial phases of liver fibrosis have not been fully investigated. The antifibrotic effects of NAC have been demonstrated in other experimental models, especially in the lung (56,57). In the current study, we observed partial protection against development of hepatic fibrosis in the NAC-treated group; however, there was no reduction in TGFβ or its downstream targets, {alpha}-SMA and PDGFr-β mRNA, both markers of HSC activation, compared with the HF group. In addition, MMP13 or TIMP-1 were not significantly affected. However, CD44, the major cell surface HA receptor, was increased in the HF group and this was prevented by NAC. CD44 plays a role in clearing fragmented HA, which accumulates during tissue injury (32). However, HA may also play a role in promoting tissue repair as the result of interactions with Toll-like receptors. In an acute lung injury model, overexpression of high molecular mass HA resulted in protection against injury in part through Toll-like receptor-mediated activation of nuclear factor {kappa}B (58). It is possible that increased HA, as the result of reduced CD44 expression in NAC-treated rats, contributed to the partial protection against fibrosis in the current model. There is a possibility that NAC was also working downstream of TGFβ to diminish the progression of fibrosis via other signaling pathways. Dietary NAC is rapidly cleared from the body (13,59). Assuming a constant infusion of NAC at 2000 mg·kg–1·d–1, steady-state plasma concentrations of NAC are calculated to be ~300 µmol·L–1. NAC has provided protection in our model, despite this relatively low predicted in vivo concentration, compared with in vitro studies where antioxidant and antifibrotic activities of NAC have been reported at levels in excess of 5 mmol·L–1 (41). The observed antifibrotic effect may be secondary to decreases in lipid peroxidation and proinflammatory cytokines, such as TNF{alpha}, both of which have been implicated in the development of fibrosis. Lipid peroxidation products can directly enhance collagen production by activated HSC (60). Consistent with a role of lipid peroxidation in steatosis-triggered fibrosis, vitamin E and C supplementation in NASH patients has been reported to improve fibrosis (61). The effects on the CD68:CD45 ratio and TNF{alpha} suggest that NAC's protective effect against liver fibrosis may be associated with its ability to inhibit infiltrating macrophages. To some degree, our data parallels in vivo studies of antifibrotic effects of other antioxidants such as curcumin, the major polyphenolic compound in turmeric. Curcumin has been shown to inhibit the development of liver fibrosis mainly due to is antiinflammatory activities and not by a direct antifibrotic effect (62).

We have shown that dietary NAC is an effective hepatic antioxidant that abolished NASH-induced lipid peroxidation, attenuated reductions in hepatic GSH, blocked NASH-associated autoimmune responses, inhibited the production of TNF{alpha}, and attenuated inflammation, leading to significant reduction in cellular damage, hepatocyte injury, and fibrosis. These effects are similar to those we have previously reported for NAC treatment in a rat model of alcoholic steatohepatitis (12). To our knowledge, this is the first report that administration of NAC partially protects against progression of liver injury, including fibrosis, in this type of dietary model of NASH. These data suggest that NAC supplementation in conjunction with other treatments may be a valuable adjuvant therapy in NASH patients. Indeed, a clinical study has recently been published in which NASH patients treated for 12 mo with 1.2 g·d–1 NAC in conjunction with metformin demonstrated significantly improved fibrosis (63).


   ACKNOWLEDGMENTS
 
We thank the following people for their technical assistance: Matt Ferguson, Jamie Badeaux, Tammy Dallari, and Michele Perry.


   FOOTNOTES
 
1 Supported, in part, by RO1 AA 12819 (M.J.J.R.), ACNC-USDA-ARS 6251-51000-005D (T.M.B.), and the National Institute for Environmental Health Sciences Graduate Student Training Grant (J.N.B.). Back

2 Author disclosures: J. N. Baumgardner, K. Shankar, L. Hennings, E. Albano, T. M. Badger, and M. J. J. Ronis, no conflicts of interest. Back

3 Supplemental Table 1 and Supplemental Figure 1 are available with the online posting of this paper at jn.nutrition.org. Back

10 Abbreviations used: ALT, alanine aminotransferase; {alpha}-SMA, {alpha}-smooth muscle actin; C, pellet-fed control; CYP2E1, cytochrome P450 2E1; GSH, glutathione; HA, hyaluronan; H&E, hematoxylin-eosin; HF, high fat; HSC, hepatic stellate cell; IL, interleukin; LOOH, arachidonate hydroperoxide; MDA, malondialdehyde; MMP13, matrix metallopeptidase 13; NAC, N-acetylcysteine; NASH, nonalcoholic steatohepatitis; PDGFrβ, platelet-derived growth factor receptor-β; ROS, reactive oxygen species; TEN, total enteral nutrition; TGFβ, transforming growth factor-β; TIMP-1, tissue inhibitor of metalloproteinases-1; TNF{alpha}, tumor necrosis factor-{alpha}. Back

Manuscript received 20 March 2008. Initial review completed 12 April 2008. Revision accepted 26 July 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Day CP, James OF. Hepatic steatosis: innocent bystander or guilty party? Hepatology. 1998;27:1463–6.[Medline]

2. Albano E. Free radical mechanisms in immune reactions associated with alcoholic liver disease. Free Radic Biol Med. 2002;32:110–4.[Medline]

3. Chawla RK, Watson WH, Eastin CE, Lee EY, Schmidt J, McClain CJ. S-adenosylmethionine deficiency and TNF-alpha in lipopolysaccharide-induced hepatic injury. Am J Physiol. 1998;275:G125–9.[Medline]

4. Osmundsen H, Bremer J, Pedersen JI. Metabolic aspects of peroxisomal beta-oxidation. Biochim Biophys Acta. 1991;1085:141–58.[Medline]

5. Mehta K, Van Thiel DH, Shah N, Mobarhan S. Nonalcoholic fatty liver disease: pathogenesis and the role of antioxidants. Nutr Rev. 2002;60:289–93.[Medline]

6. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology. 2003;37:1202–19.[Medline]

7. Pessayre D, Berson A, Fromenty B, Mansouri A. Mitochondria in steatohepatitis. Semin Liver Dis. 2001;21:57–69.[Medline]

8. Buckley NA, Whyte IM, O'Connell DL, Dawson AH. Oral or intravenous N-acetylcysteine: which is the treatment of choice for acetaminophen (paracetamol) poisoning? J Toxicol Clin Toxicol. 1999;37:759–67.[Medline]

9. Samhuaseneeto S, Thong-Ngam D, Kulaputana O, Patumraj S, Klaikeuw N. Effects of N-acetylcysteine on oxidative stress in rats with non-alcoholic steatohepatitis. J Med Assoc Thai. 2007;90:788–797.[Medline]

10. Thong-Ngam D, Samuhasaneeto S, Kulaputana O, Klaikeaw N. N-acetylcysteinine attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis. World J Gastroentrerol. 2007;13:5127–5132.

11. Pastor A, Collado PS, Almar M, Gonzalez-Gallego J. Antioxidant enzyme status in biliary obstructed rats: effects of N-acetylcysteine. J Hepatol. 1997;27:363–70.[Medline]

12. Ronis MJ, Butura A, Sampey BP, Shankar K, Prior RL, Korourian S, Albano E, Ingelman-Sundberg M, Petersen DR, et al. Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition. Free Radic Biol Med. 2005;39:619–30.[Medline]

13. De Vries N, De Flora S. N-acetyl-l-cysteine. J Cell Biochem Suppl. 1993;17F:270–7.

14. Videla LA, Rodrigo R, Orellana M, Fernandez V, Tapia G, Quinones L, Varela N, Contreras J, Lazarte R, et al. Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients. Clin Sci (Lond). 2004;106:261–8.[Medline]

15. Baumgardner JN, Shankar K, Hennings L, Badger TM, Ronis MJ. A new model for non-alcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high polyunsaturated fat diet. Am J Physiol Gastrointest Liver Physiol. 2008;294:G27–38.[Abstract/Free Full Text]

16. Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology. 1998;27:128–33.[Medline]

17. Ronis MJ, Rowlands JC, Hakkak R, Badger TM. Inducibility of hepatic CYP1A enzymes by 3-methylcholanthrene and isosafrole differs in male rats fed diets containing casein, soy protein isolate or whey from conception to adulthood. J Nutr. 2001;131:1180–8.[Abstract/Free Full Text]

18. Badger TM, Ronis MJ, Lumpkin CK, Valentine CR, Shahare M, Irby D, Huang J, Mercado C, Thomas P, et al. Effects of chronic ethanol on growth hormone secretion and hepatic cytochrome P450 isozymes of the rat. J Pharmacol Exp Ther. 1993;264:438–47.[Abstract/Free Full Text]

19. Badger TM, Crouch J, Irby D, Hakkak R, Shahare M. Episodic excretion of ethanol during chronic intragastric ethanol infusion in the male rat: continuous vs. cyclic ethanol and nutrient infusions. J Pharmacol Exp Ther. 1993;264:938–43.[Abstract/Free Full Text]

20. Badger TM, Ronis MJ, Ingelman-Sundberg M, Hakkak R. Pulsatile blood alcohol and CYP2E1 induction during chronic alcohol infusions in rats. Alcohol. 1993;10:453–7.[Medline]

21. Ronis MJ, Korourian S, Zipperman M, Hakkak R, Badger TM. Dietary saturated fat reduces alcoholic hepatotoxicity in rats by altering fatty acid metabolism and membrane composition. J Nutr. 2004;134:904–12.[Abstract/Free Full Text]

22. Saxena NK, Ikeda K, Rockey DC, Friedman SL, Anania FA. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology. 2002;35:762–71.[Medline]

23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8.[Medline]

24. Baumgardner JN, Shankar K, Korourian S, Badger TM, Ronis MJ. Undernutrition enhances alcohol-induced hepatocyte proliferation in the liver of rats fed via total enteral nutrition. Am J Physiol Gastrointest Liver Physiol. 2007;293:G355–64.[Abstract/Free Full Text]

25. Johansson I, Ingelman-Sundberg M. Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450 (IIE1) in rat and rabbit liver microsomes. Cancer Res. 1988;48:5387–90.[Abstract/Free Full Text]

26. Polavarapu R, Spitz DR, Sim JE, Follansbee MH, Oberley LW, Rahemtulla A, Nanji AA. Increased lipid peroxidation and impaired antioxidant enzyme function is associated with pathological liver injury in experimental alcoholic liver disease in rats fed diets high in corn oil and fish oil. Hepatology. 1998;27:1317–23.[Medline]

27. Yamada S, Kumazawa S, Ishii T, Nakayama T, Itakura K, Shibata N, Kobayashi M, Sakai K, Osawa T, et al. Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine. J Lipid Res. 2001;42:1187–96.[Abstract/Free Full Text]

28. Uchida K, Szweda LI, Chae HZ, Stadtman ER. Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes. Proc Natl Acad Sci USA. 1993;90:8742–6.[Abstract/Free Full Text]

29. Mottaran E, Stewart SF, Rolla R, Vay D, Cipriani V, Moretti M, Vidali M, Sartori M, Rigamonti C, et al. Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease. Free Radic Biol Med. 2002;32:38–45.[Medline]

30. O'Donohue J, Wong T, Portmann B, Williams R. Immunohistochemical differences in the portal tract and acinar infiltrates between primary biliary cirrhosis and autoimmune cholangitis. Eur J Gastroenterol Hepatol. 2002;14:1143–50.[Medline]

31. Borkham-Kamphorst E, Meurer SK, Gressner AM, Weiskirchen R. Disruption of intermolecular disulfide bonds in PDGF-BB dimers by N-acetyl-L-cysteine does not prevent PDGF signaling in cultured hepatic stellate cells. Biochem Biophys Res Commun. 2005;338:1711–8.[Medline]

32. Jiang D, Liang J, Li Y, Noble PW. The role of Toll-like receptors in non-infectious lung injury. Cell Res. 2006;16:693–701.[Medline]

33. Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis. 2001;21:27–41.[Medline]

34. Pessayre D, Fromenty B. NASH: a mitochondrial disease. J Hepatol. 2005;42:928–40.[Medline]

35. Le TH, Caldwell SH, Redick JA, Sheppard BL, Davis CA, Arseneau KO, Iezzoni JC, Hespenheide EE, Al-Osaimi A, et al. The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis. Hepatology. 2004;39:1423–9.[Medline]

36. Albano E, Mottaran E, Vidali M, Reale E, Saksena S, Occhino G, Burt AD, Day CP. Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis. Gut. 2005;54:987–93.[Abstract/Free Full Text]

37. Miele L, Grieco A, Armuzzi A, Candelli M, Forgione A, Gasbarrini A, Gasbarrini G. Hepatic mitochondrial beta-oxidation in patients with nonalcoholic steatohepatitis assessed by 13C-octanoate breath test. Am J Gastroenterol. 2003;98:2335–6.[Medline]

38. Perez-Carreras M, Del HP, Martin MA, Rubio JC, Martin A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2005;38:999–1007.

39. Hensley K, Kotake Y, Sang H, Pye QN, Wallis GL, Kolker LM, Tabatabaie T, Stewart CA, Konishi Y, et al. Dietary choline restriction causes complex I dysfunction and increased H2O2 generation in liver mitochondria. Carcinogenesis. 2000;21:983–9.[Abstract/Free Full Text]

40. Cortez-Pinto H, de Moura MC, Day CP. Non-alcoholic steatohepatitis: from cell biology to clinical practice. J Hepatol. 2006;44:197–208.[Medline]

41. Zamara E, Novo E, Marra F, Gentilini A, Romanelli RG, Caligiuri A, Robino G, Tamagno E, Aragno M, et al. 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells. J Hepatol. 2004;40:60–8.[Medline]

42. Diehl AM, Li ZP, Lin HZ, Yang SQ. Cytokines and the pathogenesis of non-alcoholic steatohepatitis. Gut. 2005;54:303–6.[Free Full Text]

43. Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis. N Engl J Med. 2000;343:1467–76.[Free Full Text]

44. Crespo J, Cayon A, Fernandez-Gil P, Hernandez-Guerra M, Mayorga M, Dominguez-Diez A, Fernandez-Escalante JC, Pons-Romero F. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology. 2001;34:1158–63.[Medline]

45. Abiru S, Migita K, Maeda Y, Daikoku M, Ito M, Ohata K, Nagaoka S, Matsumoto T, Takii Y, et al. Serum cytokine and soluble cytokine receptor levels in patients with non-alcoholic steatohepatitis. Liver Int. 2006;26:39–45.[Medline]

46. Hui JM, Hodge A, Farrell GC, Kench JG, Kriketos A, George J. Beyond insulin resistance in NASH: TNF-alpha or adiponectin? Hepatology. 2004;40:46–54.[Medline]

47. Poniachik J, Csendes A, Diaz JC, Rojas J, Burdiles P, Maluenda F, Smok G, Rodrigo R, Videla LA. Increased production of IL-1alpha and TNF-alpha in lipopolysaccharide-stimulated blood from obese patients with non-alcoholic fatty liver disease. Cytokine. 2006;33:252–7.[Medline]

48. Tomita K, Tamiya G, Ando S, Ohsumi K, Chiyo T, Mizutani A, Kitamura N, Toda K, Kaneko T, et al. Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice. Gut. 2006;55:415–24.[Abstract/Free Full Text]

49. Adams LA, Zein CO, Angulo P, Lindor KD. A pilot trial of pentoxifylline in nonalcoholic steatohepatitis. Am J Gastroenterol. 2004;99:2365–8.[Medline]

50. Koppe SW, Sahai A, Malladi P, Whitington PF, Green RM. Pentoxifylline attenuates steatohepatitis induced by the methionine choline deficient diet. J Hepatol. 2004;41:592–8.[Medline]

51. Tsukamoto H, Lu SC. Current concepts in the pathogenesis of alcoholic liver injury. FASEB J. 2001;15:1335–49.[Abstract/Free Full Text]

52. Lieber CS, Cao Q, DeCarli LM, Leo MA, Mak KM, Ponomarenko A, Ren C, Wang X. Role of medium-chain triglycerides in the alcohol-mediated cytochrome P450 2E1 induction of mitochondria. Alcohol Clin Exp Res. 2007;31:1660–8.[Medline]

53. Tsukamoto H. Oxidative stress, antioxidants, and alcoholic liver fibrogenesis. Alcohol. 1993;10:465–7.[Medline]

54. Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis. Free Radic Biol Med. 1996;20:351–9.[Medline]

55. Kawada N, Seki S, Inoue M, Kuroki T. Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology. 1998;27:1265–74.[Medline]

56. Hagiwara SI, Ishii Y, Kitamura S. Aerosolized administration of N-acetylcysteine attenuates lung fibrosis induced by bleomycin in mice. Am J Respir Crit Care Med. 2000;162:225–31.[Abstract/Free Full Text]

57. Pardo A, Ruiz V, Arreola JL, Ramirez R, Cisneros-Lira J, Gaxiola M, Barrios R, Kala SV, Lieberman MW, et al. Bleomycin-induced pulmonary fibrosis is attenuated in gamma-glutamyl transpeptidase-deficient mice. Am J Respir Crit Care Med. 2003;167:925–32.[Abstract/Free Full Text]

58. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol. 2007;23:435–61.[Medline]

59. De Caro L, Ghizzi A, Costa R, Longo A, Ventresca GP, Lodola E. Pharmacokinetics and bioavailability of oral acetylcysteine in healthy volunteers. Arzneimittelforschung. 1989;39:382–6.[Medline]

60. Parola M, Pinzani M, Casini A, Albano E, Poli G, Gentilini A, Gentilini P, Dianzani MU. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Commun. 1993;194:1044–50.[Medline]

61. Harrison SA, Torgerson S, Hayashi P, Ward J, Schenker S. Vitamin E and vitamin C treatment improves fibrosis in patients with non-alcoholic steatohepatitis. Am J Gastroenterol. 2003;98:2485–90.

62. Bruck R, Ashkenazi M, Weiss S, Goldiner I, Shapiro H, Aeed H, Genina O, Helpern Z, Pines M. Prevention of liver cirrhosis in rats by curcumin. Liver Int. 2007;27:373–83.[Medline]

63. de Oliveira CP, Stefano JT, de Siqueira ER, Silva LS, de Campos Mazo DF, Lima VM, Furuya CK, Mello ES, Souza FG, et al. Combination of N-acetylsysteine and metformin improves histological steatosis and fibrosis in patients with non-alcoholic steatohepatitis. Hepatol Res. 2008;38:159–65.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplemental Material
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baumgardner, J. N.
Right arrow Articles by Ronis, M. J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baumgardner, J. N.
Right arrow Articles by Ronis, M. J. J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Copyright © 2008 by American Society for Nutrition