www.elsevier.com/locate/jhep

Journal of Hepatology 47 (2007) 851–859

Iron preloading aggravates nutritional steatohepatitis in ratsby increasing apoptotic cell deathq

Nese Imeryuz1, Veysel Tahan1,*, Abdullah Sonsuz2, Fatih Eren1, Suleyman Uraz3,Meral Yuksel4, Sertac Akpulat5, Dervis Ozcelik6, Goncagul Haklar4, Cigdem Celikel5,

Erol Avsar1, Nurdan Tozun1

1Department of Gastroenterology, Marmara University School of Medicine, Altunizade, Istanbul, Turkey2Department of Gastroenterology, Istanbul University Cerrahpasa Medical Faculty, Aksaray, Istanbul, Turkey

3Department of Gastroenterology, Kocaeli University Medical Faculty, Kocaeli, Turkey4Department of Biochemistry, Marmara University School of Medicine, Altunizade, Istanbul, Turkey

5Department of Pathology, Marmara University School of Medicine, Altunizade, Istanbul, Turkey6Department of Biophysics, Istanbul University Cerrahpasa Medical Faculty, Aksaray, Istanbul, Turkey

Background/Aims: High serum ferritin and liver iron concentrations were found in some patients with NASH, suggesting

a role for iron as a co-factor that aggravates liver injury. The aim of this study is to investigate the effects of parenteral iron

in a rat model of NASH induced by a methionine choline deficient diet (MCDD).

Methods: Wistar rats were divided into 1 – Control, 2 – Iron (Fe), 3 – MCDD, 4 – MCDD&Fe groups. Iron dextran100 mg/kg was administered intra-muscularly in groups 2 and 4. All rats were fed MCDD, Groups 1 and 2 were supplied

with choline and methionine. Blood and tissue samples were obtained after 4 weeks.

Results: The iron injection alone did not affect the liver whereas MCDD led to steatohepatitis. Iron worsened steatosis

without any obvious effect on accompanying inflammation. It aggravated tissue injury by increasing apoptosis. Liver fibro-

sis was observed only in 3 out of 10 rats in the MCDD&Fe group.

Conclusions: Observation of liver fibrosis only in the MCDD&Fe group suggests that iron induced increase in apoptosis

contributes to the development of fibrosis at an earlier time than expected.

� 2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Liver fibrosis; Methionine and choline deficient diet; Necroinflammation; Reactive oxygen species; Nonalco-

holic steatohepatitis

1. Introduction

Since nonalcoholic fatty liver disease (NAFLD)accounts for 1/3 of all cases with unexplained liver

0168-8278/$32.00 � 2007 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2007.06.018

Received 7 February 2007; received in revised form 15 June 2007;

accepted 23 June 2007; available online 13 August 2007

Associate Editor: Y.M. Deugnierq The authors who have taken part in this study declared that they do

not have anything to disclose regarding funding or conflict of interestwith respect to this manuscript.

* Corresponding author. Tel.: +90 216 3838089; fax: +90 2164214377.

E-mail address: veyseltahan@gmail.com (V. Tahan).

enzyme elevations and progression to fibrosis occurs inonly 4–20% of patients with NAFLD [1–3], other factorsbeyond hepatic lipid accumulation are needed to induceinflammation and fibrosis. Pathologically increasedhepatocyte apoptosis, is an important mechanism con-tributing to inflammation and fibrosis of the liver [4],and plays a significant role in the in liver injury and dis-ease progression in NAFLD [5].

Although iron overloading aggravates the liverpathology of alcoholic [6] and viral [7] diseases, its rolein the pathogenesis of nonalcoholic steatohepatitis(NASH) has not been elucidated. An increased preva-lence of hemochromatosis gene mutations was reported

Published by Elsevier B.V. All rights reserved.

Fig. 1. Accumulation of iron in KCs in areas of focal necrosis located in pericentral areas of the rat liver in the MCDD group (a), and in the MCDD&Fe

group (b), accumulation of iron both in hepatocytes and KCs in the MCDD&Fe group (c), and in the cytoplasm of periportal hepatocytes in iron group (d)

(Prussian Blue; X400). Arrows show iron accumulation.

852 N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859

in NASH patients [8–10], but the effect of iron onfibrosis progression has not been proven [11–13]. Thedemonstrated role of iron in priming hepatic macro-phages – the main promoter of liver fibrosis, as well asmodulatory effect on IKK-b/NF-jb pathway [14], whichhas shown that the key mediator of liver injury in methi-onine choline deficient diet (MCDD) fed mice [15], sug-gest that iron may have a role in the progression of liverinjury in NASH. Apart from a study in which oral ironsupplementation of mothers of suckling rats fed withMCDD led to fibrosis at week 14 [16], there are no datato answer whether exposure to iron would alter theseverity of steatohepatitis. Thus, we investigated theeffects of parenteral iron supplementation on the apop-totic index, caspase activity, liver histology, and serumbiochemistry in rat nutritional steatohepatitis in thisstudy.

2. Methods

The study was approved by the Animal Research Review Commit-tee of Marmara University School of Medicine, Istanbul, Turkey.

2.1. Materials

The pelleted MCDD and the MCDD supplemented with cholinebitartrate (2 g/kg) and DL-methionine (3 g/kg) were custom-made byHarlan Teklad (Madison Ltd., WI, USA). Iron sorbitol–citric acid

Fig. 2. Normal liver architecture in the iron group (a), steatosis in zone 3 an

MCDD group (b), panacinar steatosis with prominent inflammatory foci scored

X100). Arrows show inflammatory foci.

complex (Eczacıbas�ı, _Istanbul, Turkey), Lucigenin (bis-N-methylacri-diniumnitrate) (Sigma, St. Louis, MO, USA), TdT–Nitric acid, per-chloric acid (Merck, Darmstadt, Germany), FragEL� DNAFragmentation Detection Kit (Calbiochem, USA) and antibody spe-cific for caspase cleavage site within cytokeratin 18 M30 Cyto-DEATH (Roche Diagnostics (Mannheim, Germany)) were allpurchased.

2.2. Animals

Sixty-nine male Wistar rats, between 12–14 weeks of age and 190–315 g, were obtained from Marmara University’s Animal ResearchLaboratory. The animals were kept at a constant temperature(22 ± 1 �C) with 12 h light and dark cycles. Human care was givenin compliance with the National Institutes of Health criteria for labo-ratory animals.

2.3. Iron dosage range

To determine the highest dose of iron, which does not affect liverhistology, 20, 50, 100, 200, 500 mg/kg doses of iron–sorbitol–citric acidcomplex (Jectofer ampoules, 320 mg/mL equal to 50 mg/mL elemen-tary iron) were administered intramuscularly (IM) in five rats pergroup. The rats that were injected with 200 and 500 mg/kg iron diedon the second day. Remaining 15 rats were decapitated 4 weeks afteriron injection. Liver samples were embedded in paraffin and stainedwith H&E and Prussian blue. There was neither iron accumulationwithin the hepatocytes in any of the rats nor any alteration in liverarchitecture apart from one or two inflammatory foci. Only the ratstreated with 100 mg/kg iron had slight amount of iron staining withinKupffer cells (KCs) (Fig. 1a). The iron dosage of 100 mg/kg (corre-sponding 15.6 mg/kg elementary iron) was chosen for furtherexperiments.

d 2 with rare inflammatory foci scored as grade 2 steatohepatitis in the

as grade 3 steatohepatitis in the MCDD&Fe group(c) (H&E; X40, in set

Table 1

Total body weight, weights and iron contents of liver and spleen in the study groups

Study groups Body weightat termination(g)

Change inweight(%)

Liverweight(g)

Relative liverweight(% BW)

Spleenweight(g)

Relative spleenweight(% BW)

Liver Fe(lg/g wettissue)

Spleen Fe(lg/g wettissue)

MCDD&Fe (n = 10) 197.6 ± 8.3 �17.6 ± 3.26 7.55 ± 0.66 3.8 ± 0.27 0.73 ± 0.02 0.37 ± 0.02 131 ± 4.8 336.4 ± 18MCDD (n = 10) 192.9 ± 9.4 �21.21 ± 1.48 7.29 ± 0.63 3.7 ± 0.17 0.69 ± 0.04 0.36 ± 0.03 89.3 ± 6.2 300.3 ± 8.9Fe (n = 7) 239 ± 9.49 �1.91 ± 0.91 5.86 ± 0.18 2.46 ± 0.04 0.78 ± 0.07 0.33 ± 0.04 139.6 ± 8.4 311.7 ± 25.5Control (n = 7) 236.1 ± 4.1 �1.23 ± 1.1 5.76 ± 0.16 2.44 ± 0.07 0.88 ± 0.05 0.38 ± 0.02 139.6 ± 8.4 222.7 ± 12p value a 0.005 0.0001 0.056 0.0001 0.032 NS 0.001 0.001Control vs Fe NS NS NS NS NS NS <0.05Control vs MCDD 0.05 <0.001 NS <0.001 0.05 NS <0.001 <0.05Control vs MCDD&Fe 0.05 <0.001 NS <0.001 NS NS NS <0.001Fe vs MCDD NS <0.001 NS <0.001 NS NS <0.001 NSFe vs MCDD&Fe NS <0.001 NS <0.001 NS NS NS NSMCDD vs MCDD&Fe NS NS NS NS NS NS <0.001 NS

a One-way ANOVA and Tukey’s test, NS not significant.

N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859 853

2.4. Study groups and design

Two groups of rats were fed MCDD for 4 weeks, while control ratswere pair fed with an identical diet to which choline bitartrate andmethionine were added [17]. One of the MCDD groups and controlgroups had either a single dose of 100 mg/kg of iron (Jectofer) or salineIM at the first day. Thus, 1 – the control group was given a control dietand injected saline (n = 7), 2 – the iron group was injected iron, fedwith control diet (n = 7), 3 – the MCDD group was fed with MCDD,injected saline (n = 10), 4 – the MCDD&Fe group was injected iron,fed with MCDD (n = 10).

After an overnight fast, the animals were weighed and decapitatedat the end of the 4th week. Serum samples were obtained by centrifuga-tion of trunk blood (3000 rpm, 10 min, 4 �C). The removed livers andspleens were weighed. After sampling for histological assessment, liverswere snap-frozen in liquid nitrogen. Samples were stored at �80 �C.

2.5. Tissue iron level

One gram tissue was digested by treating with 2.5 mL of 65% nitricacid at 100–120 �C and 0.5 mL of 60% perchloric acid at 150–180 �Cfor 2 h, subsequently it was diluted with deionized water after coolingand was analyzed for non-heme iron by flame atomic absorption spec-trophotometer (Shimadzu AA-680, Japan) as microgram non-hemeiron/gram of wet tissue weight [18].

2.6. Biochemical studies

Plasma transaminases, alkaline phosphatase (ALP), bilirubin, totalcholesterol, low density lipoprotein (LDL) and triglycerides weredetermined spectrophotometrically by Roche 917 R autoanalyser

Table 2

Localization and grade of iron deposition within liver in the study groups

Iron deposition (grade): Hepatocyte K

Study groups (number) 0 1 2 3 0

MCDD&Fe (n = 10) 2 5 3MCDD (n = 10) 9 1Fe (n = 7) 1 1 4 1 6Control (n = 7) 7 5pa MCDD vs MCDD&Fe 0.0867 0.pa Fe vs MCDD&Fe 1 0.pa Control vs MCDD&Fe 0.0023 0.pa Fe vs MCDD 0.0034 0.pa Fe vs control 0.02 1pa Control vs MCDD 1 0.

a Fisher’s exact test.

(Boehringer, Mannheim, Germany). Insulin was measured by ELISA(Mercodia, Uppsala, Sweden). Quantitative insulin sensitivity checkindex (QUICKI) [19] was calculated.

2.7. Histopathology

Formalin-fixed, and paraffin-embedded samples were stainedwith H&E and the periodic-acid Schiff with diastase to grade ste-atosis and inflammation. The Masson’s trichrome and Gomori’sreticulin stains were reviewed for fibrosis and architecturalchanges. The Perl’s Prussian blue stain was used to evaluate ironstorage.

For grading and staging histological lesions, Brunt et al.’ssystem was used [20]. Apart from grading inflammation, we alsodetermined the activity of inflammation by taking the averageof inflammatory cells per inflammatory foci. Fibrosis was evalu-ated by taking into account any unique zone-3 perisinusoidalfibrosis [20]; involvement of less than 33% of zone 3 foci scoredas stage 1.

Presence of iron deposition as discrete granules within hepatocytes,KCs and endothelial cells of the vessels in portal tracts was evaluatedindividually by a semi-quantitative method according to the ease ofobservation at different objectives of the light microscope (0; 1: barelydiscernable 20· but easily confirmed 40·; 2: easily confirmed 20·; 3:easily confirmed 10·) and the frequency of positive staining cells ineach compartment (0; 1: iron in <1/3 of acini/portal tract; 2: iron in1/3–2/3 of acini/portal tract; 3: iron in >2/3 of acini/portal tract).The sum of these two scores gave us the total iron scores for hepato-cytes, KCs and endothelial cells. Then, the total scores were subdividedinto 3 grades (grade 1: scores 1–2; grade 2: scores 3–4; grade 3: scores5–6). Assessment of liver injury was performed blindly using codedslides.

upffer cells Endothelium

1 2 3 0 1 2 3

3 5 2 108 2 4 3 3

1 72 7

21 0.210098 10098 10001 0.23

10001 0.23

854 N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859

2.8. Determination of apoptosis

DNA fragmentation was detected by the labeling of DNA breaksin apoptotic nuclei in paraffin-embedded tissue sections using a TdT-FragEL� DNA fragmentation detection kit (Calbiochem�, USA)[21]. Labeled cells were counted in six high power field (HPF), threeperiportal and three pericentral; and apoptotic index is given as thelabeled cells per one HPF (400·).

Additionally, caspase cleavage of cytokeratin 18 (C18) which isaffected in the early steps of apoptosis was determined by immunohis-tochemistry. M30 CytoDEATH (Roche Diagnostics, Germany) anti-bodies, which recognize a specific caspase cleavage site withincytokeratin 18 that is not detectable by the native CK18 of normalcells, were used and were diluted to 1/250 [22]. Both the intensityand the distribution of immunoreactive cells were evaluated semiquan-titatively on a scale of 0–4 in all examined sections. This evaluationwas made for all the acini identifiable in the section. The sum of theintensity and the distribution scores gave the total immunoreactivityscore (TIS). Caspase score was 1 if the TSI = 4 or less, 2 if TIS = 5–6, and 3 if TSI = 7–8.

2.9. Determination of reactive oxygen and nitrogen

species (ROS)

ROS were quantified in homogenized liver samples after the addi-tion of enhancers lucigenin (selective for superoxide radical) using aMini Lumat LB 9506 luminometer (EG&G, Berthold, Germany).Counts were obtained at 15 s intervals for a counting period of5 min and the results were given as the area under curve (AUC) of rel-ative light unit and corrected for wet tissue weight (AUC of rlu/mg tis-sue) [23].

2.10. Statistics

Results were expressed as means ± standard error of the mean(SEM). One-way ANOVA and chi square test with Yates correctionwere used to analyze ordinal and nominal data, respectively. Signifi-cant differences revealed by multi-group comparisons were further ana-lyzed by Tukey’s test. p values <0.05 were accepted as significant.

3. Results

Rats fed with MCDD lost almost 20% of their initialweight whereas body weight (BW) did not change in theiron and control groups. The relative liver weight

expressed as %BW increased in the MCDD fed ratssignificantly (p < 0.001 vs the control groups, Table 1).

Table 3

Effect of MCDD and iron on liver histopathology

Grade of the lesions: Fibrosis Inflammation S

Grade Activity G

Study groups (n) 0 1 0 1 2 3 M ± SEM 0

MCDD&Fe (n = 10) 7 3 9 1 26.4 ± 7.48MCDD (n = 10) 10 0 1 6 3 21 ± 5.6Fe (n = 7) 7 0 6 1 0 ± 0 7Control (n = 7) 7 0 6 1 0 ± 0 7p, ANOVA 0.0001�,*p, Fisher’s exact NS NS 0�p, nonparametric t NS

*Fisher’s exact test combining grades 0&1&2 vs grades 3, �MCDD vs MCsignificant.

The spleen weight reduced in MCDD group but the rel-

ative spleen weights were similar in general (Table 1).Iron pretreatment alone did not alter liver non-heme

iron content. Liver iron content decreased in the MCDDgroup significantly as compared to the other groupsprobably due to increment in the relative liver weight(p < 0.001, Table 1). Spleen iron content increased inall rats subjected to any treatment (p < 0.01 vs controls,Table 1).

In the MCDD group, iron staining was confined toKCs in areas of focal necrosis and to a lesser extent tothe endothelial cells (Table 2 and Fig. 1). Iron depositedwithin the hepatocytes and KCs in the MCDD&Fegroup. Although iron accumulation in KCs was easilyseen in low power examination in the MCDD group,the number of iron accumulated KCs was excess innumber and more widely distributed throughout theliver in the MCDD&Fe group. The pattern of irondeposition in the iron group was similar to that of theMCDD&Fe group which showed a predilection for per-iportal hepatocytes.

Iron alone leads to neither steatosis nor inflamma-tion. MCDD feeding resulted in liver steatosis(p < 0.0001, Table 3). Macrovesicular steatosis wasprominent in acinar zone 3 and spread out in a panacin-ar distribution (Fig. 2). Iron pretreatment aggravatedthe magnitude of liver steatosis in rats fed with MCDD(p = 0.02, Table 3).

There were scattered foci of inflammation in theMCDD fed rats. The activity of inflammation and gradeof steatohepatitis were higher in the MCDD fed rats(p < 0.0001 vs controls, Table 3). Iron pretreatmentdid not worsen the activity of inflammation as comparedto MCDD feeding alone, statistically.

Mean steatohepatitis grades were 2.4 and 1.7 in theMCDD&Fe and MCDD groups, respectively. Grade 3steatohepatitis was found in 5 out of 10 rats in theMCDD&Fe group whereas none were found in theMCDD group (p = 0.03, Table 3).

Masson’s trichrome stain revealed fibrosis in 3 out of10 rats in the MCDD&Fe group. Fibrosis was limited to

teatosis Steatohepatitis

rade Grade

1 2 3 M ± SEM 0 1 2 3 M ± SEM

1 9 2.9 ± 0.1 1 4 5 2.4 ± 0.227 3 2.3 ± 0.15 3 7 1.7 ± 0.15

0 ± 0 7 0 ± 00 ± 0 7 0 ± 00.0001 0.0001

.02 0.030.02 0.03

DD&Fe groups, M, mean; SEM, standard error of mean; NS, not

Fig. 3. There was no fibrosis in pericentral zone in the MCDD group (a),

but we determined pericentral minimal sinusoidal fibrosis (arrow) in 3

rats in the MCDD&Fe group (b) (Masson’s Trichrome; X200).

ig. 4. TUNEL-positive (left panel) hepatocytes with markedly brown

yknotic nuclei (brown dot) showing apoptosis in liver sections from the

ontrol (a), iron (b), MCDD (c) and MCDD&Fe (d) groups. Right panel

epicts corresponding caspase activity with low intensity and focal

istribution in control (a), moderate intensity and focal distribution in

on (b), high intensity and focal distribution in MCDD (c) and high

tensity and diffuse distribution MCDD&Fe (d) groups. Scores detected

y M30 cyto-death. (Magnification x 400).

N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859 855

the pericentral area and showed sinusoidal distribution(Table 3 and Fig. 3). Perisinusoidal fibrosis was scored

as 1 in all three rats. No portal or bridging fibrosiswas detected. None of the rats in any of the other groupsdisplayed fibrosis but there was no significant differencebetween groups.

MCDD feeding but not iron treatment alone increasedthe number of TUNEL-positive cells significantly(p < 0.01 and p < 0.001 MCDD and MCDD&Fe vs con-trol; Figs. 4 and 5). The number of TUNEL-positive cellswas significantly increased in liver specimens in theMCDD&Fe group as compared to the MCDD group(p < 0.05, Fig. 5c). The increase in apoptotic index wasmainly prominent in zone 3 as compared to zone 1 in boththe MCDD (p = 0.06) and MCDD&Fe groups (p < 0.02)(Figs. 5a and b). Immunohistochemistry demonstratedcaspase activity in TUNEL-positive specimens confirm-ing the occurrence of apoptosis. The apoptotic index cor-related well with serum ALT levels (r = 0.78, p < 0.001)and inflammatory activity (r = 0.68, p < 0.001).

Lucigenin enhanced chemiluminescence increased inthe MCDD and MCDD&Fe groups as compared to con-trols (p < 0.05) whereas the magnitude of increase in theiron group did not reach a level of statistical significance

F

p

c

d

d

ir

in

b

(p = 0.07). Addition of iron pretreatment to MCDD didnot augment the magnitude of measurement (Fig. 6a).

Iron injection alone did not affect biochemistry.Serum bilirubin (data not shown), ALP (Fig. 6b), andALT levels (Fig. 6c) were higher in the MCDD andMCDD&Fe groups as compared to control group.The magnitude of increase of ALT was higher in theMCDD&Fe group than that of the MCDD group(p < 0.001). Serum AST levels were similar in all.

Serum glucose, total cholesterol, triglyceride andLDL levels were lower in the MCDD fed groups(p < 0.0001 in all) and were associated with body weight(correlation coefficients were 0.54, 0.73, 0.34, 0.38,

Control Fe MCDD MCDD&Fe0

5

10

15

Apo

ptot

ic in

dex

Control Fe MCDD MCDD&Fe0

5

10

15

Apo

ptot

ic in

dex

PERIPORTAL

Control Fe MCDD MCDD&Fe0

5

10

15ControlFeMCDDMCDD&Fe

Apo

ptot

ic in

dex

§

†

‡

‡†

*†

PERICENTRAL TOTA L

Fig. 5. MCDD feeding increased apoptotic index in the periportal (a), and in the pericentral (b) area, as compared to the controls as well as in the total

(c), (§p < 0.05, �p < 0.01, �p < 0.001 as compared to the control group, one way ANOVA and Tukey’ test). The total apoptotic index (c) was higher in the

MCDD&Fe group as compared to the MCDD group (*p < 0.05, one way ANOVA and Tukey’s test).

856 N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859

respectively). Insulin sensitivity was lower in the MCDDfed rats but insulin levels were similar in all (Table 4).

4. Discussion

In this study we tested the hypothesis that the pres-ence of iron would aggravate steatohepatitis in rats. Asingle injection of iron, which is known not to alter liverhistopathology, aggravated steatosis, augmented apop-tosis and cell injury in male rats within 4 weeks ofMCDD feeding. Furthermore, liver fibrosis developedin 3 out of 10 rats pretreated with iron and fed withMCDD in 4 weeks which is reported to evolve in 12weeks in rat nutritional NASH model [17,24].

Although studies investigating the pathophysiologyof iron induced liver damage are based on chronic feed-ing with carbonyl iron, we chose a single intramusculariron injection to mimic clinical practice. Iron injectionled to staining in periportal hepatocytes as had beenshown [25,26]. Because Prussian blue reacts with proteinbound forms of iron [26,27] – which might be unable togenerate the mediators of iron induced liver damage-freeradicals and protein–aldehyde adducts [28,29], we mea-sured potentially active forms of iron [30]. Liver non-heme iron concentrations were well below the critical

Control Fe MCDD MCDD&Fe0

25

50

75

AU

C/rl

umg

tissu

e

LUCIGENIN

Control Fe MCDD MC0

500

1000

1500

U/L

PLA

§ § †

Fig. 6. Tissue Lucigenin (a) levels increased in all the treatment groups but r

compared to the control group (§p < 0.05). Serum alkaline phosphatase (b) and

compared to the control group (�p < 0.01, �p < 0.001). Serum ALT level wa

(**p < 0.001, One way ANOVA and Tukey’s test).

amount of iron (4000 lg/g wet weight) must be present

in the liver before the fibrotic response is triggered in ratsafter a single injection [26], confirming that this amountof iron does not account for fibrosis alone. Our iron dosewas comparable [25] or 10 times lower [26] than the

doses which were reported not to alter liver enzymes or

histology. Accordingly, liver biochemistry and histopa-

thology were normal in the iron injected groups exceptiron staining within the hepatocytes and an increase insplenic iron content.

MCDD feeding increased splenic content of non-heme iron and led to iron staining in KCs mimickingalcoholic liver disease, which causes iron accumulation[31] mainly within the hepatic and splenic macrophages[32]. Nevertheless, hepatic non-heme iron content of theMCDD group was less than that of the control groupprobably due to the diluting effect of steatosis. Rats inthe MCDD group had the heaviest livers despite losing20% of their initial body weight. Liver non-heme ironcontent of the MCDD&Fe group was similar to thatof the controls suggesting that iron injection maskedthe diluting effect of steatosis.

Feeding with MCDD resulted in steatosis usually upto 66% of hepatocytes and led to mild–moderate lobularinflammation with no evidence of fibrosis in accordancewith previous studies [17,33,34]. Serum ALT, ALP and

DD&Fe Control Fe MCDD MCDD&Fe0

100

200MCDD&FeMCDDFeControl

(U/L

)

TLA‡

†

**‡

eached statistical significance in the MCDD and MCDD&Fe groups as

ALT (c) levels were increased in the MCDD and MCDD&Fe groups as

s higher in the MCDD&Fe group as compared to the MCDD group

Table 4

Effect of MCDD and iron on serum biochemistry

Study groups Insulin (lU/mL) Glucose (mg/dL) QUICKI Cholesterol (mg/dL) Triglyceride (mg/dL) VLDL (mg/dL)

MCDD&Fe (n = 10) 0.55 ± 0.12 85 ± 4.2 0.343 ± 0.012 29.2 ± 2.16 42.8 ± 3.22 8.6 ± 0.65MCDD (n = 10) 0.3 ± 0.038 89 ± 3.9 0.363 ± 0.008 36 ± 2.28 53.8 ± 2.4 10.6 ± 0.48Fe (n = 7) 0.43 ± 0.06 100 ± 4.9 0.336 ± 0.0096 57.6 ± 5.6 56.4 ± 2.6 11.3 ± 0.52Control (n = 7) 0.68 ± 0.18 113 ± 2.8 0.33 ± 0.0226 65.1 ± 2.59 71 ± 4.37 14.3 ± 0.8p value 0.09 0.0002 NS <0.0001 0.0001 0.0001Control vs Fe NS NS NS NS <0.05 NSControl vs MCDD NS <0.01 NS <0.001 <0.01 <0.01Control vs MCDD&Fe NS <0.001 NS <0.001 <0.001 <0.001Fe vs MCDD NS NS NS <0.001 NS NSFe vs MCDD&Fe NS NS NS <0.001 <0.05 <0.05MCDD vs MCDD&Fe NS NS NS NS NS NS

VLDL: very low density lipoprotein; NS: not significant.

N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859 857

bilirubin increased as reflecting presence of liver dam-age. One of the operating mechanisms of the liver dam-age in this study was apoptotic cell death which wasshown by increased DNA fragmentation and caspaseactivity in the MCDD fed rats [35].

Liver steatosis and accompanying apoptosis wereobserved in rats fed with methionine choline and folatedeficient diet which is associated with mild inflammationbut not necrosis [36]. Steatosis may initiate apoptosiseither by inducing Fas (CD95) expression on hepato-cytes – which has been shown in other mice steatosismodels [37], or by increasing cytosolic cytochrome ccontent – which has been observed in neonatal pigletson total parenteral nutrition [38] and in cultured hepato-cytes encountered with the saturated free fatty acids [39].

Single injection of iron in a dose that did not alterliver histology by itself aggravated steatohepatitis inthe MCDD&Fe group. The mechanism of liver injuryinduced by iron injection may be due to the furtherimpairment of mitochondrial oxidation by opening ofthe mitochondrial permeability transition pores[30,40,41], which has been shown to be defective inrodent steatosis [42]. This may ultimately lead to therelease of cytochrome c and other proapoptotic factors[40,41] and to cell death. The other mechanisms of ironinduced cell injury might involve production of protein–aldehyde adducts as a result of oxidative damage withinany compartment of the cell [43]. Iron may further stim-ulate the proinflammatory and profibrotic potential ofhepatic macrophages [14,15,44,45] which are alreadystimulated by MCDD feeding alone via NF-jb path-ways [15].

We neither evaluated the efficacy of mitochondrialoxidation nor measured antioxidant capacity and lipidperoxidation products. Observation of the highest ALTlevels in the MCDD&Fe group in association withworsened steatosis and emergence of fibrosis earlierthan expected, in spite of a lesser degree of inflamma-tion as compared to the MCDD group, suggested thatiron worsened steatosis and triggered cell death bynon-inflammatory mechanisms such as apoptosis

[46,47] and necroapoptosis [41]. Presence of the highestapoptotic index in the MCDD&Fe group favoredapoptosis as one of the main mechanisms of cell deathbut did not exclude presence of other kinds of celldeath [48]. Predilection of apoptosis to the pericentralarea, which is the preferential site of injury in steato-hepatitis, instead of liver regions such as the periportalarea which is vulnerable to iron induced damage, sug-gests that prior conditioning of the liver by non-toxicamounts of iron may have an additive effect on thepathogenetic mechanisms of apoptotic cell deathinduced by steatosis.

Although considered as a mechanism of cell removalwithout consequences to the tissue, apoptosis could beinvolved in the development of fibrosis [49]. Apoptoticcells may stimulate human stellate cells either directlyor indirectly via stimulation of transforming growth fac-tor (TGF)-b1 production in hepatocytes and macro-phages [4,5]. Phagocytosis of apoptotic cells bymacrophages actively suppresses inflammatory reactionin vivo through the production of TGF-b1 [50,51]. Dualstimulation of hepatic macrophages by iron and MCDDvia convergent mechanisms could have promoted theemergence of liver fibrosis 8 weeks earlier than thatreported in previous studies [17].

In conclusion, the development of fibrosis and theworsening of steatosis only in rats subjected to a singleinjection of iron and MCDD treatment emphasize therole of iron in the progression of nutritional steatohep-atitis to the fibrotic stages of the disease. Further pro-spective clinical studies investigating outcomes of irontherapies in patients with NASH are needed to extrapo-late murine data to human beings as well as basicresearch to elucidate the operating mechanisms of fibro-genesis to prevent or even repeal development fibrosis.

Acknowledgement

The authors would like to thank David T. Thomas,M.D., for his suggestions and critical review of themanuscript.

858 N. Imeryuz et al. / Journal of Hepatology 47 (2007) 851–859

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