In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases

Journal of Hepatology 37 (2002) 56–62 www.elsevier.com/locate/jhep

In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases Shuichi Seki 1,*, Takuya Kitada 1, Takao Yamada 1, Hiroki Sakaguchi 1, Kazuki Nakatani 2, Kenichi Wakasa 3 1

Third Department of Internal Medicine, Osaka City University Medical School, 1-4-3, Asahimachi, Abenoku, Osaka 545-8585, Japan 2 Second Department of Anatomy, Osaka City University Medical School, 1-4-3, Asahimachi, Abenoku, Osaka 545-8585, Japan 3 Department of Clinical Pathology, Osaka City University Medical School, 1-4-3, Asahimachi, Abenoku, Osaka 545-8585, Japan

Background/Aims: Although oxidative stress is an important candidate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the localization and pathological significance of oxidative stress-induced cellular damage in NAFLD remains unclear. Methods: Hepatic expression of 4-hydroxy-2 0 -nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, respectively, was immunohistochemically investigated in NAFLD and the results were compared with histological findings. Results: While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE index. Regarding 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in fatty liver, 11 of 17 cases (64.7%) with NASH exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation. Conclusions: Oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Non-alcoholic fatty liver disease; 8-hydroxydeoxyguanosine; 4-hydroxy-2 0 -nonenal; Oxidative stress; Immunohistochemistry

1. Introduction Non-alcoholic fatty liver disease (NAFLD) has a spectrum ranging from fatty liver alone to non-alcoholic steatohepatitis (NASH) [1]. While non-alcoholic fatty liver is widely believed to be a benign condition with little risk of disease progression, patients with NASH can develop progressive liver disease and cirrhosis [1–8]. An aberration in the metabolism of fatty acids and triglycerides may be the common mechanism underlying the hepatic triglyceride accumulation in NAFLD [2]. NASH is histologically similar to alcoholic steatohepati-

Received 16 November 2001; received in revised form 21 February 2002; accepted 6 March 2002 * Corresponding author. Tel.: 181-6-6645-3811; fax: 181-6-6645-3813.

tis with the presence of macrovesicular steatosis, mixed inflammatory cell infiltration of the lobules, ballooning degeneration and necrosis of hepatocytes, Mallory body formation, and perisinusoidal fibrosis or cirrhosis [2–4,9]. Although the pathogenesis of NASH remains unclear, the hypothesis that excessive intrahepatic lipid accumulation could trigger a local necro-inflammatory response has recently been suggested [2,3,10–13]. Such necro-inflammation is accompanied by the production of free radicals that can result in damage of the cellular membrane and DNA [14,15]. Lipid peroxidation, a free radical-mediated mechanism, leads to oxidative destruction of polyunsaturated fatty acids constitutive of cellular membranes [16,17]. The cytotoxic products of lipid peroxidation, may impair cellular functions including nucleotide and protein synthesis [18] and may

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S01 68- 8278(02)0007 3-9

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Table 1 Clinical characteristics of the patients a

Age (year) Gender (F/M) BMI (kg/m 2) Diabetes (%) AST (IU/l) ALT (IU/l) Alkaline phosphatase (IU/l) Total bilirubin (mg/dl) Albumin (g/dl) Cholesterol (mg/dl) Triglyceride (mg/dl)

Normal liver (n ¼ 7)

Fatty liver (n ¼ 23)

NASH (n ¼ 17)

52.5 (38–72) 4/3 23.1 (21.0–25.7) 0 24 (18–32) 28 (18–39) 188 (134–198) 0.7 (0.4–1.2) 4.1 (3.7–4.6) 197 (148–236) 131 (89–166)

47.5 (37–68) 13/10 25.5 (23.1–29.5) 5 (21.7) 36 (31–45) 51 (37–63) 174 (166–203) 0.7 (0.4–1.1) 4.4 (4.1–5.0) 208 (189–272) 167 (102–325)

52 (23–71) 16/1 28.3 (20.4–32.8) 12 (70.6) 47 (38–94) 69 (47–141) 193 (150–211) 0.6 (0.5–1.3) 4.2 (3.5–4.6) 200 (178–298) 172 (143–438)

a Data are expressed as median (range). NASH, non-alcoholic steatohepatitis; BMI, body mass index; AST, aspartate aminotransferase; and ALT, alanine aminotransferase.

play a role in liver fibrogenesis by modulating the expression of the collagen gene in hepatic stellate cells [19–21]. 4hydroxy-2 0 -nonenal (HNE) is one of the major aldehydic metabolites of lipid peroxidation and is considered to be one of the most reliable markers of lipid peroxidation [17]. Increased formation of HNE adducts have been reported in liver tissues from patients with different chronic liver diseases including alcoholic liver injury, chronic viral hepatitis and hereditary hemochromatosis [22–24]. Another major target of free radicals is cellular DNA. In this context, 8-hydroxydeoxyguanosine (8-OHdG), a DNA base modified product generated by free radicals [25], is considered to be a good biomarker of oxidative DNA damage [26]. Previous studies have suggested that various carcinogens including aflatoxin B1, psychological stress and ionizing radiation increase hepatic 8-OHdG content [27– 29]. Moreover increased 8-OHdG expression has been reported in various chronic human liver diseases [30,31]. Although oxidative stress may play a major role in the pathogenesis of NAFLD [2,3,10–13], the localization and pathological significance of oxidative stress-induced lipid peroxidation and oxidative DNA damage in NAFLD remain unclear. This prompted us to investigate the localization of two parameters of oxidative cellular damage, HNE adducts and 8-OHdG, in human livers with NAFLD. The relationship between their expression and liver histologies in NASH was also investigated.

data, liver histology, and hepatobiliary ultrasound in all patients. Laboratory studies included serum liver tests (alanine aminotransferase, aspartate aminotransferase, g-GTP, alkaline phosphatase, total bilirubin, albumin, and total protein levels), hepatitis B and C serology (hepatitis B surface antigen, antibody to hepatitis B surface antigen, antibody to hepatitis B core antigen, and serum hepatitis C virus RNA, autoimmune serology (antimitochondrial antibody, antinuclear antibody), studies of iron metabolism (fasting serum iron, transferrin saturation, and ferritin levels), and ceruloplasmin and a1-antitrypsin levels. Serum glucose, cholesterol, and triglyceride levels were also obtained. Seven histologically normal liver samples were collected during surgery on patients with metastatic liver tumor. The characteristics of these patients are summarized in Table 1. Informed consent was obtained from each patient and the study was approved by the local ethics committee and was carried out according to the provisions of the Declaration of Helsinki.

2.2. Histological evaluations

2. Materials and methods

Hematoxylin-eosin, Sirius Red and Masson-Trichrome staining were performed for histological diagnosis. Liver histology was evaluated by an experienced pathologist (KW) blinded to the results of immunostaining. In NASH cases, each section was examined to grade the severity of steatosis, necro-inflammation, and fibrosis. Steatosis was scored from 1 to 3; 1 when up to 1/3 contained fat, 2 when between 1/3 and 2/3 contained fat, 3 when fat was present in .2/3 of hepatocytes. Inflammation and fibrosis was evaluated and scored according to previously published criteria with some modifications. The criteria were based on those initially developed by Brunt et al. [32] and Lee [33]. Hepatic necro-inflammation was graded from 1 to 3; one sparse or mild focal zone 3 hepatocyte injury/inflammation, two noticeable zone 3 hepatocyte injury/inflammation, three severe zone 3 hepatocyte injury/inflammation. The stage of fibrosis was assessed and scored from 0 to 4; 0 no fibrosis, one focal pericellular fibrosis in zone 3, two perivenular and pericellular fibrosis confined to zones 2 and 3 with or without portal/periportal fibrosis, three bridging or extensive fibrosis with architectural distortion but with no obvious cirrhosis, four cirrhosis.

2.1. Samples

2.3. Antibodies

Formalin-fixed, paraffin-embedded liver samples from 40 patients were used in this study. There were 23 patients with non-alcoholic fatty liver (FL) and 17 with NASH. Histological criteria for inclusion in this study were the following: the presence of macrovesicular steatosis without any necro-inflammatory changes for FL and the presence of macrovesicular steatosis with lobular inflammation with or without hepatocytes necrosis for NASH. Other causes of liver disease, including alcohol abuse (.40 g/ week), had been strictly excluded by history, family interview, laboratory

A monoclonal antibody against 4-hydroxy-2 0 -nonenal (HNE)-modified proteins (Nikken Food Co., Ltd. Shizuoka, Japan) was raised by immunizing mice with a HNE-key hole limpet hemocyanin conjugate [34]. This antibody exhibits a much higher affinity for the HNE-histidine adducts than the HNE-lysine or HNE-cysteine adducts, but do not cross-react with proteins treated with other aldehydes, such as 1-hexenal, 2-hexenal, 4hydroxy-2-hexenal, 2-nonenal, formaldehyde, or glutaraldehyde. A monoclonal antibody against 8-OHdG was purchased from the Japanese Aging

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Fig. 1. Immunohistochemical detection of HNE adducts (A–C) and 8-OHdG (D–F) in normal liver and liver with NAFLD. (A) HNE adducts staining in normal liver. No HNE adducts were detected. (B) Fatty liver. Patchy immunolabeling was observed in the cytoplasm of hepatocytes. (C) NASH. HNE adducts were detected in the cytoplasm of hepatocytes (arrow) and sinusoidal cells (arrowhead) with a predominance in zone 3. CV; central vein. (D) 8-OHdG staining in normal liver. No positive stainings were observed. (E) Fatty liver. 8-OHdG expression was also not evident in fatty liver cases. (F) NASH. Nuclear expression of 8-OHdG was mainly detected in the hepatocytes (arrow) and occasionally in sinusoidal cells (arrowhead) in areas of active inflammation.

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Table 2 Immunostaining of HNE adducts and 8-OHdG in control liver and NAFLD Liver disease

Control Fatty liver NASH

(n)

(7) (23) (17)

HNE adducts

8-OHdG

No. of positive cases (%)

Localization

No. of positive cases (%)

Localization

0 18 (78.3) 17 (100)

– Hepatocytes Hepatocytes sinusoidal cells

0 2 (8.7) 11 (64.7)

– Hepatocytes Hepatocytes sinusoidal cells

Control Institute (Shizuoka, Japan). Generation and validation of this antibody has been reported elsewhere [35].

2.4. Immunohistochemistry Liver sections (5 mm-thick) were routinely deparaffinized in xylene and rehydrated through graded ethanol. For the immunostaining of HNE, the sections were microwaved in 10 mM citrate buffer (pH 6.0) for 15 min for antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 0.3% H2O2 in methanol for 15 min. After washing with phosphatebuffered saline (PBS) three times, the sections were first treated with 10% normal goat serum in PBS for 30 min at room temperature (RT) to block non-specific bindings. Subsequently, the sections were reacted with affinity-purified mouse primary antibodies (1:100 dilution in PBS) or normal mouse serum (Dako, Glostrup, Denmark) at the same dilution overnight at 48C. For the following reactions, LSAB kit (Dako) was used according to the manufacturer’s instructions. After rinsing in PBS three times, the sections were incubated with biotinylated secondary antibody for 1 h at RT, followed by treatment with peroxidase-labeled streptoavidin for 20 min. Peroxidase activity was developed with 0.25 mg/ml 3,3 0 -diaminobenzidine tetrahydrochloride in the presence of 0.003% hydrogen peroxide in 0.05 M Tris-buffered saline at pH 7.4. Finally, the sections were counterstained for nuclei in hematoxylin (for HNE staining) or methylgreen (for 8OHdG staining).

2.5. Semi-quantitative assessment of the immunostaining Localization and intensity of staining were assessed by two investigators (SS, TK) in a simultaneous reading. The intensity of HNE-staining was scored from 0 to 3 as previously described [23] with minor modifications; zero no staining, one mild (punctuated labeling), two moderate (dense labeling in a few cells), three strong (dense and homogenous labeling in numerous cells). At least three periportal and three perivenous zones were examined in each section and the average of the scores was determined as the HNE index. For semi-quantitative assessment of 8-OHdG expression, the number of 8-OHdG-positive hepatocytes among 1000 hepatocytes was counted in at least three periportal and three perivenous zones in each section and the average of these counts was taken as the 8-OHdG index.

all cases (n ¼ 17) with NASH. In FL cases, patchy immunolabeling of HNE adducts was observed in the cytoplasm of hepatocytes (Fig. 1B). In cases of NASH, the localization of HNE adducts was in the cytoplasm of hepatocytes as well as sinusoidal cells with a predominance in zone 3 (Fig. 1C). These results are summarized in Table 2. 3.2. Immunohistochemical detection of 8-OHdG in normal liver and NAFLD In normal livers, no 8-OHdG expression was observed (Fig. 1D). 8-OHdG expression was also not evident in most cases (21 of 23 cases) with FL (Fig. 1E), however, some sporadic expression of 8-OHdG was detected in two cases (8.7%). In these two cases, the localization of 8-OHdG was in the nuclei of the hepatocytes. In contrast, 8-OHdG expression was more frequently detected in NASH (11 of 17 cases, 64.7%). The nuclear expression of 8-OHdG was mainly observed in the hepatocytes and occasionally in sinusoidal cells in areas with active inflammation (Fig. 1F). These results are also summarized in Table 2. 3.3. HNE index in NAFLD

2.6. Statistical analysis

The intensities of HNE staining were evaluated and scored in each specimen. As shown in Table 3, the HNE index in NASH cases (1.53 ^ 0.48) was significantly (P ¼ 0:024) higher than those in cases of FL (1.08 ^ 0.52). In the cases of NASH, staining intensities in the perivenous zone were significantly higher than those in the periportal zone (1.13 ^ 0.74 versus 1.73 ^ 0.67, P ¼ 0:008). However, there was no significant difference in staining intensity between the perivenous and periportal zones in FL.

Statistical analysis was performed using Spearman and Pearson’s tests and the Mann–Whitney U-test. Significance was defined as P , 0:05.

Table 3 Semi-quantitative assessment of HNE immunostaining in NAFLD a HNE index

3. Results 3.1. Immunohistochemical detection of HNE adducts in normal livers and NAFLD In normal human liver specimens, no positive immunolabeling of HNE was observed (Fig. 1A). In contrast, the adducts were widely detected in NAFLD. HNE adducts were detected in 18 of 23 cases (78.3%) with FL and in

Fatty liver (n ¼ 23)

1.08 ^ 0.52

NASH (n ¼ 17)

1.53 ^ 0.48

a

3 7 5*

Periportal

Perivenous

0.97 ^ 0.42

1.03 ^ 0.31

1.13 ^ 0.74**

1.73 ^ 0.67**

Data are expressed as mean ^ SD. The intensities of HNE staining was scored in at least three periportal. and perivenous zones in each section and the average of the scores was taken as HNE index. *The HNE index in NASH cases was significantly (P ¼ 0.024) higher than those in cases of fatty liver. **Staining intensities in the perivenous zone were significantly (P ¼ 0.008) higher than those in the periportal zone in NASH cases.

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Table 4 Correlation between HNE index or 8-OHdG index and histological parameters a Parameters

n

HNE index

8-OHdG Index

Steatosis Mild Moderate Severe

5 7 5

3 1.36 ^ 0.45 7 1.44 ^ 0.71 5 1.51 ^ 0.59

N.S.

9.2 ^ 8.1 13.8 ^ 10.2 11.3 ^ 9.6

Inflammation Mild Moderate Severe

6 7 4

3 0.98 ^ 0.34 7 1.33 ^ 0.53 5 1.79 ^ 0.88

P ¼ 0.027

8.4 ^ 5.7 16.6 ^ 7.90 19.8 ^ 6.8

Fibrosis No/mild Moderate Severe/cirrhosis

5 8 4

3 0.63 ^ 0.55 7 1.26 ^ 0.69 5 1.66 ^ 0.47

a

P ¼ 0.011

3 7 5

N.S.

3 7 5

3 11.5 ^ 8.6 7 5 10.7 ^ 6.8 15.3 ^ 12.11

P ¼ 0.039

N.S.

Data are expressed as mean ^ SD. N.S., not statistically significant; HNE, 4-hydroxy-2 0 -nonenal; and 8-OHdG, 8-hydroxydeoxyguanosine.

3.4. Correlation between HNE index or 8-OHdG index and liver histologies in NASH We next examined the relationship between HNE or 8OHdG expression and liver histologies in NASH. As shown in Table 4, the HNE index significantly correlated with the grade of necro-inflammation (P ¼ 0:027) as well as the stage of fibrosis (P ¼ 0:011). On the other hand, the 8OHdG index significantly correlated with the grade of necro-inflammation (P ¼ 0:039).

4. Discussion In the present study, we analyzed two parameters of oxidative cellular damage in order to evaluate the pathological significance of oxidative stress in human NAFLD. We demonstrated that HNE adducts, a major aldehydic metabolite of lipid peroxidation, could be widely detected in human NAFLD including simple fatty liver. The localization of HNE adducts was predominantly in the cytoplasm of hepatocytes and sinusoidal cells in zone 3 where histological damage is often observed in NASH [4,8,9]. In addition, we found that the HNE index significantly correlated with the grade of necro-inflammation and the stage of fibrosis. The correlation between the HNE index and the grade of necro-inflammation suggests that lipid peroxidation is involved in necro-inflammatory reaction in NASH. Although it remains to be clarified whether lipid peroxidation is the cause or the consequence of the liver injury, HNE is strongly chemoattractant for neutrophils [36] and oxidative stress-induced NF-kB activation may be involved in experimental Mallory body formation [37]. Thus, lipid peroxidation may be partly responsible for the pathological features observed in NASH. The HNE index also correlated with the stage of fibrosis in NASH. In this context, HNE may directly activate hepatic

stellate cells (HSCs) and up-regulate the expression of the collagen type I gene [19–21]. Activated HSCs are currently considered to be a major source of extracellular matrix proteins including collagen type I and to therefore play a central role in liver fibrogenesis [38,39]. Recently Washington et al. [40] demonstrated that HSC activation occurs not only in NASH but also in fatty liver where no apparent necro-inflammatory changes were observed. Reeves et al. [41] reported an analogous phenomenon in human alcoholic liver disease. They showed that HSC activation occurs in the absence of necro-inflammation and correlates with the severity of steatosis and speculated that an increased concentration of acetaldehyde and lipid peroxidation may play a role in HSC activation in alcoholic liver injury. Taking together these previous observations and our current findings, it seems that lipid peroxidation may play a role in the hepatic fibrogenesis in human NASH. One major finding of this study was the frequent expression of 8-OHdG in hepatocytes in NASH. The specificity of the monoclonal antibody against 8-OHdG has been established [35] and this antibody has been widely used for quantitative assessment of oxidative DNA damage in human tissues [31,35,42]. Quantitative analysis revealed that 8OHdG expression significantly correlated with the grade of necro-inflammation, but not with the severity of steatosis or the stage of fibrosis, suggesting a possible link between oxidative DNA damage and necro-inflammation in NASH. Although the precise role of 8-OHdG in vivo still remains unclear, in vitro studies have demonstrated that 8-OHdG accumulates in cellular DNA and causes mispairing, which suggests that this oxidative modification is mutagenic and carcinogenic [43–45]. Previous epidemiological studies have shown that obesity is associated with an increased cancer risk for several organs including the colon, pancreas, uterus and liver [46–48]. Although the mechanisms underlying this association are unclear, Yang et al. [49] recently demonstrated significant

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increases in hepatocyte proliferative activity with concomitant inhibition of hepatocyte apoptosis in genetically obese, leptin-deficient ob/ob mice, which are animal models for human NAFLD. Interestingly, they also showed that liver hyperplasia is evident in the earliest stage of NAFLD in these mice, supporting the concept that obesity-related metabolic abnormalities, rather than cirrhosis, initiate the hepatic neoplastic process during obesity in these mice. In this context, it has been reported that hepatic mitochondrial production of reactive oxygen species is significantly increased in ob/ob mice [50]. Although it remains uncertain whether hepatocellular carcinoma is part of the natural history of NASH or not, increased hepatic oxidative stress in human NASH has been reported [11]. Taken together, it is possible that increases in hepatic oxidative stress might be, at least in part, involved in the hepatocarcinogenesis in human NASH through the production of oxidative DNA damage. Further studies including prospective longitudinal clinical studies are required to examine this hypothesis. In conclusion, our immunohistochemical study provides evidence that lipid peroxidation and oxidative DNA damage frequently occur in liver with NAFLD, suggesting the possible role of oxidative stress in some clinical features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.

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