Immunohistochemical detection of 1,N6-ethenodeoxyadenosine in nuclei of human liver affected by diseases predisposing to hepato-carcinogenesis

Alexander Frank1, Helmut K. Seitz2, Helmut Bartsch1, Norbert Frank1 and Jagadeesan Nair1,3

1 Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany and 2 Department of Medicine, Salem Medical Center, Zeppelinstr. 11-33, D-69121 Heidelberg, Germany

3 To whom correspondence should be addressed Email: j.nair{at}dkfz.de


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Increased oxidative stress and lipid peroxidation (LPO) are implicated in multistage carcinogenesis. Recent studies have shown that LPO-derived reactive hydroxyalkenals can form promutagenic exocyclic etheno-DNA adducts in vivo. Such DNA damage was found to be increased in the liver of patients with metal storage diseases and in colon adenomas of familial adenomatous polyposis patients. We now have investigated the levels of 1,N6-ethenodeoxyadenosine ({varepsilon}dA) in human liver samples obtained from a group of patients diagnosed with hepatitis, fatty liver, fibrosis and cirrhosis, primary hemochromatosis and Wilson's disease. Using an immunohistochemical method, the relative mean pixel intensity of randomly selected nuclei was measured by imaging software; positively stained cell nuclei (arbitrary mean pixel intensity >=0.5) were counted. Prevalence of {varepsilon}dA (%) was calculated from the ratio of a number of positively stained cell nuclei over a total number of cells counted. When compared with normal livers (3.1%), the percent prevalence (means) was significantly higher in specimens of alcoholic fatty liver (15%) and fibrosis patients (50%) but not in samples with hepatitis (induced by various factors) (6.2%). The percent prevalence in alcohol fibrosis was as high as in the liver from Wilson's disease (50.7%) and hemochromatosis (33%) patients. This is the first demonstration of increased {varepsilon}dA in human liver diseases due to alcohol abuse. We conclude that excessive hepatic DNA damage, as assessed by miscoding etheno-DNA adduct in the nuclei of liver biopsies, is probably caused by alcohol-induced oxidative stress and LPO. In cancer-prone liver diseases (fatty liver, cirrhosis/fibrosis) such damage may act as a driving force towards malignancy.

Abbreviations: HCC, hepatocellular carcinoma; {varepsilon}dA, ethenodeoxyadenosine; {varepsilon}dC, 3,N4-ethenodeoxycytidine; HNE, trans-4-hydroxy-2-nonenal; IHC, immunohistochemical detection method; LPO, lipid peroxidation; PBS, phosphate-buffered saline; ROS reactive oxide species; TNF, tumor necrosis factor


    Introduction
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By 2000, hepatocellular carcinoma (HCC) was the fifth most common cancer worldwide, responsible for approximately 551 000 new cases (399 000 in men and 153 000 in women). Because of the very poor prognosis, the number of deaths (529 000) is not far short of the number of new cases, and it represents the third most common cause of death from cancer (1). In humans, liver carcinogenesis is preceded mostly by chronic hepatitis followed by liver cirrhosis of any cause (2). In the endemic areas of liver cancer such as middle Africa and South East China, HCC is caused by hepatitis B virus infection combined with exposure to aflatoxin B1, a hepatotoxic mycotoxin (3). However, in more developed countries the etiology of HCC involves alcohol abuse (4) and hepatitis C virus infection (5).

Both clinical findings and experimental results on alcohol-induced liver diseases have shown the importance of cytokine-mediated cell-to-cell interactions in the onset of ethanol-induced liver damage. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin (IL)-1 beta and IL-6, are released from Kupffer cells or infiltrating neutrophils and macrophages and elicit defensive response in parenchymal liver cells, including activation of apoptotic processes. As a consequence, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated in parenchymal cells in response to cytokine-induced stress signals and also by activation of Kupffer cells and inflammatory cells (6,7). Alcohol also can trigger oxidative stress by induction of cytochrome P450 2E1 and by depletion of mitochondrial glutathione (8). Alcohol abuse can further exacerbate hepatitis C virus infection and the associated liver damage, by causing oxidative stress and promoting fibrosis, thereby accelerating disease progression to cirrhosis (9).

The formation of DNA-adducts from carcinogens (or their metabolites) from exogenous or endogenous sources is one of the earliest damaging events to occur in the cellular genome. If not repaired, the adduct formation in a surviving cell can lead to mutations upon cell division. Once these mutations accumulate they may disrupt genomic integrity leading to cancer (10). Oxidative stress is a disturbance caused by an imbalance between the generation of ROS and antioxidant defence. It occurs when excessive ROS and RNS production overwhelms the antioxidant defence system or when the antioxidant defence is impaired. ROS can react with polyunsaturated fatty acids derived from membrane phospholipids or from dietary intake, resulting in the production of reactive aldehydes as lipid peroxidation (LPO) by-products in the body. The most abundant LPO-products are malondialdehyde (MDA) and trans-4-hydroxy-2-nonenal (HNE) (11). The latter reacts with DNA bases such as deoxyadenosine and deoxycytidine to form inter alia the exocyclic DNA adducts, 1,N6-ethenodeoxyadenosine ({varepsilon}dA) and 3,N4-ethenodeoxycytidine ({varepsilon}dC) (12). Using a sensitive immunoaffinity/32P-postlabeling method (13), elevated DNA adduct levels have been detected in the liver of patients with metal storage diseases, primary hemochromatosis and Wilson's disease (14,15) and in Long Evans Cinnamon rats (16), an animal model for Wilson's disease. Also, as shown previously in a mouse model, nitric oxide over-production led to a concomitant increase in {varepsilon}-DNA adduct levels (17). In the present study we investigated the occurrence of {varepsilon}dA, using a recently developed immunohistochemical detection method (IHC) (18) as a marker for oxidative stress and LPO-related DNA damage in human liver biopsies. The patients analyzed were diagnosed with hepatitis, fatty liver, cirrhosis and fibrosis (predominantly due to alcohol abuse). IHC was also performed in liver samples removed from patients suffering from hereditary hemochromatosis and Wilson's disease at the time of orthotropic liver transplantation (cirrhotic liver).


    Material and methods
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 Material and methods
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 Supplementary material
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Chemicals and reagents
The following products were from the sources indicated: Tween 20 (polyoxyethylene sorbitan monolaurate), Triton X-100 (t-octylphenoxypoly-ethoxyethanol), APES (3-aminopropyl-triethoxysilan), bovine albumin fract.V (BSA), Tris–HCl and Tris base from Sigma-Aldrich (Taufkirchen, Germany); hematoxylin, Kaiser's glycerine-gelatin mounting media, perhydrol, methanol p.A., KH2PO4 p.a. and Na2HPO4 p.a. from Merck (Darmstadt, Germany); biotinylated goat anti-mouse IgG and avidin–biotin–peroxidase (ABC) complex (Vectastain elite ABC kit) and 3,3'-diaminobenzidine substrate kit from Linaris (Wertheim-Bettingen, Germany); the ImmunoPure(A/G)IgG purification kit for antibody purification from Pierce (Bruchsal, Germany), Microcon microconcentrators for antibody concentration from Milipore (Eschborn, Germany) and RNase and DNase from bovine pancreas from Roche (Mannheim, Germany). The monoclonal antibody (MAb) EM-A-1 (cell culture supernatant) raised against {varepsilon}dA was provided by M.Rajewski and P.Lorenz (University of Essen, Essen, Germany).

Human liver samples
Human liver fine needle biopsy samples were collected from patients for diagnostic purposes in Salem Medical Center, Heidelberg. Frozen asymptomatic liver tissue samples from organ donors were provided by U.Mohr/H.J.Schlitt, Medizinische Hochschule, Hannover, Germany. Liver specimens from Wilson's disease and hereditary hemachromatosis patients were provided by D.H.Phillips, Sutton, UK, as reported earlier (14,15). A brief characteristic of the samples is given in Table I.


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Table I. Characteristics of patients and histopathology of liver samples

 
Immunohistochemical detection of {varepsilon}dA
The IHC was performed by a method developed in our laboratory (18) with some modifications. The main modifications were, use of MAb EM-A-1 instead of EM-A-4 omission of stain enhancement by nickel and changes in time in incubation steps. In brief 6 µm cryosections of liver tissue samples were placed on 3-aminopropyl-triethoxysilan/acetone-coated glass microscopic slides, fixed with acetone at –20°C for 10 min and air-dried. Slides were dipped in phosphate-buffered saline (PBS) for 10 min and then for a further 10 min in 0.3% H2O2 in absolute methanol to quench endogenous peroxidase activity; finally they were washed in 1% Tween/Triton X in PBS. Sections were treated with RNase (10 µg/ml in Tris–HCl), purified from DNAse in 80°C hot water for 10 min, at 37°C for 1 h to prevent antibody binding on RNA adducts. After PBS washing for 10 min, DNA was denatured by treatment with 4 N HCl for 5 min, to allow the MAb to bind on {varepsilon}dA. Slides were washed in H2O, neutralized in 50 mM Tris base for 5 min and dipped again in H2O for 5 min. To prevent primary antibody binding on collagen, non-specific binding sites were blocked with 10% BSA, 2% horse serum, 0.05% Tween and 0.05% Triton X for 20 min. The sections were incubated with the purified primary MAb for {varepsilon}dA (EM-A-1, dilution 1:20) at 4°C overnight. After washing with PBS, the sections were incubated with the biotinylated second goat anti-mouse antibody (diluted 1:400) for 1 h at room temperature and (after washing for 30 min) with the ABC complex (Vectastain ABC kit), as proposed by the manufacturer's protocol. The slides were washed again and then 3,3'-diaminobenzidine was used as a chromogen to visualize the reaction. After stopping the reaction in H2O for 5 min, slides were mounted with Kaiser's glycerin gelatin. All sections were subjected to the same procedure under standardized conditions. As negative controls, serial sections of each sample were either treated with DNase (100 µg/ml in H2O for 1 h at 37°C) before incubation with the primary MAb or with the primary MAb substituted by PBS. The specificity of the antibody against etheno-DNA adducts was reported earlier (19).

Imaging and semi-quantitative analysis of {varepsilon}dA
The images were made with a microscope equipped with a camera Axiocam (Carl Zeiss, Göttingen, Germany) for color pictures and the imaging software Axiovision (Rel. 2.05). The relative mean pixel intensity of randomly selected nuclei was measured by imaging software (Lucia G, Nikon, Düsseldorf, Germany). Positive cell nuclei that were stained having an arbitrary mean pixel density >=0.5, were counted. The ratio of positively stained cell nuclei over the total number of cells x100 was used for the expression of relative {varepsilon}dA prevalence in percent. As some of the needle biopsies were small, a minimum of 25 cells were counted per slide.

Statistical analyses
The statistical significance between the different groups was tested using the Kruskal–Wallis test; rank correlations were analyzed by Kendall's tau test. The statistical evaluation was made with the ADAM program established by the Division of Biostatistics at the DKFZ.


    Results
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 Material and methods
 Results
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We have modified the IHC protocol reported earlier by Yang et al. (19), which was developed using rodent liver, by essentially optimizing reagent concentrations and incubation times. By this it was possible to quench background staining and to detect {varepsilon}dA in the nuclei of human liver samples. This method combined with imaging software allowed the semi-quantitative estimation of {varepsilon}dA levels in the liver DNA (nuclei), including that from needle biopsies with a volume in the order of 1 mm3. Figure 1 shows photomicrographs of some representative samples with IHC staining. Exclusion of the primary antibody or treatment of the sections with DNase prior to the primary MAb treatment resulted in virtually no detectable staining of the nuclei. Both these results and the intensive staining only within the nuclei confirmed that the staining involves nuclear DNA. Human liver samples were divided into the following groups according to their histopathology and pathogenesis for their liver diseases. Asymptomatic controls (n = 15), hepatitis, miscellaneous (n = 5), fatty liver, alcoholic (n = 7), fatty liver, miscellaneous (n = 2) fibrosis, alcoholic (n = 3) fibrosis/cirrhosis, miscellaneous (n = 4), primary hemochromatosis (n = 2) and Wilson's disease (n = 6). Table I shows percent positively stained cell nuclei observed in different groups. The comparison of adduct abundance is depicted in Figure 2 where three or more samples were available. When compared with control livers, the percent positively stained nuclei were significantly higher in specimens of alcoholic fatty liver, alcoholic fibrosis and of cirrhosis/fibrosis of miscellaneous etiology ({alpha} = 0.05, P < 0.0001), but not in samples with hepatitis (Figure 2). Also, liver obtained from Wilson's disease patients had {varepsilon}dA staining as high as in cirrhosis caused by other factors and differed significantly from controls ({alpha} = 0.05, P < 0.0001). {varepsilon}dA-adduct staining showed no obvious correlation with either age or gender. Because of lack of information on alcohol consumption, a correlation with DNA-damaged cells could not be established. However, most of the liver samples in the group of fatty liver and fibrosis patients were diagnosed for liver diseases due to heavy alcoholism. A trend of increased frequency of adducts was observed: control < hepatitis < fatty liver < fibrosis/cirrhosis.



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Fig. 1. Photomicrographs of immunostained liver sections. (A) Control; (B) fatty liver; (C) cirrhotic liver; (D) liver from WD patient. See online supplementary material for colour version of this figure.

 


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Fig. 2. Box plot comparisons of sdA prevalence in liver diseases. C, control; Hep(M), hepatitis with miscellaneous etiology; FL(A), alcoholic fatty liver; FB(A), alcoholic fibrosis; FIB-CIR(M), fibrosis/cirrhosis of miscellaneous etiology; WD, Wilson's disease. *P < 0.0001 versus control. Box gives 25th and 75th percentiles, whiskers represent 10th and 90th percentiles, X are outliers and small squares represent the mean values.

 

    Discussion
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 Material and methods
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 Supplementary material
 References
 
This is the first demonstration of an immunohistochemical detection of {varepsilon}dA in nuclei of human liver specimens. With modifications of our earlier protocol (18) it was possible to reduce background staining and to stabilize the nuclei in human liver tissue sections. In our previous method, we have used mean pixel intensity for comparison of the groups. In this study we used adduct prevalence rather than mean pixel intensity, which reflects the percent of cells with high adduct levels in the tissue sections.

Our earlier studies revealed elevated etheno-DNA adduct levels ({varepsilon}dA and {varepsilon}dC), ranging from 4.5 to 8 adducts/107 parent nucleotides in the liver of patients with hereditary hemochromatosis and Wilson's disease, using a quantitative immunoaffinity 32P-postlabeling technique (14). The positive results by IHC with high nuclear staining for {varepsilon}dA reconfirm our previous data. The increase in hepatic etheno-DNA adducts in both metal storage diseases is ascribed to a persistent oxidative stress and LPO, which are induced by copper and/or iron hyper-absorption and accumulation in the liver.

A recent study in rodents showed that both acute and short-term ethanol exposures increased the hepatic concentrations of {varepsilon}dA and {varepsilon}dC in DNA ~2-fold (20) which was similar to the increase induced by carcinogens such as urethane (21). Higher levels of hepatic HNE and F2-isoprostanes, both indicators of free radical catalyzed oxidation products of arachidonic acid and oxidative stress, were observed in rats when ethanol was part of the diet. Hepatic mitochondrial HNE levels were significantly raised after repeated acute alcohol doses and this was especially seen in fetal liver (22). HNE is the major precursor aldehyde for the formation of etheno-DNA adducts (11). In rhesus monkeys the amount of alcohol consumed correlated positively with plasma LPO products, HNE and 8-isoprostane F2 alpha (23). Free F2-isoprostane and HNE were strikingly increased in individuals with alcoholic hepatitis (24). Antibodies against human serum albumin modified by reaction with MDA or HNE, were significantly higher in alcoholics as compared with non-alcoholic cirrhotic or healthy controls (25). Our results further provide evidence that oxidative stress and LPO-induced DNA damage via the HNE pathway (Figure 3) is involved in progression of these liver diseases to full neoplasia.



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Fig. 3. Pathways of formation of etheno-DNA adducts in alcoholic liver disease. SOD, superoxide dismutase; NOS2, inducible nitric oxide synthase; ROS, reactive oxygen species; RNS, reactive nitrogen species.

 
{varepsilon}-DNA adducts probably play a causal role in the initiation and progression of liver carcinogenesis as they produce base pair substitution mutations in various organisms and mammalian cells. {varepsilon}dA can lead to AT->GC transitions and to AT-> TA and AT->CG transversions (26,27). {varepsilon}dC can cause CG->AT transversions and CG->TA transitions (28,29), and N2,3-ethenodeoxyguanosine, that is also formed in vivo from LPO products, can lead to GC->AT transitions (29). These promutagenic properties of etheno-bases strongly implicate them in the initiation of carcinogenesis by vinyl chloride (30), urethane (ethyl carbamate) (31,32) and other etheno-adduct forming chemicals. Incorporation of a single {varepsilon}dA showed a similar miscoding frequency when located in either DNA strand of HeLa cells and was more mutagenic than 8-oxo-deoxyguanosine (33).

A proposed pathway for the formation of miscoding etheno-DNA adducts is presented in Figure 3. Alcohol abuse on one hand increases cytochrome P450 2E1 (in the hepatocytes) that leads to mitochondrial glutathione depletion, triggering oxidative stress (8). Cytochrome P450 2E1 is also an effective generator of ROS in the presence of an iron catalyst (reviewed in ref. 34). On the other hand chronic alcoholism leads to the production of gut endotoxin that activates pro-inflammatory signals, exerting oxidative stress on the neighboring hepatocytes (35,36). The enhanced oxidative stress generates reactive alkenals through LPO, which bind to nucleic acid bases to yield etheno-DNA adducts and other LPO-derived DNA-modifications (11,37).

In conclusion, our study strongly supports the main hypothesis that in chronic alcohol abusers reactive LPO by-products, such as HNE are involved in ROS-induced DNA damage in hepatocytes. As a consequence, mutations are generated which lead to genomic instability and progression of these cells to malignancy.


    Supplementary material
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Supplementary material can be found at: http://www.carcin.oupjournals.org/


    Acknowledgments
 
We wish to thank Dr Werner Rittgen, Division of Biostatistics, DKFZ for help in statistical analysis and Ulrike von Seydlitz-Kurzbach for technical assistance. We thank Dr D.H.Phillips, Institute of Cancer Research, Sutton, UK, for providing us liver samples from Wilson's disease and hemochromatosis patients. The secretarial help by S.Fuladdjusch is gratefully acknowledged.


    References
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 Abstract
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 Material and methods
 Results
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 Supplementary material
 References
 

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Received September 3, 2003; revised December 22, 2003; accepted January 14, 2004.





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