High urinary excretion of lipid peroxidation-derived DNA damage in patients with cancer-prone liver diseases

Mutation Research 683 (2010) 23–28

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High urinary excretion of lipid peroxidation-derived DNA damage in patients with cancer-prone liver diseases Jagadeesan Nair a , Petcharin Srivatanakul b,1 , Claudia Haas a,c,1 , Adisorn Jedpiyawongse b , Thiravud Khuhaprema b , Helmut K. Seitz c , Helmut Bartsch a,∗ a b c

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany National Cancer Institute, 268/1 Rama VI Road, Bangkok 10400, Thailand Salem Medical Center, Zeppelinstr. 11-33, 69121 Heidelberg, Germany

a r t i c l e

i n f o

Article history: Received 21 April 2009 Received in revised form 24 July 2009 Accepted 2 October 2009 Available online 12 October 2009 Keywords: Miscoding etheno-DNA adducts Urinalysis Lipid peroxidation Viral- and alcohol-induced liver disease

a b s t r a c t Chronic inflammatory processes induce oxidative and nitrative stress that trigger lipid peroxidation (LPO), whereby DNA-reactive aldehydes such as trans-4-hydroxy-2-nonenal (HNE) are generated. Miscoding etheno-modified DNA adducts including 1,N6 -etheno-2 -deoxyadenosine (␧dA) are formed by reaction of HNE with DNA-bases which are excreted in urine, following elimination from tissue DNA. An ultrasensitive and specific immunoprecipitation/HPLC-fluorescence detection method was developed for quantifying ␧dA excreted in urine. Levels in urine of Thai and European liver disease-free subjects were in the range of 3–6 fmol ␧dA/␮mol creatinine. Subjects with inflammatory cancer-prone liver diseases caused by viral infection or alcohol abuse excreted massively increased and highly variable ␧dA-levels. Groups of Thai subjects (N = 21) with chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC) due to HBV infection had 20, 73 and 39 times higher urinary ␧dA levels, respectively when compared to asymptomatic HBsAg carriers. In over two thirds of European patients (N = 38) with HBV-, HCV- and alcohol-related liver disease, urinary ␧dA levels were increased 7–10-fold compared to healthy controls. Based on this pilot study we conclude: (i) high urinary ␧dA-levels, reflecting massive LPO-derived DNA damage in vivo may contribute to the development of HCC; (ii) ␧dA-measurements in urine and target tissues should thus be further explored as a putative risk marker to follow malignant progression of inflammatory liver diseases in affected patients; (iii) etheno adducts may serve as biomarkers to assess the efficacy of (chemo-)preventive and therapeutic interventions. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Persistent oxidative and nitrative stress can trigger lipid peroxidation (LPO)-derived DNA damage. The concept that the ensued mutations and genomic instability drives inflammation induced-carcinogenesis [1,2] has now received wide acceptance. Pathological processes and mechanisms involved in disease causation have been described [3–12]. LPO-derived DNA adduct types and levels in humans have been reviewed [13]. In support of the paradigm shown in Fig. 1, our previous work revealed that

Abbreviations: ␧dA, 1,N6 -etheno-2 -deoxyadenosine; ␧A, 1,N6 ethenoadenosine; ALD, alcohol-related liver disease; CH, chronic hepatitis; CIR, cirrhosis; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HNE, trans-4-hydroxy-2-nonenal; IP–HPLC-FLD, immunoprecipitation–high performance liquid chromatography-fluorescence detection; LPO, lipid peroxidation; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +49 6221 423301; fax: +49 6221 423359. E-mail address: [email protected] (H. Bartsch). 1 These authors contributed equally to this work. 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.10.002

LPO related miscoding DNA adducts, such as etheno (␧)-adducts, increased with time in chronically inflamed target organs and preneoplastic lesions of cancer-prone patients [14,15]. These modified DNA-base adducts in human tissues are generated inter alia by reactions of DNA with LPO end products [16–19]. trans-4Hydroxy-2-nonenal (HNE) the major LPO-product yields 1,N6 etheno-2 -deoxyadenosine (␧dA), 3,N4 -etheno-2 -deoxycytidine and N2 ,3-etheno-2 -deoxyguanosine, which have been detected in vivo [16,18]. In addition HNE is able to interact with DNA to form 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)-propano2 -deoxyguanosine (HNE-dG) adducts (see Section 4). Nitric oxide via peroxynitrite-induced stress and LPO also produced ␧-DNA adducts: As demonstrated in mouse models of inflammation, NO overproduction in DNA in vivo led to a concomitant large increase in ␧-adduct levels in affected tissues [20,21]. These results suggested that the promutagenic, chemically stable ␧-DNA adducts appear to be useful markers for assessing oxidative stress- and LPO-derived DNA damage in early premalignant stages of carcinogenesis. These adducts could play a major role in the development of human cancers, especially in those whereby persistent inflammation is part of

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Fig. 1. Putative pathways for the origin of 1,N6 -etheno-2 -deoxyadenosine (␧dA) excreted in urine, after its elimination from liver DNA. Irrespective of the mechanism, ␧dA in urine is used as an indicator of damaged DNA-bases in internal organs, allowing non-invasive human biomonitoring.

their etiopathogenesis [22]. In order to provide further support for this paradigm (Fig. 1) we have developed a specific and ultrasensitive urinalysis method for quantifying ␧dA so to allow non-invasive applications in human biomonitoring. Hepatitis B and C virus (HBV, HCV) infection and chronic alcohol abuse are causally associated with increased risk of developing liver cancer in humans which progresses through chronic hepatitis and liver fibrosis/cirrhosis [23,24]. Increased oxidative and nitrative stress due to overproduction of reactive oxygen and nitrogen species (ROS, RNS) occurs during this progression phase (reviewed in [12,25,26]). Our aim of the present study was to ascertain whether ␧dA excreted in urine could serve as a DNA damage—and putative risk marker during progression of viral and alcohol-related cancer-prone liver diseases.

acetate, pH 6.8, containing 14.2% methanol, v/v) and resolved on a semi-preparative C18 column (260 mm × 8 mm i.d.) isocratically at 2.5 ml/min. Fractions containing the I.S. (8.5–12.5 min) and ␧dA (12.5–17.5 min) were collected; the I.S. fraction was counted for recovery and the ␧dA fraction was concentrated in vacuo. Then immunoprecipitation of ␧dA was performed in Tris–HCl buffer (10 mM Tris, pH 7.5, 140 mM NaCI, 3 mM NaN3 ) containing 1% bovine serum albumin and 0.1% rabbit IgG (Sigma–Aldrich, Schnelldorf, Germany) and monoclonal antibody EM A-1 (provided by Dr. M. Rajewsky, Institute for Cell Biology, University of Essen, Germany). The antigen–antibody complex was precipitated with saturated ammonium sulfate.

2. Materials and methods 2.1. Urine samples and patients Urine samples were collected from hepatitis B virus (HBV) infected male Thai patients, in Bangkok, who were diagnosed with chronic hepatitis (CH, N = 7; aged 37–52 years), cirrhosis (CIR, N = 5; 39–57 years) or hepatocellular carcinoma (HCC, N = 3; 44–65 years) and from Thai asymptomatic HBsAg carriers (NC, N = 6; 33–62 years). The samples were collected prior to any medical intervention and stored frozen at −40 ◦ C. HBV infection was ascertained by HBsAg analysis. Urine samples were also collected from male European HBV (N = 10)-, HCV (N = 14)- and alcoholrelated (N = 14) liver disease patients (Fig. 6, and Table, supplementary material) in Salem Medical Center, Heidelberg, Germany and from healthy male volunteers (N = 8; 29–59 years) and stored at −80 ◦ C. The study was approved by the Ethics Committee of the University of Heidelberg on 20.02.2003. 2.2. ␧dA determination in urine [27,28] The ␧dA levels were measured in urine samples by a modified immunoprecipitation–HPLC-fluorescence detection (IP–HPLC-FLD) method using 1,N6 -ethenoadenosine-[2,8-3 H] (␧A) as the internal standard (IS) (see Fig. 2). ␧A was synthesized by reacting adenosine-[2,8-3 H] with bromoacetaldehyde and purified by semi-preparative HPLC and then concentrated in vacuo (data not shown). Briefly, urine samples were filtered through 0.22 ␮m filter, 2 ml of the sample was spiked with the I.S. and dried in vacuo The dried samples were redissolved in methanol, centrifuged and the supernatant was carefully taken out and dried. The dried samples were dissolved in preparative HPLC buffer (25 mM ammonium

Fig. 2. Scheme for the analytical measurement of ␧dA in human urine by our developed IP–HPLC-FLD method: essential steps involve spiking of the urine sample with radiolabeled 1,N6 -ethenoadenosine (␧A) as an internal standard (I.S.) for recovery calculation, semi-preparative RP-HPLC, immuno-enrichment of ␧dA by a highly specific Mab EM-A-I and analytical HPLC-fluorescence detection of ␧dA. Detection limit is approximately 6 fmol ␧dA/injection (for details see Section 2).

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Fig. 3. (A) HPLC-FLD profiles of standard 1,N6 -ethenoadenosine (␧A) and ␧dA (top) and of three urine samples after immuno-enrichment from Thai patients shown in Fig. 5 NC, asymptomatic HBsAg carrier; CH, chronic hepatitis; CIR, cirrhosis; (B) stop-flow fluorescence scan of ␧dA-standard and ␧dA in one Thai CIR-urine sample. Excitation was at 230 nm and emission at 410 nm. The precipitate was washed and ␧dA eluted using 50% methanol/water and later concentrated in vacuo. The residue was analyzed by an HPLC system consisting of an HP1100 pump, HP1046A fluorescence detector (Hewlett Packard, Waldbronn, Germany) and 250 mm × 4.6 mm Lichrospher 100 RP 18E 5 ␮m (Bischoff, Leonberg, Germany), using a linear gradient (NH4 )3 P04 , 17 mM, pH 5, buffer/methanol 9:1–8:2 in 30 min at a flow rate of 1 ml/min. The ␧dA peak was detected at excitation 230 nm /emission 410 nm and quantified by a standard curve using an ␧dA standard (Sigma–Aldrich, Schnelldorf, Germany). The recovery of this step was also determined by adding the I.S. before starting the precipitation of the sample. The detection limit of ␧dA in this HPLC system was approximately 6 fmol/injection. All samples were assayed blindly. Urinary creatinine for normalizing the ␧dA data was measured by a picric acid-based method, using a kit provided by Sigma (Sigma–Aldrich, Schnelldorf, Germany) according to supplier’s protocol. Urine samples for which the collections were considered to be incomplete (creatinine excretion/body weight ratios less than 10.8 mg/kg) were removed from analyses. 2.3. Statistical analysis A p-value less than 0.05 (two-tail) was considered as statistically significant. SAS (version 6) and Stat-View (version S) statistics software (SAS Institute Inc., Cary, NC) were used for the analyses.

cleosides (Fig. 1). ␧dA was detected in urine samples from six asymptomatic HBsAg carriers from Thailand at a range of 1.3 to 12.4 (mean, 6.4 fmol/␮mol creatinine) (Fig. 5). The means of ␧dA levels (range in parenthesis) in chronic hepatitis patients were 127.3 (16.3–232.8), in patients with liver cirrhosis 468.3 (16.3–1474.7) and in HCC-patients 247.6 (150.9–480.4). When compared to asymptomatic HBsAg carriers used as controls the mean urinary ␧dA levels in the above three patient groups were approximately 20-fold (p < 0.01), 73-fold (p < 0.01) and 39-fold (p < 0.05) increased, respectively. To our knowledge this is the first report of a massive, highly variable ␧dA excretion in human urine as a consequence of HBV-induced chronic hepatitis, cirrhosis and liver cancer. The excretion of etheno adducts (␧dA) paralleled premalignant liver disease progression, and was in the order: asymptomatic control liver  hepatitis < cirrhosis > HCC.

3. Results 3.1. Urinanlysis of ␧dA For investigating factors influencing the formation of ethenoDNA adducts in humans we have developed a specific and sensitive urinalysis assay for ␧dA based on immunoprecipitation–HPLCfluorescence detection (IP–HPLC-FLD) with a sensitivity of about 6 fmol ␧dA/injection. Fig. 2 depicts the scheme for ␧dA analysis. The identity of ␧dA was ascertained by its isolation from the urine by a highly specific antibody and confirmed by comparing chromatographic profiles and the fluorescence excitation/emission spectra with an authentic standard (Fig. 3). Furthermore, the identification of ␧dA in urine samples was confirmed by analyzing the immunoenriched sample by HPLC–electrospray-mass spectrometry (Fig. 4). 3.2. Urinary excretion of ␧dA in HBV-infected Thai patients After their elimination from liver DNA, etheno-base adducts such as ␧dA are excreted in the urine as etheno-modified deoxynu-

Fig. 4. LC–MS confirmation of ␧dA in human urine. Standard ␧dA and an immuno-enriched Thai (CIR) urine sample was analyzed by HPLC–electrospraymass spectrometry (ES-positive mode, Hewlett Packard instruments, USA). The signal at m/z = 276 corresponds to (M+H)+ of ␧dA.

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Fig. 5. Urinary excretion of ␧dA (fmol ␧dA/␮mol creatinine) in male Thai patients diagnosed with chronic hepatitis (CH), liver cirrhosis (CIR) and hepatocellular carcinoma (HCC) due to HBV infection in comparison with asymptomatic HBsAg carriers (NC) as controls, **p < 0.01; *p < 0.05.

oxidative stress- and LPO-derived DNA damage in affected organs at early stages of carcinogenesis [15]. These miscoding adducts could play a major role in the development of human cancers, especially when persistent inflammation is part of the etiopathogenesis [22]. In order to provide further support for this paradigm (Fig. 1) we have developed a specific and ultrasensitive urinalysis method by IP–HPLC-FLD for non-invasive applications in human biomonitoring for quantifying etheno-DNA-nucleoside-adducts, i.e. ␧dA excreted in urine, following elimination from tissue DNA. The exact pathways by which ␧dA once formed in DNA in vivo is excreted in urine are not completely understood. It is likely that ␧dA originates mostly from its repair in tissue DNA. Additionally it may be formed to a minor extent by reaction of LPO-products in the cellular deoxynucleoside pool or after DNA fragmentation through apoptotic processes (Fig. 1). However there is no experimental evidence either in experimental animals or in humans for the latter pathways. Of the two currently known repair pathways for ␧dA, DNA glycosylase and the ALK B protein, neither would generate ␧dA as excision product [29–33]. ␧dA when repaired by N-alkylpurine-DNA glycosylase yields the modified base, 1,N6 ethenoadenine. As we have detected the etheno-modified deoxyribonucleoside in urine, ␧dA may originate from DNA by other pathways such as nucleotide excision repair (NER). Indeed recent work indicated that for another cyclic HNE-derived DNA adduct, 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)-propano2 -deoxyguanosine (HNE-dG), formed in addition to the simple etheno adducts (e.g. ␧dA) in human and animal tissues, nucleotide excision repair (NER) was a major repair pathway in both human and Escherichia coli cells [34,35]. We therefore hypothesize that during (long patch) NER, ␧dA may be (co-)eliminated so to end up as etheno-deoxyribonucleoside excretion product in urine. Experimental evidence for this proposal is now required, and studies on the mechanisms how ␧dA is excreted in urine are warranted. 4.2. Ultrasensitive urinalysis method for ␧dA detection

Fig. 6. Individual and mean urinary levels of ␧dA (expressed as fmol ␧dA/␮mol creatinine) in male European patients diagnosed with viral (HBV, HCV)- and ALD related cancer-prone liver diseases in comparison with healthy (virus free) control subjects (C); ALD vs. C, p < 0.02; HBV, HCV vs. C, non-significant. In over 70% of the patients ␧dA was clearly detectable by our IP–HPLC-FLD method. A value of 0.1 indicates a signal below detection limit.

3.3. Urinary excretion of ␧dA in liver cancer-prone European patients ␧dA was detected as a background level in 6 out of 8 urine samples from healthy male volunteers at a mean of 3.2 fmol ␧dA/␮mol creatinine (Fig. 6) 10 out of 10 urine samples with detectable adduct levels from HBV-related hepatitis patients had a mean value of 37.8 fmol ␧dA. 12 out of 14 samples with detectable adducts from HCV related liver disease patients had a mean ␧dA level of 20.7 fmol. Among ALD patients 10 out of 14 samples with detectable adducts had a mean of 31 fmol ␧dA in their urine. As compared to healthy and virus free controls, the respective increase in the above three patient groups was approximately 10-fold (HBV, non-significant), 7-fold (HCV, non-significant) and 10-fold (ALD, p = 0.02). Neither age nor any of the other clinical parameters listed in Table (supplementary material) showed an apparent correlation with urinary ␧dA-levels. 4. Discussion 4.1. Etheno-DNA adducts as biomarkers for oxidative stress- and LPO-derived DNA damage Previous studies suggested that the promutagenic, chemically stable ␧-DNA adducts appear to be useful markers for assessing

Our urinalysis assay for ␧dA is based on immuno-enrichment combined with HPLC-fluorescence detection. Fig. 2 depicts major steps for ␧dA analysis by IP–HPLC-FLD. The identity of ␧dA was (i) ascertained by its isolation from the urine by a highly specific monoclonal antibody, (ii) confirmed by comparing chromatographic profiles and the fluorescence excitation/emission spectra with an authentic ␧dA standard (Fig. 3) and (iii) unequivocally confirmed by analyzing the immuno-enriched ␧dA sample from human urine by HPLC–electrospray-mass spectrometry (Fig. 4). Our IP–HPLC-FLD detection method has a sensitivity of approximately 6 fmol ␧dA/injection, and has the capability to reliably detect ‘background’ values in urine samples of healthy subjects and can thus be used to monitor any disease-related increase in patients. 4.3. Chronic HBV infection, DNA damage and HCC Oxidative stress and upregulation of iNOS is a hallmark in chronic viral hepatitis (reviewed in [23–25]). Although iNOS is predominantly expressed in inflammatory cells, epithelial cells from affected tissues also express iNOS. Groups of HBV-infected Thai patients with chronic hepatitis, liver cirrhosis or HCC were analyzed for ␧dA levels in their urine and compared with asymptomatic Thai HBsAg carriers. A massive 20–73-fold increase in ␧dA concentration in urine was detected in the three above HBV-infected Thai patient groups being highest in cirrhotic patients (Fig. 5). This DNA-base damage could arise from HBV-induced chronic inflammation, overproducing NO, ROS, RNS, and DNA-reactive LPO-derived aldehydes such as HNE. As previously demonstrated in two mouse models

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of inflammation, NO overproduction in vivo via peroxynitrite, produced a large increase of etheno adducts in DNA of affected tissues [20,21]. 4.4. HNE-DNA adducts and mutational hotspot at codon 249 of TP53 In HBV and aflatoxin B1 (AFB1 )-induced hepatocellular carcinoma (HCC), a hot spot mutation, a G:C to T:A transversion in p53 codon 249 (-AGG*-), is considered to be a marker in disease pathogenesis, thought to arise from the AFB1 -dG adduct [36,37]. However, a previous study [38] showed that this p53 (codon 249) mutational hot spot in HCC is apparently not, or not alone, related to a selective formation or persistence of the AFB1 -dG adduct formed in DNA by AFB1 , a causative agent for HCC in some regions. There is now circumstantial support (see (i)–(iv)) that HNE produced by LPO may be involved in causing a mutational hotspot in codon 249 of p53 during HCC pathogenesis: (i) In a wildtype p53 cell line [39], HNE caused a high frequency of this G:C to T:A transversion at codon 249 (-AGG*-), (ii) HNE interacts with DNA to also form, in addition to simple etheno adducts such as ␧dA, an exocyclic HNE-dG adduct carrying a hydroxyhexanyl side chain. Exactly this HNE-dG adduct is preferentially formed at the third base of codon 249 (-AGG*-) in the p53 gene transfected into a plasmid [40], (iii) HNE-dG adducts, when introduced in a shuttle vector into replicating human cells, were promutagenic and G:C to T:A transversions were the most prevalent mutations induced. Furthermore, HNE-dG adducts produced a significantly higher level of genotoxicity and mutagenicity in nucleotide excision repair (NER) deficient than in NER proficient human cells, indicating that NER is a major pathway for repairing this HNE-dG adduct [35]. Thus there is a superimposition of HNE adduct formation and mutational hot spots in p53 codon 249. The biological impact of etheno-DNA adducts is further stressed by the observation that this hot spot mutation renders the cells more resistant to apoptosis [41]. Because HNE is now recognized as a reliable oxidative stress marker, a growth modulating factor, and signaling molecule [8,9,42], it could be a major player causing this specific p53 mutation. (iv) This notion is further supported by the massive increase in urinary HNE-derived ␧dA in Thai patients with HBVrelated liver diseases implicating a high LPO-derived DNA damage in the affected target organ, steadily increasing from chronic hepatitis to cirrhosis (Fig. 5). Should HNE-DNA adducts be involved in the induction of a unique mutational hotspot at codon 249, this mechanism would explain the late appearance of this specific missense mutation in the etiopathogenesis of HCC, caused by aflatoxins and HBV. Indeed, a study reported the absence of this codon 249 mutation in young Guinean children with high aflatoxin exposure, as assessed by plasma aflatoxin albumin adducts [43]. 4.5. Alcohol-induced DNA damage in human cancer-prone liver disease Oxidative stress and increased LPO byproducts persist in ALD patients [25,26,44,45]. Acute and short term ethanol feeding to mice were found to produce etheno adducts in liver DNA [46]. Using an immunohistochemical detection method [47], we previously analyzed ␧dA levels in DNA of human liver needle biopsies obtained from European patients, diagnosed with alcohol-related hepatitis, fatty liver, fibrosis, and cirrhosis. Positively stained cell nuclei were counted and the percent prevalence of ␧dA was monitored. When compared to livers from healthy controls, the (mean) percent prevalence was 16-fold higher in biopsies from fibrosis/cirrhosis patients. This increase was comparable to that found in the liver of patients with Wilson’s disease and primary hemochro-

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matosis [47], the latter a genetic disease highly predisposing to HCC. In keeping with a high DNA damage detected in the liver of ALD patients, in this study we also found 10-fold increased ␧dA levels in urine of European ALD patients when compared to healthy control urine (Fig. 6). A comparable (7–10-fold) increase was equally found in European patients with liver disease due to HBV and HCV infection (Fig. 6). Because of the small number of subjects that could be investigated and the large inter-individual variation by two orders of magnitude, the statistical significance of disease-related increase in ␧dA levels was only borderline. Nevertheless, in over 70% of the 38 patients this LPO-damage marker in urine was clearly increased above detection limit. Excess hepatic DNA damage monitored as ␧dA adducts in hepatocyte DNA and as increased ␧dA excretion in urine of ALD patients is likely caused by oxidative stress and LPO, probably by two pathways: Alcohol abuse in an early phase increases cytochrome P450 2E1(CYP2E1) in hepatocytes that leads to mitochondrial glutathione depletion triggering oxidative stress [48] and DNA adduction [47]. Later stages of chronic alcoholism lead to the production of gut endotoxin, that activates proinflammatory signals in Kupffer cells, exerting oxidative stress on the neighboring hepatocytes [49,50]. We recently could show in liver biopsies of ALD patients that strong interrelationships exist between CYP2E1 protein expression, etheno adduct levels and protein bound HNE [51]. DNA damage in hepatocytes of liver cancer-prone subjects also increases possibly because some DNA repair pathways may be impaired: repair of LPO-derived DNA adducts was shown to be inhibited by inflammatory mediators [52–54] and especially by HNE [41]. Thus adduct formation and steady state levels in DNA, generated by self-perpetuating LPO processes may be further enhanced by impaired repair in inflamed tissues. The resulting mutations may render the cells more resistant to apoptosis [41] and may drive cirrhotic/fibrotic liver cells to genomic instability and malignancy (HCC). 5. Conclusions From our findings in this pilot study we conclude: (i) high urinary ␧dA levels, reflecting massive LPO-derived DNA damage in affected liver tissue and the ensuing genetic damage may contribute to the development of HCC, possibly by inducing mutational hot spots in codon 249 of TP53. (ii) Urinary ␧dA measurements together with etheno-DNA adduct analyses in liver needle biopsies should be further explored whether they can serve as a putative risk markers to follow the malignant disease progression in patients with chronic hepatitis, liver cirrhosis and fibrosis due to viral infections or ALD. (iii) Etheno adduct biomarkers hold promise to assess the efficacy of (chemo-)preventive and therapeutic interventions, and such clinical biomonitoring trials are warranted. Conflict of interest statement None declared. Acknowledgements Research was supported by the Division of Toxicology and Cancer Risk Factors, German Cancer Research Center. The authors thank all volunteers and patients who participated in this study. We thank Susanna Fuladdjusch for skilled secretarial help and Urmila Nair and Khelifa Arab for critical reading. This article is dedicated to J. Nair who died prematurely in 2007.

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