Lipid Peroxidation and Etheno DNA Adducts in White Blood Cells of Liver Fluke-Infected Patients: Protection by Plasma α-tocopherol and Praziquantel

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1 Research Article Lipid Peroxidation and Etheno DNA Adducts in White Blood Cells of Liver Fluke-Infected Patients: Protection by Plasma α-tocopherol and Praziquantel Cancer Epidemiology, Biomarkers & Prevention Somkid Dechakhamphu 1,3,4, Somchai Pinlaor 2,3, Paiboon Sitthithaworn 2,3, Jagadeesan Nair 4, Helmut Bartsch 4, and Puangrat Yongvanit 1,3 Abstract Chronic infection by the liver fluke Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. To clarify the involvement of oxidative stress and lipid peroxidation derived DNA damage, etheno (ε)-dna adducts (εda, εdc) in WBC and plasma α-tocopherol were measured in samples collected from O. viverrini infected Thai patients (n = 50) and healthy noninfected volunteers (n = 20). εda and εdc levels were three to five times higher (P < 0.001) in infected patients than in controls; O. viverrini infection also increased two to three times in the plasma inflammatory indicators, 8-isoprostane, malondialdehyde, and nitrate/nitrite. Mean plasma α-tocopherol levels were two times lower in patients than in healthy controls (P < 0.001). Two months after a single dose to infected patients of the antiparasitic drug praziquantel, εda and εdc levels in WBC were decreased to control level (P < 0.03); plasma 8-isoprostane, malondialdehyde, nitrate/nitrite, and alkaline phosphatase (ALP) were concomitantly lowered. εda and εdc levels in WBC were positively correlated with plasma 8-isoprostane, malondialdehyde, and nitrate/nitrite levels and ALP activity, whereas plasma α-tocopherol levels showed inverse correlations. We conclude that chronic O.viverrini infection induces an accumulation of lipid peroxidation derived DNA damage through oxidative/nitrative stress, which is lowered by the plasma α-tocopherol and by antiparasitic drug therapy. Etheno adducts in WBC and urine should be explored as a risk marker for opisthorchiasis-related cholangiocarcinoma, and to assess the efficacy of preventive and therapeutic interventions. Cancer Epidemiol Biomakers Prev; 19(1); AACR. Introduction Infection by Opisthorchis viverrini, a strong risk factor for cholangiocarcinoma, probably causes cancer through growth stimuli produced by chronic inflammation. Studies from our group (1, 2) and others (3-5) have provided evidence that persistent oxidative/ nitrative stress and excess lipid peroxidation are induced by chronic inflammatory processes that lead to massive DNA damage in target organs and represent an important step in carcinogenesis. The oxidation of lipids by reactive oxygen and nitrogen species results in byproducts such as trans-4-hydroxy-2-nonenal, malondialdehyde, and crotonaldehyde. These intermediates can react with DNA bases to form exocyclic DNA Authors' Affiliations: Departments of 1 Biochemistry and 2 Parasitology, and 3 Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; and 4 Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany Note: deceased: Jagadeesan Nair. Corresponding Author: Puangrat Yongvanit, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand. Phone: ; Fax: puangrat@kku.ac.th doi: / EPI American Association for Cancer adducts (6-8). Reaction of DNA bases with the major lipid peroxidation product trans-4-hydroxy-2-nonenal yields inter alia the etheno-dna adducts 1,N 6 -etheno-2 -deoxyadenosine (εda) and 3,N 4 -etheno-2 -deoxycytidine (εdc). These etheno-modified DNA bases are highly miscoding lesions, and are thought to initiate the carcinogenic process through specific point mutations. εda and εdc, which are also produced in DNA by the human carcinogen vinyl chloride, induce specific base pair substitution mutations in mammalian cells (9, 10). The high frequency of mutations and the deregulation of cell homeostasis are events thought to play an important role in human chronic disease pathogenesis (11). Our previous work provided evidence that etheno DNA adducts may serve as potential risk markers for cancer-prone diseases caused by viral infections, chronic inflammatory processes such as chronic pancreatitis, inflammatory bowel diseases, and thalassemia-related metal storage (12-14). Previously we reported that excretion of εda and εdc in urine was highly increased in O. viverrini infected Thai patients (15). We proposed that chronic O. viverrini infection leads through oxidative/nitrative stress to a massive urinary excretion of etheno-bridged deoxyribonucleosides, reflecting high lipid peroxidation derived DNA damage in affected internal organs. These promutagenic etheno adducts, together with other lesions in the 310 Cancer Epidemiol Biomarkers Prev; 19(1) January 2010

2 Etheno-DNA Adducts in WBC During Liver Fluke Infection DNA of bile duct epithelial cells, may increase the risk of O. viverrini infected patients developing cholangiocarcinoma later in life. There is supportive evidence for a mechanistic link between oxidative stress and lipid peroxidation induced DNA damage and protection by cancer chemopreventive dietary components (16). A phase II chemoprevention trial using the oxidized DNA base 5-hydroxymethyl-2 - deoxyuridine as a damage marker, showed an inverse relationship with plasma α-tocopherol levels in female volunteers after a short-term dietary vitamin E supplementation (17). A pilot study in healthy women revealed an inverse correlation of εda levels in WBC DNA with vitamin E consumption, calculated from a food frequency questionnaire (18). Previous studies suggest that α-tocopherols (vitamin E compounds) may be inversely associated with cancer risk (19, 20). Therefore, the lipid soluble α-tocopherol seems to be effective for trapping lipid-derived free radicals in membranes and by thus inhibiting lipid peroxidation mediated adverse reactions. The major aim of this study was to clarify whether chronic O. viverrini infection can induce lipid peroxidation derived DNA damage in infested patients as a consequence of persistent oxidative/nitrative stress generated in vivo. As damage marker we measured by our ultrasensitive immunoaffinity/ 32 P-postlabeling method two lipid peroxidation derived etheno adducts (εda, εdc) in WBC samples. We hypothesized that when WBC circulates through the infected hepatobiliary tract DNA damage may occur at the site of O. viverrini infection (Fig. 1). Another aim was to verify whether a single dose of praziquantel, a known effective antiparasitic drug when administered to O. viverrini infected patients, could lower εda and εdc levels in WBC, concomitantly with those of 8-isoprostane, malondialdehyde, and nitrate/nitrite measured in plasma as inflammatory indicators. The potential of plasma α-tocopherol to lower oxidative stress and lipid peroxidation induced DNA damage in O. viverrini infected patients was also investigated. Materials and Methods Study Subjects The study was done in healthy volunteers (n = 20, 9 male and 11 female) and O. viverrini infected patients (n = 50, 26 male and 24 femal). Healthy noninfected subjects (range and mean age ± SE, and 47.6 ± 1.7 y) were recruited as persons who lived in a nonendemic area of O. viverrini infection and showed negative results (both at present and in the past) for O. viverrini egg counts in feces [range and mean egg count per gram of feces (epg) ± SE, 15-1,091 and ± epg). O. viverrini infected patients (range and mean age ± SE, and 53.0 ± 2.0 y) were persons who lived in an endemic area in Northeast Thailand (Phuvieng District near Ubonrat Dam, Khon Kaen Province) and had positive O. viverrini egg counts in feces. All individuals positive for O. viverrini infection were treated with a single dose of praziquantel (40 mg/ body weight). There were no positive O. viverrini egg counts in feces of all subjects after 2 mo of follow-up. Other chronic inflammatory processes caused by infection and chronic diseases may influence the levels of etheno DNA adducts. Therefore, persons with acute (non O. viverrini) infections showing nitrate/nitrite and positive leukocyte counts in urine were excluded; patients with chronic inflammatory conditions caused by hepatitis B virus and tuberculosis infections and those with diabetic mellitus were also excluded. An informed consent form was signed by each study subject. The study protocol was approved by the Ethics Group of the Human Research Committee (HE ), Khon Kaen University, Thailand. Plasma Sampling and DNA Isolation Peripheral 10 ml blood samples were obtained by sterilized venipuncture, collected in tubes containing acid citrate/dextrose and centrifuged at 2,000 rpm at 4 C for 15 min; plasma and buffy coat were then collected and stored at 80 C until used. The isolation of genomic DNA from buffy coats was achieved using the blood and cell culture Midi kit (Qiagen) according to the manufacturer's protocol, except with the modification of adjusting the ph to 7.4 and the NaCl concentration in the elution buffer to 1.4 mol/l. Etheno DNA Adduct Analysis εda and εdc were analyzed in DNA by an immunoaffinity/ 32 P-postlabeling method (21). In brief, 25 μg of DNA were hydrolyzed to nucleotide 3 -monophosphates using micrococcal nuclease and spleen phosphodiesterase. Normal nucleotides were quantitated by high performance liquid chromatography, and the adducts were enriched on immunoaffinity columns prepared from the monoclonal antibodies EM-A-1 (εda) and EM-C-1 (εdc). The antibodies used in this study were provided by Dr. Manfred Rajewsky (Institute of Cell Biology, University of Essen, Essen, Germany). The adducts and the internal standard deoxyuridine 3 -monophosphate was labeled with [γ- 32 P]ATP (10 μci; Amersham Buchler) and T4 polynucleotide kinase (Fermentus). The adducts were resolved on polyethyleneimine-tlc plates using two-directional chromatography [D1 = 1 mol/l acetic acid (ph 3.5); D2 = saturated ammonium sulphate (ph 3.5)]. After autoradiogram, the adduct spots and the internal standard were marked and cut, and the radioactivity was measured in a liquid scintillation counter. The absolute adduct levels were quantitated using standards, and the relative adduct level per parent nucleotides was determined with the amount of deoxycytidine (dc) and deoxyadenosine (da) obtained from high performance liquid chromatography analysis as described (21). Plasma α-tocopherol Determination Plasma α-tocopherol was measured by high performance liquid chromatography (22). Plasma (120 μl) Cancer Epidemiol Biomarkers Prev; 19(1) January

3 Dechakhamphu et al. was extracted with 120 μl of 0.1% butylated hydroxytoluene in ethanol containing 10 μmol/l α-tocopherol acetate (Sigma-Aldrich) as an internal standard. To extract α-tocopherol, 120 μl of10mmol/lsdsand3.0 ml of n-heptane were added and 2.8 ml of n-heptane supernatant containing α-tocopherol were transfered into a new tube. The supernatant was concentrated by evaporation under N 2, at 45 C. The dried residue was reconstituted with 120 μl of the mobile phase (methanol: acetonitrile:methylene chloride = 200:200:50 v/v/v) and injected into a high performance liquid chromatography device (Waters 2487) for at a flow rate of 1 ml/min. Plasma Nitrate/Nitrite Determination Nitrate/nitrite was determined by simple Griess reaction with some modifications (23, 24). Nitrate concentration in plasma and standard nitrate were measured after reducing nitrate into nitrite using the catalyst, by vanadium three chloride (VCl 3 ; Sigma-Aldrich). Briefly, a saturated VCl 3 solution was prepared from 400 mg of VCl 3 in 50 ml of 1 mol/l HCl and stored at 4 C for <2 wk. Plasma (100 μl) was deproteinized by adding 300 μl of methanol:diethyl ether (3:1 v/v), followed by incubation at 80 C for 30 min and centrifuging at 10,000 g for 10 min at 4 C. Of the supernatant, 100 μl were pipetted into a new micro centrifuge tube for nitrate/nitrite determination. The 100 μl of each supernatant, 50 μl ofn-(1- naphthyl) ethylenediamine dihydrochloride (0.1% w/v in water), and sulfanilamide (2% w/v in 5 % HCl) were thoroughly mixed and allowed to react for 20 min at 37 C. The absorbance was read against a reagent blank at 540 nm in a spectrophotometer (Spectra max GEMI- NI XPS, Molecular Devices). The sodium nitrate was used as a standard at various concentrations ranging from 6.25 to 100 μmol/l. Pooled plasma from a blood bank was used for testing within-run and between-run assay precision. A coefficient of variability of <10% was acceptable. Plasma Malondialdehyde Determination Thiobarbituric acid-reactive substances (TBARS) were measured (as reaction equivalent to malondialdehyde) with a slightly modified method by Nowak et al. (25). Briefly, 200 μl of plasma and 8.1% SDS, 1.5 ml of 0.5 mol/l HCl, 1.5 ml of 20 mmol/l thiobarbituric acid, 50 μl of 7.2% butylated hydroxytoluene in 95% ethanol, and 550 μl of deionized water were added to a screwcapped test tube. The sample was stirred and heated by boiling in a heater (90 C) for 1 h. After immediately cooling for 10 min in an ice box, the chromogen was extracted by adding 1 ml of deionized water and 5 ml of n- butanol in pyridine (15:1, v/v). The sample was then mixed thoroughly and centrifuged at 3,000 g for 15 min. The reaction product of TBARS with malondialdehyde (TBARS-malondialdehyde) was determined against reagent blank in a spectrofluorometer (Sunrise Tecan) with 520 nm excitation and 550 nm emission. The 1,1,3,3-tetramethoxypropane (Sigma-Aldrich) was used Figure 1. Proposed scheme on how inflammatory processes, triggered by O. viverrini infection of the bile duct cause oxidative stress and etheno-dna adduct formation, that act as driving force to cholangiocarcinoma (CCA). During chronic inflammation, reactive oxygen and nitrogen species (ROS, RNS) are released that lead to activation of lipid peroxidation and production of DNA-reactive byproducts such as 2-hydroxy-4-nonenal. Nitric oxide overproduction can also induce lipid peroxidation via peroxynitrite, and etheno adducts have been detected in mouse models (49, 50). A relationship between adduct markers and disease causation is further supported by the protective effect of the antiparasitic drug praziquantel both against lipid peroxidation derived DNA damage and cholangiocarcinoma. as standard. Pooled plasma from a blood bank was used for testing within-run and between-run assay precision. A coefficient of variability of <10% was acceptable. Plasma 8-Isoprostane Determination Plasma 8-isoprostane was measured by the ELISAbased method, using a commercial kit (Cayman Chemical) according to the supplier's protocol. Statistical Analysis Statistical analysis of the data was done with the Stat- View 5.0 program (SAS Institute). The comparison between O. viverrini infected and healthy control groups was done by the Mann Whitney U-test; O. viverrini infected groups before and after treatment with praziquantel were compared by Wilcoxon's rank test; the correlations between parameters were determined by using Spearman's rank test. Results To clarify the role of oxidative stress and lipid peroxidation derived DNA damage in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma, the accumulation of two lipid peroxidation derived etheno DNA adducts (εda, εdc) in WBC was measured in 20 healthy volunteers and 50 O. viverrini infected patients. WBC circulates through the infected hepatobiliary tract and damage of DNA may occur at the site of O. viverrini infection (Fig. 1). εda and εdc were quantified in WBC DNA by our ultrasensitive and specific immunoaffinity/ 32 P-postlabeling method. 312 Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 Cancer Epidemiology, Biomarkers & Prevention

4 Etheno-DNA Adducts in WBC During Liver Fluke Infection Figure 2. A-F. Levels in WBC DNA of εda and εdc, plasma 8-isoprostane, malondialdehyde, nitrate/nitrite, and α-tocopherol in O. viverrini infected patients (n = 50, before and 2 mo after treatment with praziquantel) and untreated, healthy control subjects (n = 20). P values were obtained from comparisons of O. viverrini infected patients before and after treatment versus controls (Mann Whitney U test). Levels of WBC εda (A), WBC εdc (B), plasma 8-isoprostane (C), plasma malondialdehyde (D), plasma nitrate/nitrite (E), and plasma α-tocopherol (F). Cancer Epidemiol Biomarkers Prev; 19(1) January

5 Dechakhamphu et al. Figure 2A and B shows box-plots and mean levels of εda and εdc in WBC DNA of O. viverrini infected patients as compared with healthy subjects. The means of εda and εdc levels (expressed as εda/10 8 da and εdc/ 10 8 dc, respectively) were significantly three to five times higher in O. viverrini infected patients than in healthy controls (εda, 5.66 ± 0.89 versus 1.81 ± 0.43; P =0.0002; εdc, ± 7.61 versus 5.40 ± 1.02; P = ). Several inflammatory indicators were also measured in O. viverrini infected patients and compared with those found in healthy volunteers. Figure 2C to E shows box-plots and mean levels of plasma 8-isoprostane, malondialdehyde, and nitrate/nitrite in patients versus healthysubjects.similarlytoεda and εdc levels in WBC, all above inflammatory indicators in plasma were concomitantly increased by O. viverrini infection: 8-isoprostane levels (pg/ml), ± 4.35 versus ± 4.25 (P < ); malondialdehyde (μmol/l), 0.66 ± 0.22 versus 0.58 ± 0.15 (not significant, P = 0.45); and nitrate/nitrite (μmol/l), ± 2.24 versus ± 1.33 (P = ). Plasma C-reactive protein levels did not differ between O. viverrini infected (1.33 ± 0.24 μg/dl) and healthy subjects (1.42 ± 0.55 μg/dl). Figure 2F shows box-plots and mean levels of plasma α-tocopherol in O. viverrini infected patients as compared with healthy subjects. α-tocopherol levels (μg/ml) were significantly lower in O. viverrini infected patients than in healthy controls (7.62 ± 0.35 versus ± 0.50; P = ). Table 1 compares εda and εdc levels in WBC of 8- isoprostane, malondialdehyde, and nitrate/nitrite levels in plasma of O. viverrini infected patients, before and 2 months after treatment by a single dose (40 mg/kg body weight) of the antiparasitic drug praziquantel. εda and εdc levels were significantly two times lowered by O. viverrini elimination (εda, ± 0.89 adducts/10 8 da versus 3.29 ± 0.68; P < 0.02; εdc, ± 7.61 adducts/10 8 dc versus ± 3.90; P < 0.03). Remarkably, after antiparasitic drug treatment adduct levels were not statistically different from those of healthy controls (εda, 1.81 ± 0.43 adducts/10 8 da; P = 0.058; εdc, 5.40 ± 1.02 adducts/10 8 dc; P = 0.056). Praziquantel treatment significantly decreased plasma 8-isoprostane (59.60 ± 4.35 versus ± 4.43 pg/ml; P = 0.01) but its concentration still remained higher than in healthy controls (23.70 ± 4.25; Table 1. Range, median, and mean levels of etheno adducts in WBC DNA and of inflammatory indicators in plasma of O. viverrini infected patients, before and 2 mo after treatment with praziquantel End point (unit) Before treatment After treatment P Adduct (εda/10 8 da) Range Median Mean ± SE 5.66 ± ± 0.68 Adduct (εdc/10 8 dc) Range Median Mean ± SE ± ± isoprostane (pg/ml) Range Median Mean ± SE ± ± 4.43 MDA (μmol/l) Range < Median Mean ± SE 0.66 ± ± 0.02 Nitrate/nitrite (μmol/l) Range Median Mean ± SE ± ± 1.20 α-tocopherol (μg/ml) Range Median Mean ± SE 7.61 ± ± 0.31 NOTE: P values were derived by the Wilcoxon's rank test. Abbreviation: MDA, malondialdehyde. 314 Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 Cancer Epidemiology, Biomarkers & Prevention

6 Etheno-DNA Adducts in WBC During Liver Fluke Infection Table 2. Correlations among measured end points End point εda εdc α-tocopherol 8-isoprostane MDA Nitrate/nitrite ALP εda εdc (<0.001) α-tocopherol (0.105) (0.056) 8-isoprostane (0.032) (0.048) (0.058) MDA (0.048) (0.047) (<0.001) (0.047) Nitrate/nitrite (0.008) (0.150) (0.032) (0.001) (0.049) ALP (0.009) (0.001) (0.036) (0.003) (0.065) (0.003) NOTE: Data from healthy controls and O. viverrini infected patients, before and 2 mo after treatment with praziquantel were pooled. Spearman correlation coefficients and P values (in parenthesis) from a total 120 analyzed samples are listed. P < ). Praziquantel treatment of O. viverrini infected patients significantly decreased plasma levels of malondialdehyde (0.66 ± 0.04 versus 0.45 ± 0.02 μmol/l; P < ) and of plasma nitrate/nitrite (26.56 ± 2.24 versus ± 1.20 μmol/l; P = 0.001). Plasma malondialdehyde (P = ) remained at a lower level than in controls. Treatment of patients by praziquantel led only to a marginal increase in plasma α-tocopherol (7.61 ± 0.35 versus 7.27 ± 0.31 μg/ml; P = 0.06) being still significantly lower than in healthy controls (10.12 ± 0.50; P < ). Table 2 lists the correlations among parameters measured in plasma and WBC DNA before and after treatment of O. viverrini infected patients with praziquantel. To increase statistical power data were combined with those from untreated healthy controls. Plasma ALP activity has already been reported in our previous study (15) but data were included for correlation analyses. There was a significant positive correlation between εda and εdc levels in WBC (r = 0.631; P < ). εda and εdc levels in WBC were positively and significantly correlated with 8-isoprostane, malondialdehyde, and nitrate/nitrite levels (except εdc) and with ALP activity in plasma. Positive and significant correlations were found for (a) 8-isoprostane versus malondialdehyde, versus nitrate/nitrite levels, and versus ALP activity; (b) malondialdehyde levels versus plasma nitrate/nitrite levels and versus ALP activity; and (c) nitrate/nitrite levels versus ALP activity. Plasma α-tocopherol levels were inversely and significantly correlated with 8-isoprostane and nitrate/ nitrite levels and with ALP activity. Inverse correlations of plasma α-tocopherol levels with WBC εda and with WBC εdc levels were observed but did not reach statistical significance. For all investigated parameters, neither gender- nor age-related differences were apparent; egg counts in feces and plasma C-reactive protein were also not correlated with any parameters analyzed (data not shown). The data on alcohol consumption and smoking behavior were not available in this study. Comparing data with our previous study (15), there was a significant, positive correlation between plasma malondialdehyde and urinary malondialdehyde levels (r = 0.842; P < 0.001). The plasma nitrate/nitrite levels significantly correlated with urinary levels of nitrate/ nitrite (r = 0.702; P < 0.001). The WBC εdc levels correlated significantly but negatively with urinary εdc levels (r = 0.248; P = 0.04). There was a positive correlation between the levels of WBC εda and urinary εda levels, but this did not reach statistical significance (r = 0.118; P = 0.17). Discussion Both epidemiologic and experimental evidence implicate liver fluke (O. viverrini) infection, which is endemic in the region of this study, as a major carcinogenic risk factor for cholangiocarcinoma (relative risk 5; refs. 26, 27). The main histopathologic features of opisthorchiasis both in man and in animal models are inflammatory processes causing persistent oxidative/nitrative stress and excess lipid peroxidation (28-30). We have reported a high excretion of two lipid peroxidation derived etheno DNA adducts (εda, εdc) in the urine of O. viverrini infected Thai subjects, likely reflecting high DNA damage in affected internal tissues, i.e., the hepatobiliary tract (15). As adduct levels in urine decreased significantly after a single dose of the antiparasitic and cholangiocarcinomaprotective drug praziquantel we proposed that these adducts could be explored as noninvasive risk markers for developing opisthorchiasis-related cholangiocarcinoma. In the present study, we measured the steady-state levels of εda and εdc in DNA of WBC samples by our ultrasensitive detection method. Using this procedure, the existence of variable background levels of adducts in liver and other tissues from unexposed rodents and humans could be unambiguously and quantitatively revealed (31). This background likely reflects the physiologic level of lipid peroxidation caused DNA damage from normal endogenous processes. Although our method is time consuming and labor intensive, its high sensitivity and specificity allows to reliably determine any disease-related increase in adduct levels in human WBC and tissues, requiring only a few micrograms of DNA sample for biomonitoring. WBC from healthy volunteers and O. viverrini infested patients before and after treatment by the Cancer Epidemiol Biomarkers Prev; 19(1) January

7 Dechakhamphu et al. antiparasitic drug praziquantel were analyzed. We hypothesized that when WBC circulate through the infected hepatobiliary tract, DNA damage may occur at the site of O. viverrini infection (Fig. 1). Our results indeed showed a significant increase of etheno adduct levels in WBC of O. viverrini infected patients when compared with healthy, noninfected subjects. Together with the observed significant adduct reduction after treatment with the antiparasitic drug praziquantel, our results provide strong support that O. viverrini related infection and inflammation are key events for causing lipid peroxidation derived DNA damage in vivo, which may play an important role in opisthorchiasis-associated cholangiocarcinoma. This assumption is further supported by previous results showing that O. viverrini infection in hamsters also induced other types of DNA damage, i.e., 8-nitroguanine and 8-oxo-deoxyguanosine in inflammatory and bile duct epithelial cells (32). In patients recruited from the same area as in this study, urinary levels of 8-oxodG, another oxidative damage marker were found to be significantly higher in cholangiocarcinoma patients than in O. viverrini infected and healthy subjects, and higher in O. viverrini infected subjects than in healthy subjects. Urinary 8-oxodG levels were significantly correlated with 8-oxodG levels in leukocyte DNA (33). Other results in cholangiocarcinoma patients suggested that 8- nitroguanine and 8-oxo-deoxyguanosine formation and reciprocal activation of the hypoxia-inducible factor-1α likely contribute to multiple genetic changes, tumor progression, and invasiveness of cholangiocarcinoma (34). The antiparasitic and cholangiocarcinoma-protective drug praziquantel prevents O. viverrini induced cholangiocarcinoma not only by elimination of parasites but also by its anti-inflammatory effect as an inhibitor of inducible nitric oxide synthase-dependent DNA damage (35). From our study in O. viverrini infected patients we also must assume that the reduction by praziquantel of lipid peroxidation derived etheno- DNA adducts in WBC and of the inflammatory indicators in plasma, 8-isoprostane, malondialdehyde, and nitrate/nitrite is caused by its suppression of oxidative/nitrative stress, acting together with its known antiparasitic effectiveness (Fig. 1). Three decades ago we were the first to show that praziquantel is devoid of genotoxic activity, unlike other antiparasitic drugs such as hycanthone in use at that time (36). In WBC of O. viverrini infected patients, we found a positive correlation between the levels of εda and εdc, but in all samples εdc levels were 5- to 15-fold higher than those of εda. Alkyl-N-purine-DNA glycosylase (ANPG) and AlkB protein remove εda, the mismatch-specific and AlkB protein remove εdc. Previous studies showed that εda adducts in cellular DNA in vitro are repaired more quickly by base excision repair than are εdc adducts (37-39). A third repair pathway by epoxidation of the etheno bond by the repair AlkB proteins also occurs at a faster rate for εda than εdc (40, 41). Another explanation for the higher εdc levels is provided by the hijacking of the human ANPG by εdc lesions when present in DNA (42). As a consequence ANPG that normally repairs εda, binds to εdc, suggesting that εdc cannot be rapidly repaired by thymidine-dna glycosylase and tends to accumulate. Several mechanisms by which O. viverrini infection enhances the pathogenesis of cholangiocarcinoma have been proposed by Sripa and Pirojkul (28). Moreover, immunopathologic processes and mechanical injury are thought to mediate hepatobiliary damage in opisthorchiasis (43). Our results in infected patients showing strong positive associations of WBC εda and εdc levels with plasma ALP activity are in support of the above proposed mechanism. We conclude that inflammationinduced lipid peroxidation and ensuing DNA damage together with mechanical injury in target hepatobiliary cells act as a driving force to malignancy. Using etheno-dna adducts as markers it should be possible to identify effective chemopreventive agents that protect against lipid peroxidation associated damage. We focused on α-tocopherol as dietary vitamin E intake was already found to lower etheno-dna adduct levels in WBC of healthy female volunteers (18). Also in a human hepatocellular carcinoma cell line α-tocopherol conferred protection against lipid peroxidation and oxidative DNA damage induced by ionizing radiation (44). Adequate intake of vitamin E and protein prevented an increase of oxidative damage to DNA, lipids, and protein after total body irradiation of mice (45). In our study, mean plasma α-tocopherol levels were significantly lower in O. viverrini infected patients than in healthy controls. α-tocopherol levels were inversely and significantly correlated with plasma 8-isoprostane and nitrate/nitrite levels and with ALP activity. Inverse correlations of plasma α-tocopherol levels with εda and εdc levels in WBC were observed, but did not reach statistical significance. Our results provide some support for α- tocopherol as a protective factor, and dietary supplementation may help to reduce lipid peroxidation induced damage in vivo and possibly also the risk for opisthorchiasis-associated cholangiocarcinoma. Among the parameters analyzed, the levels of plasma 8-isoprostane and α-tocopherol were well correlated and showed a statistical difference from healthy controls, even after praziquantel treatment. Supplementations with α-tocopherol have been shown to reduce the levels of isoprostane both in plasma and urine (46, 47). Therefore, plasma 8-isoprostane levels can be explored as a biomarker for verifying the efficacy of the supplementation. In conclusion, chronic O. viverrini infection of subjects via inflammation-induced oxidative/nitrative stress leads to accumulation of lipid peroxidation derived DNA damage in vivo, which may be lowered by dietary vitamin E supplementation. Promutagenic etheno adducts, together with other such DNA lesions in bile duct epithelial cells, most likely increase the risk of O. viverrini infected patients to later develop cholangiocarcinoma. A relationship between these adduct markers and disease causation is further supported by the protective effect of 316 Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 Cancer Epidemiology, Biomarkers & Prevention

8 Etheno-DNA Adducts in WBC During Liver Fluke Infection the antiparasitic drug praziquantel both against DNA damage (this study, refs. 33, 35) and against cholangiocarcinoma (48). Levels of εda and εdc in WBC and urine (15) should be explored (a) as putative risk markers for developing opisthorchiasis-related cholangiocarcinoma, (b) as biomarkers to identify disease protective agents, and (c) to assess the efficacy of preventive and therapeutic interventions in at risk populations. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments The authors thank all patients and volunteers who participated in this study. Grant Support Thailand Research Fund through the Royal Golden Jubilee Ph.D. Programme (to S. Dechakhamphu and P. Yongvanit), the Invitation Research Grant (No. i50214) from Faculty of Medicine, Khon Kaen University. Research was in part supported by the Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Heidelberg and through a grant from Dr. F.P. Armbruster (to S. Dechakhamphu) by Immundiagnostik, Bensheim, Germany (SHeP-GMS-0001). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received 8/24/09; revised 10/30/09; accepted 10/30/09; published online 1/7/10. References 1. Bartsch H, Nair J. Accumulation of lipid peroxidation-derived DNA lesions: potential lead markers for chemoprevention of inflammation-driven malignancies. Mutat Res 2005;591: Nair U, Bartsch H, Nair J. Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans. Free Radic Biol Med 2007;43: Cerutti PA, Trump BF. 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10 Lipid Peroxidation and Etheno DNA Adducts in White Blood Cells of Liver Fluke-Infected Patients: Protection by Plasma -Tocopherol and Praziquantel Somkid Dechakhamphu, Somchai Pinlaor, Paiboon Sitthithaworn, et al. α Cancer Epidemiol Biomarkers Prev 2010;19: Updated version Access the most recent version of this article at: Cited articles Citing articles This article cites 49 articles, 10 of which you can access for free at: This article has been cited by 1 HighWire-hosted articles. Access the articles at: alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.