Chronic Ethanol Consumption and Generation of Etheno-DNA Adducts in Cancer-Prone Tissues.

Chronic ethanol consumption is a risk factor for several human cancers. A variety of mechanisms may contribute to this carcinogenic effect of alcohol including oxidative stress with the generation of reactive oxygen species (ROS), formed via inflammatory pathways or as byproducts of ethanol oxidation through cytochrome P4502E1 (CYP2E1). ROS may lead to lipidperoxidation (LPO) resulting in LPO-products such as 4-hydoxynonenal (4-HNE) or malondialdehyde. These compounds can react with DNA bases forming mutagenic and carcinogenic etheno-DNA adducts. Etheno-DNA adducts are generated in the liver (HepG2) cells over-expressing CYP2E1 when incubated with ethanol;and are inhibited by chlormethiazole. In liver biopsies etheno-DNA adducts correlated significantly with CYP2E1. Such a correlation was also found in the esophageal- and colorectal mucosa of alcoholics. Etheno-DNA adducts also increased in liver biopsies from patients with non alcoholic steatohepatitis (NASH). In various animal models with fatty liver either induced by high fat diets or genetically modified such as in the obese Zucker rat, CYP2E1 is induced and paralleled by high levels of etheno DNA-adducts which may be modified by additional alcohol administration. As elevation of adduct levels in NASH children were already detected at a young age, these lesions may contribute to hepatocellular cancer development later in life. Together these data strongly implicate CYP2E1 as an important mediator for etheno-DNA adduct formation, and this detrimental DNA damage may act as a driving force for malignant disease progression.

Carcinogenic Etheno DNA Adducts in Alcoholic Liver Disease: Correlation with Cytochrome P-4502E1 and Fibrosis.

BACKGROUND: One mechanism by which alcoholic liver disease (ALD) progresses is oxidative stress and the generation of reactive oxygen species, among others due to the induction of cytochrome P-4502E1 (CYP2E1). Experimental data underline the key role of CYP2E1 because ALD could be partially prevented in rats by the administration of the specific CYP2E1 inhibitor chlormethiazole. As CYP2E1 is linked to the formation of carcinogenic etheno DNA adducts in ALD patients, a causal role of alcohol-induced CYP2E1 in hepatocarcinogenesis is implicated. The purpose of this study was to investigate CYP2E1 induction in ALD, and its correlation with oxidative DNA lesions and with hepatic histology. METHODS: Hepatic biopsies from 97 patients diagnosed with ALD were histologically scored for steatosis, inflammation, and fibrosis. CYP2E1 and the exocyclic etheno DNA adduct 1,N6 -etheno-2'deoxyadenosine (εdA) were determined immunohistochemically. In addition, in 42 patients, 8-hydroxydeoxyguanosine (8-OHdG) was also evaluated using immunohistochemistry. RESULTS: A significant positive correlation was found between CYP2E1 and εdA (p < 0.0001) as well as between CYP2E1 and 8-OHdG (p = 0.039). Both CYP2E1 (p = 0.0094) and ɛdA (p < 0.0001) also correlated significantly with the stage of hepatic fibrosis. Furthermore, a significant correlation between the fibrosis stage and the grade of lobular inflammation (p < 0.0001) was observed. However, the amount of alcohol consumed did not correlate with any of the parameters determined. CONCLUSIONS: These data suggest an important role of CYP2E1 in the generation of εdA, in the fibrotic progression of ALD, and thus in alcohol-mediated hepatocarcinogenesis. CYP2E1 may be a target in the treatment of ALD and a potential prognostic marker for disease progression.

Possible Mechanisms of Ethanol-Mediated Colorectal Carcinogenesis: The Role of Cytochrome P4502E1, Etheno-DNA Adducts, and the Anti-Apoptotic Protein Mcl-1.

BACKGROUND: Chronic alcohol consumption is a risk factor for colorectal cancer. The mechanisms by which ethanol (EtOH) exerts its carcinogenic effect on the colorectal mucosa are not clear and may include oxidative stress with the action of reactive oxygen species (ROS) generated through EtOH metabolism via cytochrome P4502E1 (CYP2E1) leading to carcinogenic etheno-DNA adducts. ROS may also induce apoptosis. However, the effect of chronic EtOH consumption on CYP2E1, etheno-DNA adducts as well as anti-apoptotic proteins in the colorectal mucosa of heavy drinkers without colorectal inflammation is still not known. METHODS: Rectal biopsies from 32 alcoholics (>60 g EtOH/d) and from 12 controls (<20 g EtOH/d) were histologically examined, and immunohistochemistry for CYP2E1 and etheno-DNA adducts was performed. Apoptosis (cleaved PARP) as well as anti-apoptotic proteins including Bcl-xL , Bcl-2, and Mcl-1 were immunohistochemically determined. RESULTS: No significant difference in mucosal CYP2E1 or etheno-DNA adducts was observed between alcoholics and control patients. However, CYP2E1 and etheno-DNA adducts correlated significantly when both groups were combined (p < 0.001). In addition, although apoptosis was found not to be significantly affected by EtOH, the anti-apoptotic protein Mcl-1, but neither Bcl-xL nor Bcl-2, was found to be significantly increased in heavy drinkers as compared to controls (p = 0.014). CONCLUSIONS: Although colorectal CYP2E1 was not found to be significantly increased in alcoholics, CYP2E1 correlated overall with the level of etheno-DNA adducts in the colorectal mucosa, which identifies CYP2E1 as an important factor in colorectal carcinogenesis. Most importantly, however, is the up-regulation of the anti-apoptotic protein Mcl-1 in heavy drinkers counteracting apoptosis and possibly stimulating cancer development.

The generation of carcinogenic etheno-DNA adducts in the liver of patients with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), in particular its more aggressive form nonalcoholic steatohepatitis (NASH) is increasingly observed as a cause of end stage liver disease and hepatocellular carcinoma (HCC). Reactive oxygen species (ROS) are an important factor in the pathogenesis of HCC. ROS can react with polyunsaturated fatty acids derived from membrane phospholipids resulting in the production of reactive aldehydes as lipid oxidation (LPO) byproducts, such as 4-hydroxynonenal (4 HNE). 4 HNE can react with DNA to form mutagenic exocyclic etheno-DNA adducts. ROS is induced by inflammatory processes, but also by induction of cytochrome P450 2E1 (CYP2E1), as seen with chronic alcohol consumption. METHODS: Immunohistochemical detection of CYP2E1, 4 HNE and hepatic exocyclic etheno-DNA adducts was performed on liver sections from 39 patients with NFLD. Spearman rank correlation was calculated to examine possible correlations. RESULTS: Exocyclic etheno-DNA adducts were detected and correlated significantly with 4 HNE, but not with CYP2E1. CONCLUSIONS: This is the first description of highly carcinogenic exocyclic etheno-DNA adducts in NAFLD patients. We could show that exocyclic etheno-DNA adducts significantly correlated with lipid peroxidation product 4 HNE, but not with CYP2E1, implying that in NAFLD ROS generation with consecutive DNA damage is rather inflammation driven through various cytokines than by induction of CYP2E1.

Detection of carcinogenic etheno-DNA adducts in children and adolescents with non-alcoholic steatohepatitis (NASH).

BACKGROUND: Carcinogenic exocyclic-DNA adducts like 1,N(6)-etheno-2'-deoxyadenosine (εdA) are formed through reactive intermediates of 4-hydroxynonenal (4-HNE) or other lipid peroxidation (LPO) products with the DNA bases A, C, methyl-C and G. High levels of hepatic etheno-DNA adducts have been detected in cancer prone liver diseases including alcoholic liver disease (ALD). In ALD εdA levels correlated significantly with cytochrome P-450 2E1 (CYP2E1) expression which is also induced in non-alcoholic steatohepatitis (NASH). We investigated the occurrence of εdA adducts in children with NASH as a DNA damage marker. METHODS: Liver biopsies from 21 children/adolescents with histologically proven NASH were analysed for hepatic fat content, inflammation, and fibrosis. εdA levels in DNA, CYP2E1-expression and protein bound 4-hydroxynonenal (HNE) were semi-quantitatively evaluated by immunohistochemistry. RESULTS: Among 21 NASH children, εdA levels in the liver were high in 3, moderate in 5, weak in 9 and not elevated in 4 patients. There was a positive correlation between CYP2E1 and protein-bound 4-HNE (r=0.60; P=0.008) and a trend for a positive relationship for CYP2E1 vs. staining intensity of εdA (r=0.45; P=0.06). Inflammatory activity and fibrosis correlated significantly (r=0.49, P=0.023). CONCLUSIONS: Our results demonstrate for the first time the presence of elevated carcinogenic etheno-DNA lesions (εdA) in the majority (17/21) of liver biopsies from young NASH patients. Our data suggest that LPO-derived etheno-adducts are implicated in NASH. Whether these adducts may serve as predictive risk markers in NASH children to develop hepatocellular cancer later in life remains to be investigated.

The role of reactive oxygen species (ROS) and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts.

Exocyclic etheno-DNA adducts are mutagenic and carcinogenic and are formed by the reaction of lipidperoxidation (LPO) products such as 4-hydoxynonenal or malondialdehyde with DNA bases. LPO products are generated either via inflammation driven oxidative stress or via the induction of cytochrome P-450 2E1 (CYP2E1). In the liver CYP2E1 is induced by various compounds including free fatty acids, acetone and ethanol. Increased levels of CYP2E1 and thus, oxidative stress are observed in the liver of patients with non-alcoholic steatohepatitis (NASH) as well as in the chronic alcoholic. In addition, chronic ethanol ingestion also increases CYP2E1 in the mucosa of the oesophagus and colon. In all these tissues CYP2E1 correlates significantly with the levels of carcinogenic etheno-DNA adducts. In contrast, in patients with non-alcoholic steatohepatitis (NASH) hepatic etheno-DNA adducts do not correlate with CYP2E1 indicating that in NASH etheno-DNA adducts formation is predominately driven by inflammation rather than by CYP2E1 induction. Since etheno-DNA adducts are strong mutagens producing various types of base pair substitution mutations as well as other types of genetic damage, it is strongly believed that they are involved in ethanol mediated carcinogenesis primarily driven by the induction of CYP2E1.

Lipid peroxidation and DNA adduct formation in lymphocytes of premenopausal women: Role of estrogen metabolites and fatty acid intake.

A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε)--DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E(2) ) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M(1) dG were quantified by immuno-enriched (32) P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M(1) dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E(2) , but not of 2-OH-E(2) , metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M(1) dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E(2) , arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E(2) instead. In conclusion, this study is the first to compare M(1) dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.

Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis.

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed.

Identification of 3,N(4)-etheno-5-methyl-2'-deoxycytidine in human DNA: a new modified nucleoside which may perturb genome methylation.

Methylation of cytidine at dCpdG sequences regulates gene expression and is altered in many chronic inflammatory diseases. Inflammation generates lipid peroxidation (LPO) products which can react with deoxycytidine, deoxyadenosine, and deoxyguanosine in DNA to form pro-mutagenic exocyclic etheno-nucleoside residues. Since 5-methyl-2'-deoxycytidine (5mdC) residues exhibit increased nucleophilicity at N3, they should be even better targets for LPO products. We synthesized and characterized 3,N(4)-etheno-5-methyl-2'-deoxycytidine-3'-phosphate and showed that LPO products can indeed form the corresponding etheno-5mdC (ε5mdC) lesion in DNA in vitro. Our newly developed (32)P-postlabeling method was subsequently used to detect ε5mdC lesions in DNA from human white blood cells, lung, and liver at concentrations 4-10 times higher than that observed for etheno adducts on nonmethylated cytidine. Our new detection method can now be used to explore the hypothesis that this DNA lesion perturbs the DNA methylation status.

Biomarkers for hazard identification in humans.

BACKGROUND: Oxidative stress enhances lipid peroxidation (LPO), which both are implicated in the promotion and progression stages of carcinogenesis, in particular under conditions of chronic inflammation and infections. Exocyclic etheno-DNA adducts, which are formed by LPO-products such as 4-hydroxy -2-nonenal, are strongly pro-mutagenic DNA lesions. METHODS: The development of ultra-sensitive detection methods for etheno-adducts in human tissues, white blood cells( WBC) and urine has provided evidence that these adducts are elevated in affected organs of cancer-prone patients, probably acting as a driving force to malignancy. RESULTS: Two recent studies that yielded some new insights into disease causation are briefly reviewed: DNA-damage in WBC of mother-newborn child pairs, and lipid peroxidation derived DNA damage in patients with cancer-prone liver diseases. Our results indicate that biomonitoring of etheno-DNA adducts in humans are promising tools (i) to better understand disease aetiopathogenesis, allowing hazard identification(ii) to monitor disease progression and (iii) to verify the efficacy of chemopreventive and therapeutic interventions. Such clinical trials are warranted.

Ethanol-mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA-lesions.

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno-DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15-600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno-DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno-DNA adducts 1,N(6)-ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N(4)-ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno-DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.

Quantifying etheno-DNA adducts in human tissues, white blood cells, and urine by ultrasensitive (32)P-postlabeling and immunohistochemistry.

Exocyclic etheno-DNA adducts are formed by the reaction of lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) with DNA bases to yield 1,N (6)-etheno-2'-deoxyadenosine (εdA), 3,-N (4)-etheno-2'-deoxycytidine (εdC), and etheno-2'-deoxyguanosine. These adducts act as a driving force for many human malignancies and are elevated in the organs of cancer-prone patients suffering from chronic inflammation and infections. Here, we describe the ultrasensitive and specific techniques for the detection of εdA and εdC in tissue and white blood cell (WBC) DNA. This approach is based on -combined immunopurification by monoclonal antibodies and (32)P-postlabeling analysis. The detection limit is about five adducts per 10(10) parent nucleotides, requiring 5-10 µg of DNA. In addition, we describe techniques for immunohistochemical detection of εdA and εdC in tissue biopsies, and the approaches for the -analysis of εdA and εdC excreted in urine. The utility of these detection methods for human studies is based on: (1) high sensitivity and specificity, (2) low amounts of DNA required, (3) capability to detect "background" levels of etheno-DNA adducts in biopsies, WBC, and urine samples of healthy subjects, and (4) reliable monitoring of the disease-related increase of these substances in patients.The described methods are useful in diagnosis and monitoring of chronic degenerative diseases, including cancer, atherosclerosis, and neurodegenerative disorders.

Polymorphisms in the apoptotic pathway gene BCL-2 and survival in small cell lung cancer.

INTRODUCTION: We investigated the single-nucleotide polymorphism C-938A in the apoptotic gene BCL-2 to assess the potential impact as a genetic marker for response to chemotherapy and outcome prediction in small cell lung cancer (SCLC) patients. Such a marker might help optimize lung cancer treatment in a tailored approach. METHODS: DNA derived from peripheral blood lymphocytes of 188 Caucasian SCLC patients treated at the Thoraxklinik Heidelberg was genotyped. Chemotherapy response, time to progression (TTP), and overall survival (OS) were evaluated using multivariable regression (unconditional logistic for response and Cox proportional hazard for TTP and OS) with odds ratios and hazard ratios (HRs) and their 95% confidence intervals (CIs) as quantitative outcome measures, respectively. RESULTS: Small cell lung cancer patients carrying the BCL-2 -938CC genotype showed significantly worse TTP than patients carrying the BCL-2 -938AA genotype (HR = 1.86; 95% CI = 1.10-3.13, p = 0.021). The same adverse effect was shown for OS (HR = 2.38; 95% CI = 1.38-4.12, p = 0.002). Also, patients with limited disease (HR = 2.57; 95% CI = 1.18-5.60, p = 0.017) showed worse OS with the BCL-2 -938CC genotype. CONCLUSION: BCL-2 -938CC genotype shows significantly worse outcome in small cell lung cancer patients. This genetic marker might particularly impact on treatment strategies using BCL-2 antisense approaches.

Aberrant repair of etheno-DNA adducts in leukocytes and colon tissue of colon cancer patients.

To assess the role of lipid peroxidation-induced DNA damage and repair in colon carcinogenesis, the excision rates and levels of 1,N(6)-etheno-2'-deoxyadenosine (epsilondA), 3,N(4)-etheno-2'-deoxycytidine (epsilondC), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondG) were analyzed in polymorphic blood leukocytes (PBL) and resected colon tissues of 54 colorectal carcinoma (CRC) patients and PBL of 56 healthy individuals. In PBL the excision rates of 1,N(6)-ethenoadenine (epsilonAde) and 3,N(4)-ethenocytosine (epsilonCyt), measured by the nicking of oligodeoxynucleotide duplexes with single lesions, and unexpectedly also the levels of epsilondA and 1,N(2)-epsilondG, measured by LC/MS/MS, were lower in CRC patients than in controls. In contrast the mRNA levels of repair enzymes, alkylpurine- and thymine-DNA glycosylases and abasic site endonuclease (APE1), were higher in PBL of CRC patients than in those of controls, as measured by QPCR. In the target colon tissues epsilonAde and epsilonCyt excision rates were higher, whereas the epsilondA and epsilondC levels in DNA, measured by (32)P-postlabeling, were lower in tumor than in adjacent colon tissue, although a higher mRNA level was observed only for APE1. This suggests that during the onset of carcinogenesis, etheno adduct repair in the colon seems to be under a complex transcriptional and posttranscriptional control, whereby deregulation may act as a driving force for malignancy.

Accumulation of miscoding etheno-DNA adducts and highly expressed DNA repair during liver fluke-induced cholangiocarcinogenesis in hamsters.

Infection by Opisthorchis viverrini, a risk factor for cholangiocarcinoma (CCA) may act through chronic inflammation, oxidative stress and lipid peroxidation (LPO)-related damage and growth stimuli. 1,N6-etheno-2'-deoxyadenosine (epsilondA), and 3,N4-etheno-2'-deoxycytidine (epsilondC), markers for LPO-derived DNA damage were highly increased in white blood cell and urine of O. viverrini-infected Thai patients. In order to investigate tissue specificity etheno adducts were measured in a cholangiocarcinogenesis model, in O. viverrini-infected hamsters that had received N-nitrosodimethylamine (NDMA, 12.5 ppm in dw) for 2 months. epsilondA- and epsilondC-levels were analyzed in paraffin-embedded liver sections by a novel immunohistochemical method, from 21 up to 180 days post-O. viverrini-infection. In inflamed areas of the liver, etheno adducts were localized in the nuclei of inflammatory cells and in the epithelial lining of the bile duct. Semi-quantitative image analysis showed higher adduct levels in the liver of O. viverrini-infected hamsters, treated with or w/o NDMA when compared with untreated controls. Levels were found highest in the liver of O. viverrini-infected plus NDMA-treated hamsters. Adducts increased in an age-dependent manner from O. viverrini-infection until CCA development. Increased adduct formation paralleled histopathological changes in plasma alkaline phosphatase (ALP) activity, bile duct hyperplasia, dysplasia, precancerous lesions, and CCA appearance. Also elevated expression of alkyladenine DNA glycosylase (AAG), which excises 1,N6-ethenoadenine (epsilonA) was linked to higher adduct formation, suggesting imbalanced repair. Our results implicate accumulation of inflammation-related, promutagenic DNA damage in target tissue and possibly imbalanced repair in the onset of cholangiocarcinogenesis.

Evaluation of the polyphenol content and antioxidant properties of methanol extracts of the leaves, stem, and root barks of Moringa oleifera Lam.

Medicinal plants have been shown to have both chemopreventive and/or therapeutic effects on cancer and other diseases related to oxidative damage. Moringa oleifera Lam., known in the Hausa and Igala languages of Nigeria as "Zogale" and "Gergedi," respectively, and drumstick in English, is a plant that is used both as food and in folkloric medicine in Nigeria and elsewhere. Different parts of the plant were analyzed for polyphenol content as well as in vitro antioxidant potential. The methanol extract of the leaves of M. oleifera contained chlorogenic acid, rutin, quercetin glucoside, and kaempferol rhamnoglucoside, whereas in the root and stem barks, several procyanidin peaks were detected. With the xanthine oxidase model system, all the extracts exhibited strong in vitro antioxidant activity, with 50% inhibitory concentration (IC(50)) values of 16, 30, and 38 microL for the roots, leaves, and stem bark, respectively. Similarly, potent radical scavenging capacity was observed when extracts were evaluated with the 2-deoxyguanosine assay model system, with IC(50) values of 40, 58, and 72 microL for methanol extracts of the leaves, stem, and root barks, respectively. The high antioxidant/radical scavenging effects observed for different parts of M. oleifera appear to provide justification for their widespread therapeutic use in traditional medicine in different continents. The possibility that this high antioxidant/radical scavenging capacity may impact on the cancer chemopreventive potential of the plant must be considered.

Genetic polymorphisms of MPO, GSTT1, GSTM1, GSTP1, EPHX1 and NQO1 as risk factors of early-onset lung cancer.

Early-onset lung cancer diagnosed up to the age of 50 is a very rare disease, with an increasing incidence rate. Differences in aetiology, characteristics and epidemiology of early and older onset lung cancer have been described previously, suggesting the importance of genetic factors in early-onset lung cancer aetiology. A case-control study was conducted to investigate the effects of genetic polymorphisms in the MPO, EPHX1, GSTT1, GSTM1, GSTP1 and NQO1 genes on the risk of early-onset lung cancer development. Six hundred thirty-eight Caucasian patients under the age of 51 with confirmed primary lung cancer and 1,300 cancer free control individuals, matched by age and sex, were included in this analysis. Seventeen single nucleotide polymorphisms and two deletion polymorphisms were genotyped. No significant association was found for any of the analyzed polymorphisms and overall lung cancer risk. Nonsignificantly decreased risk of lung cancer was observed for carriers of 1 or 2 copies of GSTM1. Subgroup analysis revealed gender- and/or smoking-specific effects of EPHX1 rs2854455 (IV-1464C > T) and rs2234922 (His139Arg), GSTT1 deletion, GSTP1 rs1695 (Ile105Val), rs947895 (+991C > A) and rs4891 (Ser185Ser) and NQO1 rs1800566 (Pro187Ser) polymorphisms. However, none of the observed effects were confirmed by interaction tests nor were they significant after Bonferroni correction for multiple testing. In summary, our study suggested a modifying effect of polymorphisms in EPHX1, GSTP1, GSTT1, GSTM1 and NQO1 genes on the risk of early-onset lung cancer. To confirm these observations and to eliminate possible bias in our analyses, larger studies are warranted.

Lipid peroxidation and etheno DNA adducts in white blood cells of liver fluke-infected patients: protection by plasma alpha-tocopherol and praziquantel.

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 (epsilon)-DNA adducts (epsilondA, epsilondC) in WBC and plasma alpha-tocopherol were measured in samples collected from O. viverrini-infected Thai patients (n = 50) and healthy noninfected volunteers (n = 20). epsilondA and epsilondC 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 alpha-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, epsilondA and epsilondC levels in WBC were decreased to control level (P < 0.03); plasma 8-isoprostane, malondialdehyde, nitrate/nitrite, and alkaline phosphatase (ALP) were concomitantly lowered. epsilondA and epsilondC levels in WBC were positively correlated with plasma 8-isoprostane, malondialdehyde, and nitrate/nitrite levels and ALP activity, whereas plasma alpha-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 alpha-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.