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1.  Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects 
Clinical Epigenetics  2012;4(1):19.
Background
Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG).
Methods
In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student’s two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount.
Results
The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate.
Conclusion
This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.
doi:10.1186/1868-7083-4-19
PMCID: PMC3579724  PMID: 23025454
MLH1; ROS; DNA methylation; Demethylation; Nutritional intervention; Diabetes; Antioxidant; Pyrosequencing
2.  In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells 
Experimental Cell Research  2013;319(4):536-545.
Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17 μM for 24 h. Addition of 300 μM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.
Highlights
► DNA-damaging effects of bile pigments have been rarely investigated. ► Thus, eight tetrapyrroles were tested for DNA-damaging effects in the comet assay. ► To assess DNA damage, cancer cells were used, and flow cytometry parameters were measured. ► Especially protoporphyrin, urobilin and stercobilin increased DNA strand breaks significantly. ► Mechanisms could include oxidative stress, cell cycle arrest and apoptosis.
doi:10.1016/j.yexcr.2012.12.003
PMCID: PMC3569715  PMID: 23246570
BP(s), bile pigment(s); BR, unconjugated bilirubin; BR-DME, Bilirubin dimethyl ester; BRf, free bilirubin; BRDT, bilirubin ditaurate; BV, biliverdin; BV-DME, biliverdin dimethyl ester; PRO, protoporphyrin; SCGE, single cell gel electrophoresis; TP(s), tetrapyrrole(s); SB, stercobilin; UB, urobilin; Stercobilin; Urobilin; Protoporphyrin; SCGE; Comet
3.  Reduced circulating oxidized LDL is associated with hypocholesterolemia and enhanced thiol status in Gilbert syndrome 
Free Radical Biology & Medicine  2012;52(10):2120-2127.
A protective association between bilirubin and atherosclerosis/ischemic heart disease clearly exists in vivo. However, the relationship between bilirubin and in vivo oxidative stress parameters in a clinical population remains poorly described. The aim of this study was to assess whether persons expressing Gilbert syndrome (GS; i.e., unconjugated hyperbilirubinemia) are protected from thiol oxidation and to determine if this, in addition to their improved lipoprotein profile, could explain reduced oxidized low-density lipoprotein (oxLDL) status in them. Forty-four matched GS and control subjects were recruited and blood was prepared for the analysis of lipid profile and multiple plasma antioxidants and measures of oxidative stress. GS subjects possessed elevated plasma reduced thiol (8.03±1.09 versus 6.75±1.39 nmol/mg protein; P<0.01) and glutathione concentrations (12.7±2.39 versus 9.44±2.45 μM; P<0.001). Oxidative stress status (reduced:oxidized glutathione; GSH:GSSG) was significantly improved in GS (0.49±0.16 versus 0.32±0.12; P<0.001). Protein carbonyl concentrations were negatively associated with bilirubin concentrations and were significantly lower in persons with >40 μM bilirubin versus controls (<17.1 μmol/L; P<0.05). Furthermore, absolute oxLDL concentrations were significantly lower in GS subjects (P<0.05). Forward stepwise regression analysis revealed that bilirubin was associated with increased GSH:GSSG ratio and reduced thiol concentrations, which, in addition to reduced circulating LDL, probably decreased oxLDL concentrations within the cohort. In addition, a marked reduction in total cholesterol concentrations in hyperbilirubinemic Gunn rats is presented (Gunn 0.57±0.09 versus control 1.69±0.40 mmol/L; P<0.001), arguing for a novel role for bilirubin in modulating lipid status in vivo. These findings implicate the physiological importance of bilirubin in protecting from atherosclerosis by reducing thiol and subsequent lipoprotein oxidation, in addition to reducing circulating LDL concentrations.
Highlights
► Bilirubin is associated with the prevention of thiol and protein oxidation in vivo. ► Gilbert syndrome subjects have higher plasma antioxidants and improved oxidative stress status versus controls. ► Elevated bilirubin is associated with improved lipid status in rodents and humans. ► Bilirubin may act via multiple pathways to prevent cardiovascular disease.
doi:10.1016/j.freeradbiomed.2012.03.002
PMCID: PMC3377994  PMID: 22521902
BMI, body mass index; CVD, cardiovascular disease; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); FRAP, ferric reducing ability of plasma; GS, Gilbert syndrome; GSH, reduced glutathione; GSSG, oxidized glutathione; HDL, high-density lipoprotein; HO-1, heme oxygenase-1; HPLC, high-performance liquid chromatography; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; TCA, trichloroacetic acid; TEAC, Trolox equivalent antioxidant capacity; UGT, uridine diphosphate glucuronosyltransferase; Cardiovascular disease; Bile pigment; Thiol; Glutathione; Bilirubin; Free radicals
4.  Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype 
Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory.
doi:10.1155/2012/701927
PMCID: PMC3312255  PMID: 22474515
5.  Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage in Workers: Results of a Multiple End Point Study 
Environmental Health Perspectives  2008;116(12):1689-1693.
Background
Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
Objective
The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory.
Methods
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Results
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
Conclusions
V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.
doi:10.1289/ehp.11438
PMCID: PMC2599764  PMID: 19079721
comet assay; cytokinesis-block micronucleus assay; DNA damage; occupational exposure; vanadium pentoxide
6.  Bilirubin and Related Tetrapyrroles Inhibit Food-Borne Mutagenesis: A Mechanism for Antigenotoxic Action against a Model Epoxide 
Journal of Natural Products  2013;76(10):1958-1965.
Bilirubin exhibits antioxidant and antimutagenic effects in vitro. Additional tetrapyrroles that are naturally abundant were tested for antigenotoxicity in Salmonella. Un-/conjugated bilirubin (1 and 2), biliverdin (4), bilirubin and biliverdin dimethyl esters (3 and 5), stercobilin (6), urobilin (7), and protoporphyrin (8) were evaluated at physiological concentrations (0.01–2 μmol/plate; 3.5–714 μM) against the metabolically activated food-borne mutagens aflatoxin B1 (9) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (10). Compound 8 most effectively inhibited the mutagenic effects of 9 in strain TA102 and 10 in TA98. Compound 7 inhibited 9-induced mutagenesis in strain TA98 most effectively, while 1 and 4 were promutagenic in this strain. This is likely due to their competition with mutagens for phase-II detoxification. Mechanistic investigations into antimutagenesis demonstrate that tetrapyrroles react efficiently with a model epoxide of 9, styrene epoxide (11), to form covalent adducts. This reaction is significantly faster than that of 11 with guanine. Hence, the evaluated tetrapyrroles inhibited genotoxicity induced by poly-/heterocyclic amines found in foods, and novel evidence obtained in the present investigation suggests this may occur via chemical scavenging of genotoxic metabolites of the mutagens investigated. This may have important ramifications for maintaining health, especially with regard to cancer prevention.
doi:10.1021/np4005807
PMCID: PMC3812704  PMID: 24156291

Results 1-7 (7)