DNA Methylation Damage Research Articles

Predicting exacerbation of renal function by DNA methylation clock and DNA damage of urinary shedding cells: a pilot study

Recent reports have shown the feasibility of measuring biological age from DNA methylation levels in blood cells from specific regions identified by machine learning, collectively known as the epigenetic clock or DNA methylation clock. While extensive research has explored the association of the DNA methylation clock with cardiovascular diseases, cancer, and Alzheimer's disease, its relationship with kidney diseases remains largely unexplored. In particular, it is unclear whether the DNA methylation clock could serve as a predictor of worsening kidney function. In this pilot study involving 20 subjects, we investigated the association between the DNA methylation clock and subsequent deterioration of renal function. Additionally, we noninvasively evaluated DNA damage in urinary shedding cells using a previously reported method to examine the correlation with the DNA methylation clock and worsening kidney function. Our findings revealed that patients with an accelerated DNA methylation clock exhibited increased DNA damage in urinary shedding cells, along with a higher rate of eGFR decline. Moreover, in cases of advanced CKD (G4-5), the DNA damage in urinary shedding cells was significantly increased, highlighting the interplay between elevated DNA damage and eGFR decline. This study suggests the potential role of the DNA methylation clock and urinary DNA damage as predictive markers for the progression of chronic kidney disease.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Label-Free and Sensitive Electrochemical Detection of DNA Methylation Damage Based on DNA Glycosylase Recognition and Excision Coupled with Exonuclease III Amplification

Because the cytotoxic DNA methylated bases are chemically inert and small in size, limited biosensing strategies especially with high sensitivity were established to detect DNA methylation damage. In this work, a label-free electrochemical assay was proposed for the sensitive detection of DNA methylation damage in dsDNA film on indium tin oxide electrode. The human alkyladenine DNA glycosylase was employed to recognize and selectively remove DNA methylated bases, generating apurinic site. Subsequently, exonuclease III not only further converted the methylation sites into strand breaks, but also progressively removed nucleotides from the 3′ to 5′ end starting from the apurinic sites. These processes could maximum amplify DNA methylation damage, also confirmed by gel electrophoresis and fluorescence measurements. The damaged DNA film bound much less electrochemical indicator, Ru(bpy)2(dppz)2+, and accompanied by an anodic current drop. As a result, DNA methylation damage produced with as low as 10 μM of methyl methanesulfonate can be detected, which was 100-fold lower than the previously reported photoelectrochemical sensing strategy. Other DNA base modification products showed much less decrease of oxidation current, demonstrating its good selectivity. This strategy can be utilized to sensitively and rapidly assess the genotoxicity of environmental pollutants.

Analysis of genome instability and implications for the consequent phenotype in Plasmodium falciparum containing mutated MSH2-1 (P513T).

Malarial parasites exhibit extensive genomic plasticity, which induces the antigen diversification and the development of antimalarial drug resistance. Only a few studies have examined the genome maintenance mechanisms of parasites. The study aimed at elucidating the impact of a mutation in a DNA mismatch repair gene on genome stability by maintaining the mutant and wild-type parasites through serial in vitro cultures for approximately 400 days and analysing the subsequent spontaneous mutations. A P513T mutant of the DNA mismatch repair protein PfMSH2-1 from Plasmodium falciparum 3D7 was created. The mutation did not influence the base substitution rate but significantly increased the insertion/deletion (indel) mutation rate in short tandem repeats (STRs) and minisatellite loci. STR mutability was affected by allele size, genomic category and certain repeat motifs. In the mutants, significant telomere healing and homologous recombination at chromosomal ends caused extensive gene loss and generation of chimeric genes, resulting in large-scale chromosomal alteration. Additionally, the mutant showed increased tolerance to N-methyl-N'-nitro-N-nitrosoguanidine, suggesting that PfMSH2-1 was involved in recognizing DNA methylation damage. This work provides valuable insights into the role of PfMSH2-1 in genome stability and demonstrates that the genomic destabilization caused by its dysfunction may lead to antigen diversification.

S-38-4: ASSOCIATION BETWEEN DNA METHYLATION AGE OR DNA DAMAGE IN URINARY SHEDDING CELLS AND EARLY CKD

Objective: Recent studies have identified that aggregate DNA methylation status of specific sites measures biological age, referred to as the DNA methylation clock. However, the relationship between kidney diseases and DNA methylation clock has been unclear. In this study, we aimed to investigate the association between DNA methylation age or DNA damage in urinary shedding cells and early CKD. Design and method: Individuals aged 45–81 years old, who visited Keio University Hospital for annual health check-up were enrolled. A total of 22 participants (14 males, 8 females) aged 61.6 ± 10.0 years old were eligible for this study. We evaluated a 71 CpG epigenetic age predictor, referred to as the Hannum's clock, which lacks 6 probes on the EPIC array using blood samples from the patient. 50 ml of urine samples were collected and centrifuged, and mRNA was extracted to analyze the expression of phenotypical markers and epigenetic modulators in urine derived cells by quantitative RT–PCR method as previously described. DNA was also extracted from urine derived cells, and DNA double strand breaks (DSBs) were evaluated by the quantitative long distance PCR method as previously described (Hishikawa, et al. Sci rep 2020). The association between biological aging (Hannum's clock age − chronological age) and clinical data was investigated. Results: Hannum's clock correlated with chronological age, and biological aging (Hannum's clock age − chronological age) was promoted in CKD patients (eGFR 43–59). DNA DSBs of the nephrin gene was also promoted in CKD patients. DNMT3 expression in urinary shedding cells correlated with biological aging. Conclusions: To our knowledge, this is the first study to demonstrate the possible association of biological aging measured by DNA methylation clock and CKD, especially in early stage. Podocyte DNA DSBs and urine DMNT3 expression may be a potential marker of biological aging.

Maternal exposure to genistein during pregnancy and oxidative DNA damage in testes of male mouse offspring.

BackgroundGenistein is a dietary supplement with phyto-estrogenic properties. Therefore, high intake of genistein during pregnancy may have adverse effects on the genetic integrity of testes and germ cells of male offspring. In this study, we examined whether maternal exposure to genistein during pregnancy induced oxidative DNA damage in the male germline at adolescence.MethodsAtm-ΔSRI mice have lower glucose-6-phosphate dehydrogenase (G6PDH) activity, which is important for maintaining levels of reduced glutathione and therefore these mice have an increased susceptibility to oxidative stress. Parental heterozygous Atm-ΔSRI mice received a genistein-rich or control diet, after which they were mated to obtain offspring. During pregnancy, mothers remained on the respective diets and after delivery all animals received control diets. Redox status and oxidative DNA damage were assessed in testes and sperm of 12 weeks old male offspring. Gene expression of Cyp1b1, Comt, and Nqo1 was assessed in testes, and DNA methylation as possible mechanism for transmission of effects to later life.ResultsIntake of genistein during pregnancy increased oxidative DNA damage in testes of offspring, especially in heterozygous Atm-ΔSRI mice. These increased DNA damage levels coincided with decreased expression of Comt and Nqo1. Heterozygous Atm-ΔSRI mice had higher levels of DNA strand breaks in sperm compared to wild type littermates, and DNA damage was further enhanced by a genistein-rich maternal diet. G6PDH activity was higher in mice with high maternal intake of genistein compared to control diets, suggesting compensation against oxidative stress. A positive correlation was observed between the levels of DNA methylation and oxidative DNA damage in testes.ConclusionThese data indicate that prenatal exposure to genistein altered gene expression and increased DNA damage in testes and sperm of adolescent male offspring. These effects of genistein on DNA damage in later life coincided with alterations in DNA methylation.

Genome-wide association and multi-omics studies identify MGMT as a novel risk gene for Alzheimer disease among women.

Increasing evidence suggests epidemiological and pathological links between Alzheimer disease (AD) and breast cancer (BC). The 17q21 locus, including MAPT encoding tau, has been identified in genome-wide association studies (GWAS) for BC in women and AD among persons lacking the APOE ε4 allele (ε4-). We hypothesized that other genes involved in tau-related pathology can be identified by GWAS in women stratified by ε4 status. We conducted a GWAS for AD among ε4- women (3,400 AD and 6,940 controls) in Alzheimer's Disease Genetics Consortium (ADGC) datasets and separately analyzed whole genome sequencing data obtained from five AD cases (all women; ε4+=4) and 26 controls (17 women, 9 men; ε4+=9) in a Hutterite kindred from South Dakota. Association tests were performed using logistic regression with covariates for age and principle components of ancestry in the ADGC datasets and for age, sex, and ε4 in the Hutterites. Potential mechanisms of top-ranked findings were investigated by integrating omics data including gene expression, DNA-methylation (DNAm), and AD-related neuropathological traits in subsets stratified by sex and ε4 status in the Religious Orders Study and Memory and Aging Project (ROSMAP; 339 AD and 229 controls). We identified genome-wide significant associations with one novel locus MGMT in each sample (ADGC: rs12775171, odds ratio [OR]=1.4, P=4.9x10-8 ; Hutterites: rs12256016, OR=2.0, P=1.9x10-14 ). Rs12775171 showed no evidence of association with AD risk in ε4+ women or in men in the ADGC datasets (P>0.05). Rs12775171 is not in linkage disequilibrium with rs12256016. At least one of these SNPs was significantly associated with increased DNAm levels near/within MGMT (best association pair: rs11016864 [proxy for rs12256016] with cg01419164; P=4.0x10-4 in women), which were also significantly associated with lower MGMT expression (P=0.03) and worse tau burden in all AD cohorts (neurofibrillary tangle; P=6.0x10-3 ), women (Braak stage; P=4.0x10-3 ), and ε4- women (neurofibrillary tangle; P=0.03), but not among ε4+ women, all men, or ε4+/ε4- men in ROSMAP. We identified a novel association of AD with the interaction of sex and MGMT. Our omics analysis confirmed that epigenetically regulated expression of MGMT (involved in DNA methylation damage repair) is significantly associated with tau in women.

UHRF1 regulates alternative splicing by interacting with splicing factors and U snRNAs in a H3R2me involved manner.

The well-established functions of UHRF1 converge to DNA biological processes, as exemplified by DNA methylation maintenance and DNA damage repair during cell cycles. However, the potential effect of UHRF1 on RNA metabolism is largely unexplored. Here, we revealed that UHRF1 serves as a novel alternative RNA splicing regulator. The protein interactome of UHRF1 identified various splicing factors. Among them, SF3B3 could interact with UHRF1 directly and participate in UHRF1-regulated alternative splicing events. Furthermore, we interrogated the RNA interactome of UHRF1, and surprisingly, we identified U snRNAs, the canonical spliceosome components, in the purified UHRF1 complex. Unexpectedly, we found H3R2 methylation status determines the binding preference of U snRNAs, especially U2 snRNAs. The involvement of U snRNAs in UHRF1-containing complex and their binding preference to specific chromatin configuration imply a finely orchestrated mechanism at play. Our results provided the resources and pinpointed the molecular basis of UHRF1-mediated alternative RNA splicing, which will help us better our understanding of the physiological and pathological roles of UHRF1 in disease development.

Potential Environmental Health Risk Analysis of Neonicotinoids and a Synergist.

The extensive use of neonicotinoid pesticides has led to their widespread presence in the environment, resulting in considerable safety risks to the ecosystem and human health. In this study, we investigated the biotransformation behavior of a cocktail of multiple neonicotinoids and piperonyl butoxide (PBO) synergist in vivo and their potential environmental health risk. It was found that neonicotinoids with a cyano group, such as acetamiprid and thiacloprid, tended to accumulate in liver and spleen tissues, while others with nitro groups (imidacloprid, thiamethoxam, clothianidin, dinotefuran, and nitenpyram) were mostly excreted in urine. In the presence of the synergist PBO, the metabolism of neonicotinoids in vivo changed, mainly through the nitro reduction pathway, while a low abundance of related metabolites was observed in the conventional hydroxylation and demethylation metabolic pathways, due to inhibition of CYP450 enzymes by the synergist. Furthermore, DNA methylation damage in vivo was exacerbated by the induction of hydroxylamine metabolites formed in the intermediate process of neonicotinoid metabolism with the synergistic effect of PBO, which resulted in a higher level of the O6-methyldeoxyguanosine (O6-medG) biomarker in the liver. Therefore, during the comprehensive evaluation of pesticide environmental risks, attention should be paid not only to the co-exposure mode under real environmental conditions but also to the potential risks of intermediate metabolism and related intermediate metabolites. This study provides a referential strategy and theoretical support for the health risk assessment of co-exposure of chemicals.

Human ALKBH6 Is Required for Maintenance of Genomic Stability and Promoting Cell Survival During Exposure of Alkylating Agents in Pancreatic Cancer.

Alpha-ketoglutarate-dependent dioxygenase (ALKBH) is a DNA repair gene involved in the repair of alkylating DNA damage. There are nine types of ALKBH (ALKBH1-8 and FTO) identified in humans. In particular, certain types of ALKBH enzymes are dioxygenases that directly reverse DNA methylation damage via transfer of a methyl group from the DNA adduct onto α-ketoglutarate and release of metabolic products including succinate and formaldehyde. Here, we tested whether ALKBH6 plays a significant role in preventing alkylating DNA damage and decreasing genomic instability in pancreatic cancer cells. Using an E. coli strain deficient with ALKB, we found that ALKBH6 complements ALKB deficiency and increases resistance after alkylating agent treatment. In particular, the loss of ALKBH6 in human pancreatic cancer cells increases alkylating agent-induced DNA damage and significantly decreases cell survival. Furthermore, in silico analysis from The Cancer Genome Atlas (TCGA) database suggests that overexpression of ALKBH6 provides better survival outcomes in patients with pancreatic cancer. Overall, our data suggest that ALKBH6 is required to maintain the integrity of the genome and promote cell survival of pancreatic cancer cells.

Protective effects of Corchorus olitorius and Butea monosperma against Arsenic induced aberrant methylation and mitochondrial DNA damage in wistar rat model.

Millions of people around the world are chronically exposed to Arsenic (As) through food and drinking water. Studies revealed that Arsenic is genotoxic and causes damage to DNA. In this study, we evaluated Corchorus olitorius and Butea monosperma for their alleviative properties against Arsenic induced genotoxicity in vivo using Wistar Rat model. Arsenic exposed rats were given C. olitorius leaf powder and B. monosperma flower powder as supplementation with normal food. Methylation status of p53 promoter was measured using Methylation Sensitive Restriction Endonuclease PCR (MSRE-PCR) assay and mitochondrial DNA (mtDNA) copy number as well as occurrence of a common deletion in mtDNA in liver and kidney tissue was determined through quantitative realtime PCR (qPCR). Arsenic exposed rats after supplementation showed relatively less severe effects of toxicity evident by significantly higher amount of (p<0.05) mtDNA copy number and reduced occurrence of deletion containing mtDNA as well as lower levels of methylation in p53 gene promoter. Histopathological analysis revealed less severe histopathological changes of liver and kidney and normal liver and kidney function parameters in supplemented rats. So, the protective properties of B. monosperma and C. olitorius against Arsenic toxicity is evident in molecular level.

PARP1-DNMT1-CTCF complex and the apoptotic-induced factor mRNA expressions in workers occupationally exposed to benzene.

Exposure to benzene is a common occupational hazard as well as a hematopoietic system intoxicant, but the entire picture of its molecular pathogenesis is still hazy. Its leukemogenic effect could be attributed to DNA damage, decreased repair capacity, altered methylation patterns, and defective apoptosis. Poly ADP-ribose polymerase1, DNA methyltransferase1, and CCCTC-binding factor (PARP1-DNMT1-CTCF) complex play an essential role in methylation maintenance and DNA damage repair response. This study aimed to assess the expression of PARP1, PAR glycohydrolases (PARG), DNMT1, CTCF, and apoptosis-inducing factor (AIF) in subjects occupationally exposed to benzene. A total of 200 subjects were enrolled in this study: 100 workers occupationally exposed to benzene (painters and decorators) and 100 unexposed office workers. Occupational exposure data were obtained. The biochemical and hematological evaluations were done. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess mRNA expression of PARP1, PARG, DNMT1, CTCF, and AIF. Both biochemical and hematological parameters were within normal limits; workplace benzene air concentration was significantly higher in exposed workers than the levels among controls (P < 0.001). Significant decrease in mRNA levels of PARP1, DNMT1, CTCF, and AIF was noticed among the exposed group (P = 0.01, P < 0.001, P = 0.004, P < 0.001, respectively) in comparison with the control group, while PARG showed non-significant difference (P = 0.16). There was a significant negative correlation between workplace benzene air concentration and expression levels of PARP1, DNMT1, and AIF. The reduced expression of PARP1, DNMT1, CTCF, and AIF observed in exposed workers may represent one of the first benzene-induced changes that might threaten erythropoiesis.

A prospective evaluation of serum methionine-related metabolites in relation to pancreatic cancer risk in two prospective cohort studies.

Deficiencies in methyl donor status may render DNA methylation changes and DNA damage, leading to carcinogenesis. Epidemiological studies reported that higher dietary intake of choline is associated with lower risk of pancreatic cancer, but no study has examined the association of serum choline and its metabolites with risk of pancreatic cancer. Two parallel case-control studies, one nested within the Shanghai Cohort Study (129 cases and 258 controls) and the other within the Singapore Chinese Health Study (58 cases and 104 controls), were conducted to evaluate the associations of baseline serum concentrations of choline, betaine, methionine, total methyl donors (i.e., sum of choline, betaine and methionine), dimethylglycine and trimethylamine N-oxide (TMAO) with pancreatic cancer risk. In the Shanghai cohort, odds ratios and 95% confidence intervals of pancreatic cancer for the highest quartile of choline, betaine, methionine, total methyl donors and TMAO were 0.27 (0.11-0.69), 0.57 (0.31-1.05), 0.50 (0.26-0.96), 0.37 (0.19-0.73) and 2.81 (1.37-5.76), respectively, compared to the lowest quartile. The corresponding figures in the Singapore cohort were 0.85 (0.23-3.17), 0.50 (0.17-1.45), 0.17 (0.04-0.68), 0.33 (0.10-1.16) and 1.42 (0.50-4.04). The inverse associations of methionine and total methyl donors including choline, betaine and methionine with pancreatic cancer risk in both cohorts support that DNA repair and methylation play an important role against the development of pancreatic cancer. In the Shanghai cohort, TMAO, a gut microbiota-derived metabolite of dietary phosphatidylcholine, may contribute to higher risk of pancreatic cancer, suggesting a modifying role of gut microbiota in the dietary choline-pancreatic cancer risk association.

The Beacon Calculus: A formal method for the flexible and concise modelling of biological systems.

Biological systems are made up of components that change their actions (and interactions) over time and coordinate with other components nearby. Together with a large state space, the complexity of this behaviour can make it difficult to create concise mathematical models that can be easily extended or modified. This paper introduces the Beacon Calculus, a process algebra designed to simplify the task of modelling interacting biological components. Its breadth is demonstrated by creating models of DNA replication dynamics, the gene expression dynamics in response to DNA methylation damage, and a multisite phosphorylation switch. The flexibility of these models is shown by adapting the DNA replication model to further include two topics of interest from the literature: cooperative origin firing and replication fork barriers. The Beacon Calculus is supported with the open-source simulator bcs (https://github.com/MBoemo/bcs.git) to allow users to develop and simulate their own models.

The Molecular Genetics of Myeloproliferative Neoplasms.

Activated JAK-STAT signaling is central to the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) and occurs as a result of MPN phenotypic driver mutations in JAK2, CALR, or MPL The spectrum of concomitant somatic mutations in other genes has now largely been defined in MPNs. With the integration of targeted next-generation sequencing (NGS) panels into clinical practice, the clinical significance of concomitant mutations in MPNs has become clearer. In this review, we describe the consequences of concomitant mutations in the most frequently mutated classes of genes in MPNs: (1) DNA methylation pathways, (2) chromatin modification, (3) RNA splicing, (4) signaling pathways, (5) transcription factors, and (6) DNA damage response/stress signaling. The increased use of molecular genetics for early risk stratification of patients brings the possibility of earlier intervention to prevent disease progression in MPNs. However, additional studies are required to decipher underlying molecular mechanisms and effectively target them.

CYP1A1 methylation mediates the effect of smoking and occupational polycyclic aromatic hydrocarbons co-exposure on oxidative DNA damage among Chinese coke-oven workers

BackgroundMultiple factors, including co-exposure between lifestyle and environmental risks, are important in susceptibility to oxidative DNA damage. However, the underlying mechanism is not fully understood. This study was undertaken to evaluate whether Cytochrome P4501A1 (CYP1A1) methylation can mediate the co-exposure effect between smoking and occupational polycyclic aromatic hydrocarbons (PAH) in development of oxidative DNA damage.MethodsWe explored the associations between smoking and occupational PAH co-exposure effect, CYP1A1 methylation and oxidative DNA damage among 500 workers from a coke-oven plant in China. Urine biomarkers of PAH exposure (1-hydroxypyrene, 1-OHP; 2-hydroxynaphthalene, 2-NAP; 2-hydroxyfluorene, 2-FLU; and 9-hydroxyphenanthren, 9-PHE) and a marker of oxidative DNA damage (8-hydroxy- 2′- deoxyguanosine, 8-OHdG) were measured by high performance liquid chromatography. CYP1A1 methylation was measured by pyrosequencing. Finally, mediation analysis was performed to investigate whether CYP1A1 methylation mediated smoking and occupational PAH co-exposure effect on oxidative DNA damage.ResultsWe observed significant associations of smoking and 1-OHP co-exposure with CYP1A1 hypomethylation (OR: 1.87, 95% CI: 1.01–3.47) and high 8-OHdG (OR: 2.13, 95% CI: 1.14–3.97). There was a significant relationship between CYP1A1 hypomethylation and high 8-OHdG (1st vs. 3rd tertile = 1.58, 95% CI: 1.01–2.47, P for trend = 0.046). In addition, mediation analysis suggested CYP1A1 hypomethylation could explain 13.6% of effect of high 8-OHdG related to smoking and 1-OHP co-exposure.ConclusionsOur findings suggested that the co-exposure effect of smoking and occupational PAH could increase the risk of oxidative DNA damage by a mechanism partly involving CYP1A1 hypomethylation.

Genoprotective Effects of Essential Oil Compounds Against Oxidative and Methylated DNA Damage in Human Colon Cancer Cells.

Essential oils (EO) are widely used in foods as flavoring and preservative agents. Many of the biological activities of EO have been attributed to major essential oil compounds (EOC) but their direct interaction with colonic epithelial cells and their genotoxic and genoprotective effects are not well established. In this study, the cytotoxicity and genotoxicity of EOC including nerolidol, thymol, geraniol, methylisoeugenol, eugenol, linalool, and a commercial blend (Agolin) were determined. Furthermore, the genoprotective effects of EOC against oxidative and methylating damage were assessed using the comet assay in HT-29 colorectal adenocarcinoma cells. The majority of EOC were cytotoxic to HT-29 cells at or above 250 ppm after 24 hr exposure. At noncytotoxic doses, none of the EOC was genotoxic in the comet assay. Genoprotection against oxidative DNA damage was observed for nerolidol (at 62.5 ppm), thymol (at 12.5 ppm), geraniol, and methylisoeugenol (both at 125 ppm), as well as linalool and Agolin (both at 250 ppm). Thymol was the most protective compound against oxidative DNA damage and geraniol (at 125 ppm) also protected cells against methylating DNA damage. This study highlights the potential of EOC such as thymol to protect the colonic epithelium against oxidative DNA damage and geraniol against methylating DNA damage. Further in vivo studies are needed to confirm these findings for safety and efficacy to exploit their potential pharmaceutical or nutraceutical uses for colonic health.

Cyclophilin D knockout protects the mouse kidney against cyclosporin A-induced oxidative stress.

Mitochondrial dysfunction and oxidative stress have been implicated in cyclosporin A (CsA)-induced nephrotoxicity. CsA interacts with cyclophilin D (CypD), an essential component of the mitochondrial permeability transition pore and regulator of cell death processes. Controversial reports have suggested that CypD deletion may or may not protect cells against oxidative stress-induced cell death. In the present study, we treated wild-type (WT) mice and mice lacking CypD [peptidylprolyl isomerase F knockout (Ppif-/-) mice] with CsA to test the role and contribution of CypD to the widely described CsA-induced renal toxicity and oxidative stress. Our results showed an increase in the levels of several known uremic toxins as well as the oxidative stress markers PGF2α and 8-isoprostane in CsA-treated WT animals but not in Ppif-/- animals. Similarly, a decline in S-adenosylmethionine and the resulting methylation potential indicative of DNA hypomethylation were observed only in CsA-treated WT mice. This confirms previous reports of the protective effects of CypD deletion on the mouse kidney mediated through a stronger resistance of these animals to oxidative stress and DNA methylation damage. However, a negative effect of CsA on the glycolysis and overall energy metabolism in Ppif-/- mice also indicated that additional, CypD-parallel pathways are involved in the toxic effects of CsA on the kidney. In summary, CsA-mediated induction of oxidative stress is associated with CypD, with CypD deletion providing a protective effect, whereas the reduction of energy production observed upon CsA exposure did not depend on the animals' CypD status.

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

To invent a biotechnology that can reprogram this process. We used Alu siRNA to direct Alu interspersed repetitive sequences methylation in human cells. We evaluated the correlation between DNA damage and Alu methylation levels. We observed an inverse correlation between Alu element methylation and endogenous DNA damage in white blood cells. Cells transfected with Alu siRNA exhibited high Alu methylation levels, increased proliferation, reduced endogenous DNA damage and improved resistance to DNA damaging agents. Alu methylation stabilizes the genome by preventing accumulation of DNA damage. Alu siRNA could be useful for evaluating reprograming of the global hypomethylation phenotype in cancer and aging cells.

Electrochemical detection of 7-methylguanosine and DNA-methylation

DNA methylation damage can be detected using a DNA-electrochemical biosensor.