Deoxyguanosine is a TLR7 agonist.

Toll-like receptor 7 (TLR7) is an innate immune sensor for single-strand RNA (ssRNA). Recent structural analysis revealed that TLR7 has an additional binding site for nucleosides such as guanosine, and is activated when both guanosine and ssRNA bind. The nucleoside binding site also accommodates imidazoquinoline derivatives such as R848, which activate TLR7 in the absence of ssRNA. Here, we report that deoxyguanosine (dG) triggered cytokine production in murine bone marrow derived macrophages and plasmacytoid dendritic cells, as well as in human peripheral blood mononuclear cells, including type I interferons and pro-inflammatory factors such as TNF and IL-6. This signalling activity of dG was dependent on TLR7 and its adaptor MyD88 and did not require amplification via the type I interferon receptor. dG-triggered cytokine production required endosomal maturation but did not depend on the concurrent provision of RNA. We conclude that dG induces an inflammatory response through TLR7 and propose that dG is an RNA-independent TLR7 agonist.

Glycoxidative damage to human DNA: Neo-antigenic epitopes on DNA molecule could be a possible reason for autoimmune response in type 1 diabetes.

Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

Selection of monoclonal antibodies against 6-oxo-M(1)dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M(1)dG in vivo.

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-ß-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.

Effects of purine nucleoside phosphorylase deficiency on thymocyte development.

BACKGROUND: Inherited or acquired defects in purine nucleoside phosphorylase (PNP) impair purine metabolism, as well as the survival and function of T lymphocytes. However, the effects of PNP deficiency on thymocyte development are not well known. OBJECTIVES: We sought to study thymocyte development in PNP-deficient (PNP-KO) mice. METHODS: Maturation, proliferation, and apoptosis were determined in thymocytes from PNP-KO mice and hematopoietic stem cells from these mice grown ex vivo into thymocyte-like cells. RESULTS: Reduced percentages of CD4(+)CD8(+) double-positive (DP) thymocytes with normal percentages of CD4(-)CD8(+) and CD4(+)CD8(-) single-positive thymocytes were found in the thymi of PNP-KO mice. Similarly, reduced DP-like thymocytes grew ex vivo from hematopoietic stem cells of PNP-KO mice. Thymi of PNP-KO mice contained increased apoptotic DP thymocytes. Increased apoptosis of PNP-deficient DP thymocytes occurred after exposure to deoxyguanosine (dGuo), although not after Fas ligation, and could be prevented by restoring PNP activity within the cells. In DP thymocytes from PNP-KO mice, dGuo caused mitochondrial membrane potential dissipation and induced release of cytochrome c from the mitochondria followed by nuclear DNA fragmentation. Inhibition of the caspase pathway prevented dGuo-induced nuclear DNA fragmentation but not mitochondrial membrane potential dissipation, indicating that PNP deficiency induces apoptosis that is initiated in the mitochondria of DP thymocytes. 5-Bromo-2-deoxyuridine incorporation demonstrated that PNP deficiency does not interfere with DP or single-positive thymocyte proliferation. CONCLUSIONS: PNP is important for the survival of DP thymocytes. Accumulation of dGuo in cases of PNP deficiency leads to mitochondria-initiated apoptosis of DP thymocytes, which can be prevented by restoring PNP activity in the cells.

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach.

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde-deoxyguanosine (M(1)dG) adduct and the O(6)-carboxymethyl-deoxyguanosine (O(6)CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer (EPIC) study (n = 162) were analyzed for M(1)dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M(1)dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n = 6) were analyzed for O(6)CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 10(7) nucleotides for the analysis of O(6)CMdG. Subsequent reanalysis for O(6)CMdG showed a weakly significant increase in O(6)CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress.

Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

An Investigation of the Effects of Curcumin on the Changes in the Central Nervous System of Rats Exposed to Aroclor 1254 in the Prenatal Period.

BACKGROUND & OBJECTIVE: Aroclor 1254 is a widespread toxic compound of Polychlorinated Biphenyls (PCBs), which can create significant nervous problems. No remedies have been found to date. The aim of this study was to reveal the damage that occurs in the central nervous system of rat pups exposed to Aroclor 1254 in the prenatal period and to show the inhibiting effect of curcumin, which is a strong anti-oxidant and neuroprotective substance. METHOD: The study established 3 groups of adult female and male Wistar albino rats. The rats were mated within these groups and the offspring rats were evaluated within the group given Aroclor 1254 only (n=10) and the group was given both Aroclor 1254 and curcumin (n=10) and the control group (n=10). The groups were compared in respect of pathomorphological damage. The immunohistochemical evaluation was made of 8-hydroxdeoxyguanosine (8-OHdG), 4-hydroxynoneal (4HNE), myelin basic protein (MBP) expressions and TUNEL reaction. The biochemical evaluation was made of the changes in the TAS-TOS and Neuron Specific Enolase (NSE) levels. Damage was seen to have been reduced with curcumin in the 8OHdG and TUNEL reactions, especially in the forebrain and the midbrain, although the dosage applied did not significantly change TAS and TOS levels. Consequently, it was understood that Aroclor 1254 caused damage in the central nervous system of the pup in the prenatal period, and curcumin reduced these negative effects, particularly in the forebrain and the midbrain. CONCLUSION: It was concluded that curcumin could be a potential neuroprotective agent and would be more effective at higher doses.

Unexpected role of the human cytomegalovirus contribute to essential hypertension in the Kazakh Chinese population of Xinjiang.

Human cytomegalovirus (HCMV) infection, chronic inflammation and oxidative stress, the renin-angiotensin system (RAS), endothelial function, and DNA methylation play roles in the pathogenesis of essential hypertension (EH); however, the mechanism by which HCMV predisposes patients to hypertension remain unclear. Our group previously demonstrated an association between EH and HCMV infection in Kazakh Chinese. Here, we investigated the relationship between HCMV infection and other clinicopathological features in 720 Kazakh individuals with or without hypertension (n=360 each; age: 18-80). Multiple linear and logistic regression analyses were used to determine the associations between HCMV infection, clinical characteristics, and EH. Notably, patients with EH, particularly those with HCMV infection, exhibited a marked increase in tumor necrosis factor-α (TNF-α) and 8-hydroxy-2-deoxyguanosine (8-OHDG) levels, but a decrease in endothelial nitric oxide synthase (eNOS) and renin levels. Similarly, elevated TNF-α and 8-OHDG levels were independent predictors of increased HCMV antibody titers, whereas eNOS and renin were negatively correlated with the latter. Moreover, serum angiotensin-converting enzyme (sACE, ACE) methylation was increased, whereas 11-ß hydroxysteroid dehydrogenase 2 (HSD11ß2; HSD3B2) methylation was decreased in patients with EH who were also infected with HCMV. A positive correlation between HSD3B2 methylation and HCMV IgG titer and blood pressure was additionally observed, whereas angiotensin-converting enzyme (ACE) methylation was inversely correlated with blood pressure. Collectively, these data indicate that HCMV may contribute to EH development in the Kazakh Chinese by increasing TNF-α and 8-OHDG levels, suppressing eNOS and renin, and manipulating HSD3B2 and ACE methylation.

Impact of iron and vitamin C-containing supplements on preterm human milk: in vitro.

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.

Preventive effect of fluvastatin on ulcerative colitis-associated carcinogenesis in mice.

An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis (UC), which displays a cycle of recurrence-remission in the colorectal mucosa. Fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, is a hypocholesterolemic agent effective in animals and humans. Repeated administration of 3% dextran sulfate sodium subsequent to a single intraperitoneal injection of azoxymethane induces chronic UC resulting in an increased incidence of high-grade dysplasia and submucosal-invasive adenocarcinomas in the mouse colorectum. The effects of fluvastatin as an antioxidant on colorectal carcinogenesis in mice with UC were investigated. Treatment with fluvastatin in mice with UC abolished the anemia caused by colorectal carcinogenesis, and markedly lowered plasma lipid levels resulting in a reduction of colitis and carcinogenesis, shown by inhibition of the decrease in colorectal length, the increased number of foci of gland loss with inflammatory cell infiltration indicating the severity of UC and incidence of colorectal dysplasia, respectively, with a reduction in anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody (a biological marker of in vivo oxidative DNA damage)-positive cells of the colorectal mucosa and the activity of the DNA-synthesizing enzyme thymidine kinase in colorectal tissues.

Immunological quantification by high-affinity antibodies of O6-ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea.

Three immunological methods [radioimmunoassay (RIA), enzyme-linked immunosorbent assay, and radioimmunosorbent technique] were established for quantification of the potentially mutagenic O6-ethyldeoxyguanosine (O6-EtdGuo) in DNA treated with the carcinogen ethylnitrosourea in vivo or in vitro. To obtain high-affinity antibodies for specific detection of low levels of O6-EtdGuo in small amounts of DNA (cells), different schemes were applied for immunization of rabbits with the hapten O6-ethylguanosine coupled to various carrier proteins (rat serum albumin, bovine serum albumin, keyhold limpet hemocyanin). Low-dose immunization with the hapten-keyhold limpet hemocyanin conjugate resulted in antibodies with an affinity constant of 1 to 2 X 10(10) liters/mol and very low levels of cross-reactivity with normal as well as other alkylated DNA components. The RIA (the most sensitive of the three assays) detects 0.05 pmol of O6-EtdGuo at 50% inhibition of tracer (O6-ethyl[8,5'-3H]-3'-deoxyguanosine)-antibody binding. This permits quantification by RIA of O6-EtdGuo at an O6-EtdGuo:2'-deoxyguanosine molar ratio of approximately 3 X 10(-7) in a hydrolysate of 100 micrograms of ethylated DNA. By chromatographic separation of O6-EtdGuo prior to the RIA, this value can be lowered to less than 5 X 10(-8).

Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay.

Calf thymus DNA was modified with the benzo(a)pyrene (BP) derivative, (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-) BPDE I], under conditions which yielded greater than 99% of the binding product in the form of trans-(7R)-N2-(10[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl) deoxyguanosine. Rabbits were immunized with modified DNA coupled to methylated bovine serum albumin, and the resulting antiserum was utilized in a competition radioimmunoassay for the quantitation of products of BP covalently bound to DNA. The antiserum was specific for both native and denatured immunogen DNA's as well as for the major isolated BP binding product, but it did not recognize BP, the tetrol of (+/-)BPDE I, or unmodified deoxyguanosine. The modified DNA was assayed in quantities as low as 2 pmol of adduct, a sensitivity sufficient to quantitate the extent of modification of cellular DNA when epidermal cell cultures were exposed either to BP or to (+/-)BPDE I. High-pressure liquid chromatographic analysis of DNA hydrolsates, obtained from epidermal cells exposed to BP or to (+/-)BPDE I, indicated that the major adduct was the same as than on the immunogen DNA. This approach should prove valuable for further studies on the mechanism of carcinogenesis and for monitoring human exposure to this ubiquitous carcinogen.

Development of a monoclonal antibody-based immunoassay for cyclic DNA adducts resulting from exposure to crotonaldehyde.

In order to develop an immunoassay for DNA modifications resulting from exposure to crotonaldehyde, monoclonal antibodies specific for the 8R,6R- and 8S,6S-stereoisomers of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxy-6 - methylpyrimido[1,2-a]purine-10(3H)one were produced. These cyclic 1,N2-propanodeoxyguanosines are formed in DNA exposed to crotonaldehyde in vitro. Three of the four antibodies were most specific for one stereoisomer while the fourth was most specific for the other stereoisomer. Fifty % inhibition of binding in an enzyme-linked immunoabsorbent assay using two of these antibodies and capable of detecting 0.5 mumol of 1,N2-propanodeoxyguanosine per mol of deoxyguanosine was developed. The method was validated by comparison to results obtained with fluorescence assay.

Antibodies against 7-methyldeoxyguanosine: its detection in rat peripheral blood lymphocyte DNA and potential applications to molecular epidemiology.

Polyclonal antibodies have been raised against the imidazole ring-open form of 7-methyldeoxyguanosine (7-mdGua). A combined high performance liquid chromatography/immunoassay method has been developed using these antibodies which provides a specific and sensitive way to quantitate 7-mdGua in DNA. Following enzyme hydrolysis and chromatographic purification of 7-mdGua, the adduct is quantitatively converted to the ring-open form and can be measured at levels as low as 0.05 pmol by immunoassay. With 1 mg of DNA a level below 1 adduct per 10(7) normal deoxynucleosides can be measured. Using DNA modified by radiolabeled carcinogens, a good correlation between 7-mdGua levels, as measured by immunoassay or radioactivity, was obtained. In rats treated with dimethylnitrosamine (0.4 and 1.0 mg/kg), both 7-mdGua and O6-methyldeoxyguanosine were detected in peripheral blood lymphocyte DNA. In addition the levels of both adducts at time points up to 48 h posttreatment were very similar to those seen in liver DNA from the same animals. The measurement of 7-mdGua, quantitatively the major methylation adduct, in small cell samples such as lymphocytes has great potential in determining the exposure of humans to environmental methylating agents such as nitrosamines.

8-hydroxy-2'-deoxyguanosine (8-OHdG) biomarker detection down to picoMolar level on a plastic antibody film.

An innovative biosensor assembly relying on a simple and straightforward in-situ construction is presented to monitor urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) down to the pmol/L level. The sensing film of the biosensor consisted of a molecularly imprinted polymer (MIP) layer for 8-OHdG assembled on a gold electrode through electropolymerization of monomer combined with the template. The analytical features of the resulting biosensor were assessed by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Some experimental parameters such as the initial concentration of the monomer and the ratio template-monomer were investigated and optimized in order to finely tune the performance of the MIP-based sensor. Under optimal conditions, the developed biosensor was able to rebind 8-OHdG with a linear response against EIS from 0.1 to 100pg/ml 3.5-3500 pM. The interference of coexisting species was tested, also with calibrations on urine samples, and good selectivity towards 8-OHdG was obtained. RAMAN spectroscopy, FTIR and SEM evaluations of the prepared films confirmed the formation of a polyphenol thin-film on the electrode surface. The presence and distribution of the imprinted cavities on the MIP layer was confirmed by confocal microscopy imaging of the film, after a post-treatment with Fluorescein Isothiocyanate (FITC) labeled 8-OHdG antibody. Overall, this label-free biosensor for urinary 8-OHdG detection constitutes a promising low-cost alternative to the conventional immunoassay approaches, due to its simplicity, stability, high sensitivity and selectivity for biological sample assays, opening new doors for other applications.

Angiotensin-converting enzyme inhibitor helps prevent late remodeling after left ventricular aneurysm repair in rats.

BACKGROUND: We reported in a previous study that the initial effects of left ventricular (LV) repair (LVR) for LV aneurysm were not long lasting. Angiotensin-converting enzyme inhibitor (ACE-I) is known to attenuate remodeling after myocardial infarction, and could be effective after LVR. METHODS AND RESULTS: Left ventricular aneurysms were developed in rats after left anterior descending artery ligation. Rats were divided into 3 groups: sham operation with ACE-I (lisinopril 10 mg/kg/d) (n=10; group A), LVR (by plicating the LV aneurysm) with placebo (n=8; group R), and LVR with ACE-I (n=10; group RA). LV function was evaluated by echocardiography and catheterization. Oxidative stress in the myocardium was estimated by immunohistochemistry for 8-hydroxy-2'-deoxyguanosine. One week after LVR, LV end-diastolic area was smaller and fractional area change was better in the 2 LVR groups. Four weeks after LVR, LV end-diastolic area, and fractional area change deteriorated in group R but not so much in group RA; E-max was higher in group RA (0.79+/-0.20 mm Hg/mL) than in groups A (0.25+/-0.03 mm Hg/mL; P<0.01) and group R (0.27+/-0.03 mm Hg/mL; P<0.01). Oxidative stress was much lower in the 2 ACE-I groups. CONCLUSIONS: LVR improved LV size and systolic function only in the early phase. Adjuvant use of ACE-I was useful for preventing redilation and maintaining LV systolic function, was associated with suppressed oxidative stress, and may make LVR a more effective surgical procedure for LV aneurysm.

Reduction of cerebral infarction in stroke-prone spontaneously hypertensive rats by statins associated with amelioration of oxidative stress.

BACKGROUND AND PURPOSE: This study aimed to clarify the effect of statins on spontaneous stroke and to examine the antioxidative effect in artificial transient middle cerebral artery occlusion (tMCAO). METHODS: Stroke-prone spontaneous hypertensive rats (SHR-SP) were treated with pitavastatin, atorvastatin, simvastatin, or vehicle for 4 weeks. Physiological parameters, serum lipids, and infarct volumes were examined. The markers for oxidative stresses on lipids and DNA were immunohistochemically detected in vehicle-treated or simvastatin-treated SHR-SP with tMCAO. RESULTS: Atorvastatin and simvastatin decreased infarct volumes, with simvastatin most effective. Simvastatin significantly reduced immunoreactivities for oxidative stress markers for lipids and DNA in neurons after tMCAO. CONCLUSIONS: The results suggest that the antioxidative properties of statins may be implicated in their beneficial effects against neuronal damage in cerebral ischemia.

Endometriotic cells exhibit metaplastic change and oxidative DNA damage as well as decreased function, compared to normal endometrium.

A widely accepted theory of the etiology of endometriosis is that it originates from the implantation and invasion of cells from retrograde menstruation to various sites in the body particularly the pelvic peritoneal cavity. Little is known of the function of these cells in ectopic sites. Normal endometrium was compared with endometriotic tissue using an antibody to Placental Cadherin (P Cadherin), a recently studied cadherin that is implicated in metaplasia and early neoplasia and also 8-hydroxyguanine, an indicator of oxidative DNA damage. Comparisons of endometrial tissue function were made using expression of transforming growth factor beta-1 (TGFbeta-1) and insulin-like growth factor-I (IGF-I). There was no labelling for anti-P Cadherin or anti-8-hydroxydeoxyguanosine in normal endometrium but marked labelling for both on the apical surface of the endometriotic epithelium. Studies of markers of normal endometrial function were all de-expressed in endometriosis. This study indicates that endometriosis cells are abnormal and exhibit oxidative DNA damage, metaplasia and markedly reduced function compared to normal endometrium.

Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice.

Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6(-/-) mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6(-/-) males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6(-/-) males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6(-/-) males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice.

Genetic dissection of systemic autoimmune disease in Nrf2-deficient mice.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with immune-complex deposition that affects multiple organs. Previous studies have suggested the involvement of oxidative stress and apoptosis in SLE, but no clear link to etiology has been established. Here we show that mice deficient in a transcription factor responsible for controlling the expression of numerous detoxification and antioxidant genes develop an autoimmune disease with multiple organ pathologies that closely resembles human SLE. Aged female mice with a knockout of nuclear factor, erythroid-derived 2, like 2 (nrf2) are prone to develop antibodies against double-stranded DNA and the Smith antigen as well as IgG, IgM, and C3 deposition in kidney, liver, heart, and brain. Prior to the development of autoimmune antibodies and organ pathology, oxidative damage occurs in the liver and kidney as indicated by the increased levels of the DNA oxidation marker 8-hydroxydeoxyguanosine and the later increase in the lipid peroxidation product malondialdehyde. Gene expression profiles demonstrate an early decrease in numerous antioxidant and detoxification genes in the livers and altered levels of cytokines and T and B cell-specific genes in the spleens of nrf2 knockout mice. These data strongly suggest that a deficiency in detoxification and increased oxidative stress can result in the development of a systemic autoimmune disease.