Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

Imbalanced oxidant and antioxidant ratio in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults and is due to trinucleotide sequence (CTG) in the 3′ UTR region of DMPK gene located at 19q13.3 chromosome. The pathogenic mechanisms of multisystemic involvement of DM1 are still unclear. The increased levels of reactive oxygen species/free radicals and lipid peroxides and decreased antioxidant levels play an important role in the pathogenesis of DM1. Present study includes 20 DM1 patients and 40 age- and sex-matched controls. Malonilaldehyde (MDA), superoxide dismutase (SOD), glutathione peroxidise (GPX), glutathione-S-transferase (GST), reduced glutathione (GSH), and TAS levels were measured and its association with clinical phenotype were evaluated. Results revealed significantly higher levels of MDA (p = 0.002), SOD (p = 0.006), and TAS p = 0.004) and lower level of GPX (p = 0.003), GST (P < 0.001) and GSH (P = 0.016) in DM1 patients. A significant negative correlation of MDA level with dyspepsia and CK-MB and GST level with serum SCK, CK-MB, and diabetes were observed. However, a significant positive correlation of SOD level with serum CK-MB, CK-MM, and diabetes and negative correlation with facial weakness were noted. Though, GSH level had significant positive correlation with learning and writing disability, speech, and languages disability yet found negative correlation with duration of disease. The GPX and TAS showed no correlation with any clinical findings. Our data further support the pathogenic role of oxidative stress in DM1 of Indian origin and support the opportunity to undertake clinical trials with antioxidants in this disorder.     

Pathogenic RNA repeats: an expanding role in genetic disease

Fragile X mental retardation and Friedreich's ataxia were among the first pathogenic trinucleotide repeat disorders to be described in which noncoding repeat expansions interfere with gene expression and cause a loss of protein production. Invoking a similar loss-of-function hypothesis for the CTG expansion causing myotonic dystrophy type 1 (DM1) located in the 3' noncoding portion of a kinase gene was more difficult because DM is a dominantly inherited multisystemic disorder in which the second copy of the gene is unaffected. However, the discovery that a transcribed but untranslated CCTG expansion causes myotonic dystrophy type 2 (DM2), along with other discoveries on DM1 and DM2 pathogenesis, indicate that the CTG and CCTG expansions are pathogenic at the RNA level. This review will detail recent developments on the molecular mechanisms of RNA pathogenesis in DM, and the growing number of expansion disorders that might involve similar pathogenic RNA mechanisms.     

The role of oxidative stress after retinal laser photocoagulation in nonproliferative diabetic retinopathy

Diabetic retinopathy (DR) represents the most common chronic complication of diabetes, and it is the leading cause of new cases of blindness in patients between 20-74 years old in developed countries. Laser photocoagulation (LF) represents an efficacious approach to the treatment of DR. Oxidative factors, such as free radicals (FR), are continuously generated in aerobic organisms as a result of different metabolic processes. It is well known that oxidative stress plays a role in the development of DR. The aim of this study was to evaluate the thermal effects of the scatter retinal laser photocoagulation technique on the production of FR. A total of 90 patients were enrolled in this study. They were divided in 3 groups: 30 diabetic patients with DR, 30 diabetic patients without DR, and 30 control individuals without diabetes mellitus (DM). Full scatter retinal LF was performed in all patients with DR. We measured the concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPOD), catalase, and total antioxidative status (TAS). Of the 30 DR patients, 13 showed the appearance or worsening of macular edema after LEF, whereas the other 17 patients showed no change. Thirty days after LF, improvement in visual acuity was observed, but this change was not statistically significant. The mean plasma or erythrocyte lysate concentrations of various antioxidants were significantly lower in the diabetic patients without DR compared to the individuals without DM and in the diabetic patients with DR compared to the individuals without DM; the diabetic patients with DR did not show lower concentrations of the antioxidants compared to the diabetic patients without DR. The concentrations of SOD, GPOD, catalase, and TAS were significantly lower in the diabetic patients with DR after retinal scatter LF, which could be the consequence of retinal oxidative stress caused by the LF thermal effect.     

Total antioxidant levels are low during active TB and rise with anti-tuberculosis therapy

In tuberculosis, oxidative stress is a result of tissue inflammation, poor dietary intake of micronutrients due to illness, free radical burst from activated macrophages, and anti-tuberculosis drugs. These free radicals may in turn contribute towards pulmonary inflammation if not neutralized by antioxidants. The total antioxidant status (TAS) of individuals is a function of dietary, enzymatic, and other systemic antioxidants and is therefore an indicator of the free radical load. Our aim was to evaluate the TAS of healthy and M. tuberculosis-infected persons from a high TB incidence community, as well as tuberculosis patients at various stages of antituberculosis drug treatment and to correlate results with plasma micronutrient levels. Blood plasma samples from TB infected patients and following antituberculosis drug treatment were assayed for TAS, vitamins A, E and Zinc. Statistical analysis of results was by one-way ANOVA and the Tukey multiple comparison post test. Active TB patients showed a significantly lower TAS (P < 0.001) compared to the community controls. We also show that TAS values increase during therapy. Results correlated with micronutrients vitamin A and zinc but vitamin E remained unaffected. We suggest that total antioxidant status of TB patients should be considered for more effective disease control and that diets low in antioxidants may render individuals susceptible to tuberculosis.     

Evaluation of oxidative stress markers in pathogenesis of diabetic neuropathy

Experimental evidences suggest that hyperglycaemia-induced overproduction of reactive oxygen species and subsequent damage to proteins, lipids and DNA may play a key role in the development of distal symmetric polyneuropathy (DSPN)-the most common complication of diabetes mellitus. The study population consisted of 51 individuals aged 52-82 years classified into 3 groups: 16 patients diagnosed with type 2 diabetes mellitus (T2DM) with DSPN, 16 T2DM patients without DSPN and 19 control subjects without diabetes and neuropathy. The study was conducted to determine the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant status (TAS) in the examined groups. An alkaline comet assay was used to determine the extent of DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites. A significant decrease of SOD (P < 0.05), GPX (P < 0.05) and nonsignificant decrease of CAT (P > 0.05), and TAS status (P > 0.05) were seen in T2DM patients with neuropathy compared to T2DM patients as well as controls. T2DM patients with or without neuropathy revealed significantly lower (P < 0.05) plasma concentration of nitrous oxide compared to the control subjects. Endogenous level of oxidative DNA damage in T2DM patients with DSPN was significantly higher compared both to the controls and T2DM patients without DSPN (P < 0.001). Moreover, lymphocytes isolated from T2DM patients with DSPN were more susceptible to oxidative DNA lesions induced by hydrogen peroxide than from T2DM patients without DSPN (P < 0.001). Our results confirm hypothesis that oxidative stress may play a substantial role in the development and progression of diabetic distal symmetric polyneuropathy.     

Physiological responses of resistant and susceptible plants to diclofop-methyl and its antagonism by 2,4-D and antioxidants

Diclofop-methyl (DM) sprayed onto 6–8-week-old plants of leafy spurge ( Euphorbia esula L.) caused senescence and abscission of older leaves, while the young leaves and apex remained attached. The phytotoxicity of DM was reversed by the antioxidant, α -tocopherol (vitamin E), in leafy spurge and DM-susceptible oat ( Avena sativa L. cv. Gary). DM and 2,4-dichlorophenoxyacetic acid (2,4-D) increased ethylene evolution in mature leaves of leafy spurge. Vitamin E reduced the DM-induced ethylene by ampproximately 50%, but had no effect on the 2,4-D-induced ethylene. DM did not increase ethylene in DM-resistant pea or tobacco, but 2,4-D induced a 3-fold increase in ethylene evolution over controls in DM-resistant tobacco. 2,4-D amppears to act at a site different from that of DM in the pathway of ethylene formation. Ethylene evolution increased in DM-treated susceptible biotypes of annual ryegrass ( Lolium rigidum L.) and wild oat ( Avena fatua L.), but not in resistant biotypes of these species. DM reduced root and shoot formation and dry weight in hypocotyl segments of etiolated leafy spurge seedlings grown in vitro. Organogenesis and dry weights were increased by the combination of DM+antioxidants. Vitamin E was a more effective antioxidant than ascorbic acid. These results sumpport the hypothesis that DM induces oxidative stress in susceptible plant tissues and that antioxidants reduce the damaging action of the phytotoxic free radicals.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

Dimethoate Induces Kidney Dysfunction, Disrupts Membrane-Bound ATPases and Confers Cytotoxicity Through DNA Damage. Protective Effects of Vitamin E and Selenium

Dimethoate (DM) is an organophosphate insecticide widely used in agriculture and industry and has toxic effects on non-target organisms especially mammalian. However, we still know little about DM-induced kidney injury and its alleviation by natural antioxidants. In the present study, selenium (Se), vitamin E, DM, Se+DM, vitamin E+DM, Se+vitamin E+DM were given to adult rats for 4 weeks. Plasma creatinine and uric acid, kidney MDA, PC, H₂O₂ and AOPP levels were higher, while Na⁺-K⁺-ATPase and LDH values were lower in the DM group than those of controls. A smear without ladder formation on agarose gel was shown in the DM group, indicating random DNA degradation and DM-induced genotoxicity. A decrease in kidney GSH, NPSH and plasma urea levels and an increase in GPx, SOD and catalase activities were observed in the DM group when compared to those of controls. Plasma cystatin C levels increased, indicating a decrease in glomerular filtration rate. When Se or vitamin E was added through diet, the biochemical parameters cited above were partially restored in Se+DM and vitamin E+DM than DM group. The joint effect of Se and vitamin E was more powerful against DM-induced oxidative stress and kidney dysfunction. The changes in biochemical parameters were substantiated by histological data. In conclusion, our results indicated a possible mechanism of DM-induced nephrotoxicity, where renal genotoxicity was noted, membrane-bound ATPases and plasma biomarkers were disturbed. Se and vitamin E ameliorated the toxic effects of this pesticide in renal tissue suggesting their role as potential antioxidants.     

Chemotherapeutic deletion of CTG repeats in lymphoblast cells from DM1 patients

Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG).(CAG) repeat in the DMPK gene on chromosome 19q13.3. At least 17 neurological diseases have similar genetic mutations, the expansion of DNA repeats. In most of these disorders, the disease severity is related to the length of the repeat expansion, and in DM1 the expanded repeat undergoes further elongation in somatic and germline tissues. At present, in this class of diseases, no therapeutic approach exists to prevent or slow the repeat expansion and thereby reduce disease severity or delay disease onset. We present initial results testing the hypothesis that repeat deletion may be mediated by various chemotherapeutic agents. Three lymphoblast cell lines derived from two DM1 patients treated with either ethylmethanesulfonate (EMS), mitomycin C, mitoxantrone or doxorubicin, at therapeutic concentrations, accumulated deletions following treatment. Treatment with EMS frequently prevented the repeat expansion observed during growth in culture. A significant reduction of CTG repeat length by 100-350 (CTG).(CAG) repeats often occurred in the cell population following treatment with these drugs. Potential mechanisms of drug-induced deletion are presented.     

Genomic instability associated with myotonic dystrophy does not involve p53 expression and activity

We tested the hypothesis that the instability of the trinucleotide CTG at the myotonic dystrophy (DM) locus could be an intrinsic DNA damage recognisable by the p53 cell-cycle checkpoint system. p53 mRNA and protein levels were assayed in muscle biopsies and fibroblast cell lines of DM patients and unaffected controls. No differences in mRNA and protein levels were found between patients and controls, regardless of their expansion size. However, in the cells treated with adryamicin, p53 protein levels were comparable in DM and control cells. We conclude that the CTG trinucleotide expansion within the myotonin gene does not activate the p53 surveillance system, at least in adult tissues. The escape of trinucleotide expansion from the p53-mediated DNA repair system could explain some of the biological characteristics of genome instability. © 1998 John Wiley & Sons, Ltd.     

Antioxidant system in Uromastyx philbyi during hibernation and activity periods

Hibernation is an extreme physiological state characterized by profound decreases in oxidative metabolism and body temperature during bouts of prolonged torpor, interrupted by brief periods of arousal with sudden increases in oxidative metabolism, with alterations in antioxidant defenses. We monitored the activities of antioxidant enzymes and oxidative stress during hibernation and activity in Uromastyx philbyi. 20 animals were used, 10 of which were collected in the hibernation season (group I) and the other 10 collected during the active period (group II). Blood, liver, brown adipose tissue (BAT) and brain samples were used to determine free radical and antioxidant levels. The results indicated a significant decrease of free radicals and increase of vitamin C, especially in serum during hibernation. In contrast, during the active period free radicals, enzymatic antioxidants as glutathione peroxidase (GPX), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) and non-enzymatic antioxidants as reduce glutathione (GSH) and vitamin E increased in all studied tissues. It can be concluded that Uromastyx philbyi has a strong antioxidant defense system that protects it from the injurious effects of free radicals either at the periods of arousal or during activity periods.     

Dominantly inherited,non-coding microsatellite expansion disorders

Dominantly inherited diseases are generally caused by mutations resulting in gain of function protein alterations. However, a CTG expansion located in the 3' untranslated portion of a kinase gene was found to cause myotonic dystrophy type 1, a multisystemic dominantly inherited disorder. The recent discovery that an untranslated CCTG expansion causes the same constellation of clinical features in myotonic dystrophy type 2 (DM2), along with other recent discoveries on DM1 pathogenesis, have led to the understanding that both DM1 and DM2 mutations are pathogenic at the RNA level. These findings indicate the existence of a new category of disease wherein repeat expansions in RNA alter cellular function. Pathogenic repeat expansions in RNA may also be involved in spinocerebellar ataxia types 8, 10 and 12, and Huntington's disease-like type 2.     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

UV induces oxy- and chromanoxyl free radicals in microsomes by a new photosensitive organic hydroperoxide, N,N'-bis(2-hydroperoxy-2-methoxyethyl)- 1,4,5,8-naphtalene-tetra-carboxylic-diimide

Low oxygen tension, a high content of reducing equivalents and endogenous vitamin E are responsible for the resistance of cancer cells to oxidative stress-based therapy. N,N'-bis(2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra- carboxylic-diimide (NP-III), capable to release radicals both in the absence and in the presence of oxygen upon UV-illumination, is a new potential anticancer agent. UV-induced reactions of NP-III in rat liver microsomes were studied under aerobic and anaerobic conditions with (i) vitamin E homologue, chromanol-alpha-C-6 having a shorter (6-carbon) hydrocarbon side chain and higher antioxidant activity, and (ii) the spin-trap 5,5-dimethyl-1-pyrroline-1-oxide, DMPO. UV-induced generation of chromanoxyl radicals was observed in the presence of NP-III under aerobic conditions, which was SOD+catalase sensitive. Hydroxyl-, superoxide- and alkoxyl-radical DMPO adducts were found upon UV-illumination of NP-III under aerobic conditions and only hydroxyl-radical adducts under anaerobic conditions. The light-dependent generation of oxy- and chromanoxyl free radicals and depletion of endogenous antioxidants suggests to be a promising strategy to overcome the inherent resistance of tumor cells to oxidative stress.     

Lipid peroxidation and antioxidant status in patients with periodontitis

Our aim was to assess the degree of oxidative stress in patients with periodontitis by measuring their levels of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and non-enzymatic antioxidants (vitamins E and C, reduced glutathione (GSH)). This study was conducted on 25 adult chronic periodontitis sufferers who were patients in Rajah Muthiah Dental College and Hospital, Annamalai University. The levels of TBARS and non-enzymatic antioxidants, and the activities of enzymatic antioxidants in the patients' plasma, erythrocytes and gingival tissues were assayed using specific colorimetric methods. The periodontitis sufferers had a significantly higher TBARS level than the healthy subjects. In the plasma, erythrocytes, erythrocyte membranes and gingival tissues of the periodontitis sufferers, enzymatic antioxidant activities were found to be significantly higher, whereas the levels of non-enzymatic antioxidants were significantly lower (except for reduced glutathione in the gingival tissues) relative to the parameters found for healthy subjects. The disturbance in the endogenous antioxidant defense system due to over-production of lipid peroxidation products at inflammatory sites can be related to a higher level of oxidative stress in patients with periodontitis.     

Autoantibodies against oxidized LDL and serum total antioxidant status in active cyclists and ex-cyclists.

There is an apparent paradox between the benefits of aerobic exercise and the potentially deleterious effects of increased free radicals and decreased circulating antioxidants generated during exercise. To assess the oxidative/antioxidative status in competitive cyclists and ex-cyclists, we measured two markers not well studied in these situations, serum total antioxidant status (TAS) and antibodies against oxidized LDL (AuAb-ox-LDL) in 18 competitive male cyclists, 10 ex-competitive cyclists and 14 healthy males. AuAb-ox-LDL was evaluated by enzyme immunoassay, and serum TAS concentration was analyzed by spectrophotometry. Ex-cyclists had serum TAS levels statistically higher than the control group and cyclists group (p = 0.001 and p < 0.001, respectively). Active sportsmen showed significantly less AuAb-ox-LDL than healthy sedentary males, while there was a non-significant trend in the ex-cyclists to have lower AuAb-ox-LDL than the corresponding control group. AuAb-ox-LDL levels were not statistically different between the groups of active and previously active sporting men. There was a positive correlation between TAS and LDL-cholesterol in active cyclists under heavy training. In the ex-cyclist group, there was a negative correlation between serum TAS and the time elapsed since they had ended the competition. Competitive cycling decreases AuAb-ox-LDL levels, suggesting that it may decrease ox-LDL levels. After ending physical training, antioxidative status remains increased for at least one year, but the effect on AuAb-ox-LDL levels is lost.