The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

Determination of effective miRNAs in wound healing in an experimental Rat Model.

The larvae of Lucilia sericata have been used for centuries as medicinal maggots in the healing of wounds. The present study aimed to screen potential microRNAs related to ES-induced wound healing in rat skin wounds and to investigate the potential mechanisms contributing to accelerated wound healing. Healthy, male, 12 weeks old Wistar albino rats weighing 250-300 g were supplied by the Animal Experimental Center. All animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Wistar albino rats were treated by ES after post wounding and the differentially expressed miRNAs in wound biopsies were screened by microarray analysis at the end of treatments for 4,7 and 10 days. In addition, bioinformatics approaches were used to identify the potential target genes of differentially expressed miRNAs and the functions of their target genes. We found a significant up-regulation of rno-miR-99a* and rno-mir-877 in response to ES treatment. Further investigation of rno-miR-99a* and rno-mir-877 and their target genes (TGFa, TNF, TAGLN, MAPK1, MMP-9) implicated in present study could provide new insight for an understanding lead to the development of new treatment strategies. The identified miRNAs can be new biomarkers for ES- induced wound healing.

Lack of association between increased mitochondrial DNA4977 deletion and ATP levels of sputum cells from chronic obstructive pulmonary disease patients versus healthy smokers.

In this study we looked at smokers with and without chronic obstructive pulmonary disease (COPD) patients in order to evaluate the incidence of 4977 base pair (bp) mtDNA (mtDNA4977) deletion and mtDNA copy number in sputum cells and in peripheral blood leukocytes (PBLs) in relation to mitochondrial function and oxidative stress status. Twenty-five COPD patients who were current smokers, 22 smokers and 23 healthy nonsmokers (for only PBLs studies) participated in this study. The 4977-bp deletion was detected in all examined samples within 40 cyles of PCR amplification, using a quantitative real time PCR. The frequency of the mtDNA4977 was significantly higher in the sputum cells of patients with COPD compared to smokers without COPD (p < 0.0001). This difference was not observed in PBLs. Levels of cellular oxidative stress were significantly higher in the sputum cells of subjects with COPD than in the smoker group. However, mtDNA copy number, mitochondrial membrane potential (ΔΨm) and cellular ATP levels in PBLs and sputum cells were not significantly different between the studied groups. The Pearson analysis revealed no correlations between the accumulation of mtDNA4977, and intracellular ATP content and ΔΨm values of the sputum cells, although there was a positive correlation between the increase in the percentage of deleted mtDNA4977 and the levels of cellular oxidative stress in COPD patients (r = 0.80, p < 0.0001). Our studies may suggest that the accumulation of mtDNA4977 in the sputum cells of smokers with COPD does not seem to have an important impact on mitochondrial dysfunction in relation to ATP production and ΔΨm when compared to those of healthy smokers.

Spatio-spectral characterization of local field potentials in the subthalamic nucleus via multitrack microelectrode recordings.

Deep brain stimulation of the subthalamic nucleus (STN) is a highly effective treatment for motor symptoms of Parkinson's disease. However, precise intraoperative localization of STN remains a procedural challenge. In the present study, local field potentials (LFPs) were recorded from three tracks during microelectrode recording-based (MER) targeting of STN, in five patients. The raw LFP data were preprocessed in original recording setup and then data quality was compared to data with common average derivation. The depth-frequency maps were generated according to preprocessing results for each patient and spectral characteristics of LFPs were explored at each depth across different tracks and different subjects. Spatio-spectral analysis of LFP was investigated to see whether LFP activity can be used for optimal track selection and STN border identification. Analysis show that monopolar derivation suffer from various artifacts and/or power line noise which makes the interpretation of target localization very difficult in most of the subjects. Unlikely, bipolar derivation helps to recover the neurological signals and investigation of signal characteristics. The frequency-vs-depth maps using a modified Welch periodogram with robust statistics, demonstrated that a median-based spectrum estimation approach eliminates outliers pretty well by preserving band-specific LFP activity. The results indicate that there is a clear oscillatory beta activity around 20 Hz in all subjects. 1/f normalization reveals the high frequency oscillations (HFOs) between 200-to-350 Hz in two subjects. It's noted that the optimal track selection is not consistent with the track having highest beta band oscillations in two out of five subjects. In conclusion, microelectrode-derived LFP recordings may provide an alternative approach to single unit activity (SUA)-based MER, for localizing the target STN borders during DBS surgery. Despite the small number of subjects, the present study adds to existing knowledge about LFP-based pathophysiology of PD and its target-based spectral activities.

Effect of donor age on the susceptibility of erythrocytes and erythrocyte membranes to cumene hydroperoxide-induced oxidative stress.

A comparative study on erythrocytes and erythrocyte membranes of healthy elderly and young adults was carried out to understand how the antioxidant defense capacity is effected by aging. The levels of endogenous malondialdehyde and Ca(2+)-ATPase activity were taken as indices of oxidative damage. In addition, chemiluminescence measurements were performed on intact erythrocytes. The susceptibility of these parameters to in vitro cumene hydroperoxide, under low oxidant level that does not induce hemolysis, was also taken as an age-related indicator of the endogenous peroxidative potential of the erythrocytes. Our data showed that the content of malondialdehyde and Ca(2+)-ATPase activity did not change with age. Furthermore, the susceptibility of intact erythrocytes to oxidative stress did not change in the elderly group. However, under the same conditions erythrocyte membranes were more susceptible to oxidative damage in the elderly than young adults. Our results also showed that antioxidant defenses were overwhelmed in intact erythrocytes of the elderly at high concentrations of cumene hydroperoxide.

Transport of glutathione conjugate in erythrocytes from aged subjects and susceptibility to oxidative stress following inhibition of the glutathione S-conjugate pump.

The aim of the present study was to investigate the effect of donor aging on the glutathione conjugate transport in erythrocytes and whether it plays a role in the resistance to oxidative stress of the erythrocytes of aging subjects. In our comparative study on intact erythrocytes of healthy aging and young adults, in which 2,4-dinitrophenyl-S-glutathione (DNP-SG) was used as model glutathione S-conjugate, we found that the efflux of DNP-SG remained unchanged in the aged subjects. This result suggests that the detoxification function is maintained against the chemical stress employed in erythrocytes of aging subjects. In the assay conditions used, which were optimized to obtain maximal inhibition of glutathione S-conjugate transport, our results also indicated that the susceptibility of erythrocytes to in vitro lipid peroxidation generated by cumene hydroperoxide was enhanced by pretreatment with DNP-SG inhibitors in both age groups. However, the difference in susceptibility was not a function of aging. Further, the results suggested that inhibition of glutathione S-conjugate pump may impair cellular protection of the erythrocytes against oxidative damage.

Effect of antiepileptic drugs on erythrocyte osmotic fragility and lipid peroxidation.

Epileptic children receiving antiepileptics were studied to investigate the effect of carbamazepine and phenobarbital therapy on erythrocyte osmotic fragility and lipid peroxidation. Significant differences between the two groups were observed in erythrocyte osmotic fragility. In addition, there was a significant increase in erythrocyte malondialdehyde release in the epileptic group compared to controls. It is suggested that the use of antioxidants in addition to antiepileptic drugs may be beneficial.

The influence of GSTM1 null genotype on susceptibility to in vitro oxidative stress.

From epidemiological studies, there is some evidence that genetic variation at the glutathione S-transferase (GST) loci GSTM1 influences individual susceptibility to disease associated with oxidative stress. The aim of this study was to elucidate the role of the GSTM1 genotype in protection against oxidant chemicals by comparing the sensitivity, genotoxicity and cytotoxicity of lymphocytes to benzo(a)pyrene (BaP)- and cumene hydroperoxide (CumOOH)-induced in vitro oxidative challenge. Malondialdehyde and protein carbonyl levels, and oxidation of 2',7'-dichlorofluorescin diacetate were used as biomarkers of oxidative stress in lymphocytes. Following supplementation with BaP or CumOOH, time-dependent increases were observed in the production of all the markers after incubation for 12-48 h. However, we could not find any differences between GSTM1 null and positive genotypes. Furthermore, dose or time response experiments indicated that GSTM1-deficient cells were not more sensitive than control cells to BaP-or CumOOH-induced cell killing and micronucleus formation, although they were hypersensitive to BaP-inhibited cellular growth. The results suggest that lymphocytes from individuals with the GSTM1 null genotype are not abnormally susceptible to in vitro induced oxidant challenge, when exposed to CumOOH.

Are individuals with glutathione S-transferase GSTT1 null genotype more susceptible to in vitro oxidative damage?

Recent epidemiological studies proposed that glutathione S-transferase (GST) T1 null genotype was correlated with an increased susceptibility to diseases associated with oxidative stress, including cancer. A comparative study using erythrocytes from individuals with GSTT1 null genotype was carried out to determine how resistance to oxidative stress is affected by lack of this gene, and whether the GST status of a person is an important factor in risk toward oxidant chemicals. Malondialdehyde and carbonyl levels and fluorescence and chemiluminescence formation were used as biomarkers of oxidative stress in erythrocytes exposed in vitro to cumene hydroperoxide (CumOOH), an oxidizing agent. When peroxidation-dependent changes in these parameters were compared between GSTT1 null genotype and controls, who are both GSTM1 and GSTT1 positive, no significant differences were found between the two genotypes, although the erythrocytes of the GSTT1 null group had lower GSTT1 activity toward CumOOH. Our results indicate that erythrocytes from individuals with GSTT1 null genotype are not abnormally susceptible to CumOOH-induced oxidant challenge.

The effect of metformin on insulin receptors and lipid peroxidation in alloxan and streptozotocin induced diabetes.

Biguanides are used for the treatment of non-insulin dependent diabetes mellitus but there is no evidence for an improving action of biguanide on the enhancement of peripheral glucose disposal in type 1 diabetes. It is known that biguanide agents reduce the oxidation of free fatty acids. Using alloxan and streptozotocin (STZ) induced diabetic rats as a model for type 1 diabetes mellitus, we measured insulin binding capacity and plasma lipid peroxidation levels before and after metformin induction. There was a significant increase in insulin binding capacity and lipid peroxidation levels in alloxan and STZ diabetes compared to controls. We examined the effect of metformin on alloxan and STZ-induced diabetic rats. In alloxan-induced diabetes metformin (Met) treatment led to an increase in insulin receptor number in liver plasma membranes (before Met: 46.50 +/- 2.69, after Met: 76.00 +/- 3.39 fmol/mg, p < 0.001) and a decrease in plasma lipid peroxidation levels compared to the non-treated group (before Met: 1.85 +/- 0.53, after Met: 1.10 +/- 0.09 nmol MDA/ml, p < 0.05). In STZ-induced diabetic rats metformin treatment did not change the lipid peroxidation levels (before Met: 1.26 +/- 0.31, after Met: 1.38 +/- 0.44 nmol MDA/ml, p > 0.05) whereas it did increase the receptor numbers (before Met: 41.60 +/- 4.33, after Met: 63.40 +/- 8.64 fmol/mg, p < 0.002).

Effects of neurotensin on the healing of experimental anastomosis of the colon.

The leakage of colonic anastomoses increases perioperative morbidity significantly. The purpose of the study was to investigate the influence of neurotensin, an intestinal trophic peptide, on the healing of colonic anastomosis. Forty-two Wistar-albino rats were separated into three equal groups: Group 1 (control group)--segmental resection of the left colon and end-to-end anastomosis; Group 2 (dexamethasone group)--resection and anastomosis, plus 0.1 mg/kg/day of dexamethasone; Group 3 (neurotensin group)--same surgical procedure plus 300 micrograms/kg/day of neurotensin. Bursting pressure and tissue hydroxyproline content were determined as parameters of the anastomosis strength and healing on the 3rd and 7th days postoperatively. On the 3rd day, mean bursting pressures were 141.4, 146.7 and 73.1 (p = 0.0001) cm of water in the control group, dexamethasone and neurotensin groups respectively. On the 7th day, bursting pressures were measured as 237.4, 100.6 (p = 0.0001) and 72.7 (p = 10(-6)) cm of water, in the control group, dexamethasone and neurotensin groups respectively. Between the 3rd and 7th days, bursting pressures were increased significantly in the control group (p = 0.0001), decreased in the dexamethasone (p = 0.048), and maintained their lowest values in the neurotensin (p = 0.96) groups. On the 7th day, mean hydroxyproline levels were measured as 9.20, 3.30 (p = 0.007), 2.86 (p = 0.007) micrograms, in the control group, dexamethasone, and neurotensin groups respectively. Between the 3rd and 7th days, tissue hydroxyproline levels were increased significantly in the control group (p = 0.004), decreased in the dexamethasone (p = 0.03), and maintained their lowest values in the neurotensin (p = 0.87) groups. The anastomosis resistance to intraluminal pressure was weak, tissue collagen content was insufficient, and healing was inadequate in the dexamethasone and neurotensin groups in respect to the control group. We concluded that neurotensin impaired the healing, and affected the strength of the colonic anastomosis.

Localization of subthalamic nucleus borders using macroelectrode local field potential recordings.

Deep brain stimulation of the subthalamic nucleus (STN) is a highly effective treatment for motor symptoms of Parkinson's disease. However, precise intraoperative localization of STN remains a procedural challenge. In the present study, local field potentials (LFPs) were recorded from DBS macroelectrodes during trajectory to STN, in six patients. The frequency-vs-depth map of LFP activity was extracted and further analyzed within different sub-bands, to investigate whether LFP activity can be used for STN border identification. STN borders identified by LFPs were compared to border predictions by the neurosurgeon, based on microelectrode-derived, single-unit recordings (MER-SUA). The results demonstrate difference between MER-SUA and macroelectrode LFP recording with respect to the dorsal STN border of -1.00 ±0.84 mm and -0.42 ±1.07 mm in the beta and gamma frequency bands, respectively. For these sub-bands, RMS of these distances was found to be 1.26 mm and 1.06 mm, respectively. Analysis of other sub-bands did not allow for distinguishing the caudal border of STN. In conclusion, macroelectrode-derived LFP recordings may provide an alternative approach to MER-SUA, for localizing the target STN borders during DBS surgery.

Relationships between levels of estradiol and testosterone in seminal plasma and GSTM1 polymorphism in infertile men.

Glutathione S-transferase M1 (GSTM1) enzyme serves as a steroid-binding protein by its ability to bind to testosterone and estradiol. The levels of total estradiol and testosterone were measured by using an electrochemiluminescence immunoassay in serum and seminal plasma from 103 subjects including 62 subfertile patients. GSTM1 polymorphism was examined using polymerase chain reaction. The estradiol and testosterone levels in seminal plasma were not different in control and subfertile subjects. No role for GSTM1 enzyme as a steroid-binding protein seemed likely as there was also no significant difference in seminal plasma estradiol and testosterone levels according to GSTM1 genotype. Significant positive correlations were found between seminal estradiol and serum estradiol in infertile males, and between seminal testosterone and serum testosterone in fertile males, independent of GSTM1 genotype. GSTM1 polymorphism is not a genetic risk factor of seminal estradiol and testosterone levels in infertile males although further studies are warranted.

Increased glutathione conjugate transport: a possible compensatory protection mechanism against oxidative stress in obesity?

OBJECTIVE: To compare glutathione S-conjugate transport in obese and nonobese persons, and how glutathione S-conjugates are involved in the antioxidant status in obesity. MATERIALS AND METHODS: The efflux of glutathione conjugates and malondialdehyde (MDA) levels were measured in erythrocytes of obese (N = 33) and nonobese (N = 28) persons at every 30 min during a 120 min incubation time in vitro. 2,4-dinitrophenyl-S-glutathione (DNP-SG) represented the glutathione S-conjugate. RESULTS: The efflux of conjugate in erythrocytes from obese subjects (708 +/- 147 DNP-SG efflux nmol/ml erythrocytes/h) was significantly higher than that of control group (490 +/- 105 DNP-SG efflux nmol/ml erythrocytes/h) (P < 0.05). At all time points measured (30-120 min), there was an increase in DNP-SG efflux in obese group (P < 0.05). This is manifested by a decrease in cellular DNP-SG levels. The susceptibility of erythrocytes to in vitro 1-chloro-2,4-dinitrobenzene (CDNB)-induced oxidative stress were greater for cells of control group (P < 0.05), although hemolysis sensitivity of these cells are not different between both groups (P > 0.05). Following CDNB pretreatment, incubation of erythrocyte with vanadate, a DNP-SG transport inhibitor, resulted in an increase of MDA in both groups. However, in this case, the difference in susceptibility was not related to obesity. On the other hand, while erythrocyte glutathione level was lower in obese subjects (79% of control) than in controls (P < 0.05), the adenosine 5'-triphosphate (ATP) levels, the enzyme activities of glutathione S-transferase (GST) and the conjugation capacities of the erythrocytes were not different between groups (P>0.05). CONCLUSION: Obesity may increase erythrocyte glutathione conjugate transport independent from ATP and GST activity that may protect against MDA formation in vitro.

Inhibition of platelet function by GSTM1-null human peripheral lymphocytes exposed to benzo(a)pyrene-induced challenge.

Recent epidemiological studies proposed that the glutathione S-transferase (GST) M1-null genotype may contribute to diseases associated with oxidative stress. The genetic polymorphism exhibited by the GSTM1 may be an important factor in risk toward oxidant chemicals. In this study, we investigated the effect of GSTM1-null genotype in lymphocyte and oxidative stress-dependent inhibition of platelet aggregation. To determine whether GSTM1 deficiency is a genetic determinant of cell toxicity toward oxidant chemicals, lymphocytes were incubated in vitro with low levels of benzo(a)pyrene (BaP), cumene hydroperoxide (CumOOH), or trans-stilbene oxide that do not decrease cell viability, and were assessed for oxidative damage and for the lymphocyte-dependent inhibition of platelet response. Malondialdehyde and carbonyl levels, and the oxidation of cisparinaric acid, were used as biomarkers of oxidative stress in lymphocytes. Following stimulation by BaP or CumOOH, when peroxidation-dependent changes in these parameters were compared between the GSTM1-null genotype and the positive genotype, no significant differences were found between the two genotypes. On the other hand, preincubation of the lymphocytes with BaP or CumOOH attenuated their inhibitory action on ADP-induced platelet aggregation. However, our results indicate that lymphocytes of individuals with the GSTM1-null genotype have greater inhibitory activity on platelet function after exposure to BaP, but not CumOOH, although they are not more susceptible to in vitro oxidative stress.

The effect of melatonin administration on ethanol-induced lipid peroxidation in rats.

This study was carried out in order to determine the role of melatonin in preventing lipid peroxidation due to acute ethanol intoxication. Male Wistar Albino rats, 2.5-3 months old, were divided into two groups. Melatonin (in 1% ethanol, 2 mg kg-1 body weight) was given intraperitoneally (i.p.) for 21 days to experimental rats whereas controls received 1% ethanol only. On day 21, 6 g kg-1 body weight ethanol was injected to half of the animals in each group and the remainder were kept as corresponding controls. Animals were killed 5 h after ethanol injection. Malondialdehyde (MDA), reduced glutathione (GSH) and the antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) were determined in liver tissue homogenates. MDA levels were increased whereas GSH levels tend to decrease following alcohol injection. Melatonin administration prior to ethanol did not alter MDA and GSH levels of tissue and among antioxidant defence enzymes studied, only CuZn-SOD was found to be increased in animals that received melatonin + ethanol. According to the findings of this study, melatonin did not appear to have any direct effect on alcohol-induced lipid peroxidation.

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance.

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance.

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.