Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms.

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H2O2 and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

Regular and Moderate Exercise Counteracts the Decline of Antioxidant Protection but Not Methylglyoxal-Dependent Glycative Burden in the Ovary of Reproductively Aging Mice.

Population aging results in urgent needs of interventions aimed at ensuring healthy senescence. Exercise often results in healthy aging, yet many molecular mechanisms underlying such effects still need to be identified. We here investigated whether the age-dependent accumulation of oxidative and methylglyoxal- (MG-) related molecular damage could be delayed by moderate exercise in the mouse ovary, an organ that first exhibits impaired function with advancing age in mammals. CD1 female mice underwent two- or four-month treadmill-based running through the transition from adult to middle age, when ovaries show signs of senescence, and markers of protection against reactive oxygen species (ROS) and MG were measured. The long-term exercise reduced the protein oxidative damage in the ovaries (P < 0.01), and this was linked to the preservation of the glutathione peroxidase protection against ROS (P < 0.001), as well as to the increased glutathione availability (P < 0.001). Conversely, even though the age-related deactivation of the MG-targeting systems was partially prevented by the long-term running programme (P < 0.001), exercised mice were not protected from the age-dependent glycative burden. In summary, lately initiated regular and moderate exercise limited some changes occurring in the ovaries of middle-aged mice, and this might help to develop nonpharmacological cointerventions to reduce the vulnerability of mammalian ovaries towards redox dysfunctions.

NAD(P)H oxidase p22(phox) polymorphism and cardiovascular function in amateur runners.

AIM: NAD(P)H system represents the major source of superoxide production at cardiovascular (CV) level. It has several genetic variants: in particular, the C242T polymorphism of its p22(phox) subunit is associated with a different oxidase activity, being the T allele related to a lower superoxide production. Although several authors investigated the protective effect of T allele in CV diseases, only few data are available on its functional role in physiological conditions. The aim of our study was to investigate the relationship between the p22(phox) C242T polymorphism and CV function in amateur runners. METHODS: Seventy-three male amateur runners were screened for CYBA polymorphism. CV analysis was performed by echocardiographic-Doppler examination and by PulsePen tonometer assessment. RESULTS: The genetic subgroups (CC and CT/TT) did not differ for VM O(2max) and cardiac dimension. Nevertheless, T carriers (n = 40) were characterized by a more efficient myocardial contraction and left ventricular (LV) filling, as evidenced by significant higher values of the midwall fractional shortening, systolic excursion of the tricuspid annular plane and of early/late diastolic wave velocities ratio and by a lower E wave deceleration time. Pulse wave velocity and augmentation index, parameters related to the arterial stiffness, were higher in CC subjects compared with CT/TT also when the analysis was adjusted for weight and diastolic pressure. CONCLUSION: In amateur runners, CYBA variants may influence both systolic and diastolic function and arterial stiffness. We suppose that the lower oxidative activity that characterizes 242T subjects may positively influence the excitation-contraction and arterial-ventricular coupling mechanisms, thus leading to a more efficient CV function.

Differential impact of acute bout of exercise on redox- and oxidative damage-related profiles between untrained subjects and amateur runners.

Despite the demonstrated exercise-induced increase in reactive oxygen species (ROS) production, growing epidemiological evidence indicates that habitual, moderate physical activity reduces the incidence of several oxidative stress-based diseases. This apparent paradox can be explained taking into account that ROS produced during repeated exercise bouts may act as mild stressors able to trigger physiological and biomolecular hormetic responses through a number of redox-sensitive transcription pathways. Unfortunately, much more limited information is available from general population-based research, which could better reflect the condition of common people interested in achieving and maintaining good fitness levels. The present work aimed at investigating whether and how exercise-related habits in non-professional regular runners (n=33) can affect the systemic anti-oxidative capacity, and the resting serum levels of typical lipid peroxidation-related by-products and oxidatively-damaged proteins, in comparison with untrained sedentary individuals (n=25). We also analyzed in both groups the redox response elicited by a modified Bruce-based maximal exercise test on the same parameters. Our findings indicated that long-term regular and moderate practice of aerobic physical activity can increase antioxidant defense systems, lower the resting protein oxidation processes and reduce the immediate up-regulation of lipid-targeting oxidative stress in response to an acute bout of exercise.

NAD(P)H oxidase and pro-inflammatory response during maximal exercise: role of C242T polymorphism of the P22PHOX subunit.

Intense exercise induces a pro-inflammatory status through a mechanism involving the NAD(P)H oxidase system. We focused our attention on p22phox, a subunit of the NAD(P)H oxidase, and on its allelic polymorphism C242T, which is known to affect the functional activity of the enzyme. We investigated whether the p22phox C242T variants exhibit systemic effects in healthy subjects by analyzing the proinflammatory and cardiocirculatory responses to physical exercise in endurance athletes. The group of study consisted of 97 long distance runners, 37 +/- 4.4 yrs of age, with similar training history. The subjects underwent a maximal stress test during which both inflammatory and cardiopulmonary parameters were monitored. Our results demonstrate that T allele deeply influences the neutrophil activation in response to intense exercise, since T carriers were characterized by significantly lower release of myeloperoxidase (MPO), a classical leukocyte derived pro-inflammatory cytokine. In addition, the presence of T allele was associated with a higher cardiopulmonary efficiency as evidenced by a significantly lower Heart Rate (HR) at the peak of exercise and, when a dominant model was assumed, by a higher maximal oxygen uptake (VO2 max). On the other hand, no effects of 242T mutation on the plasmatic total antioxidant capacity (TAC) and on the cortisol responses to the physical exercise were detected. In conclusion, our data support a systemic role for p22phox C242T polymorphism that, modifying the intensity of the inflammatory response, can influence the cardiovascular adaptations elicited by aerobic training. These results contribute to support the hypothesis of a systemic effect for the C242T polymorphism and of its possible functional rebound in healthy subjects.

Female reproductive dysfunction during ageing: role of methylglyoxal in the formation of advanced glycation endproducts in ovaries of reproductively-aged mice.

Reproductive dysfunction with ageing has been so far extensively characterized in terms of depletion of ovarian follicles and reduced ability to produce gametes competent for fertilization. Nevertheless, molecular mechanisms underlying this process are still poorly understood. In the present study we addressed the hypothesis that methylglyoxal (MG), a major precursor of Advanced Glycation Endproducts (AGE), may contribute to molecular damage occurring during ovarian ageing. Our results showed that the biochemical activity of glyoxalase 1, the main component of the MG scavenging system, is significantly decreased in ovaries from reproductively-aged mice in comparison with the young group. This effect was associated with decreased expression at protein and RNA level of this enzyme and increased intraovarian level of MG. MG-arginine adducts argpyrimidine as detected with a specific antibody was found to accumulate with ageing in specific ovarian compartments. Separation of ovarian proteins by 2D gels and Western blotting revealed an approximate 30-fold increase in the extent of protein glycation in aged ovaries along with the appearance of eight argpyrimidine modified proteins exclusive for the aged group. In conclusion, the present results show that impaired MG detoxification causing relevant damage to the ovarian proteome might be one of the mechanisms underlying reproductive ageing and/or ageing-like ovarian diseases.

Aerobic performance and antioxidant protection in runners.

Reactive oxygen species (ROS) production is known to increase as a result of muscular contractile activity and this phenomenon may perturb the fine-controlled cellular redox homeostasis within cells and tissues. We studied the possible correlations between individual aerobic performance-related factors and the oxidative stress markers profile in the serum of thirty-five endurance male runners that experienced a modified Bruce-based maximal graded exercise test. Our investigation assessed the systemic levels of malondialdehyde (MDA), protein carbonyl content (PCC) and total antioxidant status (TAS). We found that redox-related parameters and aerobic performance indicators were correlated. Indeed, significant negative associations between TAS and PCC (r-value -0.7, p<0.001) and between TAS and total protein content (r-value -0.4, p=0.005) were observed. A significant positive association between total protein and PCC (r-value 0.4, p=0.012) was also revealed. Only a trend of negative correlation between serum total protein and anaerobic threshold (r-value -0.3, p=0.07) was found. Interestingly, different responses in MDA levels were elicited by the ergometric test as a function of the individual anaerobic threshold. High aerobic capacities may be promising anthropometric factors indicative of adapted biochemical environments featuring enhanced protection against the oxidative challenge elicited by both regular endurance training and single intense exercise bouts.

Neuronal response of peroxisomal and peroxisome-related proteins to chronic and acute Abeta injury.

The central role of peroxisomes in ROS and lipid metabolism and their importance in brain functioning are well established. The aim of this work was to study the modulation of peroxisomal and peroxisome-related proteins in cortical neurons in vitro challenged with chronic or acute Abeta treatment, in order to investigate whether peroxisomes represent one of the cellular target of Abeta in these cells. The expression of peroxisomal (PMP70, catalase, acyl-CoA oxidase and thiolase), peroxisome-related (PPARalpha, insulin-degrading enzyme) and anti-oxidant (SOD1, SOD2, GSTP1) proteins was studied. The results obtained, demonstrating an early upregulation of the peroxisomal proteins during the chronic challenge, followed by their dramatic impairment after acute challenge, suggest that peroxisomes represent one of the first line of defence against Abeta-mediated oxidative injury. Our results support the notion that substances able to activate PPARalpha and/or to induce peroxisomal proliferation may constitute a novel preventive and/or therapeutic tool against neurodegenerative diseases.

Gynecological endoscopy for symptomatic endometriosis.

The best available evidence on surgery for endometriosis-associated pain has been reviewed in order to define the benefit of various interventions in the most frequently encountered clinical conditions, and discuss the robustness of the reported data in light of the quality of the relevant study design. Methodological drawbacks limit the validity of observational, non-comparative studies on the effect of laparoscopy for stage I to IV disease. The results of three randomized, controlled trials, indicate that the absolute benefit increase of destruction of lesions compared with sham operation in terms of proportion of women reporting pain relief was between 30% and 40% after short follow-up periods. The effect size decreased with time and the reoperation rate, based on long-term follow-up studies, was as high as 50%. In most case series on excisional surgery for rectovaginal endometriosis, substantial short-term pain relief was experienced by about 70-80% of the subjects who continued the study. However, at one-year follow-up approximately 50% of the women needed medical treatments. Major complications were observed in 3-10% of the patients. Medium-term recurrence of lesions was observed in about 20% of the cases, and around 25% of the women underwent repetitive surgery. Routine complementary performance of denervating procedures cannot be recommended based on the quality of the available information, as only a few symptomatic patients complain of exclusively midline, hypogastric pain. Pain recurrence and reoperation rates after conservative surgery for symptomatic endometriosis are high and probably underestimated. Clinicians and patients should be aware that the expected benefit is operator-dependent and, especially in complex conditions, acceptable results can be assured in referral centers.

The Cosmic Silence experiment: on the putative adaptive role of environmental ionizing radiation.

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to "reference" environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.

Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells.

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women > or =38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.

High levels of antioxidant enzymatic defence assure good protection against hypoxic stress in spontaneously diabetic rats.

Recent data from literature report that reactive oxygen species (ROS) seem to play a crucial role in the etiology of both types I and II diabetes. This may render diabetic individuals more prone to oxidative injury when challenged with hypoxic stress. It is in fact well known that many diabetic complications cause ischaemic episodes, with a consequent reduction in oxygen supply to various tissues and organs. To check this hypothesis, in this work we tested type I diabetic individuals' antioxidant capability towards a hypoxic-mediated oxidative challenge. In particular, spontaneously diabetic and age-matched non-diabetic biobreeding (BB) Wistar rats were submitted to chronic normobaric hypoxia, and the response of antioxidant enzymes, as well as redox-sensitive transcription factor NF-kappaB and p53, were monitored. Results show that diabetic subjects present a dramatic enhancement in the major antioxidant enzymes activities, thus supporting the notion of diabetes-related changes in cellular redox status. This allows diabetic individuals to counteract hypoxia-mediated oxidative challenge better than the non-diabetic counterpart. Also the behaviour of both the redox-sensitive nuclear transcription factor NF-kappaB and p53 protein in response to hypoxic stimulation seems to support the hypothesis of a better ROS scavenging efficiency in diabetics under hypoxic conditions. In conclusion, high levels of antioxidant enzymatic defences in diabetic BB rats reflect a positive adaptive response able to assure an efficient protection not only against chronic, diabetes-mediated reactive oxygen species (ROS) overproduction, but also versus further oxidative damage.

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes.

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.

Influence of a low background radiation environment on biochemical and biological responses in V79 cells.

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Developmental expression and distribution of amphibian glutathione transferases.

This work is aimed at detecting the expression and location of embryonic Bufo bufo GST (bbGSTP1-1) and adult B. bufo GST (bbGSTP2-2) during toad development, in order to assign a putative role to these enzymes also on the basis of their compartmentalization and to verify whether during the premetamorphic liver ontogeny the bbGSTP2-2 form appears. This study was also performed in the adult liver (the primary site of Pi class GST expression) and in the mature ovary, to discern if the embryonic form derives from maternal form. The results show that the embryos and the ovary express only bbGSTP1-1. Moreover, bbGSTP1-1 distribution is the same both in the early embryos and in the ovary: this strongly suggests that bbGSTP1-1 is of maternal origin. As development goes on, a wide distribution of bbGSTP1-1 all over the differentiating organs is observed. The embryonic liver expresses exclusively the bbGSTP1-1 form, while the adult liver is highly positive only towards the bbGSTP2-2 form. This implies that the switch towards the adult bbGSTP2-2 form occurs in metamorphic or postmetamorphic phases and that the detoxication metabolic requirements of the embryo may be completely fulfilled by the bbGSTP1-1 isoenzyme.

Antioxidant and GSH-related enzyme response to a single teratogenic exposure to the anticonvulsant phenytoin: temporospatial evaluation.

BACKGROUND: It has been proposed that the anticonvulsant drug phenytoin (PHT) requires bioactivation to reactive intermediate(s) to achieve its recognized teratogenic potential and that embryonal detoxification power may play a fundamental role in the teratogenic response. On this basis, we sought to investigate the potential effects of a teratogenic exposure to PHT on the activities of antioxidant and GSH-related detoxifying enzymes in gestational murine tissues. METHODS: Pregnant Swiss mice were injected intraperitoneally with 0 (vehicle) or 65 mg/kg of PHT on gestation day (GD) 12 (plug day = GD 1). Biochemical determinations, including activities of glutathione transferase, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, catalase, and superoxide dismutase, were carried out on maternal and embryonic/fetal livers and in placentas on GD 14 and 19. RESULTS: The major findings of this study show that (1) organogenesis-stage conceptal tissues have detectable levels of all the tested enzymes; (2) most of the embryonic liver and placental enzymes investigated undergo a significant induction within 48 hr (GD 14) after PHT administration; and (3) in the same tissues a down-regulation of enzyme activities is noted near term (GD 19). CONCLUSIONS: Overall, these findings show that teratogenic exposure to PHT is associated with a modulation of reactive-intermediates-scavenging enzyme activities, and provide further support for role of generation of reactive intermediates in PHT-induced teratogenesis.

[Multicenter study on the effectiveness of Tension Vaginal Tape (TVT) in the treatment of stress urinary incontinence]. / Studio multicentrico sull'efficacia del Tension Free Vaginal Tape (TVT) nella terapia della incontinenza urinaria da sforzo.

BACKGROUND: The aim of the study was to evaluate the efficacy of Tension Free Vaginal Tape (TVT) for the surgical treatment of stress urinary incontinence. METHODS: The design was an open multicenter study including six Italian hospitals. Between January 1998 and November 1999, 429 stress incontinent women were enrolled in the study. Before surgery subjects had been studied through their history, urine culture, physical examination, cotton swab test, cough provocation test and urodynamic evaluation including: uroflowmetry, water cystometry and urethral profilometry. Incontinence inconvenience has been quantified through a 10-grade visual analogue scale (VAS). Postoperatively patients were assessed after 6, 12 and 24 months. RESULTS: The mean age of the patients considered was 57 years (range 31-83) and 78 of them had undergone a previous operation for the treatment of stress urinary incontinence or genital prolapse. Out of the 429 patients, 371 were followed for a minimum of 6 months, 11 were lost to follow-up and 47 had been operated recently. After surgery 355 subjects (96%) were subjectively cured and no leakage of urine was observed in 97% of the patients during the postoperative cough provocation test. CONCLUSIONS: This study carried out on a great number of patients demonstrates that TVT is a safe and effective procedure for the treatment of stress urinary incontinence.

Biochemical and ultrastructural alterations in rat after hyperoxic treatment: effect of taurine and hypotaurine.

The cell ultrastructure and some detoxifying enzyme activities were studied in skeletal muscles of young rats kept for 84 h under normobaric hyperoxia (95% O2) or normoxia as control. Rat were injected i.p.. Every 12 h either with 1 ml saline, 1 ml saline+30 mg hypotaurine or 1 ml saline+30 mg taurine. Ultrastructural observation revealed an highly protective effect on tissue damages due to hyperoxia in taurine-treated rats and, at less extent, in hypotaurine-treated ones. Enzymatic assays suggest a different mechanism of the two molecules in their protective action.

Hypotaurine protection on cell damage by singlet oxygen.

Singlet oxygen (1O2), generated by irradiating methylene blue, is toxic to melanoma cell cultures. Hypotaurine is known to scavenge efficiently singlet oxygen; the addition of hypotaurine (800 microM) to the medium during irradiation of the dye produces a greater protective effect on cells than taurine added at the same concentration. The assay of some detoxifying enzymatic activities indicate a different mechanism of protection of the two molecules: taurine induces an efficient detoxifying enzymatic action with respect to the control; hypotaurine exerts its effect greatly by specifically scavenging singlet oxygen.