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.

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.

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.

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.

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.

Liposome-entrapped tyrosinase: a tool to investigate the regulation of the Raper-Mason pathway.

The effect of the entrapment of mushroom tyrosinase (EC 1.14.18.1) within liposomes on the enzyme activity and Km vs. L-3,4-dihydroxyphenylalanine is reported in the present work; the effect of cholesterol insertion within liposome membranes on the enzyme activity has also been studied. The oxidation rates of various monophenols and diphenols by free and liposome-integrated mushroom tyrosinase were measured and the oxidation latencies vs. different substrates investigated. The different substrates are apparently oxidized according to the properties of the substituents as electron donors or acceptors; the Km values vs. L-3,4-dihydroxyphenylalanine calculated on measuring O2 consumption are higher than those calculated on measuring the dopachrome production rates. It is interesting that natural substrates of tyrosinase are oxidized according to a negative catalysis by the liposome-entrapped enzyme; this point is discussed in relation to the well known cytotoxicity of some intermediates of the Raper-Mason pathway.

Age-dependent ultrastructural alterations and biochemical response of rat skeletal muscle after hypoxic or hyperoxic treatments.

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

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.

Alteration of glutathione transferase subunits composition in the liver of young and aged rats submitted to hypoxic and hyperoxic conditions.

In the present work, we have studied glutathione transferase (GST) activity and GST subunits distribution in the liver of young and aged rats kept under hypoxic or hyperoxic normobaric conditions as model of oxidative stress. A significant decrease of GST activity was detected in young hypoxic rat liver, whereas a significant increase occurred in aged hypoxic liver. No significant alteration of activity was obtained in both young and aged rat livers subjected to hyperoxic treatment. Substrate specificity measurements, SDS/PAGE analysis and reverse-phase HPLC, of GSH-affinity purified fractions were used to study the changes in the GST subunits pattern occurring in the liver of rat as a consequence of hypoxic and hyperoxic treatment. The results demonstrate that young and aged rat liver has a different constitutive GST subunit pattern which are markedly and differentially altered in hypoxia or hyperoxia. The hyperoxic treatment caused an increase of GST subunit 3 in aged, but not in young liver. In aged liver, both the hypoxic and hyperoxic treatment produced a decrease of GST subunit 4. After hypoxic treatment GST subunit 3 significantly increased in both young and aged liver. GST subunit 1a increased in both young and adult liver after hyperoxia. Following hypoxia a decrease of subunit 1a was seen in both young and aged liver. After hypoxic treatment, subunit 6 doubled in young, but not in aged, livers. It was concluded that the alterations in GST subunit expression occurring in the liver as a consequence of hypoxic or hyperoxic treatment respond to the necessity of a better protection of liver against the products of oxidative metabolism.

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.

Glyoxalases activity during Bufo bufo embryo development.

In this work we have investigated the expression of glyoxalase I (GLO I) and glyoxalase II (GLO II) activities during Bufo bufo embryo development and in some tissues of both male and female adult animals, in order to study how they correlate with cell proliferation and differentiation. The results show that both the activities are expressed at significant levels from the earliest developmental stages, reaching the highest values at the end of embryonic development (stage 25). The GLO I/GLO II ratio is very high at the beginning of the development and then gradually decreases as the development goes on. These data emphasize the importance of GLO I activity in the phases in which elevated cell division is taking place. In the differentiated tissues, a peculiar sexual dimorphism in both GLO I and GLO II activities, with higher values in female than in male, was found. GLO I embryonic activity levels are comparable to those found in female differentiated tissues, but significantly higher than those detected in male differentiated tissues. On the contrary, the GLO II activities found in the adult tissues were always higher than those found in embryos. These results further support the idea that high GLO I/GLO II ratios are a characteristic of the proliferative status, which assures a good scavenging action against the potentially cytotoxic and cytostatic effect of methylglyoxal.

Developmental aspects of Bufo bufo embryo glutathione transferases.

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Aging and detoxifying enzymes responses to hypoxic or hyperoxic treatment.

We studied the levels of antioxidant and detoxifying enzymes in the livers and lungs of young and old rats kept under hypoxic or hyperoxic conditions as models of oxidative stress. In particular, we investigated the levels of enzymes directly involved in active oxygen species scavenging (superoxide dismutase, catalase and glutathione peroxidase-selenium dependent) and enzymes challenged with detoxification processes (glutathione transferase, glyoxalase I and glutathione reductase) in order to obtain a wide comparative view of the defence strategies used with respect to the age of the animals. The results show that the responses of some protective enzymes in young rats are opposite to those of old ones. Some of the changes found appear mainly due to age, while others appear to be due only to the oxygen tensions and are independent of the aging process. The glutathione contents of the liver and lung from young and old rats under hypoxic and hyperoxic conditions were measured.

Effect of chronic hyperoxia on young and old rat carotid body ultrastructure.

Morphologic, physiological, and biochemical changes occur in the carotid body (CB) during postnatal development in relation to physiological requirements. Chronic normobaric hyperoxia attenuates the carotid chemosensory response to hypoxia. During aging there is less of a CB response to hypoxia, which results in a reduced ventilatory adaptation and chemosensory discharge. To test if the oxygen-sensitive mechanism is affected by chronic hyperoxia in an age-dependent fashion, we have studied structural and ultrastructural aspects of young and old rat CBs. Four groups of six male Wistar rats were used. One group of two-month-old rats and another of 25-month-old rats were kept at room air. The other two groups, age matched, were exposed to 98-100% O2, for 60-65 h, in a large Plexiglas chamber. The rats were anesthetized, CBs were fixed in situ with glutaraldehyde (2.5% in phosphate buffer. pH 7.4, 320 mOsm), and were prepared for electron microscopy. Young hyperoxic rats showed focal necrosis in type I cells, along with an increase of endoplasmic reticulum, Golgi apparatus, and of mitochondria volume, with loss of cristae. These changes were less pronounced in the older rat CBs compared with the young rats. In conclusion, hyperoxia seems to affect the oxygen-sensitive mechanism in the carotid body cells, and the reduced effects shown in the old rat CBs suggest an age-related decreased sensitivity to oxygen.

Effects of oral and transdermal hormone replacement therapy on lipoprotein(A) and lipids: a randomized controlled trial.

OBJECTIVE: Our purpose was to compare the effect of oral and transdermal hormone replacement therapy on lipoprotein(a) and other plasma lipids in healthy postmenopausal women. DESIGN: A total of 120 postmenopausal women were enrolled in a prospective randomized controlled study, and allocated either to transdermal 17 beta-estradiol (50 micrograms/day) or to oral conjugated estrogen (0.625 mg/day). Forty-one age-matched women were used as the reference group. Plasma lipids and lipoproteins were determined every 3 months and differences were sought by statistical analysis. RESULTS: Plasma lipoprotein(a) dropped after 3 months of treatment either with transdermal estradiol (p < 0.01) or oral estrogen (p < 0.01). Lipoprotein(a) was reduced by 12% and 22%, respectively. No further decreases were seen later on. Plasma total and low-density lipoprotein (LDL) cholesterol concentrations were decreased significantly with both treatments after 3 months of therapy. No difference was seen in the lowering effect on lipoprotein(a), LDL and total cholesterol concentrations between regimens. Plasma high-density lipoprotein (HDL) cholesterol and triglyceride concentrations increased throughout the study only in patients treated with oral estrogen. CONCLUSIONS: These data demonstrate that hormone replacement therapy reduces the concentration of lipoprotein(a) when given both orally and transdermally. The lowering effect is achieved quickly because the maximal effect is observed after 3 months of therapy.

Developmental aspects of detoxifying enzymes in fish (Salmo iridaeus).

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism. A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development. In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney. No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.

Changes of lipo-melanosome membrane leakage versus pH, charge and composition.

Liposome models of melanosomes (lipo-melanosomes) were used to investigate how phospholipid composition, charge and medium pH may affect the lipo-melanosome membrane permeability to active oxygen species or melanin synthesis intermediaries. Active oxygen accumulated only at pH 6.4 and was polarographically monitored using superoxide dismutase and/or catalase. Cholesterol appears to increase the O2- accumulation at pH 6.4 while incorporation of positive phospholipids within lipo-melanosomes results in the loss of latency with respect to tyrosinase substrate and intermediates of melanin synthesis.

Effect of methylglyoxal on Bufo bufo embryo development: morphological and biochemical aspects.

Methylglyoxal (2-oxopropanal) is a cytotoxic compound that can be formed endogenously as a by-product of glycolytic pathway; so its concentration is expected to increase when glycolysis activity increases such as during embryo development. In this work we study the effect of exogenous methylglyoxal on development and embryo viability during Bufo Bufo development and on the enzymes and cofactors involved in its detoxication process (glyoxalase I and II, reduced glutathione and glyceraldehyde 3-phosphate dehydrogenase). The results show that exogenous methylglyoxal does not affect the enzymatic pattern until stage 20, while it induces a significant activity decrease of the tested enzymes at stage 25. On the contrary methylglyoxal positively influences the reduced glutathione concentration at all the considered stages. At morphological and histological levels methylglyoxal causes a strong retardation of cell division in the early stages, that results in various abnormalities in the late development. In conclusion, methylglyoxal enters the embryo and is antiproliferative and teratogenic: the data further supports the hypothesis of the importance of the glyoxalase system in the process of cell growth and division.