A new mitochondrial probe combining pyrene and a triphenylphosphonium salt for cellular oxygen and free radical detection via fluorescence lifetime measurements.

To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP+) as a mitochondrial vector forming PB-TPP+ probes. Two pyrene isomers with the n-butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP+-based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP+ vector, a warning to the user of this well-known vector.

Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines.

OBJECTIVE: Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. RESULTS: Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1ß, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research.

A comparative study of Caesalpinia bonduc (L.) Roxb. root extracts on sexual behaviour in male Wistar rats.

Caesalpinia bonduc is among the traditionally used plant in Benin, for its enhancement of male sexual activity. This study was undertaken to investigate the potential effect of C. bonduc root extracts on sexual behaviour of male Wistar rats. For that, thirty-six rats were allocated into six groups and orally treated with dimethyl sulfoxide (control), Sildenafil citrate (standard) and C. bonduc root extracts (hexane, ethyl acetate, ethanol and methanol) orally for twenty-eight days. Sexual behaviour parameters such as intromission frequency, intromission latency, mount latency and mount frequency were evaluated on day 0, 14 and 28. After the study completion, the body and reproductive organ weights as well as testicular histology and testosterone level were recorded. C. bonduc root extracts treatments had no significant effect on the body weight of rats. Enhancement in sexual behaviour was observed in ethanolic extract treated rats. An significant increase in mount frequency and intromission frequency as well as significant reduction in mount latency and intromission latency were noticed for ethanolic extract. The same extract caused an improvement in testosterone levels, relative testes weight and histological architecture. The findings demonstrate the aphrodisiac potential of C. bonduc root and lend support to the folkloric use as aphrodisiac.

Peptide Vectors Carry Pyrene to Cell Organelles Allowing Real-Time Quantification of Free Radicals in Mitochondria by Time-Resolved Fluorescence Microscopy.

Real-time quantification of reactive nitrogen and oxygen species (ROS) in cells is of paramount importance as they are essential for cellular functions. Their excessive formation contributes to the dysfunction of cells and organisms, ultimately leading to cell death. As ROS are mostly produced in the mitochondria, we have synthesized a fluorescent probe able to reach this organelle to detect and quantify, in real time, the variation of ROS by time-resolved microfluorimetry. The new probes are based on the long fluorescence lifetime of pyrene butyric acid (PBA). Two PBA isomers, attached at their 1- or 2-positions to a peptide vector to target mitochondria, were compared and were shown to allow the measurement of free radical species and oxygen, but not non-radical species such as H2 O2 .

Synthetic Sensors for Reactive Oxygen Species Detection and Quantification: A Critical Review of Current Methods.

SIGNIFICANCE: Redox reactions play important roles in both physiological and pathological processes, highlighting the importance of quantifying and localizing intracellular redox-active components. Most research has focused on direct investigation of reactive oxygen species (ROS). Intensity-based fluorescent methods are very sensitive and easy to use, but they lack specificity and can produce artifacts. In this article, we focus on synthetic sensors, describing experimental pitfalls associated with their use. We also present alternative methods for the detection of free radicals. RECENT ADVANCES: New approaches have been developed to overcome the main artifact of intensity-based methods: spurious changes in fluorescence intensity caused by oxidation. These new approaches are based on analytical measurements of the oxidized sensors or techniques that are not susceptible to oxidation, such as electron spin resonance and fluorescence lifetime-based methods. Regardless of the approach, the need for detection of ROS on the subcellular level, especially in the mitochondria, has motivated the development of new probes. CRITICAL ISSUES: Flow cytometry systems and confocal microscopes are now available to the majority of biologists, and commercially available probes are, therefore, more widely used. The fact that these new applications are cited in thousands of publications makes these sensors even more attractive. FUTURE DIRECTIONS: The field of ROS detection by synthetic sensors continues to expand, bringing needed additional research to the development of robust techniques that are applicable both in vitro and in vivo. Antioxid. Redox Signal. 25, 520-533.

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells.

The implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 µM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro.

Some pathophysiological insights into ovarian infestation by Myxobolus sp. (Myxozoa: Myxosporea) in Clarias gariepinus (Clariids: Silurids) from Bénin (West Africa).

Mature female specimens of the catfish Clarias gariepinus originating from Ouémé River (Benin) were investigated into ovarian myxozoan parasites. Spores of Myxobolus sp. (Myxozoa: Myxosporea) were found encrusted in the whitish color oocytes which present fat dot aspect in the gonads. The pathological investigation by electron microscopy revealed that maturation and multiplication of spores induced lytic action, deformation and dysfunction of the oocyte internal structures. No host inflammatory reaction was observed, while yolk, lipid, mitochondria, and other oocyte components were degenerated inducing empty area in the oocyte and could lead to castration in case of wide infestation. The mean prevalence was 19.79%. No significant difference was observed within seasonal prevalence (χ(2) = 1.771; df = 3; p > 0.05). Though the host length classes ranging from 35 to 39 cm and 40 to 45 cm were more infected, difference was not significant (χ(2) = 2.273; df = 4; p > 0.05) within them. The spores are ovoid in shape with two polar capsules which are equal in size, pyriform, and converging in anterior part of spore with four to five polar filament turns. Spore body are (11.47 ± 0.67) × (8.19 ± 0.52) µm length by width while polar capsule size are (4.24 ± 0.25) × (3.07 ± 0.28) µm and located in the first third portion of the spore. The molecular approaches are still running for accurate identification of this parasite.

Histopathological and ultrastructural studies of a Henneguya species (Myxozoa: Myxosporea) infesting the intestine of Clarias gariepinus from Benin (West Africa).

Fish culture is the best alternative to fill the gap between natural fish catches and estimated needs of populations in animal protein consumption. In West Africa, this goal required to have suitable fishes for aquaculture which are Clariidae and Tilapia. Clarias gariepinus (Clariidae) fetches a higher price than tilapias as it can be sold alive at the market but a high infestation by Henneguya leads to decrease this commercial value. Those reasons lead us to perform studies on seasonal variations, histopathological aspects and life cycle of Henneguya sp. infecting the intestine of C. gariepinus using light and electron microscope. From November 2011 to December 2012, 339 specimens were collected from Ouémé River (Benin) and examined. An overall prevalence of 7.37 % was recorded for plasmodia of Henneguya sp. Parasite occurrence did not vary significantly between seasons (χ(2) = 12.235; df = 3; p > 0.05), nor sexes (χ(2) = 2.992; df = 7; p > 0.05) while differences were significant between classes of weight (χ(2) = 39.929; df = 5; p < 0.05). The highest prevalence was recorded in host ranging from 300 to 374 g. Histopathological analysis showed that the mass continuous development of the plasmodium produced thickening of the intestine wall and compressed neighboring tissues and destroyed villi and smooth muscle layers. The stages of the parasite development including sporogenesis, capsulogenesis, and valvogenesis were asynchronous. Investigations are still running by molecular approaches in order to identify accurately this species.

Time-resolved microfluorimetry: an alternative method for free radical and metabolic rate detection in microalgae.

Oxidative stress leads to an increase in the production of reactive oxygen species in cells and can be induced by environmental factors. To study free radical production in living microalgae, we use time-resolved microfluorimetry, a technology adopted from research on mammalian cells. In contrast to fluorescent probe-based measurements that rely on intensity changes, our sensor detects the presence of free radicals through collisional quenching, and is insensitive to most artifacts commonly observed with intensity-based methods. A new probe, 1-pyrenebutanol allows estimation of free radicals production in the green microalga Tetraselmis ssp., for the first time. In addition, our method monitors simultaneously metabolic rate (through bound-free NAD(P)H ratio). Our results show that free radical production in algal cells is correlated to algal aging, and that during cell growth phases both intracellular free radicals and metabolic activity increase. Concerning thermal stress, we observe that rapid and low temperature changes (<10°C) induce instantaneously an increase in ROS production. Our findings provide new insights into the production of free radicals in response to environmental stresses in unicellular green algae.

Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells.

We investigate the relative importance of the different mechanisms of Adriamycin, an anthracycline, and their interrelations, in particular the link between cell cycle arrest, cell death, and generation of reactive oxygen species (ROS) that is suspected to be the origin of cardiotoxic side-effects. We introduced a lifetime fluorescence based technology and used videomicrofluorometry, two efficient analytical methods. We show that depending on the doses and time after incubation, ADR will not reach the same compartments (nucleus, mitochondria, cytosol) in the cells, having consequences on the production of ROS, growth arrest pathways and cell death pathways.

Reduced metabolic rate and oxygen radicals production in stored insect sperm.

Females of internally fertilizing species can significantly extend sperm lifespan and functionality during sperm storage. The mechanisms for such delayed cellular senescence remain unknown. Here, we apply current hypotheses of cellular senescence developed for diploid cells to sperm cells, and empirically test opposing predictions on the relationship between sperm metabolic rate and oxygen radical production in an insect model, the cricket Gryllus bimaculatus. Using time-resolved microfluorimetry, we found a negative correlation between metabolic rate (proportion of protein-bound NAD[P]H) and in situ intracellular oxygen radicals production in freshly ejaculated sperm. In contrast, sperm stored by females for periods of 1 h to 26 days showed a positive correlation between metabolic rate and oxygen radicals production. At the same time, stored sperm showed a 37 per cent reduced metabolic rate, and 42 per cent reduced reactive oxygen species (ROS) production, compared with freshly ejaculated sperm. Rank differences between males in ROS production and metabolic rate observed in ejaculated sperm did not predict rank differences in stored sperm. Our method of simultaneously measuring ROS production and metabolic rate of the same sample has the advantage of providing data that are independent of sperm density and any extracellular antioxidants that are proteins. Our method also excludes effects owing to accumulated hydrogen peroxide. Our results unify aspects of competing theories of cellular ageing and suggest that reducing metabolic rate may be an important means of extending stored sperm lifespan and functionality in crickets. Our data also provide a possible explanation for why traits of ejaculates sampled from the male may be rather poor predictors of paternity in sexual selection studies and likelihood of pregnancy in reproductive medicine.

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions.

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: a comparison between hypoxic and normoxic cells.

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O(2)) and normoxic (21% O(2)) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.

Measurements of calcium with a fluorescent probe Rhod-5N: Influence of high ionic strength and pH.

We describe a new method for the spectroscopic determination of high calcium concentration using a fluorescent probe Rhod-5N. This method was investigated in order to be utilized in high ionic strength solution, such as seawater. The probe is fluorescent when bound to calcium, LM, but not as the free form L. The dissociation constant of the equilibrium (0.14mM) was determined at several ionic strengths, i.e. in the absence and in the presence of additional ions (0.7M NaCl). The influence of pH was studied. In order to correctly model the experimental data, we included a new fluorescent compound: LHM (calcium bound protonated probe). The first acidity constant (0.02muM) and the second dissociation constant (4.5mM) were calculated. A useful range for the determination of calcium concentration is provided. Such a method is fast and easy to carry out.

Variation of 1-pyrenebutyric acid fluorescence lifetime in single living cells treated with molecules increasing or decreasing reactive oxygen species levels.

1-Pyrenebutyric acid (PBA) is a fluorescent probe whose fluorescence lifetime depends on local oxygen and free radical concentrations. We propose to use PBA fluorescence lifetime to quantify reactive oxygen species (ROS) in biological samples. Time-resolved microfluorimetry was used to record the fluorescence decay of single living cells loaded with this probe. We measured intracellular PBA fluorescence lifetimes and reduced nicotinamide adenine dinucleotide phosphate intensities under various oxygen concentrations. To confirm the feasibility of the new method, CCRF-CEM cells were treated with drugs that are known to increase or decrease ROS production. After treatment with adriamycin, we observed a decrease of PBA fluorescence lifetime. This corresponded to an increase of ROS concentration (80%). After treatment with cysteamine, we observed a reduction of the ROS concentration by 67%. Moreover, addition of exogenous H(2)O(2) solution resulted in a decrease of PBA fluorescence lifetime due to a raising of the intracellular ROS concentration. These results support our hypothesis linking a part of PBA fluorescence lifetime variations to intracellular fluctuation of ROS.

Effect of adriamycin treatment on the lifetime of pyrene butyric acid in single living cells.

We investigated the fluorescence lifetime of pyrene butyric acid (PBA) using various O2 concentrations in cells. Both in living and freshly fixed cells, PBA lifetime decreased with oxygen concentration. We recorded decay curves in single cells and measured PBA lifetime and NAD(P)H intensity values. Under nitrogen atmosphere, the probe lifetime differences (199 and 209 ns in living and freshly fixed cells, respectively) suggest a supplemental pathway for the deactivation of the probe when the cell functions are not stopped. We propose reactive oxygen species (ROS) to be the additional quenchers that cause this decrease. We further studied the effect of drugs generating ROS the anthracycline doxorubicin (adriamycin). For living cells, PBA lifetime decreased after adriamycin (ADR) treatment (200 and 1000 ng/ml). This supports our hypothesis that under nitrogen atmosphere and for freshly fixed cells, PBA lifetimes increase to an unchanging value due to absence of quenchers.

Lifetime of fluorescent pyrene butyric acid probe in single living cells for measurement of oxygen fluctuation.

We study the fluorescence lifetime of the well-known 1-pyrene butyric acid (PBA) to assess oxygen concentrations in living cells. The behavior of the probe is first studied in water, ethanol, protein solution and liposome suspension. The Stern-Volmer plot of these solutions is linear, and the bimolecular reaction rate constant agrees with previous observations. In single living cells, the PBA lifetime decreases with oxygen concentration (185 to 55 ns). The probe lifetime differences between living cells and liposome suspension, especially under nitrogen atmosphere, suggest a supplemental pathway for the deactivation of the probe. We simplify further the complex living cells system by stopping the cell functions and studying freshly fixed cells. In this case, we obtained an increase of PBA lifetime under nitrogen atmosphere (215 ns).

Microfluorometric study of oxygen dependence of (1"-pyrene butyl)-2-rhodamine ester probe in mitochondria of living cells.

The access to oxygen concentration is of importance in various organelles of living cells, especially in mitochondria. A new probe, (1"-pyrene butyl)-2-rhodamine ester, was designed to target this organelle. We present here the properties of the probe in such an environment. Microspectrofluorometry confirms the localization of the probe in the mitochondrial environment at low doses (1 microM). At these doses, the cell toxicity experiments show no effect on the cell growth. The emission spectrum measured at an excitation wavelength of 340 nm (pyrene centered) indicates energy transfer from the pyrene to the rhodamine chromophore, as also observed in an ethanol solution. With excitation at 337 nm, the excited state decays biexponentially with lifetime decays of 6-9 ns and 90 ns. The first corresponds to the intrinsic fluorescence of the cell and the latter corresponds to the pyrene chromophore. In degassed conditions the pyrene lifetime decay increases up to 130 ns. Under an oxygen atmosphere the lifetime decays decrease to 62 ns. The lifetime changes with the oxygen concentration were compared with the range obtained during our previous study in ethanol solution (5-220 ns). The observed differences were interpreted by studying the lifetime of the probe in simplified environments, liposome suspensions and protein solutions. In this paper we show that the new probe can be used to measure the fluctuation of oxygen concentration in the surroundings of mitochondria.

Intervalence Electron Transfer in Mixed Valence Diferrocenylpolyenes. Decay Law of the Metal-Metal Coupling with Distance.

A series of diferrocenylpolyenes of general formula Fc(CH=CH)(n)()Fc with n = 1-6 (Fc = ferrocenyl group) has been prepared and studied from the point of view of intervalence transitions in the mixed valence state. Well-resolved intervalence transitions are observed in dichloromethane upon partial electrolytic oxidation. Comproportionation constants have been determined from redox titration data and in some cases from electrochemical wave splitting. The corrected spectra of the mixed valence species have been deconvoluted to extract the parameters (position, intensity, width) of the intervalence bands, which allowed the determination of the metal-metal coupling (V(ab)) through the bridging unit using Hush's formula. The decay of V(ab) with distance is close to an exponential law with an exponent of 0.087 Å(-)(1), constituting one of the smallest attenuations reported so far. A small deviation to the exponential law is detected.