Antiproliferative effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in human leukemia cell lines

The antiproliferative activity of lectins Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) were studied using human leukemia MOLT-4 and HL-60 cell lines. It was revealed that both ConA and ConBr were markedly cytotoxic to cells using MTT and NAC assays. The IC₅₀ values were approximately 3 and 20 μg/mL for ConA and ConBr, respectively, for both MOLT-4 and HL-60 cells. However, in normal human peripheral blood lymphocytes, the lectins were not cytotoxic, even when tested at concentrations as high as 200 μg/ml. Using comet assay, the lectins produced a rate of DNA damage exceeding 80% in MOLT-4 and HL-60 cells. Fluorescence analysis revealed the morphology characteristic of apoptosis, with low concentrations of apoptotic bodies and fragmented DNA (5 μg/ml). Flow cytometric analysis demonstrated an accumulation of cells in the sub-G1 cell cycle that is characteristic of DNA fragmentation, and a decrease in membrane integrity at high concentrations. Lastly, we evaluated the alterations in mitochondrial potential that reduced after treatment with lectins. Our results indicate that ConA and ConBr inhibited cell proliferation selectively in tumor cells and that apoptosis was the main death mechanism. Therefore, lectins can be considered a class of molecules with a high antitumor activity potential.     

Antidepressant-like effect of lectin from Canavalia brasiliensis (ConBr) administered centrally in mice

This study investigates the action of the central administration of the lectins isolated from Canavalia brasiliensis seeds (ConBr) and from Canavalia ensiformes seeds, (Concanavalin A, ConA) in the forced swimming test (FST) in mice. ConBr (1-10 micro g/site, i.c.v.), but not ConA, produced a decrease in the immobility time in the FST (observed at the time points 15, 30, 60 and 120 min after the injection), without changing the locomotor activity in the open-field test. The effect of ConBr in the FST was dependent on its protein structure integrity. ConBr (0.1 micro g/site, i.c.v.) caused a potentiation of the action of fluoxetine, a selective 5-HT reuptake inhibitor. The anti-immobility effect elicited by ConBr (10 micro g/site, i.c.v.) in the FST was prevented by the pretreatment of mice with pindolol (32 mg/kg, a 5-HT(1A/1B) receptor/beta-adrenoceptor antagonist), NAN-190 (0.5 mg/kg, a 5-HT(1A) receptor antagonist), ketanserin (5 mg/kg, a 5-HT(2A/2C) receptor antagonist), sulpiride (50 mg/kg, a D(2) receptor antagonist) or yohimbine (1 mg/kg, an alpha(2)-adrenoceptor antagonist), but not with SCH 23390 (0.05 mg/kg, a D(1) receptor antagonist) or prazosin (1 mg/kg, an alpha(1)-adrenoceptor antagonist). These results indicate that the antidepressant-like effect of ConBr in the FST is dependent on its interaction with the serotoninergic (via 5-HT(1A) and 5-HT(2)), noradrenergic (via alpha(2)-adrenoceptors) and dopaminergic (via D(2) receptors) systems. Considering the presence of lectins in the brain and based on the results, it will be important to determine a possible role of endogenous lectins in the modulation of the central nervous system function.     

Mercury at environmental relevant levels affects spermatozoa function and fertility capacity in bovine sperm

Over the last several years human sperm quality was found to be significantly reduced and the role environmental contaminants play in this phenomenon remain to be determined. Mercury (Hg) is one of the most widespread contaminants; however the correlation between metal exposure and adverse consequences on human and animals fertility are not completely established. The aim of this study was to determine the effects of direct exposure to inorganic Hg on male gametes using spermatozoa (bovine sperm) which characteristically resemble human sperm. Sperm were divided and incubated for 0.5, 1 or 2 h at low levels of Hg: i) Control: without exposure; ii) Hg8 nM: mercury chloride (HgCl₂) at 8 nM and iii) Hg8 μM: HgCl₂ at 8 μM. Sperm kinetics, morphology, sperm membrane integrity, and in vitro fertilization were assessed. In addition the levels of reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity were measured. Hg exposure for 2 h impaired sperm morphology and membrane integrity as well as kinetic parameters including curvilinear velocity and straight-line velocity, which are needed for fertilization as evidenced by the reduced fertilization rate in 8 μM Hg-treated gametes. Hg enhanced oxidative stress in male sperm as reflected by elevated levels of ROS and lipid peroxidation and decreased antioxidant capacity. Data demonstrated that low levels of Hg when incubated with spermatozoa are sufficient to increase oxidative stress, adversely affect sperm quality parameters, subsequently impairing sperm fertility capacity.     

Comparison of immunomodulatory properties of mannose-binding lectins from Canavalia brasiliensis and Cratylia argentea in a mice model of Salmonella infection

The immunomodulatory properties of mannose-binding lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) were investigated comparatively in a model of Salmonella infection. The lectins were intraperitoneally (i.p.) administered to mice daily for three days before the bacterial challenge with Salmonella enterica Ser. Typhimurium (0.2 mL i.p.; 10⁷ CFU/mL). In vivo assays have shown that both lectins induced a significant leukocyte infiltration into the peritoneal cavity of uninfected mice, which was higher in the CFL group 3 days post-infection. Total and differential cell counts in the bloodstreams have shown uninfected animals pretreated with ConBr and CFL exhibited accentuated lymphopenia. Conversely, there was an increasing population of lymphocytes following 3 days post-infection in mice pretreated with both lectins. In addition, the bacterial burden was significantly reduced into the peritoneal cavity, bloodstreams, spleen and the liver in these mice. The lectins did not induce the release of pro- or anti-inflammatory cytokines into the peritoneal fluid of uninfected animals. However, following infection, the release of TNF-α and IL-10 in the peritoneal fluid were down-regulated in mice pretreated with both lectins whereas IL-1 was only reduced in mice pretreated with ConBr. Uninfected animals pretreated with CFL exhibited high nitric oxide (NO) content in the peritoneal fluid, which was decreased after infection in comparison to ConBr group. The lectins did not alter the serum levels of NO in uninfected mice but treatments with ConBr significantly reduced the NO content in infected animals in comparison to CFL group 24 h after the bacterial challenge. Survival experiments have shown survival rates ranging from 70% to 100% in mice that received CFL or ConBr. On the other hand, untreated mice (PBS group) died 1–6 days after infection. We conclude that ConBr and CFL are prospective phytotherapeutics capable of modulate the cascade of pro-inflammatory plus regulatory cytokines and nitric oxide release derived from systemic infections.     

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: Assessment of viability,sperm chromatin structure stability,ROS production and motility

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.     

Ameliorative Effects of Taurine Against Methimazole-Induced Cytotoxicity in Isolated Rat Hepatocytes

Methimazole is used as an antithyroid drug to control the symptoms of hyperthyroidism and maintain patients in a euthyroid state. Administration of this drug is associated with agranulocytosis and hepatotoxicity, which are the two most significant adverse effects. The present investigation was conducted to study the protective role of taurine against cytotoxicity induced by methimazole and its proposed reactive intermediary metabolite, N-methylthiourea, in an in vitro model of isolated rat hepatocytes.At different points in time, markers such as cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and hepatocyte glutathione content were evaluated.Treating hepatocytes with methimazole resulted in cytotoxicity characterized by the reduction in cell viability, an increase in ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and a reduction in cellular glutathione content. Furthermore, a significant amount of oxidized glutathione (GSSG) was formed when rat hepatocytes were treated with methimazole. N-methylthiourea toxicity was accompanied by a reduction in cellular GSH content, but no significant changes in lipid peroxidation, ROS formation, GSSG production, or changes in mitochondrial membrane potential were detected. Administration of taurine (200 μM) effectively reduced the toxic effects of methimazole or its metabolite in isolated rat hepatocytes.     

In Vitro Effects of Metal Ions (Fe2+, Mn2+, Pb2+) on Sperm Motility and Lipid Peroxidation in Human Semen

The effects of divalent manganese ion (Mn²⁺), ferrous iron (Fe²⁺), and lead ion (Pb²⁺) on human sperm motility and lipid peroxidation were examined. Human semen from healthy male volunteers was incubated with 0, 5, 50, or 500 ppm divalent metal ions, and the sperm motility was determined at 0, 2, 4, 6, or 8 h by microscopy. Malondialdehyde (MDA) levels in seminal plasma was measured by high-performance liquid chromatography after 8 h of exposure. The results showed that 500 ppm Mn²⁺ or Pb²⁺ significantly inhibited sperm motility without an accompanying change in seminal MDA levels. Incubation with Fe²⁺ significantly inhibited sperm motility at 5 ppm, associated with a marked rise in MDA levels. Our results suggested that Fe²⁺ may induce lipid peroxidation to inhibit sperm motility. In the case of Mn²⁺ and Pb²⁺ there is an absence of seminal lipid peroxidation and the observed inhibition of sperm motility at high concentrations is not biologically or environmentally relevant.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytotoxicity accompanied by oxidative stress in rat Sertoli cells: Possible role of mitochondrial fractions of Sertoli cells

TCDD, as an endocrine disruptor, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underlying the testicular effects of TCDD, the potential toxicity of TCDD on Sertoli cells was investigated. Furthermore, the study aims to delineate whether mitochondrial fractions of Sertoli cells are involved in mediating the testicular effects of TCDD. Adult rat Sertoli cells were incubated with (5, 10 or 15 nM) of TCDD for 6, 12 or 24 h. Cell viability, lactate and LDH leakage into media along with lipid peroxidation, ROS generation, SOD, CAT, GPx, GR, γ-GT and β-glucuronidase activities, GSH content and Δψ_m were measured. Superoxide anion production, COX and cardiolipin content were measured in mitochondrial fractions. Cell viability was significantly decreased while lactate and LDH leakage into media were increased. ROS generation along with lipid peroxidation was also increased. SOD, CAT, GPx, GR activities and GSH content were significantly decreased. γ-GT and β-glucuronidase activities were also decreased. Superoxide anion production was increased while COX activity and cardiolipin content were decreased in mitochondrial fractions. Moreover, the Δψ_m was significantly decreased as measured in Sertoli cells. In conclusion, TCDD impairs Sertoli cell functions and this effect is, at least in part, attributed to oxidative stress. We have also found that TCDD increases mitochondrial superoxide anion production and decreases Δψ_m, COX activity and mitochondrial cardiolipin content. Our findings suggest that mitochondria may play an important role in ROS production, leading to the TCDD-induced oxidative stress response and resulting toxicological consequences in rat Sertoli cells.     

Antioxidative potential of melatonin against mercury induced intoxication in spermatozoa in vitro

Mercury is one of the most investigated natural elements and potential contaminants in the environment. Antioxidants have long been known to reduce the free radical-induced oxidative damage. Considering the antioxidant properties of melatonin, this study was aimed to evaluate the effect of melatonin on antioxidant system of rat epididymal sperm in vitro. Sperm samples were dispersed in RPS medium (pH 6.9) and incubated with mercury in the form of mercuric chloride (MC) at three different concentrations (1 μM, 10 μM, 100 μM), melatonin (MLT) at a concentration (100 μM) and mercuric chloride+melatonin (100 μM each) for 3 h at 32 °C. Sperm viability and motility were assessed every 30 min during the 3-h incubation period. An aliquot of sperm sample was homogenised, centrifuged and used for the assay of superoxide dismutase, glutathione peroxidase, glutathione reductase, TBARS assay to detect lipid peroxidation and hydrogen peroxide generation assay. Samples treated with mercury showed a dose-dependent decrease in motility while there was no significant decrease in sperm viability. In mercury-incubated sperm, the activity of superoxide dismutase, glutathione peroxidase and glutathione reductase decreased significantly while TBARS levels and H₂O₂ generation were increased in a dose-dependent manner. Co-incubation of sperm with mercury and melatonin exhibited no significant changes in the levels of motility, viability and antioxidant indices as compared to untreated controls. The results suggest that graded doses of mercury elicit depletion of antioxidant defense system in sperm without altering the viability and melatonin treatment was found to significantly inhibit oxidative damage caused by mercury.     

New insights into male (in)fertility: the importance of NO

Infertility is a global problem that is on the rise, especially during the last decade. Currently, infertility affects approximately 10–15% of the population worldwide. The frequency and origin of different forms of infertility varies. It has been shown that reactive oxygen and nitrogen species (ROS and RNS) are involved in the aetiology of infertility, especially male infertility. Various strategies have been designed to remove or decrease the production of ROS and RNS in spermatozoa, in particular during in vitro fertilization. However, in recent years it has been shown that spermatozoa naturally produce a variety of ROS/RNS, including superoxide anion radical (O₂^⋅−), hydrogen peroxide and NO. These reactive species, in particular NO, are essential in regulating sperm capacitation and the acrosome reaction, two processes that need to be acquired by sperm in order to achieve fertilization potential. In addition, it has recently been shown that mitochondrial function is positively correlated with human sperm fertilization potential and quality and that NO and NO precursors increase sperm motility by increasing energy production in mitochondria. We will review the new link between sperm NO-driven redox regulation and infertility herein. A special emphasis will be placed on the potential implementation of new redox-active substances that modulate the content of NO in spermatozoa to increase fertility and promote conception.

Linked Articles

This article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6     

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 microM duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 microM and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress.     

Suppression of oxidative stress and pro-inflammatory mediators by Cymbopogon citratus D. Stapf extract in lipopolysaccharide stimulated murine alveolar macrophages

Exploration of antioxidants of plant origin and their scientific validation for their immense pharmacological potential is emerging as an issue of intense research now-a-days.The effect of Cymbopogon citratus extract was seen on cell viability, oxidative stress markers i.e. ROS production, SOD activity, lipid peroxidation and GSH content of murine alveolar macrophages stressed with lipopolysaccharide. Modulation in release of NO and pro-inflammatory cytokine TNF-α along with alterations in mitochondrial membrane potential under stress were compared with known plant derived antioxidant quercetin. The extract was not found to be cytotoxic at any of the selected doses. At 5 and 10 μg the extract showed significant increase in SOD activity, GSH content (p < 0.001), decrease in ROS production as seen by fluorescent dye DCFH-DA and also MDA formation (lipid peroxidation marker) significantly. The extract also showed reduction in the release of pro-inflammatory mediators TNF-α and NO significantly indicating an anti-inflammatory effect. The extract was able to restore mitochondrial membrane potential as estimated by spectrofluorimetry using the fluorescent dye Rhodamine 123. The results suggest potential use of the cytoprotective, antioxidant and anti-inflammatory property of C. citratus in the form of dietary component and also in formulations against lung inflammatory diseases where oxidative stress plays an important role.     

In vitro effects of metal ions (Fe2+, Mn2+, Pb2+) on sperm motility and lipid peroxidation in human semen

The effects of divalent manganese ion (Mn2+), ferrous iron (Fe2+), and lead ion (Pb2+) on human sperm motility and lipid peroxidation were examined. Human semen from healthy male volunteers was incubated with 0, 5, 50, or 500 ppm divalent metal ions, and the sperm motility was determined at 0, 2, 4, 6, or 8 h by microscopy. Malondialdehyde (MDA) levels in seminal plasma was measured by high-performance liquid chromatography after 8 h of exposure. The results showed that 500 ppm Mn2+ or Pb2+ significantly inhibited sperm motility without an accompanying change in seminal MDA levels. Incubation with Fe2+ significantly inhibited sperm motility at 5 ppm, associated with a marked rise in MDA levels. Our results suggested that Fe2+ may induce lipid peroxidation to inhibit sperm motility. In the case of Mn2+ and Pb2+ there is an absence of seminal lipid peroxidation and the observed inhibition of sperm motility at high concentrations is not biologically or environmentally relevant.     

Vasodilator effects of Diocleinae lectins from the Canavalia genus

This study investigated and compared vascular actions of leguminous lectins obtained from the Canavalia genus (Canavalia brasiliensis, Canavalia gladiata, and Canavalia maritima) in the rat models of paw edema and isolated aorta. Paw edema was induced by subcutaneous injection of lectins (0.01–1 mg/kg) in animals pre-treated or not with indomethacin or L-NAME. In isolated aorta, cumulative concentration curves of C. gladiata or C. brasiliensis (1–100 µg/ml) were performed at the contraction plateau induced by phenylephrine or at tissue basal tonus. The mechanism of the lectin relaxant action was investigated by previous addition of L-NAME, indomethacin, or tetraethylammonium. In both models, the lectin domain involvement was evaluated by incubation of lectins with their ligand and non-ligand sugars. The lectins induced paw edema paralleled by protein leakage. The edematogenic activity elicited by C. gladiata and C. brasiliensis involves prostaglandins and nitric oxide (NO), while that of C. maritima occurs without NO interference. C. gladiata and C. brasiliensis elicited aorta relaxation involving NO and prostacyclin, while that of C. gladiata included EDHF. All lectin effects were prevented by their binding sugars. The present study demonstrated important vasodilator effects of different degrees and mechanisms in vivo and in vitro of Canavalia lectins. In vivo, the edematogenic activity was paralleled by plasma exudation, and in vitro, aorta relaxation was strictly dependent on intact endothelium. All effects occurred via interaction with lectin domains and participation of NO and/or prostanoids.     

Emodin inhibits human sperm functions by reducing sperm [Ca2+]i and tyrosine phosphorylation

Emodin, a bioactive anthraquinone widely used in Chinese traditional medicine, disrupts mouse testicular gene expression in vivo. In this study, we investigated the toxicity of emodin to human sperm in vitro. Different doses of emodin (25, 50, 100, 200 and 400 μM) were applied to ejaculated human sperm. The results indicated that 100, 200 and 400 μM emodin significantly inhibited the total motility, progressive motility and linear velocity of human sperm. In addition, sperm's ability to penetrate viscous medium together with progesterone induced capacitation and acrosome reaction was also adversely affected by emodin. In contrast, emodin did not affect sperm viability. Furthermore, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tyrosine phosphorylation, which serve as key regulators of sperm function, were dose-dependently reduced by emodin (50–400 μM). These results suggest that emodin inhibits human sperm functions by reducing sperm [Ca²⁺]_i and suppressing tyrosine phosphorylation in vitro.     

The toxic effects of microcystin-LR on the reproductive system of male rats in vivo and in vitro

The aim of this study was to investigate whether microcystin-LR, one of the most common cyanobacterial toxins has toxic effects on reproductive system in vivo or Leydig cells in vitro. Male rats were treated with MC-LR (i.p.) at a dose of 0, 5, 10 or15 microg/(kgday) for 28 days. Leydig cells were cultured with a culture medium including 0, 0.5, 5, 50 or 500 nM MC-LR. In vivo study, we observed exposure to 5 microg/(kgday) of MC-LR decreased the sperm motility, increasing the sperm abnormality rate, 15 microg/(kgday) of MC-LR led to the decrease of testis weight and sperm concentration, decreased the levels of serum testosterone, FSH and LH. The histological findings showed that the seminiferous tubules atrophied and obstructed. In vitro study evaluated MC-LR-induced toxicity and oxidative stress in Leydig cells. It was observed 50 and 500 nM MC-LR significantly decreased the cell viability, increasing the apoptotic DNA fragmentation, and increasing the ratio of necrotic cells. The Leydig cells exposed to MC-LR decreased testosterone production. 500 nM MC-LR increased ROS production, 50 or 500 nM MC-LR enhanced the lipid peroxidation. All Leydig cells exposed to MC-LR showed decreased SOD activity. The results of this study showed that the oxidative stress of MC-LR might lead to cytotoxicity, which may play an important role in cell apoptosis. Then could reduce the production of testosterone in Leydig cells and result in reproductive toxicity.     

Protective effect of quercetin in gentamicin-induced oxidative stress in vitro and in vivo in blood cells. Effect on gentamicin antimicrobial activity

We have evaluated the effect of gentamicin and gentamicin plus quercetin on ROS production, endogenous antioxidant defenses (SOD and CAT) and lipid peroxidation in vitro on human leukocytes and in vivo on whole rat blood. Gentamicin generated ROS production in human leukocytes, produced a dual effect on both enzymes dosage-dependent and generated an increase in lipid peroxidation. Quercetin, in leukocytes stimulated by gentamicin, showed more inhibitory capacity in ROS production than the reference inhibitor (vitamin C) in mononuclear cells and a similar protective behavior at this inhibitor in polymorphonuclear cells. Quercetin, in both cellular systems, tend to level SOD and CAT activities, reaching basal values and could prevent lipidic peroxidation induced by gentamicin. The results in Wistar rats confirmed that therapeutic doses of gentamicin can induce oxidative stress in whole blood and that the gentamicin treatment plus quercetin can suppress ROS generation, collaborate with SOD and CAT and diminish lipid peroxidation. Finally, flavonoid and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli, showing that changes were not generated in the antibacterial activity of gentamicin against E. coli strains, while for strains of S. aureus a beneficial effect observes. Therefore, we have demonstrated that gentamicin could induce oxidative stress in human leukocytes and in whole blood of Wistar rats at therapeutic doses and that quercetin may to produce a protective effect on this oxidative stress generated without substantially modifying the antibacterial activity of gentamicin against E. coli strains, and it contributes to this activity against S. aureus strains.     

The effect of 4-nonylphenol on semen quality, viability of gametes, fertilization success, and embryo and larvae survival in rainbow trout (Oncorhynchus mykiss)

The present study investigated in vivo and in vitro effects of environmental relevant concentrations of 4-nonylphenol (100-750 ng l(-1)) on the reproduction of rainbow trout (Oncorhynchus mykiss). To determine the effect of 4-nonylphenol on semen quality rainbow trout were exposed to three concentrations of 4-nonylphenol in a flow-through system during the spawning period (60 days). At an estimated 4-nonylphenol concentration of 750 ng l(-1) semen production was completely inhibited, at 280 and 130 ng l(-1) the semen production was significantly reduced in comparison to the control. Sperm density, sperm motility and sperm fertility were not affected. Also the development of embryos and larvae at the end of yolk sac stage was affected by 4-nonylphenol. At estimated 4-nonylphenol exposure levels of 280 and 750 ng l(-1) the percentage of eyed stage embryos was slightly but significantly lower (2-4%) than at 130 ng l(-1) 4-nonylphenol and in the control. At 4-nonylphenol concentrations of 750 ng l(-1) only 23.8 +/- 1.2% of the larvae survived to the end of the yolk sac stage, at 280 ng l(-1) 53.7 +/- 8.2%, at 130 ng l(-1) 73.8 +/- 1.5%, and in the control 70.9 +/- 1.8%. Sperm motility was not affected by 4-nonylphenol as sperm motility rate, swimming velocity, swimming pattern and motility duration were similar in water and in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol. Incubation of eggs in physiological saline solution containing of 100, 250, or 750 ng l(-1) 4-nonylphenol did not change their fertilizability in comparison to the control. Therefore, 4-nonylphenol did not affect the egg viability. Also the fertilization process (sperm egg contact) was not influenced by 4-nonylphenol as the fertilization rate (percentage of hatched larvae) was similar to the control when eggs were fertilized in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol.     

Graphene oxide had adverse effects on sperm motility and morphology through oxidative stress

Graphene oxide (GO) is a new type of graphene material, but its effects on the male reproductive system are unclear. Here, we investigated the effects of GO on human sperm in vitro. Sperms were incubated with various doses of GO (0, 10, 20, or 40 μg/mL) for different times (1, 3, or 6 h) at 37 °C, followed by analyses of the sperm motility, viability, abnormalities, and DNA fragmentations. GO exposure significantly decreased sperm motility and viability, increased sperm abnormalities, and DNA fragmentation. Moreover, GO exposure resulted in a significant reduction of sperm mitochondrial membrane potential (MMP), which was confirmed by the ultrastructural changes of chromatin and mitochondria caused by GO. These data revealed the adverse effects of GO on sperm. Further research showed that GO exposure led to a significant increase in malondialdehyde (MDA) and reactive oxygen species (ROS) in sperm cells and a significant decrease in total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px). In addition, western blot analysis showed that the levels of Nrf-2 and HO-1 protein expression in GO-treated sperm cells were significantly increased compared to the control. These results indicated that GO had adverse effects on human sperm through oxidative stress, which was associated with Nrf-2/HO-1 signaling pathway.     

Lupeol protects against acetaminophen-induced oxidative stress and cell death in rat primary hepatocytes

Drug induced hepatotoxicity is a major problem where phytochemicals hold promise for its abrogation. This study was carried out to explore cytoprotective potential of lupeol, a triterpene, against acetaminophen (AAP)-induced toxicity in rat hepatocytes. AAP exposure significantly (p < 0.05) reduced cell viability, disturbed Bcl-2 family pro/anti-apoptotic protein balance, increased ROS production and altered redox homeostasis. It also induced mitochondria-mediated hepatocellular injury by significant mitochondrial depolarization, caspase-9/3 activation and subsequent DNA fragmentation. Our results suggest that lupeol pre-treatment effectively restored antioxidant enzyme levels, decreased lipid peroxidation, inhibited ROS generation and depolarization of mitochondria. Lupeol also attenuated mitochondria-mediated signaling pathway and DNA damage as evident from TUNEL assay and cell cycle studies leading to prevention of cytotoxicity. This study confirms the efficacy of lupeol, a food derived antioxidant, in abrogating ROS generation, maintaining redox balance and providing significant protection against mitochondria-mediated cell death during AAP-induced toxicity.