Fine mapping of Ahl3 affecting both age-related and noise-induced hearing loss

A region in the vicinity of D17Mit119 on mouse chromosome 17 harbors a susceptibility gene, designated as Ahl3, to age-related hearing loss (AHL). We produced congenic lines of C57BL/6 background that substituted regions around D17Mit119 with MSM-derived ones, and examined auditory brainstem response (ABR) thresholds for their hearing capacity at 6 and 12months of age. Three congenic lines carrying the approximately 14-Mb region between D17Mit274 and D17Mit183 retained normal hearing at 12months of age whereas two congenic lines not carrying this region tended to lose hearing at that age. We also investigated noise-induced hearing loss (NIHL) in congenic lines at 1, 7 and 14days after exposure to the noise of 100dB for 1h. Most congenic mice carrying the 14-Mb region did not exhibit permanent threshold shift (PTS) whereas mice not carrying this region exhibited a strong tendency of PTS, indicating the role of Ahl3 in susceptibility to NIHL. These results indicate that Ahl3 exists within the 14-Mb region and affects not only AHL but also NIHL.     

Tadalafil alters energy metabolism in C2C12 skeletal muscle cells

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides, thereby modulating cell functions. Three highly selective PDE5 inhibitors (PDE5i), sildenafil, vardenafil and tadalafil, have been developed for treatment of erectile dysfunction (ED). Experimental evidence showed that chronic treatment with sildenafil PDE5i in a mouse model of diet-induced obesity and insulin resistance improved insulin action and decreased circulating fatty acid levels. It has recently been shown that healthy athletes use PDE5i as performance enhancers, hence in the present study we investigated whether the long-lasting PDE5i tadalafil influences energy metabolism in C2C12 skeletal muscle cells by evaluating lactate production, glucose consumption, and citrate synthase and 3-OH acyl CoA dehydrogenase activities. Our data demonstrate that tadalafil is able to modulate energy homeostasis in mouse skeletal muscle cells, depending on the treatment length and dose.     

Developmental toxicity of orally administered sildenafil citrate (Viagra) in SWR/J mice

Normal adult inbred SWR/J mice were used to investigate the teratogenic and other possible toxic effects of various dose levels of sildenafil citrate (Viagra) on fetuses. Multiple dose levels of 6.5, 13.0, 19.5, 26.0, 32.5 or 40.0 mg of sildenafil citrate/kg body weight (which correspond to the multiples of 1, 2, 3, 4, 5 or 6 of human 50 mg Viagra, respectively) were orally administered into pregnant mice on days 7–9, 10–12 or 13–15 of gestation. On day 17 of pregnancy, all fetuses were removed and examined for toxic phenomena (embryo-fetal toxicity) and for external, internal and skeletal malformations. A total of 285 pregnant mice were used in the present study.None of the dams treated with sildenafil citrate at any of the oral dose levels used in the present study died during the experimental period and all dams treated with the drug failed to reveal overt signs of maternal toxicity. Moreover, the results of the present study clearly demonstrate that none of the multiple oral dose levels of the drug at any time interval used has induced any external, internal or skeletal malformations in the fetuses obtained from treated females.However, the dose level of 40 mg/kg body weight of sildenafil citrate has a growth suppressing effect on alive fetuses when it was administered at all the time intervals used in the present study. Furthermore, the dose levels 26.0, 32.5 and 40 mg/kg of the drug have embryo-fetal toxicity when the drug is applied on days 13–15 of gestation. The possible mechanisms involved in the embryo-fetal toxicity and fetal growth suppressing effects of sildenafil citrate were discussed.The results of this study have important implications for the widespread use of this drug.     

Effect of SOD1 overexpression on age- and noise-related hearing loss

Reactive oxygen species (ROS) have been implicated in hearing loss associated with aging and noise exposure. Superoxide dismutases (SODs) form a first line of defense against damage mediated by the superoxide anion, the most common ROS. Absence of Cu/Zn SOD (SOD1) has been shown to potentiate hearing loss related to noise exposure and age. Conversely, overexpression of SOD1 may be hypothesized to afford a protection from age- and noise-related hearing loss. This hypothesis may be tested using a transgenic mouse model carrying the human SOD1 gene. Contrary to expectations, here, we report that no protection against age-related hearing loss was observed in mice up to 7 months of age or from noise-induced hearing loss when 8 week old mice were exposed to broadband noise (4-45 kHz, 110 dB for 1 h). Mitochondrial DNA deletion, an index of aging, was elevated in the acoustic nerve of transgenic mice compared to nontransgenic littermates. The results indicate the complexity of oxidative metabolism in the cochlea is greater than previously hypothesized.     

The cGMP-binding, cGMP-specific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion

The expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was studied in intestinal cells. Both PDE5A1 and PDE5A2 splice forms were cloned from the cDNA prepared from human colonic T84 cells, and PDE5 activity was dependent on increases in intracellular cGMP levels which correlated with increased phosphorylation of the enzyme. PDE5 expression was monitored in different regions of the gastrointestinal tract and nearly 50% of the phosphodiesterase activity in the duodenum, jejunum, ileum and colon was inhibited by sildenafil citrate. Administration of the stable toxin to intestinal loops resulted in activation of PDE5. Inhibition of PDE5 by sildenafil citrate led to fluid accumulation in loops, suggesting a possible explanation for the side effect of diarrhoea observed in individuals administered sildenafil citrate. Our results therefore represent the first study on the expression and regulation of PDE5 in intestinal tissue, and indicate that mechanisms to control its activity may have important consequences in intestinal physiology.     

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca²⁺-induced mitochondrial permeability transition, and hydrogen peroxide (H₂O₂) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 μM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (ΔΨ), the phosphorylation rate, and the membrane permeability to H⁺, K⁺ and Ca²⁺ were not affected either. However, sildenafil citrate decreased H₂O₂ generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O₂^•−) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 μM do not affect either rat heart mitochondrial bioenergetics or Ca²⁺-induced mitochondrial permeability transition, but it depresses H₂O₂ generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.     

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.     

Molecular Characterization of Microbial Population Dynamics during Sildenafil Citrate Degradation

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.     

Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits

Sildenafil citrate (Viagra) is the most widely used pharmacological drug for treating erectile dysfunction in men. It has potent cardioprotective effects against ischemia-reperfusion injury via nitric oxide and opening of mitochondrial ATP-sensitive K(+) channels. We further investigated the role of protein kinase C (PKC)-dependent signaling pathway in sildenafil-induced cardioprotection. Rabbits were treated (orally) with sildenafil citrate (1.4 mg/kg) 30 min before index ischemia for 30 min and reperfusion for 3 h. The PKC inhibitor chelerythrine (5 mg/kg i.v.) was given 5 min before sildenafil. Infarct size (% of risk area) reduced from 33.65 +/- 2.17 in the vehicle (saline) group to 15.07 +/- 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean +/- SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 +/- 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 +/- 2.5, which was not significantly different compared with DMSO-treated group (36.8 +/- 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-alpha, -, and -delta isoforms from cytosol to membrane after treatment with sildenafil. However, no change in the PKC-beta and -epsilon isoforms was observed. These data provide direct evidence of an essential role of PKC, and potentially PKC-alpha, -, and -delta, in sildenafil-induced cardioprotection in the rabbit heart.     

The effect of selective phosphodiesterase inhibitors,alone and in combination,on a murine model of allergic asthma

BackgroundThe anti-inflammatory effects of the selective phosphodiesterase (PDE) inhibitors cilostazol (PDE 3), RO 20-1724 (PDE 4) and sildenafil (PDE 5) were examined in a murine model of allergic asthma. These compounds were used alone and in combination to determine any potential synergism, with dexamethasone included as a positive control.MethodsControl and ovalbumin sensitised Balb/C mice were administered orally with each of the possible combinations of drugs at a dose of 3 mg/Kg for 10 days.ResultsWhen used alone, RO 20-1724 significantly reduced eosinophil influx into lungs and lowered tumour necrosis factor-α, interleukin-4 and interleukin-5 levels in the bronchoalveolar lavage fluid when compared to untreated mice. Treatment with cilostazol or sildenafil did not significantly inhibit any markers of inflammation measured. Combining any of these PDE inhibitors produced no additive or synergistic effects. Indeed, the anti-inflammatory effects of RO 20-1724 were attenuated by co-administration of either cilostazol or sildenafil.ConclusionsThese results suggest that concurrent treatment with a PDE 3 and/or PDE 5 inhibitor will reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.     

Manganese in toenails is associated with hearing loss at high frequencies in humans

Purpose: Elevated hearing thresholds from high frequencies are known to be one of the hallmarks of age-related hearing loss. Our recent study showed accumulation of manganese (Mn) in inner ears resulting in acceleration of age-related hearing loss in mice orally exposed to Mn. However, there is no evidence showing an association between Mn in non-invasive biological samples and hearing loss in humans evaluated by pure tone audiometry (PTA). In this study, we evaluated Mn in non-invasive biological samples as a possible biomarker for hearing loss in humans.

Materials and methods: We determined hearing levels by PTA and Mn levels in toenails, hair and urine with an inductively coupled plasma mass spectrometer (ICP-MS) in 145 healthy subjects in Bangladesh.

Results: Multivariable analyses showed that Mn levels in toenails, but not in hair and urine samples, were significantly associated with hearing loss at 8?kHz and 12?kHz. Moreover, our experimental study showed a significant correlation between Mn levels in inner ears and nails, but not hair, in mice orally exposed to Mn.

Conclusions: The results provide novel evidence that Mn in toenails is a possible biomarker for hearing loss at high frequencies in humans.     

Effect of sildenafil citrate (Viagra?) on trace element concentration in serum and brain of rats

As a vasodilator with good hemodynamic effects, sildenafil has been successfully used in the treatment of patients with pulmonary hypertension and cardiovascular diseases. By selectively inhibiting phosphodiestrase type 5 (PDE-5) and thus effectively reducing the breakdown of c GMP, sildenafil administration can markedly improve the erectile dysfunction. Sildenafil also elevates localized cerebral blood flow in rat brain. The objective of the present study was to investigate the effect of sildenafil on the level of trace elements (Zinc (Zn), copper (Cu), iron (Fe), selenium (Se), cobalt (Co), and chromium (Cr)) in blood and brain of rats. Sixteen male albino rats weighing 180-200 g were divided into two groups (8 rats/group). Sildenafil (Viagra, Pfizer Inc.) was dissolved in saline and administered at a dose of 10mg/kg i.p. (0.5 ml volume) to rats in the treated group every 72 h for 12 injections. Rats in the control group were administered the same volume of saline as in treated group. All rats were sacrificed 24h after the last injection. Blood samples were collected and serum was separated and stored at -20°C. Brains were dissected and stored frozen until analysis. Trace elements concentrations were determined by flame emission atomic absorption spectrophotometer. Results showed that sildenafil injection significantly (P<0.05) increased serum and brain Se and Cu concentrations. Moreover, sildenafil increased the Cr concentration in the brain tissue. It was concluded that sildenafil citrate administration increased serum Se and Cu as well as, increased brain Se, Cu, and Cr concentrations in rats.     

Ahl3, a third locus on mouse chromosome 17 affecting age-related hearing loss

Genetic variation in humans probably plays a role in determining the range of individual susceptibility to age-related hearing loss (AHL), but no contributing loci have been identified because of the difficulties of dissecting complex traits in humans. This paper reports mapping of an AHL locus using a panel of consomic mice between C57BL/6J (B6) and MSM strains, which covered more than a half of chromosome sets. B6 strain exhibited AHL beginning at 10 months of age whereas MSM strain, derived from Japanese wild mice, had normal hearing throughout life. Individuals in the panel were examined with auditory brainstem response (ABR) at various months of age, revealing that one particular strain (B6-Chr17(MSM)) substituting the chromosome 17 with the MSM-derived one showed a prominent resistance, having still good hearing at 18 months of age. Subsequent mapping using 89 individuals in the cross between B6-Chr17(MSM) and B6 was performed, which showed a significant association of ABR thresholds with loci in the vicinity of D17Mit119. These results show a novel AHL-resistant locus, designated as Ahl3, on the chromosome 17.     

Effect of chronic administration of sildenafil citrate (Viagra) on the histology of the retina and optic nerve of adult male rat

Background

Abnormal vision has been reported by 3% of patients treated with sildenafil citrate (Viagra). Although many men use Viagra for an extended period for treatment of erectile dysfunction, the implications of the long term-daily use of it on the retina and optic nerve are unclear.

Effect of sildenafil citrate (Viagra) and ethanol on the Albino rat testis: a scanning electron microscopic approach

The effects of sildenafil citrate with ethanol on the rat testis was studied using scanning electron microscopy. Male Albino rats were divided into 8 groups, each being treated for a maximum of 45 days as follows. In the 4 short-term treatment groups, control rats were administered normal saline orally, whereas experimental animals were fed sildenafil citrate (Viagra) 1 microg/g with 18% ethanol (5 g/kg body weight), which was given orally as a single dose. After 1, 2.5, 4 and 24h the rats were killed. In the 4 long-term treatment groups, daily continuous doses of drug and ethanol with a single dosage were given for 15, 30 and 45 days and the animals killed 4h after the last dosage. Changes in the testis were compared with the normal healthy rat testis. The use of a scanning electron microscope for evaluation of the changes in the testis is more suitable for observation of the surface and morphological shapes of the tissue structures.     

Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5)

Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3',5'-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown. A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K(m) of the catalytic fragment for cGMP (5.5 +/- 0. 51 microM) compares well with those of the native bovine lung PDE5 (5.6 microM) and full-length wild type recombinant PDE5 (2 +/- 0.4 microM). The catalytic fragment and full-length PDE5 have similar IC(50) values for the inhibitors 3-isobutyl-1-methylxanthine (20 microM) and sildenafil (Viagra(TM))(4 nM). Based on measured values for Stokes radius (29 A) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa). The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer.     

Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial K(ATP) channels in rabbits

Sildenafil citrate (Viagra) is the pharmacological agent used to treat erectile dysfunction in men. Because this drug has a vasodilatory effect, we hypothesized that such an action may induce a preconditioning-like cardioprotective effect via opening of mitochondrial ATP-sensitive K (K(ATP)) channels. Rabbits were treated with sildenafil citrate (0.7 mg/kg iv) either 30 min (acute phase) or 24 h (delayed phase) before 30 min of ischemia and 3 h of reperfusion. Mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was given 10 min before ischemia-reperfusion. Infarct size was measured by tetrazolium staining. Sildenafil caused reduction in arterial blood pressure within 2 min of treatment, which returned to nearly baseline levels 3 min later. The infarct size (% risk area, means +/- SE) reduced from 33.8 +/- 1.7 in control rabbits to 10.8 +/- 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 +/- 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 +/- 0.4% and 36.8 +/- 1.6% during the acute and delayed phase, respectively (P < 0.05). Similar acute and delayed cardioprotective effects were observed when sildenafil was administered orally. Systemic hemodynamics also decreased after oral administration of the drug. However, these changes were mild and occurred slowly. For the first time, we demonstrate that sildenafil induces acute and delayed protective effects against ischemia-reperfusion injury, which are mediated by opening of mitochondrial K(ATP) channels.     

Phosphodiesterase 5 inhibitors prevent 3,4-methylenedioxymethamphetamine-induced 5-HT deficits in the rat

Phosphodiesterase 5 (PDE5) inhibitors are often used in combination with club drugs such as 3,4‐methylenedioxymethamphetamine (MDMA or ecstasy). We investigated the consequences of such combination in the serotonergic system of the rat. Oral administration of sildenafil citrate (1.5 or 8 mg/kg) increased brain cGMP levels and protected in a dose‐dependent manner against 5‐hydroxytryptamine depletions caused by MDMA (3 × 5 mg/kg, i.p., every 2 h) in the striatum, frontal cortex and hippocampus without altering the acute hyperthermic response to MDMA. Intrastriatal administration of the protein kinase G (PKG) inhibitor, KT5823 [(9S, 10R, 12R)‐2,3,9,10,11,12‐Hexahydro‐10‐methoxy‐2,9‐dimethyl‐1‐oxo‐9,12‐epoxy‐1H‐diindolo[1,2,3‐fg:3′,2′,1′‐kl]pyrrolo[3,4‐i][1,6]benzodiazocine‐10‐carboxylic acid, methyl ester)], suppressed sildenafil‐mediated protection. By contrast, the cell permeable cGMP analogue, 8‐bromoguanosine cyclic 3′,5′‐monophosphate, mimicked sildenafil effects further suggesting the involvement of the PKG pathway in mediating sildenafil protection. Because mitochondrial ATP‐sensitive K⁺ channels are a target for PKG, we next administered the specific mitochondrial ATP‐sensitive K⁺ channel blocker, 5‐hydroxydecanoic acid, 30 min before sildenafil. 5‐hydroxydecanoic acid completely reversed the protection afforded by sildenafil, thereby implicating the involvement of mitochondrial ATP‐sensitive K⁺ channels. Sildenafil also increased Akt phosphorylation, and so the possible involvement of the Akt/endothelial nitric oxide synthase (eNOS)/sGC signalling pathway was analysed. Neither the phosphatidylinositol 3‐kinase inhibitor, wortmannin, nor the selective eNOS inhibitor, l ‐N5‐(1‐iminoethyl)‐l ‐ornithine dihydrochloride, reversed the protection afforded by sildenafil, suggesting that Akt/eNOS/sGC cascade does not participate in the protective mechanisms. Our data also show that the protective effect of sildenafil can be extended to vardenafil, another PDE5 inhibitor. In conclusion, sildenafil protects against MDMA‐induced long‐term reduction of indoles by a mechanism involving increased production of cGMP and subsequent activation of PKG and mitochondrial ATP‐sensitive K⁺ channel opening.