Estimation of catecholamines in daily urine by stripping voltammetry

A voltammetric method for the quantitative estimation of adrenaline (A) and noradrenaline (NA) in daily urine was developed using a carbon glass electrode. The lower detection limits for A and NA concentrations were 1 × 10⁻⁹ g/ml. The relative standard deviation for the range of concentrations 1 × 10⁻⁹ to 1 × 10⁻⁵ M was no greater than 0.1. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 2, pp. 51–53, February, 2008.     

An ion-selective electrode for quantitative analysis of therapeutic formulations of No-Shpa

An ion-selective membrane electrode (ISE) was developed for estimation of drotaverine hydrochloride by direct potentiometry and potentiometric depositional titration. The ISE reacted reversibly to changes in the potential-determining ion concentration over the range 5 × 10^{− 2} to 7.9 × 10^{− 6} M, the detection limit was (4.3 ± 0.2) × 10^{− 6} M, and the electrode function slope was 58 ± 2 mV/pC. A method for the potentiometric estimation of drotaverine hydrochloride in tablets and solutions for injection is proposed. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 5, pp. 46–49, May, 2008.     

Voltammetric Determination of Mexidol

A new voltammetric method for determining mexidol in pharmaceuticals is proposed. Using this technique, mexidol was determined in model solutions (with R _s = of 1 – 6%) and in a ready-to-use preparation. The analytical range of mexidol determination using a glassy carbon composite electrode in 0.1 M H₂SO₄ extends from 4.8 × 10⁻³ to 1.8 × 10⁻² M. The detection limit is 1.9 × 10⁻³ M. The proposed procedure can be used for the quality control in the drug production technology. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 8, pp. 51 – 52, August, 2005.     

Ion-selective electrodes for determining cephazolin in biological media

An ion-selective electrode with plasticized polyvinylchloride membrane that is based on a cephazolin—tetradecylammonium ionic associate has been developed. The ratio of components and the solubility product of the ionic associates [(2.2 ± 0.1) × 10^{− 8}] were determined. The compound is thermally stable up to 70°C. The EMF is linearly dependent on the cephazolin concentration in the range from 1 × 10 − 5 to 1 × 10^{− 1} M. The slope of the electrode function is 56 ± 2 mV/pC. The selectivity to inorganic ions allows the proposed system to be used for cephazolin determination in biological media. In particular, a method for rapid determination of cephazolin in complex saliva is developed. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 8, pp. 41–44, August, 2008.     

Ion-selective sensors for determining ampicillin and oxacillin in biological fluids and pharmaceuticals

Ion-selective sensors based on organic ion exchangers of the ampicillin (oxacillin)-tetradecylammonium (Am-TDA; Ox-TDA) type sensitive to antibiotics of the penicillin group have been developed. The ratio of components (1: 1) and the solubility products of ion associates (8.5 × 10⁻⁸ and 5.2 × 10⁻¹⁰ for Am-TDAand Ox-TDA, respectively) are determined. According to thermoanalytical data, both Am-TDAand Ox-TDAion associates are individual substances not containing water. The organic ion exchangers do not undergo changes on heating up to 70°C. It is established that the electrode functions are linear in the interval from 1 × 10 ⁻⁵ to 1 × 10⁻¹ M for all antibiotics studied. The slopes of the electrode functions for Am and Ox are 58 × 1 and 60 × 1, respectively, being close to the theoretical values for singly charged ions. Methods for the determination of Am and Ox in the blood serum and saliva and in various medicinal forms (tablets, powders in bottles, etc.) are described. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 3, pp. 53–55, March, 2006.     

β-secretase (BACE1) inhibitors from <Emphasis Type="Italic">Perilla frutescens</Emphasis> var. <Emphasis Type="Italic">acuta</Emphasis>

In the course of screening for anti-dementia agents from natural products, two β-secretase (BACE1) inhibitors were isolated from the methanolic extract of Perilla frutescens var. acuta and identified as luteolin (1) and rosmarinic acid (2) with IC₅₀ values of 5.0×10⁻⁷ M and 2.1×10⁻⁵ M, respectively. They inhibited BACE1 in a non-competitive manner with a substrate in Dixon plots, suggesting that they might bind to either β-secretase subsite or to another regulatory site. Ki values of 1 and 2 were 6.2×10⁻⁵ M and 3.9×10⁻⁵ M, respectively. They were less inhibitory against other enzymes such as α-secretase (TACE), acetylcholine esterase (AchE), chymotrypsin, and elastase, indicating that they were relatively specific inhibitors of BACE1.     

Prejunctional modulation by angiotensins of noradrenaline release from sympathetic neurons in isolated rabbit aorta

The aims of the present work were to compare the modulating effect of angiotensins I, II, III, IV and (1–7) [AI, AII, AIII, AIV and A(1–7) respectively] on stimulation-evoked noradrenaline release from post-ganglionic sympathetic nerves in rabbit isolated aorta; to examine the influence of inhibiting the neuronal and extraneuronal uptake of noradrenaline on the modulating effect of AII and thirdly, to determine the role of angiotensin converting enzyme (ACE) in the modulating effects of AI and AII and the role of aminopeptidases A and M in the effects of AII and AIII. Rings of aorta were preloaded with (–)-³H-noradrenaline and then subjected to electrical field stimulation. Cumulative addition of AI (10^–8–10^–6M), AII (3×10^–11–10^–8M) and AIII (3×10^–10–10^–6M) enhanced the stimulation-evoked ³H-overflow up to 142, 165 and 188% respectively. The order of potency was AII > AIII > AI. AIV (10^–10–10^–7M) and A(1–7) (10^–10–10^–7M) caused no change. Single concentrations (10^–9–10^–7M) of AI, AII and AIII caused initial enhancement which subsequently decreased, i.e. development of tachyphylaxis. The effect of AII was independent of stimulation frequency at 1–10Hz, but absent at 30Hz. Cocaine (3×10^–5M) plus corticosterone (4×10^–5M) did not alter the enhancing effect of AII. CaNa₂EDTA (3×10^–5M) did not alter the enhancing effect of AI. Captopril (10^–6 and 10^–5M) and lisinopril (10^–6M) attenuated the enhancing effect of AI. Captopril and lisinopril (both 10^–6 and 10^–5M) did not alter the enhancing effect of AII. Captopril (10^–7– 10^–4M) and lisinopril (10^–7–10^–4M) themselves did not alter the stimulation-evoked ³H-overflow. Amastatin (10^–5M) increased the enhancement seen with AIII (3×10^–11–10^–9M) but did not alter the enhancing effect of AII (10^–9–10^–8M). Amastatin (10^–9–10^–5M) had no effect on the stimulation-evoked ³H-overflow. It is concluded that AI, AII and AIII facilitate the stimulation-evoked ³H-noradrenaline release to various degrees (relative order of potency: AII > AIII > AI and of efficacy: AIII > AII > AI). The estimates may be compromised by the development of tachyphylaxis. The facilitation by AII was independent of the neuronal and extraneuronal uptake mechanisms. The action of AI is in part due to its conversion to AII. The effect of AIII was probably underestimated due to its degradation to AIV. AII is apparently not a substrate for aminopeptidase M. Received: 10 September 1996 / Accepted: 18 August 1997     

An in vitro approach for demonstrating the critical role of serum albumin in the detoxication of the carbamate carbaryl at in vivo toxicologically relevant concentrations

The hydrolysis of carbaryl by bovine serum albumin (BSA) was studied at toxicologically relevant concentrations (range 15–300 μM) in order to determine the role of this protein in the detoxication of the carbamate in vivo. The 1-naphthol released during the hydrolysis of carbaryl was monitored using gas chromatography coupled with mass spectrometry. BSA hydrolyzed carbaryl in a time-progressive way. The hydrolysis was also dependent of enzyme (1.0, 2.5, 5.0 and 7.0 mg ml⁻¹) and substrate (range between 15 and 1,000 μM) concentration. The estimated turnover number and Michaelis–Menten constant were 1.6 × 10⁻⁴ s⁻¹ and 430 μM, respectively. Thus, the second order rate constant was 0.37 M⁻¹ s⁻¹. At enzyme concentrations of 7.0 mg ml⁻¹ and substrate concentrations ranging between 50 and 300 μM about 80% of substrate was hydrolyzed in 3 h. At lower substrate concentrations (15 and 30 μM carbaryl) also significant hydrolysis was detected at the highest enzyme concentration, even when these substrate concentrations were 30 and 15 times lower than the Michaelis–Menten constant. Although the efficacy of the enzymatic hydrolysis is low, the extrapolation of our results to the physiological albumin high concentrations (around 40 mg ml⁻¹) suggests that the hydrolysis of carbaryl by serum albumins plays a critical role in the detoxication of this carbamate at in vivo toxicologically relevant concentrations. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Influence of quinidine on the binding of [3H]-ouabain and [3H]-digoxin by human lymphocytes

Summary To explore the molecular basis of the glycoside-quinidine interaction, the in vitro effect of quinidine on the binding of [³H]-ouabain and [³H]-digoxin to Na⁺ K⁺ ATPase receptors on human mononuclear cells was investigated. The maximum [³H]-ouabain binding capacity was 45.7±9.4×10³ molecules/cell in pure lymphocyte preparations (n=8) and 75.5±7.3×10³ molecules/cell in mixtures of mononuclear cells (n=8). These parameters were not influenced by 10⁻⁵ M quinidine. In eight equilibrium experiments with pure lymphocytes, the dissociation constant of [³H]-ouabain increased from 0.79±0.26×10⁻⁸ M in the absence of 10⁻⁵ M quinidine to 1.56±0.74×10⁻⁸ M in its presence (p<0.01), indicating that the affinity of the drug was decreased. Similar findings were observed using mixed mononuclear cells. In five uptake and release experiments, quinidine decreased the association rate constant of [³H]-ouabain from 3.15±0.36×10⁴ M⁻¹×s⁻¹ to 2.01±0.37×10⁴ M⁻¹ s⁻¹ (p<0.01), whereas the dissociation rate constant was not affected. A therapeutic concentration of quinidine does not affect the number of glycoside receptors on lymphocytes, but it does appear to reduce fractional receptor occupancy by both [³H]-ouabain and [³H]-digoxin at lower tracer concentrations. This finding is compatible with the clinical observation that quinidine reduces the distribution volume of digoxin.     

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na⁺/K⁺-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na⁺/K⁺ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na⁺ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K⁺ and 30 to 33° C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC₅₀ value) amounted to ∼ 5 × 10^–7 M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC₅₀ values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 × 10^–6 M and 1.1 × 10^–5 M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na⁺/K⁺ pump inhibition (K′_D value) were calculated to be ∼ 10^–6 M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K′_D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K′_D value for 22-allyl-digitoxigenin was even larger and amounted to ∼ 4 × 10^–5 M. The K′_D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (∼ 9 × 10² s^–1 M^–1) was smaller than the association rate constant of digitoxigenin (∼ 2 × 10⁴ s^–1 M^–1), whereas the apparent dissociation rate constants of both compounds were similar (∼ 4 × 10^–3 s^–1). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (∼ 3 × 10³ s^–1 M^–1) and a larger dissociation rate constant (∼ 8 × 10^–2 M^–1). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na⁺/K⁺ pump by affinity labeling. Received: 2 June 1997 / Accepted: 17 September 1997     

An ion-selective sensor for assay of diclofenac in medicines

A diclofenac-selective electrode with a plasticized polyvinylchloride membrane was developed. The electrode contained an ionic associate of diclofenac with neutral red and its response was linear over the diclofenac concentration range 5 × 10^–5 to 5 × 10^–2 M with an electrode function slope of (30.0 ± 1.1) - (44.0 ± 1.2) mV/pC. This membrane electrode was used as a sensor for assaying diclofenac in pharmaceutical formulations.     

Suitability of <Emphasis Type="Italic">Daphnia similis</Emphasis> as an alternative organism in ecotoxicological tests: implications for metal toxicity

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 × 10⁵ and 1 × 10⁶ cells ml⁻¹ on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL⁺ showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC₅₀ determination. LC₅₀ for D. similis was 5.15 × 10⁻⁷ mol l⁻¹ dissolved Cd without algal cells, whereas with 1 × 10⁵ cells ml⁻¹, it was significantly higher (7.15 × 10⁻⁷ mol l⁻¹ dissolved Cd). For Cr, the 48 h-LC₅₀ value of 9.17 × 10⁻⁷ mol l⁻¹ obtained for the cladoceran in tests with 1 × 10⁶ cells ml⁻¹ of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 × 10⁻⁷ mol l⁻¹ dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.     

Biopharmaceutical Characterization of the Telomerase Inhibitor BRACO19

Purpose To characterize the telomerase inhibitor and G-quadruplex stabilizing substance 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis (3-pyrrolodino-propionamido) acridine × 3HCl (BRACO19) in terms of biopharmaceutical properties such as solubility, protein binding, interaction with membrane lipids, cytotoxicity, and permeability across pulmonary epithelial cells. Methods Protein binding and interaction with membrane lipids were investigated by two high-performance liquid chromatography methods with immobilized human serum albumin and immobilized phosphatidylcholine, respectively. Cytotoxicity (methyl–thiazolyl–tetrazolium assay) and transport studies were performed with the bronchial cell lines 16HBE14o⁻ and Calu-3, primary human alveolar epithelial cells, and the intestinal cell line Caco-2. Transport experiments were also done in the presence of cyclosporin A (10 μM) and tetraethylammonium chloride (5 mM) and at low temperature (4°C). Results BRACO19 has good solubility of at least 2 mg/mL in water and in physiological buffers of pH 7.4 and below. Protein binding to human serum albumin was 38%. No interaction with membrane lipids could be found. Cytotoxicity in 16HBE14o⁻, Calu-3, and human alveolar epithelial cells was in the range of IC₅₀ = 3.5 to 13.5 μM. Caco-2 cells were not affected at concentrations up to 50 μM. No transport of BRACO19 was detected across either cell monolayer in absorptive direction. In secretory direction, permeability was very low, with P_app values in the range of 0.25×10⁻⁷ to 0.98×10⁻⁷ cm/s for all epithelial cell cultures tested. The transport was not influenced by cyclosporin A or tetraethylammonium chloride or at 4°C, indicating that no efflux/influx systems or active transport are involved. Conclusions From these results, we conclude that the very poor permeability of BRACO19 is its main biopharmaceutical limitation. Further applications will require a suitable formulation to warrant adequate delivery across cellular barriers.     

Drug-Biomacromolecule Interaction XII: Comparative binding study of sulfaethidole to bovine serum albumin by equilibrium dialysis,fluorescence probe technique,uv difference spectrophotometry and circular dichroism

Binding of sulfaethidole to bovine serum albumin was studied by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism. Equilibrium dialysis method enabled us to estimate the total number of drug binding sites of albumin molecule. For sulfaethidole, albumin had 6 primary and 40 secondary binding sites. The primary and secondary binding constants were 0.9×10⁵ M⁻¹ and 0.2×10⁶ M⁻¹, respectively. 1-Anilino-8-naphthalenesulfonate (ANS) and 2-(4′-hydroxylbenzeneazo)-benzoic acid (HBAB) were used as the fluorescence probe and the uv spectrophotometric probe, respectively. In fluorescence probe technique, results indicated that the number of higher affinity drug binding site of albumin was 1 and the number of lower affinity drug binding sites of albumin was 3, and the primary and secondary drug binding constants for bovine serum albumin were 2.15×10⁵ M⁻¹ and 1.04×10⁵ M⁻¹, respectively. In uv difference spectrophotometry, binding sites were 3 and binding constant was 1.88×10⁵ M⁻¹. The above results suggest that several different methods should be used in ompensation for insufficient information about drug binding to albumin molecule given by only one method.     

Effect of nitroxyalkyl derivatives of fullerenylproline on the activity of CA<Superscript>3+</Superscript>-ATPase of sarcoplasmic reticulum

The effect of nitroxyalkyl derivatives of fullerenylproline methyl ester on the enzymatic activity of Ca²⁺-ATPase of sarcoplasmic reticulum (SR) has been studied. It is shown that hybrid derivatives of C₆₀ fullerene are capable of inhibiting the activity of Ca²⁺-ATPase of SR. The mononitrate inhibits the hydrolytic activity of the enzyme with K _i = 1.92 × 10⁻⁶ M; active Ca²⁺ transport, with K _i = 3.79 × 10⁻⁶ M. The dinitrate inhibits ATP hydrolysis with K _i = 2.38 × 10⁻⁸ M; Ca²⁺ transport, with K _i  = 3.08 × 10⁻⁸ M. Fullerenylproline methyl ester does not affect the enzymatic activity of Ca²⁺-ATPase. Based on these data it is possible to predict the possible fields of application for hybrid fullerene derivatives as potential drugs.     

Training and characterization of a quinine taste discrimination in rhesus monkeys

 There is a limited amount of information available about taste as a discriminative stimulus in non-human primates. The objective of this study was to establish a bitter taste (quinine sulfate) as a cue for lever selection and food reward in rhesus monkeys. Training took place in a series of steps that culminated in a schedule in which five lip contacts on a spout produced either quinine solution or water, followed by an opportunity to earn a food pellet by completing 20 presses on one of two levers. Responses on one of the levers resulted in food delivery if the solution contained quinine; responses on the other lever resulted in food delivery if the solution was water. A single session consisted of 100 randomly ordered taste trials with a 60-s interval between each trial. All of the animals acquired the discrimination, and the lowest quinine concentration that maintained consistent behavior was 0.3 mg/ml. To assess the specificity of the discrimination, compounds from other human taste categories were tested. A series of compounds that are detected as ”bitter” by humans (caffeine, 1.5×10^–3 M; strychnine, 9×10^–4 M; PTC, 6×10^–5 M, denatonium benzoate, 2.24×10^–4 M; and urea, 3.0×10^–1 M) produced full generalization to the quinine sulfate discriminative stimulus, while ”sweet” (sucrose, 2.9×10^–2 M) and ”salty” (sodium chloride, 1.4 M) stimuli did not. There was individual variation among animals in response to ”sour” compounds; acetic acid did not generalize to quinine, but HCl acid produced full generalization in one of three animals. These results suggest that a ”bitter” taste cue is controlling the quinine discrimination. Received: 3 February 1998 / Final version: 14 August 1998     

Pharmacokinetics of glibenclamide and its metabolites in diabetic patients with impaired renal function

Background: Glibenclamide (Gb) may provoke long-lasting hypoglycaemic reactions, and one of the known risk factors is impaired renal function. We have demonstrated Gb to have a terminal elimination half-life of 15 h, and the main metabolites have a hypoglycaemic effect. With few exceptions, detailed studies on second generation sulphonylureas in diabetics with impaired renal function are lacking. Therefore, we analysed the pharmacokinetics of Gb and its active metabolites, 4-trans-hydroxyglibenclamide (M1) and 3-cis-hydroxyglibenclamide (M2) in this patient group. Methods: Two groups of 11 diabetic patients with impaired renal function (IRF, iohexol clearance range 7–42 ml · min⁻¹ · 1.73 m⁻²) or normal renal function (NRF, iohexol clearance range 75–140 ml · min⁻¹ · 1.73 m⁻²) were compared. A single oral 7-mg dose of Gb was administered after overnight fasting. Serum samples and urine collections were obtained over 48 h and 24 h, respectively. Concentrations of Gb, M1 and M2 were determined by a sensitive and selective high-performance liquid chromatography assay. Results: Peak serum values of M1 (24–85 ng · ml⁻¹ vs 16–57 ng · ml⁻¹), M2 (7–22 ng · ml⁻¹ vs <5–18 ng · ml⁻¹) and M1 + M2 (32–100 ng · ml⁻¹ vs 23–76 ng · ml⁻¹) were higher in the IRF group. AUC and C_max of Gb were lower and the clearance to bioavailability ratio (CL/f) was higher in the IRF group. AUC and C_max of M1 were higher and CL/f lower in the IRF group. Much lower amounts of M1 and M2 were excreted in the urine in the IRF group (7.2% vs 26.4% in 24 h). The fraction of the Gb dose excreted as metabolites (fe(met) 0–24 h), ranged between 0.005 and 0.36 and correlated significantly with renal function measured by iohexol clearance. No other pharmacokinetic differences were found. Conclusion: The differences in AUC, C_max and CL/f of Gb may be explained by a higher free fraction in the IRF group which would increase Gb metabolic clearance. The inverse findings regarding M1 may be explained by the fact that the metabolites are primarily eliminated by the kidneys. After a single dose of Gb, neither Gb, M1 nor M2 seemed to accumulate in diabetic subjects with IRF. As only small amounts of M1 and M2 were excreted in the urine, this indicates one or several complementary non-renal elimination routes, e.g. shunting of metabolised Gb to the biliary excretion route and/or enterohepatic recycling of both metabolites and unmetabolised Gb. Received: 21 April 1997 / Accepted in revised form: 14 October 1997     

Male reproductive toxicity of four bisphenol antioxidants in mice and rats and their estrogenic effect

Male mice and rats were fed a diet containing four bisphenol antioxidants, 2,2′-methylenebis(4-ethyl-6-tert-butylphenol) (ME), 2,2′-methylenebis(4-methyl-6-tert-butylphenol) (MM), 4,4′-butylidenebis(3-methyl-6-tert-butylphenol) (BM), or 4,4′-thiobis(3-methyl-6-tert-butylphenol) (TM) at levels of 0.06–0.25% for 2 months. BM and TM decreased epididymal, seminal vesicular, prostate and preputial weights, and injured seminiferous tubules in mice in a dose-dependent fashion. BM and TM also reduced sex accessory organ weights and sperm production capacity in rats, but MM and ME were more toxic to rats than BM and TM. ME and MM did not bind ER_α up to 10⁻³ M, while BM and TM competitively bound ER_α against β-estradiol (E2). Fifty percent inhibitory concentrations (IC₅₀ s) of BM, TM, and bisphenol A (positive control) against E2-binding were 7.3×10⁻⁶ M, 1.8×10⁻⁵ M, and 1.4×10⁻⁵ M, respectively. When ovariectomized (OVX) mice were sc administered TM at doses of 60 and 300 mg/kg/day for 4 days, or when OVX mice were fed BM in the diet at a level of 0.25% for 2 months, uterine weight was significantly increased. These results suggest that BM and TM are weakly toxic, possibly through an estrogenic mechanism to male reproductive organs in mice as well as rats, while MM and ME may be the direct testicular toxins in rats but not mice. A part of this study was presented at the 74th annual meeting of the Japanese Society for Hygiene held from 24–27 March 2004 in Tokyo, Japan.     

Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin

Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 × 10⁵ M⁻¹ for PFOA and 2.2 × 10⁴ M⁻¹ for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-l-proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow’s binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 × 10⁶ M⁻¹ and 2.2 × 10⁶ M⁻¹. At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 × 10⁶ M⁻¹ and 1.2 × 10⁶ M⁻¹, whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: role of RhoA/Rho-kinase and intracellular Ca2+ concentration

It is known that ceramide may play an important regulatory role in vascular tone although its effect on vascular tone and the mechanisms involved are controversial. The present study was designed to investigate the effects of ceramide and its key initial regulators, TNF-α and neutral sphingomyelinase (SMase), on vascular tone of isolated rat thoracic aortic rings and elucidate the mechanisms involved in the changes in vascular tone induced by ceramide. Contractile responses and Fura-2 Ca²⁺ signals were measured in rat thoracic aortic rings or strips. 10⁻⁵ M C₂-ceramide, 0.1 U/ml neutral sphingomyelinase (SMase), and 5×10⁻⁷ g/ml TNF-α had no effect on resting tone in rat thoracic aortic rings. However, in phenylephrine-induced pre-contracted rings, treatment with ceramide, SMase, and TNF-α evoked a gradual but sustained vasodilation. Vasodilation effect in response to 10⁻⁵ M C₂-ceramide was not significantly changed by the absence or presence of endothelium, a cyclooxygenase pathway inhibitor (10⁻⁶ M indomethacin), or PKC inhibitors (10⁻⁵ M H-7 & 5×10⁻⁷ M calphostin-C). Pretreatment with 1 μM Y-27632, a RhoA/Rho-kinase inhibitor, significantly inhibited the phenylephrine-induced contraction itself as well as the C₂-ceramide-induced vasodilation. Pre-treatment with 10⁻⁵ M C₂-ceramide had no effect on phasic rise in [Ca²⁺]_i and tension evoked by stimulation with 10⁻⁸ M phenylephrine, but post-treatment of C₂-ceramide significantly reduced the phenylephrine-induced secondary tonic [Ca²⁺]_i and tension plateau. Our results indicate that C₂-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta. Furthermore, inhibition of phenylephrine-induced activation of RhoA/Rho-kinase pathway as well as phenylephrine-induced elevations in [Ca²⁺]_i are clearly a key factors in C₂-ceramide-induced vasodilation. G.-J. Jang and D.-S. Ahn contributed equally to this work