Carbon film resistor electrode for amperometric determination of acetaminophen in pharmaceutical formulations

Flow injection analysis (FIA) with amperometric detection was employed for acetaminophen quantification in pharmaceutical formulations using a carbon film resistor electrode. This sensor exhibited sharp and reproducible current peaks for acetaminophen without chemical modification of its surface. A wide linear working range (8.0 × 10⁻⁷ to 5.0 × 10⁻⁴ mol L⁻¹) in phosphate buffer solution as well as high sensitivity (0.143 A mol⁻¹ L cm⁻²) and low submicromolar detection limit (1.36 × 10⁻⁷ mol L⁻¹) were achieved. The repeatability (R.S.D. for 10 successive injections of 5.0 × 10⁻⁶ and 5.0 × 10⁻⁵ mol L⁻¹ acetaminophen solutions) was 3.1 and 1.3%, respectively, without any memory effect between injections. The new procedure was applied to the analyses of commercial pharmaceutical products and the results were in good agreement with those obtained utilizing a spectrophotometric method. Consequently, this amperometric method has been shown to be very suitable for quality control analyses and other applications with similar requirements.     

A new hydrogel for the extended and complete prednisolone release in the GI tract

The issue of incomplete release of poorly soluble drugs from sustained-release oral formulations is addressed using prednisolone (PDS) as the model drug and a novel highly swelling hydrogel as the rate-controlling material. The hydrogel was formed by heating N-carboxymethylchitosan (CMC) to 80 °C for 24 h. Swelling, alkalimetry, FTIR, DSC, and solid-state NMR studies showed that the treatment produced physical crosslinking, i.e., polymer chain entanglement. A controlled-release system was prepared by coating an inert compacted support of ethylcellulose (50 mg; diameter, 6 mm) with a CMC layer containing dispersed PDS powder (10–50 μm). The system was heated to crosslink the CMC coating, then drug release to simulated GI fluids was studied in vitro. The drug release pattern and term were modulated via the layer mass (LM) (10 or 14 mg cm⁻²) and/or the drug–polymer wt ratio (D/P) (1:5 or 2:5). The rate parameter, K, and the time exponent, n, of the Peppas equation were: K = 26.6 ± 0.3 h⁻ⁿ, n = 0.78 ± 0.02 (LM, 10 mg cm⁻²; D/P, 1:5); K = 24.7 ± 0.7 h⁻ⁿ, n = 0.56 ± 0.02 (LM, 14 mg cm⁻²; D/P, 1:5); K = 20.7 ± 0.3 h⁻ⁿ, n = 0.76 ± 0.01 (LM, 10 mg cm⁻²; D/P, 2:5). Hydrogel swelling was faster than drug release. This was controlled, in a first stage, by drug dissolution–diffusion in the swollen gel, and subsequently, by diffusion. The drug release rate was unaffected by the GI pH variations, and slightly affected by the environmental hydrodynamics. The system promises an extended and complete release of poorly soluble drugs in the GI tract.     

Disposition kinetics and urinary excretion of levofloxacin on concomitant administration with meloxicam in cross-bred calves

The disposition kinetics and urinary excretion study of levofloxacin was conducted in 5 male cross-bred calves following its single intravenous administration (4 mg kg⁻¹) concurrently with meloxicam (0.5 mg kg⁻¹). Levofloxacin was estimated by microbiological assay. The drug levels above MIC₉₀ in plasma, were detected up to 10 h. Disposition kinetic parameters were calculated by two-compartment open model. Rapid distribution of levofloxacin was evidenced by a small distribution half-life (0.13 ± 0.01 h) and high K₁₂/K₂₁ ratio (2.21 ± 0.15). High ratio of AUC/MIC (90.2 ± 3.41) indicated good antibacterial activity of levofloxacin. The AUC, Vd_area, elimination half-life, MRT and total body clearance were 9.02 ± 0.34 μg ml⁻¹ h, 1.38 ± 0.05 l kg⁻1, 2.16 ± 0.08 h, 2.58 ± 0.11 h and 0.45 ± 0.02 l kg⁻¹ h⁻¹, respectively. About 38.4% of the administered dose of levofloxacin was excreted in urine within 24 h. A suitable intravenous dosage regimen for levofloxacin would be 1.8 mg kg⁻¹ repeated at 8 h intervals when prescribed with meloxicam in calves.     

Determination of salbutamol in syrups by capillary electrophoresis with contactless conductivity detection (CE-C(4)D)

This paper describes the separation and quantification of salbutamol in pharmaceutical products (salbutamol syrups) by capillary electrophoresis (CE) with contactless conductivity detection (C⁴D). The system was studied by micellar electrokinetic capillary chromatography (MEKC) and free solution capillary electrophoresis (FSCE), being the latter chosen in function of best resolution and sensitivity in comparison with the MEKC method. CE-C⁴D was applied to analysis of salbutamol in syrups utilizing 1.0 × 10⁻² mol L⁻¹ acetic acid/sodium acetate buffer (pH 4.9) as running electrolyte. Tetraethylammonium (TEA) solution was used as internal standard. The results obtained include a linear dynamic range from 7.0 × 10⁻⁵ to 3.0 × 10⁻⁴ mol L⁻¹ and good repeatability (R.S.D. = 4.7% for n = 10 for a 7.0 × 10⁻⁵ mol L⁻¹ salbutamol solution). The detection limit was calculated as 1.0 × 10⁻⁵ mol L⁻¹ and the limit of quantification was estimated as 3.3 × 10⁻⁵ mol L⁻¹. For syrups analysis the reproducibility presented deviations between 1.5% and 2.5% (three different days) obtained for measurements in triplicate.     

Preparation of a diclofenac potentiometric sensor and its application to pharmaceutical analysis and to drug recovery from biological fluids

A novel diclofenac ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The diclofenac complex with hexadecylpyridinium bromide is obtained in situ by soaking the PVC-membranes in a 1 × 10⁻² M diclofenac solution. Among four different solvent mediators tested, dibutyl phthalate (DBP) exhibited a proper behavior including Nernstian slope of the calibration curve, fast response time and good reproducibility of the emf values. The electrode exhibits a Nernstian slope of −59 ± 1 mV decade⁻¹ for diclofenac in the concentration range 1.0 × 10⁻⁵ to 1.0 × 10⁻² M with a limit of detection of 4.0 × 10⁻⁶ M. The electrode displays a good selectivity for diclofenac with respect to a number of common inorganic and organic species. It can be used in a pH range of 6.0–9.0. The membrane sensor was successfully applied to the determination of diclofenac in its tablets as well as for its recovery from blood serum and urine samples.     

Oral bioavailability and intestinal secretion of amitriptyline: Role of P-glycoprotein?

The aim of the study was to evaluate the influence of quinidine, a P-glycoprotein inhibitor, on oral bioavailability and on intestinal secretion of amitriptyline, a tricyclic antidepressant. Amitriptyline was administrated intravenously (5 mg/kg) and orally (50 mg/kg) to rabbits, with and without quinidine. Jejunal segments of rats were mounted on diffusions chambers and the permeation of amitriptyline was measured across the tissue in luminal–serosal (LS) and serosal–luminal (SL) directions, with and without quinidine. Finally, an in situ recirculating intestinal perfusion model was performed in rabbits to study amitriptyline permeation in LS direction with and without quinidine. Absolute oral bioavailability (F) of amitriptyline was significantly increased more than three-fold in presence of quinidine (F = 0.6 ± 0.4% versus 1.9 ± 1.1%). The apparent permeability coefficients in SL direction were significantly higher than in LS direction (P_{app (SL)} = 6.01 ± 2.42 versus P_{app (LS)} = 4.90 ± 2.73 × 10⁻⁴ cm min⁻¹). In presence of quinidine, the intestinal absorption was increased (P_{app (LS)} = 4.02 ± 2.91 versus P_{app (LS)} = 5.99 ± 2.43 × 10⁻⁴ cm min⁻¹) and the intestinal secretion was decreased (P_{app (SL)} = 4.58 ± 0.54 versus P_{app (LS)} = 3.63 ± 1.46 × 10⁻⁴ cm min⁻¹) but not significantly. In conclusion, P-glycoprotein appears to be involved in oral amitriptyline absorption but other intestinal uptake and efflux transporters maybe implicated.     

Polarographic determination of diazepam with its parallel catalytic wave in the presence of persulfate

A new method for the determination of diazepam was proposed based on its polarographic catalytic wave in the presence of persulfate. In 0.20 M NaAc–HAc (pH 4.7)–2.0×10⁻² M K₂S₂O₈ supporting electrolyte, the reduction wave of diazepam with peak potential −0.89 V (versus SCE) was catalyzed, producing a parallel catalytic wave. The peak current of the catalytic wave was 15 times higher than that of the corresponding reduction wave for 4.0×10⁻⁶ M diazepam, and was rectilinear to diazepam concentration in the range of 5.6×10⁻⁸ to 8.8×10⁻⁶ and 8.8×10⁻⁶ to 2.0×10⁻⁴ M. The detection limit was 9.6×10⁻⁹ M. The mechanism of the parallel catalytic wave of diazepam was discussed.     

Penetration of benzene, toluene and xylenes contained in gasolines through human abdominal skin in vitro

Few studies are available in literature on the risk for humans from skin exposure to gasolines. This work is focused on the in vitro skin penetration of benzene (carcinogenic substance), toluene and xylenes. We examined three commercial gasolines using the Franz diffusion cells and human abdominal full thickness skin. Gasoline composition was determined using a multi-dimensional gas chromatographic (MDGC) technique. Aromatic compounds into the receptor fluid, consisting of saline solution were quantitated by a gas chromatography technique equipped with a flame ionization detector (GC–FID) and coupled with a headspace-solid phase micro extraction system (HS-SPME).

Among the three substances, benzene showed the highest average apparent permeability coefficient (K_p = 43.8 × 10⁻⁵ cm h⁻¹) compared to toluene (K_p = 6.48 × 10⁻⁵ cm h⁻¹) and xylenes (K_p = 0.84 × 10⁻⁵ cm h⁻¹). This value could be explained by the lower boiling point and higher water solubility of benzene. Lag times were about 1 h for benzene and 2 h for toluene and xylenes. Averaged total recoveries in the receptor fluid were 0.43% of dose for benzene, 0.06% for toluene and 0.008% for xylenes.

A statistical significative difference (Student’s t-test, P < 0.05) between the fluxes calculated for the three gasolines are noted only for xylene and for toluene between gasolines #1 (richer in aromatic compounds) and #3. The obtained apparent permeability coefficient are useful for determining the permeability of these aromatics components from gasolines of a different composition.

Hands exposure risk, calculated using RfD and RfC as defined by US EPA, is critical for benzene. The risk of skin permeation of gasoline, and, in particular, of benzene, should be better evaluated for those workers who have a large potential for exposure. Adequate personal protective equipment should be used in the high exposure jobs, mainly for hands and forearms.     

Thiols in Scenedesmus vacuolatus upon exposure to metals and metalloids

Phytochelatins are intracellular metal ligands produced by algae when exposed to elevated metal concentrations. In freshwater ecosystems, algae are exposed to a wide range of metals and metalloids. The aim of this study was thus to investigate phytochelatin induction in freshwater algae upon metal and metalloid exposure. To that purpose, the unicellular green alga Scenedesmus vacuolatus, was exposed to Cu, Zn, Ni, Pb and Ag, as well as to As(III), As(V), Sb(III) and Sb(V), and examined for its thiol content (gamma-glutamylcysteine, glutathione and phytochelatins). Glutathione content was found to decrease upon the exposure to Zn and to increase upon the exposure to Pb and Ag. Phytochelatins were only induced by Cu (at [Cu²⁺] = 8 × 10⁻¹¹ M) and Pb (at [Pb²⁺] = 8 × 10⁻¹¹ to 8 × 10⁻¹⁰ M), where [Cu²⁺] and [Pb²⁺] are computed free metal ion concentrations. Glutathione content also decreased upon the exposure to Sb(V) whereas an increase was observed as a result as the exposure to As(III) and As(V). The metalloids As(III), As(V) and Sb(III) in the concentration range from 8 × 10⁻⁶ to 2 × 10⁻⁴ M (total concentrations of oxyanions) were inducing phytochelatins. Glutathione and phytochelatin content in S. vacuolatus do thus sensitively respond to exposure to a number of metals and metalloids.     

Electrochemical behavior and determination of amiloride drug in bulk form and pharmaceutical formulation at mercury electrodes

The polarographic behavior of amiloride hydrochloride has been studied in Britton–Robinson buffers of pH 1.9–11. In acidic medium at pH≤2, the dc-polarograms exhibited a single 4-electron cathodic irreversible wave, while at pH values >2, a second two-electron irreversible cathodic wave appeared at a more negative potential. The single or first wave may be attributed to the cleavage of the double bond of the ---CH=NH of the imidino amide group with the release of NH₃. While the second wave may be due to the saturation of the C=O of the carboxamide moiety. A polarographic procedure of suffocate sensitivity for the determination of bulk amiloride drug in Britton–Robinson buffer at pH 2 is described. The calibration graph was obtained over the concentration range 2.5×10⁻⁵ to 2.5×10⁻⁴ M amiloride. The limits of detection (LOD) and quantitation (LOQ) of the procedure were 1×10⁻⁵ and 3.3×10⁻⁴ M bulk amiloride, respectively. Moreover, a differential-pulse adsorptive cathodic stripping voltammetric procedure has been described to assay of the drug at lower concentration levels. The optimal conditions were: E_acc=−0.9 V, t_acc=30 s, scan rate=20 mV, pulse-height=90 mV and Britton–Robinson buffer of pH 8. The calibration graph was obtained over the concentration range 2×10⁻⁸ to 1×10⁻⁶ M for bulk amiloride. Both procedures were successfully applied to the determination of amiloride in tablets without the necessity for sample pretreatment or any time-consuming extraction or evaporation steps prior to the drug analysis.     

Determination of dipyridamole in pharmaceutical preparations using square wave voltammetry

An analytical methodology using square wave voltammetry (SWV) at a hanging mercury drop electrode (HMDE) was developed for the quantitative determination of dipyridamole (DIP), a drug used for the treatment of several cardiovascular diseases, in pharmaceutical tablets and injections of Persantin^® in phosphate buffer (pH 3.0; 0.1 M). After optimization of the parameters for SWV, analytical curves were obtained for application in the range of 1.28 × 10⁻⁶ M to 7.02 × 10⁻⁶ M. It was found a detection limit (DL) of 1.88 × 10⁻⁸ M (9.50 ng/ml). The repeatability and the reproducibility of the method were determinated by successive measurements of DIP solutions on the range of the analytical curve with a coefficient variation of 0.97% (n = 5) and 1.15%, respectively. The apparent recoveries were obtained by the IUPAC recommended procedure using the second reduction peak. Recoveries obtained by SWV were compared with the UV–vis spectrophotometric method. It was found that the determination of DIP in Persantin^® tablets gave a mean value of 75.6 ± 0.4 mg (100.8%) and 68.9 ± 0.3 mg (91.8%) for SWV and UV–vis spectrophotometry, respectively. In the case of injections, it was found 10.4 ± 0.1 mg (103.4%) and 9.9 ± 0.2 mg (99.9%) for SWV and UV–vis spectrophotometry. Both apparent recoveries for the two types of formulations are in good accordance with the declared value of 75 mg (tablets) and 10 mg (injections).     

Contribution of Rho-kinase in human gallbladder contractions

Rho/Rho-kinase-mediated pathway has been involved in a variety of physiological processes, including Ca²⁺ sensitization, which enhances smooth muscle contraction. In this study, first of all we investigated the expression of Rho-kinase (ROCK-2) and then the role of this protein in the control of smooth muscle contraction in the isolated human gallbladder. For this purpose, we examined the effects of a selective Rho-kinase inhibitor, (+)- (R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10^− 8 − 3 × 10^− 5 M) on carbachol (10^− 8–10^− 4 M), cholecystokinin-8 (10^− 8 M), endothelin-1 (10^− 8 M), histamine (10^− 5 M), neurokinin A (10^− 7–10^− 6 M), 5-hydroxytryptamine (10^− 6–10^− 5 M) and potassium chloride (KCl, 25–50 mM)-induced contractions as well as spontaneous contractile activity. Y-27632 (10^− 5 M) significantly reduced 5-hydroxytryptamine, neurokinin A and KCl-induced contractions. Moreover, this Rho-kinase inhibitor (10^− 8 − 3 × 10^− 5 M, cumulatively) relaxed the contractions produced by cholecystokinin-8, endothelin-1 and histamine in a concentration-dependent manner, being the pEC₅₀ values for Y-27632 5.74 ± 0.12, 5.33 ± 0.09 and 5.95 ± 0.18, respectively. Carbachol (10^− 8–10^− 4 M) pfroduced concentration-dependent contractions, which were also inhibited significantly by Y-27632. In addition, the spontaneous contractile activity was suppressed in the presence of Y-27632 (10^− 6–10^− 5 M). Moreover, Western blot analysis has revealed that Rho-kinase is expressed in homogenates of the human gallbladder. Taken together, these results show that Rho-kinase is expressed in the human gallbladder, and it has an essential role in agonists and depolarization-induced contractions as well as spontaneous contractile activity.     

Electrochemical study on the behavior of Morin and its interaction with DNA

Voltammetric behavior of Morin was studied in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution at glassy carbon electrode (GCE) using cyclic voltammetry (CV). Morin showed an irreversible anodic peak at 0.720 V in CV which was involving two electrons and two protons. Also, the interaction of Morin with double-stranded calf thymus DNA (ctDNA) was studied by CV at GCE with an irreversible electrochemical equation. As a result of reaction with ctDNA, the voltammetric peak of Morin was a position shift and the peak current decreased. The diffusion coefficients of both free and binding Morin (D_f = 1.1086 × 10⁻⁷ cm² s⁻¹ and D_b = 8.2544 × 10⁻⁹ cm² s⁻¹), binding constant (K = 1.7765 × 10⁷ cm³ mol⁻¹), and binding site size (s = 0.8510) of the Morin–DNA complex were obtained simultaneously by non-linear fit analysis. The results demonstrate that Morin can bind to ctDNA in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution and the ring B of Morin intercalates between the DNA base pairs.     

Electrochemical determination of Cephalothin antibiotic by adsorptive stripping voltammetric technique

A sensitive and reliable stripping voltammetric method was developed to determine Cephalothin antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then a negative sweep was initiated, which yield a well defined cathodic peak at −625 mV versus Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of Cephalothin and it shows a linear response in the range from 4×10⁻⁷ to 1.2×10⁻⁶ mol l⁻¹ (correlation coefficient=0.9995) and the detection limit (S/N=3) is 3.3×10⁻⁹ mol l⁻¹ at an accumulation time of 3 min. The developed AdSV procedure shows a good reproducibility, the relative standard deviation R.S.D.% (n=10) at a concentration level of 5×10⁻⁷ mol l⁻¹ was 0.94%. Possible interferences by other pharmaceutical drugs and surfactants have been also evaluated. The applicability of this approach was illustrated by the determination of Cephalothin in pharmaceutical preparation and biological fluids such as serum and urine.     

Catalytic action of copper (II) ion on electrochemical oxidation of metformine and voltammetric determination of metformine in pharmaceuticals

The catalytic effect of Cu(II) ion toward the oxidation of metformine (MET) have been observed in NH₃·H₂O–NH₄Cl buffer (pH 8.9; 0.1 M). The oxidation peak current of imino-group in guanidino-group of MET at 0.95 V at carbon paste electrode (C/PE) in the presence of 2.0 × 10⁻⁴ M Cu(II) ion was increased by about 20 times and the peak potential was unchanged compared with that in the absence of Cu(II) ion. Moreover, the oxidation peak current of MET at multiwalled carbon nanotube paste electrode (MWCNT/PE) was further increased by about three times compared with that at C/PE in the same medium. Based on the catalytic oxidation peak of MET by Cu(II) ion at MWCNT/PE, a voltammetric method for the determination of MET is developed. The peak current of the catalytic oxidation peak was proportional to MET concentration in the range of 2.0 × 10⁻⁷ to 1.0 × 10⁻⁵ M. The detection limit was 6.7 × 10⁻⁸ M.     

Enzyme-amplified lanthanide luminescence based on complexation reaction--a new technique for the determination of doxycycline

A new spectrofluorimetric method is described for the determination of doxycycline, based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu³⁺–doxycycline forms a ternary complex with lysozyme in close proximity and lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu³⁺ at 612 nm in doxycycline–Eu³⁺ binary complex. The enhanced fluorescence intensity is in proportion to the concentration of doxycycline. The limit of detection is 1.28×10⁻⁸ mol l⁻¹, with a linear range from 1.7×10⁻⁷ to 1.7×10⁻⁶ mol l⁻¹. Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of doxycycline in serum, urine and real samples. The mechanism of fluorescence enhancement was also studied.     

The influence of concentration on the release of drugs from gels and matrices containing Methocel

The release of propranolol hydrochloride from hydroxypropylmethylcellulose and methylcellulose matrices has been examined at constant cellulose ether contents. As the drug content decreased, the release rate of propranolol became disproportionately higher. HPMC K4M, HPMC F4M and HPMC E4M all performed similarly. However, with methylcellulose matrices, a burst release at low drug levels was apparently due to a failure of the matrix to maintain integrity. Explanations were sought on the basis of diffusional studies. Apparent diffusion coefficients were in the order of 3.1–3.8 × 10⁻⁶ cm² s⁻¹ for propranolol hydrochloride. Each of the four grades performed similarly. Using similar diffusional studies, but HPMC K15M as the polymer, an apparent diffusion coefficient of 3.6 × 10⁻⁶ cm² s⁻¹ was derived, indicating that the coefficient was independent of molecular weight. The coefficient was dependent on HPMC content decreasing from approx. 5.5 × 10⁻⁶ to 3 × 10⁻⁶ cm² s⁻¹ as the HPMC content was increased from 5 to 15% w/w. The diffusion coefficients of tetracycline hydrochloride were lower and conversion to the free base is postulated as the explanation of previously described anomolous release for this drug from matrix tablets. The tortuosity of the gels was independent of the included drug.     

Polarographic behavior of cephalexin and its determination in pharmaceuticals and human serum

Cephalexin gives a reduction wave in 0.03 mol/l HCl medium at ca. −1.24 V. With cephalexin concentration higher than 2.5×10⁻⁵ mol/l, another reduction wave is observed at ca. −0.90 V. These reduction waves are attributed to the reduction of ethylenic bond of a six-membered dihydrothiazine ring. When H₂O₂ is present, the reduction wave at ca. −0.90 V is catalyzed by H₂O₂ and its reduction intermediate hydroxyl radical √OH, producing a catalytic wave. However, the reduction wave at ca. −1.24 V remains nearly unchanged. A sensitive polarographic method for the determination of cephalexin is proposed based on the reduction wave of cephalexin. The second-order derivative peak current of the wave at ca. −1.24 V is rectilinear to the cephalexin concentration in the range 1.0×10⁻⁷ to 2.5×10⁻⁵ mol/l, and the detection limit is 5.0×10⁻⁸ mol/l. The proposed method is applied to the individual tablet dosage form and human serum.     

Effect of temperature on limitation by MK-801 of firing of action potentials by spinal cord neurons in cell culture

The anticonvulsant, MK-801, limited sustained high frequency repetitive firing of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture. Limitation was voltage- and temperature-dependent and was accompanied by decreasing rate of rise of action potentials until firing ceased during the 400 ms depolarizations. The IC₅₀ for limitation was 2 × 10⁻⁷ M at 37°C, 6.4 × 10⁻⁷ M at 35°C, and 4 × 10⁻⁵ M at 23°C. The relationship between the percentage of neurons capable of sustained repetitive firing and MK-801 concentration at 33°C was biphasic. The first phase (about 50%) of limitation had IC_50a = 1.5 × 10⁻⁷ M, and the second had IC_50b = 2 × 10⁻⁴ M; the midpoint of the connecting plateau was 10⁻⁵ M. At temperatures below 37°C, the current needed to achieve maximal firing increased. The maximal rate of rise, maximal firing frequency and sensitivity to MK-801 of action potentials elicited by 1 ms stimuli decreased at temperatures below 37°C. Passive membrane properties were unchanged. Slow firing and a temperature-sensitive conformational change in voltage-activated sodium channels could account for the higher concentrations of MK-801 required to block sodium-dependent action potentials at temperatures below 37°C.     

Herbicide effects on freshwater benthic diatoms: induction of nucleus alterations and silica cell wall abnormalities

Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5 × 10⁻⁶, 10⁻⁶, 10⁻⁷ M) for 6 h followed by a 24 h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65‰), S.D. = 4.23) and the highest concentrations (29.40‰, S.D. = 8.49 for 10⁻⁶ M and 35.96‰, S.D. = 3.71 for 5 × 10⁻⁶ M) was statistically significant (Tukey test, P < 0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10⁻⁶ and 5 × 10⁻⁶ M; Tukey test, P < 0.05). These two parameters tended to increase together (Pearson correlation = 0.702, P < 0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells.