Ondansetron and teratogenicity in rats: Evidence for a mechanism mediated via embryonic hERG blockade.

The potent hERG channel blocking drug ondansetron is used off-label for treatment of nausea and vomiting in early pregnancy. Some human epidemiological studies have associated ondansetron with fetal cardiovascular defects and orofacial clefts. This study investigated the effects of ondanestron on embryonic heart rhythm of gestational day (GD) 13 rat embryos in vitro and then integrated the results with published animal teratology, and animal and human pharmacokinetic studies to perform a risk evaluation. Ondansetron caused concentration dependent bradycardia and arrhythmia. Cardiovascular malformations in rats occurred at exposures slightly higher than those in early human pregnancy. Together the results suggest that ondansetron can have teratogenic potential in rats and humans mediated via hERG block and severe heart rhythm disturbances in the embryo. The risk may be increased in human pregnancy if additional risk factors are present such as hypokalemia.

A selective potentiometric copper (II) ion sensor based on the functionalized ZnO nanorods.

In this work, ZnO nanorods were hydrothermally grown on the gold-coated glass substrate and characterized by field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD) techniques. The ZnO nanorods were functionalized by two different approaches and performance of the sensor electrode was monitored. Fourier transform infrared spectroscopy (FTIR) was carried out for the confirmation of interaction between the ionophore molecules and ZnO nanorods. In addition to this, the surface of the electrode was characterized by X-ray photoelectron spectroscopy (XPS) showing the chemical and electronic state of the ionophore and ZnO nanorod components. The ionophore solution was prepared in the stabilizer, poly vinyl chloride (PVC) and additives, and then functionalized on the ZnO nanorods that have shown the Nernstian response with the slope of 31 mV/decade. However, the Cu2+ ion sensor was fabricated only by immobilizing the selective copper ion ionophore membrane without the use of PVC, plasticizers, additives and stabilizers and the sensor electrode showed a linear potentiometric response with a slope of 56.4 mV/decade within a large dynamic concentration range (from 1.0 x 10(-6) to 1.0 x 10(-1) M) of copper (II) nitrate solutions. The sensor showed excellent repeatability and reproducibility with response time of less than 10 s. The negligible response to potentially interfering metal ions such as calcium (Ca2+), magnesium (Mg2+), potassium (K+), iron (Fe3+), zinc (Zn2+), and sodium (Na+) allows this sensor to be used in biological studies. It may also be used as an indicator electrode in the potentiometric titration.

Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

Trapping and detection of single molecules in water.

An innovative nanoprobe-based device that can measure and adjust the pH, can mimic biochemistry, can create microscale vortices in water, and can be used to trap single molecules is presented. Because the analytes in question to trap and detect are small in dimensions, we start by presenting scaling issues and challenging limitations for miniaturized chemical nanosensors. Advantages of using nanoprobes e.g., isolated nanowires, as the components in chemical sensing are discussed. How the observation of the physical property can beneficially change with isomorphic scaling is highlighted. Some of the technology-related constrains are presented for specific sensors. Solutions to overcome such problems are also given. Different aspects, e.g., sample size and sensitivity, for chemical sensing at the nanoscale are highlighted.

Selective calcium ion detection with functionalized ZnO nanorods-extended gate MOSFET.

Zinc oxide nanorod-extended gate field effect transistor (MOSFET) is demonstrated for the detection of calcium (Ca(2+)) ions. ZnO nanorods were grown on the surface of a silver wire to produce an electrochemical nanosensor for selectively detecting Ca(2+). The electrochemical response from the interaction between the ZnO nanorods and Ca(2+) in an aqueous solution is coupled directly to the gate of a field effect transistor (MOSFET). The induced voltage change on the gate results in a measureable current response. In order to adapt the sensors for Ca(2+) ions measurements in biological fluids with sufficient selectivity and stability, a plastic membrane coating containing ionophores was applied on the nanorods. The sensor exhibited a linear response within the range of interest from 1 microM to 1 mM. This work demonstrates a simple technique for sensitive detection of Ca(2+) ions by efficient transfer of the chemical response directly to a standard electronic component producing a low impedance signal.

The region ion sensitive field effect transistor, a novel bioelectronic nanosensor.

A novel type of bioelectronic region ion sensitive field effect transistor (RISFET) nanosensor was constructed and demonstrated on two different sensor chips that could measure glucose with good linearity in the range of 0-0.6mM and 0-0.3mM with a limit of detection of 0.1 and 0.04 mM, respectively. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. For glucose measurements, negatively charged gluconate ions were gathered between the sensing electrodes. The signal current response was measured using a low-noise pico ammeter (pA). Two different sizes of the RISFET sensor chips were constructed using conventional electron beam lithography. The measurements are done in partial volumes mainly restricted by the working distance between the sensing electrodes (790 and 2500 nm, respectively) and the influence of electrical fields that are concentrating the ions. The sensitivity was 28 pA/mM (2500 nm) and 830 pA/mM (790 nm), respectively. That is an increase in field strength by five times between the sensing electrodes increased the sensitivity by 30 times. The volumes expressed in this way are in low or sub femtoliter range. Preliminary studies revealed that with suitable modification and control of parameters such as the electric control signals and the chip electrode dimensions this sensor could also be used as a nanobiosensor by applying single enzyme molecule trapping. Hypotheses are given for impedance factors of the RISFET conducting channel.

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.

Stage-specific skeletal and visceral defects of the I(Kr)-blocker almokalant: further evidence for teratogenicity via a hypoxia-related mechanism.

BACKGROUND: As a class effect, potent I(Kr)-blockers have been shown to induce stage-specific external malformations. The aim of this study was to investigate whether I(Kr)-blockers also induce stage-specific visceral and skeletal defects and to further elucidate a proposed arrhythmia-hypoxia hypothesis. METHODS: Single oral doses of the selective I(Kr)-blocker almokalant (ALM) 25-150 micromol/kg, 7-14 dams/group, were given to Sprague-Dawley rats on gestation days (GD) 10-14, and the fetuses were examined for malformations on GD 21. One group was pretreated with the spin-trapping agent, alpha-phenyl-N-t-butylnitrone (PBN), given intraperitoneally 1 hr before ALM on GD 11. RESULTS: Cardiac ventricular septum defects and vascular malformations were observed after dosing on GD 10-11 and, to a lesser degree, on GD 12-13. Urogenital defects, absence/malposition of the postcaval lung lobe, and attenuated diaphragm were observed mainly on GD 10-11. Skeletal examination showed a high incidence of vertebral abnormalities on thoracic level on GD 10, on lower thoracic to caudal level on GD 11, and sternebral defects were observed all days. On GD 13 brachy-, oligo-, and syndactyly of the forepaw were induced, and of the hindpaw on GD 14. PBN reduced the incidence of both visceral and skeletal defects. CONCLUSIONS: The stage specificity of observed visceral and skeletal defects correlates well with what has been reported in the literature after temporary interruption of oxygen supply during the same stages of development. The protective effect by PBN present further evidence that the teratogenicity of potent I(Kr)-blockers is related to induction of hypoxia- reoxygenation injury due to embryonic cardiac arrhythmia.

Principles and applications of thermal biosensors.

A review of thermistor-based calorimetric measurement is presented. The principles of thermometric measurements are highlighted in the introduction followed by the instrumentation, materials and methods. Various applications relating to enzyme activity measurements, clinical monitoring, process monitoring, multianalyte determination, hybrid sensing, environmental monitoring, non-aqueous measurements and other miscellaneous applications are described. A brief note on future developments and a detailed reference list is also included.

Development of a flow injection capillary chemiluminescent ELISA using an imprinted polymer instead of the antibody.

A flow injection competitive assay analogous to enzyme immunoassays has been developed using a molecularly imprinted polymer instead of the antibody. A glass capillary was modified by covalently attaching an imprinted polymer to the inner capillary wall. The herbicide 2,4-dichlorophenoxyacetic acid was used as a model analyte. The analyte was labeled with tobacco peroxidase, and chemiluminescence was used for detection in combination with a photomultiplier tube or a CCD camera. In a competitive mode, the analyte-peroxidase conjugate was passed together with the free analyte through the polymer-coated capillary mounted in a flow system. After a washing step, the chemiluminescent substrate was injected and the bound fraction of the conjugate was quantified by measuring the intensity of the emitted light. Calibration curves corresponding to analyte concentrations ranging from 0.5 ng mL(-1) to 50 microg mL(-1) (2.25 nM-225 microM) were obtained. A lowered detection limit by 2 orders of magnitude was obtained when detection was done in discontinuous mode and the chemiluminescence light was conducted inside the photomultiplier tube by an optical fiber bundle, thus yielding a dynamic range of 5 pg mL(-1)-100 ng mL(-1) (22.5 pM-450 nM).

Calorimetric analysis of cephalosporins using an immobilized TEM-1 beta-lactamase on Ni2+ chelating sepharose fast flow.

Two beta-lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of beta-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.

Class III antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage.

Class III antiarrhythmic drugs, like almokalant, dofetilide and ibutilide, cause a spectrum of malformations in experimental teratology studies. The pattern of developmental toxic effects is very similar to those reported for phenytoin, which is an established human and animal teratogen. The toxic effects are characterised by embryonic death, decreased fetal weights, and stage specific malformations, such as distal digital reductions, orofacial clefts and cardiovascular defects. Class III antiarrhythmics decrease the excitability of cardiac cells by selectively blocking the rapid component of the delayed rectified potassium channel (IKr), resulting in prolongation of the repolarisation phase of the action potential. Phenytoin, which decrease the excitability of neurones, has recently also been shown to block IKr, in addition to its known blockade of sodium channels. Animal studies indicate that IKr is expressed in the embryo and that the embryonic heart is extremely susceptible to IKr-blockers during a restricted period in early development. At concentrations not affecting the maternal heart, the embryonic heart reacts with bradycardia, arrhythmia and cardiac arrest when exposed to such drugs. Available studies strongly support the idea that birth defects after in utero exposure to both selective and non-selective IKr-blockers (like phenytoin) are initiated by concentration dependent embryonic bradycardia/arrhythmia resulting in 1) hypoxia; explaining embryonic death and growth retardation, 2) episodes of severe hypoxia, followed by generation of reactive oxygen species within the embryo during reoxygenation, causing orofacial clefts and distal digital reductions, and 3) alterations in embryonic blood flow and blood pressure, inducing cardiovascular defects.

Phenytoin-induced cleft palate: evidence for embryonic cardiac bradyarrhythmia due to inhibition of delayed rectifier K+ channels resulting in hypoxia-reoxygenation damage.

BACKGROUND: Phenytoin (PHT) teratogenicity has been related to embryonic arrhythmia due to the capacity of PHT to block I(K) channels pharmacologically, resulting in hypoxia-reoxygenation damage. The aim of this study was to further elucidate the proposed mechanism. METHODS: Pregnant CD-1 mice were given PHT (85 mg/kg) or saline intraperitoneally on gestational days 10-11. Embryonic heart rhythm and presence of hemorrhage in orofacial region was recorded on day 12, fetuses were examined for malformations on day 18. Embryonic heart rate was also recorded on individual days after dosing days 9-16. In addition, PHT was given at doses of 10, 25, or 85 mg/kg on day 12 for analysis of plasma concentrations. RESULTS: PTH-induced bradycardia and arrhythmia in approximately 20% of the embryos, 48% showed hemorrhage in the orofacial region; 39% of the fetuses had cleft palate. The region in which hemorrhages were visible in the embryo corresponded with the region where tissue deficiency (cleft palate) was visible in the fetus at term. None of the controls showed hemorrhages, dysrhythmia, or cleft palate. PHT affected embryonic heart rates on days 9-13, but not on days 14-16. Single dose administration on day 12, the most sensitive day, resulted in a dose-dependent decrease in embryonic heart rate (12-34%). Embryonic arrhythmia occurred at 25 and 85, but not at 10 mg/kg or in the controls. Mean maternal free plasma concentrations were 6 and 14 micromol/L in the 10- and 25-mg/kg groups, respectively. CONCLUSIONS: PHT-induced cleft palate was preceded by embryonic dysrhythmia and hemorrhage in the orofacial region. Embryonic heart rhythm was phase specifically affected, as described for selective I(Kr) channel blockers, at clinically relevant concentrations. The results support the idea that PHT teratogenicity is a consequence of pharmacologically induced dysrhythmia and hypoxia-related damage.

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance.

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.

Developmental toxicity in the pregnant rabbit by the class III antiarrhythmic drug sotalol.

The purpose of this study was to investigate the potential of sotalol to cause developmental toxicity in the pregnant rabbit. Sotalol is a beta-adrenoceptor blocking drug which also has class III antiarrhythmic properties via Ikr channel blockade. EXPERIMENT 1: Nine pregnant New Zealand White rabbits were given doses of either 300, 225, or 150 mg/kg of sotalol during gestational days, called Days, 13-16 which resulted in total litter loss. EXPERIMENT 2: A single dose of sotalol, 100 or 150 mg/kg was administered during Days 8-17 to 15 rabbits. Dosing on Day 8, 9, or 10 resulted in a slightly higher incidence of embryonic death compared to historical controls. There was marked increased embryonic death of 55-90% (four does with total litter loss), decreased number of live foetuses per litter, and elevated mean foetal weight after dosing during Days 12-16. EXPERIMENT 3: 16 pregnant rabbits were administered single doses of sotalol of either 100, 85, 75, 60 or 50 mg/kg on Day 14. The main finding was increased embryonic death, which ranged from total litter loss to approximately 30% at 50 mg/kg. At 50 mg/kg, the maternal Cmax, AUC(1-24 hr), and t1/2 were approximately 45 microM, 340 micromol x hr/l, and 6 hr, respectively. In conclusion, sotalol treatment resulted in embryonic death in the rabbit in early pregnancy in the same way as has been seen for other drugs with Ikr blocking properties (class III antiarrhythmics) in rodents. The observed developmental toxicity in the rabbit is most likely secondary to embryonic arrhythmia as has been shown in rodent studies. The results may indicate that Ikr blocking agents are developmental toxicants across species including man.

Chemiluminescence imaging ELISA using an imprinted polymer as the recognition element instead of an antibody.

An imaging assay analogous to competitive enzyme immunoassays has been developed using a molecularly imprinted polymer instead of an antibody. The antigen 2,4-dichlorophenoxyacetic acid (2,4-D) was labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. Microtiter plates (96 or 384 wells) were coated with polymer microspheres imprinted with 2,4-D, which were fixed in place by using poly(vinyl alcohol) as glue. In a competitive mode, the analyte-peroxidase conjugate was incubated with the free analyte in the microtiter plate, after which the bound fraction of the conjugate was quantified. After addition of the chemiluminescent substrates, light emission was measured in a high-throughput imaging format with a CCD camera. Calibration curves corresponding to analyte concentrations ranging from 0.01 to 100 microg/mL were obtained.

Functionalized surfaces for optical biosensors: applications to in vitro pesticide residual analysis.

Functionalized biosensing surfaces were developed for chemiluminescent immunoassay of pesticides. Two approaches to construct functionalized surfaces were tested: (i) pesticide is immobilized to the surface and interacts with a labeled antibody; (ii) antibody is immobilized and interacts with a labeled pesticide. As labels alkaline phosphatase and peroxidase were used with their corresponding substrates CSPD and luminol, respectively. Light produced by chemiluminescent substrate was detected by a thermoelectrically cooled CCD camera or a photomultiplier. The best detection limit 0.00001 ng/ml was obtained using antibodies immobilized to dextran-enhanced surface. Completely renewable surface was obtained using reversible lectin-monosaccharide interaction, one surface was used for 200 analyses without any loss of binding capacity. Most favorable stability and cost per analysis was achieved with molecularly imprinted polymer (MIP) instead of antibody. The functionalized biosensing surfaces were prepared to detect 2,4-dichlorophenoxyacetic (2,4-D) acid as a model pesticide. The developed concepts are, however, generally applicable to other pesticides and to other optical formats, e.g. optical fiber.

Biomaterials for molecular electronics development of optical biosensor for retinol.

Molecular electronics involves expertise from several branches of science. Various biomaterials and electronics are involved in the fabrication of such devices. While passive biomaterials are involved in anchoring the active biomolecules, the latter are involved in switching and/or signal transduction. In the present investigation we have used a glass-capillary-based approach to design a biosensor for retinol. The sensing element is retinol-binding protein (RBP). The affinity of retinoic-acid-horseradish peroxidase (conjugate) to RBP is tested using a surface plasmon resonance technique. A simple photomultiplier-tube-based system is exploited to monitor the chemiluminescent signal generated upon reaction of hydrogen peroxide and luminol with the conjugate bound to RBP. The photomultiplier tube is directly coupled to a computer for data logging.

Evaluation of changes in public interest concerning lipids and other cardiovascular risk factors between 1990 and 1995.

OBJECTIVE: To assess changes between 1990 and 1995 in the knowledge of cardiovascular risk factors, attitudes to lifestyle changes and to the role of primary health care in preventive work in an urban population. DESIGN: Postal questionnaire. SETTING: South-western Stockholm. SUBJECTS: 1000 randomly selected men and women aged 40 to 64 years. MAIN OUTCOME MEASURES: Knowledge of and attitudes toward cardiovascular risk factors and contacts with primary health care. RESULTS: Response rate was 67%. In 1995 69% thought it important to know one's own lipid values (75% in 1990; 95% CI for change -11, -2). Forty-two per cent thought hyperlipidaemia was a definite cause of coronary heart disease (CHD) (50% in 1990; 95% CI for change -13, -2). Sixty-one per cent thought that a reduction in hyperlipidaemia would reduce cardiovascular risk (70% in 1990; CI for change -14, -4), and 53% thought that a reduction in hypertension would do so (65% in 1990, CI for change -17, -7). Fewer people believed in the negative consequences of eating habits. A majority expected doctors to know about patients' smoking (88%) or drinking (87%) habits. CONCLUSION: Interest in hyperlipidaemia declined between 1990 and 1995, but people expected doctors to take an interest in patients' lifestyles and in prevention. This knowledge is an important working tool for physicians.

Factors influencing GPs' decisions on the treatment of hypercholesterolaemic patients.

OBJECTIVE: To study factors influencing GPs' decisions to prescribe lipid-lowering drugs and how their judgements agree with the Swedish guidelines on hyperlipidaemia. DESIGN: Postal questionnaire. SETTING: Primary health care. Authentic written case descriptions of patients, all with a cholesterol value of at least 5.5 mmol/l and with variations in seven other variables (cues) in a Clinical Judgement Analysis (CJA) design. SUBJECTS: Sixty randomly selected primary health care doctors in the south-eastern Stockholm area. RESULTS: Thirty-eight doctors answered the questionnaire. Coronary heart disease had the highest influence on judgements, followed by cholesterol. The majority of doctors used two or three of the eight cues. Doctors differed markedly in their strategies. One in four did not use coronary heart disease in their judgements, even though all patients with this risk factor present (12/40) should receive pharmacological treatment, according to the guidelines. Doctors who adhered to the guidelines in this respect were younger than those who did not. The GPs' insights into their own strategies were good. CONCLUSIONS: The results indicate that doctors use very different judgement strategies for drug prescription concerning patients with hypercholesterolaemia. A fairly large subgroup of the doctors did not include coronary heart disease in their judgements, in contrast to the present guidelines.