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A cyclam-like macrocycle with an integrated push-pull chromophore selectively detects Cu2+ inclusion through both orange-to-yellow colour change and quenching of the green fluorescence.
The syntheses and characterization of two new redox active cyclam ligands ferrocenylmethyl-(6-methyl-1,4,8,11-tetraazacyclotetradec-6-yl)-amine (L3) and 1,1'-ferrocenylmethyl-bis(6-methyl-1,4,8,11-tetraazacyclotetradec-6-yl)-amine (L4) are reported. The compounds each possess a ferrocenyl group bearing one (L3) or two (L4) appended macrocycles linked by their exocyclic amino groups and the crystal structures of both compounds have been determined. Anion binding of L3 and L4 was investigated by electrochemical titrations where H-bonding to each macrocycle causing a shift in the Fc+/0 redox potential was used as a reporter of guest binding. The Zn(II) complex of L3 has also been isolated and characterized structurally. These compounds were analysed for their capacity to electrochemically recognize anions in both aqueous and non-aqueous solution. We have found that L3, L4 and [ZnL3]2+ sense Cl- and AcO- anions in MeCN-CH2Cl2, a function that is lost in aqueous solution.
A fluorescent sensor for Cu(2+) at the nanomolar level in water has been designed by associating a BODIPY fluorophore and a selective ligand (cyclam) in ultrafine polymer nanoparticles.
The first examples of azide-bridged bimetallic trinuclear complexes ([M(cyclam)][FeL(N3)(mu1,5-N3)]2) (H2L = 4,5-dichloro-1,2-bis(pyridine-2-carboxamido) benzene) have been structurally and magnetically characterized.
The sperm of (C57BL X C3H)F1 mice were examined 1, 4, and 10 weeks after a subacute treatment with one of 25 chemicals at two or more dose levels. The fraction of sperm that were abnormal in shape was elevated above control values of 1.2-3.4% for methyl methanesulfonate, ethyl methanesulfonate, griseofulvin, benzo[a]pyrene, METEPA [tris(2-methyl-l-aziridinyl)phosphine oxide], THIO-TEPA [tris(l-aziridinyl)phosphine sulfide], mitomycin C, myleran, vinblastine sulphate, hydroxyurea, 3-methylcholanthrene, colchicine, actinomycin D, imuran, cyclophosphamide, 5-iododeoxyuridine, dichlorvos, aminopterin, and trimethylphosphate. Dimethylnitrosamine, urethane, DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane], 1,1-dimethylhydrazine, caffeine, and calcium cyclamate did not induce elevated levels of sperm abnormalities. The results suggest that sperm abnormalities might provide a rapid inexpensive mammalian screen for agents that lead to errors in the differentiation of spermatogenic stem cells in vivo and thus indicate agents which might prove to be mutagenic, teratogenic, or carcinogenic.
pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are injected with human ClC-6 RNA. Indeed, both the pICln and the ClC-6 induced current are outwardly rectifying, they inactivate slowly at positive potentials and have an anion permeability sequence NO3- > I- > Br- > Cl- > gluconate. Cyclamate, NPPB, and extracellular cAMP block the induced currents. The success rate of current expression is significantly increased when the injected Xenopus oocytes are incubated at a higher temperature (24 or 37 degrees C) prior to the analysis. In addition, the ICln current was detected in 6.2% of noninjected control Xenopus oocytes. We therefore conclude that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the ICln current in Xenopus oocytes.
For the assessment of exposure to food-borne chemicals, the most commonly used methods in the European Union follow a deterministic approach based on conservative assumptions. Over the past few years, to get a more realistic view of exposure to food chemicals, risk managers are getting more interested in the probabilistic approach. Within the EU-funded 'Monte Carlo' project, a stochastic model of exposure to chemical substances from the diet and a computer software program were developed. The aim of this paper was to validate the model with respect to the intake of saccharin from table-top sweeteners and cyclamate from soft drinks by Italian teenagers with the use of the software and to evaluate the impact of the inclusion/exclusion of indicators on market share and brand loyalty through a sensitivity analysis. Data on food consumption and the concentration of sweeteners were collected. A food frequency questionnaire aimed at identifying females who were high consumers of sugar-free soft drinks and/or of table top sweeteners was filled in by 3982 teenagers living in the District of Rome. Moreover, 362 subjects participated in a detailed food survey by recording, at brand level, all foods and beverages ingested over 12 days. Producers were asked to provide the intense sweeteners' concentration of sugar-free products. Results showed that consumer behaviour with respect to brands has an impact on exposure assessments. Only probabilistic models that took into account indicators of market share and brand loyalty met the validation criteria.
Three European directives imply for all member states an evaluation of food additives intakes. This paper describes the French approach, using a step-by-step procedure to evaluate, at a national level, the consumption of 10 food additives or groups of additives (E100, curcumin; E160b, annatto; E200-203, sorbic acid and salts; E210-213, benzoic acid and salts; E220-224, 226-228, sulphites; E249-250, nitrites; E297, fumaric acid; E473-474, sucrose esters and sucroglycerides; E491-492, stearoyl lactates; E952, cyclamic acid). The results are reassuring for the general population, except for sulphites.
2-Cyclohexene-1-one (CHX) is a cyclic alpha,beta-unsaturated ketone with broad human exposure. CHX is an environmental pollutant and is present in tobacco smoke and in soft drinks sweetened with cyclamate. Interest in the toxicity of this class of compounds is due to their structural similarity to the cytotoxin acrolein. In a pilot study, rats and mice were exposed to 0, 20, 40, or 80 ppm CHX for 6 h/day. The study was terminated after 4 days due to acute toxicity in the high-dose groups. In a subsequent 14-day study, mice and rats were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day. All animals survived exposure until terminal sacrifice. Body weights were not significantly different from controls after 14 days of exposure. Liver/body weights were increased in male and female mice exposed to 5 and 10 ppm, and in male and female rats exposed to 10 ppm CHX. Ninety-day toxicity studies were conducted to provide data required to design chronic toxicity and carcinogenicity studies of CHX if it is determined such studies are necessary. Groups of 10 male and female F-344 rats and B6C3F1 mice were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day for 13 wk. All animals survived until sacrifice. Body weights were not significantly different from controls after 13 wk of exposure. Liver weights were increased in male and female mice exposed to 5 and 10 ppm and in male and female rats exposed to 10 ppm CHX. No adverse effects on bone-marrow micronuclei, sperm motility, or vaginal cytology were observed. Microscopic lesions included hyperplasia, and squamous metaplasia in the nasal cavity in rats and mice of both sexes at all doses. Nasal-cavity erosion and suppurative inflammation also occurred in high-dose mice. Larynx and lung were not affected in either sex or species. Dose-related hepatic centrilobular cytoplasmic vacuolation was seen in male rats only. These data suggest that CHX acts as an alkylating agent primarily producing toxicity at the exposure site.
Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.
The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.
An electrophysiologic technique was used to measure changes in cell water volume in response to isosmotic luminal solution ion replacement. Intracellular Cl- activity (aCl-i) was measured and net flux determined from the changes in volume and activity. Reduction of luminal solution [Cl-] from 98 to 10 mM (Cl- replaced with cyclamate) resulted in a large fall in aCl-i with no significant change in cell water volume. Elevation of luminal solution [K+] from 2.5 to 83.5 mM (K+ replaced Na+) caused a small increase in aCl-i with no change in cell water volume. Exposure of the Necturus gallbladder epithelium to agents that increase intracellular cAMP levels (forskolin and/or theophylline) induces an apical membrane electrodiffusive Cl- permeability accompanied by a fall in aCl-i and cell shrinkage. In stimulated tissues, reduction of luminal solution [Cl-] resulted in a large fall in aCl-i and rapid cell shrinkage, whereas elevation of luminal solution [K+] caused a large, rapid cell swelling with no significant change in aCl-i. The changes in cell water volume of stimulated tissues elicited by lowering luminal solution [Cl-] or by elevating luminal solution [K+] were reduced by 60 and 70%, respectively, by addition of tetraethylammonium (TEA+) to the luminal bathing solution. From these results, we conclude that: (a) In control tissues, the fall in aCl-i upon reducing luminal solution [Cl-], without concomitant cell shrinkage, indicates that the Cl- entry mechanism is electroneutral (Cl-/HCO3-) exchange. (b) Also in control tissues, the small increase in aCl-i upon elevating luminal solution [K+] is consistent with the recent demonstration of a basolateral Cl- conductance. (c) The cell shrinkage elicited by elevation of intracellular cAMP levels results from conductive loss of Cl- (and probably K+). (d) Elevation of cAMP inhibits apical membrane Cl-/HCO-3-exchange activity by 70%. (e) The cell shrinkage in response to the reduction of mucosal solution [Cl-] in stimulated tissues results from net K+ and Cl- efflux via parallel electrodiffusive pathways. (f) A major fraction of the K+ flux is via a TEA(+)-sensitive apical membrane K+ channel.
The in vitro V79/metabolic cooperation assay measures the extent of gap-junctional transfer of metabolites from wild-type to mutant V79 cells. The assay is currently being explored as a short-term test to screen for tumor promoting chemicals, many of which inhibit metabolic cooperation. In this study, the assay was used to determine whether chemical interactions affect detection of tumor promoters in mixtures and to investigate types of interactions that may occur between chemicals. Several two-chemical mixtures were examined. The effects of phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate, two inhibitors of metabolic cooperation that operate through the same receptor-mediated pathway, were additive at concentrations below the maximally effective concentrations of either. A summation effect was observed in mixtures of two other inhibitors of metabolic cooperation, the pesticide aldrin and the principal metabolite of sodium cyclamate, cyclohexylamine. Synergistic effects were noted when PMA was combined with either aldrin or cyclohexylamine, demonstrating that chemicals in a mixture may yield a much stronger response than expected based on individual chemical exposures. Interactions were also examined between PMA, aldrin, cyclohexylamine and 2,4-diaminotoluene, a chemical that appears to enhance metabolic cooperation. 2,4-Diaminotoluene reversed effects of all inhibiting chemicals to some extent, although the pattern of response was different for each combination. In the most dramatic case, the powerful tumor promoter PMA was completely masked by 2,4-diaminotoluene. These results suggest that the V79/metabolic cooperation assay must be applied with caution in mixture testing because detection of tumor promoting chemicals can depend on other chemicals present.
The availability of many SV40 mutants, in which the ability of the virus to transform fibroblasts is variously affected, has prompted us to investigate the effect of treating SV40-infected cells with known tumor promoters on the expression of the transformed phenotype. Using mouse Swiss 3T3 cells and various SV40 tsA mutants unable to transform these cells at 39 degrees C, we have observed a dramatic effect of the potent phorbol ester promoter, 12-O-tetradecanoyl-phorbol-13-acetate, on the formation of macroscopic colonies in soft-agar at the restrictive temperature of 39 degrees C. The efficiency of other phorbol esters and various substances such as anthralin, saccharin, sodium cyclamate, mellitin, griseofulvin and benzoyl peroxide, was in agreement with their reported promoting activities suggesting that mouse Swiss 3T3 cells infected with SV40 tsA mutants could provide a quick and easy test to detect promoters.
Transforming growth factors alpha and beta (TGF-alpha and TGF-beta) isolated from normal mouse kidney induced gross morphological changes in rat urothelial cells maintained in organ culture. These morphological effects are similar to those observed after long-term treatment of rat bladder organ cultures with the carcinogen N-methyl-N-nitrosourea (MNU) or the promoting agents sodium saccharin and sodium cyclamate. Cultures were treated continuously with 5-25 micrograms/ml of Bio-Gel P-30-purified TGF containing both TGF-alpha and TGF-beta between days 1 and 14 in culture, or with 5 micrograms/ml from days 28 to 42. Controls received 1-10 ng/ml epidermal growth factor (EGF) or control medium. Untreated controls retained a normal urothelium throughout the period of study. Mature superficial-type cells covered most of the surface and less mature forms appeared on the cut sides and damaged areas where cells followed the normal pattern of urothelial differentiation. EGF at 5 and 10 ng/ml caused necrosis of the entire urothelium but at 1 and 2 ng/ml had minimal effects on histology and scanning electron microscopical appearance up to 14 days in culture. Crude P-30-purified TGFs induced a series of dose-related changes from 4 days, which were maximal at 8 days and persisted or decreased between 8 and 14 days. These included hyperplasia, loss of epithelial polarity, hyperchromasia and elongation of basal cells between the overlying cell layers to reach the culture surface. Scanning electron microscopy showed the appearance at the culture surface of immature cells with gross surface abnormalities including large numbers of blebs, stubby microvilli and long pleomorphic microvilli. Immature cells on the sides of the culture and in damaged areas developed similar features. At crude TGF doses of 10 micrograms/ml many superficial cells were rounded, some became cystic and epithelial necrosis was observed. Cultures treated with h.p.l.c.-purified TGF-beta at 80 ng/ml in the presence of 2 ng/ml EGF showed similar effects to those treated with 5 micrograms/ml P-30-purified TGF. Fully differentiated cultures treated from 28 to 42 days with crude TGF, showed changes similar to those seen in early cultures. However, histological changes, particularly basal cell elongation were more widespread and there was an abnormal development of globular processes between the membrane ridges of mature superficial cells. Neither crude TGF nor EGF stimulated growth in soft agar of isolated epithelial cells from freshly killed rats or organ cultures pretreated for 7 days with EGF or TGF.(ABSTRACT TRUNCATED AT 400 WORDS)
The purpose of this study was to determine the degree to which the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid (Na-PMP) reduced sweet intensity ratings of 15 sweeteners in mixtures. Na-PMP has been approved for use in confectionary/frostings, soft candy and snack products in the USA at concentrations up to 150 p.p.m. A trained panel evaluated the effect of Na-PMP on the intensity of the following 15 sweeteners: three sugars (fructose, glucose, sucrose), three terpenoid glycosides (monoammonium glycyrrhizinate, rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), two N-sulfonylamides (acesulfame-K, sodium saccharin), two polyhydric alcohols (mannitol, sorbitol), 1 dihydrochalcone (neohesperidin dihydrochalcone), one protein (thaumatin) and one sulfamate (sodium cyclamate). Sweeteners were tested at concentrations isosweet with 2.5, 5, 7.5 and 10% sucrose in mixtures with two levels of Na-PMP: 250 and 500 p.p.m. In addition, the 15 sweeteners were tested either immediately or 30 s after a pre-rinse with 500 p.p.m. Na-PMP. In mixtures, Na-PMP at both the 250 and 500 p.p.m. levels significantly blocked sweetness intensity for 12 of the 15 sweeteners. However, when Na-PMP was mixed with three of the 15 sweeteners (monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin), there was little reduction in sweetness intensity. Pre-rinsing with Na-PMP both inhibited and enhanced sweetness with the greatest enhancements found for monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin, which were not suppressed by Na-PMP in mixtures. The mixture data suggest that Na-PMP is a selective competitive inhibitor of sweet taste. The finding that pre-treatment can produce enhancement may be due to sensitization of sweetener receptors by Na-PMP.
The effect of soluble starch (acid-modified starch) on taste intensity was investigated in human subjects. Different concentrations of sucrose (Suc), six sweeteners, NaCl, quinine-HCl (QHCl) and citric acid (Cit) were dissolved in either distilled water (DW; standard) or starch solution (test solution). The solutions were presented to naive subjects and each subject was requested to taste and compare the sweetness intensity between the standard and test solutions based on a scale ranging from +3 (enhanced) to -3 (inhibited). A greater sweetness intensity occurred with Suc at different concentration (0.1-1.0 M) dissolved in soluble starch (0.125% to 4.0%) than with Suc in DW. Similarly, five other different products of soluble starch at 0.25 and 4.0% resulted in enhancement of sweetness for 0.3 and 1.0 M Suc. With the sole exception of the taste of 0.3 M Suc, sweet enhancement did not occur with 0.43 M fructose, 0.82 M glucose, 0.82 M sorbitol, 0.0037 M aspartame, 0.0042 M saccharin-Na or 0.016 M cyclamate. Neither the saltiness of NaCl (0.01-0.3 M), the bitterness of QHCl (0.00003-0.001 M) nor the sourness of Cit (0.0003-0.01 M) were affected by the soluble starch. These results suggest that the taste enhancing effects of soluble starch on Suc sweetness might depend not only on the taste transduction mechanism, but also on the molecular interaction between Suc and soluble starch.
The U and Gamma' models of sensory interactions, successfully applied in olfaction for several years, are tested here using data from published studies on sweetness. The models are subsequently tested on new data obtained in studies of binary mixtures of four sodium sulfamates. The U model allows for the estimation of a global interaction, whereas the Gamma' model allows for the distinction between that which is due to an intrinsic interaction in the mixture itself and that which may be due to the power function exponents in the mixture. The models give satisfactory predictions for observed phenomena of sweet taste suppression, synergism or pure additivity. Additionally, they appear to be more suitable than other models recently applied in taste, particularly the equiratio model. Application of the models to the sulfamate mixtures, reveals additivity for sodium cyclohexylsulfamate (cyclamate)/potassium cyclohexylsulfamate and sodium cyclohexylsulfamate/sodium exo-2-norbornylsulfamate, respectively; whereas for sodium cyclohexylsulfamate/sodium 3-bromophenylsulfamate, the models revealed a slight hypo addition which is simply due to the dissimilarity values of the power function exponents of the components.