The folic acid analogue methotrexate protects frog embryo cell membranes against damage by the potato glycoalkaloid alpha-chaconine.

As part of an effort to improve the safety of plant foods, a need exists to more clearly delineate the mechanisms of toxicities of glycoalkaloids, which may be present in Solanum plant species such as potatoes, tomatoes and eggplants. Alpha-chaconine is a major glycoalkaloid present in potatoes. To assess the possible influence of structure of pteridine derivatives on toxicity of potato glycoalkaloids, a previous study that demonstrated the protective effects of folic acid against the Solanum glycoalkaloid alpha-chaconine-induced toxicity on Xenopus laevis frog embryo cell membranes was extended to two folate analogues--a synthetic compound widely used as a therapeutic agent methotrexate, and naturally occurring L-monapterin. Adverse effects on embryos were evaluated by observing changes in membrane potentials with an electrochromic dye, di-4-ANEPPS, as a fluorescent probe for the integrity of the membranes. Methotrexate decreased alpha-chaconine-induced polarization, as did folic acid. This decrease may result from an alteration of membrane conformations that prevents the binding of the glycoalkaloid to the membrane receptor sites, and/or from effects on folic acid metabolism. In contrast, L-monapterin did not significantly reduce the alpha-chaconine-induced toxicity. The possible significance of these results to food safety is discussed.

Developmental toxicology of solamargine and solasonine glycoalkaloids in frog embryos.

As part of an effort to improve the safety of plant foods, a need exists to define the relative toxicities of structurally different glycoalkaloids and metabolites which may be present in Solanum plant species such as potatoes, tomatoes and eggplants. The objectives of this study were to determine the relative toxicities and the modes of action of the eggplant (Solanum melongena) glycoalkaloids solamargine and solasonine in Xenopus laevis frog embryos, using membrane potential and embryo growth and teratogenicity assays. In the cell membrane assays, adverse effects on embryos were evaluated by measuring membrane potentials using an electrochromic dye, di-4-ANEPPS, as a fluorescence probe for the integrity of the membranes. In the embryo growth and teratogenesis assays, the survival of the embryos and organ malformations was used as an index of embryo toxicity. The relative potencies of glycoalkaloids are similar for frog embryo effects (survival and teratogenicities) and for membrane effects (membrane potential). Experiments with solasonine at pH 6 and 8 suggest that the unprotonated form of the glycoalkaloids appears to be involved in the membrane effects. The nature of the carbohydrate side-chains of the steroidal glycosides governs relative potencies. The possible significance of the findings to food safety and plant physiology and possible application of the membrane assays to bacterial toxins are discussed.

Effect of alpha-tomatine and tomatidine on membrane potential of frog embryos and active transport of ions in frog skin.

alpha-Tomatine, a glycoside in which four carbohydrate residues are attached to the 3-OH group of the aglycone tomatidine, occurs naturally in tomatoes (Lycopersicon esculentum). The glycoalkaloid is reported to be involved in host-plant resistance against phytopathogens and to have a variety of pharmacological and toxicological properties in animals and humans. As part of an effort designed to establish the mechanism of action of glycoalkaloids in cells, frog embryos and frog skin were exposed to varying concentrations of alpha-tomatine and tomatidine. alpha-Tomatine increased the fluorescence-measured membrane permeability of frog embryos by about 600% compared with control values; the corresponding value for tomatidine was about 150%. alpha-Tomatine also diminished sodium-active transport in frog skin by about 16% compared with control values, as estimated from the change in the interstitial short-circuit current. Tomatidine had no effect on frog skin. As these findings complement similar results with glycoalkaloids from potatoes and eggplants, the fundamental mechanism governing their action both against fungi, insects and other phytopathogens and in animal and human cells may be disruption of cell membranes and changes in ion fluxes and interstitial currents of the membranes. The described methodologies should make it possible to define the relative potencies of both adverse and beneficial effects of glycoalkaloids and metabolites in cell membranes without the use of animals.

Measurement of DNA integrity and structure in Xenopus embryos in the presence of hydroxyurea, actinomycin-D, and triethylenemelamine using the fluorescent probe Hoechst 33258.

Cell health assay of water quality (CHAWQ) is an assay using intracellular biomarkers measured by optical techniques. CHAWQ uses embryos of the South African clawed frog, Xenopus laevis, and optical transducers of intracellular biomarkers to obtain rapid assessment of toxicity to frog embryos. Since the biomarkers are common to all cells, CHAWQ can indicate toxicity of different classes of chemicals. Among the biomarkers used are 1) the change in synthesis rate, 2) the structure, or 3) the environment of DNA. Measurement of DNA to detect genotoxicants has previously used extracted DNA or flow cytometry to detect alterations in DNA content or configuration. We report the use of viable frog embryos and the fluorescent probe Hoechst 33258 to detect the effect of three DNA-active chemicals--actinomycin-D, hydroxyurea, and triethylenemelamine (TEM)--on DNA in intact embryos. We found that we can detect changes in the DNA in the presence of toxicants at concentrations comparable to longer-term assays but following a much shorter time of drug exposure. Actinomycin-D caused a fluorescence decrease, TEM caused a fluorescence increase, whereas hydroxyurea gave a biphasic response. Hydroxyurea caused a decrease at low concentrations and an increase at higher concentrations. Concentration-response data for TEM, hydroxyurea, and actinomycin-D generated EC50 values of 0.1 mg/ml, 1.4 mg/ml, and 6.34 micrograms/ml, respectively.

Effect of 4-bromo-calcium ionophore A23187 on release of Anaplasma marginale from bovine erythrocytes in vitro.

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 microM A23187 to the cultures resulted in significant decrease in percentage of parasitized erythrocytes (PPE) by 24 hours after treatment; further development and increase in PPE was not observed. In contrast, the PPE of untreated cultures, those treated with dimethyl sulfoxide (DMSO) only and with 1 microM A23187 increased slightly during that time. Total erythrocyte count decreased in treated cultures in excess of that expected after samples of the medium were taken for analysis. The greatest cell loss and increased hemoglobin concentration in culture medium was observed in cultures treated with 10 microM A23187 and with an equivalent volume of DMSO. The DMSO appeared to cause hemolysis of some erythrocytes, but not of infected cells selectively. Release of A marginale inclusion bodies was seen by electron microscopy in samples from the 10 microM A23187-exposed cultures. At 30 minutes after treatment, free initial bodies were frequently seen. Inclusion body membranes and individual A marginale were associated with membranes of adjacent erythrocytes. Individual rickettsiae were seen in cell depressions and appeared to be entering erythrocytes. However, neither further invasion nor development of the parasite in erythrocytes was observed. Ionophore A23187 appeared to promote release of A marginale from erythrocytes, but did not enhance infection of erythrocytes or development of organisms in vitro.

Demonstration of gap junctions in frog skin epithelium.

The morphology and distribution of the intercellular junctions were investigated in isolated skin of Rana pipiens using various electron-microscopic techniques. Our evidence demonstrates the presence of gap junctions and suggests that the distribution of gap junctions is not homogeneous among the epithelial strata. Gap junctions were less frequent in the stratum corneum and stratum granulosum than in the stratum spinosum and stratum germinativum. These results support a model of widespread intercellular coupling, although the lower number of gap junctions in the stratum granulosum suggests a possible deficiency in intercellular coupling. Tight junctions were found only in two apical strata of the epithelium (stratum corneum and stratum granulosum). Desmosomes were located in all strata.

Active transport of potassium by insect midgut.

Midguts isolated from fifth-instar larvae of the insert Hyalophora cecropia actively transport potassium in the hemolymph to lumen direction. No specific co- or counter-ion is required and other alkali ions are actively transported in the same direction as potassium. No specific inhibitor of K+ active transport has been found although most metabolic inhibitors reduce the net K+ flux, potential difference, and short-circuit current to zero. The site of the epithelial active transport of potassium has been identified by microelectrode measurements of intracellular resistance as the goblet cell, one of the two major cell types in the single-layered midgut. Under certain external conditions, the neighboring columnar cells are added to the goblet cell transport route through intercellular electrical coupling that occurs after application of external depolarizing current. Tracer influx kinetics were used to establish that the fraction of exchangeable K involved in the transport route under open-circuit conditions is small, corresponding to a goblet cell pathway. Under depolarizing current conditions, virtually all of the exchangeable midgut K is involved in the transport route, corresponding to a goblet and columnar cell pathway. These results and others are used to construct a model for rheogenic active transport of potassium in insect midgut.

Identification of active cell in potassium transporting epithelium.

Epithelial ion transport regulates the environment of cells and modulates the environment inside them, making possible the evolution of eukaryotic organisms. Analysis of the transport process requires that the route taken by ions as they are actively transported across epithelia be known. The route can be determined by locating ion pumps electrically and by measuring transport pool sizes kinetically provided that the epithelial structure is simple enough to enable the results to be interpreted in cellular terms. The route by which potassium is actively transported across the lepidopteran midgut is demonstrated here by a combination of electrical and kinetic analyses. Two sorts of cells, goblet and columnar cells, are distinguished by their electrical properties. The goblet cell is identified as the site of active potassium transport, the pump being localized in its apical membrane. The goblet cells are electrically coupled with columnar cells only when midguts that have been isolated from diet-reared larvae are short circuited. A corollary to this result is that the size of the pool of potassium which is involved in transport should be small when the goblet cells are not coupled with columnar cells and should become large when they are coupled. This corollary has been confirmed by direct measurement of the transport pools using isotope tracer kinetic analysis. This is the first time that the cell type responsible for active ion transport in a polymorphic tissue has been identified directly by physiological means.

Cytochromes and gastric acid secretion. A reevaluation of mucosal acidification experiments.

Using an improved spectrophotometer, we have reinvestigated the report (Hersey, S.J. (1974) Biochim. Biophys. Acta 344, 157--203) that acidification of the mucosal surface of frog gastric mucosa produces a crossover point between flavoprotein and cytochrome b, thus identifying a site of energy coupling between the cytochrome and H+ transport systems. While we find spectrophotometric changes upon addition of HCl to the mucosal solution, we find similar changes upon addition of NaCl without pH change, but no changes when the pH is lowered by substitution of H+ for Na+ at constant osmolality. We show that osmolality changes, with consequent alteration in tissue light scattering, are responsible for these effects. Further, we can show that the pH changes used do not inhibit acid secretion, and that one cannot do so without osmolality increase. We conclude that the imputed crossover point is not demonstrated, and that models based on its existence must be revised.

Demonstration of a pump-mediated efflux in the epithelial potassium active transport system of insect midgut.

The larval midgut epithelium of lepidopteran insects (e.g., Hyalophora cecropia and Manduca sexta) actively transports potassium from hemolymph to lumen when mounted in a chamber. The potassium active transport is rheogenic and does not require the presence of other alkali ions. The transepithelial potential difference, short-circuit current, and electromotive force of active transport are rapidly diminished by anoxia. The efflux of potassium, opposite in direction to potassium active transport, dramatically increased in anoxia, whereas the effluxes of sodium, cesium, and chloride did not increase in anoxia. The increase in efflux was found to have an alkali selectivity similar to that of potassium active transport. It is concluded that the rise of efflux in anoxia is due to the change characteristics of the epithelial potassium active transport mechanism in anoxia.