Although much is known about the pathogenesis of crotoxin-induced muscle damage, the initial site and action of the toxin is still not clear. In this study we used an electrochromic fluorescent dye, Di-4-ANEPPS, to measure the changes in membrane potential of isolated murine omohyoid muscle to determine if depolarization could be one of the initial effects of crotoxin. Omohyoid isolates were pre-loaded with 1 microM Di-4-ANEPPS, exposed to various crotoxin treatments, and the change in fluorescence was recorded using either a dual-wavelength spectrofluorometer or digital imaging. Spectrofluorometry indicated that crotoxin depolarized isolated omohyoid muscles within 4 min as indicated by an increase in fluorescence to 122% of control values. Crotoxin also induced depolarization of extensor digitorum longus and soleus muscles as indicated by an increase in fluorescence of 140 and 110% of the control, respectively. Fluorescent images obtained from omohyoid muscle preparations exposed to crotoxin and Di-4-ANEPPS revealed localized areas of increased fluorescence, muscle contractions, derangement of myofibrils, and differing sensitivity to crotoxin of different muscle cells. Light microscopy results confirmed this variable disruption of muscle cell integrity and differing sensitivity to crotoxin. An increase in creatine kinase release rates confirmed damage to the plasma membrane. We conclude that plasma membrane depolarization is most likely the earliest indicator of cell damage from crotoxin and is quickly followed by hypercontraction of myofilaments, disruption of the plasma membrane, release of creatine kinase and necrosis.

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.

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.

α-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 α-tomatine and tomatidine. α-Tomatine increased the fluorescencemeasured membrane permeability of frog embryos by about 600% compared with control values; the corresponding value for tomatidine was about 150%. α-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.

To demonstrate whether folic acid can protect Xenopus embryos against reported adverse effects of the potato glycoalkaloid alpha-chaconine, the frog embryos were exposed simultaneously to the glycoalkaloid, folic acid (pteroylglutamic acid), and an electrochromic fluorescent dye, Di-4-ANEPPS, in a specially designed instrument that measures embryonic membrane potential. Folic acid decreased the chaconine-induced fluorescence, with a maximum decrease occurring at about 10 mg/L of both folic acid and the glycoalkaloid dissolved in solution. The protective effect was also operative in the frog embryo teratogenesis assay-Xenopus (FETAX), in which survival and teratogenicity of the whole embryos were the endpoints. Possible mechanisms of the protective effect and the possible significance of the results to food safety and health are discussed.

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.

To test the effect of glycoalkaloids on sodium ion active transport, frog skin was exposed to the potato glycoalkaloids alpha-chaconine and alpha-solanine in a glass Ussing chamber. It was found that the short-circuit current (ISC), the measure of transepithelial active transport of sodium, decreased up to 30% at an alpha-chaconine concentration of 10 mg/L. alpha-Solanine had a similar but smaller effect, decreasing short-circuit current by 16%. The data suggest that (a) frog skin is a useful experimental model to evaluate effects of glycoalkaloids at the cellular level and (b) the mechanism of action of the two glycoalkaloids is to modify the active transport of sodium. The possible significance of these findings to food safety is discussed.

To demonstrate whether potato glycoalkaloids can alter the integrity of membranes of frog embryo, albino frog embryos were incubated with alpha-chaconine and alpha-solanine. Di-4-ANEPPS, an electrochromic fluorescent dye, was added to measure embryonic membrane potential. Alpha-Chaconine increased the Di-4-ANEPPS fluorescence up to 1600% of control, alpha-solanine increased the fluorescence up to 400%, and solanidine had no effect. Increases in fluorescence, when plotted in a concentration-response format, produced EC50 values near published values for FETAX (frog embryo teratogenicity assay-Xenopus). Possible mechanisms and the significance of the fluorescence results to food safety are discussed.

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.