Non-specific binding of palytoxin to plastic surfaces.

The changes in blood pressure induced by palytoxin (PTX) administered intravenously through polyethylene (PE) tubing were varied, suggesting either non-specific binding of the toxin to PE or deactivation. By spectrophotometry and HPLC, we found that PTX bound non-specifically to PE tubing and that this binding was attenuated by adding 0.1% rat serum albumin. Furthermore, the chemical stability and activity of PTX were not affected by exposure to room light and/or room temperature. Biological deactivation was excluded as a cause of the observed variability because the hypertensive and lethal effects of infused PTX, delayed with simultaneous administration of sodium nitroprusside (NNP), were in full evidence when the NNP was discontinued 35 min later.

Evaluation of potential chemoprotectants against microcystin-LR hepatotoxicity in mice.

Microcystin-LR (MCLR) is a potent cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Toxic blooms of this cyanobacteria have been reported throughout the temperate world. In spite of the potential economic loss and health hazard posed by this toxin, few studies on the development of an antidote have been conducted. Thus, a number of biologically active compounds were tested in mice for effectiveness in preventing the toxicity of a lethal dose of MCLR (100 micrograms kg-1). Efficacy was evaluated based upon the percentage of surviving mice, time to death and serum lactate dehydrogenase activity 45 min after treatment with the toxin. The biologically active compounds were separated into groups based upon proposed mechanisms of action. Enzyme induction by phenobarbital but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in partial protection against toxicity. Calcium channel blockers, free-radical scavengers and water-soluble antioxidants produced little protection against toxicity. The membrane-active antioxidants vitamin E and silymarin, as well as glutathione and the monoethyl ester of glutathione, produced significant protection from lethality. Rifampin and cyclosporin-A, both immunosuppressive and membrane-active agents, which also block the bile acid uptake system of hepatocytes, produced complete protection from the toxicity of MCLR. Thus, lipophilic antioxidants provide partial protection against MCLR toxicity while cyclosporin-A and rifampin are highly effective and potentially useful antidotes. The toxicity of MCLR may depend upon stimulation of the immune system and may be mediated by membrane alterations.

Protection against microcystin-LR-induced hepatotoxicity by Silymarin: biochemistry, histopathology, and lethality.

Microcystin-LR, a cyclic heptapeptide synthesized by the blue-green algae, Microcystis aeruginosa, is a potent hepatotoxin. Pathological examination of livers from mice and rats that received microcystin-LR revealed severe, peracute, diffuse, centrilobular hepatocellular necrosis, and hemorrhage. These changes were correlated with increased serum activities of sorbitol dehydrogenase, alanine aminotransferase, and lactate dehydrogenase. Pretreatment of either rats or mice with a single dose of silymarin, a flavonolignane isolated from the wild artichoke (Silybum marianum L. Gaertn), completely abolished the lethal effects, pathological changes, and significantly decreased the levels of serum enzymes induced by microcystin-LR intoxication.

Microcystin-induced activation of prostaglandin synthesis and phospholipid metabolism in rat hepatocytes.

The effects of microcystin-LR, a trichothecene, T-2 and saxitoxin on membrane lipid mediators of inflammatory processes were evaluated in cultured rat hepatocytes using [(14)C]arachidonic acid. Microcystin-LR significantly stimulated the release of prostacyclin, measured as 6-keto PGF(1)alpha, by 38%, and thromboxane B(2) by 50%, in a concentration-dependent manner. The trichothecene toxin T-2 enhanced the release of prostaglandin F(2)alpha by 24% and arachidonic acid by 29%; while saxitoxin did not affect the release prostaglandins or arachidonic acid. Incorporation of arachidonic acid into the lipid pool was reduced by 47% by 1 mum microcystin-LR. Changes in the distribution of radioactivity derived from [(14)C]arachidonic acid within phospholipid classes indicated that prostaglandin formation induced by microcystin-LR was also due to the release of arachidonic acid from the phosphatidylinositol pool. No statistically significant effect of toxin was observed on the contribution of [(14)C]arachidonic acid release by other classes of phospholipids or neutral lipids. These effects may be important in the mechanism of microcystin-LR-induced toxicity in the liver.

Interaction of cyclic peptides and depsipeptides with calmodulin.

A variety of small peptides bind calmodulin (CaM) and inhibit CaM-dependent enzyme activity. The cyclic peptides cyclosporin A (CSA) and gramicidin-S (GRS) are shown to bind CaM and inhibit 3',5'-cyclic nucleotide phosphodiesterase (PDE) in a calcium-dependent manner. The cyclic peptide microcystin-LR (MLR) and the depsipeptides, valinomycin (VLM) and enniatin-B (ENB), bind to CaM and inhibit PDE activity. Spectral changes exhibited by the binding of MLR, VLM and ENB to dansyl-CaM as compared to that of CSA and GRS reflected different binding sites and/or different conformational changes. The apparent binding constants (Kd) for CaM-peptide were estimated and found to be 4.8 microM for CSA, 2.85 microM GRS, 12.99 microM MLR, 4.29 microM VLM and 41.26 microM ENB. Although these peptides did not inhibit baseline PDE activity, they did inhibit CaM-dependent PDE activity in a dose-dependent manner. Half-maximal inhibition (IC50) of PDE occurred approximately at 0.11 microM MLR; 0.45 microM GRS; and greater than 5 microM for ENB, CSA and VLM. This may be the first observation that these peptides (MLR, VLM and ENB) bind to a known cytoplasmic protein and inhibit an enzyme system dependent on that protein for optimal activity. Interaction of these peptides with CaM may be responsible for creating conformational-functional changes in CaM, thus altering the signal transduction mechanism required for CaM-dependent enzymes, such as cyclic nucleotidase, protein kinases and phospholipase A2.

Effect of antihepatotoxic agents against microcystin-LR toxicity in cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were used to investigate the effects of two putative therapeutic agents, dithioerythritol and silymarin on microcystin-LR-induced hepatotoxicity. Cell injury was assessed by the extent of cellular [14C]adenine nucleotides and lactate dehydrogenase (LDH) release into the medium and the extent of hepatocyte detachment from monolayers. Microcystin-LR (1 microM) induced a significant release of both 14C-labeled nucleotides and LDH from hepatocytes as well as significant detachment of cells from monolayers. Although both dithioerythritol (0.63-5 mM) and silymarin (25-200 microM) reduced the amount of marker release and cell detachment from microcystin-LR-treated wells, silymarin provided significantly greater protection than dithioerythritol at one-tenth the concentration. Furthermore, silymarin and dithioerythritol treatment prevented morphological deformations and detachment of cells.

Interaction of microcystin-LR with SuperChar: water decontamination and therapy.

Activated charcoal (SuperChar) has been recommended for therapeutic use against poisoning by several toxic agents, but it has not been tested against microcystin-LR toxicosis. Microcystin-LR, a cyclic heptapeptide isolated from fresh water blue-green algae, has been shown to be a potent hepatotoxin in animals and in man. Studies were performed to determine the degree of in vitro adsorption of microcystin-LR to SuperChar and to assess the efficacy of SuperChar as a therapeutic agent against microcystin-LR in vivo. Scatchard analysis of the in vitro data showed that microcystin-LR bound to SuperChar with a maximum binding capacity of 0.692 mM toxin/g SuperChar with a dissociation constant of 0.016 mM. The adsorption characteristics of microcystin-LR by SuperChar was applied successfully to the decontamination of water samples spiked with microcystin-LR. While an oral (po) dose of toxin mixed with SuperChar (0.31-0.36 g/kg) modulated the toxicity, an oral pretreatment with SuperChar did not prevent lethality induced by an oral or intraperitoneal (ip) dose of microcystin-LR in mice.

Quantitative relationships between structure and pharmacokinetic parameters using molecular connectivity chi indices I: Substituted 2-sulfapyridines.

Quantitative relationships between the structure and pharmacokinetic parameters of several compounds have been derived using the molecular connectivity approach. The correlation coefficients of equations obtained using the Chi indices (chi) as predictor parameters were compared favorably with those obtained by the linear free energy approach, where physicochemical parameters have been used as predictor variables. High correlation coefficients (r) for the first-order elimination rate constant (r = 0.86), total body clearance (r = 0.89), and protein binding association constant (r = 0.78) for substituted 2-sulfapyridines were obtained. However, including the pKa (indicative of electronic effects) of the compounds within the equation as a predictor variable caused an increase in the correlation coefficients.

Effect of column temperature and eluent flow rate on the high performance liquid chromatographic analysis of cyclosporin a and d.

On a reversed-phase C18 analytical column using an eluent of 70:30 acetonitrile and water, the following effects were observed with increasing column temperature (from 25 to 75°C) for Cyclosporin A (CSA) and Cyclosporin D (CSD). The peak heights and number of theoretical plates (N) increased. The height equivalent to a theoretical plate (HETP) decreased. The areas under the peaks, retention times and capacity factors (k') for both compounds did not vary with temperature. With increasing eluent flow rate (from 0.5 to 2.5ml/ min), the peak heights, peak areas, retention times and N all decreased for both compounds. A slight decrease in k' for CSA and CSD was also observed. HETP increased with increasing flow. The separation factor, α, remained relatively constant for the ranges of temperatures and flow rates investigated.

Glucosylated albumin and its influence on salicylate binding.

Human serum albumin was incubated at 37 degrees in 0.01 M phosphate buffer (pH 7.4) under sterile conditions for up to 10 days with labeled [14C]glucose (1-25 mg/ml). Glucose incorporated into albumin was calculated following extensive dialysis of the incubation mixture. The results indicated that glucose reacted with albumin by a nonenzymatic process involving Schiff base formation and Amadori rearrangement to a stable ketoamine derivative. The degree of glucosylation was dependent on the reaction time, glucose concentration, and pH. Glucosylation was enhanced when albumin was fatty acid free. Glucosylated albumin was separated from unmodified albumin by cation exchange chromatography on carboxymethylcellulose and quantitated colorimetrically with 2-thiobarbituric acid. Salicylate binding studies revealed that the glucosylated component had a decreased salicylate binding capacity accompanied by a reduction in the number of classes of binding sites.

A comparative study on the antiarrhythmic activity and acute toxicity of quinidine and four new analogs in mice.

We have compared the ED50 value for antiarrhythmic activity and the acute toxicities in mice of quinidine and 4 recently synthesized analogs. For the ED50 studies, groups of mice were treated intravenously with equally spaced logarithmic doses of 6'-methoxycinchonine (quinidine), 6'-hydroxycinchonine (cupreidine), 6'-isovaleryloxycinchonine, 6'-acetyloxycinchonine and 6'-benzoyloxycinchonine. For the actue toxicity studies, mice were treated intraperitoneally with quinidine and the 4 analogs. Mice were observed over a 24-h period, and thereafter for each additional 24-h period for a total of 120 h. Tests for parallelism of acute toxicity indicated that with the exception of the 6'-isovaleryloxy derivative the drug treatment regression lines were parallel to that of quinidine (P > 0.05). The results indicated decreases of 50% (843 mumol/kg), 52% (857 mumol/kg), and 61% (910 mumol/kg) in the acute toxicities of the 6'-acetyloxy, 6'-hydroxy, and 6'-benzoyloxycinchonine, respectively. The 6'-acetyloxy (18.5 mumol/kg) and 6'-benzoyloxy (14.6 mumol/kg) derivatives had significantly lower ED50 values than quinidine (60.1 mumol/kg). The results suggest that the 6'-acetyloxy and 6'-benzoyloxy derivatives may have much greater antiarrhythmic effectiveness than quinidine.