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Isolated rat livers were perfused for 60 min with either 0.3 or 0.5 microgram/ml (initial volume, 119 ml) of [3H]microcystin-LR at a constant flow of 10 ml/min in a recirculating system. During the 60-min exposure, toxin caused stimulation of glycogenolysis, liver engorgement, and cessation of bile flow. Electron micrographs of liver showed dilation of bile canaliculi and the space of Disse. loss of sinusoidal lining architecture, and decreased hepatocyte intercellular contacts. Although hepatocytes did not exhibit overt necrosis, mitochondria were hydropic, occasionally encircled by whorls of rough endoplasmic reticulum, and desmosomal tonofilaments were decreased on the plasma membrane lateral surface. Isolated mitochondria displayed inhibition of state 3 respiration and a 50-60% decrease in the respiratory control index, characteristic of hydropism. Distribution of radiolabel was 1.7% to bile, 79% to perfusate, and 16% to liver. Two to four percent was recovered in perfusate that leaked from the surface of the liver. Of the radiolabel found in bile and perfusate, 78 and 100% were associated with parent toxin, respectively. The radiolabel in liver, associated with the cytosolic fraction (S-100), corresponded to parent toxin (15%) and to a more-polar component(s) (85%). The elimination half-life from perfusate was 130 +/- 10 min (0.5 microgram/ml) and the hepatic extraction ratio 0.07 +/- 0.01. Although the calculated hepatic extraction ratio was low, there was a significant accumulation of microcystin in the liver. Many toxic effects of microcystin in the perfused liver mimicked those observed in the whole animal, suggesting that this model can be used as an alternative to whole animals for screening of potential therapeutic agents.

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The LD50 (25 hr, i.p.) for microcystin-LR in fed rats (122 micrograms/kg) was significantly higher than that in fasted rats (72 micrograms/kg). At doses of 100, 150 and 200 micrograms of microcystin-LR per kg, the median times to death were 31.9, 18.2 and 11.2 hr for fed rats, and 1.8, 1.7 and 1.5 hr for fasted rats. A sublethal dose of microcystin (50 micrograms/kg) afforded protection to fasted, but not fed, rats against a subsequent lethal dose (200 micrograms/kg) challenge given 72 hr later. Biochemical and ultrastructural changes resulting from microcystin-LR (100 micrograms/kg, i.p.) were compared in fed and fasted rats 1 hr after injection. In both groups, liver weight and serum levels of sorbitol dehydrogenase and glucose significantly increased. Plasma membranes, isolated from livers of fed or fasted rats, exhibited similar toxin-induced changes in associated cytoskeletal elements. Liver mitochondria from toxin-treated, fasted rats exhibited complete inhibition of state 3 respiration, while those from toxin-treated, fed rats had ADP/O ratios and respiratory control indices comparable to control values. The primary event responsible for enhanced microcystin hepatotoxicity in the fasted state has not yet been identified. Depletion of glycogen stores and a decreased respiratory capacity may, however, play significant roles in this degenerative process.

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The distribution, excretion and hepatic metabolism of [3H]microcystin-LR (sublethal i.v.) were measured in mice. Plasma elimination was biexponential with alpha- and beta-phase half-lives of 0.8 and 6.9 min, respectively. At 60 min, liver contained 67 +/- 4% of dose. Through the 6-day study the amount of hepatic radioactivity did not change whereas 23.7 +/- 1.7% of the dose was excreted; 9.2 +/- 1.0% in urine and 14.5 +/- 1.1% in feces. Approximately 60% of the urine and fecal radiolabel 6 and 12 hr postinjection was the parent toxin. Hepatic cytosol, which contained 70 +/- 2% of the hepatic radiolabel (1 hr through 6 days), was prepared for high-performance liquid chromatography analysis by heat denaturation, pronase digestion and C18 Sep Pak extraction. At 1 hr, 35 +/- 2% of the radiolabel was insoluble or C18 Sep Pak-bound; 43 +/- 3% was associated with a peak of retention time (rt) 6.6 min, and 16 +/- 3% with the parent toxin (rt 9.4 min). After 6 days, 8 +/- 1% was C18 Sep Pak-bound or insoluble; 5 +/- 0% occurred at rt 6.6 min, 17 +/- 1% with parent and 60 +/- 2% was associated with rt 8.1 min. Two other peaks, rt 4.9 and 5.6 min, appeared transiently. Analysis of hepatic cytosol by desalting chromatography under nondenaturing and denaturing conditions revealed that all of the radiolabel was associated with cytosolic components, and 83 +/- 5% was bound covalently through 1 day. By day 6 the amount of covalently bound isotope decreased to 42 +/- 11%. This is the first study to describe the long-term hepatic retention of microcystin toxin and documents putative detoxication products.

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The toxic effects of microcystin-LR, a cyclic heptapeptide isolated from the cyanobacterium Microcystis aeruginosa, were studied in the fasted rat model and in subcellular fractions from fasted, toxin-treated and control rats. Hepatotoxic effects of a lethal dose (100 micrograms/kg) were examined 15-90 min post-injection. Elevations of serum enzymes, particularly sorbitol dehydrogenase, specific for liver mitochondria, correlated with hepatic damage. Electron micrographs showed progressive cellular disruption, including dilation of rough endoplasmic reticulum, incorporation of cellular components into cytolysosomes, hydropic mitochondria devoid of electron-opaque deposits, loss of desmosome-associated intermediate filaments, disruption of sinusoidal architecture and, ultimately, lysis of hepatocytes. The appearance of hydropic mitochondria correlated with loss of coupled electron transport. Changes in plasma membrane-associated cytoskeletal filaments correlated with loss of desmosome tonofilaments. In contrast to in vivo exposure to microcystin-LR, in vitro exposure to toxin had no effect on mitochondria or cytoskeletal filaments, suggesting that the toxic effects observed in vivo were indirect and may be dependent on bioactivation of the toxin or a cascade of events not supported in in vitro models.

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Chemically tritiated microcystin-LR (spec. act. 194 mCi/mmol), purified to greater than 95% by C-18 reverse-phase high performance liquid chromatography, exhibited the same retention time and ultraviolet absorption profile as unlabeled toxin. Acid-hydrolyzed [3H]-toxin yielded tritiated glutamate and beta-methylasparate. Stability of the nonexchangeable [3H]-toxin in saline and urine was greater than 93% after 42 days stored at 22 degrees, 4 degrees or -20 degrees C. In blood, the breakdown of toxin was temperature- and time-dependent (63% at 22 degrees C, 28 days). Unlabeled toxin was stable for greater than 42 days stored at either 4 degrees or -20 degrees C in saline. The LD50 (mouse, i.p.) of [3H]-microcystin-LR and unlabeled toxin was the same [75 micrograms/kg (65-90) and 65 micrograms/kg (53-80), respectively]. From 3 to 90 min after i.p. injection of 70 micrograms/kg [3H]-microcystin-LR there was a slow absorption of toxin from the peritoneal cavity and efficient accumulation in liver. The elimination half-life of the plasma concentration curve was 29 min. Tritium distribution in tissue at death or 6 hr post injection was similar for all doses (13-101 micrograms/kg). At 101 micrograms/kg, liver contained 56 +/- 1%, intestine 7 +/- 1%, kidney 0.9 +/- 0.2% and carcass 10 +/- 1% of the injected dose. Heart, spleen, lung and skeletal muscle contained less than 1% of the radiolabel.

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The pharmacokinetics of [14C]aprophen and its distribution were determined after intravenous administration to rats. The drug was distributed rapidly with a t1/2 (alpha) of 4 min to highly perfused organs like the brain, kidney and adrenals. An elimination phase was apparent 10 min after injection with a t1/2 (beta) of 23.5 min. The high plasma clearance of the drug was due both to a large volume of distribution and to a high metabolic rate. Aprophen could be hydrolysed to diphenylpropionic acid and diethylaminoethanol in-vivo and in-vitro. Diethylaminoethanol competed with [3H]QNB binding to muscarinic receptors of N4TG1 cells, whereas diphenylpropionic acid did not. The lower plasma concentrations and lower binding activity of diethylaminoethanol compared with aprophen indicate that unchanged aprophen is largely responsible for the in-vivo actions.

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The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

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Neplanocin A, a novel carbocyclic analog of adenosine, was found to be a mixed type inhibitor of S-adenosylhomocysteine hydrolase of human red blood cells with a Ki of 2 nM and an inactivating constant, Ki, of 3 nM. When tested against Plasmodium falciparum cultured in human red blood cells, neoplanocin A inhibited malarial growth with an ED50 of 3 microM. Above 2.5 microM, some red blood cells showed morphological aberrations and became echinocytes (spiculated red blood cells). In infected red blood cells, neplanocin A caused an elevation of the concentrations of S-adenosylmethionine and S-adenosylhomocysteine in a dose-dependent manner. Concurrently, a new analog of S-adenosylmethionine, S-neplanocinylmethionine, was formed. Analysis of polyamines showed that only putrescine was decreased significantly; the others were not changed. Purine analyses showed two putative neplanocinyl nucleotides, possibly the di-and the triphosphates. Neplanocin A most likely exerted its in vitro antimalarial effect via the inhibition of S-adenosylhomocysteine hydrolase and the attendant perturbation of methylation reactions.

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Diethylaminoethyl acetate, an acetylcholine analog, was formed upon the incubation of diethylaminoethanol and acetyl-CoA with bovine brain choline acetyltransferase (acetyl-CoA: choline O-acetyltransferase; EC 2.3.1.6). The new product co-chromatographed with authentic diethylaminoethyl acetate on thin layer plates, and its formation was proportional to the duration of incubation and enzyme concentrations. When tested on guinea-pig ileum, diethylaminoethyl acetate was found to be an agonist with an ED50 of 1.3 X 10(-4) M, compared to an ED50 of 2.0 X 10(-7) M for acetylcholine. The contraction of guinea-pig ileum induced by diethylaminoethyl acetate was blocked by atropine. Moreover, diethylaminoethyl acetate induced a secretion of alpha-amylase from isolated pancreatic acini cells; this effect was also blocked by atropine. It is entirely possible that diethylaminoethyl acetate can be a false cholinergic transmitter generated in vivo when drugs such as aprophen or procaine are administered to animals, since either of these drugs can undergo enzymatic hydrolysis to generate diethylaminoethanol. A method for the synthesis of radioactive diethylamino [1,2-14C]ethyl acetate was also described.

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A simple and rapid radioisotopic assay for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase was developed, an enzyme involved in the biosynthesis of the plant hormone ethylene. The assay utilizes an AG50W-X4(NH+4) column which separates S-adenosyl-L-[carboxyl-14C]methionine (AdoMet) from the product [14C]ACC, since the latter is not bound to the resin while [14C]AdoMet is. As opposed to other assays, this procedure measures ACC directly and does not require further conversion to ethylene. When an enzyme preparation from ripe tomato fruits (Lycopersicon esculentum Mill). was assayed, an I50 of 2.5 +/- 0.8 microM for sinefungin and a Km of 27 +/- 2 microM for AdoMet were obtained; these values were in good agreement with previous determinations made with a gas chromatographic assay. When other nucleosides were tested as inhibitors, the following order of decreasing activity was found: sinefungin greater than S-adenosylhomocysteine (AdoHcy) greater than AdoHcy sulfoxide greater than S-n-butyladenosine greater than 3-deaza-adenosylhomocysteine greater than S-isobutyladenosine greater than S-isobutyl-1-deazaadenosine. In contrast, S-isobutyl-3-deazaadenosine, S-isobutyl-7-deazaadenosine, 3-deazaadenosine, and adenosine were not inhibitory.

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Using electrochemical detection, 3-deazaadenosine, a proximal inhibitor of methylation via the inhibition of S-adenosylhomocysteine hydrolase, perturbed the metabolism of catecholamines in the adrenals of rats. In adrenals of rats treated with 3-deazaadenosine, both norepinephrine and epinephrine increased significantly by about two-fold. 3-Deazaadenosine may inhibit the release of catecholamines from the adrenals by affecting membrane functions of the adrenals.

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The effect of aldosterone (Aldo) on phospholipid (PL) biosynthesis in cultured toad bladder epithelial cells was studied in cells incubated with [1,2-14C]choline and [methyl-3H]methionine over a 5-h period. Aldo (10(-7) M) did not alter the uptake of either precursor but significantly stimulated the incorporation of both labels into phosphatidylcholine (PC), the only PL labeled. 3H labeling of PC increased 29% and 14C incorporation into PC increased 34% in cells exposed to Aldo. A similar 30% increase in protein carboxymethylation occurred in cells treated with Aldo. 3-Deazaadenosine (DZA), a methylation inhibitor, abolished the Aldo-stimulated increase in PC labeling from [3H]methionine. PC labeling from [1,2-14C]choline was not affected by DZA. Basal and Aldo-stimulated protein carboxy-methylation were inhibited by DZA. DZA (300 microM) caused a mild decrease in basal short-circuit current (ISC) but completely inhibited the ISC response to 10(-7) M Aldo. Inhibition was complete when DZA was added up to 2 h following exposure to Aldo, and was reversible. Cells previously exposed to Aldo showed a significant increase in ISC within 2 h following removal of DZA. We conclude that Aldo stimulates PL methylation, protein carboxymethylation, and turnover of PC from choline. Inhibition of methylation reactions coincides with the inhibition of ISC response to Aldo.

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A shower decontamination bench model has been used to assess quantitatively the importance of several variables (water pressure and temperature, surfactant concentration in the decontamination fluid, nozzle type, and shower time) on decontamination of nontoxic chemical warfare-agent simulants diethyl malonate and thickened diethyl malonate from pig skin in vitro. Diethyl malonate was validated as a simulant for 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) by comparison of the skin penetration and decontamination of radiolabeled diethyl malonate to the radiolabeled phosphonofluoridate in shower decontamination trials of pig skin in vitro. Percutaneous penetration of diethyl malonate was significantly greater than that of the phosphonofluoridate during the 15-min period after application. However, both were less than 0.1% of the applied dose. Showering or thickener had no significant effect on the percutaneous penetration of diethyl malonate or the phosphonofluoridate. Most of the phosphonofluoridate removed by showering or scrubbing the skin was inactivated. The quantity of intact 1,2,2-trimethylpropyl methylphosphonofluoridate that penetrated through the skin was below the detection limit of the enzymatic analysis. There was no statistically significant difference between the phosphonofluoridate and diethyl malonate in efficacy of shower decontamination. The presence of thickener did not have a significant effect on decontamination efficacy.

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A high-performance liquid chromatographic method was devised that separates S-adenosylmethionine and related sulfur metabolites on a Radial-PAK SCX cation-exchange column using a four-step NH4COOH/(NH4)2SO4 elution gradient. This new procedure permits, in a single run of 60 min, the quantitative analysis of S-adenosylmethionine, S-adenosylhomocysteine (AdoHcy), 5'-deoxy-5'-methylthioadenosine, decarboxylated S-adenosylmethionine, decarboxylated AdoHcy, inosylhomocysteine, and other related metabolites. Furthermore, this method allows the detection in rat tissues of novel sulfur metabolites, S-inosylhomocysteine and decarboxylated AdoHcy. Perturbation of the levels of some of these metabolites could be detected in rat livers and spleens after the administration of 3-deazaadenosine, an inhibitor of AdoHcy hydrolase, but could not be detected in rat adrenal glands. It is notable that decarboxylated AdoHcy disappeared in the livers of rats treated with 3-deazaadenosine. HeLa cells incubated with [35S]methionine displayed the incorporation of the labeled sulfur into S-adenosylmethionine, AdoHcy, decarboxylated S-adenosylmethionine, S-inosylhomocysteine, and 5'-deoxy-5'-methylthioadenosine.

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d-Tryptophan was converted to l-tryptophan in tissue cultures of tobacco, in whole cells treated with dimethylsulfoxide, and in cell-free extracts treated by Sephadex G-25 filtration. Evidence was obtained that tryptophanase, tryptophan pyrrolase, and transaminase activities were not involved. The data were best explained by the presence of a tryptophan racemase as the enzyme catalyzing the reaction. The possible role of d-tryptophan in the biosynthesis of indoleacetic acid is discussed.

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A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.

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A 6-(gamma,gamma-dimethylallylamino) purine-like compound was found in the culture medium of Rhizopogon roseolus, which had been shown earlier to synthesize zeatin. The role of 6-(gamma,gamma-dimethylallylamino) purine as a precursor of zeatin was studied. Rhizopogon was furnished with 6-(gamma,gamma-dimethylallylamino) purine-8-(14)C. Cochromatography, oxidation studies with potassium permanganate, and bromination indicated that labeled zeatin ribonucleoside was isolated from the medium. The fungus also incorporated labeled adenine, hypoxanthine, and 4-amino-5-imidazole carboxamide into zeatin ribonucleoside.