Comparative binding and toxicity of saxitoxin and saxitoxinol in mice and in cultured cells.

The binding characteristics of saxitoxin (STX), a known voltage-gated sodium channel blocker, and its analog saxitoxinol (STXOL), were studied in neuroblastoma, peritoneal macrophage, hepatocytes and PC-12 cell lines. 3H-STXOL bound to the cell-surface sites which appear to be the same as those occupied by 3H-STX and which can, therefore, be identified as STX receptors. The relative agreement of respective Kd obtained by saturation, competition, association and dissociation kinetics for STX and STXOL suggest the absence of any artifact in binding measurements. Unlike STX, STXOL was non-toxic to mice by intratracheal instillation. The major advantage of using 3H-STXOL is that the tritium label is not exchangeable. Data from this study suggest that 3H-STXOL can be used to identify STX receptors at 37 degrees C.

Toxicokinetics of [3H]saxitoxinol in peripheral and central nervous system of rats.

This study evaluated the toxicokinetics of a saxitoxin (STX) analog, [3H]saxitoxinol (STXOL), in rats. [3H]saxitoxinol (18.9 microCi/kg body weight) was administered iv to male Wistar rats via the penile vein. After injection, [3H]STXOL disappeared rapidly from plasma (t 1/2 = 29.3 min), and 75% of the radiolabel was cleared from plasma within 2 hr. Radioactivity associated with red blood cell membranes was inversely related with the radioactivity associated with hemoglobin, suggesting internalization of STXOL. Distribution of [3H]STXOL, 48 hr after iv exposure, showed that muscle tissues retained 91.2 +/- 7.1%, liver 63.7 +/- 3.8%, heart 17.4 +/- 1.6%, and lung 9.2 +/- 0.8% of the residual dose. High-performance liquid chromatography (HPLC) analysis for saxitoxinol showed three to four major radiolabeled peaks for each of these tissues. By 48 hr, radiolabel associated with the saxitoxinol peak decreased 95% in lungs, heart, and kidneys, with a concomitant increase in unidentified, more polar peaks. No STXOL metabolites were detected in the urine from these animals. Radioactivity accumulation in the brain increased to a maximum of 162% at 8-hr postexposure, compared to values obtained at 10 min, then gradually declined to 148% by 48 hr. HPLC analysis of brain extracts showed that the relative percentage of radioactivity associated with parent toxin gradually decreased, with a concomitant rise in the levels of more polar peaks. Similar results were obtained for spinal cord. The data suggest that saxitoxinol was rapidly cleared from most of the peripheral organs and was little, if any metabolized by muscle cells.

A high-performance liquid chromatographic method for determining [3H]T-2 and its metabolites in biological fluids of the cynomolgus monkey.

High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate the trichothecin mycotoxin, T-2 (4 beta,15-diacetoxy-3 aopha-hydroxy-8 alpha [3-methyl-butyryloxy]-12,13-epoxy delta 9-trichothecin), and its metabolites in plasma and urine samples from cynomolgus monkeys treated with the toxin. A 15-min gradient elution system was developed to separate and measure radiolabeled T-2 mycotoxin and its metabolites. The HPLC technique for separating T-2 and its metabolites was compared with thin-layer chromatography. Samples from the in vitro metabolism of T-2 by plasma and urine were included as controls and as a measure of the toxin's stability in biological samples. Within 5 min, 22% of the plasma radiolabeled T-2 toxin was detected as metabolites after an intravenous administration of [3H] T-2 toxin to cynomolgus monkeys. By 24 h post-exposure, there was no parent T-2 toxin detected in plasma or urine. T-2 tetraol was the major metabolite detected in the plasma and urine of monkeys. Other metabolites observed in urine up to 5 days after exposure were 3'OH-T-2 and 3'OH-HT-2. We conclude that T-2 toxin was rapidly metabolized to more polar metabolites, which were eliminated in urine.

Oxidation of saxitoxin: detection and biological activity of its reaction products.

We characterized the oxidation products of saxitoxin by high-performance liquid chromatography and thin-layer chromatography. We observed several changes in the saxitoxin and neosaxitoxin chromatographic profile, as well as changes in the biological activities of the saxitoxin oxidized product. Toxins were heated in the presence of oxidizing agent, and at least five oxidation products were detected. Spectrophotometric measurements with Folin-Ciocalteau reagent confirmed the formation of these oxidation products. The excitation maximum of each of the oxidized components was determined by spectrophotofluorometry scans, and differed from the parent compound. The oxidized saxitoxin inhibited 3H-saxitoxin binding to neuroblastoma-glioma hybrid cells (NG108-15) to a lesser extent than saxitoxin, and had no toxic effect in mice. Formation of oxidation products suggests the possible, non-enzymatic transformation of saxitoxin and neosaxitoxin.

Effect of anti-inflammatory agents on ricin-induced macrophage toxicity.

The toxicity of ricin in susceptible cells is well characterized biochemically, but the pathophysiological implications of its toxicity and the immune response to ricin challenge in the lung are unknown. Incubating macrophage cell line with ricin (1 pM-10 nM) for 4 hours markedly inhibited 3H-leucine incorporation (acid insoluble) into protein (> 95%, at 1 nM) without affecting the acid-soluble radioactivity. In spite of increased uptake of total thymidine (141 +/- 13.5%) and total uridine (135 +/- 17.2%), DNA synthesis in ricin-treated cells was progressively inhibited although RNA synthesis was not affected. Fluocinolone (an anti-inflammatory glucocorticoid) pretreatment increased the ricin-induced inhibition of protein synthesis. The synergistic effect of fluocinolone on ricin-induced protein synthesis inhibition was due to an increased binding (167%, p < 0.01) and internalization (134 +/- 12%, p < 0.025) of ricin. Partial protection from ricin-induced inhibition of protein synthesis by indomethacin (nonsteroidal, anti-inflammatory agent) was due to decreased binding and internalization of ricin. These results show that macrophages are sensitive to ricin and that pharmacologically active drugs may regulate ricin's toxicity, perhaps by controlling synthesis and release of certain mediators of fast death.

[3H]-saxitoxinol metabolism and elimination in the rat.

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

The role of calcium ions for the expression of ricin toxicity in cultured macrophages.

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca(2+)-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.

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.

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids.

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.

Effect of organophosphates on dopamine and muscarinic receptor binding in rat brain.

The effect of Soman, Sarin and Vx, known potent cholinesterase inhibitors, on the binding of several neurotransmitter receptors in various regions of brain was studied. Vx, exhibited considerable inhibition of binding of 3H-N-methylscopolamine (3H-NMS) to muscarinic receptors and of 3H-spiperone to dopamine D2 receptors in the striatum. 3H-NMS binding was 50% inhibited at 10(-6)M and 90% at 10(-3)M Vx. Inhibition of 3H-spiperone binding by Vx in striatum had an ID50 of 10(-5)M. KD of the treatment was affected more than Bmax. Binding inhibition of both 3H-NMS and 3H-spiperone in post-mortem brain of rats pre-treated with Vx confirmed the specificity of the organophosphates effect, since other organophosphates and ligands failed to show any activity.

Effect of toxins on arachidonic acid metabolism in rat cultured pulmonary alveolar macrophages.

The action of a trichothecene (T-2), microcystin-LR and saxitoxin on arachidonic acid metabolism in cultured rat alveolar macrophages was studied. Pulmonary macrophages exposed to T-2 trichothecene were stimulated to synthesize and release large amount of thromboxane B2 (TxB2) and 6-Keto F1 alpha. Microcystin-LR induced significant release of prostaglandins F2 alpha (140%), PGE2 (175%) and TxB2 (169%) compared to controls. Saxitoxin induced TxB2 release by 37%. Arachidonic acid release was stimulated by all three toxins. The release of arachidonic acid and its metabolites in alveolar macrophages exposed to T-2 toxin was partially blocked by fluocinolone (1 microM). These results suggest that macrophages synthesize and release inflammatory mediators in response to toxin exposure, and fluocinolone may protect against T-2 toxicosis.

Thyrotropin-releasing hormone effects on phosphatidylinositol metabolism and binding in rat pituitary and retina.

This paper reveals possible similarities in the thyrotropin-releasing hormone (TRH) effects on phospholipid metabolism in pituitary and retina of the rat central nervous system. Addition of the methylated analog (MeTRH) resulted in 171 +/- 16% and 88 +/- 10% increase in 32PO4 incorporation into phosphatidylinositol (PI) in pituitary (20 min incubation) and retina (60 min incubation), respectively. There was a similar significant increase in phosphatidic cid, but with no change in phosphatidylcholine or other classes of phospholipids. The effect was concentration-dependent and the ED50 also was close to KD, suggesting the response was regulated by MeTRH receptors in membranes of both pituitary and retina.

Sex mediated lipid metabolism in human aortic smooth muscle cells.

When human aortic smooth muscle cells in culture were treated with pharmacological doses of estrogen and testosterone for 48 hrs, the rate of cholesterol synthesis measured both by acetate incorporation and the 3, hydroxy 3-methylglutaryl Co enzyme A reductase (HMG-CoA) activity declined significantly as compared to control. However, the rate of cholesterol esterification increased by 132% and 45% in response to testosterone and estrogen respectively. Also, acetate incorporation into fatty acids and fatty acid synthetase enzyme activity increased by hormonal treatment but remained in the free form especially by estrogen. Testosterone treatment resulted in more esterification (p less than .025) of fatty acid than estrogen treatment. Incubation with hormones for 48 hrs resulted in enhanced uptake of 14C-labeled cholesterol along with increased accumulation of cellular cholesterol. Increased synthesis of phospholipid and triglyceride by estrogen may be responsible for excretion of cellular sterol and fat. These results indicate that sex-hormones have an important effect on the regulation of lipid metabolism in human aortic cells.

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells.

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.

Cardiac sarcoplasmic reticulum. Effects of an atherogenic diet during the neonatal and juvenile period.

The effect on the cardiac sarcoplasmic reticulum of an atherogenic (1% cholesterol) diet fed during the neonatal vs the juvenile period of life was studied in Yorkshire swine. Male piglets were randomly assigned at birth to 1 of 4 groups: group I (control), group II (lactation feeding), group III (juvenile period feeding) and group IV (lactation and juvenile feeding). All animals were killed at 55 weeks of age and cardiac sarcoplasmic reticulum (SR) isolated for assay of calcium uptake, Ca2+-Mg2+ ATPase activity, and lipid analysis by thin-layer chromatography and gas chromatography. The amount of cholesterol/mg SR protein and the cholesterol/phospholipid ratio were higher in the animals fed during lactation (groups II and IV) and lower in those fed only during the juvenile period (group III). Phospholipid fatty acid patterns as measured by gas chromatography were unaltered in any group. Calcium uptake was markedly diminished in all experimental conditions: group II 47%, group III 65% and group IV 96%. Compared to the observed changes in calcium transport, the ATP hydrolytic activity was relatively less affected. Only in group IV a significant decrease (41%) was seen. Groups II and III show no change in ATP hydrolytic activity. The decrease in calcium uptake and altered cholesterol/phospholipid ratio without effect on ATP hydrolytic activity is consistent with an uncoupling of calcium transport related to the atherogenic diet in early life.

Lung aryl hydrocarbon hydroxylase: inhibition following cadmium chloride inhalation.

Polycyclic hydrocarbon metabolism in male Sprague-Dawley rats following inhalation of aerosolized cadmium chloride (CdCl2) was examined. Constitutive activity of microsomal aryl hydrocarbon hydroxylase (AHH) (benzo(a)pyrene substrate) was monitored in lung and liver homogenates up to 10 days after exposure. Lung AHH activity was reduced by 85% during the first 2 days following cadmium inhalation, and did not return to normal levels until 7 days after exposure. Enzyme activity in the livers of cadmium-treated animals was similarly depressed (by 65%) within 24 hr. Cadmium inhalation also inhibited (by 50%) 3-methylcholanthrene (MC) induction of lung AHH when compared with MC-treated controls. No significant effect on AHH inducibility by MC was noted in liver homogenates from cadmium-exposed animals. Nonspecific microsomal damage appeared not to occur since glucose-6-phosphatase activity in lung was unaffected by cadmium treatment. Although the mechanism of cadmium's action remains unclear, it appears not to involve a direct interaction of the metal with enzyme. The alteration of AHH activity by cadmium may result from injury to a specific cell type within the lung, which may be a major site of pulmonary AHH activity, or may result from modulation of synthesis and/or degradation of heme proteins in the lung. These results suggest that cadmium, under these conditions, markedly reduces the constitutive and inducible activity of AHH in the lung.

Disulfiram-associated hypercholesterolemia.

The rat model is used to verify disulfiram-associated hypercholesterolemia and to determine a mechanism of action. Administration of disulfiram 15 mg/kg/day for 3 wk is associated with a 25% increase in serum cholesterol which is reversible with discontinuance of the drug. The hypercholesterolemia is due in part to a fourfold increase in activity of hepatic HMG-CoA reductase, the rate-limiting step in cholesterol biosyntheses.

The influence of dietary cholesterol and fat on the homeostasis of cholesterol metabolism in early life in the rat.

Evidence has accumulated suggesting that exposure to dietary cholesterol during early developmental stages activates biochemical mechanisms that regulte cholesterol homeostasis in adult life. Pregnant Sprague-Dawley rats were fed control or 1% cholesterol [high cholesterol-high fat (HC-HF)] liquid diet during gestation, and with the same diet, offsprings were challenged at maturity. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity declined by 76%, and cholesterol 7 alpha-hydroxylase activity increased by 200%. Microsomal and serum cholesterol accumulation also increased significantly. Exposure to HC-HF diet during suckling and continued exposure with the same diet postweaning resulted in 52% decline in 3-hydroxy-3-methylglutaryl coenzyme A reductase and 280% increase in 7 alpha-hydroxylase activity. Serum cholesterol level is not affected although microsomal cholesterol accumulation and the ratio of hepatic to serum cholesterol increased significantly in the experimental group when compared to the nonchallenged group. Rats exposed to HC-HF diet during lactation alone or during gestation and lactation and then challenged demonstrated enzymatic changes of similar magnitude. Serum and microsomal cholesterol levels also increased significantly by 126 and 150%, respectively. The activity of the HMG-CoA reductase system appears to be more sensitive to modulation by diet during gestation than during lactation. However, 7 alpha-hydroxylase activity is susceptible to dietary manipulation with HC-HF diet only during lactation.

Seasonal changes in the blood parameters of two major carps, Labeo rohita (Ham.) and Cirrhina mrigala (Ham.).

Seasonal changes in blood properties of two Indian major carps, L. rohita and C. mrigala, were investigated. Haematocrit values, haemoglobin concentrations and erythrocyte numbers showed seasonal fluctuations, being high from September to November and again in April-May, and low from January to March. Males had generally higher blood values than the females.