Allylamine and beta-aminopropionitrile-induced vascular injury: an in vivo and in vitro study.

Toxic cardiovascular effects of allylamine and beta-aminopropionitrile were studied in adult male Sprague-Dawley rats given allylamine alone (AA), 100 mg/kg/day, beta-aminopropionitrile alone (beta APN), 1 g/kg/day, or both chemicals (AA + beta APN) by gavage. Rats were given a total of 10 doses in 11 days. Rats given AA + beta APN showed extensive smooth muscle cell necrosis of the aortic media not seen when either toxin was given alone. Lingual artery lesions in the form of small intracellular eosinophilic globules were seen in animals given AA and AA + beta APN treatments, but were more numerous and larger in the latter group by morphometric analysis (p less than 0.03). Myocardial necrosis was much less severe in the AA + beta APN treatment group than in rats given only AA. A long-term follow-up (47 and 180 days) after the AA + beta APN protocol above showed that rats had persistent aortic medial necrosis with striking intimal cartilaginous metaplasia. Cultured porcine aortic smooth muscle cells exposed in vitro to combined AA and beta APN showed markedly decreased viability and increased cell injury when compared to cells exposed to only one toxin, thus supporting the synergistic toxic effect seen in vivo. Our studies show a synergistic necrotizing effect of AA and beta APN on aortic vascular smooth muscle cells. A hypothesis concerning these compounds' effects on vascular amine oxidases is made to explain this toxic synergism. Synergistic toxic interactions may be important in other forms of vascular injury.

A role for a new vascular enzyme in the metabolism of xenobiotic amines.

Although it has long been thought that environmental toxins may play an underlying role in vascular diseases such as atherosclerosis, this concept is not supported by any clear-cut experimental evidence of toxic metabolism by cardiovascular enzymes. In this study, we demonstrate that allylamine, a selective cardiovascular toxin in vivo, is actively metabolized in vitro by a purified vascular enzyme (semicarbazide-sensitive amine oxidase), which has been localized recently to vascular smooth muscle cells. Oxidative deamination of allylamine to a highly toxic aldehyde, acrolein, was blocked through enzyme inhibition by semicarbazide-sensitive amine oxidase suggests that this vascular enzyme's physiological role may include metabolism of exogenous amines.

Role of benzylamine oxidase in the cytotoxicity of allylamine toward aortic smooth muscle cells.

In this study we demonstrate that by inhibiting benzylamine oxidase (BzAO) with either semicarbazide or phenelzine, aortic smooth muscle cells (ASMCs) are protected from cytolethal injury by the cardiovascular toxin allylamine. We find that although both semicarbazide and phenelzine inhibit BzAO or ASMCs grown in vitro, phenelzine is the more effective inhibitor. We further demonstrate that although semicarbazide--at concentrations inhibiting BzAO--protects ASMCs from cytolethal concentrations of allylamine, it does not fully protect ASMCs from sublethal injury as assessed by [3H]uridine uptake. In contrast, phenelzine appears to afford complete protection of ASMCs from allylamine injury. Although semicarbazide and phenelzine pretreatment does not interfere with [14C]allylamine uptake by ASMCs, retention time of the 14C-moiety from radiolabeled allylamine is less in pretreated ASMCs. Subcellular distribution studies of ASMCs exposed to [14C]allylamine demonstrate that inhibiting BzAO activity in ASMCs results in marked derangement of the distribution pattern of 14C-moiety in subcellular fractions of ASMCs, with 14C-moiety not localized to mitochondrial/endoplasmic reticulum enriched fractions.

Purification of benzylamine oxidase from cultured porcine aortic smooth muscle cells.

Soluble benzylamine oxidase (BzAO) from cell homogenates and the conditioned culture medium of porcine aortic smooth muscle cells was purified by anionic HPLC methods and characterized with regard to enzyme kinetics and inhibition by semicarbazide, phenelzine, cuprizone, diethyldithiocarbamate (DDC), and p-chloromercuriphenylsuphonate (PCMPS). BzAO from both the cell homogenates and the conditioned culture medium had an Mr of 130,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using [methylene-14C]benzylamine hydrochloride as substrate, BzAO from cell homogenates and the conditioned culture medium had Km values of 5.1 and 6.1 microM, respectfully, and Vmax values of 89 and 53 nmol.mg protein-1.h-1. Both enzymes were sensitive to inhibition by semicarbazide and phenelzine, but insensitive to inhibition by the copper chelating agent DDC. BzAO isolated from the conditioned culture medium was more sensitive to inhibition by lower concentrations of cuprizone and PCMPS than the enzyme isolated from cell homogenates. Antisera raised against BzAO from cell homogenates reacted with BzAO from the conditioned culture medium and from porcine plasma.

In vitro expression of benzylamine oxidase activity in cultured porcine smooth muscle cells.

Benzylamine oxidase (BzAO) activity was measured in cultured smooth muscle and endothelial cells derived from thoracic aorta of adult swine, and in homogenates of dissected aortic medial and intimal layers. BzAO appeared to be localized in aortic medial homogenates. Cultured smooth muscle cells (which were verified by electron microscopic characteristics and positive staining with antimyosin) showed high BzAO activity, with optimal activity at pH 7.0; no activity was detected in endothelial cells. Prolonged serial passage of smooth muscle cells resulted in increased BzAO activity, with the increase most marked by the 12th population doubling. Also, BzAO was found predominantly in the soluble portion of the culture medium of smooth muscle cells by their 12th population doubling. These findings support the concept that vascular BzAO is localized within medial smooth muscle cells, and suggest that smooth muscle cells may be capable of secreting BzAO.

Allylamine cardiovascular toxicity.

The chemistry, industrial usage, general toxicity, and experimental use of allylamine are briefly reviewed. This highly reactive unsaturated alkylamine has had industrial applications in a variety of organic processes, and continues to be utilized, although accurate data concerning production is not readily available. The general toxic effects of the freebase form are primarily related to irritation of the mucous membranes, whereas the relatively long history of experimental use of this chemical has emphasized its' extraordinarily deleterious effects on heart and vascular tissue. Allylamine has been given by a variety of routes to many species in attempts to cause lesions which mimic human acute vasculitis, acute myocardial necrosis, and atherosclerosis; examples of typical lesions are illustrated. More recent in vivo and in vitro experimental work concerning the cellular toxicity of allylamine are summarized, and possible mechanisms of this chemicals' toxic action are discussed.

Binding of [14C] allylamine to isolated mitochondria from rat heart and aorta.

This study demonstrates specific and saturable binding of [14C] allylamine to mitochondria derived from rat aorta and heart. Specific binding is linear with respect to mitochondrial concentration and has a pH optimum of 7.0. Saturation isotherms reveal anomalous kinetics of specific binding on heart mitochondria with a high affinity site (KD 16 nM) and a lower affinity site (KD 80 nM); Scatchard plots have a common intercept. Exhaustive flow dialysis in the presence of SDS demonstrates that as much as 23.5% of bound radioactive moieties in aorta mitochondria are covalently bound, and as much as 42.6% are covalently bound in heart mitochondria. Hydrolysis of heart mitochondria with phospholipase C markedly enhances saturation of [14C] allylamine, and greatly increases the quantity of covalently bound radioactive ligand. Phospholipase C hydrolysis of heart mitochondria increased monoamine oxidase B activities and unmasked a small amount of benzylamine oxidase activity, whereas hydrolysis of mitochondria with phospholipases A2 and D diminish MAO-B activity. The monoamine oxidase B inhibitor, deprenyl, significantly reduced both specific and covalent binding of the 14C-activity from [14C] allylamine to phospholipase hydrolyzed mitochondria. The benzylamine oxidase inhibitor, phenelzine, significantly decreased specific binding but had no effect on the degree of covalent binding of [14C] allylamine to phospholipase C hydrolyzed mitochondria. The benzylamine oxidase inhibitor, semicarbazide, had no effect in inhibiting [14C] allylamine binding. Covalent binding of 14C-moiety from [14C] allylamine to mitochondria--which express specific binding sites for the [14C] allylamine--and inhibition of binding by monoamine oxidase inhibitors, suggest the formation of highly reactive intermediates.

Comparative toxicity of the cardiovascular toxin allylamine to porcine aortic smooth muscle and endothelial cells.

This study supports a recent hypothesis that the cardiovascular toxin, allylamine, is toxic to smooth muscle cells of large elastic arteries (aorta). Cultures of the porcine aortic smooth muscle, endothelial, and fibroblastic cells were exposed to varying concentrations of allylamine ranging from 5 microM to 340 microM. Monitored cytotoxic and cytolytic activities demonstrated that final concentrations of 60 microM allylamine decreased cell population viability of smooth muscle cells as much as 50%. Viability decreased approximately linearly with increasing concentrations of allylamine including spontaneous lysing of smooth muscle cells at 90 microM. Endothelial cells were more resistant to lower concentrations of allylamine requiring 90 microM to decrease cell population viability by 50%. In contrast, fibroblastic cells were very resistant to lower concentrations of allylamine. The specific lytic response of these cells in culture, measured by release of [3H]thymidine, gave findings parallel to the viability studies, i.e. at 100 microM allylamine smooth muscle cells demonstrated 75% specific lysis while endothelial cells showed 29%. Growth studies of cells surviving an 8-h exposure to allylamine indicate that surviving endothelial cells have better growth characteristics than surviving smooth muscle cells; both cell lines are also apparently injured at concentrations of allylamine much lower than the CT50. These studies show that of the cellular components of the vascular wall, smooth muscle cells appear to be the most sensitive to the toxic effects of allylamine.

Phospholipase A enzymes of Entamoeba histolytica: description and subcellular localization.

Cytolysis of target cells by Entamoeba histolytica is a rapid, contact-dependent event that appears to involve calcium and amebic phospholipase A enzymes. There are two phospholipase A enzymes of E. histolytica: one calcium independent and optimally active at an acid pH, and a second calcium dependent and most active at an alkaline pH. The amebic calcium-dependent enzyme was inhibited by pharmacological antagonists that have been shown to reduce cytolysis of target cells by intact amebae: phosphatidylcholine (10(-3) M), Rosenthal's inhibitor (dimethyl-dl-2,3-distearoylpropyl-2'-hydroxyethyl ammonium acetate; 10(-4) M), quinacrine (10(-4) M), and hydrocortisone (10(-4) M). Calcium-independent phospholipase A activity was inhibited by Rosenthal's inhibitor (10(-4) M) and quinacrine (10(-3) M). The calcium-dependent phospholipase A enzyme is highly associated with plasma membrane fractions; the calcium-independent enzyme is predominantly found in soluble fractions. These findings further suggest an association of calcium-dependent phospholipase A activity with cytolytic activity of E. histolytica.

Allylamine cardiovascular toxicity: VI. Subcellular distribution in rat aortas.

The cardiovascular toxin allylamine (3-aminopropene) has been shown to concentrate in elastic and muscular tissues. In this study the 14C-moiety of [14C]allylamine was traced in aortas of adult Sprague-Dawley rats after intravenously injecting 30 microCi of [14C]allylamine (spec. act. = 0.4 mCi/mM). At 5, 10, 15 and 20 min after injection 33.3-29.8% of the 14C-moiety was sequestered in aortas; at 30 min 16.8% was still present. Subcellular fractionation of the postnuclear supernatant by isopycinic centrifugation in sucrose demonstrated that 5 min after administration of [14C]allylamine, the 14C-moiety displayed a modal density peak of 1.20 g/ml. Similar activities were observed up to 30 min exposure. This modal density was similar to the distribution pattern of mitochondria based on analysis of malate dehydrogenase activities. As early as 20 min post-exposure, mitochondrial malate dehydrogenase activities of aortic mitochondria decreased, while cytosolic malate dehydrogenase activities increased, suggesting mitochondrial membrane perturbation. We suggest that the subcellular site for allylamine injury to the aorta is the mitochondrion.

Comparison of the Cathra Repliscan II, the AutoMicrobic system Gram-Negative General Susceptibility-Plus Card, and the Micro-Media System Fox Panel for dilution susceptibility testing of gram-negative bacilli.

A comparative evaluation was done to test the accuracy of the Cathra Repliscan II agar dilution system (Diagnostic Equipment, Inc., St. Paul, Minn.), the AutoMicrobic system with Gram-Negative General Susceptibility-Plus Card (Vitek Systems, Inc., Hazelwood, Mo.), and the Micro-Media Fox Panel micro broth dilution system (Micro-Media Systems, Inc., San Jose, Calif.) in determining MICs of 12 antibiotics for 200 gram-negative bacilli. Of the 200 strains tested, 12 isolates did not grow in one of the three systems. The 188 remaining organisms included 158 members of the family Enterobacteriaceae, 20 Pseudomonas spp., 5 Acinetobacter sp., 3 Aeromonas spp., and 2 Vibrio spp. A total of 2,256 organism-antibiotic combinations were analyzed for each system. An MIC was considered correct if two of the three systems were in agreement. When disagreements occurred, correct MICs were determined by the standard agar dilution method. With this criterion, overall agreements of the Cathra Repliscan II system, AutoMicrobic system, and Micro-Media Fox Panel system were 94.7, 94.9, and 95.5%, respectively. Tetracycline (20%), nitrofurantoin (20%), and ampicillin (16%) accounted for 56% of the discrepancies observed. These results indicate that all three systems perform with a high degree of accuracy for susceptibility testing of gram-negative bacilli.

Degradation of human myelin phospholipids by phospholipase-enriched culture media of pathogenic Naegleria fowleri.

Cell-free media from cultures of virulent Naegleria fowleri were tested for phospholipase activities and their ability to degrade phospholipids of human myelin. Virulent N. fowleri selectively released lipolytic enzymes into the media at various times during growth and hydrolyzed the phospholipids of human myelin, while media from virulent-attenuated and nonpathogenic Naegleria spp. were almost totally inactive. Hydrolysis of myelin phospholipid increased concomitantly with amebal growth, and the relative rate of breakdown at pH 7.5 was sphingomyelin greater than phosphatidylcholine greater than phosphatidylethanolamine. Elevated levels of lysophosphatidylcholine and lysophosphatidylethanolamine were also noted.

Elevated levels of cellular and extracellular phospholipases from pathogenic Naegleria fowleri.

Phospholipase A, sphingomyelinase and lysophospholipase activities were examined in cell homogenates and cell-free culture media of virulent and virulent-attenuated Naegleria fowleri and nonpathogenic Naegleria gruberi. Homogenates of virulent N. fowleri contained from 3 to 250 times the lipolytic activity of virulent-attenuated and non-pathogenic Naegleria spp. Similarly, the cell-free media of virulent N. fowleri cultures contained large quantities of phospholipase A, lysophospholipase and sphingomyelinase while comparable activities in the cell-free media of virulent-attenuated and nonpathogenic Naegleria spp. were only slightly, if at all, detectable. Lipolytic enzymes accumulated in the media of virulent N. fowleri cultures at various stages during growth but not in virulent-attenuated and nonpathogenic Naegleria cultures. In general, phospholipase A and sphingomyelinase accumulated during the log phase of growth while lysophospholipase appeared only in the late stationary phase. We conclude that pathogenic Naegleria contain potent lipolytic enzymes that are released selectively into the media during growth. These enzymes could contribute to the pathogenesis of Naegleria-induced primary amoebic meningoencephalitis.